Lab Manual 1
Lab Manual 1
MICROBIOLOGY
S ECOND EDITION
by
EDITED BY
ISBN 978-955-8891-03-2
SECTION 1. BACTERIOLOGY
A. Processing of Clinical Specimens
1.1 Blood and bone marrow cultures 1
1.2 Intravascular catheters 12
1.3 Cerebrospinal fluid (CSF) 16
1.4 Sterile fluids other than CSF 25
1.5 Urine 30
1.6 Pus & wound swabs 39
1.7 Ear swabs/aspirates 46
1.8 Eye swabs and other specimens from eye 50
1.9 Lower respiratory tract specimens 55
1.10 Throat swabs 62
1.11 Swabs for diphtheria 65
1.12 Uro-genital specimens 68
1.13 Vaginal discharge 75
1.14 Faeces 78
1.15 Specimens for cholera 84
1.16 Screening for Group B streptococcal carriage 88
1.17 Screening for MRSA and Staphylococcus aureus 91
1.18 Specimens for anaerobic bacteria 94
B. Identification of organisms
1.19 Enterobacteriaceae 98
1.20 Haemophilus 101
1.21 Staphylococcus 103
1.22 Streptococcus (excluding S.pneumoniae) and enterococcus 105
1.23 Streptococcus pneumoniae 108
i
D. Antibiotic sensitivity testing
1.32 CLSI (formerly NCCLS) method 119
E. Serology
1.34 Widal test (Standard Agglutination Test – SAT) 134
1.35 Anti Streptolysin O Titre (ASOT) 138
1.36 Other serology tests 140
F. Bacteriological media
1.37 Brain heart infusion broth (BHI) 142
1.38 Amies medium 143
1.39 Alkaline peptone water (APW) 144
1.40 Blood agar 145
1.41 Blood tellurite agar 146
1.42 Cary- Blair medium 147
1.43 Chocolate agar 148
1.44 Cystine lactose electrolyte deficient (CLED) agar 149
1.45 Cooked meat medium 150
1.46 Kligler iron agar (KIA) 151
1.47 MacConkey agar 152
1.48 Mannitol salt agar 153
1.49 Mueller – Hinton agar (MHA) 154
1.50 7% NaCl nutrient broth 155
1.51 Peptone water 156
1.52 Peptone water sugars 157
1.53 Selenite F broth 158
1.54 Salmonella - Shigella agar 159
1.55 Thiosulphate citrate bile salt sucrose (TCBS) agar 160
1.56 Thioglycollate broth 161
1.57 Christensen's urea broth 163
1.58 Xylose lysine desoxycholate agar (XLD) 164
G. Stains
1.59 Albert’s stain 165
1.60 Gram stain 166
1.61 Methylene blue stain 168
1.62 Ziehl- Neelsen stain 169
ii
SECTION 2. VIROLOGY
2.1 Guidelines for collection and transport of blood and CSF for 171
serology
SECTION 3. MYCOLOGY
SECTION 4. PARASITOLOGY
iii
C ONTRIBUTORS – B ACTERIOLOGY
D R . S A M A N M A L E E G U N A SE K A R A -CONSULTANT MICROBIOLOGIST,
GENERAL HOSPITAL KURUNEGALA.
iv
DR. DUSHANI JAYAWARDHANA -SENIOR REGISTRAR, COLOMBO SOUTH TEACHING HOSPITAL.
v
M R . S . A .L. P . S U R A W E E R A - TUTOR, SCHOOL OF MLT,
MEDICAL RESEARCH INSTITUTE, COLOMBO.
C ONTRIBUTORS - I MMUNOLOGY
DR. SEPALI GUNAWARDANE -CONSULTANT IMMUNOLOGIST, MEDICAL RESEARCH INSTITUTE,
(COORDINATOR) COLOMBO
C ONTRIBUTORS – P ARASITOLOGY
PROF. NILANTHI DE SILVA -PROFESSOR OF PARASITOLOGY, FACULTY OF MEDICINE,
(COORDINATOR) UNIVERSITY OF KELANIYA.
vi
C ONTRIBUTORS – V IROLOGY
DR. SUNETHRA GUNASENA -CONSULTANT MEDICAL VIROLOGIST,
(COORDINATOR) MEDICAL RESEARCH INSTITUTE, COLOMBO
P R O F . N .P . S U N I L -C H A N D R A -VIROLOGIST
C ONTRIBUTORS – M YCOLOGY
DR. PREETHI PERERA -CONSULTANT MYCOLOGIST,
(COORDINATOR) MEDICAL RESEARCH INSTITUTE, COLOMBO
vii
FOREWORD
Sri Lanka College of Microbiologists has once again contributed to an echoing need in the
laboratory service in the country by preparing the second edition of the ‘Laboratory
Manual in Microbiology’.
Today, healthcare activities are very much dependent on laboratory test results. Thus
reliable laboratory results have become an asset for patient management and control of
diseases in hospitals and community.
The guidelines provided by the second edition of the ‘Laboratory Manual in Microbiology’ is
timely and appropriate as patients seeking healthcare have increased in numbers, as well
as in their quest for quality. Therefore laboratory staff should be properly qualified and
correctly guided to elicit reliable laboratory data. In keeping with Mahinda Chinthana,
the Government has taken several actions to boost the tourism industry. Health-tourism
is one such area identified, where foreigners come for affordable and reliable medical care.
Our hospitals should be geared to meet such future challenges.
I take this opportunity to congratulate the College for a work well done.
Dr. U. A. Mendis
Director General of Health Services,
Ministry of Healthcare & Nutrition,
Colombo 10
viii
PREFACE
Microbiology laboratories play a vital role in screening and confirming the diagnosis of
infectious diseases both in the hospital settings as well as in the community. Data
generated are utilized for patient management and for disease specific control activities.
Almost ten years ago the Sri Lanka College of Microbiologists developed a ‘Laboratory
Manual in Microbiology’ for hospital laboratories as a standard practice guideline for
conducting most of the common and epidemiologically relevant tests in state and private
sector laboratories. Since then several developments have taken place in health-care
management in the country. Requirements and expectations for quality have become
wider, especially as laboratories are seeking accreditation for better performance. National
External Quality Assurance Schemes in Microbiology have brought about inter-laboratory
comparisons, placing less supervised laboratories at a disadvantage. The necessity of an
updated standard operating procedure guideline was strongly felt. The Sri Lanka College of
Microbiologists appreciating this current need undertook the task of revising and updating
the existing manual with the participation of all stake-holders. This manual is the result of
a collective collaboration of the microbiology stake-holders in the country. The manual will
be available for use by hospital laboratories, university laboratories, microbiologists, MLT
schools and post-graduate trainees in microbiology.
On behalf of the College I wish to express my sincere gratitude to all contributors and
especially the devoted editorial board comprising of Drs Kumudu Karunaratne, Malka
Dassanayake, Thamara Wijesuriya and Kanthi Nanayakkara together with Ms Priyanga
Opatha, secretary, SLCM who worked tirelessly to complete this large task.
I am very grateful to the Ministry of Health for providing the funds for printing of the
manual to be available for state sector laboratories and to Dr U. A Mendis, Director
General of Health Services for the continued support extended to the College.
November 2011
ix
BACTERIOLOGY
BLOOD AND BONE MARROW CULTURES
Bone marrow culture should be reserved for culture for specific pathogens such as Brucella, Salmonella,
Listeria, Mycobacteria and fungi. It adds a little to detection of most other bacteria in the blood. Routine
cultures of bone marrow without a specific indication should be discouraged.
Introduction
Laboratory diagnosis of bacteraemia and fungaemia depends on blood cultures, which are probably the
most important cultures performed by the microbiology laboratory. Because the culture methods are so
sensitive, the procedure of collection must be aseptically performed to prevent contamination of specimen
with skin commensals.
Drawing blood from two different sites will help to identify contamination. Ideally one set of blood culture
refers to one anaerobic blood culture bottle and aerobic blood culture bottle inoculated with blood from a
single venepuncture.
Likelihood of recovering a pathogen increases as the volume of blood increases. Optimal blood to broth
ratio of 1:5 to 1:10 should be maintained. Addition of SPS (Sodium Polyanethol Sulphonate) at a
concentration of 0.025 to 0.05% will increase isolation rate but it can be inhibitory to certain bacteria.
IMPORTANT:
Fungal blood culture
service is available at the When acute sepsis or another infection (osteomyelitis, meningitis,
Mycology Department, pneumonia, pyelonephritis) requires immediate institution of
MRI antimicrobial therapy, draw two blood cultures of maximum volume
Tel:011 2698725 from different anatomical sites before starting antibiotics.
REJECTION CRITERIA
In 1&2, inform ward / responsible clinician before discarding the specimen. Document persons
involved and action taken and request repeat sample.
Labeled blood cultures are not rejected even if medium is expired, volume or number of bottles is
insufficient, or bottles were received >12 hours after collection. Document deficiency, as well as the
effect on the reliability of culture result, in report. Educate the staff to ensure that cultures are collected
appropriately.
DAY 3-7 2. Re-incubate all bottles which are negative and inspect daily.
If evidence of growth detected, subculture and identify.
Further reports will be issued only on positives.
Note:
It is very important to follow the manufacturer’s instructions in processing the blood cultures by
automated blood culture systems.
Safety
Always wear gloves, because blood cultures may contain blood borne pathogens.
Take precautions to prevent needle stick injuries. Never recap sharps. Dispose needles and syringes
in puncture proof containers.
Incubation
For manual cultures, perform at least one blind subculture to solid agar from visually negative
bottles. This has to be performed essentially after overnight incubation.
Note: Blind subculture of automated systems has little clinical utility.
Coagulase test
Use fresh rabbit/ human plasma - haemolysis - haemolysis no haemolysis
(No MacConkey growth) Group- if grouping kit available
optochin on blood Bacitracin 0.04 U
agar plate
Positive Negative
R S
S R S. pyogenes
S. pneumoniae Perform bile solubility
Staphylococcus ** Coagulase negative
aureus staphylococcus (CNS) Bile soluble Bile insoluble
S. pneumoniae viridans streptococci
MacConkey Agar
no growth
growth+
Haemophilus spp.
Lactose Fermenter (LF) Non Lactose Fermenter (NLF) Blood agar- satellitism
Do identification with Oxidase
API 20 E if available or
Biochemical tests IMVC + Motility
Negative Positive Identify
using x,v &
xv discs
Gram stain
If these are not available
Report as coliform Gram negative bacilli Negative cocci/ coccobacilli
KIA no reaction
ABST
Do primary sensitivity if organisms are seen in Gram stain of broth culture. Repeat ABST following day
with isolate.
REPORTING PROCEDURE
Microscopy Microscopy results should be informed to the ward by telephone if positive.
Note:
a) Infective endocarditis
Most likely isolate: viridans streptococci
Do not do routine sensitivity testing.
MIC for penecillin required – if facilities are not available, contact MRI.
b) ABST should be done and recorded on all blood culture isolates. However,
coagulase negative staphylococci, diphtheroids, aerobic spore bearers are
frequent contaminants. Inform MO to check on clinical details. Report ABST
only if clinically significant.
Coliforms ( Enterobacteriaceae)
1st line 2nd line
Ampicillin Cefepime
Gentamicin Ticarcillin-clavulanic acid
Amoxicillin-clavulanic acid Piperacillin-tazobactam
Cefuroxime Imipenem
Ciprofloxacin Meropenem
Netilmicin Amikacin
Cefotaxime or ceftriaxone Aztreonam
Ceftazidime Ertapenem
Important:
Screening and confirmatory tests for ESBLs in Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca
and Proteus mirabilis are indicated.
Screening and confirmatory tests for suspected carbapenemase production is indicated.
Acinetobacter spp.
1st line 2nd line
Gentamicin Ticarcillin-clavulanic acid
Ceftazidime Piperacillin-tazobactam
Cefepime Amikacin
Ciprofloxacin Imipenem
Trimethoprim-sulfamethoxazole Meropenem
Tetracycline Cefoperazone – sulbactam
Netilmicin Ampicillin - sulbactam
Colistin – recommended method in CLSI is MIC
Burkholderia pseudomallei
Amoxicillin – clavulanic acid Doxycycline
Ceftazidime Tetracycline
Imipenem Trimethoprim – sulfamethoxazole
Burkholderia cepacia
Ceftazidime
Ticarcillin – clavulinic acid – recommended method in CLSI is MIC
Levofloxacin - recommended method in CLSI is MIC
Meropenem
Trimethoprim – sulfamethoxazole
Chloramphenicol - recommended method in CLSI is MIC
Stenotrophomonas maltophilia
Ceftazidime- recommended method in CLSI is MIC
Ticarcillin – clavulinic acid - recommended method in CLSI is MIC
Chloramphenicol - recommended method in CLSI is MIC
Levofloxacin
Trimethoprim – sulfamethoxazole
Staphylococcus spp.
1st line 2nd line
Penicillin Ciprofloxacin
Cefoxitin /oxacillin / methicillin (preferably Vancomycin ( recommended method in CLSI is MIC)
cefoxitin) - report as cloxacillin Teicoplanin
Erythromycin Tetracycline
Clindamycin Chloramphenicol
Trimethoprim-sulfamethoxazole Gentamicin
Linezolid
Daptomycin – recommended method in CLSI is MIC
Important:
Screening tests for ß – lactamase production, detection of MRSA and inducible clindamycin resistance in
Staphylococcus spp. and ß – haemolytic streptococcci are indicated
Enterococcus spp.
1st line 2nd line
Penicillin Tetracycline
Ampicillin Vancomycin
Gentamicin 120 µg – for high level resistance Linezolid
screening in endocarditis Teicoplanin
Daptomycin – recommended method in CLSI is MIC
Important:
Testing ß lactamase production is indicated in selected cases
Screening tests for high – level aminoglycoside resistance (HLAR) and vancomycin resistance may be
indicated
Streptococcus pneumoniae
1st line 2nd line
Oxacillin (report as penicillin) Vancomycin
Erythromycin Clindamycin
Cefotaxime/Ceftriaxone Tetracycline
Co-trimaxazole Linezolid
Chloramphenicol Levofloxacin
Meropenem
Important:
Isolates of pneumococci with oxacillin zone sizes of ≥ 20 mm are susceptible to penicillin (MIC ≤ 0.06
µg/ml).
Penicillin and cefotaxime or ceftriaxone or meropenem MICs should be determined for those isolates with
oxacillin zone diameter ≤ 19 mm because zones of ≤ 19 mm diameter occurs with penicillin resistant,
intermediate or certain susceptible strains.
For isolates with oxacillin zones ≤ 19 mm, do not report penicillin as resistant without performing a
penicillin MIC.
β-haemolytic Streptococcus
Penicillin Clindamycin
Erythromycin Cefotaxime or ceftrixone
Important:
Strains with penicillin MICs of greater than 0.12 µg / ml have not been observed yet. Therefore, if the
isolate is resistant to penicillin reidentify, repeat ABST and inform Microbiologist.
References
1. Henry D. Isenberg .Clinical microbiology procedures hand book ,Volume 1, 2nd ed. 2004.
2. Clinical and Laboratory Standards Institute. CLSI (formerly NCCLS) Performance Standards for
Antimicrobial Susceptibility Testing; 21st Informational Supplement, M100-S21 Vol 31 No.1 January
2011.
3. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology, 9th
Edition 2007. ASM Press. Washington, DC.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Safety Cabinet for sub culturing
Microscope
Bunsen Burners
CO2 jar
CONSUMABLES
Glassware
Blood culture bottles - a) In house blood culture bottle with 60 ml of BHI ( with SPS) in 100 ml
for adults medical flat bottle made up of clear glass with a hole at the centre of the
screw cap with diaphragm
b) Blood culture bottles for automated systems
c) Other commercial blood culture bottles
Blood culture bottles a) In house blood culture bottle with 30 ml of BHI ( with SPS) in 50 ml
for children medical flat bottle made up of clear glass with a hole at the centre of the
screw cap with diaphragm
b) Blood culture bottles for automated systems
c) Other commercial blood culture bottles
Blood culture bottles a) In house blood culture bottle( universal bottle) with 10ml of BHI
for neonates b) Blood culture bottles for automated systems
Glass slides
Other items:
Disposable syringes / needles preferred for subculturing
Wire loops may be substituted if disposable syringes/needles are unavailable
Stains
Gram stain
Introduction
Both local and systemic infections can result from contamination of IV devices. These include cellulitis,
abscess formation, septic thrombophlebitis, device associated bacteraemia and rarely, endocarditis.
Colonization can occur as early as 24 hours after insertion of the catheter. The skin insertion site is the most
common route of colonization and related infections.
A link has been demonstrated between the number of organisms on the catheter surface and the risk of
infection/disease associated with these catheters.
Tip of catheter
When specimen is
collected on removal of 1. Clean skin at insertion site using 70% alcohol.
catheter 2. Allow surface to dry.
3. Remove catheter aseptically using sterile forceps.
4. Avoid contact of tip of catheter with skin.
5. Using a sterile pair of scissors, cut the distal 5-6 cm (area under skin) of
the catheter.
6. Place the cut portion in a dry sterile screw capped bottle.
REJECTION CRITERIA
1. Catheter tips :
a. Specimen sent in non sterile container
b. Major delay in receipt of sample (> 1 day)
2. For blood cultures refer SOP on blood culture.
SPECIMEN PROCESSING
Culture
Catheter tip 1. Using a sterile pair of forceps, roll the catheter tip 4-5 times on a blood
agar plate using whole plate, without touching the edge.
Semiquantitative 2. Incubate at 35C overnight in 5-10 % CO2.
method
(Maki method) Reading
1. Note presence / absence of growth.
2. If growth present - count the number of colonies for each colony type.
3. < 15 colonies – not significant
4. Any species ≥15 colonies – significant
5. Mixed growth, <15 colonies of each type – not significant
6. Identify and perform ABST on all species with ≥ 15 colonies/plate
No growth No growth.
Interpretation of 1. If blood cultures are negative or isolate different from those isolated from
results catheter tip
comment - Catheter colonization
2. If blood culture positive with same species as that isolated from catheter
tip
comment - Catheter related blood stream infection
3. If blood culture positive with same species that was isolated from catheter
tip but <15 colonies or different isolate from catheter tip
comment - Bacteraemia not associated with IV catheters
Blood culture 1. If blood culture through the catheter becomes positive 2 or more hours
before the peripheral blood culture with the same organism
comment - IV catheter associated blood stream infection
2. If only blood through the line is positive and not the peripheral blood
comment - Catheter colonization
3. If both blood cultures through the catheter and the peripheral blood
become positive with the same organism but the time gap is less than 2
hours or peripheral blood culture becomes positive first.
comment - Bacteraemia not associated with IV catheters
REQUIREMENTS
FOR COLLECTION OF -
catheter blood through catheter port
70% alcohol 70% alcohol
Sterile forceps Sterile disposable needle/syringe
Sterile pair of scissors Blood culture bottle
Dry sterile screw capped bottle
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen burners
Sterile forceps
Sterile pair of scissors
CO2 jar
CONSUMABLES
Glassware
Sterile screw capped glass bottles
Glass slides/petri dishes
Other items:
Disposable syringes / needles preferred for sub culturing
Wire loops may be substituted if disposable syringes/needles are unavailable
Stains
Gram stain
References:
1. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical Medical
Microbiology, 14th Edition 1996. Churchill Livingstone.
2. IDSA guideline 2009 on CVC infection control
3. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology,
9th Edition 2007. ASM Press. Washington, DC.
4. Henry D. Isenberg .Clinical microbiology procedures hand book ,Volume 1, 2nd ed. 2004.
This SOP describes the examination of CSF for the presence of pathogens causing bacterial meningitis.
Likely pathogens
SPECIMEN PROCESSING
Test selection 1. Cell count / Differential count refer annexure at the end of this SOP.
2. Protein estimation
3. Sugar estimation
CSF culture should be 4. Microscopy ( if culture is done from the same sample do culture before
available in all hospitals microscopy)
with a paediatric and a) Gram stain
medical consultant b) Ziehl-Neelsen clinician request or if tests 1-3 abnormal
service c) Wet preperation in compromised patients or if
clinically indicated -examine for protozoa and spirochete
d) Dark ground microscopy if clinically indicated
e) India ink preparation
5. Culture
a) Acute bacterial meningitis
b) Tuberculous meningitis
c) Cryptococcal meningitis
6. Antibiotic sensitivity
7. Antigen detection
8. Special tests
a) Antibody detection - HSV/JE/rabies
b) PCR - HSV/TB
c) Culture for enteroviruses
Sample < 1 ml
a) Place one drop of uncentrifuged CSF onto slide. Allow to dry.
b) Repeat with 2 more drops of CSF.
c) Fix by gentle heat.
d) Examine as given above.
*If Chocolate agar plate NOT quality controlled for growth of H. influenzae, make
several stabs on the Blood agar plate with Staphylococcus aureus.
Contact Deposit - Retain at room temperature or incubator for further cultures / PCR
Reference 1. Mycobacterial culture and/or PCR – store in refrigerator (4 - 80C)
Laboratory 2. Culture for fungi (if clinically indicated)
Choice of antibiotics
REPORTING PROCEDURE
Macroscopy Colour - Colourless / clear yellowish (xanthochromia) / clear red
Turbidity- clear / turbid / Blood stained Clot + fibrin / blood
NORMAL VALUES
Cell count Predominant Protein (g/l) Glucose (% of blood
cells sugar)
Neonates <30 / mm3 Neutrophils 0.4 – 1.2 60%
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Safety cabinet
Centrifuge Speed 3000 x g and 1500 x g
Microscope
Bunsen Burner
CO2 jar / incubator
CONSUMABLES
Glassware
Glass slides New (unused)
Sterile 1-2 ml screw capped containers For collection of CSF
Sterile 5 ml screw capped conical tubes For centrifugation
Sterile pasteur pipettes
Fuchs Rosenthal chamber
(or Improved Neubauer chamber)
Stains
Gram stain
Ziehl-Neelsen stain
Leishman stains
MAINTENANCE OF RECORDS
References
1. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical Medical
Microbiology, 14th Edition 1996. Churchill Livingstone.
2. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology,
9th Edition 2007. ASM Press. Washington, DC.
3. Clinical and Laboratory Standards Institute. CLSI (formerly NCCLS) Performance Standards
for Antimicrobial Susceptibility Testing; 21st Informational Supplement, M100-S21 Vol 31 No.1
January 2011.
8. If a dye (Toludine blue / Methylene blue) is used, multiply the number of cells by
the dilution factor to get the final cell count.
1. Dilute CSF 1:10 with 2% acetic acid (1 drop CSF + 9 drops acetic acid).
2. Final count = cells counted x 10 (dilution factor)
Differential Perform if WBC (pus cells) > 5 / mm3 using Leishman stain
count
Introduction
This SOP describes the examination of fluids obtained from normally sterile sites.
SPECIMEN PROCESSING
Test selection Cell count / Differential count – perform on all sterile fluids received refer
annexure page 24: Performing cell counts in CSF and sterile fluids
1. Microscopy
a) Gram stain
b) Ziehl-Neelsen stain
c) Wet preparation - if indicated
2. Culture
a) bacterial culture
b) for M. tuberculosis if requested or indicated by cell count
c) for fungi if requested or indicated by history/cell count
3. Antibiotic sensitivity on all isolated organisms
4. Antigen detection - if indicated
5. Special tests
a) PCR ( TB)
b) Adenosine deaminase (pleural fluid for TB)
Staining Sample > 1 ml
a) Decant fluid into a sterile screw capped conical tube.
b) Centrifuge at 1500 x g for 15 minutes.
Gram stain c) Carefully decant supernatant into a sterile container.
d) Resuspend deposit in 5 drops of fluid.
e) Using sterile Pasteur pipette, place 1 drop of fluid deposit on to a new
(unused) slides. Do not spread. Air dry.
f) Fix using gentle heat.
g) Perform Gram stain on one slide.
h) Examine stained smear using x100 magnification (oil immersion).
i) Using battlement technique, scan whole smear (should take approximately 20
minutes).
