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

a. Kinyoun Method – cold method , use high conc. detergent

Acid fast stain
Purpose Ziehl-Neelsen Kinyoun Auramine-
(HOT) (COLD) Rhodamine
(Fluorochrome)
Primary
Mordant
Decolorizer
Counterstain 0.5% Potassium
permanganate
Result AFO – AFO – AFO – Y
NAFO – NAFO – NAFO –
 Special stains
• Capsule – negative stain
• Spore – Dorner, Wirtz Conklin, Schaeffer Fulton
• Metachromatic granules – Albert’s, LAMB, Neisser
• Flagella – Leifson, Gray’s
• Nucleic acid – Fuelgen
• Polar bodies – Wayson
• Rickettsia – Gimenez, Macchiavelo
• Spirochetes – Levaditi, Fontana Tribondeau

Nonstaining method – String test (3% KOH)

INDIA INK STAIN
3 KOH STRING TEST
Leiffson stain
 Types of Microscopy
1. Brightfield
2. Darkfield
3. Phase contrast
4. Fluorescent
5. Electron
a. TEM
b. SEM
Darkfield microscopy
Phase-contrast microscopy
Fluorescent microscopy
Magnification
Resolution
Resolving power
 UNIVERSAL PRECAUTIONS
• Consider all type specimens infectious
• Barrier protection
• Color coding for waste disposal
• Black
• Green
• Yellow
• Yellow with black band
• Red
Classification of Biologic agents
1. Biosafety Level 1 – No risk
2. Biosafety Level 2 – Moderate Risk
3. Biosafety Level 3 – High risk, with Treatment
4. Biosafety Level 4 – High risk, NO Treatment
Bioterrorism categories
• Category A – highly infectious
• Category B – Moderate morbidity,
low mortality
• Category C – emerging pathogens
 Biosafety cabinet classes
•Uses HEPA filter, negative pressure
 CLASS I
BIOLOGICAL SAFETY CABINET
 100% Exhaust
 Inflow velocity 75 fpm minimum
 BSL 1 –3 Usage
 Personnel protection only
 CDC/NIH recommends a glove-port
panel for use with small amounts of
radionuclides when exhausted
 Typical uses today: Toxic powder
weighing, necropsy
 Maybe thimble/air gap or hard
connected to a exhaust system when
Filtered Exhaust Air
proper precautions are taken
Room Air

LabGard 813 Air Flow

CLASS I AIR FLOW
 CLASS II – TYPE A

 30% Exhaust, 70% Re-circulate

 Negative pressure plenum (Changed 2007)
 Inflow velocity 75 fpm minimum
 BSL 1 –3 Usage
 Personnel and Product protection
 Minute amounts of non-volatile toxic
chemicals and radionuclides if
canopy/thimble exhausted
 Typical uses today: Bacterial, Viral,
Fungal, Parasitic
VERTICAL LAMINAR FLOW
WORK STATION
 CLASS II – TYPE A

70%

Console Bench Top

 CLASS II – TYPE B

 70% Exhaust, 30% Re-circulate

 Negative pressure plenum
 Inflow velocity 100 fpm minimum
 BSL 1 –3 Usage
 Personnel and Product protection
 Minute amounts of volatile toxic chemicals
and radionuclides
 Must be hard connected with typical exhaust
requirement being 300-500 CFM at 1.0” w.g.
 Must have interlocked internal blower with
audible and visual alarm for exhaust failure
 Typical uses today: Bacterial, Viral, Fungal,
Parasitic, Arbor-viruses
CLASS II – TYPE B
 CLASS III – “GLOVE BOX”

 100% Exhaust Glove Box

 Negative Pressure at 0.5” w.g. minimum
 Double HEPA Filter Exhaust
 BSL 4
 Personnel and Product Protection
 Small amounts of volatile toxic chemicals and
radionuclides
 Must be hard connected with typical exhaust
requirement being 50-100 CFM at 0.5 w.g.
 Must have negative pressure alarm for cabinet
or exhaust failure
 Typical uses today: Toxic Powders, BSL 4
Agents
 BIOLOGICAL SAFETY CABINET
CLASS / TYPES
 Class I: Personnel Protection Only
 100% exhaust
 Inflow velocity 75 fpm minimum
 Class II: Personnel and Product Protection
Type A1 - 30% exhaust, 70% re-circulate
 Negative Pressure Plenum (Changed 2008)
 Inflow velocity 75 fpm minimum
Type A2 - 30% exhaust, 70% re-circulate
 Negative Pressure Plenum
 Inflow velocity 100 fpm minimum
 BIOLOGICAL SAFETY CABINET
CLASS / TYPES
 Class II: Personnel and Product Protection
Type B1 - 70% exhaust, 30% re-circulate
 Negative Pressure Plenum
 Inflow velocity 100 fpm minimum
Type B2 - 100% exhaust
 Negative Pressure Plenum
 Inflow velocity 100 fpm minimum
 Class III: Personnel and Product Protection
 100% exhaust
 Negative Pressure at 0.5” w.g. minimum
 BSC Class I BSC Class II BSC Class III
Filters exhausted air a.k.a. Vertical Laminar Entirely sealed
only Flow BSC Accessible through
Product Filters exhausted & glove ports
contamination is recirculated air Filters supplied air &
possible IIa = exhausts air in exhausted air
the room
IIb = exhausts air
outside the building
 Culture Media (Table 7-1)

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