Monday was an interestingly long class day. We went to the microscopy center in the basement of life sciences to see if we could see anything interesting about our isolates. Each group took 2 of their 26 isolates that were chosen by Dr. Rainey before he had left down to the center to see what they looked like under light microscopy, DIC, and Electron microscopy with negative stain. DIC is a type of microscopy that has to do with the phase shift that the bacterial cell causes in the transmitted wave of light. Light microscopy worked to some degree my group had a little trouble actually finding any cells could be we didn’t put enough cell mass into the suspension. However, when Ms. Cindy did find some cells in our AT03-34-2 slide she said it looked like Deinococcus. The other groups didn’t fair that much better a few pictures were taken which we got back Thursday. One was of a black pigment that she thought was a heavy metal substance. Upon conversation with Manish I find out that melanin in general has a high Iron content so she could have been right. We tried DAPI stain which is a fluorescent stain that binds to nucleic acids and fluoresces blue under UV light, but none of our organisms seemed to take up any of the stain. This could be do to the facts that because of the environment that they live in the organisms are not that permeable to water. This lack of permeability would stop the DAPI from crossing into organisms. After it was concluded that our strains would not pick up the DAPI we were shown an example slide that was made from a biofilm she had in the lab. Next on our list was TEM with negative stain again the first procedure did not work, and it needed to be changed for our organisms. Originally she had floated the copper grid on top a drop of the cell suspension before treating it with uranium acetate. The corrected procedure was with the cell suspension being placed on top of the copper grid instead and this worked better. There were some globs of things to be seen and a flagellum of one organism. It was said that with a little work and tweaking of procedures we could get a better showing.
Wednesday we ran the gel of the PCR products that was performed last Wednesday. Our gel showed that 6 DNA samples from the original isolates amplified very well with the GEO primers this reaffirms what we originally expected that these isolates are of the Geodermatophilaceae family. The other 16 samples from the gel are of the isolates that were obtained from the irradiated soil serial dilution plating. All except for 2 had no band. The two that had a weak band were the two isolates named N97-6 and N97-19 because of the presence of at least a weak band further testing will be done to these isolates they will first be included in the 16s rRNA amplification and sequence coming in the next few classes. The rest of the class was spent grading the remaining temperature test that was plated on the Wed after Mardi Gras along with the Avicell, Granular Cellulose, and Xylan test plated on the same day. Avicell is a type of cellulose. All of the cellulose and Xylan plates had good growth on them, but after analyzing their cellulose or xylan degradation it was clear just because they grew well didn’t mean they degraded the polymer. To test if the organisms degraded the two types of cellulose Congo Red at a concentration of 1g/L was added to the plates after 15min it was poured off and 1M NaCl was added for 15min after dumping off the NaCl the zone of hydrolysis would be measured anything over 2mm is a positive result. For those interested the paper that this procedure comes out of, according to Dr. Rainey, was Dr. Rainey’s 13th paper. Xylan had the same parameters as the cellulose except instead of Congo Red Iodine was used. Sadly the class result was negative for degradation of all polymers by all isolates.
Image 1 is of the Gel containing only the DNA samples from the Serial dilution isolates as you can see there are some bands present and most are weak.
Wednesday we ran the gel of the PCR products that was performed last Wednesday. Our gel showed that 6 DNA samples from the original isolates amplified very well with the GEO primers this reaffirms what we originally expected that these isolates are of the Geodermatophilaceae family. The other 16 samples from the gel are of the isolates that were obtained from the irradiated soil serial dilution plating. All except for 2 had no band. The two that had a weak band were the two isolates named N97-6 and N97-19 because of the presence of at least a weak band further testing will be done to these isolates they will first be included in the 16s rRNA amplification and sequence coming in the next few classes. The rest of the class was spent grading the remaining temperature test that was plated on the Wed after Mardi Gras along with the Avicell, Granular Cellulose, and Xylan test plated on the same day. Avicell is a type of cellulose. All of the cellulose and Xylan plates had good growth on them, but after analyzing their cellulose or xylan degradation it was clear just because they grew well didn’t mean they degraded the polymer. To test if the organisms degraded the two types of cellulose Congo Red at a concentration of 1g/L was added to the plates after 15min it was poured off and 1M NaCl was added for 15min after dumping off the NaCl the zone of hydrolysis would be measured anything over 2mm is a positive result. For those interested the paper that this procedure comes out of, according to Dr. Rainey, was Dr. Rainey’s 13th paper. Xylan had the same parameters as the cellulose except instead of Congo Red Iodine was used. Sadly the class result was negative for degradation of all polymers by all isolates.
Image 1 is of the Gel containing only the DNA samples from the Serial dilution isolates as you can see there are some bands present and most are weak.
Image 2 is of the Gel containing the PCR products from the 16 serial dilution samples plus the 6 samples from the original 26 isolates the bright bands are the original isolate samples the 2 weak bands are from serial dilution isolates giving the conclusions stated earlier that our 6 isolates are highly probable to be GEO’s and the serial dilutions are highly unlikely to be
Is uranyl acetate the same thing as uranium acetate?
ReplyDeleteyes it is
ReplyDeleteI wonder if the dark metal good be manganese. Manganese is though to be in high concentrations in radiation resistant bacteria because it prevents iron dependent protein oxidation.
ReplyDelete