HEMA + BACTE QUIZ AUGUST 29.pdf

BACTE LEC Shapes and Arrangements 1. Cocci (spherical): - Shape: Round, spherical bacteria. - Arrangements: - Chains (Streptococci): Formed when cocci divide in one plane and remain attached. - Clusters (Staphylococci): Form irregular clusters resembling grapes, formed by division in...

BACTE LEC Shapes and Arrangements 1. Cocci (spherical): - Shape: Round, spherical bacteria. - Arrangements: - Chains (Streptococci): Formed when cocci divide in one plane and remain attached. - Clusters (Staphylococci): Form irregular clusters resembling grapes, formed by division in multiple planes. - Pairs (Diplococci): Cocci arranged in pairs after division, common in certain pathogens (e.g., Neisseria). - Tetrads: Groups of four cocci arranged in a square. - Sarcinae: Cubic arrangement of eight cocci. 2. Bacilli (rod-shaped): - Shape: Cylindrical or rod-like. - Arrangements: - Single Bacilli: Occur singly. - Diplobacilli: Pairs of bacilli. - Streptobacilli: Chains of bacilli resulting from division in one plane. - Coccobacilli: Short, stubby rods that can appear similar to cocci. 3. Spirilla (spiral): - Shape: Spiral or helical. - Arrangements: - Single Spirilla: Typically appear as single spiral-shaped cells. - Spirochetes: Flexible, corkscrew-shaped cells with axial filaments allowing for twisting motion. - Vibrios: Comma-shaped, curved rods that are a subset of spiral bacteria. Structures 1. Cell Wall: - Gram-Positive: - Structure: Thick peptidoglycan layer (up to 90% of the cell wall), with teichoic and lipoteichoic acids. - Staining: Retains crystal violet stain, appearing purple in a Gram stain. - Function: Provides structural support and protection; less complex than Gram-negative walls. - Gram-Negative: - Structure: Thin peptidoglycan layer (about 10% of the cell wall) surrounded by an outer membrane containing lipopolysaccharides (LPS). - Staining: Does not retain crystal violet; appears pink/red after counterstaining with safranin. - Function: Outer membrane provides additional protection and contains endotoxins (LPS). 2. Capsule: - Composition: Polysaccharide layer surrounding some bacteria. - Function: Protects against phagocytosis, aids in adherence to surfaces, and contributes to pathogenicity by evading the immune system. - Significance: Visualized using capsule stains (e.g., India ink or negative staining techniques). 3. Flagella: - Structure: Long, whip-like appendages made of flagellin protein. - Function: Responsible for bacterial motility, enabling movement towards or away from stimuli (chemotaxis). - Types: - Monotrichous: Single flagellum at one end. - Lophotrichous: Cluster of flagella at one or both ends. - Amphitrichous: Single flagellum at both ends. - Peritrichous: Flagella distributed all over the bacterial surface. 4. Pili (Fimbriae):  - Structure: Short, hair-like appendages made of pilin protein. - Function: Primarily involved in attachment to surfaces (e.g., epithelial cells), aiding in colonization and biofilm formation. - Sex Pili: Specialized pili used during bacterial conjugation to transfer DNA between cells. 5. Endospores: - Structure: Highly resistant, dormant structures formed by some Gram-positive bacteria (e.g., Bacillus and Clostridium species). - Function: Ensure survival in harsh environmental conditions (e.g., extreme heat, desiccation, radiation). - Formation: Sporulation occurs when nutrients are scarce, leading to the formation of a spore that can germinate into a vegetative cell under favorable conditions. Bacterial Classification 1. Gram Staining: - Purpose: Differentiates bacteria based on cell wall composition. - Gram-Positive: - Characteristics: Have a thick peptidoglycan layer (up to 90% of the cell wall), retain crystal violet stain, appearing purple under the microscope. - Examples: Staphylococcus aureus, Streptococcus pneumoniae. - Gram-Negative: - Characteristics: Possess a thin peptidoglycan layer (about 10% of the cell wall) and an outer membrane containing lipopolysaccharides (LPS). Do not retain crystal violet but take up the counterstain (safranin), appearing pink or red. - Examples: Escherichia coli, Neisseria meningitidis. 