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Microbiology Review

• This review covers microbiology, focusing on microorganisms, prokaryotic and eukaryotic cells, bacterial cytology, Gram stains, and acid-fast stains.

  MICROORGANISMS

  • Microorganisms are categorized based on their cellular structure into two primary groups: acellular, which lack cells entirely, and cellular, which contain one or more cells. This classification is fundamental in microbiology as it helps in understanding the biological complexities and processes associated with different types of microorganisms.
  • Viruses are classified as acellular entities. They exist in a unique state, neither fully alive nor entirely dead, depending on a host cell to replicate and propagate. Unlike cellular organisms, they do not display characteristics such as metabolism or cellular division.
  • Prokaryotes encompass organisms like Eubacteria, Cyanobacteria, and Archaebacteria. These single-celled entities are fundamentally distinct as they possess genetic material but lack a defined, membrane-bound nucleus, which classifies them separately from eukaryotic microorganisms.
  • Eukaryotes include diverse organisms such as Protozoa, Fungi, and Algae. These organisms are characterized by their complex cellular organization, which includes membrane-bound organelles and, critically, a true nucleus that houses their genetic material, contributing to their overall complexity and functionality.

Prokaryotic vs. Eukaryotic Cells

• Prokaryotes:
  • Nucleoid is not bound by a membrane, consists of one chromosome (DNA) and histone-like proteins.
  • Cell division occurs via binary fission.
  • Cell wall composition varies, but some EubACTERIA contain peptidoglycan (except Mycoplasma and Ureaplasma), while Archaebacteria have cell wall resembling peptidoglycan.
  • Cytoplasmic membrane is a fluid phospholipid bilayer without CHO and sterols (except Mycoplasma and Ureaplasma, which have CHO and sterols).
  • Lacks cell organelles.
  • Site of energy production is the cytoplasmic membrane.
  • Site of protein synthesis is free ribosomes.
• Eukaryotes:
  • Nucleus is bound by a membrane; consists of one or more pairs of chromosomes (DNA) and histones.
  • Cell division occurs via mitosis.
  • Cell wall composition varies (animals - no cell wall, plants - cellulose wall, fungi - chitin wall).
  • Cytoplasmic membrane is a fluid phospholipid bilayer with CHO and sterols.
  • Contains cell organelles.
  • Site of energy production is mitochondria.
  • Site of protein synthesis is the rough endoplasmic reticulum (RER) and free ribosomes.

Bacterial Cytology

• Bacterial cell wall: The main component is Peptidoglycan, also known as murein, is a structural component of the bacterial cell wall, providing rigidity and strength to maintain the cell's shape. It is a polymer consisting of sugars and amino acids, forming a mesh-like structure that surrounds the cell membrane in most bacteria. This unique structure not only protects bacteria from environmental stresses and lysis due to osmotic pressure but also plays a critical role in the classification of bacteria into two major groups: Gram-positive and Gram-negative. In Gram-positive bacteria, the peptidoglycan layer is thick and retains the crystal violet dye during staining, giving these bacteria their characteristic purple color. In contrast, Gram-negative bacteria have a much thinner peptidoglycan layer, which is located between the inner cytoplasmic membrane and the outer membrane, leading to a pink color after Gram staining due to the counterstaining process. Understanding the composition and function of peptidoglycan is crucial for the development of antibiotics, as many target the biosynthesis of this essential component, effectively inhibiting bacterial growth., which protects internal structures and gives bacteria their shape.
  • Some components (e.g., M protein in Streptococcus pyogenes, mycolic acid in Mycobacterium tuberculosis) are virulence factors.

Gram Stain

• Gram stain differentiates bacteria based on cell wall properties.
• Gram-positive: Retains crystal violet, appears purple.
• Gram-negative: Loses crystal violet, appears pink/red after a counterstain.
  • The key difference lies in the thickness of the peptidoglycan layer.

