microbiology

• Microbiology


  The study of microorganisms which are large and diverse group of microscopic organisms that can occur as either single cell or in cluster arrangement including viruses which are microscopic but acellular

  
• Bacteriology


  The study of bacteria
• Bacterial morphology


  • Coccus (round/circular)
  • Bacillus (rod shaped)
  • Spirochetes (spiral shaped)
  • Pleomorphic (vary in shape)

  
• Bacterial envelope


  • Capsule
  • Outer Membrane
  • Cell wall
  • Cell Membrane
  • Attachment protein
  • Organ of locomotion
• Capsule


  Outermost layer, if present
• Virulence factor (VF) of capsule


  K antigen → prevents phagocytosis
• Capsule identification


  India ink stain
• Outer membrane


  Major permeability barrier for Gram-negative bacteria, contains lipopolysaccharide (LPS) → endotoxin
• Periplasmic space


  Found between the outer membrane and cell wall
• Cell wall


  Also known as Peptidoglycan (PG) layer, contains O antigen
• Differences between Gram-positive and Gram-negative bacteria

  • Gram reaction (Gram-positive: blue/violet, Gram-negative: red/pink)
  • PG layer thickness (Gram-positive: thicker 7-8, Gram-negative: thinner 1-2)
  • Teichoic acid (Gram-positive: present, Gram-negative: absent)
  • Periplasmic space (Gram-positive: absent, Gram-negative: present)
  • Toxin produced (Gram-positive: exotoxin, Gram-negative: endotoxin)
• Gram staining


  Most commonly used differential stain
• Gram staining steps


  1. Crystal Violet (primary stain)

  2. Gram's Iodine (mordant)

  3. Alcohol (decolorizer)

  4. Safranin O (secondary stain/counterstain)
• Gram-positive cocci exceptions


  • Neisseria
  • Veillonella
  • Moraxella
• Gram-negative bacilli exceptions


  • Bacillus
  • Actinomyces
  • Nocardia
  • Streptomyces
  • Corynebacterium, Clostridium
  • Erysipelothrix
  • Listeria, Lactobacillus
  • Mycobacterium
  • Propionibacterium

  
• Bacteria that cannot be seen on Gram staining

  • Mycoplasma (absent cell wall)
  • Cryptosporidium (protozoan)
  • Chlamydia
  • Legionella micdadei
  • Legionella pneumophila
  • Isospora (protozoan)
  • Rickettsia
  • Nocardia (weakly acid fast)
  • Treponema
  • Mycobacterium (Gram-variable)

  
• Cell membrane


  Phospholipid bilayer embedded with protein, functions as selective barrier for solutes and energy metabolism (ATP production)
• Attachment proteins


  • Fimbriae (attachment to host cell)
  • Pili (attachment to another bacteria, conjugation - virulence factor)
• Organ of locomotion


  • Axial filament (corkscrew motion of spirochetes)
  • Flagella (forward locomotion of bacilli, H antigen)
  • Atrichous (no flagella)
  • Monotrichous (one flagellum on one end)
  • Lophotrichous (tufts of flagella on one end)
  • Amphitrichous (flagella on both ends)
  • Peritrichous (flagella all over the surface)
• Internal bacterial structure


  • Nucleoid
  • Plasmid
  • Ribosomes
  • Endospores
• Nucleoid


  Also known as nuclear region/nuclear body/chromatin region/chromatin body, circular double stranded (ds) DNA which contains essential genetic information
• Plasmid


  Extrachromosomal circular dsDNA which contains non-essential genetic information like resistance, conjugation, toxin production, virulence, and transposons
• Methods of gene transfer

  • Transformation (free uptake of naked DNA in solution)
  • Transduction (bacteriophage-mediated)
  • Conjugation (via sex pili - F plasmid)
  • Transposition (via transposons)
• Ribosomes


  Site of protein synthesis, 70s = 30s + 50s (potential sites for antibacterial agents)
• Endospores


  Main composition is calcium dipicolinate/dipicolinic acid, important for survival (resistance to heat, chemicals, dehydration), identified by Malachite Green staining
• Examples of endospore-forming bacteria

  • Bacillus
  • Clostridium

  
• Stages of microbial growth

  • Lag phase (no replication, increased size and synthesis)
  • Log phase/Maximum exponential phase (increased replication, increased antibiotic susceptibility)
  • Stationary phase (number of replications = number of deaths)
  • Death phase/Decline phase (number of deaths > number of replications)
• Reasons for microbial death

  • Decreased nutrients
  • Increased toxins
• Physical requirements for microbial growth

  • Temperature (psychrophiles/cryophiles, mesophiles, thermophiles)
  • pH (acidophiles, neutrophiles, alkaliphiles/basophiles)
  • Osmotic pressure (halophiles)
• Acid-fast staining


  Carbol-Fuchsin (primary stain), heat (physical mordant), acid alcohol (decolorizer), Methylene blue (secondary stain/counterstain)
• Energy sources for microbes

  • Phototrophs (light)
  • Chemotrophs (redox potential - medically important)
• Carbon requirements for microbes

  • Heterotrophs/Organotrophs (organic carbon - medically important)
  • Autotrophs/Lithotrophs (inorganic carbon)
• Oxygen requirements for microbes

  • Obligate aerobes (require O2 for survival - PNBMC)
  • Obligate anaerobes (grow only in absence of O2 - ABCEFP)
  • Microaerophiles (require decreased O2, increased O2 toxic - Campylobacter, Helicobacter)
  • Aerotolerant (tolerate O2 but cannot replicate in its presence - LP)
  • Facultative anaerobes (can thrive with or without O2 - THE REST)
• Composition of culture media

  • Chemically defined medium (exact composition known)
  • Complex/Not chemically defined medium (exact composition unknown)
  • Tissue culture (for growth of viruses)
• Physical state of culture media

  • Liquid (no solidifying agents)
  • Semi-solid (0.5-1.0% agar)
  • Solid (1.5-3.0% agar)
• Functions/uses of culture media

  • Simple media (for unfastidious organisms)
  • Enriched media (fortified with vitamins, nutrients, etc. for fastidious organisms)
  • Reducing media (for obligate anaerobes)
  • Selective media (for specific organisms)
  • Differential media (subdivides a group of microorganisms)
• Enriched media examples


  • Blood Agar Plate (BAP) for organisms requiring high nucleic acid
  • Milk agar for organisms requiring high protein
  • Chocolate Agar Plate (CAP) for organisms requiring molybdenum from hemoglobin
• Selective media examples


  • MCA, EMB for Enterobacteriaceae
  • Colistin-Nalidixic Agar (CNA) for Gram-positive organisms
  • Thayer-Martin for Neisseria spp.
  • Lowenstein-Jensen for Mycobacterium tuberculosis
  • Loeffler's Serum Medium, Tellurite Medium for Corynebacterium diphtheriae
  • Campy BAP, Skirrow's agar for Campylobacter jejuni
  • Bordet-Gengou Medium for Bordetella pertussis
  • Thiosulfate citrate bile salts sucrose (TCBS) for Vibrio spp.
• Differential media examples


  • Blood Agar Plate for Streptococcus spp. (α, β, γ-hemolysis)
  • MacConkey agar (MCA) and Eosin Methylene Blue (EMB) for Enterobacteriaceae (lactose fermenters vs non-lactose fermenters)
• Gram-positive cocci


  • Staphylococcus (catalase positive)
  • Streptococcus (catalase negative)