Applied Microbiology SCH2235 Practical Skills Test Prep

Applied Microbiology Practical Skills Test Overview

Date and Time: Wednesday 13/05 @ 8:30AM.
Coverage: Content from Practicals 1–8.
Test Components:
Practical Component: Rotation through stations. Tasks include Gram stains, API interpretation, antimicrobial sensitivity reading, and biochemical test interpretation.
Written Component: Multiple-choice and short-answer questions based on lab content.
Laboratory Learning Outcomes: Comprehensive assessment of standard microbiological techniques and theoretical underpinnings.

Practical 1: Microbiological Safety and Foundations

Purpose of Microbiological Safety:
Prevent contamination of yourself, other people, cultures/samples, and the laboratory environment.
Ensure the accuracy of experimental results.

Personal Protective Equipment (PPE):
Lab coat must be fully buttoned.
Closed shoes are mandatory.
Long hair must be tied back.
Gloves must be used when required.
Safety glasses must be worn if needed.

General Laboratory Rules:
No eating or drinking.
Hands must be washed before and after lab sessions.
Bench surfaces must be disinfected with $70\%$ ethanol before and after practical work.
Personal items and bags must be kept away from benches.
Mouth pipetting is strictly prohibited.
Labelling Requirements: All cultures must clearly state name, date, organism/sample, and orientation.

Waste Disposal Protocols:
White Waste Bin: Used for general contaminated lab waste and agar plates/cultures.
Yellow Sharps Bin: Used for needles, blades, and broken glass.
Hazardous Waste: Includes contaminated gloves, aprons, and bench coat material.

Aseptic Technique:
Purpose: To prevent contamination of cultures and the environment.
Examples: Flaming the inoculating loop, opening plates only briefly, working near a flame, avoiding touching sterile surfaces, and sterilizing the bench.

Flame Sterilisation:
Purpose: Kill microorganisms on inoculating loops/needles before and after use.
Procedure:
1. Hold the loop like a pencil.
2. Place the loop into the blue flame (the hottest part of the Bunsen burner).
3. Heat from base to tip until the loop becomes red-hot.
4. Allow to cool before touching a culture (prevents killing bacteria during inoculation).
Errors:
Loop not cooled: Bacteria are killed.
Loop not fully heated: Contamination is possible.
Touching sterile loop: Loss of sterility.

Preparing and Heat-Fixing a Bacterial Smear:
Purpose: Spread bacteria thinly on a slide for staining and viewing.
Steps:
1. Label the slide.
2. Add a drop of sterile water (only if using solid media).
3. Transfer a small amount of bacteria.
4. Spread into a thin film.
5. Air dry completely.
6. Pass the slide quickly through the flame $2-3$ times (Heat Fixing).
Function of Heat Fixing: Kills bacteria, makes them stick to the slide, and preserves cell shape. The slide must be completely air-dry first; excessive heating will distort cells.

Simple Stain Using Methylene Blue:
Purpose: Increase contrast for visibility.
Mechanism: Methylene blue is a basic, positively charged dye that binds to negatively charged bacterial cells.
Procedure: Flood a heat-fixed smear with dye for approximately $1 \text{ minute}$ , rinse with water, and blot with bibulous paper.

Microscopy and Metrics:
Magnification Table:
$4\times$ objective = $40\times$ total magnification.
$10\times$ objective = $100\times$ total magnification.
$40\times$ objective = $400\times$ total magnification.
$100\times\text{ oil immersion}$ objective = $1000\times$ total magnification.
Oil Immersion Strategy: Oil has a similar refractive index to glass, preventing light bending/refraction. This allows more light to enter the lens, improving resolution (the ability to distinguish detail).

Hanging Drop Preparation:
Purpose: Observe living microorganisms and natural motility.
True Motility vs. Brownian Motion:
True Motility: Directional, independent movement.
Brownian Motion: Random vibration caused by water molecule collisions.

High-Yield Practice Q&A (Practical 1):
Q: Why use $70\%$ ethanol? A: For disinfection of benches.
Q: Why must hair be tied back? A: To prevent it from catching fire or contaminating samples.

Practical 2: Gram Staining and Colony Characterization

Gram Staining Fundamentals:
Purpose: Differentiate bacteria into Gram-positive and Gram-negative based on cell wall structure.
Gram-Positive: Thick peptidoglycan layer; retains crystal violet; appears purple/violet.
Gram-Negative: Thin peptidoglycan layer and an outer membrane; loses crystal violet during decolourisation; takes up safranin; appears pink/red.

Reagents and Functions:
Crystal Violet: Primary stain.
Iodine: Mordant (forms crystal violet-iodine complex).
Alcohol/Acetone: Decolouriser (the most critical step).
Safranin: Counterstain.

Critical Step:
Decolourisation:
Over-decolourisation: Gram-positive appear Gram-negative.
Under-decolourisation: Gram-negative appear Gram-positive.

