Illustrated Medical Microbiology Satish Gupte
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5BACTERIOLOGY
General Bacteriology
Systemic Bacteriology
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General Bacteriology

 
BACTERIA
  • Unicellular
  • Free living
  • Without chlorophyll
  • Having both RNA and DNA
  • Prokaryotic
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Shape of Bacteria
  1. Cocci (Spherical)
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  2. Bacilli (Rod-shaped)
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Structure of Bacteria
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Capsule
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  • Gelatinous secretion of bacteria
  • Gets organised as a thick coat around cell wall
  • May be composed of polysaccharide (e.g. Pneumococci and Klebsiella) or polypeptide (e.g. Bacillus anthracis) or hyaluronic acid (Streptococcus)
  • Have no affinity for dyes
  • Demonstrated by India ink, special capsule staining using copper as moderant and Quellung reaction
  • Functions are protective and inhibit phagocytosis.
 
Cell Wall
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  • Outermost supporting layer of bacteria
  • 20 to 25 nm thick (14 to 23 nm in Gram-positive and 10 to 15 nm in Gram-negative)
  • Composed of mucopeptide (muerin), scaffolding formed by N: acetyl glucosamine and acetyl muramic acid molecules alternating in chain cross-linked by peptide chain.
  • Demonstrated by plasmolysis, microdissection, reaction with specific antibody and electron microscope.
  • Functions include protection, conferring shape and rigidity to bacterial cell, division of bacteria and resistance to harmful effects of environment.
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Cytoplasmic Membrane
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  • Thin semipermeable membrane lying just beneath the cell wall
  • 5 to 10 nm in width
  • Consists of lipid with small amount of protein
  • May be demonstrated by plasmolysis and electron microscope
  • Functions are: Control of inflow and outflow of metabolites to and from protoplast with enzymes manufacturing substance of cell wall and extracellular structure, also provides little mechanical strength to bacterial cell.
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Envelope of gram-positive organisms
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Envelope of gram-negative organisms
Character
Gram-positive organisms
Gram-negative organisms
Thickness
15 to 24 nm
10 to 15 nm
Variety of amino acids
Few
Several
Aromatic and sulfur Containing amino acids
Absent
Present
Lipids
Low (4%)
High (20%)
Techoic acids
Present
Absent
 
Cytoplasm Contains
 
RIBOSOMES (Ribonucleoproteins)
Diameter 100 to 200 A°
Sedimentation coefficient: 70 Svedberg units
Function: Sites of protein synthesis
 
Polysome
Group of ribosomes in beads of chain
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Mesosome
  • Vesicular
  • Convoluted/multilaminated
  • Invagination of cytoplasmic membrane
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Intracytoplasmic Inclusions
  • Volutin granules (metachromatic granules)
  • Lipid inclusions
  • Polysaccharide inclusions
  • Vacuoles
 
Nucleus
Long DNA filament coiled tightly in cytoplasm (1 mm long)
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Flagellum
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  • Long
  • Sinuous
  • Contractile
  • Antigenic
  • Possess protein with properties of keratin and myosin
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Arrangement of Flagella in Bacteria
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Fimbriae (Pili)
  • Filamentous
  • Short
  • Thin
  • Straight
  • Projects from cell surface
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Functions
  • Adhesion
  • Conjugation
  • Hemagglutination
  • Pellicle formation
  • Pellicle from cell surface
 
Bacterial Spore
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  • Spores are highly resistant state of bacteria (Bacillus and Clostridium)
  • Not killed by ordinary methods of sterilization
  • Autoclavation at 15 lbs pressure and 121 °C can kill spores
  • Have 5 to 20% dipicolinic acid
  • When transferred to favorable condition
    • Spore loses refractility
    • Swells up
    • Spore wall is shed
    • Germ cell appears by rupturing spore coat
    • Germ cell elongates to form vegetative bacterium
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Sporulation Process
  • Exact stimulus for sporulation is not known
  • May be related to depletion of nutrient (Exogenous)
  • Sporulation is initiated by appearance of clear area near one end of cell which becomes more opaque (for spore)
 
