Handout 2. Examination of Microorganisms.pdf

MCB 11 Lecture Section ST 1st Sem., 2024 – 2025 Handout #2 Examination of Microorganisms Microscopic examination of microorganisms is vital for identify...

MCB 11 Lecture Section ST 1st Sem., 2024 – 2025 Handout #2 Examination of Microorganisms Microscopic examination of microorganisms is vital for identifying, diagnosing, and understanding microbial structure, behavior, and interactions. It plays a key role in medicine, research, and environmental monitoring. I. Observing Living Microorganisms Observing living microorganisms under the microscope provides insight into their natural behavior, motility, and interactions, without the distortions caused by fixation or staining. However, living cells are often transparent and difficult to see, requiring special techniques to enhance visibility. Advantages:  Natural Behavior: Observing living microorganisms allows you to study their natural behavior in real time, including: o Motility (movement using flagella, cilia, or pseudopodia) o Reproduction (binary fission, budding) o Interactions with other microorganisms and the environment  Physiological Activities: You can observe essential biological activities such as: o Feeding (phagocytosis in amoeba, for example) o Cellular processes like respiration or metabolism in live cells o Responses to environmental changes, such as pH or temperature shifts  No Artifacts: In live observation, microorganisms are free from distortions or artifacts that might be introduced during staining and fixing processes. This ensures that the observed structures and activities are more accurate representations of their true state.  Useful for Delicate Cells: Some microorganisms, particularly certain protozoa and algae, may be sensitive to staining and fixation processes. Observing them alive preserves their morphology and function.  Methods used: 1. Wet Mount  Purpose: To observe live microorganisms in their natural, fluid environment.  Procedure: A drop of the microbial culture is placed on a glass slide, covered with a coverslip, and observed under a light microscope.  Advantages: o Simple and quick. o Allows observation of natural movement (motility), size, and shape of cells.  Disadvantages: o Drying occurs quickly, limiting observation time. o Contrast is low because microorganisms are mostly transparent. 2. Hanging Drop Technique  Purpose: To observe motility and natural behaviors of microorganisms in a liquid environment over an extended time.  Procedure: A drop of microbial suspension is placed on a coverslip, which is then inverted over a concave depression on a glass slide, creating a sealed chamber with an air pocket. This prevents drying and allows the organisms to move freely.  Advantages: o Longer observation time without drying. o Better for observing motility, as organisms have more space to move. o Reduces the risk of squashing or disturbing the cells.  Disadvantages: o Slightly more complex than a wet mount. o Requires special slides with a depression. II. Observing Stained Microorganisms Staining microorganisms for microscopic observation is a crucial technique in microbiology that enhances the visibility and differentiation of microbial cells and their components. Advantages:  Enhanced Visibility and Contrast: Many microorganisms are transparent and difficult to visualize under a microscope without staining. Staining makes these cells and their components more visible, enhancing contrast.  Differentiation Between Structures: Staining techniques, particularly differential stains like Gram staining or acid-fast staining, help in distinguishing between different types of microorganisms or cellular components (e.g., nucleus, cell wall, spores).   Ease of Identification: Stained preparations provide key visual clues (color and morphology) that aid in the identification of microorganisms. For example: o Gram-positive vs. Gram-negative bacteria o Acid-fast vs. non-acid-fast bacteria  Longer Observation Time: Stained microorganisms are fixed onto the slide, meaning they do not move, allowing for longer, more detailed observations of morphology without being affected by the organism’s motility.  Permanent Records: Stained slides can be preserved for extended periods, allowing them to be studied later, compared, or shared with others. This is essential for teaching and reference purposes.  Structural Detail: Some structures, like bacterial endospores or capsules, are not easily visible in living cells. Stains that bind to these structures (e.g., endospore stains, capsule stains) allow for more detailed observations. Stages of Staining Microorganisms for Microscopic Observation Staining microorganisms involves a series of steps designed to enhance the visibility and differentiation of microbial cells and their structures under a microscope. These stages can vary depending on the staining technique used (e.g., simple, differential, or special stains), but the general process involves four main stages: 1. Preparation of the Microbial Smear The first step in staining is creating a microbial smear, which involves transferring microorganisms onto a microscope slide and preparing them for staining. Steps:  Collection of Sample: A small sample of the microorganism is obtained from a liquid culture, a solid medium, or a natural sample (soil, water, etc.).  Application to Slide: The sample is placed on a clean glass slide. If working with a liquid culture, a drop is placed directly on the slide. If working with solid media, a small amount of the colony is mixed with a drop of water on the slide to create a suspension.  Spreading the Smear: Using a loop or another sterile tool, the microorganism is spread thinly across the slide to ensure even distribution.  Air Drying: The smear is allowed to air dry completely. This removes any excess water and ensures that the cells adhere well to the slide in the next step. Importance: A well-prepared smear ensures even staining and prevents clumping of cells, which can hinder observation. 