Microbial Metabolism and Biochemical Testing: Lab Exercises 11–15

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Microbial Metabolism and Biochemical Testing

Exercise 11: Colony Morphology and Pure Culture Identification

Colony morphology is a fundamental technique in microbiology for identifying and describing bacterial colonies. Observing colonies on plates and slants helps determine if a culture is pure or mixed.

Colony Description: Record size, shape, color, margin (edge), and elevation (height).
Pure Culture: If all colonies appear identical, the culture is considered pure. A pure culture contains only one type of microorganism.
Example: Escherichia coli colonies are typically round, smooth, and off-white.

Exercise 13: Starch Hydrolysis Test

The starch hydrolysis test determines whether bacteria can break down starch using the enzyme amylase. This is a key metabolic trait.

Procedure: Flood the starch agar plate with Gram's iodine, which binds to starch and turns it dark.
Interpretation:
- Positive Result: A clear ring around the bacterial streak indicates starch breakdown (amylase activity).
- Negative Result: Dark color up to the streak means starch remains; no amylase activity.
Example: Bacillus subtilis is typically positive for starch hydrolysis, while E. coli is negative.

Exercise 13: Oxidation-Fermentation (OF) Glucose Test

The OF-glucose test distinguishes between oxidative and fermentative metabolism of glucose in bacteria.

Procedure: Compare color changes in open (aerobic) and sealed (anaerobic) tubes.
Interpretation:
- Both tubes yellow: Fermentative metabolism (can ferment glucose anaerobically).
- Open tube yellow only: Oxidative metabolism (requires oxygen).
- Neither tube changed: Neither oxidative nor fermentative metabolism.
Example: Pseudomonas aeruginosa is oxidative; E. coli is fermentative.

Exercise 14: Sugar Fermentation, MRVP, and Citrate Utilization Tests

These tests assess the ability of bacteria to metabolize different sugars and utilize citrate as a carbon source. The MRVP test differentiates between mixed acid and butanediol fermentation.

Sugar Fermentation Tubes: Glucose, lactose, and sucrose tubes each contain a Durham tube to detect gas production.
MRVP Broth: Used for the Methyl Red and Voges-Proskauer tests in the next class.
Citrate Slant: Tests if bacteria can use citrate as the sole carbon source. Shared tubes for E. coli and Enterobacter.
Setup: Label tubes, inoculate with loop, streak citrate slants, and incubate.
Example: Enterobacter can utilize citrate; E. coli cannot.

Exercise 15: Gelatin Hydrolysis and Urea Hydrolysis Tests

These tests evaluate the ability of bacteria to hydrolyze gelatin and urea, important for identifying metabolic capabilities.

Gelatin Hydrolysis: Tests for the enzyme gelatinase. Gelatin is stabbed with a needle and incubated at room temperature (gelatin melts above 28°C).
Urea Hydrolysis: Tests for the enzyme urease. Urea agar is streaked with a loop.
Litmus Milk: Not performed in this lab session.
Example: Proteus vulgaris is positive for urease.

Summary Table: Biochemical Tests and Their Purposes

Test	Purpose	Key Indicator	Example Organism
Colony Morphology	Identify pure culture	Uniform colony appearance	E. coli
Starch Hydrolysis	Detect amylase activity	Clear ring after iodine	B. subtilis
OF-Glucose	Oxidative vs. fermentative metabolism	Yellow color change	P. aeruginosa, E. coli
Sugar Fermentation	Fermentation of glucose, lactose, sucrose	Acid/gas in Durham tube	E. coli
MRVP	Mixed acid or butanediol fermentation	Color change after reagents	Enterobacter
Citrate Utilization	Use citrate as sole carbon source	Growth/color change	Enterobacter
Gelatin Hydrolysis	Detect gelatinase activity	Liquefaction of gelatin	B. subtilis
Urea Hydrolysis	Detect urease activity	Pink color change	Proteus vulgaris

Key Concepts and Definitions

Fermentation: Anaerobic breakdown of sugars producing acids and gases.
Oxidation: Aerobic metabolism of sugars.
Amylase: Enzyme that hydrolyzes starch.
Gelatinase: Enzyme that hydrolyzes gelatin.
Urease: Enzyme that hydrolyzes urea to ammonia and carbon dioxide.
Citrate Utilization: Ability to use citrate as sole carbon source.

Relevant Equations

Urea Hydrolysis:

Starch Hydrolysis:

Glucose Fermentation (general):

Additional info:

Litmus milk is a common test for protein and lactose metabolism, but was not performed in this session.
MRVP (Methyl Red and Voges-Proskauer) tests are used to distinguish between mixed acid and butanediol fermentation pathways in enteric bacteria.