Acid-Fast Stain

• A method used to identify acid-fast bacteria.
  • Mycobacterium is the only genus in this group, which is important since the cell wall contains mycolic acid that prevents decolorization with acid alcohol.
• Specific stains used are carbol fuchsin and methylene blue or malachite green.

Gram-positive vs. Gram-negative Bacilli

• Gram-positive (aerobes): This category includes several prominent genera such as Bacillus, known for its ability to produce endospores, which contribute to its resilience in adverse environmental conditions. Corynebacterium is often associated with diphtheria, highlighting its clinical significance. Erysipelothrix is notable for its role in conditions such as erysipeloid, while Lactobacillus plays a crucial role in fermentation processes important for food production, particularly dairy. Listeria is a pathogen of concern, particularly in immunocompromised individuals. The genera Mycobacterium and Nocardia are significant for their associations with chronic granulomatous diseases and respiratory infections.
• Gram-positive (anaerobes): This group includes genera such as Actinomyces, which is involved in human infections and is part of the normal oral flora. Clostridium, known for its pathogenic species like C. difficile, causes a range of infections including tetanus and botulism. Propionibacterium is typically found on skin and associated with acne. Meanwhile, Bifidobacterium is a beneficial bacterium important for gut health and is commonly used as a probiotic. Eggerthella is less well-known but has been identified in various clinical samples.
• Gram-negative (aerobes): The Enterobacteriaceae family is a large and diverse group that includes clinically significant examples such as Escherichia coli, which can cause gastrointestinal infections, and Citrobacter, known for its opportunistic infections. Genera such as Pseudomonas thrive in moist environments and are notorious for their resistance to antibiotics, frequently impacting immunocompromised patients. Acinetobacter is increasingly recognized for its role in hospital-acquired infections. Other notable genera include Burkholderia and Stenotrophomonas, both known for their opportunistic pathogenicity. The HACEK group comprises slow-growing bacteria commonly associated with endocarditis, highlighting their clinical relevance.
• Gram-negative (anaerobes): This group primarily consists of genera such as Bacteroides, which dominate the human gut microbiota and are essential for processing polysaccharides. Bilophila is increasingly recognized, especially in the context of inflammatory bowel disease. Porphyromonas is significant in periodontal disease, while Prevotella is associated with various infections, including those in the oral cavity. Lastly, Fusobacterium is known to play a role in both oral and systemic infections.

Enterobacteriaceae

• Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ewingella, Hafnia, Klebsiella, Morganella, Plesiomonas, Proteus, Providencia, Salmonella, Serratia, Shigella, Yersinia.

HACEK Organisms

• Bacteria associated with subacute bacterial endocarditis. Include: Haemophilus spp., Aggregatibacter, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae.

Bacterial Cell Wall

• Gram-positive bacteria have a thick peptidoglycan layer, which traps the crystal violet stain.
• Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane.

Gram-positive vs. Gram-negative Cocci

• Gram-positive (aerobes): Micrococcus, Staphylococcus, Streptococcus
• Gram-positive (anaerobes): Peptococcus, Peptostreptococcus, Sarcina
• Gram-negative (aerobes): Branhamella, Neisseria, Moraxella
• Gram-negative (anaerobes): Veilonella, Acidaminococcus, Megasphaera

Acid-Fast Stains

• Two main types: Ziehl-Neelsen and Kinyoun.
• Used to identify and stain bacteria that resist decolorization with acid-alcohol.
• Include procedures such as selecting the ideal size and removing adherent sputum and appropriate use of stains.

Mycobacterium

• The only genus considered acid-fast (which means cell walls retain carbol fuchsin even with acid-alcohol decolorization).
• Contains mycolic acid in its cell walls.
• Important example is Mycobacterium tuberculosis.

Mycobacterial Characteristics

• Cell walls contain mycolic acid, leading to acid-fast nature.