Sample < 1 ml
1. Place one drop of uncentrifuged fluid onto a slide. Allow to dry.
2. Repeat with 2 more drops of fluid.
3. Fix by gentle heat.
4. Examine as given above.
Clotted sample
a) Use the clot to prepare a smear.
b) Perform Gram stain.
Supplementary
Ziehl-Neelsen 1. Ziehl-Neelsen stain for AFB
stain a) Decant fluid into sterile screw capped conical tube.
b) Centrifuge at 3000 x g for 15 minutes (2).
c) Decant supernatant into sterile container.
d) Resuspend deposit in a few drops of fluid.
e) Place one drop of resuspended deposit on a clean dry slide.
f) Do not spread. Air dry.
g) Place another drop over the dried first drop and allow to dry.
h) Repeat the process once more.
i) Fix using gentle heat.
j) Stain.
k) Examine the whole smear using the battlement technique.
* For joint fluids from children and pleural fluids , if chocolate agar plate is not
quality controlled for growth of H. influenzae– make several stabs on the Blood agar
plate with Staphylococcus aureus.
Supernatant - Refrigerate
Antigen detection if required
a) S. pneumoniae (from pleural and pericardial fluids)
b) H. influenzae (from joint fluids in children and pleural fluids)
REPORTING PROCEDURE
Microscopy Gram stain- Presence of organisms.
Presence of pus cells.
Microscopy results should be telephoned to ward as soon as available.
Gram stain results should be informed on same day.
Inform microbiologist of positive results.
Bacteria / fungi / pus cells seen
Culture
Negative No growth at 48 hours
No growth after 48 hours
a) Send final Report
incubation
b) If enrichment cultures are done include “Further report will follow if positive
on enrichment”.
c) Consider antigen detection on supernatant (presence of pus cells).
d) Consider need for Mycobacterial culture/fungal culture.
e) Consider PCR.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burner
CO2 jar or incubator
Centrifuge Speed 3000 x g and 1500 x g
CONSUMABLES
Glassware
Glass slides New (unused)
Fuchs Rosenthal chamber
(or Neubauer chamber)
Sterile screw capped 10 ml containers For collection of samples
Sterile screw capped 5 ml conical tubes For centrifugation
Sterile Pasteur pipettes
Stains
Gram stain
Ziehl-Neelsen stain
Leishman stains
MAINTENANCE OF RECORDS
References
1. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical Medical
Microbiology, 14th Edition 1996. Churchill Livingstone
2. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology,
9th Edition 2007. ASM Press. Washington, DC.
Types of specimens
Midstream urine Surgical sample
Clean catch urine Catheter specimen
Suprapubic aspirate Nephrostomy urine
Possible pathogens
Complicated UTIs
E. coli - commonest
Klebsiella, enterobacter - usually associated with instrumentation or catheterisation
Pseudomonas aeruginosa - associated with structural abnormality or long-term
catheterisation
S. aureus- rarely causes UTI. Associated with renal abnormality or as a secondary
infection due to bacteraemia, surgery or catheterisation. It is also seen as a
contaminant due to perineal carriage.
Coagulase negative staphylococci - may cause complicated infections
Candida species - Candida albicans is the most frequently isolated species. Bladder
colonisation is associated with indwelling catheters but may also be present as
contamination from the genital tract.
Correct specimen type Correct instruction of patient on how to collect urine is essential. Written
and method of collection instructions in all 3 languages should be available in laboratory.
Screw capped wide mouth sterile bottle should be used for collection of
urine for culture.
Midstream sample – Instruct patient to clean the genital area with soap
and water and begin passing urine into the toilet bowl. Collect the mid
part of the urine flow into a sterile container after the initial flow of the
urine has been passed.
Clean catch sample (paediatric sample) – Following breast/ bottle
feeding, baby should be kept without a nappy (perineal cleaning is
recommended).Urine is collected straight from the stream into the
container (preferably midstream).
Supra-pubic aspirate – Aseptic collection of urine directly from urinary
bladder using a needle and syringe. Needs to be indicated on the request
form.
Indwelling catheter – Clamp the tube and collect the urine aseptically
using a needle and syringe. Insert the needle in head to toe direction.
REJECTION CRITERIA
Specimens kept at room temperature for more than two hours
Specimens transported without ice if transport has taken more than two hours
Specimens refrigerated for more than 24 hours
Unlabelled specimens
Specimens taken from a receptacle (catheter bag or bed pan)
Catheter tips
Unsterile container
Leaking specimens
SPECIMEN PROCESSING
Time of processing
Urine samples should ideally be processed on arrival to the laboratory. If a delay is unavoidable sample
should be refrigerated until processed.
(i) (ii)
Assumption
If the field diameter of high power (X 40) is 0.44 mm (measured by a slide micrometer),
the area of the HPF is 0.15 mm3. If the size of cover slip is 22 X 22 mm and the depth of
the film examined is 0.1 mm, the volume of urine observed in an HPF will be 0.015
mm3. Under these conditions finding of 1 leucocyte per 7 HPF corresponds with 10 4
leucocytes per ml (10 pus cells / mm3). Finding clearly larger numbers than this indicate
significant pyuria.
Method
1. Mix the urine sample carefully.
2. Transfer 0.05 ml of urine on to the middle of a microscopic slide.
3. Immediately apply a cover slip (22 X 22 mm) avoiding trapped bubbles.
(Film should show a small excess of fluid along the edges of the cover slip and
then be about 0.1 mm in depth)
4. Examine under high power (X 40) and interpret as follows.
Reporting
Observation Interpretation
This is a less reliable method than using a counting chamber. Urine is centrifuged for 5
minutes at 2000 rpm and then the sediment is examined under high power. With this
method, 5 to 10 leukocytes/high-power field in the sediment is the upper limit of normal.
©SLCM / 2011
> 100 Pure growth of ≥105 CFU/ml Yes *................... isolated Not necessary
Colony count ≥105 CFU/ml
Mixed growth of 2 organisms with ABST for Mixed growth with Interpret with clinical details
predominance of one organism (organism predominant predominant growth of
Between Pure growth of * 104 - 105 CFU/ml Yes 104 - 105 CFU/ml of Not necessary
10-99 *………….……. Isolated.
34
Mixed growth of ≥2 organisms No Mixed growth Please repeat if clinically indicated
< 10 Any growth < 104 CFU/ml No No significant growth Not necessary
Any Any growth taken as significant in supra- Yes Report the isolates with colony Not necessary
growth pubic aspirates, urine taken from the renal count
pelvis, ureter or bladder during surgery
* Identification of organisms may be important in recurrent infections. Discuss with Microbiologist.
URINE
URINE
ABST ABST
Select 5 or 6 antibiotics from list in consultation with Microbiologist/
clinicians.
Do not report second line antibiotics unless clinically indicated.
Pseudomonas species
1st line 2nd line
Ciprofloxacin Amikacin
Gentamicin Aztreonam
Ceftazidime Meropenem/ Imipenem
Norfloxacin Netilmicin
Ticarcillin/Clavulanic acid
Cefepime/ cefpirome
Piperacillin tazobactam
Cefoperazone- sulbactam
Enterococcus species
Ampicillin
Nitrofurantoin
Norfloxacin
Vancomycin (for resistant strains only and after consultation with Microbiologist)
REPORTING PROCEDURE
Uncentrifuged urine -
Microscopy > 10 pus cells / mm3 (significant pyuria)
or
< 10 pus cells/ mm3
Organisms to be
reported as
Gram Negative Bacilli
REQUIREMENTS
EQUIPMENT
Incubator 35°C
Refrigerator 2°C - 8°C
Microscope
Bunsen Burner
CONSUMABLES
Glassware (or disposables)
Screw capped wide mouthed sterile containers (10-20ml) For collection of urine
Screw capped wide mouthed sterile containers (10-20ml) with For collection of urine
1.8% boric acid
50 µl pipette, Sterile pipette tips For microscopy
Glass slides For microscopy
22 x 22 mm cover slips
Other items:
Calibrated wire loops – 0.001ml (1µl)
Stains
Gram stain
MAINTENANCE OF RECORDS
Unique records register for urine culture
References
1. 1.Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical
Medical Microbiology, 14th Edition 1996. Churchill Livingstone
2. Mandell GL, Bennette J, Dolin R. Mandell, Douglas and Bennett’s Principles and Practice of
Infectious diseases, 7th Edition 2010. Churchill Livingstone
Introduction
This SOP describes the examination of following types of specimens for the presence of bacterial
pathogens.
Anaerobic Culture
*Contact Anaerobic
Transfer swabs or aspirate directly into anaerobic transport
Reference Laboratory,
medium or send the sealed syringe itself.
Medical Research
Institute
Tel: 0112 693532,
Ext 344/332,
0112691350
Additional specimens Blood culture should be taken when patients show systemic signs of sepsis.
REJECTION CRITERIA
General
1. Unlabelled specimen
2. Unsterile container
3. Leaking specimen
4. Improper transport time
Note:
1. If a specimen is unacceptable inform Microbiologist before rejecting.
2. If a specimen is rejected, a responsible individual must be notified immediately and request another
sample of good quality.
3. Names of persons involved and action taken should be documented.
4. Do not discard samples which may be unrepeatable eg: pus from a brain abscess.
SPECIMEN PROCESSING
Processing of tissue samples prior to microscopy and culture
Grind under sterile conditions using sterile mortar and pestle or tissue grinder or
stomacher (best method).
or
Cut into small pieces using a sterile scalpel.
Microscopy 1. Gram stain should be performed on all specimens. Perform AFB when necessary.
Wound swabs – If two swabs are received prepare a smear for Gram stain using
one swab. Use the other swab for culture.
2. Examine Gram stain smear under 10 x objectives.
Record microscopy as follows:
- Number of pus cells 0, occasional, <10, 10-24, ≥25
Enrichment culture - pus/tissue from ‘inaccessible’ sites such as brain abscess should
be enriched in BHI broth up to 5-7 days. Subculture as above.
REPORTING PROCEDURE
Microscopy 1. Pus cells
0 Nil
When pus cells are not seen in every field Occasional
<10 / LPF 1+
10-24 / LPF 2+
≥25 / LPF 3+
2. Organisms -
Culture
Report all primary and potential pathogens. Quantify growth as
Wound swabs scanty 1+, 2+, 3+, 4+
Scanty – a few colonies only
1+ - growth in primary inoculum only
2+ - growth up to first streak
3+ - growth up to second streak
4+ - growth up to limits of streaking
a) Primary pathogens
– Staphylococcus aureus
– -haemolytic streptococci (group A,C,G)
Report with ABST.
b) Other organisms
Significance is considered depending on clinical details, site of infection and
the Gram stain result.
If there is any doubt regarding the significance of an organism discuss with
Microbiologist.
*Pure growth
1. Report Gram negative organisms including pseudomonas which are moderate
or heavy (2+,3+,4+) with ABST
Pseudomonas species from burns patients and plastic surgery patients–report with
ABST.
Pus/tissue Report and do ABST for all isolates from a single sample. Deep seated abscesses are
samples generally of polymicrobial aetiology.
1st line
Choice of # Cefoxitin / methicillin / oxacillin (Report as cloxacillin)
antibiotics Clindamycin –check for inducible resistance
Erythromycin
Select Co-trimoxazole
antibiotics Tetracycline/doxycycline
depending
on previous 2nd line
ABST Ciprofloxacin – selective reporting only. Discuss with Microbiologist.
profiles, Fusidic acid
availability *Vancomycin
and clinical *Teicoplanin
use *Linezolid
Enterococcus species
Penicillin
Ampicillin/ Amoxycillin
Tetracycline
2nd line
*Vancomycin
*Linezolid
*Teicoplanin
Streptococcus species
1st line
Penicillin
Erythromycin
Clindamycin – check for inducible resistance
Tetracycline
2nd line
Vancomycin (Report if penicillin resistant)
Pseudomonas spp.
1st line 2nd line
Ceftazidime Amikacin
Ciprofloxacin Aztreonam
Gentamicin Cefipime
Ticarcillin-clavulanic acid Cefeperazone-sulbactam
Piperacillin-tazobactam Meropenem
Imipenem
Colistin (MIC interpretive standards
only)
Polymixin
Acinetobacter spp.
1st line 2nd line
Gentamicin Amikacin
Ceftazidime Piperacillin-tazobactam
Ciprofloxacin Cefipime
Ampicilln-sulbactam Cefeperazone-sulbactam
Ticarcillin-clavulanic acid Meropenem
Meropenem Imipenem
Doxycycline
Colistin (MIC interpretive standards
only)
Polymixin B
References
1. Bowler P.G, Duerden B.I and Armstrong D.G 2001, Wound Microbiology and Associated
Approaches to Wound Management. Clinical Microbiology Reviews 14.2 244-269
2. Clinical and Laboratory Standards Institute. CLSI (formerly NCCLS) Performance Standards
for Antimicrobial Susceptibility Testing; 18th Informational Supplement, M100-S18 Vol 28 No.1
January 2008.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burner
CO2 jar
Anaerobic jar Only if anaerobic cultures undertaken in laboratory
CONSUMABLES
Glassware
Glass slides
Introduction
This SOP describes the examination of pus / exudate collected from the ear canal or fluid aspirated from
the middle ear for the presence of aerobic bacterial pathogens causing otitis media or otitis externa.
Possible pathogens:
2. For perforated ear drum, collect the fluid using a sterile swab on flexible shaft
inserted via an auditory speculum. Rotate swab & allow fluid to collect on
swab. Obtain 2 swabs (one for Gram stain and the other for culture).
Otitis Externa
1. Initially remove any debris using a moist swab.
2. Obtain sample by firmly rotating a fresh sterile swab in the outer canal.
Specimen transport Specimens should be processed within 2 hours (to prevent drying of swabs).
& storage Maintain specimens at room temperature if there is a delay. If a transport
medium is used storage time can be ≤ 24 hours at room temperature.
REJECTION CRITERIA
General
1. Unlabelled specimen
2. Unsterile container
3. Improper transport time
SPECIMEN PROCESSING
Laboratory 1. Perform Gram stain and a wet smear if an aspirate or a separate swab is
procedure available. If only one swab is sent prepare smears after inoculating plates.
2. Inoculate as follows:
Blood agar plate - incubate overnight at 350C in 5-10 % CO2
MacConkey agar plate - incubate overnight at 350C aerobically
Chocolate agar plate - incubate overnight at 350C in 5-10 % CO2
(Anaerobic cultures may be indicated for tympanocentesis fluid)
3. Inoculate two Sabouraud’s dextrose agar plates if hyphae are present on wet
smear. Incubate one plate at room temperature and one at 35oC.
(Refer SOP on Mycology- laboratory procedures.)
Reading &
Interpretation
Gram stain Note the presence of pus cells, bacteria and yeast cells.
Culture Read after overnight incubation. If there is no growth incubate cultures for further
24 hours & tympanocentesis fluid up to 4 days,
Note the colony appearances and identify.
Tympanocentesis fluid: Any growth is considered significant.
M.catarrhalis S.pneumoniae
Ampicillin Chloramphenicol
Trimethoprim-sulfamethoxazole Oxacillin*2
Erythromycin Erythromycin*3
Co- amoxiclav Cefuroxime
Cefuroxime
REPORTING PROCEDURE
Gram stain Pus cells, bacteria and yeast cells
Wet smear fungal hyphae
Culture & ABST Acute Otitis Media
Report the primary pathogens (S. pneumoniae, S.aureus, Moraxella catarrhalis,
β-haemolytic streptococci, H.influenzae) with antibiotic sensitivity.
Tympanocentesis fluid- report any growth as significant with ABST.
Otitis Externa
S. aureus, Group A streptococci, Pseudomonas spp. – Report with Antibiotic
sensitivity
Fungal isolates –Discuss with ENT surgeon. May need to send to reference
laboratory. See SOP on mycology.
Candida spp. should be reported after a germ tube test.
In mixed cultures organisms of normal skin flora may be listed without ABST.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burner
CO2 jar
CONSUMABLES
Glassware
Glass slides
Stains
Gram stain
MAINTENANCE OF RECORDS
References
1. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology,
9th Edition 2007. ASM Press. Washington, DC.
2. Clinical and Laboratory Standards Institute. CLSI (formerly NCCLS) Performance Standards
for Antimicrobial Susceptibility Testing; 21st Informational Supplement, M100-S21 Vol 31 No.1
January 2011.
Introduction
This SOP describes the examination of pus, discharge or aspirate from the eye for the presence of aerobic
bacterial pathogens.
Possible pathogens
1. Bacteria
Staphylococcus aureus
Streptococcus pneumoniae
Haemophilus spp.
Neisseria gonorrhoeae
Pseudomonas spp.
Coagulase Negative Staphylococci
Moraxella catarrhalis (Acute conjunctivitis)
Moraxella lacunata (Chronic conjunctivitis)
-haemolytic streptococci
Coliforms
2. Fungi
3. Viruses
4. Chlamydia spp.
5. Parasites
Corneal scrapings are collected (as given above) after anaesthetic drops are
instilled. If swabs are collected specimens need to be collected prior to
instillation of anaesthetic drops.
Intraocular fluids
1. Always collected by an eye surgeon using a sterile needle & a syringe.
2. Bedside inoculation is ideal. If not transport the fluid in the syringe
itself (as the amount of sample is scanty).
Corneal button
1. Sample is collected by the attending surgeon at the time of surgery.
2. Send to the lab in a sterile screw capped container or directly inoculate
into BHI at the bedside.
REJECTION CRITERIA
General
1. Unlabelled samples
2. Unsterile container.
3. Dried out specimens and swabs.
4. Duplicate specimen on same day for same request.
Note:
Inform Microbiologist before rejecting the sample.
1. If a specimen is rejected, a responsible individual must be notified immediately.
2. Names and persons involved and action taken should be documented.
3. Do not discard corneal scrapings/aqueous or vitreous humour which may be unrepeatable. Discuss with
Microbiologist and Clinician.
SPECIMEN PROCESSING
1. Inoculate/and incubate media according to the table given below.
2. After inoculation of the plates, smear the swab/ exudates on to a clean microscopic slide to prepare a
smear for Gram stain.
3. Prepare a wet film for fungi in 10% KOH.
Reading & Note the colony appearances and identify the possible pathogens as in SOP on
identification.
Interpretation Corneal scraping/aspirate: Any growth is significant.
ABST
Staphylococcus spp. Streptococcus spp. Pneumococcus Coliforms
Choice of
Chloramphenicol Chloramphenicol Oxacillin(report as Chloramphenicol
antibiotics
Gentamicin Penicillin penicillin) Gentamicin
Cefoxitin* Chloramphenicol Neomycin
Discuss Ciprofloxacin Co-trimaxazole Cefuroxime
with eye Fusidic acid Cefuroxime Coamoxyclav
surgeons Norfloxacin Ciprofloxacin Cefotaxime
and modify Levofloxacin
as required Moxifloxacin
Gentamicin Amoxycilin
Ceftazidime Chloramphenicol
Ciprofloxacin Cefotaxime
Some antibiotics are included in the ABST due to the availability of preparations
for local therapy (typed in bold). This may be mentioned on report as topically
used antibiotics can be used irrespective of ABST result.
In endophthalmitis - second line antibiotics (see SOP for blood culture for choice
of antibiotics))
REPORTING PROCEDURE
Report When urgent microscopy is requested or when significant microscopic result found
inform microbiologist/MO. Immediately inform the relevant ward by phone.
Intraocular
aspirate
corneal
scrapings Report any growth with ABST
corneal
buttons
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burners
CO2 jar
CONSUMABLES
Glassware
Glass slides
Other items:
Wire loops
Swabs for collection of specimens
Sterile bottles
Stains
Gram stain
MAINTENANCE OF RECORDS
References
1. www.hpa-standardmethods.org.uk/documents/bsop/pdf/bsop2.pdf -
2. Mallett, J and Dougherty, L (2008). The Royal Marsden Manual of clinical Nursing Procedures.
Oxford: Blackwell Science.
Introduction
This SOP describes the examination of sputum and other specimens obtained from the lower respiratory
tract, for common pathogens. Further discussion with microbiologist/clinician is required regarding BAL
sent from immuno-compromised patients, as additional tests other than given in this SOP may need to be
carried out.
Time between specimen The specimen should be transported and processed in the laboratory without
collection and processing delay. Ideally, specimens should be processed within 2 hours of collection.
This maximizes recovery by reducing overgrowth of commensal oral flora
and maintaining the viability of fastidious organisms.
REJECTION CRITERIA
1. Salivary sample
2. Swabs of ET secretion
3. Tips of endotracheal tubes
4. Samples taken > 24hours ago
5. Sample in unsterile container
6. Repeat samples taken on same day
Note: Specimens should not be rejected solely on macroscopic appearance. Inform microbiologist
before rejecting a specimen.
1. If a specimen is rejected, a responsible individual must be notified immediately.
2. Names and persons involved and action taken should be documented.
SPECIMEN PROCESSING
All specimens should preferably be handled inside a class 1 or 2 biological safety cabinet (BSC).
Macroscopic mucoid / muco purulent/ muco salivary / salivary / blood stained/ purulent
appearance
Microscopy Gram Stain
a) Select purulent or muco-purulent portion of the sample to do a Gram stain.
b) Observe under low power ( x 10 magnification) for cells.
c) Under oil immersion, look for intra cellular bacteria and note the predominant
micro organism.
d) Assess the quality of the specimen – refer Tables 1 & 2
Table 1: Murray and Washington’s grading system for assessing the quality of
sputum samples2
Grade Epithelial cells per low Pus cells per low power (x10)
power (x10) field field
Group 1 ≥25 <10
Group 2 ≥25 10-25
Group 3 ≥25 ≥25
Group 4 10-25 ≥25
Group 5 <10 ≥25
Accept the samples that belong to *Murray & Washington Group 3, 4 & 5 for
processing.
Reject the samples that belong to *Murray & Washington Group 1 & 2
Table 2: Screening ET & BAL specimens requested for routine bacterial culture
to ensure quality1
Reading and The growth on the whole area of the plate of 25 or more colonies of the same
interpretation potential pathogen will then indicate that 106 or more of that pathogen were present in
each ml of the original sputum.
REPORTING PROCEDURE
Distinction between tracheo-broncheal colonization & true pulmonary infection is difficult. Hence Gram
stain & culture together with the clinical condition of the patient need to be considered.
ABST
Streptococcus pneumoniae
Choice of 1st Line: 2nd Line:
antibiotics Oxacillin (report as penicillin) Vancomycin
Levofloxacin Chloramphenicol
Trimethoprim-sulfamethoxazole Tetracycline
Erythromycin Linezolid
Clindamycin
H. influenzae
1st Line: 2nd Line:
Ampicillin*1 Cefepime
Co-amoxiclav Imipenem
Cefuroxime Meropenem
Cefotaxime or ceftriaxone Ertapenem
Erythromycin Tetracycline
Trimethoprim-sulfamethoxazole Cefixime
M.catarrhalis
1st Line 2nd Line
Ampicillin*1 Cefotaxime or ceftriaxone
Trimethoprim-sulfamethoxazole Levofloxacin
Erythromycin
Tetracycline
Co-amoxiclav
Cefuroxime
Acinetobacter spp.
1st Line: 2nd Line:
Cefotaxime or ceftriaxone Ticarcillin-clavulanic acid
Gentamicin Piperacillin-tazobactam
Ceftazidime Amikacin
Cefepime Imipenem
Levofloxacin Meropenem
Ciprofloxacin Netilmicin
Trimethoprim-sulfamethoxazole Cefoperazone/sulbactam
Tetracycline
Ampicillin-sulbactam
Pseudomonas aeruginosa
1st line 2nd line
Ceftazidime Imipenem
Gentamicin Meropenem
Cefepime Cefoperazone/sulbactam
Ciprofloxacin
Ticarcillin-clavulanic acid
Aztreonam
Piperacillin-tazobactam
Coliform
1st Line 2nd Line
Ampicillin Cefepime
Gentamicin Ticarcillin-clavulanic acid
Amoxicillin-clavulanic acid Piperacillin-tazobactam
Cefuroxime Imipenem
Ciprofloxacin Meropenem
Netilmicin Amikacin
Cefotaxime or ceftriaxone Aztreonam
Ceftazidime Ertapenem
Important:
Screening and confirmatory tests for ESBLs in Escherichia coli, Klebsiella
pneumoniae, Klebsiella oxytoca and Proteus mirabilis are indicated.
