Microbiology/Bacteriology PDF

Microbiology/ Bacteriology Dr. Yasir W. Issa ❖ Microbiology Study of microscopic organisms, such as bacteria, viruses, fungi, protozoa, and algae. In Medicine: Disease prevention, treatment, and diagnostics. History of Microbiology...

Microbiology/ Bacteriology Dr. Yasir W. Issa ❖ Microbiology Study of microscopic organisms, such as bacteria, viruses, fungi, protozoa, and algae. In Medicine: Disease prevention, treatment, and diagnostics. History of Microbiology Key Historical Figures Antonie van Leeuwenhoek: First to observe microorganisms using a microscope. Louis Pasteur: Developed pasteurization and vaccines disproved spontaneous generation. Robert Koch: Established Koch’s postulates, linking specific microbes to diseases. Types of Microorganisms 1. Bacteria Prokaryotic cells, diverse metabolic capabilities. Usually has cell wall, plasma membrane, cytoplasm, nucleoid, ribosomes. 2. Viruses Non-cellular entities, require a host for replication. The viruses have capsid, genetic material (DNA or RNA), sometimes an envelope. 3. Fungi Eukaryotic, include yeasts and molds. 4. Protozoa Single-celled eukaryotes, often motile. 5. Algae Photosynthetic eukaryotes, aquatic environments. 1|P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa 6. Parasites They are Single-celled organisms: include Plasmodium (causes malaria), Giardia (causes giardiasis), and Entamoeba histolytica (causes amoebiasis). Or Helminths: Worm-like parasites. (multi- cellular parasites). A. Bacteria Bacteria are classified based on various characteristics including their shape, cell wall structure, metabolism, and genetic makeup. Here's an overview of the major classification criteria: 1. Shape: Cocci: Spherical bacteria (e.g., Streptococcus, Staphylococcus). Bacilli: Rod-shaped bacteria (e.g., Escherichia coli, Bacillus). Spirilla: Spiral or corkscrew-shaped bacteria (e.g., Spirillum). Vibrios: Comma-shaped bacteria (e.g., Vibrio cholerae). Spirochetes: Flexible, spiral-shaped bacteria (e.g., Treponema pallidum). 2|P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa 2. Gram Staining: Gram staining is a method to classify bacteria based on the composition of their cell walls. Gram-Positive: Bacteria with thick peptidoglycan cell walls that retain the crystal violet stain (e.g., Staphylococcus aureus, Streptococcus pneumoniae). Gram-Negative: Bacteria with thin peptidoglycan cell walls and an outer membrane, which do not retain the crystal violet stain but take up the counterstain (safranin) (e.g., Escherichia coli, Neisseria gonorrhoeae). Protocol for gram staining Material required (Bacterial culture, Glass microscope slides, Inoculating loop, burner, Crystal violet stain (primary stain), Iodine solution (mordant), 95% ethanol or acetone-alcohol (decolorizer), Safranin or fuchsine (counterstain), Distilled water, Staining rack, Bibulous paper or paper towels) Microscope 1. Preparation of the Smear: Place a small drop of water on a clean glass slide using a sterile loop. Aseptically transfer a small amount of bacterial culture to the drop of water, spreading it thinly to create a smear. Allow the smear to air dry completely. Once dry, pass the slide through a flame (smear-side up) several times to heat-fix the bacteria to the slide. Heat-fixing kills the bacteria and makes them adhere to the slide. 2. Application of Crystal Violet (Primary Stain): Cover the fixed smear with crystal violet stain. Let it sit for about 1 minute. 3|P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa Rinse the slide gently with distilled water to remove excess stain. 3. Application of Iodine Solution (Mordant): Cover the smear with iodine solution. Let it sit for about 1 minute. Iodine forms a complex with crystal violet, which helps retain the dye within the cell. Rinse the slide gently with distilled water. 