Microbio Pracs Reviewer Bacteriology 2017

Bambam2017
Made by AMFValle UST-FMS SecD Batch 2017

Bambam2017
 Procedure (from Broth Cultures):

 Sterilize Inoculating Loop.
▪ Hold it over the bunsen burner flame in a vertical position until it
is red hot. Then, hold it away from flame and let it cool.
 With sterilized inoculating loop, fish a loopful of bacterial
suspension and spread evenly on marked surface on slide.
 Spread smear gently to make a thin film.
 Let smear dry in air. Sterilize loop after use.
 Fix the smear briefly by passing slides through the flame,
film side up, 3x.
Sources: Notes from UST-FMS Micro Lab Manual, Pictures from UST-FMS 2017 Sec D & Internet
Bambam2017
 Utilizes only ONE dye

 (+) charged stains so that it will bind to
the (-) bact. cell wall
 Identify bacteria based on morphology
 Size
 Shape
 Arrangement
 Procedure:
1. Flood smeared area with METHYLENE
BLUE stain
2. Allow stain to stay for 1-2 minutes
3. Wash stain with gentle stream of tap
water
4. Blot dry using sheets of blotting paper
Credits to L. Viray for the pictures
Bambam2017
 Procedure:
1. Flood smeared area with CRYSTAL VIOLET stain
(initial stain) for 1 minute
2. Pour off stain, wash with gentle flow of water
3. Apply GRAM’s IODINE (mordant) for 1 minute, then
rinse off with water
4. Decolorize using ACETONE-ALCOHOL solution or
95% ETHYL ALCOHOL for 20 seconds
5. Apply SAFRANIN (counter stain) for 30 seconds, then
wash off with water
 Differentiates Gram(+) and Gram (-)

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Gram (+) Gram (-)
Credits to L.Viray for the pictures

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 For Acid Fast bacteria

(Mycobacterium tb)
 Procedure (KINYOUN’s METHOD):
1. Cover smear with Kinyoun’s
CARBOL FUCHSIN stain (initial
stain) for 2-3 minutes, then rinse
with tap water
2. Flood smear with 3% ACID
ALCOHOL (decolorizer) for 30-45
seconds, then rinse with tap water
Bacterium Background
3. Cover smear with METHYLENE
BLUE (counter stain) for 20-30 Pink Blue
seconds, then rinse with tap water
and blot dry
Credits to K. Erguiza SecB 2014 for the picture
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 Report as follows:
Reporting Acid Fast bacilli in Sputum specimen
No AFB in at least 100 0 or (-)
fields
1-9 AFB in 100 fields +n (report actual AFB
count), Suggest repeat
collection
10-99 AFB in 100 fields 1+
1-10 AFB in at least 50 2+
Bacterium Background
fields
>10 AFB/field in at least 3+ Pink Blue
20 fields

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 For Bacillus subtilis and other

sporeformers
 Procedure:
1. Flood slide with 5% Aqueous
solution of MALACHITE GREEN
and heat to gentle steaming for
2-3 minutes. Avoid overheating
and drying.
2. Pour off excess stain and rinse
with water
3. Flood smear with SAFRANIN for Spore Vegetative
30 seconds cell
Green Pink
4. Rinse again with tap water and
blot dry
Credits to L. Viray for the picture
Bambam2017
 For Klebsiella pneumoniae, etc.

 INDIRECT METHOD (Congo Red):
 Also called negative or relief stain
1. Using a sterile inoculating loop,
fish out a small colony of K.
pneumoniae and mix it with a
drop of CONGO RED STAIN and
spread 2 cm diameter.
2. DO NOT HEAT
Capsule Background
3. Cover the smear with 1 % ACID
colorless Blue
ALCOHOL until smear turns blue
4. Air dry
Bambam2017
 For Klebsiella pneumoniae,

etc.
 INDIA INK Method
1. Place a loopful of bacteria
on a slide
2. Add a small amount of
India ink and immediately
cover with coverslip to
Capsule Background
allow fluid to spread as a
colorless India Ink
thin film

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 Physical and Chemical Disinfectant
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 Different Methods of Streaking

1. Simple streaking
2. 4 quadrant streaking – isolation of colony
3. Overlapping streaking – sensitivity testing
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 KIRBY-BAUER method – Disk Diffusion Method

 Introduced by William Kirby and Alfred Bauer in 1966
 Consists of newly seeded lawn of bacterium in a
nutrient medium (MUELLER-HINTON AGAR) and filter
paper disks impregnated with antibiotics
 Culture is incubated for 16 to 18 hours
 However, in the procedure given in the manual, we
incubate for 18-24 hours at 35 degrees Centigrade
 Measure ZONE OF INHIBITION (diameter)
 Diameter of zone ≈ Rate of DIFFUSION