Screening and confirmatory tests for suspected carbapenemase production is
indicated.
S.aureus
1st line 2nd line
Cefoxitin / methicillin / oxacillin Ciprofloxacin*2
(Report as cloxacillin) Fusidic acid
Clindamycin –check for inducible Vancomycin*3
resistance Teicoplanin
Erythromycin Linezolid
Fusidic acid
Co-trimoxazole
Tetracycline/doxycycline
References
1. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology, 9th
Edition 2007. ASM Press. Washington, DC.
2. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and Textbook of
Diagnostic Microbiology, 6th Edition 2006. Lippincott-Raven Publishers. Philadelphia, Pennsylvania.
3. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical Medical
Microbiology, 14th Edition 1996. Churchill Livingstone.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Safety cabinet Class 1 or 2 Biological safety cabinet
Microscope
Bunsen Burners
CO2 jar
Vortex mixer
Calibrated loops 0.005ml/0.001 ml or 0.01 ml
CONSUMABLES
Sterile wide mouthed screw capped (leak proof) For collection of samples
containers
Glass slides
Stains
Gram stain
Ziehl-Neelsen stain
Types of specimens Swabs from posterior pharynx or any other inflamed area in the throat, tonsils
Introduction
Most sore throats are caused by viruses. This SOP describes the examination of throat swabs for
bacterial pathogens causing sore throat.
Specimen transport 1. Swabs for Neisseria gonorrhoeae should be directly plated on selective
media for gonococci or placed in to charcoal containing transport
medium e.g. Amies transport medium.
REJECTION CRITERIA
1. Unlabelled specimen
2. Delay in receipt of sample > 24 hours
In both instances, inform ward / responsible clinician to ensure that a second specimen is sent.
REPORTING PROCEDURE
Positive Report results as one of the following statements.
streptococcus
If bacitracinisolated.
or grouping kit not available
Negative
- haemolytic streptococci not isolated
Note:
Only Group A, C and G -haemolytic streptococci are known to be causally
related to sore throat.
The throat is often colonized with other organisms eg: coliforms,
which should NOT be reported and for which ABST should NOT be done.
S. aureus may be significant in peritonsillar abscesses and pustular tonsillitis.
Interpretation of pathogens in throat swabs from immuno-compromised patients
should be done in consultation with Microbiologist.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Microscope
Bunsen Burners
CONSUMABLES
Glassware
Glass slides
Other items
Wire loops
Swabs For specimen collection
Tongue depressor Only if specimens are collected in the laboratory
Stains
Gram stain
MAINTENANCE OF RECORDS
This SOP describes the examination of specimens for the presence of Corynebacterium diphtheriae.
This may be carried out in the event of a patient presenting with suspected diphtheria (faucial, nasal,
cutaneous) or for contacts of a proven case of diphtheria (usually throat swabs). Clinical infection in other
sites such as vagina, ear or conjunctiva can be seen rarely.
Types of specimens
Throat swabs Nasal swabs
Ulcer swabs Vaginal, ear or eye swabs
SPECIMEN PROCESSING
Culture 1. Inoculate the following media using the swab.
a) Blood Tellurite (BT) agar plate - incubate overnight at 350C in 5-10% CO2.
b) Blood agar plate - incubate overnight at 350C in 5-10% CO2.
Reading & 1. Pick raised black or grey colonies on BT medium for further study.
preliminary 2. Gram stain all the different colony types that appear on the BT agar plate.
identification 3. Subculture any suspicious colony from BT agar plate on to a Loeffler's serum
slope as soon as possible.
4. Incubate aerobically at 350C for 18 hours.
5. Stain smears (from Loeffler) as follows:
a) Gram stain
b) Methylene blue
c) Albert’s stain
6. Look for Gram positive pleomorphic club shaped rods with chinese letter
arrangement on Gram stain and typical coryneform bacilli with metachromatic
granules when stained with Methylene blue or Albert’s stain.
7. If there are no typical colonies on BT / Blood agar after overnight incubation, re-
incubate a further 24 hours and re-examine all the plates.
8. Send Loffler’s serum slopes to MRI for further study.
Identification Final identification of C. diphtheriae and detection of toxin production will be carried
out at Reference Laboratory, Medical Research Institute, Colombo.
ABST Penicillin
Erythromycin
REPORTING PROCEDURE
Preliminary 1. Telephone report
Report Inform Clinician the preliminary result if characteristic colony morphology
together with microscopic morphology are present.
Corynebacterium spp. isolated.
2. Written report Isolate sent to Reference Laboratory,
Medical Research Institute for confirmation
and toxigenicity testing. Final report follows.
Final Report
Corynebacterium spp. not isolated
negative If there are no typical colonies
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burner
CONSUMABLES
Glassware
Glass slides
Other items:
Wire loops
Stains
Gram stain
Methylene blue stain
Albert stain
MAINTENANCE OF RECORDS
References
1. Greenwood D, Slack RCB, Peutherer JF. Medical Microbiology, 16th Edition 2006.
Churchill Livingstone
2. Mandell GL, Bennette J, Dolin R. Mandell, Douglas and Bennett’s Principles and Practice of
Infectious diseases, 7th Edition 2010. Churchill Livingstone
3. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology,
9th Edition 2007. ASM Press. Washington, DC.
Rejection criteria
1. Unlabelled specimens
2. Specimens without accompanying request forms
3. Improper transport for isolation of N. gonorrhoeae
If collected into transport medium and kept for more than 48 hours
If collected into transport medium and refrigerated
If discharge sent on dry swab without transport medium
4. Duplicate specimen from patient for same investigation without justification
SPECIMEN COLLECTION
Optimal time of Before antibiotic treatment
specimen collection
Specimens For gonococcal infection
Urethral swab from male and female patients
Endocervical swab from female patients
Anorectal swab
Throat swab
Urine
1. Clean the meatus with a swab moistened in normal saline or sterile water.
2. Insert a swab 2 - 3 cm into the urethra and rotate, making sure the swab is
in contact with the urethral wall.
3. Remove the swab and place it in transport medium.
Endo-cervical swab Preferably 2 swabs should be taken (for culture and preparation of a smear).
1. Cervical specimens should be collected by a medical officer.
2. Vaginal speculum may be moistened with sterile warm water if necessary.
No lubricant cream or antiseptic should be used.
3. Any cervical discharge, mucus or pus should be wiped away with a sterile
swab.
4. Take endocervical swab by inserting the swab into the cervical canal for
1-2cm.
5. Rotate for 10 seconds in canal and withdraw swab without touching
vaginal wall or secretions.
6. Inoculate the specimen directly on suitable media at collection site or
place the swab in a suitable transport medium and send for culture.
Ano-rectal swab and These swabs should be collected if suspected of gonococcal infection in the
throat swab throat or rectum in addition to the swabs from the primary sites as above.
Rectum – Insert a cotton swab 3cm into the anal canal and rotate it for 10
seconds to collect exudates from the crypts just inside the anal ring. If faecal
contamination occurs, discard the swab and use another to obtain the
specimen.
Oropharynx – Swab the region of the tonsillar crypts and the posterior
pharynx.
For N. gonorrhoeae
First voided urine sample can be used after centrifugation for culture.
Transport media can be 2. Ideally, the urethral or cervical discharge should be inoculated at the
obtained from any STI bedside directly onto a selective culture medium such as Thayer Martin
clinic in the country medium for the isolation of Neisseria gonorrhoeae. Inoculated plates
should be sent to the laboratory immediately.
3. If culture media are not available locally, transport medium can be used.
4. Bottles of Amies transport medium can be obtained from any STI clinic
in the country. However it should be remembered that even with the use
of transport media, results are not satisfactory if transport takes more than
48 hours.
5. When inoculating, insert the swab into a container of Amies transport
medium. Break off the swab stick aseptically and allow the bottle top to
be replaced tightly.
Culture and PCR Use the special swab in the collection kit of chlamydia antigen detection test.
SPECIMEN PROCESSING
Microscopy Staining the direct smear for examination
Direct microscopic examination is not recommended for rectal and
pharyngeal infections.
Culture 1. A selective culture medium such as Thayer Martin medium gives best
isolation rates for Neisseria gonorrhoeae.
2. If selective media are not available inoculate on to chocolate agar and
incubate for 2 - 5 days for the colonies to appear. Do a Gram stain and the
oxidase test on typical colonies.
3. For further identification and confirmation of gonococci send the isolate
to a STI laboratory.
4. Isolation rate is low in comparison to the use of selective media.
Investigation of Syphilis
Tzanck test 1. Samples should be taken from a fresh vesicle, rather than a crusted one, to
ensure the yield of a number of virus infected cells.
Method used to 2. The vesicle should be unroofed or the crust removed, and the base gently
demonstrate cellular scraped with a swab.
changes caused by the 3. The material thus obtained is smeared onto a microscopic slide, allowed to
herpes group of air dry, and stained with Giemsa stain.
viruses 4. The slide should be clean, since cells will not adhere to an unclean slide.
5. The stained nuclei may vary in colour from reddish blue to purple to pink.
The cytoplasm stains bluish.
REPORTING PROCEDURE
DIRECT SMEAR Number of pus cells per oil immersion field :
(Gram stain for
N. gonorrhoeae) No pus cells - Not seen
occasional ( 1) - Occasional
few (1 – 4) - +
Moderate (5 – 8) - ++
numerous (> 8) - +++
Positive smear
Intracellular Gram negative diplococci seen. (or Intracellular and extracelluar Gram
negative diplococci seen.) Diagnostic of gonococal infection.
or
Please note in patients with chlamydial infection pus cells will be present with no
organisms seen in the smear.
References
E. Van Dyck, A.Z Meheus, P.Piot. Laboratory Diagnosis of Sexually Transmitted Diseases, WHO
Geneva1999.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burners
CO2 jar or preferably CO2 incubator
CONSUMABLES
Glassware
Glass slides
Cover slips
Other items:
Inoculating loop
Sterile swabs
Stains
Gram stain Note that the counter stain for gonococci is
saffranin
Giemsa stain
MAINTENANCE OF RECORDS
Introduction
Specimens of vaginal discharge are sent for microbiological investigation in the following situations.
1. Investigation of vaginal discharge
2. Post-operative & post partum infections
3. Premature rupture of membranes (PROM)
Less commonly, gonococcal infection may present with a vaginal discharge in pre-pubertal
girls. Refer SOP on urogenital specimens.
REJECTION CRITERIA
Candida infection Prepare a smear on a clean slide using a separate vaginal swab.
1. Stain with Gram stain.
2. Examine for yeast cells and pseudohyphae.
Bacterial vaginosis 1. Look for ‘clue cells’ in the Gram stain or wet smear - Clue cells are
vaginal epithelial cells coated with ‘Gram-variable’ short bacilli which
obscures the borders of the cells.
2. Note the absence of (or a decrease in) lactobacilli.
3. Note the absence of ( scanty) pus cells.
Culture Specimens should be inoculated onto,
Blood agar Incubate overnight at 35°C in 5-10% CO2.
MacConkey agar Incubate overnight at 35°C aerobically.
Chocolate agar Incubate overnight at 35°C in 5-10% CO2.
Sabouraud’s Glucose agar Incubate 48 hours at 35°C.
For processing, identification and ABST refer SOP for Pus.
REPORTING PROCEDURE
Report
Gram stain Pus cells - not seen / scanty / + / ++ / +++
Clue cells - seen / not seen
Lactobacilli - seen / not seen
Trichomonas - seen / not seen
Yeasts / pseudohyphae - seen / not seen
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burners
CO2 jar or preferably CO2 incubator
CONSUMABLES
Glassware
Glass slides
Cover slips
Other items
Inoculating loop
Sterile swabs
Stains
Gram stain Note that the counter stain for gonococci is saffranin
Giemsa stain
MAINTENANCE OF RECORDS
Unique records register for Swabs.
Introduction
Diarrhoea is caused by a large number of organisms, which include bacteria, viruses and parasites. This
SOP describes the methods of detection of four frequently encountered bacterial pathogens. The
absence of these pathogens in watery or bloody faeces does not exclude an infective cause of diarrhoea.
Bacterial
o Enteropathogenic E. coli
o Salmonella species
o Shigella species
o Vibrio cholerae (refer SOP on Cholera)
o Campylobacter species – culture done at MRI ( 011-2691350)
Laboratory diagnosis: detection of toxins A & B produced by Clostridium difficile and anaerobic
cultures are done at Anaerobic laboratory, MRI (011-2693532/3/4 ext. 344). Specimen should be
collected after 48hrs of diarrhoea into a sterile container and transported in ice.
Viral
Watery diarrhoea in children is most likely to be caused by rota virus.
Laboratory diagnosis: Antigen detection is the preferred method but electron microscopy is also
available (refer SOP on virology)
Parasites
(Refer SOP on Parasitology)
Optimal time of Early in the course of diarrhoea, before starting antibiotic treatment
specimen collection
Correct specimen type 1. Faeces specimen - If it is blood and mucous diarrhoea collect om a
(in order of preference) portion of faeces containing blood and mucous. Collect into a clean,
and wide mouthed, disinfectant free, screw capped container or leak proof
Method of collection container with a tight fitting lid (eg: Marmite bottle).
REJECTION CRITERIA
SPECIMEN PROCESSING
Microscopy Make a saline smear on a slide and examine microscopically (x 40 objective) for pus
cells, red cells and parasites (amoebae, ova and cysts). (Refer SOP on Parasitology).
Day 2
1. Examine plates.
2. Look for typical colonies on these media (refer SOP on media).
3. Inoculate typical colonies into Kligler iron agar (KIA) or TSI agar slants and
urea slants. Incubate overnight at 350C.
4. Subculture from Selenite F broth on SS or XLD agar plate. Incubate at 350C
overnight.
Day 3
1. Look for the typical Kligler patterns as given below.
2. Identify using serology as given below.
3. If no pathogens are isolated from the primary plate proceed with SS or XLD
plates inoculated from Selenite F.
Identification
Kligler pattern Urease
slant butt gas H2S
Salmonella spp. K A + + -
Shigella spp. K A - - -
(Shigella flexneri 6
may produce gas)
E. coli A A + - -
Salmonella typhi K A - + (little along -
the stab)
Salmonella paratyphi K A + - -
K – alkali A –acid
(S. typhi and S. paratyphi A rarely present with diarrhoea. Usually requested in the
diagnosis of enteric fever).
Isolates with a typical Kligler pattern which do not give agglutination with
shigella antisera – perform oxidase test to exclude cholera
SERO TYPING
Idealy before Salmonella serotyping, isolate should be confirmed with lysine, indole
and motility tests.
O H H (phase 2)
(phase 1)
Salmonella typhi 9 d - (Vi +)
Salmonella paratyphi A 2 a -
Salmonella paratyphi B
or S. java 4 b 1,2 (send isolates to reference lab for
differentiation)
REPORTING PROCEDURE
Wet prep Direct smear - Pus cells – absent / scanty/ numerous
RBC -- absent / scanty/numerous
Time frame for AOC --
Culture report Direct smear result should be sent on the same day.
Negative report –
Day 3-4 Positive culture: Report as ……………………. isolated
Positive report – (ABST will follow)
Day 3 – 5
Report ABST when available
(Note: issue
preliminary Negative culture: Report as Shigella and Salmonella not isolated
report as early as
possible and final
report later)
When EPEC is looked for: Report as Shigella , Salmonella and EPEC
not isolated
In patients with
HUS – contact Note:
Enteric Reference
Laboratory, MRI
E. coli can cause diarrhoea by several mechanisms
Colombo 8
Tel. 0112 691350 a) Enterotoxigenic E. coli (ETEC) is the commonest. This cannot be identified in
a routine laboratory.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator Maintained at 2C - 8C
Microscope
Bunsen Burners
CONSUMABLES
Glassware
Glass slides
Wide mouthed sterile screw capped containers For collection of samples
Bijou bottles For Selenite F broth, Urea slant
Test tubes For KIA / TSIA
MAINTENANCE OF RECORDS
References
1. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical Medical
Microbiology, 14th Edition 1996. Churchill Livingstone.
2. The Oxoid Manual. 9th Edition 2006.
REJECTION CRITERIA
Note:
1. If a specimen is unacceptable inform Microbiologist before rejecting.
2. If a specimen is rejected, a responsible individual must be notified immediately and request
another sample of good quality.
3. Names of persons involved and action taken should be documented.
SPECIMEN PROCESSING
Culture Day 1
1. Inoculate a loopful on TCBS medium (primary medium).
2. Inoculate a loopful in 5–10 ml of Alkaline peptone water (APW-enrichment
medium).
3. Incubate the plates at 350C overnight.
4. Incubate alkaline peptone water for 5-8 hours at 350C.
5. Do not shake or mix APW before subculturing.
6. Streak a loopful (of the culture taken from the top most portion) of APW on
TCBS. Incubate plates at 350C overnight.
Day 2
Send 1. Examine the TCBS plate for typical colonies. (V.cholerae – flat, golden yellow
preliminary colonies. Gram stain –small Gram negative curved rods)
report 2. If characteristic colonies are seen – inform the ward. Inoculate Kligler and urea
slopes.
Inform 3. If APW could not be subcultured after 5-8 hrs of incubation, after overnight
MOH incubation at 350C subculture to a fresh APW. Incubate for 5-8hrs and then
* subculture to TCBS.
Send first
isolate to (Do not perform oxidase test or serotyping from growth on TCBS agar)
Reference
Laboratory Day 3
for 1. Kligler pattern K/A, no gas, no H2S. Urease negative.
confirmation 2. Perform oxidase test and string test on isolates with typical kligler pattern.
3. String test - Make a suspension of growth from Kligler in 0.5% sodium
deoxycholate on a glass slide and look for mucus string formation.
SEROTYPING
1. Mix one drop of organism suspension and one drop of antiserum O1 on a glass
slide. (Usually equal volumes of antisera and growth suspension are mixed)
2. Rotate slide for one minute and observe agglutination.
3. If positive with O1 antiserum –send to MRI for further typing (subserotyping
and biotyping).
4. If it is not agglutinating with O1 antiserum , test with O139 antiserum.
5. In an epidemic only initial few isolates of V. cholerae O1 need to be sent to MRI
for biotyping/ subserotyping.
REPORTING PROCEDURE
Final report
Negative cultures Negative for Vibrio cholerae.
Reference Laboratory Reference laboratory for enteric pathogens, Medical Research Institute.
References
1. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology, 9th
Edition 2007. ASM Press. Washington, DC.
2. CDC laboratory method for diagnosis of epidemic dysentery and cholera, 1999
WHO/CDS/CSR/EDE/99.8
http://www.cdc.gov/ncidod/DBMD/diseaseinfo/cholera_lab_manual.htm
3. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical
Medical Microbiology, 14th Edition 1996. Churchill Livingstone.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burners
CONSUMABLES
Glassware
Clean wide mouthed for collection of specimen
screw capped bottles
Glass slides
Other items:
Sterile screw capped vials /containers with nutrient agar slopes for transport of isolates to
reference laboratory
Wire loops
Stains
Gram stain
MAINTENANCE OF RECORDS
Specimen register for recording specimens / isolates sent to reference laboratory
Unique records register for faeces
Introduction
Neonatal group B streptococcal disease has become the major infectious cause of illness and death among
newborns. Up to 30% of normal women are colonized with group B streptococcus (GBS) in the vagina or
rectum.
Studies have shown that the accuracy of prenatal screening for identification of GBS colonization can be
enhanced by timing of cultures, the sites swabbed and the microbiological method used for culture.
Collection of vaginal & rectal swabs between 35 and 37 weeks of gestation is recommended to improve the
sensitivity and specificity of detection of colonization at the time of delivery.
Selective enrichment broth is recommended to increase the isolation of GBS and to avoid overgrowth of
normal flora.
REJECTION CRITERIA
General:
1. Specimens without a label.
2. Unsterile container
3. Duplicate specimens.
Note:
1. If a specimen is unacceptable inform Microbiologist before rejecting.
2. If a specimen is rejected, a responsible individual must be notified immediately and request
another sample of good quality.
3. Names of persons involved and action taken should be documented.
SPECIMEN PROCESSING
Test selection Laboratory procedure: Screening by enrichment
REPORTING PROCEDURE
Report
Positive Group B streptococci isolated.
References:
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burners
CO2 jar
a) Screening of an individual patient in the event of recurrent staphylococcal sepsis (MSSA and MRSA
are both reported).
b) If MRSA is isolated from one site in in-patients, for decolonization purposes.
c) Screening for MRSA carriage in the event of a hospital outbreak (eg.post operative sepsis) which may
be performed on patients as well as on health care workers.
d) Screening carried out before high risk surgery such as cardiac, orthopaedic and neuro surgery (MSSA
and MRSA are both reported).
e) Screening of patients from high risk units or hospitals on admission, depending on the local policy.
Nasal swab: Swab both nostrils with a single swab in a circular motion.
Throat swab: A swab of the back of the throat is taken by rotating a swab as it is moved
gently back and forth across the throat.
Perineum or groin swab: Swab from the perineum or groin area may be taken by the patients
themselves following instructions given by the nursing staff about the
technique, if the patient is able to. Again, the swab should be rotated whilst
being brushed across the area.
Other sites e.g. axilla, skin Swab using the same technique.
lesions etc
Note: Swabs could be collected into 7% NaCl nutrient broth or manitol salt
broth.
The request form should clearly state that the swabs are for “MRSA / Staphylococcus aureus screen”.
Note: A separate form is not required for each swab.
REJECTION CRITERIA
General:
1. Specimens without a label
2. Unsterile container
3. Dried out specimens and swabs
4. Duplicate specimens
SPECIMEN PROCESSING
Test selection Laboratory procedure: Screening by enrichment:
Enrichment method
1. Inoculate swabs into selective enrichment broth (mannitol salt broth or 7% NaCl
MRSA/ nutrient broth).
2. Incubate overnight at 35 0C.
MSSA 3. Subculture onto mannitol salt agar. (if not available use blood agar)
screening
Selective media which can be used for MRSA screening are:
Blood agar with methicillin - Incubate for 24 hrs at 350C aerobically.
MRSA Chrome agar
REPORTING PROCEDURE
Report
S. aureus isolated.
Positive culture Methicillin sensitive S. aureus report as:
If methicillin resistant report as: MRSA isolated
Report Mupirocin susceptibility results.
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burners
CONSUMABLES
Glassware
Glass slides/petri dishes
Other items:
Wire loops
Cotton swabs
Stains
Gram stain
Reference
Health Protection Agency Guidelines; UK
Types of specimens
Aspirated material from abscesses, sinus tracts and pus from beneath skin flaps
Aspirated sterile fluids (peritoneal fluid, pleural fluid, joint aspirate)
Supra pubic bladder aspirate
Blood and bone marrow
CSF
Biopsy of tissue or bone
Material obtained by transtracheal aspiration
Material obtained by culdocentesis
Introduction
Proper selection of specimens for anaerobic culture is important in order to minimize contamination with
endogenous micro flora. It is the task of the laboratory personnel to educate those responsible for selection,
collection and transport of specimens for anaerobic bacteriology.
Method of Specimen should be collected using aseptic techniques, and taking precautions to
collection minimize contamination with endogenous microflora.
The best specimens would be those collected with a needle and syringe and
biopsies taken during surgery.
Specimen transport All specimens must be transported as rapidly as possible with minimum
& storage exposure to oxygen.
Use anaerobic transport containers if available.
If they are not available, transport pus in a sterile screw capped container.
Biopsies in sterile saline in a screw capped container.
Stools for C.difficile toxin and culture in a wide mouthed container with a lid. If
a delay in transport is anticipated transport in ice.
REJECTION CRITERIA
1. Unlabelled specimen
2. Unsterile container
3. Unacceptable specimens
Throat , nasal or oral swabs
Expectorated sputum
SPECIMEN PROCESSING
Laboratory Processing a specimen of pus for anaerobic culture
procedure
Macroscopic Note : Colour
examination Odour
Consistency
Presence of granules
Culture Culture of specimens for anaerobes should be performed immediately after the
specimen reaches the laboratory. A delay may result in death of obligate
anaerobes.