4. Decolorization: Hold the slide at an angle and apply 95% ethanol or acetone-alcohol drop by drop until the runoff is clear (usually 10-20 seconds). This step is critical as over-decolorizing can remove the stain from gram-positive cells, while under-decolorizing may not sufficiently remove the stain from gram- negative cells. Rinse immediately with distilled water to stop the decolorization process. 5. Application of Safranin (Counterstain): Cover the smear with safranin stain. Let it sit for about 1 minute. Safranin stains the decolorized gram-negative bacteria. Rinse the slide gently with distilled water to remove excess stain. 6. Drying: Gently blot the slide dry with bibulous paper or allow it to air dry. 7. Examination: Place a drop of immersion oil on the stained smear. Examine the slide under a microscope using an oil immersion objective (usually 100x magnification). 4|P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa Results Interpretation: Gram-Positive Bacteria: Appear purple or blue due to the retention of the crystal violet-iodine complex in their thick peptidoglycan layer. Gram-Negative Bacteria: Appear pink or red due to the uptake of the counterstain (safranin) after the crystal violet is washed out of their thinner peptidoglycan layer and outer membrane. 5|P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa Feature Gram-Positive Bacteria Gram-Negative Bacteria Cell Wall Structure Thick peptidoglycan layer Thin peptidoglycan layer Outer Membrane Absent Present Teichoic Acids Present in cell wall Absent Lipopolysaccharides Present in outer membrane Absent (LPS) Gram Stain Color Purple or blue (retains crystal violet) Pink or red (retains safranin) Decolorization Resistant to decolorization Decolorized easily by ethanol Periplasmic Space Absent or very small Present Sensitivity to Penicillin Generally, more sensitive Generally less sensitive Porins Absent Present in outer membrane Escherichia coli, Salmonella, Examples Staphylococcus, Streptococcus, Bacillus Neisseria Sometimes present, when present usually Sometimes present, often Flagella several flagella one or few Some species can form endospores (e.g., Generally do not form spores Spore Formation Bacillus, Clostridium) Primarily endotoxins (part of Toxins Produced Primarily exotoxins LPS) 6|P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa 3. Oxygen Requirement: Aerobic: Require oxygen for growth (e.g., Mycobacterium tuberculosis). Anaerobic: Grow in the absence of oxygen (e.g., Clostridium species). Facultative Anaerobes: Can grow with or without oxygen (e.g., Escherichia coli). Microaerophilic: Require reduced oxygen levels (e.g., Helicobacter pylori). 4. Metabolic Characteristics: Phototrophs: Obtain energy from light (e.g., Cyanobacteria). Chemotrophs: Obtain energy from chemical compounds. Lithotrophs: Use inorganic compounds as energy sources. Organotrophs: Use organic compounds as energy sources. 5. Genetic Characteristics: DNA Sequencing: Modern classification increasingly relies on genetic sequencing to determine phylogenetic relationships. 16S rRNA Gene Sequencing: Commonly used genetic marker for identifying and classifying bacteria. 6. Other Structural Characteristics: Flagella: Presence, number, and arrangement (e.g., monotrichous, lophotrichous, peritrichous). Capsule: Presence of a polysaccharide capsule around the cell wall (e.g., Streptococcus pneumoniae). Endospores: Ability to form resistant spores (e.g., Bacillus, Clostridium). Common Bacterial Phyla: Proteobacteria: A diverse group that includes many gram-negative bacteria (e.g., Escherichia, Salmonella). 7|P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa Firmicutes: Mainly gram-positive bacteria (e.g., Bacillus, Clostridium). Actinobacteria: Gram-positive bacteria (e.g., Mycobacterium, Streptomyces). Bacteroidetes: Gram-negative, non-spore-forming bacteria (e.g., Bacteroides). Cyanobacteria: Photosynthetic, oxygen-producing bacteria (e.g., Anabaena). The Structure of Bacteria 8|P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa Bacterial Isolation and Culture: 1. Sample Collection: Obtain a sample from the environment, clinical specimens, food, water, or other sources using sterile techniques. 