Bacte PPT 4 - Detection Methods
Bacte PPT 4 - Detection Methods
Smear Preparation
1. Tissues
2. Swabs
3. Aspirates and body fluid
a. Single-Drop smear
b. Centrifuged smear
c. Layered smear
Additives
•Albumin
•Mucolytic agents
Staining
1. Simple staining
2. Differential staining
3. Special staining
4. Indirect/Negative staining
a. India Ink stain test
b. Nigrossin method
Staining
1. Gram stain
• Two groups:
• Cell wall
Reagent Purpose G(+) cell wall G(-) cell wall
1. Crystal Violet
2. Gram’s Iodine
3. Acetone Alcohol
4. Safranin Red
Errors
Gram (+) becomes Gram (-)
1. Overdecolorization, old, dying
2. Use of acidic iodine as mordant
3. Penicillin. Omit iodine
Gram (-) becomes Gram (+)
1. Underdecolorization
2. Thick smear
Gram stain general rule:
• All cocci are gram (+) except:
Neisseria, Veilonella, Moraxella
• All bacilli are gram (-)
Mycobacteria, Corynebacteria, Bacillus, Norcardia, Clostridium,
Actinomyces, Lactobacillus, Listeria, Erysipelothrix
• All spirals organisms are gram (-)
• Yeast are gram (+)
2. Acid-fast stain
• Used primarily to detect Mycobacterium species
a. Ziehl-Neelsen Method - hot method, uses heat to
facilitate staining
70%
Types of culture
1.Pure culture – ID and AST
2.Mixed culture – >2 org
3.Stock culture -QC
Culture Media (Table 7-1)
• Media – contains nutrients for growth, isolation and ID, can be
classified based on:
Physical state or consistency
• Solid
• Semi-Solid
• Liquid (broth)
• Biphasic – both liquid and soli(Castañeda bottle)
Composition
• Synthetic/Commercial
• Non-synthetic
• Tissue culutre
How it is dispensed
•Plated
•Tube media
a. Broth
b.Agar slant
c. Agar slant and butt
d.Agar butt
According to USE
1. Non-selective – supports non fastidious bacteria
2. Selective – support the growth of one type of bacteria not
the other, may contain inhibitory substances
3. Differential – allows the grouping of bacteria on the basis of
diff. characteristics displayed on medium, may be selective
or non-selectivereplication while keeping the bacteria cells
viable
4. Enriched media – growth enhancers have been
added, isolate fastidious bacteria
5. Enrichment broth – growth for particular
organism, while suppressing the other flora present
6. Transport media – prevents
• Routine Culture media
1. BAP (Blood agar plate)
2. CAP (Chocolate Agar Plate)
3. TMA (Thayer Martin Agar)
4. Modified TMA
5. Martin Lewis Agar
6. EMB – lactose fermentation, inhibits g+
7. MacConkey –same use with EMB, inhibits g+
8. MSA (Mannitol Salt Agar) – S. aureus, 7.5% NaCl,
9. PEA (Phenyl Alcohol Agar) – g+ isolation, PE inghibits G-
10. Selenite F – S and S
Inoculation Techniques
• Streaking – accurate colony count, 2 purpose
a. Quantitative
b. Isolated colony
• Pour plate
•Utilization of Colonial Morphology as Diagnostic
Tool
1. Presumptive Diagnosis
2. Reduce Cost
3. QC on automated mechanisms
•Key Colony Characteristics
1. Colony size
2. Colony pigmentation
Staphylococcus
Pseudomonas
E. Coli
Serratia
Salmonella
Staphylococcus
Pseudomonas
Escherichia coli
Serratia
Salmonella
3. Colony shape (elevation and margin)
4. Surface appearance
5. Changes in agar media
Hemolysis
6. Odor – pseudomonas = grapelike odor
Antimicrobial Therapy
Antibiotic
Antimicrobial
therapy
Streptomyces
– common source of
antibiotics
•Study box 11-1, Table 11-2
•Chapter 11 page 153
Effects of antibiotic
resistance
•Synergy = 2>1or 1+1 =3
•Antagonism = 1>2 or
1+2=2
•Additive = 1+1=2
•Indifference = no effect
on combination
Basic activities of antimicrobial agents
•Interruption on structural integrity
•Interruption of basic metabolic functions
Cell wall inhibitors
1. Penicillin
2. Cephalosporin
3. Vancomycin
4. Bacitracin
5. Cycloserine, Imepinem, Carbapenems
6. Penicillinase resistant = Methicilin,
cloxacillin, nafcillin
Cell membrane inhibitors
1.Colistin,
Polymixin
2.Amphotericin B,
Nystatin,
Imidazole,
Clotrimazole
Ribosome inhibitors
• Aminoglycosides – gentamicin,
kanamycin, tobramycin,
netilmicin, amikacin
• Tetracycline
• Chloramphenicol
• Erythromycin
• Clindaymycin, Lincomycin
Nucleic Acid inhibitors
• Mitomycin, Quinolones
• Metronidazole
• Sulfonamide-Trimetoprim (SXT)
• Rifampin