Specimen is inoculated into non selective and selective media.
Non selective media: Blood agar(BA),Brucella Blood Agar with Hemin and Vit
K (BBA),
Selective agar: Neomycin Blood Agar(NBA), Bacteroides Bile Esculin
Agar(BBE)
1. Incubate anaerobically for 48 hours or longer.
2. Examine plates with a hand lens and record the colony morphology of each
colony.
3. Perform Gram stain.
4. Do aerotolerance testing.
5. Subculture each colony on BBA.
Aero tolerance Aero tolerance testing is done to differentiate a true anaerobe from a facultative
Testing: anaerobe.
BA and a BBA plate are inoculated simultaneously from a single test colony,
using a straight wire.
Incubate the BA plate aerobically and the BBA plate anaerobically.
After overnight incubation check the aerobically incubated plate for growth.
If growth is present, the isolate is not a true anaerobe.
Those isolates which show no growth when incubated in air will be considered as
true anaerobes and be subjected to further testing.
Reading &
Interpretation
Culture Read after 48 hrs of incubation. If there is no growth incubate cultures for a
further 48 hours. If Actinomycosis is suspected, incubate for a total of 5-7 days.
Note the colony appearances and identify.
Identification If aero tolerance testing confirm that the isolate is a true anaerobe Identify the
isolate by Rapid Identification kits(Rapid ANA II)
REPORTING PROCEDURE
Macroscopy
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Microscope
Bunsen Burners
Anaerobic jars
CONSUMABLES
Glassware
Glass slides
Stains
Gram stain
MAINTENANCE OF RECORDS
IDENTIFICATION OF ORGANISMS
Procedure Appearance
Gram stain Gram negative bacilli
Oxidase
Negative Positive
KIA or TSI
No change
Non motile
Gram negative cocci
Report as Coliforms Acinetobacter spp. Pseudomonas spp.
Shigella/Salmonella pattern
Note:
This is not a very accurate identification but serves the purpose of deciding on antibiotic discs. Precise
identification should be done if clinically required eg. multi-resistant organism from ICUs / isolates from
blood, CSF and sterile sites and hospital cross infections etc. Discuss with the MO/ Microbiologist
regarding the need for precise identification.
If precise identification is required, use biochemical tests or commercial kits (eg. API).
If these are not available store the isolates in nutrient agar slopes and send to MRI for identification.
Record the date of dispatch of specimen to MRI and enter date of receipt of results and identification.
However, oxidase negative Gram negative bacilli could be further identified with some degree of
accuracy by the following tests. Citrate utilization, indole production, urease production, phenylalanine
deaminase production, motility and Kligler or TSI reaction pattern.
Identification of Enterobacteriaceae
*Slant/Butt
#
K – alkaline, A- Acid
+ > 90% of strains positive, - > 90% of strains negative, +/- 50 – 90 % of strains positive, -/+ 50-90% of
strains negative
References
1. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and Textbook
of Diagnostic Microbiology, 6th Ed. 2006. Lippincott-Raven Publishers. Philadelphia,
Pennsylvania.
2. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical Medical
Microbiology, 14th Edition 1996. Churchill Livingstone.
Satellitism test
1. Plate a blood agar plate heavily with the suspected colonies.
2. Stab/streak the plate with Staphylococcus aureus.
3. Incubate at 35C overnight and look for colonies growing round
S. aureus streaks/stabs.
2. Apple plate
X factor dependence
This is best tested with the porphyrin test.1
Reference
1. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and
Textbook of Diagnostic Microbiology, 6th Ed. 2006. Lippincott-Raven Publishers.
Philadelphia, Pennsylvania.
2. Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the Identification of Medical
Bacteria, 3rd Edition 1993. Cambridge University Press.
Chocolate agar Usually white to cream while some strains Clinical samples except urine.
yellow to orange
PBP2 latex test mecA gene positive Detects PBP2, the product of
staphylococci including MSRA mecA gene
isolates gives a positive result
References
1. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology,
9th Edition 2007. ASM Press. Washington, DC.
2. Henry D. Isenberg 2004, Clinical Microbiology Procedure Hand book, p.1.0.1 – 4.12.6, Second
Ed. American Society for Microbiology, Washington, D.C.
Lancefield grouping
Group B streptococci
Enterococci
Group Group Group Group Group Group
Group D streptococci
A B C D F G Some viridans streptococci
Some Streptococcus milleri
Positive Negative
PYR Test
Growth in 6.5% NaCl broth Tiny colonies with
Survival at 60 0C for 30 min caramel smell
Streptococcus milleri
Positive - Negative -
Enterococci Group D streptococci
SXT – Co- trimoxazole, CAMP – Christie,
Atkins & Munch-Petersen, PYR – Pyrrolidonyl arylamidase
Laboratory Manual in Microbiology 106
©SLCM / 2011
STREPTOCOCCI AND ENTEROCOCCI
2. Penicillin resistance has not been reported in Group A streptococci. Re-check identity,
repeat sensitivity test & inform Microbiologist.
4. All enterococcal isolates from blood & CSF should have a direct beta-lactamase test.
Strains testing positive should have penicillin & ampicillin reported resistant, irrespective
of disc diffusion readings.
5. All viridans streptococci isolated from sterile body sites (blood, CSF and bone) should
have penicillin MIC done.
6. Enterococci with intermediate zones to vancomycin should have MIC done to exclude
VRE.
8. All enterococcal isolates from patients with infective endocarditis should have high level
gentamicin resistance (HLAR) assessed with gentamicin 120 µg disc.
References
1. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical
Microbiology, 9th Edition 2007. ASM Press. Washington, DC.
2. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and
Textbook of Diagnostic Microbiology, 6th Edition 2006. Lippincott-Raven Publishers.
Philadelphia, Pennsylvania.
4. Stokes EJ, Ridgway GL, Wren MWD. Clinical Microbiology, 7th edition. 1993.
2. CLSI does not recommend disc diffusion testing for -lactams other than penicillin.
3. CLSI advocates direct performance of MIC for penicillin, ceftriaxone etc for CSF & blood isolates of
patients with meningitis.
4. Isolates that are oxacillin resistant (zone diameter ≤19 mm) should have penicillin MIC done.
Reporting other -lactams (ceftriaxone) in such cases should be based on MIC.
References
1. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology,
9th Edition 2007. ASM Press. Washington, DC.
2. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and Textbook
of Diagnostic Microbiology, 6th Edition 2006. Lippincott-Raven Publishers. Philadelphia,
Pennsylvania.
3. Clinical and Laboratory Standards Institute. CLSI (formerly NCCLS) Performance Standards
for Antimicrobial Susceptibility Testing; 21st Informational Supplement, M100-S21 Vol 31 No.1
January 2011.
PURPOSE
Helps differentiate S. pneumoniae, which is soluble in bile and bile salts, from viridans streptococci
which are insoluble. The addition of bile salts activates the autolysins and accelerates the natural lytic
reaction observed with cultures of pneumococci.
REQUIREMENTS
10% sodium deoxycholate3
2% Sodium deoxycholate
Sterile saline
Positive control – S. pneumoniae
Negative control – S. agalactiae1
A. PLATE METHOD
Method
1. Touch a suspected colony on the primary plate with a loop charged with 10% sodium
deoxycholate solution.
2. Incubate the plate for 15 min at 35ºC.
Interpretation
Colonies of pneumococci disappear leaving an area of α-haemolysis on blood agar
Note: The use of 2% sodium deoxycholate in this method has been documented in some literature.
B. TUBE TEST
Method
1. Prepare a milky suspension (McFarland No. 1) from an overnight culture in 1 ml of saline.
2. Divide the suspension in two tubes (test and control) of 0.5 ml.
3. Add 0.5 ml of 2% sodium deoxycholate (bile salts) to the test tube and 0.5ml saline to the
control tube.
4. Vortex and incubate at 35ºC up to 2 hours (inspect after 10 – 30 minutes initially).
Interpretation
- If it is S. pneumoniae, suspension will be completely transparent without any turbidity.
- Any other α-haemolytic streptococci will remain turbid after 2 hrs of incubation.
Note:
In some reports 10% sodium deoxycholate has been used in this procedure.
Bile solubility test should be preformed only for optochin resistant isolates.
The test should not be performed on old cultures, as the active enzyme may not be present.
References
1. Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the identification of medical
bacteria, 3rd Edition 1993. Cambridge University Press.
2. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical
Medical Microbiology, 14th Edition 1996. Churchill Livingstone.
3. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical
Microbiology, 9th Edition 2007. ASM Press. Washington, DC.
CAMP TEST
PURPOSE
Used in the identification of Streptococcus agalactiae (Group B streptococcus) which produces a factor
called the CAMP factor which enhances the effect of the -lysin produced by S. aureus. This
phenomenon is seen with both haemolytic and non-haemolytic isolates of group B streptococci.
REQUIREMENTS
-lysin producing Staphylococcus aureus (NCTC 7428)2
Blood agar plate containing 5% sheep blood
Positive control – S. agalactiae
Negative control – E. faecalis2
METHOD
1. Streak a -lysin producing Staphylococcus aureus down the center on the 5% sheep blood agar
plate.
2. Make a single streak of the test isolate perpendicular to the staphylococcal streak.
3. Leave about a 1cm space between the two inoculation lines.
4. Negative and positive controls should be inoculated on the same plate.
5. Incubate the inoculated plates for 24 hours at 35ºC.
INTERPRETATION
An area of enhanced haemolysis occurs where the lysin produced by the Staphylococcus aureus and
the CAMP factor produced by the group B streptococcus intersect and may appear in the shape of an
arrow head.
Note:
Some group A streptococci will be CAMP test positive if incubated in a candle jar or in a CO 2
atmosphere.
References
1. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and
Textbook of Diagnostic Microbiology, 6th Edition 2006. Lippincott-Raven Publishers.
Philadelphia, Pennsylvania
2. Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the identification of medical
bacteria, 3rd Edition 1993. Cambridge University Press.
3. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical
Medical Microbiology, 14th Edition 1996. Churchill Livingstone.
CATALASE TEST
PURPOSE
Used to differentiate bacteria which produce catalase from non-catalase producing bacteria.
REQUIREMENTS
Sterile wooden sticks or glass rods
Hydrogen peroxide - 3% H2O2 stored in a brown bottle under refrigeration
An 18 -24 hr culture of the organism to be tested
Positive control – Staphylococcus species
Negative control – Streptococcus species
Note: Hydrogen peroxide reagent must be tested with positive and negative control organisms
immediately before unknown bacteria are tested. Shaking the reagent before use will help expel any
dissolved oxygen. False positive results may occur if the H2O2 contains dissolved oxygen.
METHOD
1. Pour 2-3ml of the H2O2 solution into a test tube.
2. Using a sterile wooden stick or glass rod, remove a good growth of the test organism and
immerse in the H2O2
3. Look for immediate bubbling (within 20 seconds).
If the organism is on an agar slope, 1 ml of H2O2 can be poured onto the slope. Release of bubbles
indicates a positive result.
INTERPRETATION
Positive result – active bubbling
Negative result – no bubbling
Some bacteria possess enzymes other than catalase that can decompose H2O2. Therefore, a few tiny
bubbles forming after 20 – 30 seconds is not considered a positive test
Note:
Performing the test on a slide is not recommended for safety reasons (production of aerosols).
A nichrome wire should not be used as it may give a false positive result.
Blood agar and other blood containing media are unsuitable for this test. When test is done
from colonies on blood agar, false positive results can occur. Catalase is present in red blood
cells and care must be taken to prevent the carryover of red blood cells with the colony material
if the test is done from colonies on blood containing media.
References
1. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and
Textbook of Diagnostic Microbiology, 6th Edition 2006. Lippincott-Raven Publishers.
Philadelphia, Pennsylvania.
2. Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the identification of medical
bacteria, 3rd Edition 1993. Cambridge University Press.
COAGULASE TEST
PURPOSE
Test should be done on fresh isolates (less than 24 hrs old) from a non selective medium. Test
cannot be performed from growth on mannitol salt agar.
Reagent is either rabbit or human plasma. Use EDTA as anticoagulant (citrated blood can give false
positive results). Rabbit plasma is preferable as it gives better clotting, is free from inhibitors and is
safe.1
Store the plasma in small aliquots at -200C and keep a stock of in-use plasma at 40C, bringing into
room temperature before use.
Quality control (QC) of plasma
Do not use plasma that has not been stored refrigerated or frozen or that appears turbid.
Perform QC on new lots of plasma prior to putting them into use.
QC Organisms
Staphylococcus aureus ATCC 25923 – coagulase positive
Staphylococcus epidermidis ATCC 12228 or ATCC 14990 – coagulase negative
Method
1. Emulsify a staphylococcal colony in a drop of water or saline on a microscope slide with a
minimum of spreading to make a smooth milky suspension. If clumps occur and organism does
not suspend in water do not proceed with the test.
2. Make similar suspensions of positive and negative control strains to confirm the proper
reactivity of the plasma.
3. Dip a flamed and cooled straight inoculating wire into the undiluted plasma at room
temperature, withdraw and stir the adhering traces of plasma into the staphylococcal suspension
(not a loopful of plasma).
Tube method 11
1. Prepare a 1 in 6 dilution of the plasma in saline (0.85% NaCl). (e.g. 2 drops of plasma and 10
drops of saline).
2. Emulsify a colony of staphylococcus under test.
3. With each batch of tests include tubes with positive and negative controls and a tube of
uninoculated diluted plasma to confirm that it does not clot spontaneously.
4. Incubate at 35ºC and examine at 1, 2 and 4 hrs for clot formation by gently tilting the tube
through 900.
5. Leave negative tubes at room temperature overnight and re-examine.
Note:
Do not leave the tube coagulase test at 35ºC for more than 4 hrs.
If 4 hrs of incubation is inconvenient the test is most sensitive when incubated at 250C for the
entire time, but the clot may take longer to form.2
This method can be used for rapid detection of S.aureus from positive blood cultures containing
Gram positive cocci in clusters.
References
1. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical
Medical Microbiology, 14th Edition 1996. Churchill Livingstone.
2. Henry D. Isenberg, Clinical Microbiology Procedure Hand book, 2nd Edition. 2004.
American Society for Microbiology, Washington, D.C.
PURPOSE
Used to differentiate Staphylococcus aureus which produces the enzyme DNase from other
staphylococci which do not produce this enzyme. It is particularly useful if plasma is not available or
when results of the coagulase test are difficult to interpret. When HCl is added to the DNase agar plate
unhydrolyzed DNA is precipitated and produces a white opacity or cloudiness in the agar. Positives are
surrounded by a clear zone.
REQUIREMENTS
DNase agar plate (up to 6 organisms may be tested on one plate)
Hydrochloric acid 1 mol/L (HCl)
Positive control - Staphylococcus aureus
Negative control - Staphylococcus epidermidis
METHOD
1. Divide the DNase agar plate into the required number of sections by marking the underside of
the plate with a marker pen.
2. Using a sterile loop or swab, spot inoculate the test and control organisms.
3. Make sure each test area is clearly labeled.
4. Incubate the plates at 35ºC overnight.
5. Flood the surface of the plate with 1 mol/L HCl solution to precipitate unhydrolyzed DNA.
6. Tip off excess acid.
7. Look for clearing around the colonies within 5 minutes of adding the acid.
8. Positive result – clearing around colonies.
9. Negative result – no clearing around colonies/inoculation line.
LIMITATIONS
Some MRSA strains do not give positive reactions and some coagulase-negative staphylococci
(S. schleiferi) give weak reactions.
Reference
Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical Medical
Microbiology, 14th Edition 1996. Churchill Livingstone.
OPTOCHIN TEST
PURPOSE
REQUIREMENTS
Optochin (ethylhydrocuprein chloride) 5g discs
Blood agar plate
Positive control – S. pneumoniae
Negative control – E. faecalis
METHOD
1. Inoculate a blood agar plate with light broth suspension (or a heavy inoculum) of the test
organism.
2. Place an optochin disc in the inoculated area and gently press down the disc so that it
adheres firmly to the agar surface.
3. Incubate at 35ºC overnight in 5-10% CO2
INTERPRETATION
Zone of inhibition of 14mm or more around a 6 mm disc or 16 mm or more around a
10 mm disc presumptively identifies the test organism as Streptococcus pneumoniae.
Zones smaller than these- Perform bile solubility test. If bile soluble, organism is identified
as Streptococcus pneumoniae.
No zone of inhibition - viridians streptococci.
Reference
Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and
Textbook of Diagnostic Microbiology, 6th Edition 2006. Lippincott-Raven Publishers.
Philadelphia, Pennsylvania.
OXIDASE TEST
PURPOSE
This test is used to differentiate bacteria that produce intracellular oxidase enzymes from those that do
not produce these enzymes. It is an important test in differentiating members of the genus pseudomonas
from other non fermentative Gram negative bacteria.
REQUIREMENTS
Freshly prepared oxidase reagent (1% solution of tetramethyl-p-phenylene-diamine
dihydrochloride)
Positive control - P. aeruginosa
Negative control - E. coli2
METHOD
Interpretation
Positive result – deep purple colour developing within 5-10 seconds
Delayed positive result – colour development in 10- 60 seconds
Negative result – no colour or colour developing after 60 seconds
B. PLATE METHOD
1. Cultures are made on suitable solid growth medium (a medium free from glucose and
nitrate).
2. Pour a freshly prepared 1% solution of the oxidase reagent on the plate to cover the surface.
3. Decant excess reagent.
Interpretation
Positive result – purple colonies
Negative result – no change in colour
Important
The oxidase reagent is unstable. Fresh preparations must be used for reliable results.
Test isolate must be transferred to filter paper using a clean platinum loop, wooden stick or a
glass rod. Traces of iron can catalyze the reaction and give false positive results.
References
1. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical
Medical Microbiology, 14th Edition 1996. Churchill Livingstone.
2. Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the identification of medical
bacteria, 3rd Edition 1993. Cambridge University Press.
UREASE TEST
PURPOSE
Urease is an enzyme possessed by many species of microorganisms that can hydrolyze urea producing
ammonia and CO2. This is an important test in differentiating enterobacteria. Proteus species are strong
urease producers. Salmonella and shigella do not produce urease.
REQUIREMENTS
Urea Medium
Positive control – Proteus vulgaris
Negative control – Escherichia coli
METHOD
1. Using a sterile straight wire, inoculate a tube of sterile urea medium with the test organism.
2. Incubate at 35ºC
INTERPRETATION
Examine for development of pink colour in the medium after 4h and after overnight
incubation2.
Positive result – pink colour
Negative result – no change in colour (yellow)
Note:
No tube should be reported negative until after 4 days of incubation.
References
1. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and
Textbook of Diagnostic Microbiology, 6th Edition 2006. Lippincott-Raven Publishers.
Philadelphia, Pennsylvania.
2. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical
Medical Microbiology, 14th Edition 1996. Churchill Livingstone.
The CLSI method is a disc diffusion test which is stringently standardized. The zone diameters obtained by
this method have an approximately linear relationship with the log of MIC.
The test uses materials which are easily available. All steps of the technique are strictly standardized. The
correct use of this method allows inter-laboratory comparison of results. It also enables the user to become
part of surveillance programmes which use CLSI method.
Standardized antibiotic sensitivity testing is the goal. Laboratories which aim to use this test method must
ensure that all test requirements are met. The validity of the results cannot be assured if there is deviation
from the described method.
REQUIREMENTS FOR TEST
Media 1. Mueller Hinton Agar (MHA).
2. Mueller Hinton Blood Agar – Add 5% defibrinated sheep blood
for fastidious organisms (Streptococcus spp. & N. meningitidis.)
3. Haemophilus Test Medium (HTM) should be used for
Haemophilus spp.
Dry plates with lids ajar until there are no drops of moisture on the agar
surface (incubator set at 35C for 15 minutes could be used for this
purpose). Air drying is the best. Caution: over drying inhibit growth of
organisms.
INOCULUM
Preparation of inoculum It is essential that an approximately standard number of bacteria are
tested (1-2 x 108 bacteria/ml).
QUALITY CONTROL
1. Maintenance of QC strains
a) Maintain permanent stock cultures in brucella broth (or validated alternative such as glycerol blood
broth or skimmed milk) at -70C for up to 3 years. Subculture twice to Blood agar prior to testing.
b) Maintain working stock culture on Trypticase Soy Agar slants (or validated alternative) at 2C-8C
for up to 2 weeks. Subculture once to Blood Agar plate prior to testing.
2. Frequency of QC testing
a) Perform QC daily or each time a patient test is requested.
b) Frequency of performing QC testing can be reduced from daily to weekly if a laboratory can
document proficiency in performing this test as follows:
i. perform QC testing daily (or each day patient tests are performed) until results
from 30 consecutive days of testing have been obtained
ii. proficiency in performing QC tests is confirmed, if for each drug, no more than 3
of 30 results are outside the accuracy limits
c) Document proficiency each time a new drug is added to the testing protocols.
d) Perform QC testing prior to or concurrent with testing of patient isolates each time a new lot or
new shipment of materials (MHA, discs) is put into use.
Save records of documenting proficiency indefinitely.
4. Corrective action
Inform supervisor and proceed with corrective action as follows when out of range results are
observed with QC strains.
a) Review records.
b) Check test materials (including reference strains).
c) Check equipment (refrigerators, incubators etc.).
d) Review technical aspects of test performance with individuals performing the tests.
CONTROL ORGANISMS
Escherichia coli ATCC 25922
Pseudomonas aeruginosa ATCC 27853 Source of control organisms
Haemophilus influenzae ATCC 49247 Department of Bacteriology, MRI.
Haemophilus influenzae ATCC 49766 Colombo 8
Staphylococcus aureus ATCC 25923 Tel. 0112 691350
Enterococcus faecalis ATCC 29212
Escherichia coli ATCC 35218
S. aureus ATCC BAA-977 and S. aureus ATCC BAA-976
Refer CLSI performance standards M100 – S21 for antibiotics to be tested and Q ranges.
