Another Very Busy Week

This week was extremely busy and frustrating, at times, but our group accomplised a lot. On Monday we counted our irradiated soil dilution plates, completed the streaking of our starch plates, checked for and recorded growth in our liquid cultures (made from our stock cultures), selected 10 colonies from the irradiated plates that we thought could be Modestobacter and restreaked them onto new plates, and lastly we streaked 10ul from our diluted (-1,-2,-3) irradiated cultures onto media plates. When we counted our irradiated dilution plates we found that the most growth occurred on 1/100 PCA, with an average of 132 colonies per plate. We found this very interesting because this average was even higher than the average for the unirradiated 1/100 PCA plate (which had an average of 58 colonies per plate). We think this might be because one organism was inhibiting the growth of a particular species on the unirradiated plates. However, when the samples were irradiated, this inhibitor was killed, which allowed other species to flourish. The second highest growth occurred on 1/10 PCA (76.3 average colonies per plate) followed by PCA (average of 11 colonies per plate) and finally MA (6.6 colonies per plate). After counting the colonies, we took pictures of -1,-2,-3 1/10 PCA to show the effect of dilution on colony count. We also took pictures of -1 PCA, -1 MA, -1 1/10 PCA, and -1 1/100 PCA in order to show the effect of different media on colony count. Lastly, we calculated the cfu/mL for each type of media using each -1 plate and calculated the percent survival by dividing the irradiated cfu/mL by the unradiated cfu/mL. Our results are as follows:

Media Type--Unradiated cfu/mL--Irradiated cfu/mL---% Survival

---1/10 PCA---------1.02 x 10^4---------------7.63 x10^4----------74.8%--------

---1/100 PCA--------5.8 x 10^3---------------1.32 x 10^4---------227.6% (!!!!)-

----PCA------------2.633 x 10^3--------------1.1 x 10^3------------41.78%--------

-----MA-------------9.7 x 10^3-------------------666-------------------6.87%--------

From these results, we found that, in general, radiation reduces cfu/mL and that all of the groups' percentage survival ranged from 0-227.6%. From this dilution series we chose 10 colonies that we thought might be Modestobacter and attempted to isolate them by streaking them onto new plates. We chose black, peach, orange, and pink colonies from the -1 dilutions of 1/100, 1/10, PCA, and MA. We streaked two colonies per plate and incubated them at 25°C. We also finished streaking our strains onto the starch plates from our stock plates (strains: 53, 55, 56, 57, 59, 67, 70, 75, and 74). We then parafilmed these plates and incubated them at 25°C. Then, we removed our liquid stock cultures from the 25°C incubator to check for and record growth. To do this, we vortexed the tubes and then observed them for turbidity. We recorded the amount of growth using a scale of 4+ for most turbid (most growth), 1+ as the least turbid and 0 as no growth. All of our tubes did have some growth. We found a range of 1+ to 4+ when examining these tubes for growth. We did not have a liquid culture for 68 (our stock culture was contaminated on the day we made these liquid cultures), so we made a new one using PCB. We incubated this new tube and we will check it for growth in 20 days. Lastly, we removed our liquid dilution series of the irradiated samples (-1, -2, -3) and streaked 10 uL onto plates. We plated these samples onto the type of media each particular strain grows the best on. We then parafilmed and incubated them at 25°C. On Wednesday we observed and recorded growth/color of the colonies on the varying NaCl and temperature plates. Again, we used 4+ as the most growth and 1+ being little growth. These plates were compared to the 25°C temperature plates, which we used as a control. Every strain that was put under varying conditions was compared against the control plate to see if it had less or more growth (Ex: strain 53 on 1% NaCl was compared against strain 53 that was incubated at 25°C). The results our group recorded can be found on AirSet. We found that the majority of our strains preferred 1% or 3% NaCl (most had 3+ to 4+ growth). However, some of our strains were able to grow at 6% NaCl and even 9% NaCl (although the growth was very small, mostly 1+ and a few 2+). We also found that the colonies of each strain tended to change color depending on the amount of NaCl they were exposed to. For example, a colony that grew bright orange on 1% NaCl was a pastel orange on 6% NaCl. I found on the internet that this color change could be because of secondary metabolites that are produced when excess salt is present. I'm not sure if this is the case for our strains, though. For the temperature portion of the experiment, we found that most strains preferred a range from 25°C to 37°C, but there were some strains that were viable at 10°C and 42°C, as well (even some with as much as 4+ growth!!). We did not notice as much of a color change due to varying temperatures. There were a few strains that had a slight color variation correlating to temperature increase, but it was not as dramatic as color changes due to changing salt concentration.Unfortunately, the strains of 59 and 69 that were put under varying temperatures were contaminated with a fuzzy, white growth. We replated these for each temperature (using the stock plate) and we will observe these plates in 20 days. We also had to replate strain 68 for both the temperature and the salt experiment. We had to do this because this strain was not plated to begin with (the original stock plate had contamination on it. We had to restreak the stock culture onto a new plate. On the day this experiment was performed, the new stock culture had not grown yet). The last thing that we did on Wednesday was restreak our stock cultures using the plates that we had incubated at 25°C for the temperature experiment. We did this in order to keep new colonies growing, since the colonies on the original stock plates were getting old and difficult to remove. I'm sure they were also undergoing phenotype and chemotype changes due to their age.Below you can see the effects of dilution on our irradiated 1/10 PCA plates. Notice how the number of colonies drastically decreases with increasing dilution. These pictures were taken on Monday.