Microbiology Project, Staining, and Lab Safety: Key Concepts and Procedures

What is the overall purpose of the entire semester-long microbiology project?

Inoculation, Incubation, Examination, and Identification.

What does 'Inoculation' mean in the project?

Introducing the environmental sample to growth media.

What does 'Incubation' mean?

Allowing the bacteria time to grow under controlled conditions.

What does 'Examination' include?

Microscopy, staining, serial dilution, VT/PT, colony morphology, biochemical tests.

What does 'Identification' include?

API strip results, Bergey's Manual, all test interpretation.

What four items are required to enter microbiology lab?

Lab coat, long pants, closed-toe shoes, goggles.

What goes in the regular biohazard bin?

Non-sharp items that touched bacteria (plates, tubes, towels, gloves).

What goes in the sharps biohazard bin?

Sharp items that touched bacteria (slides, broken tubes).

What goes in the clean sharps bin?

Sharp items that did not touch bacteria.

What goes into micropipette waste?

All pipette tips (used or unused).

What goes into stain waste?

Dyes and staining liquids.

What goes into regular trash?

Non-biohazardous items like hand towels, wrappers, tape.

What is NOT allowed in the lab under any circumstances?

Eating, drinking, chewing gum, or ingesting anything.

How must the microscope be carried?

Two hands: one on arm/neck, one supporting base.

What does coarse adjustment control?

Large focusing movements.

When can coarse adjustment be used?

Only at 4× and 10×.

Why is coarse adjustment banned at 40× and 100×?

It may break the slide or lens.

What does fine adjustment do?

Small, precise focusing.

What is the total magnification formula?

Ocular (10×) × objective lens.

What magnification requires oil immersion?

100× objective.

What is true motility?

Directional, purposeful movement (hanging drop).

What is Brownian motion?

Random vibration due to water molecules (wet mount).

Why do we stain bacteria?

To increase contrast; most are transparent.

What are the two major classes of stains?

Simple and differential.

What is the primary stain in Gram Staining?

Crystal violet.

What is the mordant in Gram Staining?

Iodine.

What is the decolorizer in Gram Staining?

Gram's decolorizer (alcohol).

What is the counterstain in Gram Staining?

Safranin.

Why do Gram-positives stain purple?

Thick peptidoglycan traps CV-iodine complex.

Why do Gram-negatives stain pink?

Outer membrane dissolves → CV removed → safranin stains them.

What is the primary stain in Acid-Fast Staining?

Carbol fuchsin.

What is the mordant in Acid-Fast Staining?

Heat/steam.

What is the decolorizer in Acid-Fast Staining?

Acid alcohol.

What is the counterstain in Acid-Fast Staining?

Methylene blue.

What is the acid-fast appearance?

Pink/red rods.

What is the non-acid-fast appearance?

Blue.

What is the primary stain in Endospore Staining?

Malachite green + heat.

What is the counterstain in Endospore Staining?

Safranin.

What color are spores in Endospore Staining?

Green.

What color are vegetative cells in Endospore Staining?

Pink.

Why do we heat-fix slides?

Kills bacteria, adheres to slide, prevents washing off during staining.

What is the dilution formula?

Dilution = added volume ÷ total volume.

What is the dilution factor?

Reciprocal of dilution (10⁻³ → DF = 10³).

What does CFU stand for?

Colony Forming Unit — one viable cell or clump.

What does VT stand for?

Viable titer.

What is the valid CFU counting range?

20-200 CFUs.

What does <20 CFUs mean?

Not statistically valid → do NOT calculate.

What does >200 CFUs mean?

TNTC → do NOT calculate.

If a plate has 150 CFUs and DF = 10⁵, what is the VT?

VT = 150 × 10⁵ = 1.5 × 10⁷ CFU/mL.

If a plate has 12 CFUs, can you calculate VT?

No — below valid range.

If a plate has 287 CFUs, can you calculate VT?

No — TNTC.