Disc diffusion testing- acceptable limits (mm) for quality control strains
Staphylococcus Pseudomonas
Antimicrobial agent Escherichia coli Escherichia coli
aureus aeruginosa
ATCC 25922 ATCC 35218
ATCC 25923 ATCC 27853
Amikacin 30 µg 19-26 20-26 18-26
Cefixime 5 µg 23-27
Clarithromycin 15 µg 26-32
Clindamycin 2 µg 24-30
Daptomycin 30 µg 18-23
Erythromycin 15 µg 22-30
Linezolid 30 µg 25-32
Oxacillin 1 µg 18-24
Rifampicin 5 µg 26-34
Teicoplanin 30 µg 15-21
Vancomycin 30 µg 17-21
Antimicrobial disc R I S R I S R I S R I S R I S
Amikacin 30 µg ≤14 15 – 16 ≥17
Ampicillin 10 µg ≤28 - ≥29 ≥24 ≤16 - ≥17
Cefepime 30 µg ≤14 15 – 17 ≥18 ≥24 ≤21 22 - 23 ≥24
Cefotaxime 30 µg ≤14 15 – 22 ≥23 ≥24 ≤25 26 - 27 ≥28
Cefoxitin 30 µg ≤24 ≥25
Ceftriaxone 30 µg ≤13 14 – 20 ≥21 ≥24 ≤24 25 - 26 ≥27
Chloramphenicol 30 µg ≤12 13 – 17 ≥18 ≤20 - ≥21 ≤17 18 - 20 ≥21 ≤12 13 - 17 ≥18 ≤17 18 - 20 ≥21
Ciprofloxacin 5 µg ≤15 16 – 20 ≥21 ≤15 16 - 20 ≥21
Clarithromycin 15 µg ≤13 14 – 17 ≥18 ≤16 17 – 20 ≥21 ≤16 17 - 20 ≥21 ≤16 17 - 20 ≥21
Clindamycin 2 µg ≤14 15 – 20 ≥21 ≤15 16 – 18 ≥19 ≤15 16 - 18 ≥19 ≤15 16 - 18 ≥19
Erythromycin 15 µg ≤13 14 – 22 ≥23 ≤15 16 – 20 ≥21 ≤15 16 - 20 ≥21 ≤13 14 - 22 ≥23 ≤15 16 - 20 ≥21
Gentamicin 10 µg ≤12 13 – 14 ≥15
Levofloxacin 5 µg ≤15 16 – 18 ≥19 ≤13 14 – 16 ≥17 ≤13 14 - 16 ≥17 ≤13 14 - 16 ≥17 ≤13 14 - 16 ≥17
Linezolid 30 µg ≤20 - ≥21 - - ≥21 - - ≥21 ≤20 21 - 22 ≥23 - - ≥21
Minocycline 30 µg ≤14 15 – 18 ≥19 ≤14 15 - 18 ≥19
Netilmicin 30 µg ≤12 13 – 14 ≥15
Nitrofurantoin 300 µg ≤14 15 – 16 ≥17 ≤14 15 - 16 ≥17
Norfloxacin 10 µg ≤12 13 – 16 ≥17 ≤12 13 - 16 ≥17
Oxacillin 1 µg ≤10 11 – 12 ≥13 - - ≥20
Penicillin 10 units ≤28 - ≥29 ≥24 ≤14 - ≥15
Rifampicin 5 µg ≤16 17 – 19 ≥20 ≤16 17 - 18 ≥19 ≤16 17 - 19 ≥20
Teicoplanin 30 µg ≤10 11 - 13 ≥14 ≤10 11 - 13 ≥14
Tetracycline 30 µg ≤14 15 - 18 ≥19 ≤18 19 - 22 ≥23 ≤18 19 - 22 ≥23 ≤14 15 - 18 ≥19 ≤18 19 - 22 ≥23
Vancomycin 30 µg - - - - - ≥17 - - ≥17 ≤14 15 - 16 ≥17 - - ≥17
Stenotrophomonas
Enterobacteriaceae Pseudomonas spp. Acinetobacter spp. Haemophilus app. Burkholderia cepacia maltophilia
Antimicrobial disc R I S R I S R I S R I S R I S R I S
Amikacin 30 µg
≤14 15 – 16 ≥17 ≤14 15 – 16 ≥17 ≤14 15 - 16 ≥17
Amoxicillin-clavulanic
acid 20/10 µg
≤13 14 – 17 ≥18 ≤19 - ≥20
Ampicillin 10 µg
≤13 14 – 16 ≥17 ≤18 19 - 21 ≥22
Aztreonam 30 µg
≤17 18 – 20 ≥21 ≤15 16 – 21 ≥22 - - ≥26
Cefepime 30 µg
≤14 15 – 17 ≥18 ≤14 15 – 17 ≥18 ≤14 15 - 17 ≥18 - - ≥26
Cefixime 5 µg
≤15 16 – 18 ≥19
Cefotaxime 30 µg
≤22 23 – 25 ≥26 ≤14 15 – 22 ≥23 ≤14 15 - 22 ≥23 - - ≥26
Cefoxitin 30 µg
≤14 15 – 17 ≥18
Ceftazidime 30 µg
≤17 18 – 20 ≥21 ≤14 15 – 17 ≥18 ≤14 15 - 17 ≥18 - - ≥26 ≤17 18 - 20 ≥21
Ceftriaxone 30 µg
≤19 20 – 22 ≥23 ≤13 14 – 20 ≥21 ≤13 14 - 20 ≥21 - - ≥26
Cefuroxime 30 µg (oral)
≤14 15 – 22 ≥23 ≤16 17 - 19 ≥20
Cefuroxime 30 µg
(parenteral)
≤14 15 – 17 ≥18 ≤16 17 - 19 ≥20
Cephalothin 30 µg
≤14 15 – 17 ≥18
Chloramphenicol 30 µg
≤12 13 – 17 ≥18 ≤25 26 - 28 ≥29
Ciprofloxacin 5 µg
≤15 16 – 20 ≥21 ≤15 16 – 20 ≥21 ≤15 16 - 20 ≥21 - - ≥21
Clarithromycin 15 µg
≤10 11 - 12 ≥13
Colistin 10 µg
≤10 - ≥11
Ertapenem 10 µg
≤15 16 – 18 ≥19 - - ≥19
Stenotrophomonas
Enterobacteriaceae Pseudomonas spp. Acinetobacter spp. Haemophilus app. Burkholderia cepacia maltophilia
Antimicrobial disc R I S R I S R I S R I S R I S R I S
Gentamicin 10 µg
≤12 13 – 14 ≥15 ≤12 13 – 14 ≥15 ≤12 13 - 14 ≥15
Imipenem 10 µg
≤13 14 – 15 ≥16 ≤13 14 – 15 ≥16 ≤13 14 - 15 ≥16 - - ≥16
Levofloxacin 5 µg
≤13 14 – 16 ≥17 ≤13 14 – 16 ≥17 ≤13 14 - 16 ≥17 ≥17 ≤13 14 - 16 ≥17
Meropenem 10 µg
≤13 14 – 15 ≥16 ≤13 14 – 15 ≥16 ≤13 14 - 15 ≥16 - - ≥20 ≤15 16 - 19 ≥20
Minocycline 30 µg
≤12 13 – 15 ≥16 ≤12 13 - 15 ≥16 ≤14 15 - 18 ≥19 ≤14 15 - 18 ≥19
Nalidixic acid 30 µg
≤13 14 – 18 ≥19
Netilmicin 30 µg
≤12 13 – 14 ≥15 ≤12 13 – 14 ≥15
Nitrofurantoin 300 µg
≤14 15 – 16 ≥17
Norfloxacin 10 µg
≤12 13 – 16 ≥17 ≤12 13 – 16 ≥17
Piperacillin-tazobactam
100/10 µg
≤17 18 – 20 ≥21 ≤17 - ≥18 ≤17 18 - 20 ≥21 - - ≥21
Polymyxin B
≤11 - ≥12
Rifampicin 5 µg
≤16 17 - 19 ≥20
Tetracycline 30 µg
≤11 12 - 14 ≥15 ≤11 12 - 14 ≥15 ≤25 26 - 28 ≥29
Ticarcillin-clavulanic
acid 75/10 µg
≤14 15 – 19 ≥20 ≤14 ≥15 ≤14 15 - 19 ≥20
Trimethoprim-
sulfamethoxazole
1.25/23.75 µg ≤10 11 – 15 ≥16 ≤10 11 - 15 ≥16 ≤10 11 - 15 ≥16 ≤10 11 - 15 ≥16 ≤10 11 - 15 ≥16
Note : Please refer the latest CLSI performance standards for zone diameters.
REPORTING PROCEDURE:
ABST report 1. Test and report appropriate antibiotics (as in SOP for specimens /
identification).
Turn around time 2. Test and report second line antibiotics only if there is resistance
Normal – 48 hours to first line antibiotics or if patient is on a particular antibiotic (as
Unusual circumstances – stated in the request).
longer 3. Do not report antibiotic sensitivity of colonizing flora.
(always inform ward / 4. If antibiotic disc not available –indicate on report. If clinically
clinician by telephone if important, consider sending the isolate to Reference laboratory
results are delayed) for testing.
3. Salmonella isolates from extra-intestinal sites should be tested for nalidixic acid and if it is resistant
to nalidixic acid, even though sensitive to ciprofloxacin, physicians should be informed that
ciprofloxacin may be associated with clinical failure or delayed response
Salmonella spp., Shigella spp. 1st and 2nd gen cephalosporins, aminoglycosides
Oxacillin resistant Staphylococcal spp. Penicillins, β- lactam/β-lactamase inhibitor combinations,
cephalosporins, carbapenems
Enterococcus spp. Aminoglycosides (except high level resistance),
cephalosporins, clindamycin and co-trimoxazole
Coliform Carbapenem -R or I
References
1. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical Medical
Microbiology, 14th Edition 1996. Churchill Livingstone.
2. Clinical and Laboratory Standards Institute. CLSI (formerly NCCLS) Performance Standards
for Antimicrobial Susceptibility Testing; 19th Informational Supplement, M100-S18 Vol 29 No.1
January 2009.
REQUIREMENTS
EQUIPMENT
Incubator 35 C
Refrigerator 2C - 8C
Vortex mixer
Bunsen Burners
CONSUMABLES
Other items
Wire loops
Straight wire
Calipers, dividers or millimeter ruler
Sterile swabs
Sterile Pasteur pipettes
MacFarland 0.5 turbidity standard
Sterile saline suspension in bijou bottles
Control organisms
MAINTENANCE OF RECORDS
Introduction
The Stokes method is a disc diffusion method, where comparison of the inhibition zone of the test isolate
with the inhibition zone of a control organism forms the basis for interpretation of sensitivity. Variations in
the test are controlled by inoculation of the test organism and the control strain at the same time on the
same plate. It is assumed that any variation in the test affects the test and control strains equally and thereby
cancels them out. It is therefore termed a “comparative” approach.
This method allows more flexibility in the use of media, disc strengths and inoculum than standardized
methods. However, lack of standardization makes inter laboratory comparison difficult and the reliability of
results obtained by this method has been questioned. Information on the reliability of this method in testing
newer antibiotics is hard to find. Where control organisms are very sensitive to the newer antibiotics,
incorrect reports of resistance or intermediate resistance may be issued by laboratories using this method.
Newer mechanisms of resistance such as ESBL production cannot be detected using this method. Therefore
many laboratories worldwide have shifted to standardized methods. Most of the recent published literature
on antibiotic sensitivity testing and resistance patterns is based on standardized ABST testing methods.
Therefore it is recommended that, wherever possible, standardized methods of ABST are used. However,
where the precise requirements of the standardized methods cannot be followed because of constraints in
the supply of the required media / discs, this method may still be useful.
CONTROL ORGANISMS
Control strain Test organism
Escherichia coli NCTC 10418 Coliform organisms
Pseudomonas aeruginosa NCTC 10662 Pseudomonads
Haemophilus influenzae NCTC 11931 Haemophilus species
Staphylococcus aureus NCTC 6571 Other organisms that will grow aerobically
(Oxford staphyloccus)
Escherichia coli NCTC 11560 β lactam / β lactamase inhibitor combinations(co-
amoxiclav)
Amikacin 30 30
Ampicillin/Amoxycillin
a) Enterobacteriaceae /enterococci 10 25
b) Haemophilus,Moraxella and
Staphylococcus species 2 2
Amoxycillin-clavulanic acid
a) Enterobacteriaceae /enterococci 20/10 20/10
b) Haemophilus,Moraxella and
Staphylococcus species 2/1 2/1
Azithromycin 15 -
Cefuroxime 30 30
Cefotaxime 30 30
Ceftazidime 30 30
Cephradine1 30 30
Cephalexin1 30 30
Cephaloridine 5 30
Chloramphenicol
a) Enterobacteriaceae 30 -
b) Haemophilus, Pneumococcus
meningococcus 10 -
Ciprofloxacin (Ofloxacin) 1 1
Clindamycin 2 -
Co-trimoxazole1 1.2/23.8 1.2/23.8
Erythromycin (Clarithromycin) 10 -
Fusidic acid 10 -
Gentamicin 10 10
Imipenem 10 10
Kanamycin (Neomycin) 30 30
Methicillin 5 -
Metronidazole 5 -
Mupirocin 5 -
Nalidixic acid - 30
Netilmicin 10 10
Nitrofurantoin - 50
Oxacillin2 (for testing of pneumococci) 1
Penicillin
a) Staphylococcus spp *2 *2
b) meningococcus *0.25 *0.25
gonococcus
Piperacillin 30 30
Rifampicin 5 -
Tetracycline 10 30
Ticarcillin 75 75
Trimethoprim 1.25 5
Vancomycin 30 30
*International Units
Sensitive Zone radius equal to, wider than or not more than 3 mm smaller than the control
Intermediate Zone radius greater than 2mm but smaller than the control by more than 3mm
Exceptions 1. Penicillinase producing staphylococci show heaped up, clearly defined zone
edges and should be reported as resistant irrespective of zone sizes.
3. With organisms that swarm, the zone edge should be measured as for other
organisms and swarming should be disregarded.
REPORTING PROCEDURE
ABST report 1. Test and report appropriate antibiotics (as in SOP for specimens).
2. Test and report second line antibiotics only if resistant to first line
Turn around time antibiotics or if patient is on a particular antibiotic as stated in the request.
Normal – 48 hours 3. Do not report antibiotic sensitivity of colonizing flora.
Unusual circumstances – 4. If antibiotic discs not available –indicate on report. If clinically important,
longer consider sending the isolate to a Reference laboratory for testing.
(always inform ward /
clinician by telephone if
results are delayed)
REQUIREMENTS
EQUIPMENT
Incubator 35C
Refrigerator 2C - 8C
Bunsen Burners
CONSUMABLES
Media and reagents
Media MHA, DST or AST
Antibiotic discs Use validated discs from a single manufacturer
Bulk stock of discs should be stored at -20C
In use discs may be stored up to a week at 2C-8C
In use discs should be removed from the refrigerator and left at
room temperature for ½ hour before the container is opened to
minimize deterioration due to condensation of moisture.
Other items:
Wire loops
Sterile swabs
Straight wire
Calipers, dividers or millimeter ruler
Control organisms
MAINTENANCE OF RECORDS
Reference
SEROLOGY
STANDARD OPERATING PROCEDURE FOR
WIDAL TEST (STANDARD AGGLUTINATION TEST – SAT)
______________________________________________________________________________
Introduction
This SOP describes method of performing the Widal test (SAT) using antigens provided by the Medical
Research Institute (MRI). Though Widal test is done for the diagnosis of enteric fever it is not a very
reliable diagnostic test. False positives and false negatives and interpretation problems are frequently
encountered. Therefore encourage requesting clinicians to send blood for culture as isolation of Salmonella
typhi & Salmonella paratyphi A is the most reliable method of diagnosing typhoid fever.
Commercial kits (slide agglutination and tube agglutination) are available. The use of only a slide
agglutination test is not recommended. Commercial agglutination kits should be used according to the
manufacturer’s instructions.
Optimal time of specimen Acute and convalescent stages of the disease. (10 – 14 days apart)
collection
REJECTION CRITERIA
1. Incubate all tubes at 370C for 2-4 hours (water bath preferable if available).
2. Remove from water bath and leave at 40C overnight.
3. Raise tube and inspect the bottom (without disturbing the sediment).
4. Control tube should have a clear button.
Reading 5. The presence of granular deposit indicates a positive result .
6. If all 3 tubes positive, repeat test with further dilutions up to
1: 1600 as follows (a) – (h)
i) Incubate the tubes at 370C and follow the same steps as in the earlier
test.(4-9).
j) Run a positive control and a negative control along with the test samples.
Test method for Arrange 2 sets each of 3 dryers tubes each in a metal rack.
H antigen
1. Label one set for S. typhi (set 1) and the second set for S. paratyphi A
H (set 2) antigens
2. Add 1.25ml of ‘in use’ S. typhi H antigens to each dryer’s tubes
in set 1 and S. paratyphi A H antigen to each tube in set 2.
3. Add serum as indicated to each of the 2 sets:
a) 0.025ml neat serum to tube 1
b) 0.05ml of 1:4 serum dilution to tube 2
c) 0.025ml of 1:4 serum dilution to tube 3
Final dilutions 1 2 3
antigen 1.25 1. 25 1. 25
serum neat 0.025ml 1:4 dilution -0.05ml 1:4 dilution-0.025ml
1:50 1:100 1:200
Reading
1. Incubate in a water bath at 560C for 2-4 hours.
2. Remove from water bath and leave tubes at room temperature for 30 minutes .
3. Examine tubes for the presence of flocculation (seen as uneven clumps /
feathery appearance /cotton wooly appearance).
4. If floccules in all 2 tubes of a set, make serial dilutions of serum up to
1:1600 and re do test from D 2-9 .
5. Titre: highest dilution at which flocculation present
REPORTING
Interpretation 4 fold rise between acute and convalescent samples is indicative of active
infection
REQUIREMENTS
.
EQUIPMENT
Water bath 370C
Water bath 560C
Refrigerator 20C - 80C
Freezer -200C
CONSUMABLES
Glassware
Sterile screw capped 5 ml containers- For collection of samples
Graduated pipettes/ Microtitre pippette
Kahn tubes
Dryers tubes
Test tube racks
Wasserman tubes
Control sera
Positive and negative sera maintained at -200C
MAINTENANCE OF RECORDS
Introduction
The ASOT is based on the immunochemical reaction between anti-streptolysin O antibodies and
streptolysin O coated latex particles. Presence of anti streptolysin O antibodies results in visible
agglutination.
Commercial kits are readily available for this test. Perform the test in accordance with the manufacturer’s
instructions.
REJECTION CRITERIA
1. Inadequate serum for the test
2. Decomposed sample (when sent by post)
3. Unlabelled specimen
Note: If a specimen is unacceptable inform Microbiologist before rejecting.
1. If a specimen is rejected, a responsible individual must be notified immediately and request another
sample of good quality.
2. Names of persons involved and action taken should be documented.
TEST METHOD
Reagents 1. Record details of kit used
a) Manufacturer
b) Lot number
c) Expiry date
2. Store reagents at 2-8C (do not freeze).
3. Allow reagents to reach room temperature before use.
Processing Follow manufactures instructions.
REPORTING PROCEDURE
ASOT Upper limit of normal is 200 Todd units.
A 4 fold or greater rise in anti-streptolysin titre is significant in all age groups
REQUIREMENTS
EQUIPMENT
Incubator 35C - 37C
Refrigerator 2C - 8C
Centrifuge for lipaemic specimens
Shaker Optional
CONSUMABLES
Glassware
Sterile screw capped 5 For collection of samples
ml containers
Reagents
ASOT kits
Template
MAINTENANCE OF RECORDS
Reference Laboratory
MRI, Colombo 8
Tel No: (011) 2693532, 2693533,
2693534
Separation of serum
1. Keep the blood sample in room temperature for 2 hours undisturbed for the formation of a clot.
2. Centrifuge at 1500 x g for 10 min to separate the serum.
3. Transfer the separated serum in to a new tube under aseptic conditions.
4. Separated serum need to be sent in an ice box.
5. If transport of this specimen is needed and if sterile conditions are not available, do not separate the
serum. Send blood directly to the Reference Laboratory in an ice box.
6. Acute specimen should be processed on receipt.
7. On receipt of the convalescent sample acute and convalescent specimens to need to be run in parallel.
Test Specimen required Optimal time of Time taken for Other remarks
specimen results to be (supplementary
collection available tests etc...)
Mycoplasma Serum or 2ml Acute and 2 days
antibody test blood convalescent
Legionella urinary Urine sent in Acute sample Please check with
antigen test sterile container lab as tested in
at 40 C batches
Leptospira Serum or 2ml Acute and Same day For culture
Microscopic blood convalescent contact lab.
agglutination test
(MAT)
Leptospira IgM Serum or 2ml Acute and Please check with
and IgG ELISA blood convalescent lab as tested in
batches
BACTERIOLOGICAL MEDIA
1. DESCRIPTION
Nutritious infusion medium recommended for the cultivation of fastidious and nonfastidious
microorganisms, including aerobic and anaerobic bacteria, from a variety of clinical materials.
The medium is recommended for blood culture work.
2. SUPPLEMENTS
Addition of sodium polyanethol sulfonate (SPS or Liquoid) 2.5 gram per Litre will improve isolation
rate.
3. METHOD
a) Suspend the weight of the medium as specified by the manufacturer in 1 litre of distilled
water. Mix well and distribute the reconstituted broth into final containers (for blood culture
use bottles with perforated screw caps and rubber liners).
b) Autoclave with caps closed tight at 121C for 20 minutes.
c) Cover exposed area of the liner in the perforation with a foil cap before autoclaving.
d) Brain heart infusion broth which is not used on the day that it is sterilised should be placed in
a boiling water bath for several minutes to remove absorbed oxygen, and cooled rapidly
without shaking just before use.
4. QUALITY CONTROL 1
Negative control:
Uninoculated medium No change
Reference
1. The Oxoid Manual. 9th Edition 2006.
AMIES MEDIUM
1. DESCRIPTION
This medium is used to preserve the viability of anaerobes, Neisseria gonorrhoeae and other
pathogens.
2. METHOD
1. Suspend 20g of medium in 1litre of distilled water. Bring to boil to dissolve the agar completely.
2. Distribute into small screw capped bottles stirring to keep charcoal suspended. Completely fill the
bottles. Tighten the screw-caps.
3. Autoclave at 121C for 15 minutes.
4. Allow to cool.
5. During cooling, invert the bottles to ensure an even distribution of charcoal.
6. Date the medium and give it a batch number. Record the expiry date (9 months from preparation,
provided there is no change in volume or appearance of the medium) on each bottle.
7. Store in a cool place away from direct light.
3. QUALITY CONTROL 1
Negative control:
Uninoculated medium No change
Reference
1. DESCRIPTION
APW is a broth medium used as the enrichment medium for Vibrio cholerae and other vibrio species.
Sodium chloride in the medium favours the multiplication of vibrios while the alkalinity (pH 8.6-9.0)
inhibit the growth of faecal commensals. The vibrios grow on/ just below the surface of the medium.
It may be used as a transport medium providing the transportation time does not exceed 10 hours.
2. METHOD
Prepared medium – Clear, straw coloured solution without any precipitate. Can keep up to one
month at room temperature.
4. QUALITY CONTROL
Negative control:
Uninoculated medium No change
References
BLOOD AGAR
1. DESCRIPTION
2. SUPPLEMENT
3. METHOD
a) Suspend the weight of blood agar base as specified by the manufacturer in 1 litre of distilled
water.
b) Bring to the boil to dissolve completely.
c) Sterilize by autoclaving at 1210c for 15 minutes.
d) Cool the base to 500c and add sterile sheep blood to make a concentration of 7%.
e) Mix with gentle rotation and pour into petri dishes. Pour about 20ml per plate.
f) Move a flame over the surface of the agar to remove air bubbles as a final step.
Note: Human blood should be used only if sheep blood is not available. (Citrated human blood from
blood banks is unsuitable as citrate inhibits the growth of β-haemolytic streptococci. The presence of
even small amounts of glucose may alter the haemolytic reaction).
4. QUALITY CONTROL1
Negative control:
Uninoculated plate No change
Reference
1. DESCRIPTION
This medium is used for isolation, differentiation and identification of Corynebacterium diphtheriae.
Other organisms are generally inhibited. The medium is enriched by the addition of blood and made
selective by the addition of potassium tellurite which inhibit the growth of Gram negative organisms,
Staphylococcus and Streptococcus.
2. FORMULATION
K2TeO3 ( Pottasium tellurite), 2% aq. Solution - 16 ml
Sterile blood - 50 ml
* Infusion Agar1 - 1000 ml
* MRI use blood agar base instead of infusion agar.
3. METHOD1
a) Melt the Infusion Agar medium, cool to 500C and aseptically add the blood and sterile
tellurite solution.
b) Medium must not be heated after addition of the tellurite.
c) Mix and pour into petridishes.
Note:
Some Staphylococcus spp. Gram negative bacilli and yeasts may overcome inhibition and grow on
this medium
References
1. Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the Identification of Medical
Bacteria, 3rd Edition 1993. Cambridge University Press.
2. PHLS MEDIA SPECIFICATION - Technical Services, PHLS ,UK
1. DESCRIPTION
This is a transport medium used for the collection and transport of clinical specimens. The low
nutrient content of the medium and utilisation of phosphate as a buffering agent prevents bacterial
overgrowth of several enterobacteria. The low oxidation-reduction potential of the medium ensures
bacterial survival over long periods. This medium is used to preserve the viability of enteric
pathogens in faecal specimens.
2. METHOD
4. QUALITY CONTROL
Negative control:
Uninoculated medium No change
References
1. DESCRIPTION
This medium is suitable for the growth of fastidious organisms such as Haemophilus influenzae,
Pneumococci and Neisseriae. During heating the red cells are ruptured and nutrients are liberated.
