Capsule and Endospore Staining, Microbiology PDF

# Capsule Stain - Glycocalyx: A layer consisting of non-ionic polysaccharides that is found on the outer surface of the cell wall in some bacteria species. - Not all bacterial species have capsules. - All bacterial species have a cell membrane. - In some bacterial species, the capsule layer is...

# Capsule Stain - Glycocalyx: A layer consisting of non-ionic polysaccharides that is found on the outer surface of the cell wall in some bacteria species. - Not all bacterial species have capsules. - All bacterial species have a cell membrane. - In some bacterial species, the capsule layer is made of amino acids. - In some bacterial species, the capsule layer is made of D-glutamine amino acids. - Bacillus anthracis is an example of this. - In other bacterial species, the capsule is made of a mixture of polysaccharides. - Capsule: It is made of thick and dense layer of polysaccharides that is firmly attached to the cell wall. - Slime layer: It is a layer of polysaccharides that forms an unorganized layer and is loosely attached to the cell wall. - Bacteria that have a capsule are characterized by their colonies being sticky (mucoid) or mucoid-like (smooth colonies). - Examples: - *Klebsiella pneumoniae* - *Streptococcus pneumoniae* - *Bacillus anthracis* - Bacteria that lack a capsule are characterized by their colonies being rough. ## Importance of the Capsule The capsule is considered a major virulence factor. - It mediates attachment to host tissues. - It interferes with phagocytosis (anti-phagocytic). - Some bacterial species have capsules that are made of polysaccharides that are similar in structure to those found in host tissue. This leads to an inability for the host immune system to recognize the bacteria. - Bacteria use the capsule to overcome a lack of nutrients (starvation). - Bacteria use the capsule as a source of water when dehydrated. - Because the capsule is highly hydrated. ## Staining the Capsule - The capsule is a non-ionic polysaccharide. This means it cannot be directly stained with acidic or basic stains. - **Maneval's Capsule staining method (indirectly):** This method involves the use of acidic and basic stains. - **Acidic stain**: Congo Red - **Basic stain**: Acidfuchsin ## How the Staining Process Works 1. **Acidic stain (Congo Red):** - The bacterial cell will not stain because it has a negative charge. - The capsule will not stain because it is neutral. - The background will stain because it has a positive charge. 2. **Basic stain (Acidfuchsin):** - The bacterial cell will stain because it has a negative charge. - The capsule will not stain because it is neutral. - The background will not stain because it has a positive charge. - When using the basic stain, the bacterial cell will be stained and the capsule will appear as **halo spaces** around the bacterial cells. ### Klebsiella pneumoniae - *Klebsiella pneumoniae* can be used to demonstrate the capsule staining method. ## Using Cheat Fixation - Cheat fixation is a method that is not used for capsule staining. This is because it is not suitable for capsule staining. - Cheat fixation will destroy the capsules. - Cheat fixation will dehydrate the bacterial cells. - Cheat fixation shrinks the capsule and does not allow for acidic stain to be absorbed properly. - Cheat fixation can result in bubbles forming in the capsule. ## Endospore Staining - Fungi produce spores for reproduction. - Bacteria produce spores for protection and survival under extreme environmental conditions. - Lack of nutrients - Inappropriate temperature - Inappropriate pH - The process of endospore formation (sporulation) takes time. 6-8 hours. - Endospores are formed inside the mother cell. - When the endospore is finished forming, the mother cell breaks down and releases the endospore into the environment. - Bacterial endospores are defined as metabolically inactive (dormant) cells. - Endospores are resistant to harsh chemical conditions like desiccation, radiation, and extreme temperatures. - Endospores can remain viable for thousands of years. - When environmental changes make the environment suitable for bacterial growth, the endospore will germinate, becoming active and vegetative again. - Endospores can be sterilized using autoclaving. - Examples of bacteria that produce endospores are *Bacillus* and *Clostridium*. - Genus *Bacillus* examples: - *Bacillus anthracis* - *Bacillus cereus* - Genus *Clostridium* examples: - *Clostridium tetani* - *Clostridium botulinum* - *Clostridium difficile* - *Clostridium perfringens* - When endospores are detected in clinical specimens, this is a sign that there is an infection from the *Bacillus* or *Clostridium* bacteria. ## Endospore Location - The location of the endospore in a bacterial