Knock, Knock…who’s there...Lionel…Lionel who…Lionel get you nowhere, better tell the truth!! On Monday, surprisingly, we left almost an hour early! That never happens. But before we left we set up a carbon utilization test. This test uses 16 carbon sources plus a control to see what types of carbon each organism uses. In the Luedemann paper he said that he used basal media for his carbon test. The media we used contains 2.5 g/L of yeast extract (this is the buffer that sucks up the acid that the organism makes), 1g/L of calcium carbonate, 1% NaCl at 10g/L, then the carbon sources were added at 1g/L. Note that we added NaCl because most of the organisms that we are working with require salt to grow. We are hoping for more growth on the plates to which the carbon source was added, opposed to the control. To set up this experiment, we spotted 6, 10uL of each organism on the control and also on each carbon source plate. Eugene used stock plates to prepare the organisms for this experiment. Since the organisms were already growing on some type of media in which carbon was already added he had to wash them with saline, centrifuge them, wash them again with saline, and pipette off the saline to remove the carbon source from the plates that they were growing on.
On Wednesday we had ALOT to do. We started off by rating our pH plates that we had done a few weeks before. In looking at our plates we noticed some contamination, however, it wasn’t enough that we could not tell the amount of growth. In looking at the plates there wasn’t a common growth trend and this really seemed weird since the organisms we are working with are from the same family, Geodermatophilaceae. Although not all, but some organisms did show some sort of a growth trend. You can see that at pH’s 4-5 the organism 31 did not grow.

This is probably because the media was too acidic and prohibited the organism from growing. At pH’s 6,7, and 9 it grew well and did not grow at pH 8. Because the organism grew at pH 9 and not 8, it is a chance that those two plates were mixed up. So the pH 9 plate is probably the pH 8 plate, and vice versa. In looking at our plates we figured the optimum pH for these organisms is around 7-7.5. In reading the papers, this was confirmed.

After that, we started our DNA extraction which took FOR-EV-ER, FOR-EV-ER (as they said on the Sandlot)!! We used the MOBIO UltraClean Mega Soil DNA Kit. The items in this kit are much larger than those in the other DNA kit we used before. This is probably because this kit is used to extract DNA from soil, not directly from plated cultures. We put 10 g of our soil sample in the bead tube and vigorously vortexed it to mix the contents. We also vortexed our tube for an additional 30 minutes. While the tubes were being vortexed for 30 minutes, we enjoyed our lovely treat, Krispy Kreme and Milk from the Promiseland, courtesy of Dr. Rainey. OH, and I can’t forget the delicious potatoes that Manish’s wife made. After all of that we were near completion of the DNA extraction. We spinned (is this even a word) the Spin Filter 3 times, not 2, this was to get as much DNA on the filter as possible. We also added 4 mL of the MD5 solution instead of 8 to make the DNA “stronger.” After completion of our kit, we froze our DNA for later use on “next day,” as Dr. Rainey would say.

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.

here you go frederico!

On Monday, we conducted carbon utilization test on our 78 strains. Eugene prepared basal media prior to our lab meeting for us. Thank you Eugene! The basal media consist:
1) 11 g Yeast Extract (2.5g Yeast Extract/ Liter)
2) 1g Calcium Carbonate/Liter
a. The calcium carbonate is a buffer that neutralizes the acid produced by the organisms
3) 1% NaCl (10g NaCl/ Liter)
We added one of 16 carbon sources to this media. In addition, one set of plates was deemed the control and had no carbon source added to the basal media. We will use this plate as a comparison to see if there was more growth on the control plates or on the media containing the carbon sources. The carbon sources that we used are:
1. Control C
2. Arabinos 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
By the way, I hope that all of you are aware of the fact that I made a mistake when typing up the carbon sources for everybody. The C is CONTROL not carbon.
We sported 10 ul of each strain onto each carbon sources. There are 6 strains/plate. Cells were washed with saline prior to spotting to remove any remnants of the previous media. The plates were then incubated at 25 C.
Also on Monday, we used our stock plates to streak strains that grew at 10C and incubated them at 4 C. All of our group’s 26 strains grew at 10 C and therefore were restreaked and incubated at 4 C except for strains 54, 58, 68, and 78.
On Wednesday, we observed the results of our pH plates. The strains were streaked on media at pH 4-10. A special thanks to everyone we came in over Mardi Gras to prepare these plates for us. We had some pretty interesting results and they are located on airset. Most notably, color changes were observed in several strains at the various pH’s. I am really excited about strain 69. At pH 6 it was a smooth black colony. However as the pH increased (7-10) it began to form a cream/peach color around its parameter. This made me think of Modestobacter versicolor. However, in the paper pH did not induce a color change. Nutrient conditions elicited the color change. Who knows! I’m still excited. Our plates containing strains 67,69,70, 75, and 76 are awesome. The color of the colonies of these plates look like it could be all three genera of interest. There is a orange colony (maybe Blastococcus), two black colonies (Geodermatophilus), a cream colony (Modestobacter multisepatus/versicolor), and a colony that is centrally dark with a cream parameter (Modestobacter versicolor),

pH 7 plate containing 67,69, 70, 75, and 76

Lastly on Wednesday, we extracted DNA from our three soil samples using the protocol on airset. 10g of each soil was used.
Remind me to tell yall on Monday a really great story that happened at the Circle K last night.

Monday we.......

Monday we spot plated our isolates on carbohydrate utilization media which took the majority of the class period. We also began a 4oC temperature test any isolate that grew at 10oC was plated and put in the 4oC cold room. Any isolates that had become contaminated over time were also streaked onto new stock plates. The carbohydrate utilization media contains only inorganic nutrients, chemicals the cell can’t make on its own like certain vitamins and a single carbon source. The inorganic nutrients and unmakeable chemicals were provided in the media’s Yeast Extract component. Calcium Carbonate was also added this was to absorb any acid that was made in the carbohydrate utilization process. A clearing around the cell mass will be seen if acid was actually produced. Salt was added to the media because some of our organisms grow best on MA and require at least a little salt and all of the organisms will tolerate a little salt. Finally a carbohydrate was added as a carbon source. Taking all this into consideration the Final Media consisted of .25% Yeast Extract .5% CaCO3, 1%NaCl and 1g/L carbohydrate. The media is a modified version that Leudemann used in his paper “Geodermatophilus, a New Genus of Dermatophilaceae ( Actinomycetales)”. 16 carbohydrates and 1 control were tested this day. Results will give a large amount of data to be used further our knowledge about these isolates.
Wednesday was a busy day we extracted DNA from organisms that lived in Soil Samples. The Soil was taken from the same location that our previous Soil experiments was taken from in my case it is sample N97 which is from Nevada. The extraction process was performed with a 10g sample of soil and was fairly similar to the process of DNA extraction from cells. Before we began extracting DNA we graded our pH test that was started after the midterm examination. All already described species have a wide range of possible pHs they grow at. This experiment can help us further align our isolates with which described species it best resembles. The majority of my group’s results showed a decent growth from ph6-10 with a few exceptions. These exceptions are not surprising in that they could be one of the Modestobacter sp. that have a range of pH 3-12. A color change at higher pH occurred usually around pH 9 and maintaining through pH 10. There was one exception which is shown in the provided pictures and that was isolate number 7 it usually has a dark orangey center with a light cream to pinkish ring around the outside and as the pH increased the pinkish ring got small and the orangey middle got darker, but at pH 9 it changed to just the cream pink color and reverted back to the orange and pink morphology at pH 10. As one can see in the pH6 photo this isolate seems to have a mixture of colony colors the pink and orange so the normal morphology is not a far stretch of the imagination. However, what would be the reason for only the growth of one colony morphology between pHs.
I would like to thank Cristi for her help in jogging my memory about Monday.

pH4 no growth

pH5 no growth pH6 growth notice the two colony morphologies of isolate 7 pH7 decent growth pH8 decent growth pH9 notice color change of isolate 7 pH10 again color change of isolate 7

So this was the best week ever, by far!!