2. Inoculation: Transfer the sample to a suitable growth medium. This can be done using a sterile loop, swab, or pipette. Common types of growth media include nutrient agar, blood agar, MacConkey agar, and selective or differential media designed for specific bacterial groups. Culture Media 1. Nutrient Agar: Composition: 5% Peptone: Provides nitrogen 3% Beef Extract: Supplies vitamins, carbohydrates, nitrogen, and salts. 1.5% Agar: A solidifying agent derived from seaweed. 0.5% Water: Solvent for the nutrients. Purpose: 9|P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa General-purpose medium used to grow a wide variety of microorganisms. Use: Often used for routine cultivation of bacteria. 2. Blood Agar: Composition: Base Agar: Typically, nutrient agar or tryptic soy agar. Sheep Blood (5-10%): Provides growth factors and allows for hemolysis detection. Purpose: Enriched medium that supports the growth of fastidious organisms requiring additional growth factors. Differential medium for detecting hemolytic activity. Types of Hemolysis: Alpha Hemolysis: Partial hemolysis producing a greenish discoloration around colonies (e.g., Streptococcus pneumoniae). Beta Hemolysis: Complete hemolysis creating a clear zone around colonies (e.g., Streptococcus pyogenes). Gamma Hemolysis: No hemolysis or discoloration (e.g., Enterococcus faecalis). 10 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa 3. MacConkey Agar: Composition: Peptone: Nutrient source. Lactose: Differentiates lactose fermenters. Bile Salts and Crystal Violet: Inhibit gram-positive bacteria. Neutral Red: pH indicator. Agar: Solidifying agent. Purpose: Selective and differential medium for isolating and differentiating Enterobacteriaceae and other gram-negative rods. 11 | P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa Use: Selective: Bile salts and crystal violet inhibit the growth of gram-positive bacteria. Differential: Lactose fermentation produces acid, turning colonies pink/red due to neutral red (e.g., Escherichia coli). Non-lactose fermenters produce colorless colonies (e.g., Salmonella). Bacterial growth on MacConkey Agar 12 | P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa 4. Chocolate Agar: 1. Composition: Base Agar: Similar to nutrient agar or tryptic soy agar. Hemoglobin or Blood: Heated to 80°C to lyse the red blood cells, releasing intracellular nutrients. Additional Supplements: May include factors like NAD (nicotinamide adenine dinucleotide, also known as V factor) and hemin (X factor) to support the growth of specific bacteria. 2. Purpose: Enriched medium used for the cultivation of fastidious organisms that require specific growth factors. Supports the growth of bacteria that cannot grow on standard blood agar due to their need for lysed blood cells. 3. Uses: Haemophilus Species: These bacteria require both X factor (hemin) and V factor (NAD) for growth. Haemophilus influenzae is a common pathogen isolated on chocolate agar. Neisseria Species: Neisseria gonorrhoeae and Neisseria meningitidis also require NAD and additional nutrients found in chocolate agar. Other: Certain strains of Streptococcus pneumoniae, and other respiratory pathogens. 13 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa 5. Selective Media: Composition: Contains inhibitors that suppress the growth of unwanted organisms while allowing the growth of desired organisms. Examples: Mannitol Salt Agar (MSA): High Salt Concentration (7.5% NaCl): Selective for Staphylococcus species. Mannitol: Differential ingredient for mannitol fermentation. Phenol Red: pH indicator that turns yellow in the presence of acid from mannitol fermentation (e.g., Staphylococcus aureus). 