And it’s time for yet more agar

Day four started off with everyone splitting into their separate groups. There was a flurry of activity to start with, until we had to wait for agar and broths to be mixed.

Laura and Keiran were set to work with P.acnes (the bacterium that causes Acne rashes)  on TYG agar in an anaerobic environment (this involved using a cabinet which was kept anaerobic (under a nitrogen and not an oxygen environment).

Hayley and Sarah prepared agar plates infused with different antibiotics in order to test the resistance of the E.coli strains in the lab. A new type of agar was to be used instead of the standard nutrient agar which was called plate count agar (PCA). This was to ensure a greater growth of bacteria once the antibiotics had been mixed in and left to set, with the E. coli streaked out and ready to grow. The antibiotics of choice for this experiment were kendomycin (Km), tetracyclin (Tc), ampicillin (Ap) and cloramphenicol (Cm). This preparation involved some very delicate weighing techniques; for Km and Ap, only 0.5 grams was needed for every 10ml of squeeky clean, distilled water. In order to keep everything as sterile as possible, a sterile syringe and a filter were also used. Tc and Cm, however, were even more tricky, this was due to the fact that it was necessary to dissolve their complex structures in 70% ethanol (not to be used for a tasty tipple, nightcap or anything along those lines), they also required only a mere 0.1 grams per 10ml of ethanol. A slight problem arose when Cm accidentally had water added to it instead of ethanol (no idea who did that…), but this was swiftly corrected by adding another 0.1 grams of Cm and 1oml of ethanol to the test tube. After all this work though, the agar was not quite ready to pour, so it was time for a break. But here is a picture of the antibiotic tetracyclin, in its neon yellow glory:

"Ooh yellow" - tetracyclin

 

Sophie and Tammy were given the task of investigating if samples of   E. faecium, which had been  stored in glycerol at the university,  were still alive and could be regrown and stored.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2010.04932.x/full

To achieve this we first needed to check each sample to see if the organism was alive. This required us to first grow them on agar plates suitable for the nutrient requirements of the microorganism, in this case M17 agar. The samples were then incubated and checked for signs of growth. We could then recorded the findings and the live samples were then stored correctly in a mixture of M17 agar broth and glycerol before being frozen and stored for at -86 degrees centigrade in a special low temperature refridgerator for future use.

Their first job was to make the M17 agar for bacteria to grow on. This was achieved by suspending 48.25 grams of M17 powderd agar into 950ml of distilled water and then bring it to the boil on a hot plate with a magnetic stirrer to blend the mixture. The mixture was sterilised by autoclaving and then left to cool. Just before use 50ml of sterile lactose solution (10%w/v) was added and they were good to go.  They poured the agar solution into the agar plates and left to cool, to be used later for producing streak plates.

Dry ice sublimating

After lunch, the PCA  made by Sarah and Hayley was ready to be poured. A small amount of antibiotic was pipetted into an empty petri dish, and it was then covered with a layer of molten PCA. After this, it was time for the swirling to begin, thoroughly mixing the antibiotic through the plate. This task took relatively little time, so whilst the plates were left to set, it was time for the dry ice fun to begin!

Dry ice is actually the solid form of carbon dioxide, and as seen in the picture on the left, the ice has been placed in a beaker of water and undergoes the process of sublimation (changing directly from a solid to a gas – without being a liquid), producing the strange fog/mist favoured by progressive rock bands the world over.

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