MORPHOLOGY of BACTERIA. SHAPE and SIZE of BACTERIAL CELLS. SIMPLE STAINS of BACTERIA. MICROSCOPIC EXAMINATION TECHNIQUES Prelab conference. Topics for discussion: 1. Rules of work in the microbiological laboratory. Biosafety rules. 2. Classification of

### Prelab Conference: Topics for Discussion #### 1. Rules of Work in the Microbiological Laboratory. Biosafety Rules - **Personal Protective Equipment (PPE)**: Always wear gloves, lab coats, and safety goggles. Depending on the risk level of the work, additional protection such as face shields or respiratory masks may be necessary. - **Hand Hygiene**: Wash hands thoroughly with soap and water before and after laboratory work or use hand sanitizer. - **Disinfection**: Surfaces should be disinfected before and after use. All spills should be cleaned immediately with appropriate disinfectants. - **Waste Disposal**: Biohazard waste must be disposed of in designated biohazard bins. Glassware should be disposed of in specific sharps containers. - **Equipment Use**: Understand how to properly use all equipment before starting experiments. This includes microscopes, autoclaves, and incubators. - **Emergency Procedures**: Be familiar with the location of safety equipment, such as eyewash stations, safety showers, and fire extinguishers. Know the emergency procedures for accidents or spills. #### 2. Classification of Microbes: Main Principles, Taxonomy, Binomial Nomenclature - **Taxonomy**: The science of classification of living organisms. It involves hierarchical levels: domain, kingdom, phylum, class, order, family, genus, and species. - **Binomial Nomenclature**: A system for naming species using two names — the genus name (capitalized) and the species name (lowercase), both italicized (e.g., *Escherichia coli*). - **Principles of Classification**: Classification can be based on various criteria, including morphology, genetic characteristics, biochemical properties, and ecological niche. #### 3. Shape and Arrangement of Bacterial Cells - **Cocci**: Spherical bacteria, which can exist singly (monococcus), in pairs (diplococci), chains (streptococci), or clusters (staphylococci). - **Bacilli**: Rod-shaped bacteria, which can also appear singularly, in chains, or in palisades. - **Spiral Bacteria**: These can be further classified into vibrios (comma-shaped), spirilla (rigid, wave-like), and spirochetes (flexible, corkscrew-shaped). - **Filamentous Bacteria**: These are long, thread-like structures. Examples include mycelial organisms such as Actinobacteria. #### 4. Methods of Microscopic Examination - **Brightfield Microscopy**: The most common form of microscopy using visible light. Stains are typically required to visualize the cells clearly. - **Phase Contrast Microscopy**: Enhances the contrast of transparent, colorless objects by converting phase shifts into amplitude shifts. - **Fluorescence Microscopy**: Utilizes fluorescent dyes that emit light when exposed to specific wavelengths, allowing for visualization of specific structures within cells. #### 5. Techniques of Microscopical Examinations: Brightfield Microscopy Under Immersion Oil Objective - **Immersion Oil**: Used to reduce light refraction and increase resolution at high magnifications (usually 100x). The oil must be placed between the slide and lens. - **Procedure**: 1. Start with the lowest magnification to locate the specimen. 2. Move to the high power (40x) objective lens, and focus using the coarse and fine focus knobs. 3. Switch to the oil immersion lens (100x). Place a drop of immersion oil on the slide. 4. Carefully rotate the oil immersion lens into position without allowing the lens to touch the slide. 5. Adjust focus as necessary to observe details. #### 6. Preparation of Smears: Procedure of Simple Staining - **Preparation of Smears**: 1. Label a clean microscope slide. 2. Using a sterile loop, place a small drop of sterile water on the slide. 3. Transfer a small amount of bacterial culture into the drop and spread it to create a thin, even film. 4. Allow the slide to air dry completely. 5. Heat-fix the bacteria by passing the slide briefly through a flame to adhere bacteria to the slide. - **Simple Staining Procedure**: 1. Flood the heat-fixed slide with a simple stain (e.g., crystal violet, safranin, or methylene blue) and let it sit for 1-2 minutes. 2. Rinse gently with water to remove excess stain. 3. Gently blot dry with bibulous paper. 4. Observe the stained smear under a microscope, noting the shape, size, and arrangement of cells. This prelab conference will help prepare participants for practical work in the microbiology lab by ensuring they understand safety protocols, classification methods, and microscopic techniques fundamental to studying bacteria.