Final Lab Practical Study Guide: Microbiology Lab Techniques and Identification

Viral Plaque Assay

Introduction to Viral Plaque Assay

The viral plaque assay is a quantitative method used to measure the number of virus particles present in a sample. It is commonly used in microbiology to study bacteriophages and their interaction with host bacteria.

• Bacteriophage: A virus that infects bacteria.

• Plaque: A clear zone on a bacterial lawn caused by virus-induced cell lysis.

Key Points

• Purpose of Diluting Bacteriophage: Dilution allows for counting individual plaques, which represent single infectious virus particles.

• Calculation of T-4 Virus Titer: The titer is calculated by multiplying the number of plaques by the dilution factor and dividing by the volume plated.

Formula:

$\text{Titer (pfu/mL)} = \frac{\text{Number of plaques} \times \text{Dilution factor}}{\text{Volume plated (mL)}}$

Example: If 50 plaques are counted on a plate from a 10-6 dilution and 0.1 mL was plated, the titer is $\frac{50 \times 10^6}{0.1} = 5 \times 10^8$ pfu/mL.

Fungi

Macroscopic and Microscopic Appearance of Mold

Fungi can be identified by their colony morphology and microscopic structures.

• Macroscopic Appearance: Includes surface and reverse pigment, texture, and growth rate on agar plates.

• Microscopic Appearance: Involves spore arrangement, hyphae structure, and reproductive features.

Example: Aspergillus species produce conidial heads, while Penicillium forms brush-like conidiophores.

Streptococci Identification

Differentiation of Streptococcus Species

Streptococci are classified based on hemolysis patterns and biochemical tests.

• Hemolysis on Blood Agar:

  • Alpha hemolysis: Partial hemolysis, greenish discoloration (e.g., Streptococcus pneumoniae).

  • Beta hemolysis: Complete hemolysis, clear zone (e.g., Streptococcus pyogenes).

  • Gamma hemolysis: No hemolysis (e.g., Enterococcus faecalis).

• Bacitracin Sensitivity: Used to distinguish S. pyogenes (sensitive) from other beta-hemolytic streptococci.

• Optochin Sensitivity: Used to identify S. pneumoniae (sensitive).

Table: Hemolysis Patterns and Key Tests

Species	Hemolysis	Bacitracin	Optochin
S. pyogenes	Beta	Sensitive	Resistant
S. agalactiae	Beta	Resistant	Resistant
S. pneumoniae	Alpha	Resistant	Sensitive
Enterococcus faecalis	Gamma	Resistant	Resistant

Additional info: Correct spelling and hemolysis classification are essential for accurate identification.

Enterococcus Identification

Differentiation from Other Alpha Hemolytic Streptococci

Enterococcus species are distinguished from other alpha hemolytic streptococci by their ability to grow in high salt concentrations and hydrolyze esculin.

• Bile Esculin Test: Enterococcus faecalis hydrolyzes esculin, turning the medium black.

• Salt Tolerance: Growth in 6.5% NaCl broth is characteristic of enterococci.

Gram-Negative Bacilli and Enterobacteriaceae Identification

Differentiation of Gram-Negative Bacilli

Gram-negative bacilli are identified using biochemical tests and selective media.

• CNA and MacConkey Agar: CNA selects for Gram-positive cocci; MacConkey selects for Gram-negative rods and differentiates lactose fermenters (pink colonies) from non-fermenters (colorless colonies).

• Biochemical Tests: IMViC tests (Indole, Methyl Red, Voges-Proskauer, Citrate), Urease, Oxidase, and KIA (Kligler Iron Agar) are used for further identification.

Example: Escherichia coli is indole positive, methyl red positive, citrate negative, and produces acid/gas in KIA.

Mixed Unknown Identification

Identification in Mixed Cultures

Mixed cultures require the use of selective and differential media, as well as biochemical tests, to identify individual bacterial species.

• Colony Morphology: Observe differences in colony size, shape, color, and hemolysis.

• Gram Stain: Distinguishes between Gram-positive and Gram-negative bacteria.

• Biochemical Key: Use a dichotomous key to systematically identify unknown bacteria based on test results.

Additional info: Accurate identification requires careful observation and correct interpretation of test results.