Microbiology Lab Exam Study Guide

Pure Culture

• A pure culture contains a single, unadulterated species of cells.

Media

• Agar-agar is a solidifying agent added to media.
• Semisolid medium: less than 1% agar.
• Solid medium: more than 1% agar.
• Agar liquefies and solidifies at specific temperatures (details not fully specified - "know the temperature").
• Example of semisolid medium: motility slant.
• Motility tubes: solid medium.

Sterilization

• Autoclaving is a key sterilization technique.

Plate Incubation

• Plates are incubated in an inverted position (lid on the bottom).
• This prevents condensation from dripping onto the culture, which could induce contamination.

Microscope

• Type used in lab: bright field microscope.
• Components:
  • Stage: where the slide is placed.
  • Illumination.
  • Condenser: condenses light onto the slide.
    • Equipped with an iris diaphragm: regulates the amount of light reaching the specimen.
• Lenses:
  • Objective lenses (4x, 10x, 100x, etc.). Closer to the object.
  • Ocular/eyepiece lenses (typically 10x).
• Total magnification: ocular magnification $(\times)$ objective magnification.
  • Example: 10x ocular lens with 10x objective lens = 100x total magnification.
  • Example: 10x ocular lens with 100x objective lens = 1,000x total magnification.
• Resolving power: ability of a lens to show two adjacent objects as discrete entities.
• Working distance: distance between the objective lens and the slide.

Pure Culture

• Arises from a single cell.

Gram Stain

• Know the ingredients, steps, and duration of each step.
• Understand the differences between gram-positive and gram-negative cell walls.
• Iodine acts as a mordant, binding to the crystal violet dye to form an iodine-crystal violet complex.
• Know the decolorizer and counterstain used.

Bacteriological Media Categories

• Supportive media: know the definition and one example.
• Differential media: know the definition and one example.
  • MacConkey agar: differential medium.
    • Contains lactose sugar.
    • Lactose-fermenting organisms produce pink colonies.
    • Non-lactose-fermenting organisms produce pale-colored colonies.
    • Contains an indicator that changes color based on fermentation.
• Selective media: know the definition and one example.
  • Shigella agar is a selective medium.
  • Mannitol salt agar is a selective medium.
• Enrichment medium: know the definition and one example.
  • GN broth is an example.
  • Typically broth-based (liquid media).
  • Used for stool samples to selectively enrich pathogens like Salmonella.
    • The pathogen is allowed to multiply while suppressing normal flora.

MacConkey Agar

• Contains crystal violet: inhibits the growth of gram-positive organisms.
• Contains bile salts: prevents the growth of gram-positive organisms.

Media pH

• Most bacteria grow between pH 7.2 and 7.4.
• Sabouraud Dextrose Agar (Sabdex) for fungi has an acidic pH to inhibit bacterial growth.

Selective Media with Antibiotics

• Thayer-Martin medium: selective for Neisseria gonorrhoeae.

Enriched Media

• Sheep blood agar: blood is added (5-10% concentration).

Autoclaving Conditions

• Steam under pressure.
• 15 pounds pressure, 15 minutes at 121°C.

Other Sterilization Methods

• Inspiration (for egg-based media).
• Dry heat: 160°C for 2 hours or 180°C for 1 hour.

MacConkey Agar Specifics

• Contains bile salts and crystal violet.
• pH indicator: neutral red.
  • Acidic environment (lactose fermentation) causes the indicator to turn pink to red.
• Contains lactose sugar.
• Gram-positive organisms do not grow due to bile salts and crystal violet.
• It is both a differential and selective medium.
  • Selective: suppresses gram-positive organisms.
  • Differential: differentiates based on lactose fermentation (pink vs. pale colonies).

Mannitol Salt Agar

• Selective due to high salt concentration.
• Contains mannitol sugar.
• Staphylococcus aureus can grow and ferment mannitol, producing yellow colonies.
• Other Staph species may grow but not ferment mannitol, producing pink colonies.
• Indicator: phenol red.
  • Mannitol fermentation produces yellow colonies.
  • No fermentation results in red colonies (alkaline conditions).

Catalase Test

• Principle: detects the enzyme catalase.
• Reagent: 3% hydrogen peroxide $(H<em>2O</em>2)$.
• Positive result: brisk bubbling.
• Negative result: no bubbling.
• Staphylococcus aureus: positive.
• Streptococcus pyogenes: negative.

Oxidase Test

• Enzyme: cytochrome oxidase.
• Reagent: tetra-methyl-p-phenylenediamine dihydrochloride (lengthy name).
• Positive result: dark purple color.
• Negative result: no color development.
• Pseudomonas aeruginosa: positive.
• Morganella morganii/E. coli: negative.

Indole Test (part of IMViC reactions)

• Principle: detects the enzyme tryptophanase.
• End product: indole.
• Broth: tryptophan broth.
• Reagent: Kovac's reagent (2-3 drops).
• Positive result: red ring development.

VP Test (part of MRVP)

• Principle: detects the formation of neutral end product acetoin (acetylmethylcarbonyl).
• Reagents: added in sequence (know the sequence and names).
• Incubation: 10 minutes.
• Positive result: pink/red color development.
• Negative result: yellow color.

Citrate Test

• Principle: tests the ability to use citrate as the sole carbon source.
• Inoculation: fishtail the slant only (do not stab).
• Incubation conditions (unspecified).
• Positive vs negative (unspecified).

Phenylalanine Test

• Enzyme: phenylalanine deaminase.
• Reaction: converts phenylalanine to phenylpyruvic acid.
• Morganella should test positive.
• Reagent: ferric chloride $(FeCl_3)$.
• Positive result: deep green color on the slant.

Urease Test

• Enzyme: urease.
• Reaction: urea is split into ammonia $(NH_3)$, increasing alkalinity.
• Inoculation: fishtail the slant only (do not stab).
• Positive result: red color development due to increased pH.