2. SUPPLEMENT
7% blood (preferably sheep blood)
3. METHOD
a) Suspend the weight of blood agar base as specified by the manufacturer in 1 litre of distilled water.
b) Bring to the boil to dissolve completely.
c) Sterilize by autoclaving at 1210C for 15 minutes.
d) Cool in water bath up to 500C and add the blood to make a final concentration of 7%.
e) Heat it in a water bath to 800C with frequent mixing until the medium has a chocolate colour.
f) Pour into petri dishes. Pour about 20ml per plate.
g) Move a flame over the surface of the agar to remove air bubbles as a final step.
4. QUALITY CONTROL1
Reference
1. E. Joan Stokes, G.L. Ridgway, M.W.D. Wren. Clinical Microbiology, 7th edition 1993. Edward Arnold
Publishers, UK.
1. DESCRIPTION
CLED medium is a valuable non inhibitory diagnostic medium for urinary bacteria as it supports the
growth of all urinary pathogens. It is recommended for diagnostic urinary bacteria. It gives good colonial
differentiation and clear diagnostic characteristics. The absence of electrolytes inhibits the swarming of
Proteus species. Cystine is added for growth of organisms that require it. Differentiation of lactose
fermenters and non lactose fermenters is achieved by using Bromothymol blue as a pH indicator.
2. METHOD
a) Suspend the weight of the medium as specified by the manufacturer in 1 litre of distilled water.
b) Bring to the boil to dissolve completely.
c) Sterilize by autoclaving at 1210C for 15 minutes.
d) Pour into petri dishes (about 20ml per plate).
e) Move a flame over the surface of the agar to remove air bubbles as a final step.
3. COLONIAL CHARACTERISTICS
4. QUALITY CONTROL1
Negative control:
Uninoculated medium No change
Reference
1. DESCRIPTION
Cooked Meat Medium is used for the cultivation of anaerobic microorganisms. It initiates growth
from small inoculums and maintains viability of micro-organisms for long periods of time. In
mixed cultures, slower growing organisms survive without being displaced. Products of growth do
not rapidly destroy inoculated organisms. Therefore it is an excellent medium for storage.
This medium can be used to differentiate saccharolytic from proteolytic Clostridium spp.
Saccharolytic species rapidly form acid and gas without digesting meat. Proteolytic species break
down meat to amino acids and form sulphur compounds (blackening and putrid smell).
2. METHOD
3. QUALITY CONTROL
Negative control:
Uninoculated medium No change
Inoculate specimen deep into meat particles (bottom of the tube). Tissue specimens should be
ground prior to inoculation. Typically growth is visually observed in media by turbidity and/or
presence of gas bubbles.
Reference
1. Robertson, M. Notes upon certain anaerobes isolated from wounds. J. Pathol. Bacteriol.
1916 ;20:327.
2. The Oxoid Manual. 9th Edition 2006.
1. DESCRIPTION
A differential slope medium used to assist in the identification of Salmonella, Shigella and other
enteric bacteria. This is based on double sugar (glucose & lactose) fermentation and H2S production.
2. METHOD
3. KLIGLER REACTIONS
4. QUALITY CONTROL 1
Negative control:
Uninoculated medium – No
change
Reference
1. The Oxoid Manual. 9th Edition 2006.
MacCONKEY AGAR
1. DESCRIPTION
This is a useful selective, differential primary plating medium for the cultivation of enterobacteria. It
contains bile salt to inhibit non intestinal bacteria and lactose with neutral red to distinguish lactose
fermenting from non-lactose fermenting organisms. The omission of NaCl from the medium prevents the
spreading of Proteus species.
2. METHOD
a) Suspend the weight of the medium as specified by the manufacturer in 1 litre of distilled
water.
b) Bring to the boil to dissolve completely.
c) Sterilize by autoclaving at 1210C for 15 minutes.
d) Pour into petri dishes (about 20ml per plate).
e) Move a flame over the surface of the agar to remove air bubbles as a final step.
Note: Different formulations for separate purposes are available. It is important to select the correct
formulation for the required purpose.
For faeces - MacConkey agar with added bile salts/crystal violet
- Medium is inhibitory to Gram positive cocci
3. COLONIAL CHARACTERISTICS
Organism Colour of colony Comments
Escherichia coli Dark pink Non mucoid
Enterococcus spp. Pink (magenta) Very small round colonies
Staphylococci spp. Pale pink Opaque colonies
Pseudomonas spp. Pale /translucent -
4. QUALITY CONTROL 1
Negative control:
Uninoculated medium No change
Reference
1. The Oxoid Manual. 9th Edition 2006.
1. DESCRIPTION
Selective and differential primary culture medium for the isolation of Staphylococcus from specimens
containing mixed flora (detection of nasal and skin Staphylococcus carriage / food). The high sodium
chloride level (7.5%) inhibits most organisms except Staphylococcus spp. and some halophilic
organisms such as Vibrio species.
Addition of egg yolk emulsion enables the lipase activity of Staphylococci to be detected. The high
salt concentration in the medium clears the egg yolk emulsion and lipase production is detected as a
yellow opaque zone around colonies that produce the enzyme.
2. SUPPLELMENT ( optional)
5% egg yolk emulsion
3. METHOD
a) Suspend the weight of the medium as specified by the manufacturer in 1 litre of
distilled water.
b) Bring to the boil to dissolve completely.
c) Sterilize by autoclaving at 1210c for 15 minutes.
d) Pour into petri dishes (about 20ml per plate).
e) Move a flame over the surface of the agar to remove air bubbles as a final step.
4. COLONY CHARACTERISTICS
Note:
Confirmation of the identity of Staphylococcu. aureus must be performed.
If supplemented with egg yolk emulsion, Staphylococcus aureus colonies will show an opaque halo
due to lipase activity.
After inoculation plates should not be discarded until 48 hrs of incubation as Staphylococcus aureus
may be slow in mannitol fermentation.
5. QUALITY CONTROL1
Negative control:
Escherichia coli ATCC 8739 No growth
Reference
1. The Oxoid Manual. 9th Edition 2006.
1. DESCRIPTION
Muller-Hinton agar is a transparent medium that is recommended for antimicrobial susceptibility testing.
The composition of the medium may vary from batch to batch which could affect zones of inhibition.
Quality control of MHA is essential if CLSI method of antimicrobial susceptibility testing is carried out.
2. SUPPLEMENTS
5% defibrinated sheep blood for testing of Haemophilus influenzae, Streptococcus pneumoniae and
Neisseria species.
3. METHOD
a) Suspend the weight of the medium as specified by the manufacturer in 1 litre of distilled water.
b) Bring to the boil to dissolve completely.
c) Sterilize by autoclaving at 1210C for 15 minutes.
d) Pour into petri dishes ( about 25-30 ml per 90mm plate ). The depth of the medium should be 4mm.
e) Move a flame over the surface of the agar to remove air bubbles as a final step.
4. QUALITY CONTROL1
Negative control:
Uninoculated medium No change
Reference
1. DESCRIPTION
Nutrient broth is an economical medium for cultivation of non fastidious organisms. The addition
of 7% NaCl (analytical grade) makes it a suitable enrichment and selective medium for
Staphylococcus aureus.
2. METHOD:
Prepare nutrient broth from a good quality commercial base. Add NaCl to make a 7% broth. The
sodium chloride concentration should be reduced if local prevalent strains are known to be
inhibited by 7% sodium chloride.
3. QUALITY CONTROL1
Reference
1. Microbiology Laboratory Manual - Sri Lanka College of Microbiologists. 1st Edition. 2001
PEPTONE WATER
1. DESCRIPTION
Peptone water is used as a growth medium or as the basal medium for carbohydrate fermentation media. It is
also used to obtain overnight cultures for biochemical testing or antibiotic sensitivity testing.
2. METHOD
a) Suspend the weight of the medium as specified by the manufacturer in 1 litre of distilled water.
b) Mix well and distribute into final containers.
c) Sterilize by autoclaving at 1210C for 15 minutes.
3. QUALITY CONTROL1
Negative control:
Uninoculated medium No change
Reference
1. The Oxoid Manual. 9th Edition 2006.
1. DESCRIPTION
Peptone water is used as the basal medium for carbohydrate fermentation media. It may be modified
for use in fermentation tests by the addition of Andrade’s indicator.
2. METHOD
* Prepare 10% sugar stock solution by dissolving 2.5 grams of the required sugar in 25 ml of
distilled water. Sterilize by filtration or tyndallisation. (Tyndallisation is steaming for 30 minutes
for 3 days).
a) Add the sterile sugar solutions aseptically to the sterile peptone water and mix well.
(At MRI, unsterilized sugar is added to sterilized peptone water with indicator and then the whole
solution is sterilized by tyndallisation).
b) Dispense aseptically 4 ml amounts into sterile Bijou bottles (containing an inverted Durham tube
which was sterilized with the bottle, to look for gas production in glucose tube).
c) Date the medium and code the sugars to identify them.
d) Store in a cool dark place.
3. QUALITY CONTROL
Test for performance using control micro-organisms of known positive and negative fermentation
reactions.
Reference
1. Collee J. G, Fraser A. G., Marmion B. P., Simmons A. Mackie & McCartney Practical Medical
Microbiology, 14th Edition 1996. Churchill Livingstone
2. The Oxoid Manual. 9th Edition 2006
SELENITE F BROTH
1. DESCRIPTION
This medium is used for selective enrichment of Salmonella species. Sodium biselenite is used as a
selective agent. Selenite inhibits coliforms and other organisms present in faeces such as faecal
streptococci. Inhibited strains may eventually overgrow pathogens.
Lactose in the medium maintains a uniform pH. As selenite is reduced by the growth of bacteria alkali is
produced. An increase in pH would reduce the toxicity of selenite and result in overgrowth of organisms.
As lactose is fermented acid is produced which serves to maintain a neutral or slightly decreased pH.
Phosphate serves to maintain a stable pH and lessen the toxicity of the selenite.
2. FORMULATION
Selenite broth base ( Lactose) - 19g
Sodium biselenite - 4g
Distilled water - 1 litre
3. METHOD
4. QUALITY CONTROL1
Negative control:
Escherichia coli ATCC 29222 Inhibited or No growth
Subculture to MacConkey agar
Reference
1. The Oxoid Manual. 9th Edition 2006.
SALMONELLA-SHIGELLA AGAR
1. DESCRIPTION
Salmonella-Shigella (SS) agar is a differential selective medium used to isolate salmonella and some
strains of shigella species from faecal specimens. Gram positive and coliform organisms are inhibited by
the action of a specially prepared bile salts mixture. Thiosulphide in combination with iron act as an
indicator for sulphide production which is indicated by blackening in the centre of the colonies.The
medium is inhibitory and it is important to ensure by quality control that the expected pathogens can be
isolated by the medium used in the laboratory.
2. METHOD
a) Suspend the weight of the medium as specified by the manufacturer in 1 litre of distilled
water.
b) Bring to boil to dissolve completely.
c) Sterilize by autoclaving at 1210c for 15 minutes.
d) Pour into petri dishes (about 20ml per plate).
e) Move a flame over the surface of the agar to remove air bubbles as a final step.
3. COLONIAL CHARACTERISTICS
4. QUALITY CONTROL1
Reference
1. The Oxoid Manual. 9th Edition 2006.
1. DESCRIPTION
A selective isolation medium for Vibrio species. TCBS agar can also be used to differentiate
various Vibrio species depending on their ability to ferment sucrose. The high pH of TCBS
encourages the growth of Vibrio species while inhibiting other Gram negatives. Most
enterobacteriaceae other than Vibrio species are suppressed for at least 24 hrs. Bile salts inhibit
Gram-positive organisms. When sucrose is fermented it produces acid which changes the pH. This is
indicated by bromothymol blue and thymol blue. The medium is alkaline which enhances the
recovery of Vibrio cholerae.
2. METHOD
3. COLONIAL CHARACTERISTICS
4. QUALITY CONTROL1
Negative control:
Eschericia coli ATCC 25922 No growth
Reference:
1. The Oxoid Manual. 9th Edition 2006.
THIOGLYCOLLATE BROTH
1. DESCRIPTION
The use of thioglycollate broth permits growth of anaerobic and aerobic bacteria. No paraffin or
special seal is required. An anerobic jar is also not required for the growth of anaerobes in this
medium. It is well buffered so that acid or alkaline inocula produce negligible alteration in the
reaction of the medium.
Thioglycollate broth contains sodium thioglycollate, a reducing agent that creates anaerobic
conditions when it reduces molecular oxygen to water. Dyes such as resazurin or methylene blue
are usually added to the broth to provide a visual indication of the presence of oxygen. Resazurin is
pink when oxidized and colorless when reduced. Methylene blue is blue when oxidized and
colorless when reduced. A pink/blue/blue band near the top of the broth results when oxygen
diffuses in. Strict aerobes will grow only in the pink/blue band, microaerophiles will grow near the
bottom of the band where the concentration of oxygen is lower. The absence of pink/blue in the
rest of the tube indicates the absence of oxygen and a suitable environment for strict anaerobes.
Both facultative anaerobes and aerotolerant anaerobes will grow throughout the tube; however,
facultative anaerobes will grow most densely where oxygen is present.
2. METHOD
a) Best and most economically prepared using commercially available dehydrated medium.
b) Suspend the weight of the medium as specified by the manufacturer in 1 litre of distilled water.
c) Dispense the well mixed medium in 50 ml amounts in screw capped bottles with a central hole
and rubber liner.
d) Sterilize by autoclaving (with caps loosened) at 121C for 15 minutes.
e) When cool, tighten bottle caps.
f) Cover each bottle with a foil cap or other protective covering (previously soaked in 70% v/v
ethanol).
g) Label the bottles.
h) Store in a cool dark place, preferably between 200C and 300C.
i) If at any time, more than a narrow band at the surface appears pink, this indicates oxidation and
the medium should not be used. Reuse after carrying out procedure given below.
Place the bottle in a container of boiling water (with caps loosened) for 15 minutes to
expel dissolved oxygen.
3. QUALITY CONTROL2
Negative control:
Uninoculated plate No change
References
1. DESCRIPTION
Christensen's modified urea broth is a biochemical fluid medium used to test microorganisms for
urease production. It can be prepared from a urea broth base obtained in dehydrated form from a
commercial source. To this base, a sterile solution of urea is added.
If preferred, urea agar (prepared from urea agar base) can be used as a slope or stab. When used as a
stab, only 1 ml of medium is needed, providing a narrow bore tube is used. A stab technique gives
more rapid results.
a) Prepare urea broth base according to manufacturers instructions and sterilize by autoclaving.
b) Cool urea broth base to 50C - 55C and aseptically introduce 5 ml of sterile 40% urea
solution.
c) Mix well and dispense aseptically, 3 ml amounts into sterile bijou bottles or screw capped
tubes. Store in a cool dark place or at 2C - 8C.
d) Shelf life: up to 6 months providing there is no change in the volume or appearance of the
medium to suggest contamination or alteration of pH (pH 6.6-7.0 at room temperature).
3. QUALITY CONTROL 1:
Negative control:
Escherichia coli ATCC 25922 No colour change; urease negative
Reference
1. The Oxoid Manual. 9th Edition 2006.
1. DESCRIPTION
A selective and differential medium for the recovery of Salmonella and Shigella species. It is low in
nutrients and contains a small amount of sodium desoxycholate for selectivity. Relies on Xylose
fermentation, lysine decarboxylation and production of H2S for the primary differentiation of
Salmonellae and Shigellae from non pathogenic bacteria. Most enteric organisms except Shigella,
Providencia and Edwardsiella ferment xylose to produce acid. Salmonella also decarboxylate lysine
which keeps the pH neutral or slightly alkaline. At this pH Salmonella species can produce hydrogen
sulphide from the reduction of thiosulphate. This is indicated by ferric ammonium citrate producing
black or black-centred colonies. Some organisms, such as Citrobacter, can also decarboxylate lysine.
However, they ferment lactose and sucrose which keeps the pH too acid for hydrogen sulphide to be
produced.
2. METHOD
a) Suspend the weight of the medium as specified by the manufacturer in 1 litre of distilled water.
b) Heat with frequent agitation until the medium boils.
c) Do not overheat.
d) Transfer immediately to water bath at 500C.
e) Pour into petri dishes as soon as medium has cooled. Pour about 20ml per plate.
3. COLONIAL CHARACTERISTICS
Organism Appearance
Salmonella, Edwardsiella Red colonies with black centres
Shigella, Providencia, Red colonies
H2S negative Salmonella
Escherichia, Enterobacter, Klebsiella, Yellow, opaque colonies
Citrobacter
Proteus , Serratia
Note :To enhance blackening of salmonella colonies,XLD agar should be incubated at 350C for 24 hours
and in ambient air up to 48 hours.
Shigella dysenteriae and Shigella flexneri may occasionally be inhibited on XLD agar.
4. QUALITY CONTROL1:
Reference
1. The Oxoid Manual. 9th Edition 2006.
ALBERT’S STAIN
1. DESCRIPTION
2. METHOD
Dissolve the dyes in the ethanol. Mix the acid with the water and add to the dye solution.
Allow to stand for 24 hours and then filter.
b) Lugol's iodine
Iodine 5g
Potassium iodide 10 g
Distilled water 100 ml
Dissolve the iodide and iodine in some of the water, and adjust to 100 ml with distilled
water. For use dilute 1/5 with distilled water.
3. PROCEDURE
1. Fix the air dried smear by passing slowly through the flame three times.
2. Satin with Albert’s stain for 3-5 minutes.
3. Wash with water and blot to dry.
4. Stain with Lugol’s iodine solution for 1 minute.
5. Wash with water and drain or blot to dry.
4. INTERPRETATION
Reference
Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the Identification of Medical
Bacteria, 3rd Edition 1993. Cambridge University Press.
GRAM STAIN
1. DESCRIPTION
The Gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphology
and Gram reactions. In a clinical microbiology laboratory, it is a critical test for the rapid
presumptive diagnosis of infective agents and serves to assess the quality of clinical specimens.
Several modifications of the original Gram stain have been proposed. The Preston and Morrell
modification is given below.
2. METHOD
Solution B
Ammonium oxalate 1% aq. soln
For use, mix 20 ml of solution A and 80 ml of solution B. Filter the solution before use.
b) Lugol’s Iodine
Iodine 5g
Potassium iodide 10 g
Distilled water 100 ml
Dissolve the iodide and iodine in some of the water, and adjust to 100 ml with distilled
water. For use dilute 1/5 with distilled water.
c) Acetone-iodine solution
Strong iodine solution
Iodine 10 g
Potassium iodide 6g
Distilled water 10 ml
Ethanol (90%) to 100 ml
Dissolve the iodine and potassium iodide in the water and adjust to volume with ethanol.
Acetone-iodine mixture
Strong iodine solution 3.5 ml
Acetone 96.5 ml
Mix well before use.
Mix the phenol and fuchsin and if necessary heat gently to dissolve the phenol. Add the
ethanol and distilled water and filter into a stoppered bottle. Propanol may be substituted
for ethanol.)
3. PROCEDURE
1. Fix the air dried smear by passing slowly through the flame three times.
2. Apply ammonium oxalate-crystal violet stain for 30 seconds
3. Wash off thoroughly with Lugol’s iodine solution*
4. Apply Lugol’s iodine for 30 seconds
5. Wash thoroughly with Iodine-acetone*
6. Apply fresh iodine acetone for 30 seconds
7. Wash thoroughly with water
8. Counter stain with dilute carbol fuchsin for 30 seconds**
9. Wash thoroughly with water and drain or blot dry
*Water can be used as a substitute for reagents in routine laboratories for washing purposes.
**Safranin is used as a counter stain for specific organisms. (Eg: Neisseria gonorrhoeae)
It is important to flood the whole slide with each reagent in turn and that previous reagent is
thoroughly removed at each step.
4. INTERPRETATION
References
1. Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the Identification of Medical
Bacteria, 3rd Edition 1993. Cambridge University Press.
2. Murray PR, Baron EJ, Jorgensen JH, Pfaller AM, Yolken RH. Manual of Clinical Microbiology,
9th Edition 2007. ASM Press. Washington, DC.
1. DESCRIPTION
This stain is useful to demonstrate beading and metachromatic granules of Corynebacterium spp.
when grown on Loffler serum slopes. With sporing organisms spores appear as unstained bodies
within blue cells.
2. METHOD
Staining solution
KOH 1% aq. soln 1 ml
Distilled water 99 ml
Ethanolic methylene blue 30 ml
Mix in order; this reagent must be ripened by oxidation, a process taking several months to
complete, but ripening can be hastened by aeration. Bottles should not be more than half-full, the
stopper replaced by a light cotton-wool plug and the bottle shaken frequently. The stain improves
with keeping and batches sufficiently large to last for 5-10 years may be prepared. This ripened
stain is called Polychrome methylene blue.
3. PROCEDURE
1. Fix the air dried smear by passing slowly through the flame three times.
2. Cover the slide with stain solution for 1minute.
3. Wash thoroughly with water.
Reference
Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the Identification of Medical
Bacteria, 3rd Edition 1993. Cambridge University Press.
ZIEHL-NEELSEN STAIN
1. DESCRIPTION
This method is a modification of Ehrlich’s (1882) original method for the differential staining
of tubercle bacilli and other acid fast bacilli. It incorporates modifications suggested
successively by Ziehl and Neelsen. Ordinary aniline dyes do not readily penetrate the substance
of the tubercle bacillus and are therefore unsuitable for staining. However, by the use of a
powerful staining solution that contains phenol, and the application of heat, the dye can be
made to penetrate the bacillus. Once stained, the tubercle bacillus will withstand the action of
powerful decolourizing agents
2. METHOD
Phenol 85 g
Basic fuchsin 15 ml
Ethanol 250 ml
Distilled water 1250 ml
Mix the phenol and fuchsin and if necessary heat gently to dissolve the phenol. Add the ethanol
and distilled water and filter into a stoppered bottle. Propanol may be substituted for ethanol.
b) Acid-alcohol
Conc. HCl 3 ml
Ethanol (95%) 97 ml
Staining solution
KOH 1% aq. Soln 1 ml
Distilled water 99 ml
Ethanolic methylene blue 30 ml
Mix in order; this reagent must be ripened by oxidation, a process taking several months to
complete, but ripening can be hastened by aeration. Bottles should not be more than half-full,
the stopper replaced by a light cotton-wool plug and the bottle shaken frequently. The stain
improves with keeping and batches sufficiently large to last for 5-10 years may be prepared.
3. PROCEDURE
1. Fix the air dried smear by passing slowly through the flame three times.
2. Flood the slide with strong carbol fuchsin and heat until steam rises ( DO NOT BOIL).
3. After 3-4 minutes apply more heat until steam rises again; do not let the stain dry on the
slide.
4. About 5-7 minutes after the first application of heat wash the slide thoroughly under
running tap water.
5. Decolourize in acid-alcohol until all traces of red have disappeared from the film.
Decolourization should not be attempted in one stage; there should be intermittent
washings in water and re-application of acid-alcohol.
6. Wash well in water when decolourization is complete.
7. Counter stain with Loeffler’s methylene blue or 0.5% malachite green for 1 minute.
8. Wash and stand on end to drain; DO NOT BLOT.
Acid fast organisms are red; other organisms are blue or green depending on the
counter stain.
Do not use staining jars as tubercle bacilli from positive samples may become detached
and float about in the staining fluid or decolourizing agent. This could cause false
positive results.
Reference
Barrow GI, Feltham RKA. Cowan and Steel’s Manual for the Identification of Medical
Bacteria, 3rd Edition 1993. Cambridge University Press.
Note: The Medical Research Institute is open 24 hours for receipt of samples.
References
1. Steven Specter, Richard L Hodinka, Stephen A Young. Clinical Virology Manual, 3rd Edition 2002.
2. Douglas D Richman, Richard J Whitley, Fredrick G Hayden Clinical Virology, 2nd edition 2003.
3. Sirimali Fernando. A Handbook on Collection and Transport of Specimens for Microbiological
Investigations. 2001.
