REQUIRED PRACTICAL 6 - GROWTH OF BACTERIA/ASEPTIC TECHNIQUES

• aseptic techniques are used to avoid contamination of the sample from outside substances, like microorganisms • important to get reliable and repeatable data

• wipe down surfaces with antibacterial cleaner before and after experiment • use a bunsen burner in the work space so that convection currents draw microbes AWAY from the culture • flame the wire loop before using it to transfer bacteria • flame the neck of any bottles before using them to prevent any bacteria from entering the vessel • keep all vessels containing bacteria open for the minimum amount of time • close windows and doors to limit air currents

• bacteria sample • disinfectant • bunsen burner • heatproof mat • ethanol • wire hoop • pipette • forceps • plastic spreader • prepared agar plate • antibiotic ring • ruler

• carry out the aseptic techniques • use a sterile pipette or wire hoop to transfer bacteria from broth (distilled water, bacterial culture or nutrients) to the agar plate • spread bacteria evenly over the plate using a sterile plastic spreader • use sterile forceps to place a multi disc antibiotic ring on the plate, the ring should only be moved by holding the centre NOT the arms • lightly tape a lid on, invert and incubate at 25 degrees C for 48 hours • for this ^, do not tape around the entire dish as this prevents oxygen entering and promotes the growth of more harmful anaerobic bacteria • sterilise equipment used to handle bacteria and disinfect work surfaces • post incubation, measure the diameter of the inhibition zone (clear circle) for EACH antibiotic however do not remove the lid from the agar plate • work out the area of the inhibition zone

A = pi x diameter^2/4

• disinfectant • biohazard • naked flame

flammable

keep away from the naked flame

• contamination • infection

• use disinfectant • wash hands with soap after dissection • do not incubate at human body temp • do not open agar plate post incubation

• fire hazard • burns

• keep away from flammable materials • tie up long hair • wear goggles

• if there is a LARGER inhibition zone around the antibiotic, it has killed more bacteria • the larger the inhibition zone, the better the antibiotic zone • some antibiotics will have no/very little inhibition zone, this shows that bacteria are resistant to this antibiotic and are NOT killed by it

• bacteria may outcompete bacteria being investigated or may be harmful to humans/pathogenic

• allows oxygen in, preventing growth of ANAEROBIC bacteria as they are more likely to be pathogenic/harmful to humans

condensation drips onto the lid, instead of the surface of agar

repeat readings in different positions and calculate a mean

most bacteria are killed so clear zones may overlap

as that is below human body temp and this prevents the growth of pathogens