On Monday, we set up carbon utilization test, incubated our strains at four degree celsius, and restreaded out stock cutures which were contaminated. The carbon utilization test consist of 16 carbon sources and 1 control. Each strain was spotted on each of the different carbon sources and on the control. The experimental plates consist of 2.5 g/L of yeast abstract, 1g/L of calcium carbonate, 10 g/L of 1% NaCl, and 1g/L of the carbon source. The control plates were similar; however, they lacked the carbon source. The yeast which was in the basil medium was necessary because the organism needs some type of nutrient to grow on; carbon alone would not be sufficient. We spotted 10 uL of the strains onto the plate (6 strains per plate). This was a very simple procedure however some how we managed to screw things up a little. You heard right! While spotting the plates the lids were taken off so that the spots would be able to dry; however, we (me and one of my lab partners) forgot which lid went with which plate so we had to toss the plates and start again. Other than that the experiment was quite successful, so far! Next we reviewed our temperature charts to see which strains grew at 10 degrees. Those plates which grew were streaked unto new plates and incubated at 4 degrees. This would allow us to see which strains could sustain these low temperatures. After reviewing, those which could not sustain these temperatures will be incubated at 6 degrees, but this is another story for another day. Any how, next we restreaked our stock cultures which were contaminated. That pretty much wrapped up Monday! Wednesday, was the big shin dig!!! First we checked our pH plates for growth. Through our findings I concluded that the plates were labeled wrong. For example strain 28 had an optimum growth at a pH of 7, none at 8, and a large colony of growth at 9. This does not seem very consistent; therefore, I believe that the 8 and 9 plates were labeled incorrectly. Next we performed DNA extraction. This ran rather smoothly! The best part of it all was when we took our little break while the tubes were in the water bath. Rainey bought us doughnuts with WHOLE milk :), and Manish bought some potato balls :)!! I must say this week was worth all of the hassle Rainey gives!! Just joking Rainey, we know you dont really think were bloody people haha!

I love doughnuts!

This was a productive week! I finished extracting DNA from all of the 4126 collection strain organisms that we did not have 16S for on Wednesday morning as part of my BIOL 3999 activities. Monday afternoon we set up for carbohydrate utilization tests on basal media plates containing 17 (correct me if I’m wrong I forgot to write them all in my book) varying types of carbohydrates. The media contained no carbon source yet 1% NaCl was added due to the fact that all the strains grew well when tested at this salt concentration. 2.5g of yeast extract/L along with 1g of CaCO3, a buffer that soaks up acid produced, made up the rest of the media. We spotted 10 µl of a liquid suspension of our strains that had been rinsed in saline to get off any residue from its previous media that could possible aid in its growth. We also streaked our strains that grew at 10°C onto plates of their optimal media to be incubated at 4°C in the cold room. 22 out of 26 of our strains grew at 10°C so I am excited to see which, if any, can grow at 4°C. Wednesday we extracted DNA from our soil samples using the Ultra Clean Mega Soil DNA Kit. It was fun and I found it easier than the regular small bead beating kit but maybe because it wasn’t so many small tubes. We also observed our pH plates. We had a couple of strains that had some interesting results. For strain 7 we observed a lighter pink band around the outside with a darker center beginning at pH 6 but then at pH 9 is suddenly appeared cream then it was right back to pink at pH 10. Strain 8 was also dark in the center but had a light, almost white, band around the edge at pH 6 and 7 but lost it and the entire colony appeared light pink. Strain 16 was our only strain that displayed strong growth at all pH levels tested. Strain 24 grew the best at pH 4 then had no growth at pH 5 then moderate growth from pH 6- pH 10. I am not sure what happened at pH 5 but we might need to test it again. Overall our strains did not grow well at pH 4 or 5. Only 4 grew at pH 4 and 3 at pH 5. pH’s 6- 10 had growth for all the strains. Below you can see our pH plates for strains 1,2,4,5 and 7. None of these grew at pH 4 and 5 and all grew from 6-10. Strain 7, mentioned above, can be seen in this picture. The best part about the week was our party Wednesday… we should do that more often!!

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

This week was very productive and fun!

On Monday, we started our carbon utilization tests (using 16 different carbon sources and a control). Each type of plate started with a basal (or is it basic?) medium which contains yeast extract, calcium carbonate, and 1% NaCl. The salt was added since some strains grow best on MA and all strains grew on the 1% salt plates. To this basal medium a carbon source was added (1 g/L). We are hoping that we will find more growth on media with a carbon source than on the basal medium (which does not contain a carbon source). Does this mean that organisms growing on the basal medium are autotrophs? Anyway, we spotted 10 uL of sample on each type of plate (17 types of plates). We put 6 samples on each plate. Before we used the sample, it was washed with saline to remove other carbon sources. Also, we restreaked strains that grew at 10C onto new plates and incubated them at 4C. Surprisingly, most of our strains (21 out of 26) grew at 10C. This was surprising to me since the optimum temperatures for Geodermatophilus, Blastococcus, and Modestobacter species is around 25-30C.On Wednesday, we analyzed our pH experiment plates and recorded their growth in a spread sheet. Very few of our strains grew at 4 and 5 pH, but most grew at 9-10 pH…this shows that our strains prefer more basic pH rather than a more acidic pH. We also extracted DNA from our Gobi soil sample on Wednesday. We followed the protocol given by the MoBio kit. This procedure took a long time, but it wasn’t bad since we had YUMMY donuts and got to socialize :-) . Hopefully, we performed a good DNA extraction.

Below you can see a picture of our suspected M. versicolor strain! We love this strain! It seems to change color in the media from coral to green/black and we think this is because it may be running out of nutrients (since this species is coral on nutrient rich media but black/green/brown on low nutrient media)! This is a picture from our pH 7 plate (strain 69 is in bottom right corner):

BLOG.......

On Monday we tested our cultures for carbon utilization. First, a basal media was prepared based on the Luedemann paper, which included 25g/L of yeast abstract, 1g/L of Calcium Carbonate, and 10g/L of NaCl. The 1% NaCl was added to the media because some the stains grew best on marine agar, and also, all the strains had at least a 1% salt tolerance. Control plates were made with just the basal media. Then to the basal media, 16 different carbons sources were added at 1g/L, which included: arabinos (AR),dulictol (DL), galactose (GA), glucose (GU), glycerol (GY), inositol (IN), lactose (LA), mannitol (MN), melezitose (MZ), melibiose (MB), raffinose (RF), rhannose (RH), ribose (RI), sucrose (SU), xylose (XY), and xylan (XN). 10µl of each strain in liquid media was spotted onto the seventeen different plates. The liquid media containing the cultures was prepared by scrapping the cultures from the stalk plates, then washing them with saline to remove any remnants of the previous media including the carbon sources. The growth of the culture on the control will be compared to the different carbon sources. Next, plates were streaked from the stalk plates of the stains that grew at 10ºC to test for growth at 4ºC.
On Wednesday, we first recorded the results from the pH tests. We had many interesting finds. Many of the strains were more tolerant to higher basic pHs than acidic pHs. Also, a few of our strains actually changed color with changing pH. Then we extracted DNA from the dirt samples we used previously in the class. The MoBio soil DNA extraction kit was used with 10g of soil, and the protocol was followed. These DNA samples will be used in a PCR with GEO primers to see if our dirt samples contain any of the studied genera.
The pictures of our pH plates (see those in post of Yellow Rive below) show how stain #69 actually changes color with increasing pH.

