Micro Lab Exam

Spread Plate Isolation

What is it?

Technique to isolate individual colonies on agar.

Purpose

• Obtain pure cultures

• Count bacteria

• Separate mixed organisms

Steps

1. Pipette 0.1 mL diluted sample onto agar.

2. Sterilize spreader.

3. Spread evenly across surface.

4. Incubate.

Results

• Isolated colonies = successful isolation.

• Lawn growth = sample too concentrated

Dilution Plate Count

What is it?

Method to determine bacterial concentration.

Purpose

Calculate CFU (Colony Forming Units)

Steps

1. Make serial dilutions.

2. Plate diluted sample.

3. Incubate.

4. Count colonies (30–300).

5. Calculate:

CFU/mL = Colonies × Dilution Factor ÷ Volume plated

Results

Higher colony count = higher bacterial concentration.

Nutrient Agar Preparation

What is it?

A general-purpose growth medium that supports the growth of many bacteria.

Purpose

• Grow bacteria

• Maintain bacterial cultures

• Observe colony morphology

Steps

1. Weigh nutrient agar powder.

2. Add distilled water.

3. Heat until dissolved.

4. Sterilize in autoclave (121°C, 15 psi, 15–20 min).

5. Cool to about 50–55°C.

6. Pour into sterile Petri dishes.

7. Allow to solidify.

Results

• Growth present: organism can grow on nutrient agar.

• No growth: organism requires special nutrients or conditions.

Simple Stain

What is it?

Uses one basic dye.

Purpose

• Observe

  • Shape

  • Arrangement

  • Size

Steps

1. Prepare smear.

2. Heat fix.

3. Add crystal violet or methylene blue.

4. Rinse.

5. Dry.

6. Observe.

Results

Entire cell stains one color.

Gram Stain

Purpose

Differentiate bacteria by cell wall.

Principle

• Gram (+) thick peptidoglycan

• Gram (-) thin peptidoglycan + outer membrane

Steps

1. Heat fix

2. Crystal violet (1 min)

3. Iodine (1 min)

4. Alcohol/acetone (10–20 sec)

5. Safranin (1 min)

Results

Result	Color
Gram +	Purple
Gram -	Pink/Red

Mannitol Salt Agar (MSA)

Purpose

• Selective and differential medium.

Selective because:

• 7.5% NaCl inhibits most bacteria except Staphylococcus.

Differential because:

• Detects mannitol fermentation.

Steps

1. Streak plate.

2. Incubate.

Results

Organism	Growth	Color
Mannitol fermenter (S. aureus)	Yes	Yellow
Non-fermenter (S. epidermidis)	Yes	Pink/Red
Salt-sensitive bacteria	No growth	—

MacConkey Agar

Purpose

• Selective and differential.

Selective

• Inhibits Gram (+).

Differential

• Lactose fermentation.

Steps

• Streak plate and incubate.

Results

Result	Appearance
Lactose fermenter	Pink colonies
Non-fermenter	Colorless colonies
Gram +	No growth

EMB Agar (Eosin Methylene Blue)

Purpose

• Selective for Gram (-)

• Differential for lactose fermentation.

Results

Organism	Result
Strong lactose fermenter (E. coli)	Metallic green sheen
Weak fermenter	Pink/Purple
Non-fermenter	Colorless

Blood Agar

Purpose

• Detect hemolysis.

Steps

• Streak plate and incubate.

Results

Hemolysis	Appearance
Alpha (α)	Green halo (partial)
Beta (β)	Clear zone (complete)
Gamma (γ)	No change

Glucose Fermentation

Purpose

• Determine ability to ferment glucose.

Steps

1. Inoculate glucose broth.

2. Durham tube inside.

3. Incubate.

Results

Result	Meaning
Yellow	Acid produced
Bubble	Gas produced
Red	No fermentation

Catalase Test

Purpose

• Detect catalase enzyme.

• Catalase breaks:

  • Hydrogen peroxide → Water + Oxygen

Steps

1. Place colony on slide.

2. Add H₂O₂.

Results

Result	Meaning
Bubbles	Positive
No bubbles	Negative

Oxidase Test

Purpose

• Detect cytochrome c oxidase.

Steps

1. Add oxidase reagent.

2. Rub colony onto reagent.

Results

Result	Meaning
Purple within 30 sec	Positive
No color	Negative

SIM Test

SIM =

• Sulfur

• Indole

• Motility

Steps

Stab inoculate deep.

Incubate.

Add Kovac's reagent.

Results

Sulfur:

• Black = Positive

Indole:

• Red ring = Positive

Motility:

• Cloudy growth away from stab = Positive

MR-VP Test

MR (Methyl Red)

Purpose:

• Mixed acid fermentation.

Add

• Methyl Red.

Results

• Red = Positive

• Yellow = Negative

VP (Voges-Proskauer)

Purpose:

• Detect acetoin.

Add

• VP reagents A & B.

Results

• Red = Positive

• Copper/Brown = Negative

Citrate Test

Purpose

• Determine if bacteria use citrate as sole carbon source.

Medium

• Simmons Citrate Agar

Steps

• Streak slant.

• Incubate.

Results

Result	Meaning
Blue	Positive
Green	Negative

Starch Hydrolysis Test

Purpose

• Detect amylase.

Steps

1. Streak starch agar.

2. Incubate.

3. Flood with iodine.

Results

Result	Meaning
Clear halo	Positive
Blue-black	Negative

PCR (Polymerase Chain Reaction)

Purpose

• Amplify DNA.