cell can be: - Central - Sub-terminal - Terminal ## Schaeffer-Fulton Staining Method - This method is used to stain bacterial endospores. - Bacterial endospores are highly resistant to regular staining procedures. - The stain used to stain endospores is non-ionic. - Examples: - *Malachite green* - It is heat-fixated (gently heated). - Because the stain used is non-ionic, it does not stain the cytoplasm of the bacterial cell. The cytoplasm has a negative charge and the stain has no charge. However, the endospore will stain green. - The bacterial cytoplasm is counter-stained with a basic stain, such as *Safranin* (Color Pink or Red). ### Performing a Schaeffer-Fulton Staining - Use a 18-36 hour old bacterial culture. - Heat the bacterial smear for 10-15 minutes (use a steamer). ## Enumeration of Bacteria - The growth of a microbe is defined as an increase in the number of individuals in its population. - Microbial growth depends on many chemical and physical factors. - Nutrient availability - Oxygen concentration - pH - Temperature - There are several techniques for estimating the number of bacterial cells in a given solution: - **Counting the bacterial cells:** - This can be done using a light microscope or an electronic particle counter. - **Measuring the mass of the cells:** - Measuring the dry weight of the cells of the microbial population. - Measuring the total nitrogen content of the cells. - **Measuring the turbidity of the microbial population:** - The greater the turbidity, the greater the number of microbes. - It is essential to use dilution before starting the process of bacterial enumeration. - This ensures that the sample is not too concentrated, which would prevent accurate counts. - **Preparation of a serial dilution**: - Take **0.1 ml** of the bacterial culture. - Add **0.9 ml** of a normal saline solution. - After mixing thoroughly, you will have diluted the original sample **10x** (1:10 dilution factor). - Continue this process until the culture has been diluted to the desired level. ## Determining Colony Forming Units (CFU) - **Agar plate method:** This is a common method for counting bacteria. - Spread the diluted bacterial culture on an agar plate. - Incubate the plates at 37°C for 24 hours. - Count the colonies. - The CFU (Colony Forming Unit) is the number of live bacterial cells in 0.1 ml. - To calculate the concentration of bacteria in the original sample, you need to consider the dilution factor. - Use the equation: CFU/ml = Number of colonies × *10* × Dilution factor ## Advantages of the Agar Plate Method - Easy to use. - Allows you to see individual colonies. - Can be used to screen and select different bacterial strains. ## Disadvantages of the Agar Plate Method - Overgrowth of colonies can occur (too many colonies). - Microaerophilic bacteria may be affected. - Some colonies may be hidden inside the agar. - Heat-labile organisms will die. # Antibiotics - **Antimicrobial agents** are chemical substances that kill or inhibit the growth of microorganisms without causing significant harm to the host. - Bacterial pathogens are remarkably capable of developing resistance to antibiotics. - It is important to test the sensitivity of bacteria to antibiotics before treatment. ## Methods Used for Antibiotics Susceptibility Testing - **Conventional methods:** - **Broth dilution:** - **Agar dilution:** - **Disk diffusion:** - **Commercial methods** - **Antibiotic strips** - **Computerized automated systems** - **Minimal Inhibitory Concentration (MIC):** This is the lowest concentration of an antibiotic that prevents the growth of the tested bacterium in vitro. - **Minimal Bactericidal Concentration (MBC):** This is the lowest concentration of an antibiotic that kills the tested bacterium in vitro. ## Disk Diffusion Method - The *Disk Diffusion Method* (Kirby-Bauer test) is a commonly used test in medical microbiology for determining the antibiotic susceptibility profile of bacterial pathogens. - 1. Prepare the bacterial culture - use a McFarland standard (0.5 for most bacteria). The standard is equivalent to 1.5 × 10⁸ CFU/mL. - 2. Inoculate Mueller-Hinton agar plates using a sterile cotton swab. - 3. Place antibiotic disks onto the agar plates. - Disks should be placed 2.5 cm apart. - 4. Incubate the plates at 35°C for 16-18 hours. - 5. Measure the zone of inhibition around each disk. - The zones of inhibition will appear as clear areas around the antibiotic disks. There is no bacterial growth in these areas. - Zones of inhibition can be used to determine whether the bacteria being tested is susceptible, sensitive or resistant to a particular antibiotic. - The results from the disk diffusion method are interpreted according to standards set by the Clinical and Laboratory Standards Institute (CLSI).