New data and more to come......

On Monday we started our carbon source utilization tests. We did this using the medium described by Ludeman (1968). Eugene had prepared 16 different carbon sources plus basal medium without an added carbon source (this will act as the control). We spotted 10ul of a cell suspension of each of our strains. The cells had been washed with 0.9% saline to remove any residual carbon source from the initial growth medium.

For the strains that had shown good growth at 10C we streaked these (from the stock plate – grown at 25C) on the medium they grow best on and incubated them at 4C. If these grow at 4C after 20 days we will reduce them to 2C.

The pH plates which had been incubated for 20 days at 25C were scored on Wednesday and we found that some strains grew through the range pH 4 to pH10. Others did not grow at either end of the range tested. We scored these as 1-4 + so as we can say what the optimum pH for growth was as well as the range. See a very nice example of the plates from our pH experiment below. Interestingly these strains did not grow at pH 4 or pH 5 but did grow at pH 6 through pH 10. Also of interest is the change in color of strain 69 for example (bottom right corner in pictures below) at different pH values. At pH6 strain 69 is black while at 8, 9 and 10 it is orange/tan.

We need to look in our strain excel sheet and see which soils samples these strains (67, 69, 70, 75 and 76) came from. Did they all come from the same sample and what was the pH of the original sample. We should measure the pH of some of the original soil samples which Rainey will have in his lab.

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9 pH 10

In a previous class we demonstrated that we could use the Geo specific primers (Salazar et al) to test if a strain is a member of the Geodematophilaceae. We used these on some strains from our strain collection as well as on strain we had isolated from the 3 soil samples we used in class. This week we extracted DNA from these same 3 soils (without radiation) using the MOBIO Soil DNA Extraction Kit. We used the kit that starts with 10g of soil. The reason for using 10g is that these desert soils have low numbers of CFUs/g and so will probably have a low DNA yield. We also attempted to maximize the DNA amount we recovered by doing the final step (the elution) using half the amount of MD5 (4ml instead of 8ml). Next week we will use this DNA in PCR using the Geo specific primers to demonstrate the presence of members of the family Geodermatophilaceae in an environmental sample without having to isolate the actual organisms.

Growth doesn’t mean degradation

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.

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

blog.......

On Monday we began class by grabbing our strain 37 and 40 and heading to the basement of Life Sciences to the Microscopy Lab. When we got there we suspended our cultures in DI water. To make the necessary slides for Light Microscopy, we pipeted 2 micro liters of suspension solution onto a cover slip and then allowed the slide and cover slip to suction together. We then applied a drop of DAPI stain to the side of the cover slip. DAPI stain intercalates in between basepairs which causes a conformational change in the DAPI. This change allows it to glow when UV light is shined on it.
Our strain 37 was uniformly shaped ovals clumped together into groups of 3 to 4. We took photos of this organism under the light microscope. Our strain 40 showed possible signs of motility based on the twitching motion observed. Strain 40 varied between elongated ovals to round in shape, occurring in pairs. We took photos of this organism under the light microscope as well. We observed no DNA on both of our slides; probably do to the cells needing to be treated to allow the DAPI in.
An example of how the Dapi works was given by gathering biofilm on a slide. Under UV, lots of DNA could be seen with the Light Microscope.
The professor of the lab prepared our slides for the TEM. On the first go-round neither of our cells appeared, but we she changed the order of making the TEM slides, our organisms appeared. It was possible she didn’t get any cells on the copper/carbon plate. Our strain 37 occurred in clumps and appered as lumpy ovals whose membranes lacked rigidity. Our strain 40 appeared as elongated ovals with singular flagella. Images of both strains were taken
The next class we made a gel to run our PCR products in. We used size marker 3 and we were looking for fragments of about 550 basepairs. We had positive results for strains 34, 44 and 45. Possible reasons why no bands appeared for strains 27, 29 and 48 could be no DNA was extracted to begin with or an error occurred in the PCR. We also had positive results for LRH-4, 9, 11, 12 and 14. . Possible reasons why no bands appeared for LRH-1, 2, 5, and 13 could be no DNA was extracted to begin with, the samples were not the correct organism (we used specific primers), or an error occurred in the PCR. No contamination of the PCR Master Mix was indicated by the lack of bands in the DNA (-) lane. We recorded a photo of our PCR.

Goooooodmooooorning....

Hello, how was your week? Great, mine was interesting as well. On Monday Dr. Rainey was in Mexico so we went to the Microscopy lab in the basement of Life Science. It was very interesting at first when Mrs. Cindy was telling all of the things that we were going to do. We were did DAPI stains, looked at our organisms under the light microscope, and also under the TEM. To prepare our cells for viewing under the light microscope we had to do a little preparation; that involved suspending the cells in 0.5 mL of water and then mix them. We also noted that if any of our organisms were adapted to high salt content that they may explode when added to the water because the osmotic pressure may be too high outside the cell. Lucky for us, this wasn’t the case. We then transferred only 2 uL of the organismal mix onto a slide for viewing. We only used 2uL because if more than that was used the cells may “swim” around the slide even if they aren’t motile. The water would cause them to move around. Before viewing the cells we stained them with DAPI, 4',6-diamidino-2-phenylindole, a florescent stain that intercalates between DNA. To load the DAPI into the cells we added at one end of the slide and that way it worked its way through the entire slide. Below are the pictures from the light micrscope, numbers 37 and 40, respectively. In looking at 37 you can see lots of cells uniformly shaped. They also look lumpy. Also, in looking at 40 we saw the cells moving around. This organism is probably motile because there were cells moving in a different direction than the cells that were all drifting in one direction because of all the water that was used. You can see that some cells look like spirochetes and some occurred in pairs. It was hard to get a good picture because they were moving so fast.When looking at our cells under the TEM we didn’t see very many. So, she made a slide from some gooey fish tank water that she had, and boy, did it have lots of things to see! To prepare the slide for viewing under the TEM we put a collidian on the slide along with the cells. The collidian has 2 sides, a light color and also a dark copper color. We then touched the collidian with the liquid (uranium acetate) which creates a halo around the bacteria. It is useful to use the TEM when you need a higher resolution. You can actually see the difference between two spots on the TV like screen located on the microscope. The resolution is improved so much in the TEM because electron beams improve resolution because the wavelength is shorter. We also added liquid nitrogen to the TEM to keep the vacuum cool. The liquid nitrogen does this by evaporating all of the vapors. Take a look below to see what we saw. In 37 we saw oval, lumpy shaped organisms again. In 40 we saw oval shaped organisms with flagella, and other weird shapes. We also could see some of the flagella that were separated from the organism.On Wednesday were supposed to go and get Jambalaya from outside of Williams and Choppin at 230. I thought Dr. Rainey remembered and that he would tell us what time we could go. Unfortunately, he didn’t remember so we missed the Jambalaya. So for that, he’s bringing us Krispy Kreme on Monday. On a heavier note, since DAPI didn’t work very well with our cells we learned that it could be because they are hydrophobic. To prepare these cells for viewing with DAPI you would have to get rid of the polysaccharide wall. Things that be used are toluene, alcohol, and formaldehyde.We also looked at our temperature, cellulose, xylan, and avircell plates. We stained the xylan plates with iodine and the AC and CG plates with Congo Red. Then once they the stains were there for 15 minutes we poured it off of the plates and added NaCl for 15 min. None of our plates tested positive. While DP and I were doing the hard work, Larry was over on the other side of the room running our PCR out on a gel. Once he was done we could see that our organisms ran out at about 560. This is right on target for Geodermatophilaceae. Look at the red arrows below. They are pointing to some of the organisms that ran out at 560.