The shape and position of the spore in bacterial cell
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Staining of Bacteria
Gram's stain
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Ziehl Neelsen Stain
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CULTURE TECHNIQUES
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Stab Culture: Used for maintaining stock culture
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Description of Colonies of Bacteria
  • Shape
  • Surface size : 0.5 to 3 mm
  • Elevation
  • Edge
• Entire
E. coli
Klebsiella
Staphylococcus
  • Fimbriated B. subtilus
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Color
Pigmented colonies: Staphylococcus aureus, Pseudomonas
Opacity
Transparent, Translucent, Opaque
Consistency
Hard: Mycobacterium tuberculosis
Friable: B. subtilus
Soft: E. coli
Emulsifiability
Emulsifiable: E. coli, Salmonella
Not emulsifiable: Neisseria catarrhalis
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Description of Growth in Liquid Media
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STERILIZATION
Sterilization is the process of freeing the article from all living organisms including spores
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Methods of Sterilization
 
Heat
  1. Dry heat: Sterilization by dry heat is attributed to
    • Denaturation of proteins
    • Coagulation of proteins
    • Oxidative damage
    • Elevated levels of electrolytes
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  2. Red heat: It is useful for sterilizing:
    • Inoculation loop
    • Spatula
    • Forceps
 
Flaming
Used for:
  • Sterilization of mouth of culture tubes
  • Cotton wool plugs
  • Glass slides
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Temperature required: 160°C for 1 hour
Uses: Sterilization of
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MOIST HEAT
 
Vaccine Bath
Vaccine is treated with moist heat for one hour. Useful in killing non-sporing becilli
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Inspissator
Slow solidification of serum or egg containing media is done at 80°C in an inspissator (Lowenstein Jensen medium, Loeffler serum slope)
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Steam Sterilizer (100°C)
Cheap method of sterilization where free steam is used to sterilize (100°C)
Culture Media
 
Two types of sterilizers available
  1. A Koch steamer
  2. Arnold steamer
 
Autoclave
15 lb/sq inch, 121°C for 20 minutes
Used for Sterilization of
  • Culture media
  • Rubber goods
  • Syringes
  • Instruments
  • Gown
  • Dressing
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Best method of sterilization because of wet heat under pressure
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Filtration: Sterilization of
  • Antibiotic solution and sera
  • Carbohydrate solution
  • Toxins
Drawback: Viruses and Mycoplasma may pass through filter.
Sintered glass filter
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Membrane Filter Apparatus
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Bacterial colonies on membrane filter after placing on culture medium and incubation
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Radiation
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Anaerobiasis
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IDENTIFICATION OF BACTERIA
 
Microscopic Examination: Gives information
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After Gram staining we get information like
  • Gram positive (violet color)
  • Gram negative (red color)
  • Size and shape
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  • Spores
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Hanging drop preparation shows whether organism is motile or nonmotile
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Resistance
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Culture Character
Straphylococcus aureus (pin-head colonies with beta hemolysis on blood agar)
Streptococcus pyogenes (pin-point colonies with beta hemolysis on blood agar)
Streptococcus viridans (pin-point colonies with alpha hemolysis on blood agar)
 
Biochemical Reactions
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ANTIGENIC ANALYSIS
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  • Streptococci (Grouped into Lancefield groups)
  • Pneumococci (Various capsular types)
  • Confirmation of
    • Salmonella species
    • Shigella species
 
BACTERIOPHAGE TYPING
Useful in the correlation of bacteriophage type and epidemic (Staphylococcus, Salmonella)
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PATHOGENICITY IN LABORATORY ANIMALS
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Route of Inoculation
  • Subcutaneous
  • Intramuscular
  • Intraperitoneal
  • Intracerebral
  • Intravenous
  • Oral
  • Nasal spray
 
RESISTANCE TO ANTIBIOTICS
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Sensitivity pattern useful in
  • Providing range of antibiotics in treating patient
  • Epidemiologic marker
  • Identification of bacteria (Pneumococci sensitive to optochin, streptococci (A) sensitive to bacitracin
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NUTRITIONAL AND OTHER REQUIREMENTS OF BACTERIA
 
Minerals (required in traces)
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Gas
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Obligate Anaerobes
Grow only in absence of oxygen (Clostridium tetani and bacteriodes)
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Carbon dioxide
Enhances the growth (Neisseria gonorrhoeae, Brucella)
 