14 | P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa MSA Agar Eosin Methylene Blue (EMB) Agar: Eosin Y and Methylene Blue: Inhibit gram-positive bacteria and react with lactose fermenters. Lactose: Differentiates between lactose fermenters (e.g., E. coli produces a metallic green sheen) and non-fermenters (colorless or pale colonies). 15 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa (EMB) Agar 6. Differential Media: Composition: Contains specific substrates and indicators to differentiate organisms based on metabolic properties. Examples: Xylose Lysine Deoxycholate (XLD) Agar: Xylose: Fermentable sugar. 16 | P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa Lysine: Allows detection of lysine decarboxylation. Sodium Thiosulfate and Ferric Ammonium Citrate: Detect hydrogen sulfide production. Phenol Red: pH indicator. Use: Differentiates Salmonella (red colonies with black centers due to H2S production) from Shigella (red colonies without black centers). 17 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa 7. Selective Enrichment: Alkaline Peptone Water (APW): Composition: Peptone (10 g/L), Sodium Chloride (10 g/L), pH adjusted to 8.5. Purpose: Enhances the growth of Vibrio species by providing an alkaline environment. Procedure: Inoculate the sample into APW and incubate at 35-37°C for 6-8 hours to enrich Vibrio species. Selective Plating: Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar: Composition: Contains thiosulfate, citrate, bile salts, sucrose, and bromothymol blue. Purpose: Selective and differential medium for Vibrio species. Procedure: Streak the enriched sample from APW onto TCBS agar and incubate at 35-37°C for 18- 24 hours. Colony Appearance: V. cholerae: Yellow colonies due to sucrose fermentation. Non-sucrose fermenting Vibrio species: Green colonies. 18 | P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa 3. Streak Plate Method (Isolation): Streaking is a technique used to isolate individual bacterial colonies from a mixed sample. Use a sterile loop to spread a small amount of the sample over the surface of an agar plate in a series of streaks. Incubate the plate under appropriate conditions (temperature, oxygen levels) to allow bacteria to grow. 4. Incubation: 19 | P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa Incubate the inoculated media at the appropriate temperature, typically 37°C for human pathogens, though other bacteria may require different temperatures. Incubation time varies depending on the bacterial species, usually ranging from 24 to 48 hours. 5. Isolation of Pure Cultures: After incubation, examine the agar plate for isolated colonies. Each colony arises from a single bacterial cell or a group of identical cells and represents a pure culture. Select well-isolated colonies with distinct morphologies for further analysis. 6. Subculturing: Transfer a single colony to a new sterile agar plate or broth to obtain a pure culture. Use aseptic techniques to prevent contamination. 7. Factors effects the growth of Bacteria: Physical Factors: 1. Temperature: 2. pH: 3. Oxygen Availability: 4. Moisture: 5. Osmotic Pressure: 20 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa 6. Light: Some bacteria are sensitive to UV light, which can cause DNA damage and inhibit growth. Chemical Factors: 1. Nutrients: Carbon: Essential for cellular structures and energy. Nitrogen: Needed for proteins, nucleic acids, and other cellular components. Sulfur and Phosphorus: Essential for amino acids, vitamins, and nucleic acids. Trace Elements: Required in small amounts for enzyme function (e.g., iron, magnesium, zinc). Vitamins and Growth Factors: Some bacteria require additional growth factors like vitamins or amino acids. Biological Factors: 1. Competition: Bacteria compete with other microorganisms for nutrients and space. Competitive exclusion can limit bacterial growth. 2. Symbiosis: Some bacteria benefit from symbiotic relationships with other organisms (e.g., nitrogen-fixing bacteria with plants). 