Microbiology Lab Practical

4.24.26

What lens do you use for bacteria?

Oil immersion (100x objective → 1000x total)

Total magnification formula?

Ocular (10x) × objective

Coarse vs fine focus?

Coarse = big movements (low mag), Fine = precise (high mag)

Purpose of isolation streak?

Separate single cells → isolated colonies

Successful streak looks like?

Dilution gradient → isolated colonies in later streaks

Gram + vs Gram − colors?

• Positive = purple

• Negative = pink

Over-decolorizing does what?

• Gram + → false Gram −

Under-decolorizing does what?

Gram − → false Gram +

What is the Gram stain actually testing?

Cell wall structure (thick peptidoglycan vs thin + outer membrane)

What is the Acid-Fast test detecting?

Mycolic acid (waxy cell wall → resistant to decolorization)

Positive result of an Acid-Fast?

Bright red/pink cells

Negative result of an Acid-Fast?

Blue cells

What does Capsule Stains detect?

Presence of a capsule (virulence factor)

What is a positive result for a capsule stain?

Clear halo around cell

What is a negative result for a capsule stain?

No halo, just stained cells

What is the endospore stain testing

Ability to form endospores (protective, survival structure)

What is a positive result for an endospore stain?

Green spores inside pink cells

What is a negative result for an endospore stain?

Only pink cells

What does the aerotolerance test measure?

Oxygen requirements

What is an obligate aerobe and what does it look like?

An organism that requires oxygen to grow

• growth only at the top

What is an obligate anaerobe and what does it look like?

Organisms that are unable to grow in the presence of oxygen, even small amounts of oxygen are lethal

• Growth only at bottom

What is an facultative anaerobe and what does it look like?

Capable of growing in the presence of oxygen OR in the absence of oxygen

• Growth everywhere, more at top

What is an aerotolerant anaerobe and what does it look like?

Only perform fermentation for energy and do not use oxygen, but they are not harmed by it

• Growth everywhere, equally throughout

What is the motility test testing?

Ability to move (flagella)

What is the positive result of the motility test?

Diffuse/cloudy growth spreading from stab

What is the negative result of the motility test?

Growth only along stab line

What are two qualities of motility media that help determine motility (general)?

1. Soft (semi-solid) agar → allows bacteria to physically move through it

2. Indicator dye (TTC) → turns red where bacteria grow, making movement visible

What is the selective ingredient in MSA?

High salt concentration (NaCl)

• gram negative bacteria can not withstand the osmotic pressure created

(MSA) What is the differential ingredient?

Mannitol (with pH indicator)

• Detects fermentation

(MSA) What does it mean if there is no growth on an MSA plate? What does MSA select against?

No growth = salt-sensitive organism, likely gram negative

(MSA) What does it mean if there is growth on an MSA plate? What does MSA select for?

Growth = staphylococci halophilic bacteria (salt-tolerant organism)

(MSA) What does it mean if an MSA plate stays red?

Not S.aureus

• No mannitol fermentation or acid production

(MSA) What does it mean if an MSA plate turns yellow around the growth?

possibly S.aureus

• Mannitol fermentation → acid production

(EMB) What are the (general) selective ingredients?

Methylene blue and eosin

• Dyes that inhibit Gram-positive bacteria

(EMB) What is the main differential ingredient?

Lactose/Sucrose (sugars)

(EMB) What does it mean if there is no growth on an EMB plate? What does EMB select against?

No growth = Gram-positive bacteria

(EMB) What does it mean if there is growth on an EMB plate? What does EMB select for?

Growth = Gram-negative bacteria

(EMB) What does it mean if growth on an EMB plate does not change color?

No lactose fermentation

(EMB) What does it mean if growth on an EMB plate turns green/metallic/dark purple?

gram negative; coliform

(EMB) What are coliform bacteria and why do we monitor environmental samples for coliform growth?

Gram-negative lactose fermenters and acid producers

(MSA/EMB) What happens when you forget the selective ingredient?

Everything grows → no gram positive/negative selection

(MSA/EMB) What happens if you forget the differential ingredient?

No color change → cannot distinguish metabolism

(Indole) What is the name of the enzyme that breaks down tryptophan?

Tryptophanase

(Indole) What does a positive indole test look like? What does this tell you about the organism?

reagent turns red, red ring.