2. Major Groups of Bacteria: - Proteobacteria: - Characteristics: A large and diverse group of Gram-negative bacteria. Includes many medically important genera. - Examples: Escherichia, Salmonella, Vibrio, Helicobacter. - Firmicutes:  - Characteristics: Primarily Gram-positive bacteria with a thick peptidoglycan layer. Some members form endospores. - Examples: Bacillus, Clostridium, Lactobacillus, Staphylococcus. - Actinobacteria: - Characteristics: Gram-positive bacteria with high guanine and cytosine content in their DNA. Many are soil-dwelling and known for producing antibiotics. - Examples: Mycobacterium, Streptomyces, Corynebacterium. - Bacteroidetes: - Characteristics: A phylum of Gram-negative, anaerobic, and non-sporulating bacteria. They are significant in the human gut microbiome. - Examples: Bacteroides, Prevotella. 3. Identification Methods: - Biochemical Tests: - Used to identify bacterial species based on metabolic and enzymatic activities. - Examples: Catalase test, oxidase test, carbohydrate fermentation, urea hydrolysis. - Molecular Methods: - Include techniques like Polymerase Chain Reaction (PCR), DNA sequencing, and ribotyping to detect genetic markers specific to bacterial species. - Used for more precise identification, especially for bacteria that are difficult to culture. - Culture Characteristics: - Observation of colony morphology, growth patterns, pigmentation, and hemolytic activity on various culture media (e.g., blood agar, MacConkey agar) helps in identifying bacteria. - Examples: Staphylococcus aureus shows beta-hemolysis on blood agar, while Escherichia coli ferments lactose on MacConkey agar, producing pink colonies. Bacterial History 1. Key Figures: - Louis Pasteur (1822–1895): - Contributions: Developed the germ theory of disease, which proposed that microorganisms are the cause of many diseases. Pasteur’s experiments disproved the theory of spontaneous generation and supported the concept that microorganisms originate from other microorganisms. - Milestones: Developed pasteurization to prevent spoilage of food and beverages, and created vaccines for rabies and anthrax. - Robert Koch (1843–1910): - Contributions: Known as the father of modern bacteriology, Koch identified the causative agents of tuberculosis (Mycobacterium tuberculosis), cholera (Vibrio cholerae), and anthrax (Bacillus anthracis). - Milestones: Developed Koch's postulates, a set of criteria used to link a specific pathogen to a specific disease. He also introduced staining techniques that greatly enhanced the visualization of bacteria under the microscope. - Joseph Lister (1827–1912): - Contributions: Pioneered antiseptic surgery by promoting the use of carbolic acid (phenol) to sterilize surgical instruments and clean wounds, significantly reducing post-operative infections. - Milestones: His work established the importance of aseptic techniques in medical procedures and revolutionized surgery, leading to modern sterile practices. 2. Milestones in Bacteriology: - Development of Germ Theory: - Shifted the understanding of disease causation from supernatural explanations and miasma theories to microbial causes. This theory was pivotal in developing effective public health measures and medical treatments. - Advancements in Staining Techniques: - The introduction of Gram staining by Hans Christian Gram in 1884 allowed for the differentiation of bacteria into Gram-positive and Gram-negative groups based on their cell wall structure. Other staining techniques, such as acid-fast staining and spore staining, were developed to visualize specific bacterial features, aiding in the identification of pathogens. - Discovery of Specific Pathogens:  - Following the establishment of germ theory, specific bacteria were identified as the causative agents of various diseases. Robert Koch's discoveries were instrumental in this, as well as other breakthroughs such as the identification of Neisseria gonorrhoeae by Albert Neisser and Yersinia pestis by Alexandre Yersin as the cause of the plague. Bacterial Physiology & Metabolism 1. Cell Structures: - Cell Wall: - Provides shape, protection, and structural integrity to bacteria. - In Gram-positive bacteria, the thick peptidoglycan layer offers resistance to osmotic pressure. - In Gram-negative bacteria, the cell wall includes an outer membrane with lipopolysaccharides, offering protection from environmental stress and antibiotics. - Cell Membrane: - Composed of a phospholipid bilayer, it regulates the entry and exit of substances. - It plays a critical role in energy production (especially in bacteria lacking mitochondria) by housing components of the electron transport chain. - Cytoplasm: - The internal matrix of the cell where metabolic reactions occur. - It contains enzymes, nutrients, and the genetic material (nucleoid region) necessary for bacterial growth and reproduction. - Ribosomes: - The site of protein synthesis, where translation of mRNA into proteins takes place. - Bacterial ribosomes are 70S in size, made up of 50S and 30S subunits, which are targeted by certain antibiotics (e.g., tetracyclines, aminoglycosides). 