3. Antibiotics and Antimicrobials: The presence of antibiotics or antimicrobial agents can inhibit bacterial growth or kill bacteria. 21 | P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa 8. Identification and Characterization: Perform various tests to identify the isolated bacteria. These may include: Gram Staining: To determine if bacteria are gram-positive or gram-negative. Biochemical Tests: Such as catalase, oxidase, and fermentation tests. Molecular Methods: Such as PCR, sequencing, and MALDI-TOF mass spectrometry. Antibiotic Sensitivity Testing: To determine susceptibility to antibiotics. 22 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa Bacterial infection 1. Entry into the Host: Bacteria can enter the host through various routes: Respiratory Tract: Inhalation of airborne bacteria (e.g., Mycobacterium tuberculosis, Streptococcus pneumoniae). Gastrointestinal Tract: Ingestion of contaminated food or water (e.g., Salmonella, Vibrio cholerae). Skin and Mucous Membranes: Through cuts, abrasions, or direct contact (e.g., Staphylococcus aureus, Treponema pallidum). Urogenital Tract: Through sexual contact (e.g., Neisseria gonorrhoeae, Chlamydia trachomatis). Vector-Borne: Through insect bites (e.g., Borrelia burgdorferi from ticks, Yersinia pestis from fleas). 2. Adherence to Host Cells: Bacteria must adhere to host cells to avoid being washed away by bodily fluids. Adhesins: Surface molecules on bacteria that bind to specific receptors on host cells (e.g., fimbriae/pili in Escherichia coli). Biofilms: Communities of bacteria that adhere to surfaces and are embedded in a self-produced extracellular matrix (e.g., Pseudomonas aeruginosa in cystic fibrosis patients). 23 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa 3. Invasion and Colonization: Some bacteria can invade host tissues and cells to establish infection. Enzymes: Bacteria produce enzymes like hyaluronidase and collagenase that degrade host tissues and facilitate invasion. Intracellular Pathogens: Certain bacteria can enter and survive within host cells (e.g., Listeria monocytogenes, Salmonella). 4. Evasion of Host Immune System: To establish infection, bacteria must evade or resist the host’s immune defenses. 24 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa Capsules: Polysaccharide capsules prevent phagocytosis by immune cells (e.g., Streptococcus pneumoniae). Antigenic Variation: Changing surface antigens to avoid immune detection (e.g., Neisseria gonorrhoeae). Inhibition of Phagolysosome Fusion: Preventing the fusion of phagosomes with lysosomes in phagocytic cells (e.g., Mycobacterium tuberculosis). Secretion of Toxins: Producing toxins that kill immune cells or disrupt immune functions (e.g., Staphylococcus aureus produces leukocidins). 5. Toxin Production: Many bacteria produce toxins that damage host tissues and contribute to disease symptoms. Exotoxins: Proteins secreted by bacteria that can cause damage to specific tissues (e.g., botulinum toxin from Clostridium botulinum, diphtheria toxin from Corynebacterium diphtheriae). Endotoxins: Components of the outer membrane of gram-negative bacteria (lipopolysaccharides, LPS) that can trigger strong immune responses (e.g., septic shock caused by Escherichia coli). 6. Nutrient Acquisition: Bacteria need nutrients from the host to grow and multiply. Siderophores: Molecules secreted by bacteria to scavenge iron from the host (e.g., enterobactin in Escherichia coli). Enzymatic Degradation: Breaking down host tissues to release nutrients (e.g., proteases and lipases). 25 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa 7. Spread within the Host: Bacteria may spread to different parts of the body to establish systemic infections. Bloodstream: Bacteremia can lead to sepsis if bacteria spread through the bloodstream (e.g., Staphylococcus aureus, Streptococcus pyogenes). Lymphatic System: Bacteria can spread via the lymphatic system to other tissues and organs. 