Method: plating out

In the next section of this prac (next week) you will be identifying bacteria that you grow from this session. In order to prepare for this session you need to grow the bacteria from your sample in the media provided. This is the first stage of the culture and identification procedure for bacterial typing.

1. Put on the gloves provided.


2. Turn on the Bunsen Burner and light it. Adjust the collar (at the base) to produce a blue flame. Warning: when the Bunsen burner is adjusted the flame is hard to see. If you are not using it immediately either turn it off or adjust the collar to produce a yellow flame.


3. Place the MacConkey with Salt (MACS) media on the bench in front of you with the base (containing the media) facing up i.e. the lid is in contact with the bench top.


4. Label each plate (the part with the media which should be facing up) with your name and sample ID and the date.


5. Using your smallest finger (‘pinkie’) of your free hand, grasp the lid of the milk sample tube and rotate and remove the cap. Keep the cap securely between your small finger and the edge of your hand. Place the opened tube on the bench.


6. Take one of the loops in the other hand and pass it over the blue flame of the Bunsen burner until the round tip turns red (flaming the loop). Dip the loop into milk, stir it around slightly and withdraw it.


7. Pick up the media plate and slowly wipe the loop over a 3 cm section of the media near the edge (Figure 1). Wait for a few seconds for the fluid to permeate the media. Return the plate to the lid. Flame the loop. Keeping the sample tube lid in your finger, replace it onto the milk sample tube.


8. Pick up the plate again and flame the loop. Using four smooth but gentle strokes with the loop at a slight angle to the surface, slide the loop from the area of the milk sample to the circumference about a quarter way around the plate. Each stroke is separated by about 1 cm (Figure 3). Flame the loop.


9. Rotate the plate a little less than a quarter turn. Take another loop and repeat the strokes, this time crossing the later part of the four streaks you made previously (Figure 4). Flame the loop.


10. Rotate the plate another less than quarter turn, take another loop and make four more streaks, crossing the streaks from the previous step (Figure 5). Flame the loop.


11. Rotate the plate another less than quarter turn and take another loop and make four more streaks, crossing the streaks from the previous step. Make sure you do not touch the areas previously streaked (Figure 6). Flame the loop.


12. Make a “squizzle” across the last four lines into the centre of the plate (Figure 7). Flame the loop.


13. Return the plate to the lid.


14. Repeat the ‘streaking-out’ procedure with the Mannitol Salt media (MSA) and then the Sheep blood agar with aesculin media (AES) and then all three plates with the second sample.


15. Make sure you have labelled all 6 plates (not the lid) with your name, the sample number and the date.


16. Place the 6 media plates in the provided container. It will be transferred to an incubator (at 37oC) by staff. They will remain in the incubator for 48 hours and then be removed and stored in a cool room until the next prac class.