2. Antigen detection
Respiratory viruses [Adenovirus, Influenza virus A & B, Parainfluenza virus, Respiratory Syncitial
Virus (RSV)]*
Herpes simplex virus*
Cytomegalovirus*
Rabies virus
3. Virus isolation
4. Molecular diagnosis*
* Special tests
General guidelines
Samples should be collected within the first 4 days of illness .(Exception: AFP surveillance – Refer
SOP for AFP surveillance)
Samples should be collected into dry, sterile, screw capped containers. (Exception: AFP
surveillance – Refer SOP for AFP surveillance)
All samples should be collected into Viral Transport Medium (VTM). (Exception: Blood, CSF,
Stools)
VTM can be collected from Department of Virology, Medical Research Institute, Colombo 8.
The samples should be transported within 48 hours of collection.
Samples should be stored and transported at +40C in reverse cold chain box with frozen ice packs.
The samples should be properly labelled with date of collection.
The samples should be accompanied with duly filled request form.
Rejection criteria
Leaking sample
Visible contamination of sample
Improperly collected sample
Unlabelled samples
Samples sent by post
Specimen not transported in VTM (Viral Transport Media) & in ice
Samples not collected during the acute stage (within 4-5 days of onset)
Samples unaccompanied by a request form
Unmatching patient details on request form and label
SPECIMEN COLLECTION
Respiratory Nasal swabs
specimens Insert flexible, fine-shafted swab into the post-nasal space.
Rotate swab and let swab rest in place for several seconds to absorb secretions.
Use separate swabs for each nostril.
Place both swabs in the bottle of viral transport medium (VTM).
Throat swabs
Vigorously swab both tonsil areas and place swab in VTM.
Use tongue depressor to depress tongue so that contamination of swab with saliva
is prevented.
Note: Both nasal swabs and one throat swab from the same patient should be
transported in one bottle of VTM
Note:
The Medical Research Institute is open 24 hours for receipt of samples.
2. Polio virus isolation from stools of AFP patients and typing and intra - typic differentiation by Real
Time PCR and Real Time VDPV screening PCR.
REJECTION CRITERIA
Refer to general guidelines for sample collection and transport for virus detection.
REPORTING
AFP surveillance Reports will be available from 21 days after arrival of sample at the
laboratory.
Enterovirus isolation Reports will be available 14 days after receipt of sample.
SPECIMEN COLLECTION
Required Specimens and 3-5 ml of blood in a dry, sterile container or 2-3 ml of serum in a sterile
optimal time for container after 3-5 days of onset of symptoms
collection Congenital CMV infection – cord blood or blood sample within 3-5
days of birth
REJECTION CRITERIA
Refer to guidelines for collection and transport of blood and CSF for serology.
REPORTING
Test Time for results
CMV IgG/IgM Up to 7 days
EBV IgG/IgM Up to 7 days
Measles IgG/IgM Up to 7 days
Mumps IgG/IgM Up to 7 days
SPECIMEN COLLECTION
Required Specimens and 3-5ml of blood or 2-3 ml of serum in a sterile container.
optimal time for
collection 1. Serum for antibody tests
Single sample for IgM / IgG – collected after the 5th day of illness
Paired serum samples for IgM / IgG – acute and convalescent
samples collected 10 - 14 days apart
2. Antigen detection/virus isolation/PCR*
Acute samples collected within 4 days of illness
REJECTION CRITERIA
Refer to general guidelines for sample collection and transport for virus isoaltion.
Refer to guidelines for collection and transport of blood and CSF for serology.
REPORTING
Test Time for results
IgG** (HAI test) Up to 7 days
IgM** (EIA test) Up to 7 days
All these serological tests are performed using commercial test kits. The manufacturer’s instructions should
be followed when performing the test.
SPECIMEN COLLECTION
Required Specimens and 3-5 ml of blood in a dry, sterile container or 2-3 ml of serum in a
optimal time for sterile container
collection
REJECTION CRITERIA
Refer to guidelines for collection and transport of blood and CSF for serology.
REPORTING
Test Time for results
HAV IgM Up to 7 days
HBsAg Up to 7 days
HbsAb Up to 7 days
HCV antibody Up to 7 days
Hepatitis B core (total, IgM) antibody Varies depending on availability of test kits
HBeAg, HBeAb Varies depending on availability of test kits
Hepatitis E IgM and IgG Varies depending on availability of test kits
Hepatitis A IgG Varies depending on availability of test kits
* All tests are batch tests and are carried out once a week.
Tests available
Reference Laboratory for STD & HIV
1. HIV 1 & 2 antibody (EIA, PA, rapid tests) National STD/AIDS Control Programme,
2. HIV-1 antigen detection* Colombo 10.
3. CD4/CD8 counts* Tel. 011 266 7163
4. Viral load assay*
Validated EIA is the preferred method for detection of HIV-1 & 2 antibody. These tests are performed
using common test kits. The manufacturer’s instructions should be followed when performing the test.
SPECIMEN COLLECTION
Required Specimens and 3-5 ml of blood in a dry, sterile, screw capped container
optimal time for (preferably in a vacuum tube)
collection Sample should be collected 6 months after a suspected exposure.
REJECTION CRITERIA
Refer to guidelines for collection and transport of blood and CSF for serology.
REPORTING
Screening tests are performed daily. Reports will be available 1-2 days after receipt of sample.
CONFIRMATORY TESTING
When screening test is positive a second sample should be sent to the National Reference
Laboratory with duly completed Health 1214 form for confirmation of results.
Confirmatory tests will be performed once a week and the report will be available on the same day.
Special tests
Other special tests such as antigen detection tests, CD4 counts and viral load assay will be
performed only with prior arrangement with the reference laboratory.
SPECIMEN COLLECTION
Required Specimens and
optimal time for 1. Serum for antibody test (IgM) – after 5th day of illness
collection 2. CSF for antibody test (IgM) – after 5th day of illness
Storage and transport Refer to general guidelines collection and transport of blood and CSF for
serology.
Relevant clinical details and details of JE vaccination is necessary.
REJECTION CRITERIA
Refer to guidelines for collection and transport of blood and CSF for serology.
REPORTING
Test Time for results
JE - IgM* Up to 10 days
Tests available
National Respiratory Virus Reference
1. Direct fluorescent test (DFT) for respiratory viral Laboratory
antigen detection (Influenza A&B, Adeno virus, Medical Research Institute, Colombo 8
Parainfluenza & Respiratory Syncitial Virus) Tel: 011 269 3532-4 (Ext. 462),
011 269 7280
2. Virus Isolation, typing & subtyping
3. Molecular testing, typing & subtyping
SPECIMEN COLLECTION
Required specimens and Refer to general guidelines on virus isolation
optimal time for Nasal swabs
collection Throat swabs
Nasopharyngeal aspirate (NPA)
Broncho-alveolar lavage (BAL)
Tracheal aspirate
Lung Biopsy ( PM)
Samples should be collected within first 4 days of illness
REJECTION CRITERIA
Refer to general guidelines for sample collection and transport.
REPORTING
Respiratory viral Ag Within 1-2 days of arrival of sample at the laboratory
detection by DFT
Virus Isolation, typing & 2-3 weeks
sub typing
Molecular typing Carried out as a batch test. Results available in 1-5 days
REJECTION CRITERIA
Refer to general guidelines for sample collection and transport for virus detection.
Refer to guidelines for collection and transport of blood and CSF for serology.
REPORTING
Test Time for report
IgG (HAI) Up to 7 days
IgM (EIA) Up to 7 days
SPECIMEN COLLECTION
Optimal time of 1 antemortem
specimen collection 2-3 post mortem
Specimen 1. Serum / CSF – anti-rabies antibody
Animal head If transported more than 8 hours after death, the specimen should be packed
in ice and transported in a leak proof container. A short history would be
obtained at the time of receipt of specimen.
Note:
MRI is open 24 hours, for receipt of specimens.
REJECTION CRITERIA
Refer to general guidelines for sample collection and transport.
Full animal carcass
REPORTING.
TEST RESULT TIME FOR RESULT
Human brain (FAT) Positive /negative for rabies Within 72 hours
antigen
Animal head Direct smear (DS): Negri Within 24 hours
bodies positive / Inconclusive
References
1. Meslin FX, Kaplan MM, Koprowski H. Laboratory techniques in rabies. WHO 1996. 4th edition.
page 271 – 77
2. WHO Expert committee on Rabies. WHO Technical Report Series. 8th report. 1992
3. WHO first report of the expert consultation on rabies 2004. WHO Technical Report Series. 931, 2005
a) Skin Clean area with 70% alcohol, sterile saline or distilled water.
Scrape the edge of the lesion, with blunt scalpel and collect material
onto clean paper or slide.
When there is minimal scaling as in Pityriasis, clear sticky tape
(cellotape) is used to remove material by pressing the sticky side onto
lesion and then placing it sticky side down on a slide containing a drop
of lactophenol cotton blue or Parker’s stain.
In moist lesions, a swab or pieces of skin can be taken with forceps.
b) Hair Scalp should be scraped with a blunt scalpel to obtain hair stubs and skin
scales. Hairs should also be plucked with intact roots using forceps and
transported in paper or between two slides.
Piedra – pieces of hair with nodules should be collected.
d) Mucous Take scrapings with a plastic / wooden spatula from epithelial surfaces
membranes of vagina or mouth or a high vaginal swab.
Take scrapings from cornea with a sterile plastic or wooden spatula.
e) Eye specimens Immediately transfer material at the bed side, to two Sabouraud’s
glucose agar (SGA) slants or plates and to a glass slide for microscopy.
Corneal buttons removed during keratoplasty can be sent in a sterile
bottle without additives.
Intra ocular fluid, vitreous humour when collected should be sent in a
sterile bottle without additives.
f) Ear specimens Scrapings from external auditory meatus are preferred to a swab.
Transfer material onto a slide or paper.
May be transported by post, if necessary.
SUBCUTANEOUS MYCOSES
g) Pus Collect dried crusts into a folded square paper, sterile petri dish or sterile
bottle. Pus from undrained subcutaneous abscesses or sinus tracts or
grains is collected aseptically with a sterile needle and a syringe.
h) Biopsy specimens Biopsies from chronic ulcers, sinuses and subcutaneous lesions are
preferred to swabs containing pus.
Specimens should be taken from deep within the lesion as close as
possible to healthy tissue.
Place one tissue sample in a sterile bottle containing sterile normal saline
and the other in formol saline and send to the laboratory.
Refrigerate the sample in sterile normal saline if there is a possible delay
in transport, to prevent decomposition.
SYSTEMIC MYCOSES
i) Abscesses, ulcers Pus from undrained abscesses or expressed pus is collected aseptically
(skin) with a sterile needle and syringe and transported as such.
Biopsy specimens are taken from ulcerated lesions of skin and mucosa.
For collection and transport see “h” above.
k) Bone marrow 2-3 ml of bone marrow aspirate is placed in a sterile container with
0.5 ml of 1:1000 sterile heparin.
Send within 24 hours to the laboratory.
m) Fluids Pleural, abdominal and synovial fluids are collected aseptically into a
sterile bottle containing 1:1000 sterile heparin in a ratio of 10:1.
Drain fluid from patients on continuous peritoneal dialysis should be
collected into a sterile screw capped container without heparin.
Receiving of specimens 1. A designated area outside the laboratory should be allocated for receipt
of all specimens.
Maintenance of specimen 2. All specimens received for fungal studies are accepted by a MLT/MLA,
records and should be recorded in a separate register with details such as date,
type of specimen, patient details, clinical diagnosis and test required.
3. Those that can be processed in the laboratory should be identified, a
special number given and sent to the laboratory. The same number
should be exhibited clearly on the sample as well.
4. Those that have to be referred to another laboratory should be forwarded
to a separate area and such details should be entered in the register.
Specimen
no. receipt no. requested Age Name Clinical in house or referred to
/sex BHT no. history referred & date of
dispatch
References
SUPERFICIAL MYCOSES
MICROSCOPY
Skin Transfer a portion of scrapings in the paper packet on to a clean glass slide.
Add 1-2 drops of 10% potassium hydroxide (KOH) * (appendix I) to scrapings on the
slide and apply a cover slip.
Keep for 15 minutes at room temperature (on the bench), and press down on the cover
slip with the help of a filter paper to make a thin layer of the specimen.
Remove the extra KOH on the slide by blotting with filter paper.
Examine initially under x10 magnification with reduced illumination.
Any suspicious area is re-examined under x40 magnification.
Look for fungal filaments, spores and budding yeasts.
Nails The nails should be cut into very small pieces under sterile conditions.
After addition of 10% KOH to small pieces of nail and debris, place the slide in
incubator at 350C-370C for 30 minutes or keep on bench for 1-2 hours.
Blot excess KOH with a filter paper.
Firmly press down on the cover slip and examine as for skin scrapings.
Note: For nails and skin 30% KOH can be used with less exposure time.
CULTURE
Skin, hair Inoculate Sabouraud’s glucose agar (SGA) with chloramphenicol and gentamicin
and nail (appendix II) and SGA with chloramphenicol, gentamicin and cyclohexamide
specimens (actidion) (appendix II) incorporated into it.
Incubate at 260C or room temperature for up to 2 weeks.
IDENTIFICATION
Candida and Examine for growth from day 2 onwards to detect Candida.
dermatophytes To detect dermatophytes examine cultures from day 5 onwards, every other day.
If a contaminant is seen and likely to overgrow the pathogen, sub-culture the likely
pathogen, onto another appropriate culture media.
1. When a filamentous fungal growth is detected, the culture is identified as
follows:
a. Appearance of the colony eg. colour of obverse and reverse sides,
texture, grooves and furrows, adherence to the medium etc.
b. A teased mount preparation is done by picking up a small portion
from the mid area of the colony, and teasing it in a drop of Lacto-
phenol cotton blue (appendix II) on a slide. Apply cover slip and
examine under microscope x10 and x 40 for morphology.
c. Slide culture only for moulds (appendix III).
REPORTING
PRELIMINARY REPORT
FINAL REPORT
APPENDIX I – EQUIPMENT
STAINS
1. Lactophenol Lactophenol: 20 g of phenol crystals, 20 ml lactic acid, 40 ml of glycerol
cotton blue and 20 ml of distilled water. Heat gently to dissolve. Keep in a dark bottle
away from direct light.
Lactophenol cotton blue: 100 ml of lactophenol and 0.075 g of cotton blue.
Allow to stand for a few days and filter.
2. Parkers stain Equal volumes of Parkers Quink (blue black) and KOH reagents are mixed
together.
REAGENTS
1. Potassium Potassium hydroxide in concentrations varying from 10 – 30% is normally
hydroxide used.
(For 10% KOH -Potassium Hydroxide crystals 20 g and distilled water 200
ml, dissolve and use).
MEDIA
1. Sabouraud’s In house method:
Glucose agar Peptone (mycological) 10 g
(SGA) with Glucose 40 g
antibiotics Agar 20 g
Distilled water 1 litre
Chloramphenicol 50 mg is added to media prior to autoclaving1.
1. Slide culture A Petri dish containing a microscope slide, two cover slips and a bent glass rod
support and small filter paper is autoclaved.
At the time of preparing the slide culture, the slide is placed on the glass rod
support which is kept on the filter paper.
With a sterile needle or scalpel blade cut a small square of solid Sabouraud’s
glucose agar medium, measuring 0.75 - 1cm.
Transfer aseptically the agar block on to the center of the slide in the Petri dish.
Inoculate the center of each side of the agar block with the fungus by means of a
sterile needle.
Place the sterile cover slip on top of the inoculated agar block.
Add adequate sterile water to soak the filter paper in the Petri dish, to ensure that
the agar block does not dry out.
Incubate at 250C – 300C for two-weeks (for dermatophytes). If mycetoma is
suspected incubate for 3-4 weeks.
Add more sterile distilled water if required.
When sufficient growth has occurred, remove the cover slip, and place it on the
bench with the growth upwards. Add a drop of absolute alcohol to the center and
allow it to spread outwards to dissipate air from the fungal growth and allowed
to evaporate.
Add a drop of lacto phenol cotton blue to the center of cover slip and place a
new clean glass slide over it and turn over.
Examine microscopically x10 and x40.
The agar block can be removed from the original slide, and this slide can be used
to make a second preparation (as above).
2. Germ tube test This test provides a rapid and presumptive identification test of 95% – 97% of
Candida albicans.
It is carried out on primary isolates or purified cultures.
Place 0.5 ml of serum (human, fetal calf or horse) in a small test tube.
Emulsify a small portion of the yeast colony to be tested in the serum.
Incubate at 37 0C, for 2 hours.
Remove a drop of serum onto a slide, place a cover slip and examine (x10 or
x40) microscopically for germ tubes, which appear as cylindrical filaments
originating from blastospores, without any constriction at point of origin and
without obvious swelling along the length of the filament.
Boil rice in a beaker for 45 minutes and filter the solution with a piece of gauze.
Pour it to a measuring cylinder and top up with distilled water up to the 1000 ml
mark.
Weigh Tween 80 and agar and put into a 2000 ml flask.
Add the above mixture to the same flask and boil until everything is dissolved.
Pour into tubes with screw cap lids and sterilize by autoclaving at 1210C for 15
minutes.
Store at 4-80C.
When required re-heat in beaker with water and pour to petri dish.
Reference
Evans EGV, Richardson MD, Medical Microbiology a practical approach. 1989 . page 50. Oxford
University press England.
Introduction
This SOP describes the examination of faeces for the presence of cysts and trophozoites of Entamoeba
histolytica, Giardia intestinalis and oocysts of Cryptosporidium species. Trophozoite stages are most
often found in diarrhoeic faecal specimens and cysts are typically found in formed faecal specimens.
Several other protozoans that are generally considered as non pathogens can also be seen in faeces. If
any unusual protozoans are seen please contact a reference laboratory.
SPECIMEN COLLECTION
1. Patient should be provided with a suitable container for specimen collection such as an 80 or
100 ml wide-mouthed plastic cup with tight fitting, leak-proof lid and spoon (such as those
used to sell ice-cream or yoghurt).
2. Patient should be instructed to defecate onto a paper or a large leaf such as a banana leaf (that
may be disposed of later). A small quantity of faeces (at least half a teaspoon) should be
transferred immediately into the container using the spoon. Remaining faeces should be
disposed of in a sanitary latrine.
3. Faeces should not be mixed with urine or dirt.
4. The container should be labeled clearly with the patient’s name, date of collection and the time
the patient passed faeces.
SPECIMEN PROCESSING
Specimens should be examined as soon as possible after defaecation. If a number of specimens are
received at the same time, first examine specimens containing mucous or blood as they may contain
motile trophozoites of Entamoeba histolytica. Motile trophozoites are seen only in wet saline smears of
specimens examined within 30 minutes of passing stools. [Wet films can be warmed slightly to
stimulate motility of non-motile trophozoites.]
2. Place a drop of saline in the centre of the left half of the slide and place a drop of Lugol’s
iodine solution in the centre of the right half of the slide.
3. With an applicator stick, pick up a small portion of the faecal specimen and FIRST mix with
the drop of saline. Pick up another small portion of faeces and mix with the drop of iodine.
In patients with dysentery, make smears from ‘suspicious looking’ areas of the sample
containing blood or mucous.
4. Cover each drop with a coverslip. Hold the coverslip at an angle, touch the end of the drop and
lower gently onto the slide, to reduce the chance of including air bubbles in the mount.
5. Examine the slide initially using the low power (x10) objective, with reduced illumination. Any
suspicious object should be re-examined using the high power (x40) objective.
6. Look for cysts and trophozoites of Entamoeba histolytica/ Entamoeba dispar and Giardia
intestinalis. Focus the objective on the top left-hand corner of the coverslip and move the slide
systematically backwards and forwards or up and down so that the entire coverslip area is
examined.
IDENTIFICATION
For identification of protozoa, especially cysts, size is very important as otherwise the morphology
could be similar (Refer Figure 1- page 220). Ideally the microscope should have a calibrated eyepiece.
If this is not available make an assessment of the size based on the size of a red cell (7.5µm) under the
same magnification or a common faecal finding such as a hookworm or round worm ova.
Trophozoites- Saline smear is useful for identification of trophozoites. Apart from size and shape the
characteristic motility helps in identification. Amoebic trophozoites, unlike those of flagellates, are
fragile and as such E. histolytica trophozoites can be identified with certainty only in a fresh faecal
sample in saline when the characteristic directional motility with rapidly thrusting, clear, finger-like
pseudopodia is seen. The nucleus is small and ingested red cells may be seen as refractile bodies. The
trophozoites of G intestinalis have a tumbling down motility like falling leaves. (Refer Figure 2- page
220) – Trophozoites in saline smears
Note: Macrophages also can show sluggish ‘amoeboid’ movement but their nucleus is proportionately
much larger.
REPORTING
The report should identify the parasite(s) seen, by scientific name, indicating whether it is the cyst,
trophozoite or oocyst stage which was identified.
Entamoeba species
Cysts of E. histolytica and E. dispar are indistinguishable. Therefore Entamoeba cysts and trophozoites
without ingested red blood cells should be reported as E. histolytica / E.dispar. Only trophozoites with
ingested red blood cells, should be reported as E. histolytica.
Blastocystis hominis
Report should indicate whether the numbers seen are light/moderate/ heavy.
Non-pathogenic protozoa
Protozoa considered as nonpathogenic such as Entamoeba coli, Entamoeba hartmanii, Endolimax nana
and Iodamoeba butschlii should also be reported as their presence in stool indicates fecal contamination
of food or water.
Cryptosporidium oocysts are oval to round (diameter 4-6µm) and are acid–fast, irregularly staining pink
to red. Cyclospora cayetanensis oocysts are larger (diameter 8-10µm). (Refer Figures 8 & 9 page 223 )
Note: About half of all Cyclospora oocysts do not take the stain.
1. Using a rod or stick, emulsify about 1 g (pea size) faeces in 4ml of 10% formalin in a screw
capped bottle or tube.
Note: include in the sample, faeces from the surface and several places in the specimen.
2. Add a further 3-4ml of 10% formalin, cap the bottle/tube and mix well by shaking.
3. Sieve the emulsified faeces using a nylon tea strainer/ 2 layers of gauze and collect the sieved
suspension in a beaker.
4. Transfer the suspension to a conical centrifuge tube.
5. Add 3-4ml of diethyl ether or ethyl acetate.
Caution: Ether is highly flammable and ethyl acetate is also flammable, therefore use well away
from an open flame. Ether vapour is anaesthetic therefore make sure the laboratory is well-
ventilated.
6. Vortex for 15 seconds without closing the tube. If no vortex is available, stopper the tube with a
cotton wool plug/screw cap and mix well for 1 minute.
Note: Do not use a rubber bung or a cap with a rubber liner as ether attacks rubber.
7. Loosen the stopper and centrifuge at 750-1000 g (approximately 3000 rpm) for 1 minute.
8. Using a stick (ekel) loosen the layer of faecal debris from the side of the tube and invert the
tube to discard the ether, faecal debris and formalin (Refer Figure 3 page 221). The sediment
will remain.
9. Return the tube to its upright position and allow the fluid from the side of the tube to drain to
the bottom. Tap the bottom of the tube to re-suspend and mix the sediment.
10. Add a drop of Lugol’s iodine to a slide and mix a drop of sediment with the iodine.
11. Cover with a coverslip and examine under low power (x10) and high power (x40).
7. Centrifuge immediately at low speed RCF 300-400 g (1000 rpm) for 1 minute.
8. Using a plastic bulb pipette or Pasteur pipette, carefully remove the entire column of fluid
below the faecal debris and ether, and transfer this to another centrifuge tube. (Refer Figure 3
page 221)
9. Add 10% formalin to make the volume up to 10-15ml. Centrifuge at 750-1000 g
(approximately 3000 rpm) for 5-10 minutes.
10. Remove the supernatant. Tap the bottom of the tube to re-suspend and mix the sediment.
11. Transfer the sediment to a slide, stain with modified Ziehl-Neelsen method and examine under
high power (x40) and oil; immersion (x100) objectives.
DISPOSAL OF SPECIMENS
1. Add enough 10% formalin to the container to cover the faeces left in the container. This will
kill any parasites that may be present. Allow to stand for 1 h or more before discarding or
washing.
