Things Remembered............

This week we performed some experiments that I am fairly familiar with. Over Monday and Wednesday the class with the help of Dr. Manish performed radiation and desiccation experiments. On Monday Rachel, Eugene and Manish took our isolates and suspended them in their respective media broths and dispensed them equally into 4 tubes. These tubes were exposed to various levels of Gamma radiation 0, 3, 6, and 16 kGy. These samples were then used to inoculate their media plates using the spot technique with 10ul of each sample. The 0kGy plates are the control for this experiment and the desiccation experiment that was also started. 10ul of the 0kGy samples was taken and put into a well on a 24 well micro titer plate which will stay in a desiccator until 20 days before the final class date.
The reason we are doing these experiments is there is a relationship between desiccation resistance and Gamma radiation resistance. Over evolution as organisms lived in a desiccated environment they developed ways to protect themselves from that environment. This includes developing methods to repair double stranded DNA breaks and Oxidative damage. It just so happens that these types of damage are also caused by Gamma Radiation. So because the isolates come from the dessert soil which is a desiccated environment they in theory should also be able to survive some level of Radiation. These experiments will show if they really are desiccation resistant and to approximately what level Radiation resistant they are.

Also to wrap up the week we counted our unirradiated soil sample serial dilutions, and plated the majority of our isolates for a starch hydrolysis test. The starch hydrolysis test will be graded in 20 days. Counting the serial dilution plates tells us the CFU's per gram of soil from our sample site. It also shows us possible nutrient availability in the soil. This can be deduced from the media in which your sample grew best. 10 colonies were taken from these counted plates and restruck on new plates to have some new isolates to further study.Our sample was N97-2 and the plates tell a story. When looking at the -1 dilution plates over all the media it can be seen that on the higher nutrient plates there was one major colony morphology. This shows that one mainly one type of organisms liked these plates however on the MA plate the colonies are smaller than the PCA plate. To me this shows that even though the organisms like higher nutrients they can withstand 3% salt but doesn't necessarily like it. The lower nutrient Medias have the better diversity but 1/100th PCA was definitely the most diverse. But 1/10th had more colonies in the color range we were looking for. These plates show that the N97-2 sample came from a site with low nutrients and possibly some salt but not very high amount.

N97-2 -1 dilution on PCA:

N97-2 -1 dilution on MA:

N97-2 -1 dilution on 1/10 strength PCA:

N97-2 -1 dilution on 1/100 strength PCA: