Monday we started the second step of the three step process of our desiccation experiment as some might remember about 6 weeks ago we added 10ul drops of cell suspensions to 24 well plates and left these plates in an environment that has less than 5% humidity. After this long drying out process we needed to rehydrate them to remove them from the wells and plate them. We modified the typical procedure that Manish explained because we did not add the typical 100ul of suspension in the original set up. So instead of rehydrating with 1ml of media we rehydrated with 500ul this was to try and keep the concentration of cells at a decent level. We rehydrated for 30min and mixed the media and cells for 30sec being careful not to allow the suspension to aspirate into the barrel of the pipette. Then 10ul of this newly formed suspension was plated on the media each isolate grew best on. The third part of this experiment reading the results will come in 20days. Also the 9kGY gamma radiation test was read only 2 of our 26 isolates grew after the dose one was really good growth and the other not so good some cells survived but the majority probably different they will be in the pictures below. It will be interesting to see the results of the desiccation experiment because in theory since all of these isolates came from desiccated environments they should have no problem surviving to some degree the desiccation.
Wed was a large amount of spotting another round of carbon sources was prepared in the same manner as the previous set. 17 sources plus one control were plated. There was a mistake Eugene’s handwriting is not the best and his L’s look like C’s and vice versa so when he designated alanine as AL and acetate as AC no one including him could distinguish those plates from one another. They will have to be remade. There was another mistake that is easily overcome. The sorbitol plates were labeled with two designations SO and SB no other carbon sources have either of those designations so there is no mix up. The cell suspensions that were used to spot the plates were the same suspensions used last Monday when spotting the first set of carbohydrate utilization plates. For my group there was one exception the number 12 isolate suspension was lost and a new one had to be remade. It was remade in the same cell washing protocol as the first set. After spotting all of the plates we watched as Group three preformed a PCR clean up of one of our PCRs I believe it to be a 16s PCR to finish identifying all of the isolates but I’m not sure. Once the clean up was complete a 1%agarose gel was run and picture taken once the computer program was fixed. Some of the PCR product was lost so they will be done again. Hope everyone has a good spring break.
Wed was a large amount of spotting another round of carbon sources was prepared in the same manner as the previous set. 17 sources plus one control were plated. There was a mistake Eugene’s handwriting is not the best and his L’s look like C’s and vice versa so when he designated alanine as AL and acetate as AC no one including him could distinguish those plates from one another. They will have to be remade. There was another mistake that is easily overcome. The sorbitol plates were labeled with two designations SO and SB no other carbon sources have either of those designations so there is no mix up. The cell suspensions that were used to spot the plates were the same suspensions used last Monday when spotting the first set of carbohydrate utilization plates. For my group there was one exception the number 12 isolate suspension was lost and a new one had to be remade. It was remade in the same cell washing protocol as the first set. After spotting all of the plates we watched as Group three preformed a PCR clean up of one of our PCRs I believe it to be a 16s PCR to finish identifying all of the isolates but I’m not sure. Once the clean up was complete a 1%agarose gel was run and picture taken once the computer program was fixed. Some of the PCR product was lost so they will be done again. Hope everyone has a good spring break.
9kGy isolate 5 some cells survived most didn’t
9kGy isolate 25 did fairly well
In the bottom picture...the spot that is orange and black...did it start out as black or orange only? I know some of our strains had black dots in the orange spot, so I just supposed that it started one color but after experimentation (radiation, temp...whatever) it changed, but that does not seem to be the case here. So it must be two different colonies, but why... this is interesting!
ReplyDeleteThis answers my question from another blog I asked about. Here is a perfect example of desication and irradiation resistance.
ReplyDeletei figured that some of the colonies that seem to be two colors just were contaminated with liquid cultures from other strains or something..
ReplyDelete