Moisture
A requirement for growth of bacteria
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Growth Factors
Gonococcus requires glutathione
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C. diphtheriae requires B. alanine
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Staphylococcus aureus requires nicotinic acid and thiamine
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Temperature
Psychrophilic organisms (water and soil bacteria): 0°C to 25°C
Mesophilic organisms (producing human diseases) 20°C to 44°C
Thermophilic organisms (Bacillus): 45°C to 60°C
 
pH : Optimum pH 7.4
↑TH (alkaline) required, e.g. Vibrio cholerae
↓pH (acidic) required, e.g. Lactobacilli
 
Osmotic Pressure
Isotonic environment (0.5% addition of sodium chloride) is ideal for the growth of organisms
 
Light
  • Usually darkness favours growth of bacteria (sensitivity to UV rays of sunlight)
 
Vibrations
  • Mechanical and sonic vibrations cause disintegration of cell wall and rupture
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Growth Curve
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  1. Lag: Increase in size of bacteria, ↑ metabolism, adaptation to new environment
  2. Log: High metabolism, bacteria sensitive to antibiotic
  3. Stationary: Depletion of nutrient, accumulation of toxic products, sporulation
  4. Decline: Nutrition depletion, accumulation of toxic products, autolytic enzymes, involution
  5. Survival: Few bacteria manage to survive
 
Continuous Culture
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Here rate of growth is controlled by rate of addition of fresh nutrients, which in turn controls rate of removal of cells by overflow device into collector vessel.
This process continues indefinitely.
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ANTIMICROBIAL AGENTS
Chemotherapeutic agents have
  1. Lethal or inhibitory effect on microbes
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  2. In therapeutic dose no toxic action on tissue
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Antibiotics are substances produced by living organisms and kill other living organisms
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Mode of Action
Site of Action
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Mechanism of Action
  • Competition with natural substrate for the active site of enzyme, e.g. of action of PABA with PAS.
  • Combination with an enzyme close to the active site as to interfere with its enzymatic function, e.g. vancomycin, bacitracin.
  • Combination with non-enzymatic structural components, e.g. drugs which inhibit protein synthesis and drugs which act by damaging cytoplasmic membrane.
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Sites and mode of action of antibiotics
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INFECTION
Infection is lodgement and multiplication of organisms in tissue of host
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Classification of Infection
Primary infection is initial infection with organism in a host. Reinfection is subsequent infection by same organisms
Secondary infection is the infection set up by new organisms in pre-existing infectious diseased host.
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Indirect airborne Direct airborne Dust borne Contact via hands or other objects.
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Methods of Transmission
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Inoculation
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Insect
  • Malaria (Mosquito)
  • Dysentry (Housefly)
 
Congenital
  • Congential syphilis
  • Rubella
  • Toxoplasma
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Cross Infection: If occurs in a person having certain infection and acquiring new infection from another person or external source
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Patient of malaria (recovering) is visited by a patient of whooping cough
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Infection of whooping cough in this patient
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Hospital infection or nosocomial infection is cross infection occurring in hispital
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Specifically Neutralized by Antitoxin
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Enzymatic Action
 
Specific Affinity for Tissue
Tetanus toxin –––––– → Neural tissue (ganglioside of grey matter)
Highly antigenic; can be toxoided
 
Endotoxin
Protein lipid polysaccharide complex and heat stable
Obtained only by cell lysis
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  • Does not diffuse into the medium as it forms part of cell wall
  • No enzymatic action
  • Non-specific action
  • Active only in large doses
  • Weakly antigenic
  • Cannot be toxoided
  • Produced by Gram-negative organisms
Communicability is the ability of organisms to spread from host to host
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OTHER BACTERIAL PRODUCTS
  • Coagulase
  • Fibrinolysin
  • Hyaluronidase
  • Leucodins
  • Hemolysin
  • Capsule
  • (Pneumococci, Klebsiella)
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Route of Infection
Vibrio cholerae (produce disease when taken orally but no disease when induced parenterally)
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Vertical section of human skin showing structural arrangement and sites of certain infection
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Mechanism of Fever