• tryptophanase degrades tryptophan to produce indole

(Indole) What does a negative indole test look like? What does this tell you about the organism?

No red ring

• no tryptophanase enzyme; cannot produce indole

(MR) What does a positive MR test tell you about the bacteria?

Mixed acid fermentation

• tube turns red

(VP) What does a positive VP test tell you about the bacteria (general)?

2,3-butanediol fermentation → acetoin produced

(MR-VP) How are these two tests similar?

Both test glucose fermentation pathways

(Citrate) What 2 results indicate a positive citrate test? What does it tell you about the organism?

Royal blue slant color + growth

• CItrate is utilized as sole carbon source

(Citrate) What does a negative citrate test look like? What does it tell you about the organism?

no color change; no growth

• citrate is not utilized

(Urease) What is the enzyme that breaks down urea?

Urease

What does a positive urease test look like? What does this tell you about the organism?

Pink

• rapid urea hydrolysis; presence of urease

What does a negative urease test look like? What does this tell you about the organism?

Peach/yellow/no change

• no urea hydrolysis; urease enzyme is not present

What does a positive and negative catalase test look like? What does each result tell you about the organism?

Positive = bubbles → has catalase

Negative = no bubbles → lacks catalase

(Catalase) What is the reagent added in the catalase test? In the presence of catalase, what two products does the reagent get broken down into?

3% hydrogen peroxide

• two products: H2O (water) + O2 (oxygen

(Oxidase) What does a positive and negative test look like? What does each result tell you about the organism?

Positive = dark blue → has cytochrome c oxidase
A: Negative = no color change, oxidase not present

(TSI) Know what the various red/yellow color changes mean as far as sugar fermentation, and the symbols used to indicate this.

Yellow (A) = fermentation

Red (K) = no fermentation

Black (H2S) = Sulfur Reduction

Cracks or lifting of agar (G) = Gas production during fermentation

No Change (NC)

(TSI) If an organism produces gas while it ferments, how will you know?

Cracks or lifting of agar

(TSI) If an organism reduces sulfur how will you know?

Black precipitate (H₂S)

(Blood Agar) What is alpha, beta, and gamma hemolysis?

Alpha = partial

Beta = complete

Gamma = none

(Blood Agar) Know what these types of hemolysis look like on Blood Agar plate.

Alpha = green halo

Beta = clear zone

Gamma = no change

(Blood Agar) What type of hemolysis does S. pyogenes do?

Streptococcus pyogenes → beta hemolysis

(Food Micro) Be able to calculate dilution factors and write as a ratio and scientific notation.

Example: 1:10 = 10⁻¹

(Food Micro) Calculate original cell concentrations.

(CFU ÷ volume plated) × dilution factor

(Food Micro) Be able to make a 1:10 dilution.

1 mL sample + 9 mL diluent

(Kirby-Bauer) Be able to measure and interpret zones of inhibition.

Larger zone = more effective antibiotic

(Kirby-Bauer) Know the meaning of resistant and susceptible.

Susceptible = large zone

Resistant = small/no zone

(Handwashing) Know how to recognize/count abundance and diversity.

Abundance = number of colonies

Diversity = different colony types

(Handwashing) Know how to interpret abundance and diversity.

High diversity = contamination/poor hygiene

(Epi) What is the difference between endemic, pandemic, epidemic?

Endemic = constant

Epidemic = regional spike

Pandemic = global

(Epi) What is the prevalence of a disease?

Total cases

(Epi) Be able to use the formula used to calculate prevalence.

(Total cases ÷ population) × K

(Epi) What is the incidence of a disease?

New cases

(Epi) Be able to use the formula used to calculate incidence.

(New cases ÷ population) × K

(Epi) What is “K”?

Scaling factor (100, 1,000, 100,000)

(Experiment Design) Know the concept of false negative and false positive.

False + = says yes when no

False − = says no when yes

(PCR) What is PCR and what does it do?

Amplifies DNA

(qPCR) Be able to interpret a qPCR graph.

Threshold = detection line

(qPCR) How can you tell if there is more or less starting sample?

Lower Ct = more starting DNA

(RT-PCR) What is RT-PCR and why do we need reverse transcriptase?

Converts RNA → DNA for amplification