Required Practical Activity 2 - Culturing Microorganisms Flashcards
Paper 1 - B1
Method to investigate the effect of antiseptics or antibiotics on bacterial growth using agar plates and measuring zone inhibition:
Preparing the bacterial plate:
1) Clean an area of desk with disinfectant
2) Remove the lid from the bacterial culture and quickly pass the neck of the bottle through a yellow Bunsen flame to sterilise it
3) Remove a drop of culture using the sterile pipette
4) Lift the lid of the Petri dish just enough to drop the bacteria over the surface. Quickly replace it. Put the pipette into the disinfectant.
5) Sterilise the spreader by dipping in disinfectant and then placing in Bunsen flame. Then allow it to cool.
6) Spread out the bacteria to cover the surface using the spreader. Do not lift lid more than needed.
7) Put the spreader back into the disinfectant.
Adding the antibiotics:
8) Sterilise the forceps by dipping in disinfectant and then placing in Bunsen flame. Then allow it to cool.
9) Soak the paper discs in different types/concentrations of antibiotics and place on an agar plate evenly spread with bacteria.
10) One disk should be a control, soaked in sterile water
11) There should be no death of bacteria with this disc - showing only the type of antibiotic affects the size of the inhibition zone
12) Lift the lid of the Petri dish and place an antibiotic disk about 2cm away from the edge.
13) Add the other discs in the same way, including the disk dipped in water.
14) Put the forceps into disinfectant
15) Tape the lid on the dish with stick tape in two places (do not tape all the way around)
16) Leave the dish at a temperature of 25 degrees C for 2 days
17) After 2 days measure the area that bacteria are killed (inhibition zone) for each antibiotic by measuring the diameter - bigger it is, the more bacteria are killed and therefore the more effective the antibiotic is
18) The cloudy agar shows where the bacteria has grown
19) Calculate the area of the inhibition zones (clear areas growing no bacteria) by measuring the diameter of the inhibition zone and then calculating the area using πr²
Apparatus used to investigate the effect of antiseptics or antibiotics on bacterial growth using agar plates and measuring zone inhibition:
-petri dish containing agar jell
-sterile pipette
-spreader
-bacterial culture
-marker pen
-different antibiotic disks
-filter paper disk dipped in water
-stick tape
-disinfectant
-eye protection forceps
How are microorganisms studied by scientists?
-microorganisms are very small, so in order for scientists to study them they need to grow many of them in the lab using nutrients (culturing them)
What are uncontaminated cultures of microorganisms needed for?
uncontaminated cultures of microorganisms are required for investigating the action of disinfectants and antibiotics
What are the two ways to grow microorganisms in the la
1) In nutrient broth solution
2) On an agar gel plate
How are microorganisms grown in a nutrient broth solution?
-involves making a suspension of bacteria to be grown and mixing with sterile nutrient broth (the culture medium), stoppering the flask with cotton wool to prevent air from contaminating it and shaking regularly to provide oxygen for the growing bacteria
How are microorganisms grown on agar gel plate?
-The agar acts as a culture medium and bacteria grown on it form colonies on the surface
Making the plate:
-Hot sterilised agar jelly is poured into a sterilised Petri dish, which is left to cool and set
-Wire loops called inoculating loops are dipped in a solution of the microorganism and spread over the agar evenly
-A lit is taped on and the plate is incubates for a few days so the microorganism can grow (stored upside down)
How and when do bacteria multiply?
If bacteria have a supply of nutrients and a suitable temperature, bacteria can multiply quickly by binary fission as fast as every 20 mins
How can you calculate the number of bacteria?
-bacteria at the beginning × 2to the power of number of divisions = bacteria at end of growth period
-you can calculate the number of bacteria in a population after a certain time, if given the mean time division time for bacteria
-to calculate the number of divisions dive the time the population is left for by the mean division time for the bacteria
-the number of bacteria at the end of the growth period can be very large (so common for it to be left in standard form)
If the microorganisms are bacteria what can they used be test?
-Can be used to test the effect different antibiotics have on their growth
-investigation involves soaking paper disks in different antibiotics which are placed on an agar plate
-after leaving the plate, the size of the clear area around the discs shows how many bacteria have died and therefore how effective the antibiotic is
Explain the reason for there being a disk of water (in investigation of the effect of antiseptics or antibiotics on bacterial growth using agar plates and measuring zone inhibition):
control to see that the antibiotics are what’s killing the bacteria
Why may a certain antibiotic may not be the best to threat a throat infection?
may not be best treatment as don’t know if throat infection is a bacterial infection or if the bacteria of the Petri dish is the same as the bacteria for her infection
Important steps in the investigation of the effect of antiseptics or antibiotics on bacterial growth using agar plates and measuring zone inhibition
-petri dishes and culture media must be sterilised before use, often done by an autoclave (an oven) or UV light
-inoculating loops must be sterilised by passing them through a flame
-the lid of the Petri dish should be sealed (but not completely) with tape
-the Petri dish should be stored upside down
-the culture should be incubated at 25 degrees C
Why must petri dishes and culture media be sterilised before use, often done by an autoclave (an oven) or UV light?
-likely to be contaminated with other microorganisms if this does not take place
-the contamination could be harmless but will compete with the desired bacteria for nutrients and space, or they could be harmful (e.g. through a mutation taking place), potentially producing a new pathogen
Why must inoculating loops be sterilised by passing them through a flame?
kills unwanted microorganisms as otherwise they would compete with the desired bacteria for nutrients and space, or they could be harmful (e.g. through a mutation taking place), potentially producing a new pathogen
