Microbiology

Three bacteria shapes:

•  Bacillus (rod shape) 
• Coccus (spherical)
• Spirillum (spiral shape) 

Gram staining technique:

• Dye slide of bacteria with Crystal violet 
• The dye stains to bind to peptidoglycan layer staining all types of bacteria 
• Add Lugol's iodine to fix the stain 
• Decolourise with alcohol (which removes unbound stain and lipopolysaccharide layer on gram negative bacteria) 
• Gram negative becomes colourless 
• Gram positive remains purple 
• Counterstain the slide with safranin which penetrates the cell surface membrane of both types and stains them red 
• Gram negative is now red 
• Gram positive is now purple 

• The absence of lipopolysaccharide layer allows them to bind stain more efficiently 
• Makes them more succeptible to antibiotic, penicillin and the enzyme lyzosyme 
• Bacteria constantly make and break chemical links in their cell walls. The antibacterial enzyme lysozyme hydrolyses the bonds holding the peptidoglycan molecules together
• Penicillin prevents the bonds inter-linking peptidoglycan molecules from forming. This is especially significant when the bacteria make new cell walls when they divide. Penicillin therefore makes the cell walls structurally weak and prone to collapse. Water uptake by osmosis bursts the cells. 

• Have a more chemically complex cell wall. 
• Outer membrane is supplemented with large molecules of lipopolysaccharide which protext the cell and excluse dyes like crystal violet, so they appear red or pink under the microscope
• Outer lipopolysaccharide layer protects peptopolyglycan below so they are not effected by lysozyme and are resistant to penicillin.
• To control them requires a different class of antibiotics that interfere with the cell's ability to make proteins. 
• Eukaryotic cells also make proteins, but the protein- making cellular machinery is different from that in bacteria, so these antibiotics do not harm them. 

• Nutrience 
  • A carbon for energy source, usually glucose 
  • Nitrogen for amino acid synthesis, in organic moleucles and in inorganic form, such as nitrate ions. 
• Growth factors 
  • Vitamins 
  • Biotin and mineral salts 
• Temperature 
  • A bacterial metabolism is regulated by metabolism a range of 25-45 degrees is sutible 
  • Optimum for mammalian pathogens is around 37 degrees
• pH
  • Most bacteria are favoured by slightly alkaline conditions whereas fungi gorw bettwe in neutral to slightly acidic conditions 
• Oxygen
  • many micro-organisms require oxygenfor metabolism and are obligate aerobes
  • Some grow best in the presence of oxygen but some can survive in the absence are called facultive anaerobes. 
  • those what cannot grow in the presence of oxygen oligate anaerobes. 

• A defined medium contains only known ingredience 
• An undefined medium contains components that are not all known because they include yeast extract or beef peptone
• A selective medium only allows certain bacteria to grow e.g. media containing tetracycline only allow tetracycline-resistant bacteria to grow. 

Aseptic technique: Laboratory practice that maintains sterility in apparatus and prevents contamination of the equiptment and the enviornment

Ways to prevent contamination of pure cultures and apparatus by bacteria from the enviornment:

• Sterilise apparatus and media before use to prevent initial contamination 
• Handle culutures carefully, flaming the necks of culture vessels before opening and closng and use equiptment such as sterile loops to prevent subsequent contamination

Ways to prevent contamination to the enviornment by bacteria being used in experiments:

• Sterilise the work surface before and after an experiment using a disinfectant
• Use the corect handling techniques to prevent the contamination of personnel and the immediate enviornment by the organisms being cultured. When carrying out the process of innoculation:
  • Hold the culture bottle in one hand: remove cap with the little finger of the other hand. Do not place the cap down in the work surface. 
  • Flame the mouth of the bottle for 2 or 3 seconds
  • Pass the inoculating loop through a flame until its red hot, and allow it to cool in the air. 
  • Lift the lid of the Petri dish just enough to allow entry of the inoculating loop
  • Secure the Petridish with two pieces of adhesive tape. Do not seal all the way round as this could create anaerobic conditions and, potentially, encourage the growth of pathogenic species
  • Do not open Petri dishes after incubation
• In a Laboratory the preffered method of sterlilisation is to use an autoclave. This is a sealed container in which glass and metal equiptment is heated at 121 degrees in steam and under pressure for 15 minutes after the required pressure has been reached. 
• After use disposable materials, such as plastic petri dishes, can be sealed inside autoclaveable plastic bags, autoclaved and then placed in the dustbin. 

Size of a population can be counted in liquid culture by: 

• Directly, by counting cells
  • Viable count is only living cells 
  • Total count is living and dead cells
• Indirectly, by measuring the turbidity of the culture. This method can be used as a field work technique, using light absorption by a sample of river water to indicate the number of bacteria present.