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 Can be reported as follows

1. Resistant – no zone of inhibition
2. Intermediate
3. Susceptible – large zone of inhibition

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 Differentiated by
hemolysis, mannitol salt
agar growth, and
coagulase test
 S. aureus
 Deep gold to pale cream
or white colonies
 Clear zone of hemolysis
 S.epidermidis
 Porcelain white colonies
 Non-hemolytic
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 CATALASE TEST (slide

method)
1. Place a drop of 3% H2O2
(hydrogen peroxide) on slide
2. Get a loopful of colony, mix
with the drop of 3% H2O2 on
the slide
3. Observe for bubble
formation (gas liberation)
 (+) Staphylococcus
 (-) Streptococcus
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 MANNITOL
FERMENTATION Test
1. Streak on the Mannitol
Salt Agar plate
2. Observe color of colonies
▪ Yellow colonies =
fermentation of mannitol
3. Wrap plate and incubate
for 18-24 hours
7.5% NaCl (Salt) – inhibits

growth of other organisms
 S. aureus – yellow colonies
 S. epidermidis – pink
colonies
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 COAGULASE TEST
I. Slide Method
1. Place 2 drops of sterile saline on slide
2. Pick a colony using loop, mix with the saline
3. Add 1 drop citrated fresh normal plasma
4. CLUMPING = positive!
 (+) S. aureus
 (-) S. epidermidis

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 COAGULASE TEST
II. Tube Method
1. Get tube containing 0.5 mL
citrated fresh plasma
2. Suspend colonies on tube
3. Incubate for 37 deg C in water
bath and read results at 4 hours
and 24 hours if still negative
4. COAGULATION (by tilting tube) =
positive!
5. Often, a clot will stick to the
bottom of the tube even if it is
inverted
 (+) S. aureus
 (-) S. epidermidis Credits to K. Erguiza SecB 2014 for the picture
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 Differentiated by type of HEMOLYSIS

 TYPES OF HEMOLYSIS
1. Beta – complete hemolysis
 Completely decolorized Hgb
 More marked when the plate has been incubated in candle jar (5-10% CO2)
 Streptococcus pyogenes
2. Alpha – partial or incomplete hemolysis

 Green-brown color (reduced Hgb)
 S. pneumoniae and Viridans
3. Gamma – non-hemolytic, may have slight discoloration in the

medium
 Enterococcus fecalis
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 Gram (+) cocci in chains

 Streptococcus pyogenes
 Group A, Beta hemolytic,
susceptible to bacitracin
 Streptococcus pneumoniae
 Group B, Alpha hemolytic,
lancet-shaped, susceptible to
optochin in chocolate agar
plate (CAP)
 POSITIVE QUELLUNG
REACTION
 Viridans streptococci
 Alpha hemolytic
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 TOLERANCE to 6.5% NaCl

1. Inoculate to a tube of 6.5% NaCl broth (Brain
Heart Infusion Broth supplemented with NaCl to
6.5% concentration)
2. Incubate at 35 deg C for 24-48 hrs
3. TURBID = positive growth!
 (+) Group D Strep i.e. Enterococci
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 CATALASE TEST
1. Place a colony on a clean glass slide
2. Add a drop of 3% H2O2
3. Observe for bubble formation

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 Test for Susceptibility (Bacitracin & Optochin)

1. Place the disk on the blood agar culture
2. Incubate at 35 deg C for 18-24 hours
3. Zone of Inhibition = Susceptible!
 BACITRACIN TEST (TAXO A)

 Susceptible: Group A Strep i.e. S. pyogenes
 OPTOCHIN TEST
 Susceptible: Group B Strep i.e. S. pneumoniae
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 Urethral smear: Gram negative

diplococci with adjacent sides flattened
(kidney-bean shaped)
 Usually found inside pus cells
 Intracellular in acute gonorrhea, in early
infections or in chronic gonorrhea
 But they may also be found extracellularly
 Modified THAYER MARTIN agar culture

 Sugar Fermentation test to
differentiate:
 N. gonorrhea and N. meningitides
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 Sugar Sugar N. menigitides N. gonorrhea

Fermentation Glucose + +
Test Lactose - -
▪ Both are Glucose Maltose + -
fermenters
▪ Only N.
meningitides is
maltose
fermenter
▪ Yellow (+)
▪ Red (-)
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I. NON-SPOREFORMER BACILLI
 Corynebacterium diphtheria
II. ANAEROBIC SPOREFORMER