What are the colony morphology descriptors?

Shape, size, surface, color, opacity, elevation, margin.

Why is colony morphology important?

Provides physical characteristics for comparison in early identification.

Where on a streak plate are isolated colonies found?

Tail region (3rd/4th quadrant).

Why must cultures be pure before biochemical testing?

Mixed cultures give false results.

If two colonies look identical but stain different colors, are they the same organism?

No — color must also match.

What is the purpose of the OF test?

Determine if organism metabolizes glucose oxidatively, fermentatively, or both.

What is the indicator dye in the OF test?

Bromothymol blue.

What does yellow indicate in the OF test?

Acid (positive).

What does green indicate in the OF test?

No reaction.

What does blue indicate in the OF test?

Alkaline (negative).

What does yellow in the aerobic tube only indicate?

Oxidizer.

What does yellow in both tubes indicate?

Facultative fermenter.

What is the growth requirement for the OF test?

Must have visible bacterial growth.

What sugars are present in TSI?

0.1% glucose, 1% lactose, 1% sucrose.

What is the indicator in TSI?

Phenol red.

What does a red slant/yellow butt indicate in TSI?

Glucose only fermented.

What does an all yellow slant/butt indicate in TSI?

Lactose and/or sucrose fermentation.

What does an all red slant/butt indicate in TSI?

No sugar fermentation (obligate aerobe).

What does black precipitate indicate in TSI?

H₂S production.

What do cracks or lifting of agar indicate in TSI?

Gas production.

What is the purpose of the Nitrate Reduction Test?

Detect nitrate → nitrite → further reduction products.

What are the reagents used in the Nitrate Reduction Test?

NIT-1 (sulfanilic acid) + NIT-2 (alpha-naphthylamine).

What reagents are used for nitrate reduction test?

NIT-1 (sulfanilic acid) + NIT-2 (alpha-naphthylamine)

What does a red color after reagents indicate in the nitrate reduction test?

Positive for nitrate → nitrite

What does no color after reagents and red after zinc indicate?

Negative; nitrate still present

What does no color after zinc indicate in the nitrate reduction test?

Positive for full reduction to N₂ or NH₃

What does gas in the Durham tube indicate?

Denitrification → N₂ gas

What is the purpose of gelatin hydrolysis test?

Detect gelatinase

What indicates a positive result in gelatin hydrolysis test?

Liquefaction after ice bath

Why must the gelatin tube be chilled?

Gelatin melts at warm temperatures even without bacteria

What is the purpose of the urease test?

Detect urease enzyme

What indicator is used in the urease test?

Phenol red

What color indicates a positive result in the urease test?

Hot pink (alkaline from ammonia)

What color indicates a negative result in the urease test?

Yellow

What is the purpose of starch hydrolysis test?

Detect alpha-amylase

What reagent is used in starch hydrolysis test?

Iodine

What indicates a positive result in starch hydrolysis test?

Clear zone around growth (starch hydrolyzed)

What indicates a negative result in starch hydrolysis test?

Plate turns purple with no clear area

What is the purpose of the Methyl Red (MR) test?

Detect mixed acid fermentation

What color indicates a positive result in the Methyl Red test?

Red

What color indicates a negative result in the Methyl Red test?

Yellow

What is the purpose of the Voges-Proskauer (VP) test?

Detect acetoin (2,3-butanediol pathway)

What reagents are used in the Voges-Proskauer test?

VP-1 (alpha-naphthol) + VP-2 (KOH)

What color indicates a positive result in the Voges-Proskauer test?

Brownish-red

What color indicates a negative result in the Voges-Proskauer test?

Brownish-green/yellow

What is the purpose of the catalase test?

Detect catalase enzyme

What reagent is used in the catalase test?

Hydrogen peroxide (H₂O₂)

What indicates a positive result in the catalase test?

Bubbles (O₂ released)

What is the purpose of the oxidase test?

Detect cytochrome c oxidase