Lab 1: Microbiology Lab Orientation

Lab 1: Orientation to the Laboratory

Lab 1 orientation to microbiology laboratory.
Instructor: Dr. Mohammed Ali Eid, Assistant Professor of Microbiology
Date: 06/10/2024

Laboratory Layout

Sample Receipt and Storage: Includes procedures for receiving and storing samples.
Microscopy: Discusses the use of microscopes for visualizing microorganisms.
Culturing: Covers using incubators, autoclaves, and other equipment to grow and isolate microorganisms.
Biohazard Disposal: Includes details on proper biohazard waste disposal.
Handwashing Stations: Locations of handwashing stations are specified.
Emergency Equipment: Locations of fire extinguishers, first aid kits, safety showers, emergency wash stations, and emergency exits.

Familiarization with Common Lab Equipment

Microscopes: Primary tools for visualizing microorganisms.
Autoclaves: Used for sterilizing lab equipment and waste.
Bunsen Burners: Used for heat-related procedures.
Centrifuges: Used to separate different components of a sample.

Familiarization with Common Lab Equipment (Continued)

Incubators: Provide controlled environments for growing microorganisms.
Pipettes: Used for precise measurement and transfer of liquids.
Personal Protective Equipment (PPE): Includes lab coats, gloves, safety glasses, and other equipment used for personal protection.

General Laboratory Safety Rules

Personal Protective Equipment (PPE): No eating, drinking, handling chemicals or biological materials improperly.
Handling Chemicals and Biological Materials Safely: Proper handling of chemicals and biological materials.
Dispose of Waste Properly: Instructions on the disposal of waste.
Know Emergency Procedures: Knowing emergency procedures is vital.
Use Equipment Correctly: Use equipment according to instructions.
Maintain Cleanliness: Keeping the lab clean is important.
No Unauthorized Experiments: Avoid performing unauthorized experiments.
Report Accidents and Unsafe Conditions: Reporting incidents is imperative.
Do Not Work Alone: Working in groups is advisable.

Microscopic Techniques

Light Microscopy: Provides magnification up to 1000-2000 times, used in routine tasks like identifying bacteria and observing cell structures.

Parts of a Microscope and Their Functions

Eyepiece (Ocular Lens): The lens you look through, usually providing 10x or 15x magnification.
Objective Lenses: Lenses closest to the sample, providing various magnifications (e.g., 4x, 10x, 40x, 100x)
Stage: The flat platform where the slide with the sample is placed.
Stage Clips: Hold the slide in place on the stage.
Light Source (Illuminator): Provides the light to view the sample.
Condenser: Controls the amount of light reaching the sample.
Coarse and Fine Focus Knobs: Move the stage up and down to bring the sample into focus.
Body Tube: Holds the eyepiece and objective lenses.
Base and Arm: Support the microscope and its parts.

Lab 2: Sterilization and Disinfection

Sterilization: The killing or removal of all microorganisms, including bacterial spores.
Disinfection: The killing of most microorganisms, mainly pathogenic ones.
Disinfectants: Vary in their tissue-damaging potential, ranging from corrosive phenol-containing compounds to less toxic materials like ethanol and iodine.
Antiseptics: Chemicals used to kill microorganisms on the skin and mucous membranes.

Classification of Sterilizing Agents

1-physical agents: heat, filtration, radiation.
2- Chemical agents: Chemical disinfection methods.

Physical Sterilization

I. Heat:
- A. Dry Heat:
  - Direct flame (simplest method)
  - Used for metal instruments (e.g., wire loops, forceps).
  - Incineration for disposable material.
- B. Hot air: Sterilizing dry glassware and metal instruments in a hot air oven (160-180°C for 1 hour).
C. Moist heat:
- 1. pasteurization: Uses heat (63°C at 30 sec) to kill organisms like Brucella or Salmonella. Alternative method involves raising the temperature to 72°C (161°F) for 15-20 seconds (flash process).
- 2. Steam under pressure (High pressure steam): This method using autoclaves or pressure cookers at 121°C (15 lbs) for 15 minutes.
II. Filtration: Passing materials through special filters that hold back bacteria. Useful for sterilizing heat-sensitive materials like sera, plasma, vitamins, and antibiotics.
III. Radiation: Used commercially to sterilize large amounts of pre-packed items like plastic syringes and catheters. Ultraviolet rays induce thymine dimmers in DNA and disrupt replication.

Chemical Sterilization

1-Alcohols: Ethyl alcohol or isopropyl alcohol (70% aqueous solution); antiseptic for thermometers.
2-Phenols: Sterilize surgical instruments, bathrooms, and hospital floors; chlorohexidine is a skin disinfectant.
3-Heavy metal ions (metallic salt): Mercuric salts used in preservation and as a vaccine and silver salts as eye drops for preventing infections (e.g., Neisseria gonorrhea).

Lab 3: Isolation and Purification of Bacteria

Isolation Techniques

Serial Dilution Method: Diluting a sample through steps to reduce cell concentration, crucial in quantifying bacteria for scientific investigations.
Filtration Method (Membrane Filter [MF]): Separates bacteria based on size using filters with specific pore sizes for isolating bacteria from mixed populations, purifying cultures, and sterilizing heat-sensitive solutions.
Streak Plate Method: Isolating pure bacterial colonies by spreading diluted samples on agar plates. Includes four quadrant streak and continuous streak.
Enrichment Culture: Creating selective growth conditions to favor the target microorganism, while inhibiting the growth of others (e.g. manipulating nutrients, pH, temperature, oxygen levels, or adding selective agents).

Purification Methods

Colony Picking: Isolating specific colonies and culturing them separately ensures purity.

Table of Techniques

Technique | Description | Application
--- | --- | ---
Streak Plate | Isolate colonies | Pure cultures
Serial Dilution | Reduce cell concentrations | Quantitative studies
Filtration | Separate based on size | Water treatment
Enrichment Culture | Enhance specific bacteria | Research studies

Isolation from Different Sources

Soil: Diluting soil samples, plating dilutions on appropriate media and using selective media.
Water: Filtering water, placing the filter on agar plates.
Air: Exposing agar plates to air for set periods.
Food: Homogenizing, diluting and plating food samples on selective media.
Clinical samples Using sterile swabs for collection and streaking on selective media.
Plant surfaces: Washing plant parts, plating wash solution.
Animal sources: Collecting and culturing samples.

Lab 4: Colony Description, Selection, and Subculturing

Purpose of Colony Description

Bacterial species often form colonies with unique sizes and appearances.
These characteristics help microbiologists identify and distinguish different types of bacteria.
Identified colonies allow for obtaining pure cultures of specific bacterial species.

Factors Affecting Colony Morphology

Nutrient Composition and Availability: Different growth patterns based on nutrient availability.
Temperature: Different temperatures influence bacterial growth rates, and overall structure.
Oxygen Availability: Aerobic and anaerobic bacteria exhibit different types of colonies based on oxygen requirements.
pH Level: The acidity or alkalinity of growth medium affects bacterial growth, and consequently colony morphology; some bacteria prefer alkaline environments while others prefer acidic ones.
Moisture Content: Some bacteria produce mucoid or slimy colonies under high-moisture conditions.
Incubation Time: Longer incubation periods may lead to larger colonies, and growth patterns.
Genetic Factors: Different strains of the same bacteria exhibit distinct characteristics in terms of morphology.
Bacterial Behavior(Quorum Sensing) and Cooperative Behavior: Bacteria can exhibit unique structures in response to environmental conditions.
External Influences: Physical barriers, other microorganisms, and antimicrobial agents affect colony morphology.