BACILLI
 Clostridium
III. AEROBIC SPOREFORMER

 Bacillus

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Mott – Mycobacterium other than Mtb

NTM – Nontuberculous mycobacterium
 LOWENSTEIN JENSEN (LJ) Medium

 Culture for M. tuberculosis
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 International Union Against Tb and Lung Disease
Reporting Acid Fast bacilli in Sputum specimen

No AFB in at least 100 0 or (-)
fields
1-9 AFB in 100 fields +n (report actual AFB
count), Suggest repeat
collection
10-99 AFB in 100 fields 1+
1-10 AFB in at least 50 2+
fields
>10 AFB/field in at least 3+
20 fields
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 EMBA (Eosin Methylene Blue

Agar)
▪ Selective and Differential
 Lactose fermenter – greenish
metallic sheen in reflected light
and blue black center on
transmitted light; may be
mucoid with grayish brown
centers
 Nonlactose fermenter –
translucent, colorless to slightly
amber colored colonies
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 EMBA (Eosin
Methylene Blue
Agar)
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 MacConkey Agar
 Lactose fermenter
– large, red colonies
surrounded by
turbid zones
 Nonlactose
fermenter –
colorless and
translucent colonies
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 MacConkey Agar
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 MacConkey Agar
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 Biochemical
Tests Procedure
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A. TRIPLE SUGAR IRON

 Tests the ability to utilize
glucose anaerobically with
formation of acid and products
(fermentation)
 Indicator: Phenol Red
▪ Basic  Acidic
 Sugars
▪ Glucose/Dextrose
▪ Lactose
▪ Sucrose
 Tests: Sugar fermentation, H2S
production, Gas production
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A. TRIPLE SUGAR IRON
Acid slant yellow Alkaline slant red Alkaline slant red Blackening of Cracks, bubbles
Acid butt yellow Acid butt yellow Alkaline butt red butt or displacement
of medium
All sugars GLUCOSE No sugar Produce H2S Produce gas
fermented fermented fermented (aerogenic)
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B. LYSINE IRON AGAR

 Decarboxylation and Deamination
 Decarboxylation of amino acids
(lysine, ornithine, arginine) resulting
in alkalination of the media
 Indicator: Bromcresol purple
▪ Basic  Acidic
 Tests: Deamination, Lysine
decarboxylation
▪ Manner of Inoculation
▪ Stab the butt
▪ Streak the slant
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B. LYSINE IRON AGAR

 Lysine Deamination (SLANT)
 Deamination occurs in the presence of
O2, kaya sa slant part (exposed to O2)
 Deamination produces acidic species
 Red slant (+), acidic
 Purple slant (-), basic
 Lysine Decarboxylation (BUTT)
 Decarboxylation produces basic species
(you remove the carboxyl end, natira si
amino which is basic)
 Purple butt (+), basic
 Yellow butt (-), acidic
Bambam2017
C. MOTILITY INDOLE ORNITHINE

 Indicator: Bromocresol Purple
 Motility Indole Ornithine Medium (MIO)
▪ Tests: Motility, Indole production, Ornithine
decarboxylation
▪ Inoculation: Stab the butt
 For Motility and Decarb testing;

1. The portal of entry should be the portal of exit
to eliminate false (+) results
2. Stab the medium up to the bottom because
decarboxylation occurs at the deep butt
3. Do not shake. Medium is semisolid in
consistency.
4. Read the motility and decarboxylation result
before adding Kovac’s Reagent!
Bambam2017
C. MOTILITY INDOLE ORNITHINE

 Indicator: Bromocresol Purple
 Motility Indole Ornithine Medium (MIO)
▪ Tests: Motility, Indole production, Ornithine
decarboxylation
▪ Inoculation: Stab the butt
 For Motility and Decarb testing;

▪ (+) Motile = diffuse growth around stabbing line
▪ (+) Decarboxylation = purple butt
 For Indole Production;

▪ Add 2-3 drops Kovac’s reagent
▪ Tryptophanase: Tryptophan  Indole
▪ (+) Indole Production = red ring upon addition
of Kovac’s modification of Erhlick’s reagent
(paradimethylaminobenzaldehyde)
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D. CITRATE UTILIZATION TEST

▪ Tests ability to synthesize complex
carbon compounds from an inorganic
carbon source like citrate
▪ (+) for Autotrophs
▪ Many soil bacteria DO NOT have this ability
 Indicator: Bromothymol Blue
 Simmon’s Citrate Agar
▪ Test: Citrate Utilization Test
▪ Inoculation: Streak the slant
 (+) Blue Slant