I'm a paint master!

On Monday we did microscopy with Mrs. Cindy. Strains 14, 17, 37, 40, 66, and 75 were selected for microscopy. Cindy first showed us how to make slides for microscopy. Cells were scraped off of the plates and innoculated into 2ul of water in a tube. The risk of the water is that it could cells to lyse; therefore, cells would not be visualized during the microscopy. The slides are covered with collodian which helps the cells to stick to the plates. 0.5 ul of the cell solution was pipetted onto a coverslip and the glass slide was placed on top. DAPi was added to the slides as well. DAPi is a fluorescent stain that allows you to see DNA because it binds in between bases in the DNA which causes the DAPi to change conformation. The location of DNA can be visualized using DAPi because it glows blue when viewed with UV light.
The results of our light microscopy were not phenomenol. However we are able to obtain five images of our strains. No image was obtained for strain #14 using light microscopy.
IMAGE 1: Strain 17
· Little and large round cells
· Pairs and tetrads
· Cells congregated at the edge of the water
IMAGE 2: Strain 37
· Cells clumped together
· These cells were larger than strain 17
IMAGE 3: Strain 40
· Possible motility
· Oval shape
· Variety of sizes and shapes
· Some looked like spirochetes
IMAGE 4: Strain 66
· Various shapes and sizes
· Irregular shape
· Big clumps
· Sort sort of dark metal present in cell solution
IMAGE 5: Strain 75
· Motile
· Rods
· Vibrio
The DAPI stain was unsuccessful using the light microscope. This may be due to the fact that DAPI could not penetrate the membranes of the cells. This is probably due to the fact that the cells are hydrobic because they have an extracellular polysaccharide layer. Treating the cells with a chemical such as toluene that would dissolve this polysaccharide layer could make cells permeable to DAPI.
Next, we performed transmission electron microscopy on our select strains. The slide preparation for electron microscopy is very different from light microscopy. A copper grid is used which contains is coated with carbon on one side. The copper acts as a thin film for maintaining cells on the grid. I’m not exactly sure why the copper is used? Somebody might comment and let me know. Anyways, a small drop of cells is placed on the parafilm. The carbon side of the grid is placed on top of the drop. The grid is then removed from the drop using tweezers and dabbed on filter paper. Next the grid is placed on a drop of uranyl acetate and dabbed again on the filter paper. The uranyl acetate is used to visualize the cells. Electrons cannot penetrate through uranyl acetate so it makes everything around the cells dark. It is kind of like a negative stain. After the grid is prepped it is placed onto the electron microscopy. The resolution of the TEM is higher than light microscopy because the wavelength of electrons is smaller than that of the white light used in electron microscopy. Liquid nitrogen is placed inside the TEM to condense the vapor in the vaccume. The vaccume is used because of the small wavelengths of the electrons. A beam of electrons is show down onto the sample. The screen gives off green light when excited by electrons. I’m not sure of the images number for TEM.
37: No images obtained; cells may have exploded
17: tetrads and pairs
14: very small; looked like collapsed soccer balls; long crystals
66 big black blog (some sort of metal precipitate)
75: no images taken
40: oval with flagella; lots of flagella present; detached flagella present; cells are joined together
On Wednesday we read temperature results temperature results for 10C, 45C, and 50C. None of our strains grew at 50C and only three grew at 45C. Most of our 26 strains grew at 10C. Two of the strains that grew at 45C were cream and the other one was black. Distinct color changes were observed in some strains at various temperatures. I’m not exactly sure what may cause this color change.
We also tested xylan, granular cellulose and avicell hydrolysis. The xylan plates were inoculated with iodine. None of our strains hydrolyzed xylan. The granular cellulose and avicell plates were inoculated with congo red for 15 minutes and for a salt solution for 15 minutes. The purpose of the salt solution was to wash away the ubound dye. Our results seem somewhat ambiguous because it was unclear if some of our plates were positive or not. We recorded several strains as being weakly positive for hydrolysis of granular cellulose and avicell.
Sean Michael rain the gel of our PCR results using the Geo specific primer. Our results were not satisfactory. However, I did learn how to interpret a gel. We used latter three. All our 6 strains from Dr. Rainey ran, but only one of our 6 soil isolates ran on the gel. The results are found in the image below. If anybody sees that I labeled anything wrong please let me know because I’m still learning how to do this.

while some were in mexico...we worked hard......

So, this week Rainey decided to abandon us kids and go on a vacation! Luckily for us Cindy invited us to the microscopy lab. In the microscopy lab we worked with the light microscope and the electron microscope. In order to view the organisms under the light microscope we had to follow a few procedures. First we put the colonies into the tube with water. This step could have caused the cell to explode through osmotic pressure, which could have ended really badly. Next, we put a small amount (2 micro liters) of the solution on the cover slip of a slide. If we had put too much of the solution the cells would have moved around even if they were not motile. We then turned the slide upside down and let the cover slip adhere to the slide. We then put water on the slide so it wouldn’t dry out. After, we added DAPI on one side of the slide. DAPI staining intercalates between DNA bases; this form of fluorescent staining works with UV light. The DAPI cannot absorb all of the light without giving off visible light. After this was complete we moved to the room with the light microscope. Here Cindy gave us some tips on using the microscope and focused in on the organisms for us. Some things that we learned about the microscope was that focusing on the word on the slide would help us find the cells better, Also, we were told that a decrease in magnification cause the lens to appear further away from the slide. We used differential contrast and 80X magnification. Under the light microscope the strains appeared as follows:
14- nothing specific showed
17- resembled Deinococcus, suspect Modestobacter, moved a lot because of the liquied, aggregated at the edge of the water, Image 1
37- uniformly shaped cells; a lot present, lumpy in appearance, in pairs and clumps, motile (moved opposite directions, twitching), bigger than strain 17, image 2
40- twitching, possible motile, various shapes from round to long to short, looks like spirochetes, image 3
66- big clumps, irregularly shaped, lots of cells, big piles of cells, looks like Deinococcus, various shapes and sizes, blackish brown colored, looks like its outside the cells, looks stressed or dead, no fluorescent used, Image 4
75- motile, twitching, no fluorescence, vibrio shaped, moves really fast, not Deinococcus, Image 5
This completed part one of our day, next we worked with a biofilm found from the bottom of an aquarium. For this experiment collodion was on the slide to stick the bacteria to it so that it cannot move. We also added Dapi to the slide. This was examined under the light microscope under low magnification. On the slide you can see that the DNA fills up cells. This was apparent from the blue dots present which represented the DNA. The cells moved very quickly so taking a picture of it was quite a task. This is Image 6.
Part three of our experiment consisted of using the TEM (transmission electron microscopy). For this experiment our liquid solution was put on parafilm. The copper was placed dark side down on the liquid sample strain. A drop of uranium acetate was put on the slide. The copper was then picked up with a tweezer and put on filter paper. The uranium acetate formed a coat around the bacteria capsule so that the cells would be visible under the microscope. The TEM shined an electron beam down through the sample; however, we cannot see the electrons. The advantage of using this microscope was that we could tell the difference between two different spots close together because this type of microscope offered better resolution by making wavelength shorter, so the sample appeared more detailed. The screen gives off visible light when excited by electrons. The TEM utilized liquid nitrogen so that the vapors that are given off are condensed. The strains appeared as follows:
37- no image; nothing found
17- no image; nothing found
14- small, balloon like , round shaped
66- big crystal arrangements; dense; Image 7
After these strains appeared to be giving off very little Cindy used a different method. She soaked the strains in uranium acetate.
40- flagella present (1), oval shaped, lots of different cell shapes and sizes
14- occasional coccoids, various sizes some long, tiny, and thin, and some rectangles
66- clumps of cells together
75- nothing present
17- cells together; looks like dumbbells; four cells in a ring formation
37- group of cells stuck together, oval shaped, lumpy
That pretty much sums up Monday! Wednesday Rainey came back, we immediately got back to work. Our assignments consist of: analyzing temperature plates, analyzing and testing the xylan, avacel crystalline, and granular cellulose plates, and running our gel. Majority of the strains did not grow on 45 or 50 degree plates. We ran test on our xylan plates by putting iodine on it to test for the hydrolysis of amylase. We put congo red for 15 minutes than salt for 15 minutes on the CG and AC plates. Our plates test negative for both tests. By running our gel we found out that our isolates were approximately 564 base pairs. Those which showed up were 34, 44, 45, LRH 4, 9, 11, 12, and 14

BLOGGG........