Bacterial Cytology (continued)

• Cytoplasmic membrane: Selectively permeable membrane involved in energy production.
• Mesosomes: Involves extension of cytoplasm membrane; site of attachment for chromosomes.
• Ribosomes: Involved in protein synthesis.
• Inclusions: Storage of nutrients—examples are much granules (contain lipids in MTB), Babes-Ernst/Metachromatic/Volutin granules (contain polyphosphates), and bipolar bodies (characteristic of Y.pestis).
• Endospores: Highly resistant resting cells formed by certain bacteria. Contain Calcium dipicolinate for increased resistance to adverse conditions. Common genera include Bacillus and Clostridium.

Bacterial Structural Components

• Capsule/slime layer: Capsules are organized layers; slime layers are unorganized; both increase a pathogen's virulence by helping it evade phagocytosis.
• Pili: Help bacteria adhere to host cells or for bacterial conjugation.
• Flagella: Provide motility; different types based on arrangement (monotrichous, lophotrichous, peritrichous).
• Axial filaments: Specialized flagella enabling movement in spirochetes.

Bacterial Virulence Factors

• Adherence factors: pili
• Antiphagocytic factors: capsule, some cell wall components,
• Enzymes: e.g., coagulase, fibrinolysin, hyaluronidase.
• Toxins: exotoxins and endotoxins

Exotoxins vs. Endotoxins

• Exotoxin:
• Source: both Gram (+) and Gram (-)
• Release: metabolic product released from a living cell.
• Composition: proteins or peptides
• Heat stability: labile
• Immunologic: can be converted to toxoids; can be easily neutralized with anti-toxin
• Endotoxin:
• Source: Gram (-) only
• Release: released upon cell lysis
• Composition: lipids (Lipid A)
• Heat stability: stable
• Immunologic: not easily converted to toxoids; not easily neutralized with antitoxins

Tests to Detect Endotoxins

• Limulus lysate test: detects endotoxin in body fluids, surgical instruments.
• Positive result: clumping

Bacterial Growth Factors

• Nutrients: Carbon, nitrogen, minerals. Examples of specialized requirements include: halophiles (salt tolerance), and those needing specific nutrients like protein, X and V factors, or cysteine/cystine for growth.
• Oxygen: Obligate aerobe (requires oxygen; postive for catalase and SOD), obligate anaerobe (requires absence of oxygen; negative for catalase and SOD), facultative anaerobe (growth in or without oxygen), aerotolerant anaerobe (growth in presence of oxygen), microaerophile (requires reduced amount of oxygen), capnophiles (require high CO2).
• Temperature: Psychrophiles/Cryophiles (below 10C), mesophiles (20-40C), thermophiles (above 50*C).
• pH : Acidophiles (below pH 3.0), alkalophiles (above pH 8).

Growth Cycle

• Lag phase: adjustment to new environment, little/no growth
• Log / Exponential phase: maximum growth rate
• Stationary / Plateau phase: growth ceases due to nutrient exhaustion or accumulation of toxic byproducts.
• Death / Decline phase: viable count decreases

Culture Media

• Types:
  • Liquid (broth): No solidifying agent
  • Semisolid (contains 0.5% agar): Red algae, Gelidium; often for motility or transport studies.
  • Solid (contains sufficient agar, typically 2-3%): Liquefiable or non-liquefiable (chopped meat, rice grain).
• Classification by composition:
  • Synthetic/Chemically-defined: Exact composition is known.
  • Complex/Non-synthetic: Contains at least one component not chemically defined (blood, serum, plant/animal/yeast extract, tissue).
• Classification by function/use:
  • Simple/Basal/Supportive/General Isolation: Supports various bacteria.
  • Enriched: Supports fastidious bacteria (blood, chocolate agar).
  • Enrichment: Enhances the growth of organisms in the cultures (selenite, tetrathionate broth, alkaline peptone water)
  • Selective: Selects the growth of particular organisms (inhibitors like Crystal violet, bile salt).
  • Differential: differentiates based on distinct colonial appearances.