 (-) Green Slant
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E. UREASE TEST
▪ Detects ability to split urea
to ammonia (basic!) and CO2
by the enzyme urease
 Indicator: Phenol red
 Medium: Urea Broth
▪ Test: Urease Test
 (+) Fuschia Pink

 (-) Salmon Pink/Yellow
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E. UREASE TEST
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Selective for
S. typhi
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A. Water Collection
1. Clean the mouth of faucet with 70% EtOH
2. Collect 100 mL of chlorinated (tap water) by
allowing water to flow about 1-5 minutes
3. Alternative: unchlorinated (deep well) water
4. Refrigerate while waiting to transport
5. Transport in ice (4 degrees Celsius)
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B. Methods of Water Analysis

1. Heterotrophic Plate count: Pour Plate Method
2. Multiple Tube Fermentation Technique (MTFT)
for Coliform group of organisms
a) Presumptive test
b) Confirmatory test
c) Completed test
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1. Heterotrophic Plate Count: Pour Plate Method
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MTFT: Presumptive test

Medium: Triple Strength
Lauryl Tryptose Broth
Incubation:
 24 hours
 35 deg C
(+) Coliform bacteria

(+) Gas formation and
turbidity
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MTFT: Confirmatory Test

Medium: Brilliant Green
Lactose Broth (BGLB)
Incubation:
 48 hours
 35 deg C
(+) Total coliforms
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MTFT: Confirmatory Test

Medium: EC Broth
Incubation:
 24 hours
 44.5 deg C + 0.2 deg C
(+) Fecal coliforms
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 Computation of MPN
(Most Probable Number)
Reviewer by Bambam2017

Microbio Pracs Reviewer Bacteriology 2017

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Bambam2017

Made by AMFValle UST-FMS SecD Batch 2017

 Procedure (from Broth Cultures):

 Utilizes only ONE dye

 Differentiates Gram(+) and Gram (-)

Gram (+) Gram (-)

Credits to L.Viray for the pictures

 For Acid Fast bacteria

Credits to K. Erguiza SecB 2014 for the picture

 For Bacillus subtilis and other

 For Klebsiella pneumoniae, etc.

 For Klebsiella pneumoniae,

Credits to K. Erguiza SecB 2014 for the picture

 Physical and Chemical Disinfectant

 Different Methods of Streaking

 KIRBY-BAUER method – Disk Diffusion Method

 Diameter of zone ≈ Rate of DIFFUSION

 Can be reported as follows

Credits to K. Erguiza SecB 2014 for the picture

Credits to K. Erguiza SecB 2014 for the picture

 CATALASE TEST (slide

7.5% NaCl (Salt) – inhibits

Credits to K. Erguiza SecB 2014 for the picture

 Differentiated by type of HEMOLYSIS

2. Alpha – partial or incomplete hemolysis

3. Gamma – non-hemolytic, may have slight discoloration in the

 Gram (+) cocci in chains

 TOLERANCE to 6.5% NaCl

 (+) Group D Strep i.e. Enterococci

Credits to K. Erguiza SecB 2014 for the picture

 Test for Susceptibility (Bacitracin & Optochin)

 BACITRACIN TEST (TAXO A)

 Urethral smear: Gram negative

 Modified THAYER MARTIN agar culture

 Sugar Sugar N. menigitides N. gonorrhea

II. ANAEROBIC SPOREFORMER

III. AEROBIC SPOREFORMER

Credits to K. Erguiza SecB 2014 for the picture

Mott – Mycobacterium other than Mtb

 LOWENSTEIN JENSEN (LJ) Medium

 International Union Against Tb and Lung Disease

Reporting Acid Fast bacilli in Sputum specimen

 EMBA (Eosin Methylene Blue

A. TRIPLE SUGAR IRON

A. TRIPLE SUGAR IRON

B. LYSINE IRON AGAR

B. LYSINE IRON AGAR

C. MOTILITY INDOLE ORNITHINE

 For Motility and Decarb testing;

C. MOTILITY INDOLE ORNITHINE

 For Motility and Decarb testing;

 For Indole Production;

D. CITRATE UTILIZATION TEST

 (+) Blue Slant

 (+) Fuschia Pink

B. Methods of Water Analysis

1. Heterotrophic Plate Count: Pour Plate Method

MTFT: Presumptive test

(+) Coliform bacteria

MTFT: Confirmatory Test

(+) Total coliforms

MTFT: Confirmatory Test

(+) Fecal coliforms