Microbiology

Microbiology

1. Classification of bacteria

   • By Shape
     • 3 Main shapes
       • Bacillus (Rod-shaped)
       • Coccus (Spherical)
       • Spirillum (Corkscrew-shaped)
     • Further differentiation done by how they group
       • I.e In pairs/Chains/Clusters
   • Gram stain
     • Method of staining of cell walls, allow for identification
       • Difference in composition > Different stains > Which are identifiable
     • Before = Colourless
       • After gram-positive = Violet, Gram-negative = Red
     • Method
       • 1) Crystal violet: Basic dye: Binds to peptidoglycan, all bacteria stain purple
         • 2) Lugol's iodine: Mordant: Binds crystal violet to peptidoglycan more strongly
           • 3) Acetone-Alcohol: Decolouriser: Removes unbound crystal violet & lipopolysaccharide
             • Gram negative > Lose stain > Colourless
             • Gram positive > Stays purple
             • 4) Safranin: Counter-stain
               • Gram negative: Stain red
               • Gram positive: Stays purple
     • Gram-positive
       • After staining, Gram staining = Violet/Purple
         • Absence of liposaccharide layer > Allows Stain to bind more efficiently
           • Lysosomes hydrolyse bonds holding peptidoglycan molecules together
             • Penicillin prevents the formation of inter-peptidoglycan bonds
               • Making cell wall structurally weak > Explodes
           • Makes them more susceptible to penicillin & lysosomes
     • Gram-negative:
       • Outer cell wall made of lipopolysaccharide > Additional protection
         • Stops crystal violet from binding
           • Not affected by lysozymes/Penicillin
             • Antibiotics that mess w/protein making stuff controls population
               • Bacteria and eukaryotes have different protein making structures
                 • Therefore eukaryotic cells not affect by antibiotics

2. Conditions needed to culture bacteria

   • Microbes undergo binary fission, reproduce quickly
   • Microbes need:
     • Nutrients
       • Provided in nutrient media
         • Cultured in either
           • Liquid medium (Nutrient broth)
           • Medium solidified w/agar
         • Media provides:
           • Water
           • Carbon + Energy source
           • Nitrogen in form of organic/inorganic ions
             • For amino acid syntheis
     • Growth factors:
       • Vitamins Biotin & Mineral salts
     • Optimum Temp:
       • Metabolism catalysed by enzymes, optimum temp needed for enzyme action
     • pH
       • Fungi: Neutral to slight acidic conditions
       • Bacteria: Slightly alkaline environment (pH 7.4)
     • Oxygen
       • Depends on the type of bacteria
   • Ways to describe culture media
     • Defined Medium: Contains known ingredients
     • Undefined Medium: Some of the ingredients are unknown
     • Selective Medium: Allows certain bacteria to grow

3. Principle of aseptic technique

   • Laboratory practise, Maintain sterility in apparatus & prevents contamination of the equipment & the environment
   • To prevent contamination of pure cultures/apparatus
     • Sterilise all equipment before use
       • Prevents initial contamination
     • Carefully handle cultures
       • Flaming culture vessels before opening/closing
   • To prevent contamination into the environment
     • Sterilise work surface before/after experiment
     • Use correct handling techniques, E.g Inoculation
       • 1) Hold culture in 1 hand > Remove cap w/little finger > Don't put cap on surface
         • 2) Flame mouth of bottle for 3 sec
           • 3) Pass inoculating tube thru flame until red hot, let it cool in air
             • 4) Lift lid of petri dish just enough to pass through the inoculating tube
               • 5) Loosely secure petri dish w/tape, too tight > Anaerobic > Growth of pathogens
                 • 6) Incubate at 27', as 37' is optimum for many pathogenic species
                   • 7) Don't open dish after incubation
     • In lab, sterilisation done by autoclave
       • Sealed container in which plastic/metals heated to 121' in steam + pressure for 15min
     • Disposable materials placed in autoclave bags > autoclaved > Binned

4. Methods of measuring growth

   • Directly
     • Viable counts: Living cells only
     • Total counts: Living & dead cells
     • Plating & counting colonies
       • Sample of culture is taken, diluted down & measured
         • Relies on bacteria forming colones
           • Assumed that 1 cell gives rise to 1 colony
             • Thus result is normally an underestimate
           • Hence it's a viable count
     • Haemocytometer
       • More accurate than colony counting
         • Uses specialised microscope slide (Called haemocytometer)
           • Not possible to distinguish between living/dead cells
             • Hence it's a total count
     • Dilution done by
       • Solution transferred to new container that contains a pure solvent (Water)
   • Indirectly
     • Turbidmetry
       • Colorimeter used to measure the cloudiness/turbidity of culture as cell No. increases
         • Population size derived by
           • 1) Finding the absorbance of the suspension
             • 2) Comparing it to a standard graph of light absorbance vs. No of bacterial cells
               • Not possible to distinguish between living/dead cells
                 • Hence it's a total count

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