Microbiology

• The lipopolysaccharide layer of the Gram-negative bacteria provides protection from attack by lysozyme and penicillin antibiotics, making them more difficult to control in medical settings.
• Microbes, such as bacteria, can be cultured in Petri dishes or in larger containers, generally grown on agar, a jelly like substance, to which the required resources can be added.
• Bacteria require a source of carbon for growth, generally an organic compound such as glucose.
• Nitrogen needs to be provided for synthesis of nitrogenous compounds and can be provided as organic or inorganic compounds.
• Growth factors, such as water, vitamins and mineral salts, can also be added to the media.
• The optimum temperature of the bacteria you wish to grow should be taken into account and it is good practice to not grow bacteria at 37 o C (body temperature) unless attempting to grow bacteria that live in the human body, e.g. human pathogens.
• The optimum pH of the bacteria should be considered when making media and acid or alkali can be used to change the pH.
• Obligate aerobe bacteria can only reproduce or metabolise in an oxygen-rich environment.
• Facultative anaerobe bacteria thrive in an oxygen-rich environment but are also able to respire anaerobically.
• Obligate anaerobe bacteria are unable to reproduce or metabolise where there is oxygen present.
• Bacteria can be classified as Gram positive (purple) or Gram negative (red) according to how they react to the Gram stain.
• To count microbes reliably, it may be necessary to dilute the original sample to give between 20–200 colonies which grew from the bacteria in the original sample.
• Colonies are not merged so it can be presumed that each colony is derived from 1 bacterium in the original sample.
• Gram negative bacteria have a lipopolysaccharide layer that is washed away with the alcohol along with any crystal violet stain.
• A reliable count has between 20–200 colonies easily countable
• Viable count includes only living cells.
• Gram-negative cells then stain pink or red with safranin.
• Total count includes living and dead cells.
• Serial dilution is a method used to determine the concentration of a substance in a solution.
• In serial dilution, the concentration of a substance is diluted by a factor of 100 or 10,000.
• Gram positive bacteria have a thick peptidoglycan (murein) cell wall that retains crystal violet stain and so appears violet or purple.
• Inoculated agar plates are incubated at 25°C in school laboratories for 24–48 hrs, which encourages growth of the culture without growing pathogens.
• Aseptic technique must be used to prevent contamination of the environment by the microbes being handled and prevent contamination of the cultures by microbes from the environment.
• Petri dishes and nutrient agar should be sterilised before the agar is poured.
• An inoculating loop is used to transfer bacteria and should be sterilised before and after use by heating in a Bunsen flame.
• Only lift the Petri dish lid slightly as this prevents microorganisms from the air contaminating the culture and vice versa.
• After inoculation, the lid of the Petri dish should be secured in place by strips of adhesive tape labelled and dated.