This week began with Dr. Rainey soaking up the rays in Mexico while we were trapped in a dark basement looking at slides J JK! We very much enjoyed our field trip to the basement to do some microscopy. We started off by suspending some cells from our selected strains in .5ml H20 using a wooden stick (unsterilized, eww). We then pipetted 1.5µl of the liquid onto a coverslip and placed the slide onto the coverslip. We were explained to that you don’t want to have more than 2µl of liquid on the coverslip or everything will just be swimming around and hard to focus and take pictures of under the microscope. We also stained the slides with DAPI, 4'-6-Diamidino-2-phenylindole, which forms fluorescent complexes with natural double-stranded DNA. When viewed under UV light you are able to see the organisms’ DNA. Unfortunately we did not have much luck with this stain. We believe this is due to the hydrophobic and membrane characteristics of these organisms. We did however view our slides under the light microscope using differential interference and were able to see our organisms. Strain 17, AT03-34-2, from our group appeared to be little round cells in groups of two that were trying to get to the edge of the water/air line. (sorry cant get image to insert properly but its image 1)
We also attempted to view our organisms using the TEM or transition electron microscope. TEMs use electrons as a light source and have a much lower wavelength making it possible to get a resolution significantly better than with a light microscope. We prepared a copper grid by dipping it into our cell/h20 mixture and then into uranyl acetate. Liquid nitrogen was added to the TEM to condense the molecules. The screen we viewed the slides on gives off visible light by electron excitation. For most of the slides we were not able to see anything. She decided to set up the slides differently by collecting the water from the bottom of the drop rather than the top in hopes of being able to see more. Strain 40 ended up having a cell with a very long, defined flagellum. We were also able to view strain 17 again and saw the same pairing of cells. Overall it was interesting and a fun day! I also highly enjoyed myself at the Hornets game that night!!
Wednesday morning I began the process of extracting DNA using the MOBIO bead beating kit for strains that we do not have 16S information for. In class that afternoon we ran our PCR from last week on gel and recorded data for our temperature and cellulose plates. To the xylan plates we added iodine and to the avicell and granular cellulose plates we added congo red let it sit for fifteen minutes then added NaCl. Unfortunately the results for all strains all tests was negative L Our PCR was semi successful. The first six were strains from our collection and were all positive. The rest were our N97 soil dilution colonies. Only two, N97-6 and N97-19 were weakly positive and suspected Geo’s due to the presence of a band at about 564bp. We will use 16S primers for these. In the image below you can see our gel. The first six are from the strain collection and row one column 11 is N97-6 and row 2 column 9 is N97-19.

Microscopy! PCR!

On Monday, we spend the whole day in the microscopy lab! Our group used strains KR-65 (suspect of Geodermatophilus) and MCC-66 (a B. jujuensis suspect) to look at in the microscopy lab. In order to look at our samples under the microscope, we had to scrape colonies off of our plate and suspend them in .5 mL of water. We looked at a small amount of these suspensions (about 2 uL) on a slide under light microscopy (which allows us to veiw live cells). We stained these slides with DAPI, which allows one to see where the DNA is in the cell. The DAPI, which is a dye that intercalates between base pairs, fluoresces under UV light. This technique did not work (probably because the cells are resistant to the DAPI--remember, the are hydrophobic!! They might need to be treated with something else first, like toluene, which makes the cells permeable). We also viewed the slides using DIC microscopy. Some interesting things we saw using DIC included: strain #40 which showed motile, twitching cells, #66 which showed clumps of cells that were irregularly shpaed and looked stressed/dead, and #75 which also showed motile, twitching, fast moving cells. Lastly, we viewed our samples using a TEM microscope. The electrons that shine down on the slide when using this technique allow us to see the difference between closely spaced objects (in other words, it increases resolution). We also added liquid nitrogen to a tank on the side of the microscope. This is used to help keep a vacuum going and to condense vapor. To use this microscope, we put 50uL drop of cell suspension on parafilm and floated a copper-looking circle on top of the drop. The we dropped the copper circle on top of uranium acetate. However, when we viewed the circle (what is the actual name for this object? didn't she call it a grid, or something?) under the microscope, we couldnt find anything. Dr. Henk tried a different technique and we finally saw a dense, irregular blob of something from strain 66. All in all, this technique was not very sucessful.On Wednesday, we analyzed our strains that we placed in 10C, 45C and 50C. Most of our strains grew at 10C, some grew at 45C, but none grew at 50C. We also analyzed our strains that were plated on media containing xylan. To analyze these results, we covered the plate with iodine. A clearing in the iodine indicates a positive test (NONE of our strains were positive). Does this test for the presence of xylanase? Next, we analyzed our strains that had been plated on media containing avicell and granular cellulose. We first poured Congo Red on the plate, let it sit for 15 minutes, and then poured it off. Then, we poured a NaCl solution on the plate, let it sit for 15 minutes, and then poured it off into a beaker. A positive test shows up as a clearing in the red dye. A very small number of our strains were weakly positive and the rest were negative. Lastly, we ran our PCR products on an agarose gel. From our collection, strains 53, 54, 55, 60, 65, 73 showed up on the gel at band 565. GB20 also appeared on the gel. The rest of our strains did not show up on the gel, which most likely indicates improper extraction of the DNA.

Microscopy

On Monday of this week, we went to the microscopy lab while Dr. Rainey was in Mexico. Each group took two of their strains to observe using two different techniques. We first looked at our strains using light microscopy. To prepare slides, we first scraped of cells into a tube containing water. Then we place 1.5µl of the liquid culture onto a cover slip. Then the labeled slide was placed onto the cover slip. DAPI (4',6-diamidino-2-phenylindole) was then added to the slides. DAPI is a fluorescent stain illuminated by U.V. light that binds to the DNA by passing through the intact cell membrane. Next the slides were viewed using Differential Interference Microscopy. It works by separating a polarized light source into two beams that pass through the sample. When the beams recombine, it is gives a 3D image. Pictures were taken of each sample, then we attempted to view the DAPi under U.V. light. None our samples showed any illumination from DAPI. This may have been because it was unable to pass through our highly hydrophobic samples. We probably needed to get rid of the polysaccharides so the DAPi could inter the cell.
Then we view was our samples using the transmission electron microscope (TEM). The sample were prepared by placing a drop of the liquid culture on a small copper grid. The grid contains a thin carbon film that holds the bacteria between the grid. Then grid was placed onto a drop of uranium acetate. The sample was placed into the TEM. TEM works by shining a beam of electron through the bacteria in a vacuum. The vacuum is maintained by adding liquid nitrogen to the machine which condenses the vapor within the machine. The image is formed by the interaction of electrons through the sample which is magnified and focused. We were able to observe the bacteria and find structures such as flagella on the bacteria. Pictures were taken of each sample on the TEM also.
On Wednesday, we recorded our results from the xylan, Avicel, and granular cellulose. We also recorded the result from the 45ºC, 50ºC, and second 10ºC temperature trials. We found that in some of our samples the strains appear a slightly different color at different temperatures. Iodine was added to the xylan plates to look for clearing. Congo red was added to the Avicel and granular cellulose plates to look for clearings also. Then our PCR results were run on a 1% agrose gel. We found that the bands that did appear were about the same size (approximately 565 base pairs) as reported in the paper.

