WJEC A-level Biology 3.4 Microbiology

• What are bacteria?

  Prokaryotes, smallest cellular organisms.
• How are they classified?

  By shape
• What are the classifications and what do they look like?

  Cocci (spherical), Bacilli (rod-like) and spirilla (spirals)
• What is helicobacter?


  A single bacteria
• What is diplo?

  A pair of bacteria
• What is strepto?

  A chain of bacteria
• What is staphylo?

  A cluster of bacteria
• How is grain staining done?

  Gram staining is done by heat fixing a smear of bacteria. Staining it with crystal violet stain. Fix the stain with iodine and decolourise with alcohol. Finally, counterstaining with safranin.
• What are the results of gram staining?

  Gram-positive comes out purple, and gram-negative is red.
• Why are gram positive purple?

  Gram-positive bacteria don't have the lipopolysaccharides in their cell walls and retain the crystal violet
• Why are gram negative red?

  Gram-negative bacteria have walls which are more chemically complex. The peptidoglycan is supplemented by large molecules of lipopolysaccharide, which protect the cell. They do not retain the dyes.
• What does penicillin do?

  Inhibits cell wall synthesis. They are unable to divide and the cell wall ruptures.
• What conditions do bacteria need to survive?

  They need the proper nutrients (energy source, nitrogen for amino acid synthesis), vitamins, temperature (enzymes regulate metabolism; the optimum is 37), pH (favour alkaline conditions), and oxygen (needed for metabolism).
• What is obligate aerobes?

  They require oxygen. Only aerobic growth. Bacteria growth where high concentrations of oxygens in the medium.
• What is the effect of oxygen on obligate aerobes?

  Presence of enzymes catalase and superoxide dismutase allows toxic forms of oxygen to be neutralised, can use oxygen.
• What is facultative anaerobes?

  Both aerobic and anaerobic growth. Better growth with oxygen. Growth occurs throughout the tube.
• What is the effect of oxygen on facultative anaerobes?

  Presence of enzymes catalase and SOD allows toxic forms of oxygen to be neutralised, can use oxygen.
• What is obligate anaerobes?

  Only anaerobic growth. Stop in the presence of oxygen. Growth in the bottom away from oxygen.
• What is the effect of oxygen on obligate anaerobes?

  Lacks enzymes to neutralise harmful forms of oxygen. Can't tolerate oxygen.
• Why are aseptic techniques used?

  To keep the apparatus free of microorganisms.
• What aseptic techniques were used before starting?

  Hands should be washed, and benches should be sterilised with a disinfectant. Lab coats and goggles must be worn.A Bunsen burner on a roaring flame is used to make a convection current, and all work should take place close to this - so tie your hair back.All equipment used must be sterile. Disposable Petri dishes are sterile when closed as they have been sterilised by the manufacture heat. Glass spreaders are sterilised by dipping in alcohol, and the alcohol is burned off. Inoculating loops are heated until they glow red. Other equipment can be sterilised in an autoclave, which heats the equipment up to over 120˚C.
• What aseptic techniques are used during transfers?

  Lids of agar bottles or cultures should be held in the crook of the little finger and not placed on benches.The necks of agar bottles and cultures must be 'flamed' on opening and before closing.Loops must be sterilised and cooled before insertion into cultures and sterilised afterwards.Lids of Petri dishes should be tilted and not lifted off when the agar is poured in or while the plate is being inoculated.
• What aseptic techniques are used after inoculation?

  The Petri dish should be sealed with a cross of tape. This discourages the growth of human pathogens and allows oxygen into the culture.The plates should be incubated upside down at 25˚C for 24 hours.Plates must not be re-opened and should be autoclaved after observations have been made.
• Explain a bacterial growth curve.

  Lag phase- The first stage is very small as it is a log graph. It won't show up on the graph. Double every generation phase if there are enough nutrients and limited waste.Log phase- Doubling in every unit of time, so a constant line. There are enough nutrients and things the bacteria need.Stationary phase- Limiting factors like oxygen, food, running out of nutrients, and increasing amount of waste. Number of new cells = number of cells dying.Death Rate- Cells only live for a particular time; the number of living bacteria decreases. They incest, they just stay there. When in the right conditions, they will come back. It goes back to a dormant state to survive.
• How can bacteria been counted?

  Directly- Counting each cell. Indirectly- Measuring turbidity.
• What is turbidity?

  Cloudiness of culture with a colourimeter.
• How are direct counts done?

  Total count of living and dead cells. Viable counts only count living or actively growing cells and estimated population size.
• What is a haemocytometer?

  A specialised microscope that can count the total count.
• How is turbidity used?

  It is compared with known standards.
• What is the method for serial dilution?

  1. Fill 5 test tubes with 9 cm3 sterile water using a sterile pipette.2. Add 1 cm3 of your sample to the first tube; this is a 1 in 10 dilution or 10 -1.3. Mix the 10 -1 dilution thoroughly and pipette 1 ml into the second test tube; this is a 1 in 100 or 10 -2 dilution.4. Repeat this process until you reach a 1 in 10 000 dilution (10 -4).5. Plate each dilution on nutrient agar using aseptic techniques.
• How can you tell a good count using serial dilution?

  Plates with good separation (isolated) of serially diluted individual bacterial colonies - colonies are easily counted on such a plate.Too many to count - merged colonies (overlapping) cause non-reliable results for statistical analysis. There are too few colonies to provide reliable results.
• How do you find the viable count?

  Number of colonies multiplied by dilution factor.
• What are direct counts?

  Total count of living and dead cells.
• What are viable counts?

  Count living or actively growing cells and estimated population size.