Microbiology- Diagnostic Techniques

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Light Microscopes

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Brightfield: Uses 2 lenses (ocular and objective) to light up specimen

Darkfield: Condenser is modified so light is directed obliquely causing light to deflect off specimen. Specimen is bright and background is dark

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Fluorescent

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UV light source
Fluorescent dyes absorb UV light at one wavelength and reflect it back at another
Specimens stained will glow against a dark background

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Phase Contrast

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Special objectives and condensers that allow light to refract differently through cell components which vary in intensity

Makes edges of the specimen more refractile

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Confocal Laser Scanning

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Used to create a 3D image of the specimen by optically sectioning the specimen with the use of lasers

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Electron Microscope

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Uses magnets and electron beams

2 Types
Transmission: Can cross section specimen
Scanning: Gives 3D look to the outside of the specimen

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Gram Stain

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Step 1: Crystal Violet (primary stain)

Step 2: Gram Iodine (Mordant)

Step 3: 95% Ethanol (Decolorizer)

Step 4: Safranin (Counterstain)

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Acid Fast Stain

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Used primarily to identify Mycobacterium sp.

Heat FIx
Carbol-fuchsin (Primary Stain)
HCl/Alcohol (Decolorizer)
Methyl Blue (Counterstain)

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Capsule Stain

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Skim Milk Medium

Heat Fix
Stain (Congo Red & Crystal Violet)

Background will stain as well as bacterial cell

Capsule will not stain leaving a halo around the cell

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Liefson's Flagellar Stain

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Mordant used in conjunction with Stain (encrusts on to flagella to increase thickness)

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Endorespore Stain

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Stain with Malachite Green (heat fix to allow penetration)
Rinse
Secondary Stain with Safranin

Spores appear green
Vegetative cells turn pink/red

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PEA Agar Media

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Phenylethyl Alcohol Agar

Supports Gram (+) bacteria

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Columbia CNA Blood Agar

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Colistin, Naladixic Acid

Grows Gram (+) bacteria

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Mannitol Salt Agar

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Selective for Staphyloccus sp. (salt-tolerant)
Differential for Aureus (mannitol fermentor)

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Tinsdale Medium

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Thiosulfate-tellurite based medium

Primary isolation of Corynebacterium diptheria

Inhibits Gram (+)

Forms black colonies with gray halos

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Egg Yolk Medium

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Rich in phospholipids

CLOSTRIDIUM sp. with lecthinase activities are differentiated

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Thayer-Martin Agar

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Blood agar base

Antibiotics placed on dish to help control contamination

Selective for NEISSERIA GONORRHEA, NEISSERIA MENINGITIDIS

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MacConkey Agar Medium

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Selects for Gram (-)

Contains bile salts to inhibit Gram (+)

Specific for E. coli (turns red because it ferments lactose)

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Hektoen Enteric Agar

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Contains lactose

Helps to distinguish Salmonella, Shigella

Contains thiosulfate which Salmonella reduces to hydrogen sulfide

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Xylose Lysine Desoxycholate Agar

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Primary isolation of Salmonella vs. Shigella

Xylose fermentation, H2S production, and lysine decarboxylation distinguish Salmonella from Shigella

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BG Agar

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Bordet-Gengou agar

Selective for Bordetella pertussis (whooping cough)

Toxins produced by organism

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BCYE Agar Medium

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Buffered charcoal yeast extract agar

Selective for Legionella sp. (legionaires)

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TCBS Agar Medium

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Thiosulfate citrate-bile salts

Selective Differential medium for Vibrio sp. (Cholera)

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Cefsulodin-Irgasan-Novobicin (CIN) Agar

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Selective for Yersinia sp. (Plague)

Inhibits Gram (+ and -)

Appears as a red "bullseye" in each colony

48 hours @ 25 degrees C

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Lowenstein-Jensen Agar

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Egg based medium

Isolation of Mycobacterium sp.

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Middlebrook Agar

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Cultivate Mycobacterium sp.

More chemically defined than LJ

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MES (Ureaplasma Agar)

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Isolation of Ureaplasma urealyticum

Contains horse serum that supplies cholesterol to stabilize cell wall

Fried Egg colony morphology

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Catalase

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Selective between Staph and Strep

Staph will be positive and bubble
Strep will be negative and not do anything

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Coagulase

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Differentiate between Staph aureus and other species

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Gelatin Liquifaction test

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Selective for bacterium that secrete gelatinases to liquify gelatin. Positive bacteria will degrade gelatin and it will be uniform throughout

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Oxidase Test

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Detects activity of the cytochrome oxidase enzyme

Selective for Neiserria

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Indole Test

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Detects activity of tryptophanase enzyme on tryptophan

End product indole reacts with Kovacs reagent turning it a red color

Selective for E. Coli

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Urease Test

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Detects activity of urease

Selective for Proteus sp.

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Citrate Utilization Test

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Ability to use citrate as sole carbon source

pH rises turning the bromthymol blue indicator from green to bright blue

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Bile Esculin Test

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Enterococcus (Group D streptococci)

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DNAse Test

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DNAse catalyzes depolymerization of DNA

Medium contains methyl green which binds to polymerized DNA

Exoenzyme breaks down the DNA into smaller fragments releasing it from the methyl green dye

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Optochin Test

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P disc

Selective for Streptococcus pneumoniae

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Bacitractin Test

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A disc

Differentiate group A Streptococci

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Direct microscope examination

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Treat specimen with KOH prep

Stain with either crystal violet or lactophenol cotton blue

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KOH w/ calcofluor white

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Fluorescent dye added to KOH prep that will bind to polysaccharides present in chitin and will fluoresce under light

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Motility Test

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Soft agar deeps are used

Stabbed with an inoculating needle

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India Ink

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Negative Stain

Used to see Cryptococcus neoformans (found in CSF and causes meningitis)

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Periodic acid-Schiff stain

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Used in detection of fungal elements in tissue specimens

Causes them to turn red/pruple to make them stand out

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Grocott-Gomori Methenamine-Silver Nitrate Stain

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Stains fungal elements black

Stains strains of Candida, Histoplasm, Blastomyces

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Giemsa stain

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Used for blood smears

Detects Histoplasma capsulatum in blood or bone marrow

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Masson-Fontana Stain

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Stains melanin in the cell wall a brown color