2. Slides and coverslips should be put into separate jars of disinfectant (e.g. sodium hypochlorite),
because coverslips break easily, especially if they are discarded together with slides. An
applicator stick can be used to push to coverslip off the slide into the disinfectant
References
1. District laboratory practice in tropical countries - Monica Cheesebrough 1998
2. Manual of basic techniques for a health laboratory (2nd edition) WHO 2003
3. Practical Medical Microbiology – Mackie and McCartney 1989
4. Diagnostic Techniques in Medical Parasitology – Fleck & Moody. Butterworth-Heinemann
1988
Equipment
1. Microscope slides
2. Coverslips
3. Marker pens or wax pencil for labeling slides
4. Applicator sticks (10cm ekel sticks)
5. Plastic tea strainer or gauze
6. Screw- capped bottles/tubes
7. Conical centrifuge tubes
8. Vortex mixer (optional)
9. Centrifuge
10. Plastic bulb pipettes or Pasteur pipettes
11. Compound microscope with x10, x40 and x100 objectives and adjustable condenser
Reagents
1. 10% formalin
2. Diethyl ether or ethyl acetate
3. Immersion oil
Introduction
This SOP describes the examination of faeces for the presence of helminth eggs (ova of nematodes,
cestodes and trematodes). Examination of a wet mount in saline is sufficient to diagnose a clinically
significant infection with any of the intestinal helminths.
SPECIMEN COLLECTION
1. Patient should be provided with a suitable container for specimen collection such as an 80 or
100 ml wide-mouthed plastic cup with tight fitting, leak-proof lid and spoon (such as those
used to sell ice-cream or yoghurt)
2. Patient should be instructed to defecate onto a paper or a large leaf such as a banana leaf (that
may be disposed of later). A small quantity of faeces (at least half a teaspoon) should be
transferred immediately into the container using the spoon. Remaining faeces should be
disposed of in a sanitary latrine.
3. Faeces should not be mixed with urine or dirt
4. The container should be labeled clearly with the patient’s name, date of collection and the time
the patient passed faeces
SPECIMEN PROCESSING
1. The specimen should be examined microscopically using a saline wet mount prepared directly
from the faecal sample.
2. Write the patient’s name or number on one end of the microscope slide, using a marker pen or
wax pencil
3. Place a drop of saline in the centre of the slide
4. With an applicator stick, pick up a small portion of the faecal specimen and mix with the drop
of saline
5. Cover the drop of saline with a coverslip. Hold the coverslip at an angle, touch the end of the
drop and lower gently onto the slide, to reduce the chance of including air bubbles in the
mount.
6. Examine the slide initially using the low power (x10) objective, with reduced illumination. Any
suspicious object should be re-examined using the high power (x40) objective.
7. Look for ova of intestinal nematodes, cestodes and trematodes, as well as larvae of hookworms
or Strongyloides spp., while examining the entire coverslip area. Focus the objective on the top
left-hand corner of the coverslip and move the slide systematically backwards and forwards or
up and down.
8. Quantification of infection should not be attempted on examination of direct wet smears.
REPORTING
The report should identify the parasite(s) seen, by scientific name, except in the case of hookworm
(which cannot be identified at species level by the examining the morphology of eggs or L1 larvae).
Other helminth eggs that are commonly seen in Sri Lanka include those of Ascaris lumbricoides,
Trichuris trichiura and Enterobius vermicularis.
Hookworm larvae are not usually present if the faecal sample is fresh, but it may be necessary to
distinguish these from the larvae of Strongyloides stercoralis if an old sample is examined.
Cestode and trematode infections are uncommon. If such infections are suggested, send a sample
preserved in formol-saline to any of the reference laboratories listed in the section on identification of
adult worms. Refer Figure 4 (page 221 ).
DISPOSAL OF SPECIMENS
1. Add enough 10% formalin to the container to cover the faeces left in the container. This will
kill any parasites that may be present. Allow to stand for 1 h or more before discarding or
washing.
2. Slides and coverslips should be put into separate jars of disinfectant (e.g. sodium hypochlorite),
because coverslips break easily, especially if they are discarded together with slides. An
applicator stick can be used to push to coverslip off the slide into the disinfectant
References
1. Manual of basic techniques for a health laboratory (2nd edition) WHO 2003
2. Basic laboratory methods in medical parasitology. WHO, 1991.
Introduction
This SOP describes the procedure for examination of perianal swabs for the detection of the eggs of
Enterobius vermicularis. Although these eggs may be occasionally seen in wet smears of faeces,
examination of a peri-anal swab is the more sensitive technique when enterobiasis is suspected.
SPECIMEN COLLECTION
1. Fold a strip of transparent adhesive tape over the end of a glass slide so that the sticky surface is
outermost.
2. Wearing disposable gloves, separate the patient’s buttocks with one hand and press the end of
the slide covered with the tape against the skin around the anus in several places.
3. Fold back the adhesive tape so that the sticky surface now rests on the glass slide.
4. Label the slide using a marker pen or wax pencil, with the patient’s name or number
5. The swab should be taken early in the morning before the patient defaecates or bathes, in order
to increase the chances of picking up eggs. At least 2 swabs should be taken on 2 consecutive
days before excluding a diagnosis of enterobiasis.
SPECIMEN PROCESSING
1. Add a drop of toluene under cellotape to aid visualization (this is not essential)
2. Examine the slide microscopically, initially using the low power (x10) objective, with reduced
illumination. Any suspicious object should be re-examined using the high power (x40)
objective.
3. The entire slide should be examined systematically, starting at the top left-hand corner and
moving systematically backwards and forwards, or up and down.
DISPOSAL OF SPECIMENS
1. The tape-slide should be discarded into a disinfectant and left immersed for at least 1 h before
removing the tape and washing the slide for re-use.
1. Microscope slides
2. Transparent adhesive tape (such as Cellotape) of 25 mm width
3. Toluene
4. Marker pens or wax pencils for labeling slides
5. Compound microscope with x10 and x40 objectives and adjustable condensor
References
1. Manual of basic techniques for a health laboratory (2nd edition) WHO 2003
2. Basic laboratory methods in medical parasitology, WHO, 1991.
Introduction
Adult worms and segments can be seen macroscopically in faeces or on passage from the anus. Adult or
immature worms can also be seen macroscopically in surgical specimens of lymph nodes (lymphatic
filarial parasites) or in subcutaneous nodules (zoonotic filarial worms). This SOP describes how to send
such worms that may be passed out from the anus, or found in tissues, to a reference laboratory, for full
identification.
Adult worms should not be put directly into 10% formalin since this will cause the worm to contract
and curl up. If no other preservative is available, put the worm in normal saline and send to reference
laboratory without delay.
In Sri Lanka, the most commonly seen intestinal worms are nematodes: Enterobius vermicularis,
Ascaris lumbricoides, Necator americanus (during upper GI endoscopy) and Trichuris trichiura (during
sigmoidoscopy or colonoscopy). Cestode proglottids are also seen occasionally. Intestinal trematode
infections are rare.
Adult worms of lymphatic filarial worms (Wuchereria bancrofti) or zoonotic filarial worms (Dirofilaria
repens) are seen in surgical specimens.
Reagents required
Alcohol glycerin
Buffered formalin
References
Garcia LS and Bruckner DA. Chapter 33. Fixation and special preparation of fecal parasite specimens
and arthropods In: Diagnostic Medical Parasitology, 3rd ed. Washington DC, ASM Press, 1997.
Introduction
This SOP describes the examination of blood for the presence of malarial parasites. Prompt and
accurate diagnosis of malaria is critical for effective patient management. Microscopic examination of
thick/thin blood smears remains the operational gold standard for diagnosis of malaria infections but
rapid diagnostic tests (RDTs) could be used as an alternative when microscopy is unavailable or is
impractical.
SPECIMEN COLLECTION
1. Collection of capillary blood:
Wear gloves and follow safety procedures.
With the patient’s left hand palm upwards, select the middle finger (big toe can be used
for children)
Clean the finger with a swab soaked in alcohol
Puncture the ball of the finger with a sterile lancet using a quick rolling action
Apply gentle pressure to the finger, express the first drop of blood and wipe it away
with a swab of cotton wool. Re-apply pressure, express blood and collect a single
small drop of blood on to the middle of the slide. This is for the thin film. Apply
further pressure to express more blood and collect two or three larger drops on to the
slide about 1 cm from the drop intended for the thin film.
2. Timing: The sample of blood for identification of malarial parasites should be collected as soon
as possible and before starting chemotherapy. In febrile patients timing of blood collection in
relation to febrile episode is irrelevant. If the result is negative in the first instance, the test
should be repeated a few hours later, and daily at least for 2 or 3 days.
3. If the preparation of slides is done immediately, use of anticoagulants is not necessary.
Otherwise, a clean, leak-proof container with sufficient anticoagulant (dipotassium salt EDTA
1.5 mg/mL blood or heparin 15-30 mg/mL blood) should be used for collection of blood.
4. The container or slides should be labeled clearly with the patient’s name, code/identification
number and date of collection. The dry thin film could be labeled with a soft lead pencil by
writing the patient’s name, number and date across the thicker portion of the thin film.
months, travel history (if from non-endemic areas), treatment given and antimalarial
prophylaxis) should be enclosed.
2. Blood can be transported and stored at room temperature, thick/thin blood smears should be
made as soon as possible, preferably within 2 hours of sample collection.
SPECIMEN PROCESSING
1. Fixing: Fix the thin film by dipping it in a container of pure methanol for 30 seconds. The thick
film should not be fixed in order to permit dehaemoglobinization. Therefore, avoid methanol or
methanol vapour touching the thick film.
REPORTING
Species, stages (asexual/sexual) should be indicated together with the parasite count or parasitaemia
(number of parasites per micro liter of blood or percentage of infected red cells).
Calculation of % parasitaemia:
Number of parasites counted x 100 (%)
Number of fields examined (200) Number of RBC per field (200)
Number of parasites per micro liter of blood = % parasitaemia x red cell count
100
[Refer Figure 10 (10A – 10E), pages 224 - 226 for different stages of vivax and falciparum parasites in
thin and thick smears]
Further investigations:
1. Rapid diagnostic tests for malaria antigen detection. If a kit is available in hospital laboratory,
this should be performed according to the manufacturer’s instructions
2. Molecular biological methods for species typing, genotyping, bar coding etc.
Detailed information could be obtained from:
Malaria Research Unit, Department of Parasitology in the Faculty of Medicine, University of
Colombo, Kynsey Road, Colombo 8. Tel. 0112699284
Introduction
This SOP describes examination of a thick smear made of peripheral blood for the presence of
microfilariae of lymphatic filarial parasites prevalent in Sri Lanka, Wuchereria bancrofti & Brugia
malayi. This is applicable for investigation of patients with clinical features suggestive of lymphatic
filariasis, as well as for screening of asymptomatic individuals resident in endemic areas.
SPECIMEN PROCESSING
1. Place the slides vertically in the staining trough filled with clean water and leave for 10
minutes. The haemoglobin sinks to the bottom.
2. Take the slides and drain them
3. The dehaemoglobinised smear may be stained with Giemsa or Delafield’s haematoxylin
Microscopic examination
Examine the smear under low power using the x 10 objective
When a microfilaria is found, centre it in the field, cover the smear with a thin film of
immersion oil and examine using x10 x100 magnification (oil immersion objective) for
species identification.
REPORTING
The report should identify the species of the microfilaria seen, by scientific name.
Further investigations:
1. Rapid diagnostic tests for filarial antigen detection. If a kit is available in hospital laboratory,
this should be performed according to the manufacturer’s instructions
2. Filarial Fluorescent Antibody Test (FFAT): antibodies are detected using the indirect
fluorescent antibody technique at the Dept of Parasitology, Medical Research Institute,
Colombo 8.
References
1. World Health Organization. Part II A. Parasitology. In: Manual of basic techniques for a health
laboratory, Geneva 1980.
SPECIMEN COLLECTION
1. If there are any skin lesions, they should be cleaned with soap and clean tap water. Any
scab/crust should be gently removed during the procedure. Then gently wipe off the lesion with
70% alcohol.
2. Lesion aspiration: aspirate the active edge of the lesion or prominent nodule using 0.2 to 0.3
ml of sterile saline injected to the site using a 22 or 23 gauge sterile needle. Place one drop of
the aspirate on a clean glass slide. Make a smear by spreading the aspirate drop using the
needle.
3. Slit-skin scrapings: make a slit over the active edge of the cleaned wound using a sterile no. 10
scalpel. Take tissue scrapings from both edges of the slit to the scalpel by gently moving the
scalpel blade along the slit. Immediately smear the scrapings on to a clean glass slide using the
scalpel.
4. Both lesion aspirates and slit skin scrapings can be smeared on to one slide as shown in Figure
5 (Refer page 222).
5. For PCR: Two millimeter (2mm) punch biopsies should be taken from the active edge of the
ulcers or the prominent and active sites of nodules, using a sterile 2 mm sterile punch biopsy
needle, according to standard dermatological procedures. Put the biopsy in to a sterile
Eppendorf tube (usually provided by the reference lab) without formalin, containing 1ml of
sterile normal saline so that the sample can be used for PCR and culture
SPECIMEN PROCESSING
1. All slides should be clearly labeled with patient ID and the sample type (especially if two
smears are made on a single slide) before staining.
2. Smears should be fixed in methyl alcohol for 30 seconds.
3. Fixed smears should be stained with 10% Giemsa in buffered water. Single slides can be
stained face downwards in a staining plate for 20-30 minutes. If large numbers of slides are
used, a staining trough can be used.
4. Stain should be gently washed off using water and air dried by placing them upright at an
angle.
Giemsa stained smears are examined under x10 x100 with the oil-immersion lens. Leishmania
amastigotes may be found inside macrophages or extra-cellularly. The whole smear should be
examined systematically before issuing a negative report as low parasitaemias can be easily missed by
light microscopy. Species identification is not possible with light microscopy.
REPORTING
The report should identify the parasites by the scientific name up to the genus level. (i.e., Leishmania
sp. parasites seen/not seen). Please note that it is not possible to establish species identification by light
microscopy, culture or routine PCR. (Refer Figure 11 page 226)
References:
National Action Plan for Leishmania control. Proceedings of the Leishmaniasis Colloquium, 2009
Specimen: In the female – a high vaginal swab from the posterior fornix
In the male - urethral discharge/ a sample of urine sediment
Introduction:
This SOP describes the ‘wet mount’ preparation and direct microscopy for diagnosis of trichomonal
infection, a sexually transmitted parasitic disease caused by a flagellated protozoan Trichomonas
vaginalis. Female patients (50-75%) will present with a frothy greenish-yellow vaginal discharge with a
characteristic fishy odour. Males may present with a urethral discharge, but majority will be
asymptomatic. The wet mount is rapid, easy to perform, accurate, and costs little to carry out.
SPECIMEN COLLECTION
In the female:
1. A high vaginal swab should be collected by a medical officer, using a cotton-tipped applicator
stick, under a speculum examination, from secretions in the posterior fornix.
In the male:
1. Urethral discharge can be collected with an applicator stick similarly, at the urethra, or
2. Urine: collect the early morning first catch fresh urine sample.
1. The specimen should be examined within 20 minutes of collection. Delays will make the
parasite immotile and die and the diagnosis will be impossible. Do not refrigerate.
2. This is preferably performed as a bed side test in the laboratory and then the sample can be
directly applied on the slide with a drop of saline.
3. If the sample is to be dispatched to the laboratory, the applicator stick has to be inserted into a
test tube containing 0.5-1 ml of 0.9% N. saline and be sent to the lab as quickly as possible.
SPECIMEN PROCESSING
Diagnosis
In a wet mount, T. vaginalis trophozoites will appear as motile (with a rapid rolling movement), pear-
shaped, 10 µm by 7 µm organisms, with visible flagellae (5) and a posterior axostyle (Refer Figures 6
& 7 page 222) (in T. vaginalis life cycle only trophozoite form has been identified). The field will also
have many polymorphonuclear neutrophils (PMNs) and epithelial cells. If the specimen dries up the
parasites can be confused with PMNs.
REPORTING
The report should be given as “wet mount positive/negative for Trichomonas vaginalis”. A minimum
of 10 fields should be examined before giving a negative report.
DISPOSAL OF SPECIMENS
Specimen and the applicator stick have to be considered as potentially infectious waste.
References
1. Cook G.C (2003) Trichomonal infections In: Manson’s Tropical Diseases (ed G.C. Cook & A.
Zumla) 21st edition, 1427-29pp, Saunders, Elsevier Science, Jamestown Road, London.
2. Arlene C. Senã AC, Miller WM, Hobbs MM, Schwebke JR, Leone PA et al ( 2007)
Trichomonas vaginalis Infection in Male Sexual Partners: Implications for Diagnosis,
Treatment, and Prevention. Clinical Infectious Disease, 44:13–22
Toxoplasmosis is caused by the intracellular parasite Toxoplasma gondii. Due to the non-specific nature
of symptoms and the presence of asymptomatic /sub-clinical infections in the community, diagnosis is
frequently based on serology. This SOP describes how to send specimens for the detection of
Toxoplasma antibodies by ELISA, which is the commonly used technique in Sri Lanka.
SPECIMEN COLLECTION
1. 3ml of blood should be collected into a plain dry sterile screw capped container
2. Send to Reference Laboratory packed in ice.
3. If specimen is to be posted to the reference laboratory, it is preferable to send serum.
1. Blood should be sent to the reference laboratory preferably within two hours with
accompanying request form giving patient identification details and clinical details.
2. Serum sample - if it is not possible to post on the same day, store it in a refrigerator in the
freezer compartment and transport the labeled specimen packed in ice.
1. There is a possibility of getting false negative and false positive IgM results in specimens with
extremely high Rheumatoid factor and high autoimmune antibodies.
2. Lipemic, hemolyzed, icteric or heat inactivated sera may cause erroneous results.
3. As with other serological assays, the results of these assays should be used in conjunction with
information available from clinical evaluation and other diagnostic procedures.
Introduction
Toxocariasis (also known as Visceral Larva Migrans) is caused by the larval stages of Toxocara spp.
Since parasitological diagnosis is usually not possible, diagnosis is frequently based on serology. This
SOP describes how to send specimens for the detection of Toxocara antibodies using Excretory –
Secretory Antigen of second stage larva of Toxocara spp. (TES-ELISA)
SPECIMEN COLLECTION
1. 5ml of blood should be collected into a plain dry sterile screw capped container
2. Send to Reference Laboratory packed in ice.
3. If specimen is to be posted to the reference laboratory, it is preferable to separate serum.
ALCOHOL-GLYCERIN
95% ethyl alcohol 70 ml
Distilled water 25 ml
Glycerin 5 ml
Formaldehyde is normally purchased as a 37% HCHO solution; however, for dilution, it should be
considered to be 100%.
Dissolve the haematoxylin crystals in absolute ethanol. Add a few drops at a time to the saturated
aluminium ammonium sulfate solution. Leave this solution unstoppered in direct sunlight or in a
37◦C incubator for 3-4 months to oxidize the haematoxylin to haematin.
Stopper and label the bottle and write the date. When reopened, filter and add the glycerol and
methyl alcohol, and the stain is ready for use. Keep stoppered to prevent evaporation. The stain will
remain good for 18 months.
Use a chemically clean and dry dark glass or polyethylene bottle of suitable size.
1. Put the glass beads in the bottle; pour in the measured amount of methanol and add the
stain powder.
2. Tightly stopper the bottle. Allow the stain powder to sink slowly through the methanol until
it settles to the bottom. Shake the bottle in a circular motion for 2-3 minutes.
3. Add the measured amount of glycerol and repeat the shaking process. Continue to shake for
2-3 minutes at half-hourly intervals for at least six times.
4. Leave the bottle for 2-3 days, shaking it 3-4 times each day until the stain is thoroughly
mixed.
5. Filter.
Each newly prepared batch of stain should be properly labelled, including date of preparation and
should be tested for stain quality prior to routine use. Always keep the bottle tightly stoppered, in a
cool place, away from sunlight.
LEISHMAN’S STAIN
1. Add 1.5 g of dry Leishman’s powder to 1000 ml of absolute methanol in a glass bottle.
2. Add a few glass beads.
3. Keep the bottle lightly capped and shake well occasionally throughout the day.
4. The stain is ready for use after 24 hours.
LUGOL’S IODINE
Iodine 1g
Potassium iodide (KI) 2g
Distilled water 100ml
Weigh the iodine in a porcelain dish or watch glass. Grind the dry iodine and potassium iodide in a
mortar. Add water a few milliliters at a time, and grind thoroughly after each addition until the
iodine and iodide dissolve. Put the solution into an amber glass bottle with the remainder of the
distilled water. Sunlight will decolourize the stain.
Add about a ¼ of the water to the dye and mix until the dye is fully dissolved. Then add the
remainder of the water and mix well. The stain is stable for several months.
NORMAL SALINE
Sodium chloride (NaCl) 8.5 g
Distilled water 1000 ml
Weigh out the NaCl. Measure the distilled water into a clean, glass-stoppered bottle. Dissolve the
NaCl in the water and mix thoroughly. Label the bottle ‘NORMAL SALINE’ and write the date.
Put a piece of string or a narrow strip of paper between the glass stopper and the neck of the bottle
to keep the stopper from sticking. Pour some saline into a dropping bottle or dispensing bottle for
daily use.
STRONG CARBOL-FUCHSIN
Solution A
Basic fuchsin 10g
Absolute Ethanol (100%) 100ml
Mix and dissolve in a stoppered bottle and keep at 350C-370C overnight
Solution B
Phenol 5g
Distilled water 100ml
Mix and dissolve
References
1. World Health Organization. Annex 2. Reagents and solutions and their preparation. In: Basic
Laboratory methods in Medical Parasitology Geneva, WHO, 1991.
Figure 4. Comparative sizes and morphology of helminth eggs found in human faeces
221
Label Aspirate Slit skin
smear smear
Figure 5. Preparing skin smears for examination for Leishmania parasites
222
Oocysts in faecal smears stained with Ziehl-Neelsen stain
223
Figure 10 (10 A- 10 E) - . Malaria and Leishmania parasites
Figure 10 A. Plasmodium vivax ring trophozoites in thin blood film stained with Giemsa (x1000
magnification)
Figure 10 B. Plasmodium vivax amoeboid trophozoites in thin blood film stained with Giemsa (x1000
magnification)
224
Figure 10 C. Plasmodium vivax schizont in thick blood film stained with Giemsa (x1000 magnification)
Figure 10 D. Plasmodium falciparum ring trophozoites in thick blood film stained with Giemsa (x1000
magnification)
225
Figure 10 E. Plasmodium falciparum gametocyte in thick blood film stained with Giemsa (x1000
magnification)
226
IMMUNOLOGY
IMMUNOLOGICAL DIAGNOSTIC TESTS
INTRODUCTION
This section lists investigations suitable for local laboratories, and tests which are available at the
Immunology Reference Laboratory, MRI.
i. WBC/DC
ii. C- reactive protein (CRP)
iii. Rheumatoid factor (RF)
CRP and RF levels are measured using commercial assays (slide agglutination).
Please follow the manufacturers’ instructions on performing the tests.
I. ANTIBODIES
II. COMPLEMENT
TEST By Patient to be Specimen Points to note Availability of
appointment sent details report
Complement NO NO 2ml of blood in Test
components a plain bottle commences on
C3,C4 Tuesdays and
report issued
on Fridays of
same week
IV. LYMPHOCYTES
a) Quantitative assay
*for evaluation of
immunodeficienci
es
including
HIV/AIDS
Other relevant CD
markers for
classification of
leukaemias
Refer rejection
criteria
b) Qualitative assay
Skin biopsy sample needs to be sent in Michel’s medium. Medium can be obtained from Department of
Immunology, MRI.
Samples will be accepted only if the clinical history is written in the form provided by the Department.
1. It is IMPORTANT to note that all specimens should be accompanied by a duly filled request form with
the patient's age etc. and a brief history. (Age is important because normal range of immunoglobulins
vary in different age groups)
Note: History is essential for interpretation of results.
2. Note that appointments are required for some tests. If an appointment cannot be kept, please ensure that
the laboratory is informed.
REJECTION CRITERIA
Reference
Rose N. R., de Macario E C., Folds J. D., Lane H. C., Nakamura. R. N. Manual of Clinical Laboratory
Immunology, 5th Edition 1997, American Society of Microbiology