Colony Morphology Features:
Size: Small, medium, large.
Shape/Form: Circular, irregular, filamentous.
Margin: Entire (smooth), undulate (wavy), lobate (lobed).
Elevation: Flat, raised, convex (dome-shaped).
Texture/Opacity: Smooth, rough, mucoid; Transparent, translucent, opaque.

Streak Plating for Single Colonies:
Purpose: Dilute bacteria across the agar to obtain isolated colonies (growth from one single bacterial cell).
Principle: Each successive streak spreads fewer bacteria until individual cells are separated.

Skin Flora on Blood Agar:
Blood Agar Type: Enriched and differential media.
Haemolysis Types:
Beta: Clear zone; complete destruction of red blood cells.
Alpha: Green/brown zone; partial haemolysis.
Gamma: No change; no haemolysis.
Incubation Note: Plates are always incubated upside down to prevent condensation from dripping onto and spreading colonies.

Practical 3: Specialized Staining and Pure Cultures

Pure Culture Definition: A culture containing only one species of microorganism. Indicated by colonies of uniform colour, size, and morphology.
Ziehl–Neelsen (Acid-Fast) Stain:
Purpose: Differentiate bacteria with mycolic acid in their cell walls.
Acid-Fast Bacteria (e.g., Mycobacterium): Resist decolourisation by acid-alcohol due to a waxy, hydrophobic lipid layer. They appear red/pink.
Procedure Reagents: Carbol fuchsin (primary), Heat (softens wax/drives stain in), Acid-alcohol (decolouriser), Methylene blue (counterstain).

Capsule Staining:
Capsule Definition: Gel-like polysaccharide outer coating that increases virulence by protecting against phagocytosis and dehydration.
Appearance: Capsules are non-ionic and repel stains; they appear as a clear halo against a dark background and a coloured cell (Negative Staining).
Note: Heat fixing is avoided as it may shrink or destroy the capsule.

Practical 4: Skin Flora and Identification Integration

Common Skin Flora Organisms:
Staphylococcus epidermidis: Gram-positive cocci in clusters; white smooth colonies.
Micrococcus species: Gram-positive cocci in tetrads/clusters; yellow pigmented colonies.
Corynebacterium species: Gram-positive rods; small dry colonies.
Mixed Culture Recognition: Indicated by multiple colony types (different colours, sizes, shapes).
Mixed Smear Utility: Staining a smear with both Gram-positive and Gram-negative organisms tests the accuracy of the decolourisation technique.

Practical 5: Growth Requirements and Media Types

Atmospheric Growth Requirements:
Obligate Aerobes: Require Oxygen ( $O_2$ ).
Facultative Anaerobes: Grow with or without $O_2$ , but better with it.
Obligate Anaerobes: Oxygen is toxic.
Microaerophiles: Require low levels of $O_2$ .

Physical Requirements:
Temperature: Psychrophiles (cold), Mesophiles ( $20-45\,{^\circ}C$ ; includes human pathogens), Thermophiles (heat).
Salt/Osmotic Conditions: High salt causes water to leave cells via osmosis (plasmolysis). Halophiles are salt-loving.

Specific Media Examples:
MacConkey Agar (MAC): Selective for Gram-negative (bile salts/crystal violet); differential for lactose fermentation (pink = positive).
Mannitol Salt Agar (MSA): Selective for Staphylococci (high salt); differential for mannitol fermentation (yellow = positive).

API 20E System: A strip containing multiple biochemical tests used to identify enteric Gram-negative rods (Enterobacteriaceae). Mineral oil is added to specific wells to create anaerobic conditions.

Practical 6: Biochemical Testing and Quality Control

Catalase Test:
Reaction: $2H_2O_2 \rightarrow 2H_2O + O_2$ .
Result: Bubbling indicates catalase. Differentiates Staphylococcus (positive) from Streptococcus (negative).

Coagulase Test:
Function: Converts fibrinogen to fibrin, causing plasma clotting.
Result: Differentiates Staphylococcus aureus (positive) from S. epidermidis (negative).

Oxidase Test:
Purpose: Detects cytochrome c oxidase in the electron transport chain.
Result: Dark purple/blue is positive (e.g., Pseudomonas, Neisseria). Enterobacteriaceae are negative.

QA vs. QC:
Quality Assurance (QA): Standardising procedures to prevent defects.
Quality Control (QC): Using known control organisms to detect defects.

Practical 7: Clinical Application and Sensitivity

Urinary Tract Infections (UTIs):
Pathogens: E. coli (most common), Proteus (swarming), Klebsiella (mucoid).
Media: CLED agar (prevents Proteus swarming).

Antimicrobial Sensitivity (Kirby–Bauer Method):
Mueller-Hinton Agar: Standardised medium used for consistent antibiotic diffusion.
Zone of Inhibition: Clear area around a disc. A large zone indicates the organism is Sensitive (S); a small/no zone indicates Resistance (R).

Antibiotics:
Broad Spectrum: Targets many bacteria.
Narrow Spectrum: Targets specific bacteria; preferred to reduce resistance and preserve normal flora.