Clinical Specimens

• Blood cultures: Antiseptic (alcohol-iodine), anticoagulant (SPS); Ratio of blood to culture media varies by age.
• Throat/Nasopharyngeal cultures: Include anaerobic conditions if Beta Streptococcus is suspected and use of specific media/techniques for fluorescence microscopy.
• Sputum cultures: Samples should be examined immediately for the presence of epithelial cells and white blood cells, as specimens with excessive epithelial cells are generally unsuitable for culture.
• Urine cultures: Midstream clean-catch, catheterized, or suprapubic aspirations; use appropriate loops for counting (1 ul/10 ul).
• CSF cultures: Examine immediately, centrifuge; note the number and types of cells in the sediment.

Specimen Storage

• Appropriate temperature conditions are critical for specimen integrity and the survival of pathogens.
  • CSF: 37 degrees Celsius
• Urine, stool, swabs, viral specimens, sputum, foreign bodies: 4 degrees Celsius.
• Serum: -20 degrees Celsius for up to one week.
• Tissues and specimens for long-term storage: -70 degrees Celsius.

Antimicrobial Agents

• Cell wall synthesis inhibitors: beta-lactams (penicillins, cephalosporins, carbapenems), glycopeptides (vancomycin, teicoplanin), cycloserine, bacitracin.
• Protein synthesis inhibitors: Aminoglycosides (gentamicin, tobramycin, kanamycin), tetracyclines, chloramphenicol, erythromycin, fusidic acid.
• Nucleic acid synthesis inhibitors: sulfonamides, trimethoprim, quinolones, rifampicin.
• Cell membrane function inhibitors: polymyxins (colistin), antifungals (amphotericin B).

Beta-Lactamase

• Beta-lactams are often neutralized by bacterial enzymes like beta-lactamases (ESBL, ampC, carbapenemase).
• Different tests are used such as the chromogenic cephalosporin assay and the nitrocefin assay and modified Hodge test to detect presence of beta lactamases and carbapenem resistance

Sterilization

• Completely destroys all microbial life (pathogens and nonpathogens–vegetative cells and spores).
• Methods include moist heat (autoclaving, fractional sterilization), dry heat (oven, incineration), ionizing radiation, and filtration.
• Autoclaving: Steam under pressure.
• Fractional sterilization: Alternating heating and incubation cycles (more complex than autoclaving, potentially needed for heat-sensitive substances like proteins)
• Dry heat sterilization: Heated in an oven at 160-180˚C.
• Incineration: Used for infectious wastes. Burned under high temperature conditions; not utilized in healthcare settings due to environmental concerns.
• Ionizing radiation: Use of gamma rays to destroy microbes; used for heat sensitive substances.

Disinfection

• Destruction of pathogens on non-living surfaces.
• Physical methods: Boiling (100˚C, 15 minutes); Pasteurization (varies with temperatures and duration dependent on bacterial type, food product), non-ionizing radiation (like UV light).
• Chemical methods: Alcohol (considered an antiseptic), Quaternary ammonium compounds (Quats), Halogens (iodine and chlorine), Heavy metals (mercury, silver nitrate), phenolic compounds (phenol coefficient is a measure of bactericidal power against those types).

Biological Safety Cabinets

• Class I: Open-fronted; air flow is unidirectional - airborne microorganisms move out of the BSC
• Class II: Provides a flow of air entering and leaving the cabinet; different types are available: A1/A2/B1/B2
• Class III: All air entering and leaving the cabinet is sterilized with a HEPA filter. Personnel use only sealed rubber gloves, and the cabinet operates under negative pressure.

Additional Notes

• Specific examples of organisms associated with various infections and cultures are listed on each slide.
• Culture media specifics (composition, indicators, inhibitors) for different specimen types are covered.
• Quality control methodologies and common errors are also covered.
• The slides contain figures depicting different processes, such as gram staining or methods to determine the growth/presence of microbes in various mediums.