Microbial Growth Media and Isolation Techniques

What are the three typed of media

Growth media, selective/differential, and enriched

Selective Media

Media capable of restricting the growth of some microbes while promoting the growth of others. It is best used to isolate a specific target from a mixed sample

Differential Media

Media that distinguishes between two (often related) microbes growing on the same plate, usually through color changes based on biochemical/metabolic differences

Enriched Media

Media designed to grow fastidious microorganisms by providing specific, essential nutrients (e.g., blood, vitamins) that the organism cannot produce itself

Fastidious Microbe

An organism with complex growth requirements; if specific nutrients are absent, it will not grow

Agar

A polysaccharide derived from seaweed (algae) used to create a solid, smooth surface for microbial growth. It is a hardening agent, not a nutrient source itself

Plating

The process of spreading a bacterial culture onto a petri dish (agar plate) to isolate colonies

Pure Culture

A culture containing a single species of organism, free of contaminants, usually obtained by isolating a single colony

Hemolysis

The lysis (breakdown) of red blood cells. It is a key differential trait for Streptococcus species on Blood Agar

LB Agar & TSA

Multi-purpose, non-selective, non-differential. Used for general expansion (e.g., E. coli)

Blood Agar (BAP)

Enriched and Differential (hemolysis)

Alpha Hemolysis

Partial digestion (Greenish-brown zone of BAP)

Beta Hemolysis

Complete digestion (Clear zone of BAP)

Gamma Hemolysis

No digestion (No change/White zone of BAP)

Columbia CNA

Enriched, Selective (suppresses Gram-negative), Differential (hemolysis). Isolate Gram-positives

Chocolate Agar

Enriched, Non-selective, Non-differential. Contains lysed (cooked) red blood cells, not actual chocolate. Used for Haemophilus and Neisseria

MacConkey Agar

Selective (Gram-negative only) and Differential (Lactose fermentation)

Lac+ (Fermenter)

Pink/Red colonies on MacConkey Agar (e.g., E. coli)

Lac- (Non-fermenter)

White/Tan colonies on MacConkey Agar

SMAC Agar

Variant of MacConkey. Detects pathogenic E. coli O157:H7

Standard E. coli

Ferments sorbitol (Pink) on SMAC agar

Pathogenic O157:H7

Cannot ferment sorbitol (White) on SMAC agar

EMB Agar

Selective (Gram-negative) and Differential (Lactose)

E. coli produces what distinct color on EMB agar?

Metallic green

Mannitol Salt Agar (MSA)

Selective (Gram-positive/Staph) and Differential (Mannitol)

Staph aureus

Ferments mannitol (Yellow) on MSA

Non-pathogenic Staph

No fermentation (Red) on MSA

Advantage of Plates

Bacteria are "fixed" in place, allowing the formation of individual colonies (clones of a single cell)

Quadrant Streak (Phase Dilution)

Used to isolate a pure culture, by establishing a dilution gradient

What is the most important factor in creating a quadrant streak

Using a new sterile loop between each phase

Incubation

Standard is 37°C for 12–24 hours. Lower temperatures (25°C) restrict pathogenic growth; higher temps/longer time encourage growth

What is the protocol for unknowns

Expand and select/differentiate

Expand

Use non-selective media (like LB) first to increase the population

Select/Differentiate

Re-streak onto specialized plates to identify

What is the primary goal of selective media

Restrict growth of unwanted microbes; promote growth of target microbes

What is the primary goal of differential media

Distinguish between different microbes growing on the same plate

How dose selective media work

Uses additives like antibiotics, salt, or bile salts to kill/inhibit specific groups

How dose differential media work

Uses indicators (dyes, pH sensors) to show metabolic differences (e.g., fermentation)

What is the visual result of selective media

Growth vs. No Growth

What is the visual result for differential media

Color change in the colony or the agar itself