8. Inducing Host Damage: Bacterial infections can cause direct damage through toxin production and enzyme activity, as well as indirect damage by triggering excessive immune responses. Inflammation: Bacterial components can trigger inflammation, leading to tissue damage (e.g., pneumolysin from Streptococcus pneumoniae). Immune-Mediated Damage: Some bacterial infections cause immune responses that damage host tissues (e.g., post-streptococcal glomerulonephritis). 26 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa Pathogenic Bacteria Gram Stain Characteristics Virulence Factors Human Organ System Staphylococcus Protein A, coagulase, hemolysins, Skin, respiratory system, Gram-positive Cocci (clusters) aureus toxins (e.g., TSST-1) bloodstream Streptococcus M protein, streptolysins, Gram-positive Cocci (chains) Throat, skin, bloodstream pyogenes hyaluronidase, exotoxins Pili, toxins (e.g., Shiga toxin, Gastrointestinal tract, Escherichia coli Gram-negative Rod enterotoxins), capsule urinary tract Type III secretion system, Gastrointestinal tract, Salmonella spp. Gram-negative Rod endotoxin, invasion proteins bloodstream Comma-shaped Cholera toxin (CTX), TCP pili, Vibrio cholerae Gram-negative Gastrointestinal tract rod hemagglutinin protease Urease, flagella, adhesins, Spiral-shaped Helicobacter pylori Gram-negative cytotoxin-associated gene A Stomach rod (CagA) Neisseria Capsule, pili, endotoxin (LOS), Nervous system Gram-negative Diplococci meningitidis IgA protease (meninges), bloodstream Neisseria Gram-negative Diplococci Pili, Opa proteins, IgA protease Urogenital tract gonorrhoeae Mycobacterium Acid-fast (Ziehl- Mycolic acid, cord factor, Respiratory system Rod tuberculosis Neelsen stain) intracellular survival (lungs) Spore-forming Clostridium difficile Gram-positive Toxins A and B, spores Gastrointestinal tract rod Clostridium Spore-forming Nervous system Gram-positive Botulinum toxin botulinum rod (neuromuscular junction) Spore-forming Nervous system Clostridium tetani Gram-positive Tetanus toxin (tetanospasmin) rod (peripheral nerves) 27 | P a g e  Microbiology/ Bacteriology Dr. Yasir W. Issa Outer surface proteins (Osps), Skin, joints, nervous Borrelia burgdorferi Gram-negative Spirochete motility system Treponema Urogenital tract, skin, Gram-negative Spirochete Outer membrane proteins pallidum nervous system Legionella Type IV secretion system, Respiratory system Gram-negative Rod pneumophila intracellular survival (lungs) The biochemical characteristics of various pathogenic bacteria: Pathogenic Bacteria Gram Stain Example Tests and Results Staphylococcus aureus Gram-positive Catalase (+), Coagulase (+), Mannitol Salt Agar: yellow colonies Streptococcus pyogenes Gram-positive Catalase (-), Beta-hemolysis on Blood Agar Escherichia coli Gram-negative MacConkey Agar: pink colonies, Indole (+), MR (+), VP (-) Salmonella spp. Gram-negative MacConkey Agar: colorless colonies, H2S (+) on TSI, Urease (-) Vibrio cholerae Gram-negative TCBS Agar: yellow colonies, Oxidase (+), Indole (+) Helicobacter pylori Gram-negative Urease (+), Catalase (+), Oxidase (+) Neisseria meningitidis Gram-negative Oxidase (+), CTA Glucose (+), CTA Maltose (+) Neisseria gonorrhoeae Gram-negative Oxidase (+), CTA Glucose (+) Mycobacterium tuberculosis Acid-fast Acid-fast stain (+), Niacin (+), Nitrate reduction (+) Clostridium difficile Gram-positive Cytotoxin assay for Toxin A/B, PCR for toxin genes Clostridium botulinum Gram-positive Mouse bioassay for toxin, PCR for toxin genes 28 | P a g e Microbiology/ Bacteriology Dr. Yasir W. Issa Clostridium tetani Gram-positive Mouse bioassay for toxin, PCR for toxin genes Borrelia burgdorferi Gram-negative Serology (ELISA, Western Blot), PCR Treponema pallidum Gram-negative Dark-field microscopy, Serology (RPR, FTA-ABS) Legionella pneumophila Gram-negative Catalase (+), Oxidase (+), Growth on BCYE 29 | P a g e