Lab class at high altitude…..

Monday saw Rainey on Pico de Orizaba at some 4700m looking for Martians! Check out this link for a small explanation of “Why Pico de Orizaba” – http://www.hightreeline.blogspot.com/. Some above refer to Rainey catching some rays – and yes that was very true as the uv at such high altitudes is significantly higher than below sea level back in the swamp. The application of some very good sun screen is a must to protect against uv and wind burn on Pico de Orizaba. A number of strains that we are using in class came from this site they have the PO designation.

Monday was microscopy day in the Socolofsky Microscopy Center in the basement of Life Sciences Building. The plan was to look at 6 of our strains (2 strains per group) using light microscopy and Transmission Electron Microscopy (TEM). Reportedly there were some problems with staining the cells with DAPI and will getting the cells attached to the copper grids for TEM. These problems are most likely caused by the properties of the organisms and not by anything we were doing wrong.

Wed, temp repeats 10C plus new 45 and 50C. Since we had some that grew at 42 we had gone to 45 and 50. Those plates were incubated in plastic bags to stop them drying out. The 37C plates just had parafilm and some dried out. We sent our results to Rainey who will update the spread sheet on AirSet. More data on the temperature range for growth of these strains. Thsose strains that grew at 10C we will now try at a lower temperature like 4C (this is the temperature of the cold room). We will set these 4C tests up on strains that grew at 10C this Monday.

We also checked the results of xylan, Avicel and Granular Cleeulose degradation. The plates were stained as follows: Xylan –iodine, Avicell (crystalline cellulose) and Granular Cellulose – Congo red. In eth case of Congo red we applied it to the plates for 15 mins then poured it off and applied saline solution for 15 mins then observed the plates for zones of clearing around the colonies.

All of our strains tested –ve for hydrolysis of these compounds. This is not too surprising as this is the case for the type strains of the species of the genera to which they belong. There is still the option to test them using filter paper in liquid culture. The cellulose of filter is easier to digest than crystalline cellulose by some organisms.

We also ran out the PCR that we set up last Wednesday using the Geodermatophilaceae specific primers from the paper of Salazar et al 2006 (at AirSet). Some groups got strong positive bands for their strains from the class collection. For the isolates from the soil samples pre and post irradiation there were both positive and negative results. A positive result is a band around 550bp. There is some debate about the amount of DNA being recovered from the strains using the MOBIO kit. Some groups have good DNA while others seem to have little or no DNA.
Those isolates that gave a positive band with the Geo primer PCR will have their 16S rRNA gene sequence to determine which of the Geodermatophilaceae these isolates belong. We will set up 16S rRNA gene sequence PCR on those isolates next Monday.

More DNA......

At the beginning of this week, we ran the DNA we extracted from 15 Gobi samples on agarose gel (we extracted this DNA last week). Unfortunately, when we looked at our gel under fluorescent light, there were no DNA bands to be found (except the ladder). This could be because we performed the extraction protocol incorrectly or because we loaded the gel incorrectly. So, we ran our DNA again…and it failed to show up…again. We also spotted controls and samples that had 9 kGys of radiation (from our 26 strains) onto optimal media. In our lecture, we discussed the 16s rRNA sequence, which has about 1500 nucleotide positions. We work with the 16s sequence because it has variable regions (enough variations to separate species). Primers are made from highly conserved regions (because that way they can work for many organisms). Lastly, we compared some of our strains in EzTaxon and BLAST to see what organisms they are most closely related to.On Wednesday, we restreaked our stock plates and put them in the 25C incubator. We put the old stock plates in the cold room (we restreaked two stock plates for KR-65 and MCC-66 so we could use them to take to the microscopy lab). We also set up PCR using the DNAs we had extracted so far (meaning 21 separate DNAs). We used a premix that Dr. Rainey so kindly made for us. In lecture, we talked about PCR in depth. We learned that the critical part in PCR is annealing temperature which determines whether or not the primer will bind to the DNA. We also learned that we must mix our DNA with buffer, primer 1 and 2, taq polymerase, dNTPs, and water (we made a 50 uL reaction using 49.5 uL of premix—which is everything listed except the DNA---and .5 uL of DNA). We also had to make a control which just contained premix (no DNA). Our group was not able to use our GB16 strain because we rain out of premix (we spilled some, accidentally..oops!). All in all, it was a good, productive week. We will all be sad when Dr. Rainey is not in class on Monday.

Polymerase chain reaction and 16S

This week, we learned a lot about the 16S RNA sequence and about running a PCR. The 16S RNA sequence contains about 1500 Nucleotide bases. These fairly large sequences contain highly conserved regions that show their relationship to specific genera and families, but there is enough variation to distinguish between species. Whereas the 5S is too highly conserved and too small to differentiate between species. The PCR technique uses forward and reverse primers to bind to conserved regions and amplifies the 16S gene. A good replicated gene sequences contains about 600 to 900 bases.
On Monday, we spotted 10µl of a control and the 9kGy treated stains on the media they grew best on. Then we ran a 1% agrose gel on fifteen DNA extractions from March 3. We had to run the gel twice and neither gel showed a strong presence for DNA. Either something went wrong during the DNA extraction or the wells weren’t loaded properly. After, we used to EzTaxon and Blast to find which species the strains that were previously sequenced were most closely related to. Between the three groups, we had at least one stain from each genus.
On Wednesday, we re-streaked new stalk plates and streaked six stains (at least one from each genus) twice which we will use for microscopy next Monday. Then we prepared to run a PCR on the extracted DNA from the class up to this point. Our group has 21 different DNAs. Dr. Rainey mixed the correct amounts 10XBuffer, dNTPs, Primer I, Primer II, water and Taq (a heat-stable DNA polymerase) to make the pre-mix. We added 49.5 µl of the pre-mix and 0.5 µl of DNA to the PCR tubes. One tube contained only the premix which was used for the negative control. The tubes were placed into the PCR machine. The PCR machine contains a heated lid to prevent condensation forming on the lids of each tube. The machine was started and if went through 40 cycles of heating and cooling. It heats up to melt the DNA and separate the stands, then it cools down to the annealing temperature to allow the primer to bind to the DNA. The annealing temperature is found by adding 2ºC for every A-T pair and 4ºC for every G-C pair in the primer. We found the annealing temp to be 50ºC for primer I and 60 to 62ºC for primer II. After the forty cycles of temperature changing, the machine maintains a temperature of 72ºC for 10 min to make sure all the genes are elongated. Then process is complete and the machine keeps the DNA at 4ºC until you take them out.

blog

This week went by so fast! Biochemistry took over my life as well as copying and pasting EzTaxon and BLAST results! On Monday we ran our our selected strains from our radiated and non irradiated soil samples on agrose gel. We will used these for PCR Wednesday. We also spotted a control and 9 kGy radiated sample of each of our 26 strains. Strains that grew after 6 kGy and not 16 kGy will show whether they can grow at levels above 6 kGy. We also began putting our known 16S RNA sequences into EzTaxon and BLAST to see which organisms they are most similar to. Our group had the most hits of Blastococcus sp. Wednesday we began lab by streaking new stock plates. I noticed that colonies that were pink/peach were much easier to get off of the agar and streak than the cream/white colonies which seemed to be growing stiffly on top of and into the agar. We grew an extra plate of possible Modestobacter and Blastococcus aggregates (strains AT03-34-2 and AT04-158-8) to be used for microscopy on Monday. There we will view cellular morphology and hopefully mobility accessories of these organisms. We also preformed PCR for our soil DNA as well as our 6 strains chosen from our strain collection. We used a master mix and learned as a class to be careful with our distribution of master mix so that there will be enough for a negative control. I am looking forward to viewing our strains under the microscopes Monday and getting to see what they look like on something other than their normal agar!! Hope Mexico is treating you nicely Rainey!

