1. Bacteriology

Question 1

principles of aseptic technique

Answer 1

1. the growth medium and its container should be sterilised as soon as its prepared
2. the mouth of the container should be covered to keep unwanted microbes out
3. instruments and fluids which touch the inside of the container must be sterile
4. you must not contaminate the work area with cultures

Question 2

streak plates

Answer 2

• using an inoculating loop and aseptic technique
• plate is incubated upside down at 30C
• dilutes culture so individual colonies can be studied

Question 3

preparation of an air-dried smear

Answer 3

1. place a drop of water on a clean microscope slide with a flame-sterilised inoculating loop
2. resterilise loop, allow to cool and transfer a small quantity of a colony to the drop of water which should become slightly cloudy
3. resterilise loop to burn off excess culture, allow to cool and use to emulsify the bacteria in the drop and then spread out over the slide, resterilise the loop
4. allow film to dry at room temperature, excess heat will distort the bacterial morphology
5. fix the bacteria to the slide by passing through the bunsen flame a few times, killing the bacterial cells so the dyes can penetrate more easily and making the cells stick tightly to the slide
6. allow to cool before staining

Question 4

gram staining

Answer 4

• distinguishing between gram positive and gram negative bacteria without genetic sequencing
• gram positive bacteria are purple (crystal violet), gram negative are red (safranin)
  1. prepare air dried smear
  2. stain with Crystal Violet (primary stain) for 2 minutes
  3. wash with water
  4. apply Gram’s iodine (mordant) for 1-2 minutes
  5. wash with water
  6. decolourise with 95% ethyl alcohol (flooding or dripping)
  7. wash with water
  8. counterstain with dilute safranin 10-25 seconds
  9. wash and dry

Question 5

Gas vacuoles

Answer 5

• provide buoyancy to planktonic cyanobacteria and bacteria
• made up of gas vesicles, permeable so contains air at atmospheric pressure
• bright refractile granules
• disappear when subjected to moderate pressure
• cyanobacteria can regulate their vertical position with respect to light, high light intensities cause an increase in cell turgor pressure, causing the cell to collapse a proportion of their vesicles and sink

Question 6

disruption of the gas vacuole in Anabaena flos-aquae

Answer 6

1. fill 3 universal bottles to the brim with a suspension of bacteria
2. insert rubber stopper in 2 of them
3. place one stoppered bottle in corking device provided and hit stopper firmly with a hammer, leave the other unhit as a control
4. the bacteria in the stoppered bottle sunk to the bottom