Microbiology Lab Master Final

[Microscopy] What is resolution?

Ability to distinguish two points as separate; improved by shorter wavelengths or using immersion oil.

[Microscopy] How do you improve resolution?

Shorten wavelength or use immersion oil.

[Microscopy] Why does oil immersion improve resolution?

It reduces refraction/scattering of light by matching refractive index of glass.

[Microscopy] What does parfocal mean?

If in focus at low power, it stays in focus at higher powers.

[Microscopy] What do you adjust if you see cells at 4x but not 100x?

Adjust diopters and fine focus.

[Microscopy] How do you improve contrast?

Dim the light or stain the cells.

[Microscopy] Why use iris diaphragm?

Controls amount of light to help contrast.

[Microscopy] Basic stain definition

Positively charged dye attracted to negatively charged bacteria (colors the cell).

[Microscopy] Acidic stain definition

Negatively charged dye repelled by cell wall; stains background.

[Aseptic] Purpose of aseptic technique

Prevent contamination of cultures and yourself.

[Aseptic] Examples of aseptic technique

Flaming loop, wearing lab coat, wiping bench with alcohol.

[Aseptic] Why heat fix?

Adheres cells to slide and kills bacteria.

[Aseptic] Why NOT heat fix capsule stain?

Heat destroys capsule structure.

[Smear] Smear from broth?

Do NOT add water; use two loopfuls of broth.

[Smear] Smear from plate?

Add one loopful of water and emulsify colony.

[Gram Stain] Primary stain?

Crystal violet.

[Gram Stain] Mordant?

Gram's iodine.

[Gram Stain] Decolorizer?

Alcohol/acetone.

[Gram Stain] Counterstain?

Safranin.

[Gram Stain] Gram-positive color?

Purple.

[Gram Stain] Gram-negative color?

Red/pink.

[Gram Stain] Why Gram positives stain purple?

Thick peptidoglycan retains crystal violet-iodine complex.

[Gram Stain] Why Gram negatives stain red?

Thin peptidoglycan + outer membrane loses CV-I during decolorization.

[Gram Stain] Structural difference: Gram+

Thick peptidoglycan, no outer membrane.

[Gram Stain] Structural difference: Gram-

Thin peptidoglycan, outer membrane with LPS.

[Acid Fast] Acid-fast stain primary stain?

Carbolfuchsin.

[Acid Fast] Why use carbolfuchsin?

Dissolved in phenol to penetrate waxy mycolic acid.

[Acid Fast] Acid-fast decolorizer?

Acid-alcohol (HCl).

[Acid Fast] Counterstain?

Methylene blue.

[Acid Fast] Acid-fast positive color?

Hot pink/fuchsia.

[Acid Fast] Non-acid-fast color?

Blue.

[Acid Fast] Cell wall component responsible?

Mycolic acid.

[Acid Fast] Clinically important acid-fast genera?

Mycobacterium species.

[Acid Fast] Diseases caused by acid-fast organisms?

Tuberculosis, leprosy, Buruli ulcer.

[Acid Fast] Why acid-fast organisms resist disinfectants?

Waxy, hydrophobic cell wall.

[Spore Stain] Primary stain?

Malachite green (7.5%).

[Spore Stain] Counterstain?

Safranin.

[Spore Stain] Why heat in spore stain?

Drives stain into tough spore coat.

[Spore Stain] Endospore color?

Green.

[Spore Stain] Vegetative cell color?

Red/pink.

[Spore Stain] Conditions causing sporulation?

Harsh conditions (temp, pH, starvation).

[Spore Stain] Spore-forming genera?

Bacillus (aerobic), Clostridium (anaerobic).

[Spore Stain] Disease examples of spore-formers?

C. diff, tetanus, botulism, gas gangrene.

[Capsule Stain] Stains used?

Congo red (acidic) + Maneval's stain (basic).

[Capsule Stain] Do you heat fix?

No — destroys capsule.

[Capsule Stain] Capsule appearance?

Clear halo around cell; background stained.

[Capsule Stain] Capsule composition?

Polysaccharides or polypeptides.

[Capsule Stain] Capsule functions?

Prevent desiccation, resist phagocytosis, help attachment.

[Capsule Stain] Capsule-producing organism example?

Klebsiella pneumoniae.

[Culture Concepts] What is a pure culture?

One species only.