Back to the Daily Grind

This week’s class began with a group discussion that covered the importance of using the 16sRNA gene sequence to classify organisms. This ~1500bp sequence is highly conserved region of DNA makes it perfect for our comparisons. We also discussed the 5sRNA and 23sRNA and why those sequences are not used. The 5sRNA at ~100-150bp has too little variation, too small and too conserved to be useful. The 23sRNA at ~3000bp never quite took off because there was such a massive amount of 16sRNA sequenced and to sequence the all those organisms’ 23sRNA would be quite a task.
The importance of the high conserved areas on the gene became quite apparent when the discussion of primers began. The sections “27 forward” and “15-25 reverse” are two conserved areas in the 16sRNA that we can exploit by making primers that can replicate the DNA of our three genera. These primers allow the DNA to be copied in both directions to generate many copies of the 16sRNA gene sequence when using Polymerase Chain Reaction Method. To get the complete sequence if our replication pauses, we can use other primers set to start at other areas in the sequence (fox forward at ~519bp). A good amplification is ~900bp and the average is ~600bp
Our first task involved spot plating 10 microliters of our twenty-six strain suspensions exposed to 9 kGy of radiation. We divided the plates into 6 quadrants because we are measuring a qualitative characteristic of our organisms. Each was plated of their optimal media. The second task involved the production of a gel and buffer solution to run our Little Red Hill DNA extract samples (LRH-1, 2, 4, 5, 9, 11-17). Unfortunately when I tried to remove our first gel it broke into many pieces. I made a second gel and re-ran the electrophoresis. When placed under the UV light, only a few faint bands appeared which could mean there was no DNA or not enough DNA was present (we will test this by utilizing PCR). The last task of the day involved looking up the forward 16sRNA sequences on BLAST and EzTaxon. This provided us with a good idea of the relationship of our strains to our three genera.
The next began with a group discussion on what organisms we will bring down to the Microscopy lab on Monday based on the BLAST and EzTaxon results. The two strains we chose were: AT04-166-14 (strain 40) and AT03-37-7 (strain 37). We then moved on to a discussion about PCR which involved discussing the process to figure out the correct annealing temperature for our strains (the most important step in PCR is the annealing). We then discussed the proper order of making PCR Master Mix: buffer 10 µl, dNTPs 5 µl, primer one 0.5 µl, primer two 0.5 µl, water 38.5 µl, Taq polymerase 0.25 µl and your DNA 0.5 µl. The mix is added to the tube first and then the DNA is added, centrifuged and then placed in the PCR machine. We made the PCR solutions using the DNA we’ve extracted so far in the semester (labeled tubes red and renumbered according this order: strain 18, 27, 29, 34, 44, and 48: LRH-1, 2, 4, 5, 9, 11-17).
Our next task involved making new stock plates of our twenty-six strains. Two of our stock plates were contaminated so we tried to pick around the contamination, if that fails will we have to restreak from the strain vials. We made duplicates of AT04-166-14 (strain 40) and AT03-37-7 (strain 37) so we can bring them to the lab on Monday. The next step involved determining the tests that we will run again because either the sample was contaminated, was missing or had dried out due to poor parafilming technique. On Friday my group is tentatively scheduled to meet and re-do some of these corrupted tests.

The wonders of Molecular biology

Monday was an interesting day. First was a lecture about 16srRNA and why it is used as the main way to determine if organisms are different species. I found out that 16s is around 1500 nts long. We were shown were on the sequence some of the more common primers would go and in which way they would amplify the sequence during PCR. Upon returning to the lab some of us began spotting the remaining 10kGy gamma radiation treatment along with its unirradiated control. Others began preparing to run out their recently isolated DNA from their serial dilution isolates. Upon completion of my groups gel there were some isolations that worked fine and others that appear to not have worked this was observed by the presence or absence of a band in the image. We can not completely say they did not work until after the PCR is checked since we could have just isolated too little DNA to show on the gel. I have to go back and check but there were some isolates that were really difficult to remove from the agar and I believe that those might be the ones that possibly did not work. I did not get to it during class but we were also given the task of taking all the available DNA sequences for our group’s main 26 isolates and putting them into EZtaxon and BLAST to see what organisms they were most related too. The biggest difference between EZtaxon and BLAST is that EZtaxon compares against type strain sequences only and BLAST compares the sequence to any sequence in the data base. My results show that of the 10 isolates that we do have sequences for at this time 9 are of the Genus Blastococcus and 1 is of the Genus Modestobacter.
Wednesday we had a lecture about PCR. During the lecture we had a brief overview of PCR and were given the amounts of the constituents of a 50μl PCR reaction. The constituents for this PCR reaction are Buffer which already contains the MgCl, dNTPs, diH2O, Primers, Taq, and DNA. When making a master mix always make 2 extra then the amount of DNA sample you have. One of these extra reactions will be your negative control the other is for mishaps when pipetting. Along with the set up of the PCR we also streaked stock plates for our continuing experiments along with an extra 2 plates that are Isolates to be used during Microscopy on Monday My groups Isolates were ones that had come up during the EZtaxon search as Blastococcus aggregatus and Modestobacter versicolor.
On Thursday afternoon was the awards ceremony were I officially received my award.

Yah Eugene!!

First of all, I wanted to apologize for missing Monday. I hate to miss this lab because I feel like I’m making everyone else pull my weight. I assure you that I would have been of no use on Monday had I came. I’m going to the doctor again today to try to get a handle on these headaches.
The Salzar paper is very interesting. It’s research is very helpful to use in our attempt to discover new species in the family Geodermatophilaceae. I will not focus on its stone sample findings, but rather on the design of PCR primers specific for the family Geodermatophilaceae. I feel that it is also to mention that the paper refers to two probes for specific detection of Geodermatophilus and Modestobacter using fluorescence in situa hybridization. These probes may be useful for us down the road. The Salzar paper designed a forward primer called Geosp2 for positions 156 and 174 and a reverse primer called Geosp1 for positions 725 and 743 on the 16S rRNA gene. specific for the detection of the three genera of the Geodermatophilaceae family. The reason it is specific is because the primers are for conversed only in the Geodermatophilaceae family. They tested the specificity of the primer by comparing its oligonucleotide sequence to all of the sequences available in the GenBank using the FASTA program. The PCR procedure was 40 cycles of 30 s at 93C, 30s at 49C, and 2 min at 72 C, followed by 72C. They compared the amplified products by observing the melting temperature which showed that all of the clones had similar melting temperature.
The sequence of the primers should complete hybridization with species of Geodermatophilus. However, one mismatch was found between one of the primers and Modestobacter multisepatus.Three mismatches were found between the two Blastococcus species and the reverse primer, and one mismatches were found between the Blastococcus species and the forward primer. This is not of great concern because in other families there are many more mismatches than observed between the primers and the Geodermatophilaceae family.
` On Monday, the 16s rRNA gene sequence of a few of our strains was put into BLAST and EZTaxon to compare its relatedness to other species within the Geodermatophilaceae family. The data from this test enabled us to choose two species from our 26 strains to examine using microscopy. We chose the strains K465 (Geodermatophilus) and NC66 (B. jejuensis).
As reported from Sean Michael, on Monday we also made a 1% agarose gel and ran the DNA from the last class. The gel was electrophoresed for about 20 minutes at about 75 volts. The gel was ran twice but neither time produced good results. We also made 10ul spots of the unirradiated control and the irradiated at 9 kGY on the media at which it grew best. 6 strains were put on each plate.
Sean Michael redid the test which had strains that were contaminated. The data from these tests will be added to the master spread sheet.
On Wednesday, we prepared the samples for PCR. A PCR reaction mixture was made. 49.5 ul of the reaction mixture was added to the tubes for adding 0.5 ul of DNA. The DNA is spon down in the centrifuge before being added to the reaction mixture. We did not have enough reaction mixture to run GB-16. Our blank is labeled with a B. The blank will allow us to see if there were any contaminants in our reaction mixture that may have affected our PCR results. The most important step in PCR to avoid primer mismatch is to make sure that the annealing temperature is as high as possible. The higher the annealing temperature the more specific the primer/DNA hybridization will be. The annealing temperature for the forward primer is 50C and the annealing temperature for the reverse primer is 60-62 C. The M is the reverse primer means that a A,C, or G can be present.
We restreaked all our stock plates once. We restreaked KR65 and NC66 twice to be used for microscopy.