Used for

• Disease diagnosis

• Detect pathogens

• Research

• Genetics

Steps

1. DNA extraction

2. Denaturation (95°C)

3. Annealing (50–65°C)

4. Extension (72°C)

5. Repeat 25–40 cycles

Results

• DNA band detected = Target DNA present.

• No band = Negative.

Antigen Test

Purpose

• Detect pathogen proteins (antigens).

Used for

• Rapid diagnosis.

Examples:

• COVID

• Influenza

• Strep

Steps

1. Collect specimen.

2. Add extraction buffer.

3. Apply sample.

4. Wait.

Results

• Control line only = Negative

• Control + Test line = Positive

• No control = Invalid

Kirby-Bauer Test

Purpose

• Determine antibiotic susceptibility.

Steps

1. Swab bacterial lawn.

2. Place antibiotic disks.

3. Incubate.

4. Measure zones.

Results

• Large zone = Sensitive

• Medium = Intermediate

• Small/no zone = Resistant

Gas Fermentation

Purpose

• Determine gas production during fermentation.

Method

• Durham tube.

Results

• Bubble = Gas positive

• No bubble = Negative

H₂S Production

Purpose

• Detect hydrogen sulfide production.

Common test

• SIM agar.

Results

• Black precipitate = Positive

• No black = Negative

Indole Test

Purpose

• Detect tryptophanase enzyme.

Steps

• Grow in tryptophan medium.

• Add Kovac's reagent.

Results

• Red ring = Positive

• Yellow = Negative

Motility Test

Purpose

• Determine bacterial movement.

Method

• SIM or motility agar.

Results

• Diffuse/cloudy growth = Motile

• Growth only along stab = Nonmotile

RBC Hemolysis

Performed on Blood Agar.

Alpha (α)

• Partial hemolysis

• Green discoloration

Examples:

• Streptococcus pneumoniae

• Viridans streptococci

Beta (β)

• Complete hemolysis

• Clear zone around colony

Examples:

• Streptococcus pyogenes

• Staphylococcus aureus

Gamma (γ)

• No hemolysis

• No color change

Examples:

• Enterococcus faecalis

• Staphylococcus epidermidis

Summary Table

Test	Detects	Positive Result
Simple stain	Cell morphology	Cells stain one color
Gram stain	Cell wall type	Purple (Gram +), Pink (Gram -)
MSA	Mannitol fermentation	Yellow medium
MacConkey	Lactose fermentation	Pink colonies
EMB	Lactose fermentation	Metallic green (E. coli)
Blood agar	Hemolysis	α = green, β = clear, γ = none
Catalase	Catalase enzyme	Bubbles
Oxidase	Cytochrome c oxidase	Purple
SIM	Sulfur	Black precipitate
SIM	Indole	Red ring
SIM	Motility	Diffuse growth
MR	Mixed acid fermentation	Red
VP	Acetoin production	Red
Citrate	Citrate utilization	Blue medium
Starch hydrolysis	Amylase	Clear halo
Glucose fermentation	Acid production	Yellow broth
Gas fermentation	Gas production	Bubble in Durham tube
Kirby-Bauer	Antibiotic susceptibility	Large zone = sensitive
PCR	Target DNA	DNA amplification/band present
Antigen test	Pathogen proteins	Test line + control line

What is the total magnification of a specimen that is being viewed with a standard ocular lens and a 40⨯ objective lens?

400X

Which part of the light microscope controls the intensity of light entering the viewing area?

Diaphragm

Which of the following is an accurate statement of the function of immersion oil in standard light microscopy:

decreases the refraction of light

What is the main purpose of 4-way streaking?

to isolate single colonies of microorganisms from a larger sample.

You are performing a serial dilution as part of an experiment.

1. In Tube 1, you start by preparing a 1:10 dilution of your original sample .

2. Next, you take 100 µL from Tube 1 and transfer it into 900 µL of distilled water in Tube 2, creating another dilution.   How many times you have diluted your sample from tube 1?

10 times

You are performing a serial dilution as part of an experiment.

1. In Tube 1, you start by preparing a 1:10 dilution of your original sample.

2. Next, you take 100 µL from Tube 1 and transfer it into 900 µL of distilled water in Tube 2, creating another dilution.

3. Finally, you take 100 µL from Tube 2 and plate it onto an agar plate.

Question:
What is the total dilution factor of the sample that is plated on the agar plate compared to the original undiluted sample?

1/1000

If you need to take 15.5 µL  which pipette do you use?

P20

MacConkey agar is an example of

Selective media and differential

Which one/s apply to differential staining: 

• The purpose is to to distinguish between different types of microorganisms or to highlight specific cellular structures by using multiple stains that react differently with various cell components

• Gram staining is differential staining

• Capsule staining is differential staining

Put these steps in correct order for gram staining

1. Prepare Smear

2. Air Dry

3. Heat Fixing

4. Adding Crystal Violent

5. Adding Iodine

6. Decolorization

7. Using Safranin

Pick media that are both selective and differential.

• EMB

• MSA

Pick all that applies to EMB.

• Lactose fermenters produce colored colony

• Only Gram negative will grow on it

What is the most common gas collected in the Durham tubes?

CO2

Indole positive means:

The bacteria is able to produce Tryptophanase.

What reagent is added to the starch plate to read the results?

Iodine

What is the purpose of the wash steps in ELISA?

To remove all unbound molecules

In Kirby Bauer experiment, the zone of inhibition (ZOI) and is due to…

Bactericidal effect

Gel electrophoresis is used to separate DNA fragments on the basis of which property?

Size

What is/are the purpose of using SYBR Safe in gel electrophoresis?

To visualize DNA

Which one is not used in PCR?

Helicase