W6 Pre-Lab Material

Based on W6 Lab Simulations

3 Types of Approaches for Microbial Identification

1. Phenotypic — Observing characteristics of microbes (shape, colour, growth, biochemical tests)

2. Genetic — Using DNA sequencing

3. Immunologic — Using antibodies

Traditional phenotypic identification of microbes involves…

• Use microscopy, staining, and biochemical tests

• Analysis of results from any of the above processes helps assign traits to bacteria

Agar Plate

Petri dish containing agar — nutrient medium used for microbial growth.

Biochemical Testing

• Using chemical reactions to identify bacterial traits

• Specialized growth media and reagents reveal bacterial metabolic characteristics.

• Results are based on visible changes such as:

  • Color shifts

  • Growth patterns

  • Appearance of colonies

Colony

• Visible cluster of microbial cells — typically bacteria that originates from single progenitor cell 

• Grows on solid medium, forming a mass

Culture

Growth of microbes under controlled conditions.

Differential Medium

Medium used to distinguish b/w organisms based on biochemical test reactions.

Phenotypic Characteristics

Observable physical or metabolic trait.

Sterile

Any object/being completely free of living microbes.

Selective Medium

Supports growth of certain molecules while inhibiting others.

Why is microbe identification essential in microbiology (real-world application)? 

To be able to…

• Diagnose patients

  • Treat successfully patients based on the specific microbial infection they have (diff microbes require diff types of medications; e.g. viruses treated with antiviral meds v.s. bacterial infection treated with antibiotics)

• For tracking disease outbreaks & managing pandemics 

Name & briefly describe the 3 main approaches to identifying microbes.

1. Phenotypic —> Observing microbes’ traits

2. Genetic —> DNA sequencing 

3. Immunologic —> Antibody detection

Phenotypic Identification

(a) What does it entail/involve? 

(b) What methods are used? 

(a) What does it entail/involve? 

• Observing physical & biochemical traits such as shape, colour, and growth characteristics

(b) What methods are used? 

• Microscopy

• Staining 

• Biochemical tests

When is phenotypic identification often used? 

When genetic or immunologic tests are too costly or unavailable.

What kind of results do phenotypic tests produce?

A: Visual outcomes such as color changes, growth patterns, or changes in appearance.

What is the purpose of biochemical testing in microbiology?

A: Color shifts, growth patterns, and changes in colony appearance.

Q: What do carbohydrate metabolism tests determine?

A: Whether bacteria can metabolize carbohydrates such as lactose.

Q: What kind of media can distinguish bacteria based on lactose metabolism?

A: Selective and differential media.

Q: What is MacConkey agar used for?

Q: What is MacConkey agar used for?

Q: What type of medium is MacConkey agar?

A: Both selective and differential.

Q: What are the selective agents in MacConkey agar?

A: Bile salts and crystal violet dye — they inhibit gram-positive bacteria and allow gram-negatives to grow.

Q: What are the differential components in MacConkey agar?

A: Lactose (carbohydrate source) and neutral red (pH indicator).

Q: How does MacConkey agar differentiate between bacteria?

A: Based on whether they ferment lactose, which changes the color of colonies.

Q: What happens when bacteria ferment lactose on MacConkey agar?

A: Acid is produced → pH decreases → neutral red turns pink/red.

Q: What happens when bacteria do not ferment lactose?

A: No acid is produced → pH remains neutral → colonies stay colorless or clear.

Q: What does MacConkey agar select for and against?

A: Selects for gram-negative bacteria and inhibits gram-positive bacteria.

Q: Why is MacConkey agar both selective and differential?

A: It inhibits gram-positive growth (selective) and distinguishes lactose fermenters from non-fermenters (differential).

Q: How does MacConkey agar support public health efforts?

A: By helping correctly identify bacteria for diagnosis, outbreak tracking, and treatment decisions.

Q: When is phenotypic identification commonly used?

A: When genetic or immunologic methods are unavailable or too costly.

What is Mannitol Salt Agar (MSA)?

A selective & differential medium.

What makes MSA a selective medium.

It contains 7.5% NaCl which inhibits most bacteria but allows salt-tolerant staphylococci to grow.

What makes MSA a differential medium?

It contains mannitol & phenol red, allowing distinction b/w mannitol fermenters and non-fermenters.

What does no growth on MSA indicate?

Organism cannot tolerate high salt concentrations (its not salt-tolerable staphylococci).

What happens when bacteria ferment mannitol on MSA?

They produce acid —> lowers pH —> phenol red turns yellow around the colonies.

What result indicates that bacteria didn’t ferment mannitol?

Medium remains red or pink because no acid is produced.

A yellow medium around growth indicates what?

A: Mannitol fermentation (acidic result) — e.g., Staphylococcus aureus.

Q: Red or pink medium with growth indicates what?

A: No mannitol fermentation — e.g., Staphylococcus epidermidis.

Q: No growth on MSA indicates what?

A: The bacterium is not salt-tolerant (e.g., E. coli).

Q: What is the purpose of the MSA lab test?

A: To determine if a Staphylococcus species can ferment mannitol.

Q: How long should the plate be incubated before reading results of MSA test?

A: 24 hours.

Q: Define selective medium.

A: A medium that supports growth of some organisms while inhibiting others.

A: A medium that supports growth of some organisms while inhibiting others.

A: A medium that shows visible differences between organisms based on metabolic activity.

Q: Define phenotypic characteristic.

A: An observable trait like colony color, shape, or growth behavior.

Q: Define sterile.

A: Completely free of all living microorganisms.

Q: What does MSA test for? What does it measure?

MSA = 

Measure of the ability of bacteria (especially staphylococci) to ferment mannitol.

Q: How do S. aureus and S. epidermidis differ on MSA?

A: S. aureus → yellow medium (ferments mannitol);
S. epidermidis → red medium (no fermentation).

Why is MSA both selective and differential?

Selective —> for salt-tolerant staphylococci

Differential —> based on mannitol fermentation (color change).

Some staphylococcus species are pathogenic. Especially in…

• immunocompromised individuals

• situations where bacteria brach skin or mucosal barriers

Species differentiation b/w staphylococci are based on:

1. Antibiotic sensitivity (e.g., to novobiocin)
   
   

2. Hemolytic activity (blood cell lysis on blood agar)
   
   

3. Enzyme presence (e.g., catalase, coagulase)
   
   

4. Metabolic abilities (e.g., mannitol fermentation)

Describe each of the components of the MSA and their functions.

Component	Function
7.5% NaCl (high salt)	Makes the medium selective — inhibits most bacteria, allowing salt-tolerant staphylococci to grow
Mannitol (sugar)	Provides a carbohydrate source to test for fermentation
Phenol red (pH indicator)	Detects acid production from mannitol fermentation

Describe the possible results on an MSA plate & what each means.

🧫 Results Interpretation (MSA Plates)

Observation	Interpretation	Example
Yellow medium around growth	Mannitol fermented → acid produced	Staphylococcus aureus
Growth but medium remains red/pink	No mannitol fermentation	Staphylococcus epidermidis
No growth	Cannot tolerate salt (not Staphylococcus)	E. coli or other non-halophiles