.....impt of staying awake in BIOL 101...........

So this week went well! On Monday we received our test grades which were fair! The tasks which were performed included: making agarose gel, irradiated our organisms at 9kGy, and discussed gene sequencing. First, we started with making agarose gel and running our DNA. Actually, Rainey made the agarose gel and my lab partner ran the DNA. Our DNA results didn’t go as planned. The agarose gel broke and the experiment didn’t go as planned. There was no sign of any DNA present. Next was the irradiation. For this we used a control (no irradiation) and organisms which were irradiated at 9kGy. We spotted six spots per plate (3 controls and 3 irradiated organisms). This was a continuation of a previous experiment. For the previous experiment we irradiated organisms at 0, 3, 6, and 16kGy. There were colonies present at 0, 3, and 6kGY but none at 16kGy, so this experiment was performed to see if there would be any growth between 6 and 16kGy. Next, there was the DNA sequencing discussion. This was very informative! Of some things that I learned were the 3 ribosomal genes which are: 16S, 23S and 55S. The reason that 16S is so often used is because at this level there are variable regions and also highly conserved regions. These highly conserved regions are found in bacteria and certain species so 16S is specific. Also, we have a 16S description for every described species, so there is more information available for 16S sequencing. I also learned that a partial 16S sequence was 650-900 base pairs where as a full 16S sequence was 1900 base pairs. This knowledge was beneficial because next we used online databases such as eztaxon and BLAST to sequence some of our strains. Most results from these databases turned up many results which varied in there percentages of similarities. WE used the result with the highest percentage to classify our organism.
On Wednesday we streaked new stock culture plates, analyzed our old stock culture plates, and set up PCR using all DNA extracted thus far. Streaking our new stock culture plates was nothing new; however, we got a chance to find out which ones were contaminated which may have led to some of our other contaminations (in my opinion). Next we set up the PCR experiment. From Monday and Wednesday discussions I learned quite a few things about PCR primers. PCR binds to DNA and amplify it. The most important role of primers are annealing. The annealing temperature of primers are: for every G+C 4 degrees and for every A+T 2 degrees. Sorry back to what we did! We used 18 strains to sequence and one control. Our PCR premix was 50 micro liters for each. It included 95 micro liters of buffer, 95 micro liters of dNTPs, 9.5 microliters of primer 1 and 2, 731.5 microliters of water and 4.75 micro liters of Taq, and .5 micorliters of DNA. These calculations were pretty exact; however, we still managed to NOT have enough premix. This was disappointing because we were not able to perform the experiment on all of our strains; however, I did learn how to correctly use the pipette FINALLY!! After preparing the premix we centrifuged our tubes, then add the DNA. We were supposed to start our sequencing, but time did not allow ((so sad))!! That was our week in a nutshell J

yeah YEAH yeahhhhh........

On Monday Dr. Rainey told us that our blogs weren’t inventive enough…he also said that we needed to add more detail. I mean, I thought everyone was inventive in their own way, however, I can admit that not all of us went into enough detail…myself included. So, since this week was somewhat of a “slow” week, I’ll save the inventiveness and details for next week’s blog. By slow I mean that we didn’t do too many things, however, the things that we did were very time consuming. For instance, on Monday we had to melt down our agarose gel and run out our DNA (Larry did this twice..the first time he broke the gel and second time he found that we had NO DNA PRESENT). In addition to that, DP and I found similarities to each our strains using EZTAXON and BLAST. We also learned that scientist use the 16S Rrna because it has enough variation, unlike 5S that doesn’t have enough and 55S that has too much. We also use the 16S because it has variable regions and highly conserved regions. The highly conserved regions are those that are found in a species or phylum. We now have a 16S rRNA for every validly described species. In addition, we also use it because the database actually exists and we have things to compare it to. I also learned that there are 2 primers needed to sequence DNA, a forward and reverse primer. The forward sequences one strand while the reverse sequences the other (hopefully everyone knows that DNA is doubled stranded).

Unfortunately, Rainey-Bug sent out a mass email to the class saying that even though many our results had been contaminated, we were still responsible for them so that they can be included in the paper. So that’s why I’m here in the lab at 1045 am…early huh? I KNOW!!! I guess it doesn’t matter anyway since my dog got me up at 8am. Anywho, on Wednesday we had to re-streak our stock plates. This is to ensure that the cultures remain pure. Keeping them longer than 20 days makes Rainey-Bug weary!! We also set up PCR using all DNA that we had extracted thus far (with the help of the ALMIGHTY…haha!). Lucky for us there was a pre-mix so we didn’t actually have to make it. We actually have the recipe to make the pre-mix, so if you’d like it, just respond to this blog and leave your name and email and I’ll be sure to get that to you as soon as possible.

Rainey: Class, what is the most critical step in PCR?
Class: (in unison) The annealing temperature

The annealing temperature is very important because it determines if a primer will bind to DNA. The higher the G+C content, the lower the annealing temperature. To the determine the annealing temperature you will need to know that G & C equals 4 degrees and every A & T equals 2 degrees. If the temperature is set too low then the primer probably will not bind to everything.

Sorry guys, there are no pictures to share with you, check back next week (this is to our BLOG followers…if we have any!).

The week that was? Hard to remember at >4000m…………..

It is hard for me to actually remember what we did this past Monday and Wednesday as it now seems a lifetime. I am currently at a conference in Mexico and working on Pico de Orizaba. I will post a longer entry will be posted when I reach lower altitudes…………………

Congratulations to Eugene !

Eugene Becker

a student in the BIOL4126 class is the 2009 winner of the

Marion D. Socolofsky Award

The award in recognition of Eugene's research activities in the RaineyLab and scholarly

activities at LSU will be presented at the College of Basic Sciences Honors Convocation.

The image above shows the site in Mexico from which the Herbaspirillum strains that Eugene is working to characterize were isolated. The strains are shown in the 16S rRNA gene sequenced based phylogeny indicating their novelty within the previously described species of the genus Herbaspirillum. Pico de Orizaba is the 3rd highest peak in North America with one of the highest treelines in the world. We are studying this site as it represents a terrestrial analog of Mars in the past and in the future as a terraformed planet.