2. Metabolic Pathways: - Aerobic Respiration:  - Oxygen is used as the final electron acceptor in the electron transport chain (ETC), which occurs in the cell membrane. - The ETC generates a proton gradient across the membrane, driving ATP synthesis through oxidative phosphorylation. - It is the most energy-efficient metabolic pathway, producing up to 38 ATP molecules per glucose molecule. - Anaerobic Respiration: - Similar to aerobic respiration, but instead of oxygen, other molecules like nitrate, sulfate, or carbon dioxide serve as the final electron acceptors. - This pathway produces less ATP than aerobic respiration but is essential for bacteria that live in oxygen-deprived environments. - Fermentation: - Occurs when no external electron acceptors are available. - Organic molecules (e.g., pyruvate) act as electron acceptors, leading to the production of various end products such as lactic acid, ethanol, and carbon dioxide. - Fermentation generates only 2 ATP per glucose molecule, much less than respiration, but is crucial for survival in anaerobic conditions. 3. Energy Production: - Glycolysis: - The first step in energy production, where one glucose molecule is broken down into two pyruvate molecules. - This process yields a net gain of 2 ATP and 2 NADH molecules. - Glycolysis occurs in the cytoplasm and does not require oxygen, making it the primary pathway for both aerobic and anaerobic bacteria. - Krebs Cycle (Citric Acid Cycle): - Occurs in the cytoplasm of bacteria (since they lack mitochondria). - Pyruvate is further broken down into carbon dioxide, generating ATP, NADH, and FADH2.  - These high-energy electron carriers then feed into the electron transport chain. - The Krebs cycle provides key precursors for biosynthesis and generates additional energy. - ATP Generation: - The majority of ATP in bacteria is produced via oxidative phosphorylation during aerobic respiration or anaerobic respiration. - In fermentation, ATP is generated only through substrate-level phosphorylation during glycolysis. BACTE LAB Aseptic Techniques 1. Principles: - Prevention of Contamination: Ensures sterile environments and samples by avoiding the introduction of unwanted microorganisms. Critical in preventing infections in medical and research settings. - Use of Sterile Equipment: Involves sterilizing tools and materials before use to eliminate contaminants. Methods include autoclaving and chemical sterilants. - Proper Handwashing: Essential for minimizing the transfer of microorganisms. Includes thorough washing with soap and water or using alcohol-based hand sanitizers. - Handling Procedures: Involves careful handling of sterile items and maintaining sterility, especially in controlled environments like biosafety cabinets. 2. Techniques: - Sterilization: - Autoclaving: Utilizes pressurized steam (121°C for 15-20 minutes) to kill all microorganisms, including spores. It is the standard for sterilizing medical instruments and media. - Filtration: Removes microorganisms from heat-sensitive liquids and gases using filters with pore sizes typically 0.22 micrometers. - Other Methods: Includes dry heat, radiation (gamma rays, UV light), and chemical sterilants (ethylene oxide) for specific needs. - Disinfection:  - Alcohol: Ethanol or isopropyl alcohol (70%) is effective for disinfecting surfaces and skin by denaturing proteins and disrupting membranes, though it does not kill spores. - Bleach: Sodium hypochlorite (10% bleach) oxidizes cellular components and is effective against a wide range of pathogens, including spores. Used for surfaces and spills. - Other Disinfectants: Quaternary ammonium compounds, hydrogen peroxide, and phenolics are also used in various settings. Methods of Studying Bacteria 1. Streak Plate Method: - Purpose: Isolates pure bacterial colonies from mixed samples on solid media. - Procedure: Bacterial sample is spread on an agar plate in a streaking pattern to dilute the sample and achieve isolated colonies. - Significance: Isolated colonies can be used for further testing and identification. 2. Gram Staining: - Purpose: Differentiates bacteria based on cell wall composition. - Procedure: 1. Crystal violet (primary stain). 2. Iodine (mordant). 3. Decolorization with alcohol or acetone. 