Rain, Rain Go Away….

I don’t know about the rest of the class, but I am tired of this nasty weather that’s been plaguing us the past week. The storm of Wednesday caused a good bit of damage to the trees around campus and the Metro area. Hopefully the weather next week will be bright and sunny so I can enjoy my final few weeks as an LSU undergraduate.
On Monday we began by discussing how utilization of a carbon source can be used to characterize and differentiate bacteria. We were assigned to plate our 26 strains on to media containing two and a half grams of yeast extract per liter, one gram of calcium carbonate per liter (basic component), ten grams of salt per liter (most of the bacteria grew on Marine Agar which contains ~3% salt) and one gram of carbon source per liter (control plate didn’t have one of the sixteen carbon sources we were testing). We spot-plated ten microliters of a cell suspension solution (six spots per plate) made by Eugene on to a control and the sixteen plates that contain only one carbon source each. I had to learn a difficult lesson, when plating so many plates I figured I could do all of them at once, the problem was I confused my lid order of the plates. This effectively made it impossible to tell which plate contained which carbon source. One of my fellow group members also did the same thing, go figure. For my second go-round, I only did six plates at once to prevent any confusion and decrease chance of contamination. Then we parafilmed the plates and placed them in the incubator. The different plates/carbon sources and their labels are:
1. Control C
2. Arabinose AR
3. Dulictol DL
4. Galactose GA
5. Glucose GU
6. Glycerol GY
7. Inositol IN
8. Lactose LA
9. Mannitol MN
10. Melezitose MZ
11. Melibiose MB
12. Raffinose RF
13. Rhannose RH
14. Ribose RI
15. Sucrose SU
16. Xylose XY
17. Xylan XN

We consulted our master chart to see which of our 26 strains grew at 10 C; we gathered the stock plates of those organisms to be plated on new optimal media (two organisms per plate) to be placed in the 6 C incubator. A single streak was used to inoculate the media. We plated strains: 30, 31, 32, 34, 35, 36, 37, 38, 47, 42, 44, 45, 46, 47, 48, and 51. The plates were then parafilmed and placed in the 6 C incubator. Our last task of the day involved plating a new stock strain 40 plate because it had contamination on it.
Our next lab began with the class observing and recording (scoring growth with pluses and noting the pigmentation) the results of the 26 strains growth on pH plates (4 to 10 pH). This will hopefully give us an idea of the optimal pH for each organism. At pH 4, no growth of the 26 strains was observed. At pH 5, only five of our 26 strains grew. At pH 6, six organisms had between three and four plus growth and the majority of the rest had one plus growth. At pH 7, eight organisms had between three and four plus growth and the majority of the rest had one plus growth. At pH 8, eight organisms had three plus growth and the majority of the rest had one plus growth. At pH 9, nine organisms had between three and four plus growth and the majority of the rest had one plus growth. At pH 10, ten organisms had no growth and the majority of the rest had one plus growth. We selected strain 31 to take pictures because it showed how pH can affect the growth of an organism as well as the pigmentation.

pH 4: 0+, no growth

pH 5: 0+, no growth
pH 6: 3+, light orange (BEST GROWTH)
pH 7: 2+, light orange

pH 8: 2+, light orange
* For some reason our pH 8 plate picture was missing, this may be due to the initial media label being incorrect (based on visual comparison to other plates by Dr. Rainey) and this may not have been considered when the pictures were taken. Based on my recorded notations, these pictures should be the correct.

pH 9: 2+, white
pH 10: 0+, no growth

Our final task of the day was DNA extraction from our Little Red Hill soil sample. We are trying to get a general idea of the Geodermatophilaceae present in our soil via extraction coupled with gel electrophoresis. We used a different extraction method (MOBIO’s Ultra Clean™ Mega Soil DNA Kit) this time because we were dealing with soil instead of just media plates of organisms (method is located on Airset and within my notebook). During the extensive down-time during the DNA extraction, Dr. Rainey was kind enough to bring some delicious doughnuts and milk to snack on while Manesh made a traditional Indian dish. After we finished the final step we placed our DNA in the refrigerator to prevent any damage/ breakdown of our DNA. In the next lab, we will perform gel electrophoresis on our sample DNA; we are looking for DNA bands approximately 550 to 560 in length which is characteristic of our target organisms.