2. Heat Fixation Once the smear is dry, it needs to be heat-fixed to the slide to prevent the microorganisms from washing off during staining. Steps:  The slide is passed quickly through a flame (usually 2-3 times) with the smear side facing up. Alternatively, it can be placed on a hot plate.  This process kills the microorganisms, making them safe to handle.  Heat fixation causes the proteins in the microbial cells to coagulate, helping the cells adhere to the slide. Importance: Heat fixation ensures that the microorganisms are immobilized on the slide and retain their morphology during the staining process. 3. Staining Application The main stage of the staining process involves applying a dye or series of dyes to the fixed smear. The choice of stain depends on the type of staining technique being used (simple, differential, or special). Steps:  Simple Staining: A single stain is applied (e.g., methylene blue or crystal violet). The slide is covered with the stain for a specific time (usually 30 seconds to 1 minute) and then rinsed with water.  Differential Staining (e.g., Gram staining, acid-fast staining): Involves the sequential application of multiple stains to distinguish different types of microorganisms. 1. Primary Stain: The first dye is applied (e.g., crystal violet in Gram staining). 2. Mordant: A mordant, such as iodine in Gram staining, is added to enhance the binding of the primary stain to the cell. 3. Decolorization: The smear is treated with a decolorizing agent (e.g., alcohol or acetone). Depending on the cell type, some cells will retain the primary stain, while others will lose it. 4. Counterstain: A second dye (counterstain) is applied to provide contrast (e.g., safranin in Gram staining or methylene blue in acid-fast staining).  Special Staining: Special staining procedures for bacteria are used to highlight specific structures or features that are not visible with simple staining methods. 1. Capsule Staining; Visualizes bacterial capsules, which are difficult to stain. 2. Flagella Staining:: Stains bacterial flagella, which are too thin to be seen otherwise. Importance: Staining enhances the visibility of microorganisms, differentiates between cell types, and highlights important cellular structures.  4. Washing, Drying, and Observation After applying the stains, excess stain is washed off, the slide is dried, and it is ready for observation under the microscope. Steps:  Washing: The slide is gently rinsed with water to remove excess stain. Care is taken not to wash too forcefully, which could remove the cells from the slide.  Blotting Dry: The slide is blotted dry with absorbent paper (e.g., bibulous paper) to remove excess water. This step should be done gently to avoid disturbing the smear.  Observation: The stained slide is now ready for microscopic examination. Immersion oil may be used with the 100x objective lens to improve resolution and clarity during observation. Importance: Proper washing and drying prevent over staining or under staining, which can obscure the details of the microorganisms under the microscope. Summary of Staining Stages Stage Key Steps Purpose Ensures even distribution of 1. Preparation of - Transfer sample to slide. microorganisms and allows proper Smear - Spread thinly and air dry. interaction with stains. - Pass slide through flame or Immobilizes and kills cells, making 2. Heat Fixation place on a hot plate to fix cells to them safe to handle and ready for the slide. staining. - Apply stain(s), mordant, Enhances contrast, visualizes structures, 3. Staining decolorizer, and counterstain (for and differentiates between cell types Application differential staining). (e.g., Gram +/−). - Rinse slide to remove excess 4. Washing, Drying, stain. Ensures clear visualization of stained and Observation - Blot dry and observe under a microorganisms. microscope. Types of Stains: Stains can be classified based on the charge of the chromophore (the colored part of the dye molecule) into three main types: 1. Basic (Cationic) Stains Chromophore Charge: Positive (+) Mechanism: Bacterial cell surfaces generally have a net negative charge. Therefore, basic stains, which carry a positively charged chromophore, are attracted to and bind to the negatively charged bacterial cell, effectively coloring the cell itself. Examples: Crystal violet (used in Gram staining) Methylene blue Safranin (used as a counterstain in Gram staining) Malachite green (used in endospore staining) Usage: Basic stains are most commonly used in simple staining, Gram staining, and other differential staining methods where the goal is to color the bacterial cells to make them visible. 2. Acidic (Anionic) Stains Chromophore Charge: Negative (–) Mechanism: Since bacterial cell surfaces are negatively charged, acidic stains are repelled by the cell. As a result, these stains color the background instead of the cells. This technique is used for negative staining, where the background is stained and the bacteria appear as clear, unstained silhouettes. Examples: Nigrosin India ink Eosin Congo red Usage: Acidic stains are used primarily for negative staining, which helps in visualizing delicate structures like bacterial capsules and overall cell morphology without subjecting the cells to heat fixation. 3. Neutral Stains Chromophore Charge: Both positive (+) and negative (–) Mechanism: These are composed of both acidic and basic stains, which result in neutral compounds. They can stain both bacterial cells and the background, depending on the dye composition and the charge distribution. Examples: Neutral red (used in metabolic staining) Usage: Neutral stains are less commonly used for bacterial staining but can be useful in special circumstances to stain specific components of cells or tissues. References 1.Pelczar, M.J., Chan, E.C.S., & Krieg, N.R. (2008). Microbiology. McGraw Hill. 2.Cappuccino, J.G., & Sherman, N. (2017). Microbiology: A Laboratory Manual. Pearson Education.