[Culture Concepts] What is a mixed culture?

More than one species present.

[Culture Concepts] Why isolated colony doesn't guarantee purity?

Multiple species can produce isolated colonies.

[Culture Concepts] How to obtain pure culture from mixed broth?

Isolate colony → streak plate.

[Growth Media] What is selective media?

Promotes some organisms, inhibits others.

[Growth Media] What is differential media?

Shows visible differences between organisms.

[Growth Media] CNA-BAP selective for?

Gram positives (colistin + nalidixic acid inhibit Gram-).

[Growth Media] CNA-BAP differential for?

Hemolysis patterns.

[Growth Media] MAC selective for?

Gram negatives (bile salts + CV inhibit Gram+).

[Growth Media] MAC differential for?

Lactose fermentation (pink = positive).

[Plate Count] Why use viable plate count?

Counts only living cells.

[Plate Count] 30-300 rule?

Use plates with 30-300 colonies for accuracy.

[Plate Count] Units for plate counts?

CFU/mL.

[Plate Count] Why report as CFU?

Each colony arises from one viable cell.

[Plate Count] OCD formula?

OCD = (colonies × dilution factor) / volume plated.

[Plate Count] Dilution theory concept?

Each step reduces concentration based on ratio added to total.

[Plate Count] What is turbidity measurement?

Indirect count using spectrophotometer and standard curve.

[Dilutions] What happens in a 1:2 dilution?

Half stock, half diluent.

[Dilutions] How to make 1:4 dilution from 1:2?

Dilute 1:2 sample again by 1:2 → multiply dilutions.

[Dilutions] Example of 1:8 dilution?

1 mL into 7 mL total volume.

[Dilutions] How to dilute 6 mL stock by 1/2?

Mix 3 mL stock + 3 mL diluent.

[Temperature] Psychrophile definition

Grows best in cold temps.

[Temperature] Mesophile definition

Grows between ~10-50°C, includes pathogens.

[Temperature] Thermophile definition

Loves high temperatures.

[Temperature] Hyperthermophile definition

Very high temp range (archaea).

[Osmotic] Osmophile definition

Thrives in high osmotic pressure.

[Osmotic] Why high salt inhibits bacteria?

Water leaves cell → plasmolysis → dehydration.

[Osmotic] Halophile definition

Requires high salt.

[Osmotic] Halotolerant definition

Tolerates but doesn't require salt.

[Aerotolerance] Aerobic definition

Requires O₂.

[Aerotolerance] Anaerobic definition

Does not require O₂; may die in O₂.

[Aerotolerance] Facultative anaerobe definition

Uses O₂ if present but can grow without.

[Aerotolerance] Microaerophile definition

Needs reduced O₂.

[Aerotolerance] Capnophile definition

Requires high CO₂ (candle jar incubation).

[Aerotolerance] Thioglycolate broth purpose

Test oxygen requirements.

[Thioglycolate] What removes oxygen?

Sodium thioglycolate; resazurin indicates presence of oxygen.

[Pour Plate] How to inoculate pour plate?

Add loopful of bacteria to molten agar (50°C), mix, pour into plate.

[Pour Plate] Why molten agar at 50°C?

Warm enough to stay liquid; cool enough not to kill bacteria.

[Pour Plate] Labeling plates?

Initials, date, species, section.

[Microscopy Specific] Why E. coli stains red in spore stain?

It doesn't form spores → only takes safranin.

[General Stains] Differential stain examples

Gram, Acid-fast, Spore, Capsule.

[General Stains] Simple stain purpose

Stains all cells for visibility.

[Fermentation] Purpose of fermentation tubes?

Test carbohydrate fermentation and gas production.

[Fermentation] What is the pH indicator in fermentation tubes?

Bromocresol purple.

[Fermentation] Acidic fermentation color?

Yellow.

[Fermentation] Alkaline reversion explanation?

Sugars depleted → organism metabolizes proteins → alkaline end products → purple color returns.

[Fermentation] Gas detection method?

Durham tube bubble.

[Fermentation] What does gas bubble indicate?

Fermentation pathway producing CO₂.

[Fermentation] Sugar concentration in tubes?

1% sugar.

[Fermentation] E. coli fermentation pattern?

Gas with glucose, lactose, mannitol; NO sucrose fermentation.

[Fermentation] Klebsiella fermentation pattern?

Ferments all sugars with gas.