4. Safranin (counterstain). - Results: - Gram-Positive Bacteria: Appear purple due to a thick peptidoglycan layer. - Gram-Negative Bacteria: Appear pink/red after losing crystal violet and taking up safranin. - Significance: Provides initial classification and guides further testing. 3. Other Staining Techniques: - Acid-Fast Staining: - Purpose: Identifies bacteria with waxy cell walls (e.g., Mycobacterium species). - Procedure: Ziehl-Neelsen method with carbol fuchsin, acid-alcohol decolorization, and counterstaining.  - Results: Acid-fast bacteria retain red color; non-acid-fast bacteria appear blue/green. - Endospore Staining: - Purpose: Detects endospores in bacteria (e.g., Bacillus, Clostridium). - Procedure: Schaeffer-Fulton method with malachite green and safranin. - Results: Endospores appear green; vegetative cells appear red/pink. - Capsule Staining: - Purpose: Visualizes the protective capsule surrounding some bacteria. - Procedure: Negative staining (e.g., India ink) shows a clear halo around the bacterium. 4. Culture Methods: - Media Types: - Solid Media: Agar plates (e.g., blood agar, MacConkey agar) are used for isolating and differentiating bacterial colonies. - Liquid Media: Broths for growing large numbers or enriching specific bacteria. - Conditions: - Temperature: Bacteria grow optimally at temperatures specific to their type (mesophiles at 37°C, thermophiles at higher temperatures, psychrophiles at lower temperatures). - Oxygen Levels: - Aerobes: Require oxygen. - Anaerobes: Grow without oxygen and may be harmed by it. - Facultative Anaerobes: Grow with or without oxygen. - Microaerophiles: Require low oxygen levels. - Capnophiles: Need elevated carbon dioxide levels. - Significance: Culture methods enable the growth, isolation, and identification of bacteria based on their characteristics. HEMA LAB Hematocrit 1. Procedure:  - Definition: Hematocrit (PCV) measures the percentage of blood volume occupied by red blood cells (RBCs). - Method: - Centrifugation: Blood is collected in an EDTA tube, placed in a capillary tube, and centrifuged at 10,000 to 12,000 RPM (revolutions per minute) for 5 minutes. This separates the blood into plasma (top), buffy coat (middle), and packed RBCs (bottom). - Measurement: The hematocrit value is calculated as the percentage of the total volume occupied by RBCs. Automated analyzers may also calculate hematocrit from RBC count and mean cell volume (MCV). 2. Normal Values: - Men: 40-50% (0.40-0.50 in SI units) - Women: 36-44% (0.36-0.44 in SI units) - Values can vary with altitude, age, and specific population norms. 3. Clinical Significance: - Anemia: Low hematocrit indicates reduced RBCs, which can be due to iron deficiency, chronic diseases, or bone marrow disorders. It helps in diagnosing and classifying anemia. - Polycythemia: Elevated hematocrit suggests increased RBCs, potentially due to polycythemia vera or secondary causes like chronic hypoxia or dehydration. - Hydration Status: Hematocrit can be influenced by hydration levels; dehydration can elevate it, while overhydration can lower it. Hemoglobin 1. Measurement Techniques: - Spectrophotometry: - Principle: Measures hemoglobin concentration by quantifying the light absorbed at a specific wavelength, usually around 540 nm (nanometers). - Procedure: Blood is hemolyzed, and the absorbance of the hemolysate is measured. This method is quick and used in automated analyzers. - Cyanmethemoglobin Method: - Principle: The reference method where hemoglobin is converted to cyanmethemoglobin using Drabkin's reagent. Absorbance is measured at 540 nm. - Procedure: Blood is mixed with Drabkin’s reagent to form cyanmethemoglobin, which is then measured spectrophotometrically.  - Advantages: Highly accurate and measures all forms of hemoglobin. It is the gold standard for hemoglobin measurement. 2. Normal Values: - Men: 13.8-17.2 g/dL (grams per deciliter) or 138-172 g/L (grams per liter) - Women: 12.1-15.1 g/dL or 121-151 g/L - Reference ranges may vary with laboratory, altitude, and patient population. 3. Clinical Significance: - Oxygen-Carrying Capacity: Hemoglobin reflects the blood’s ability to carry oxygen. Low levels indicate anemia, while elevated levels may suggest conditions like polycythemia. - Anemia: Low hemoglobin levels point to anemia, which can be due to deficiencies, chronic diseases, or blood loss. - Hemoglobinopathies: Essential for diagnosing conditions like sickle cell disease and thalassemia, which involve abnormalities in hemoglobin. - Monitoring: Regular monitoring in chronic conditions or treatments affecting RBC production.