:-( Last Class :-(

Well we all knew this roller coaster of a ride would end sometime. On Monday, we were lectured on qPCR by Mr. Jun. I learned a few things that I had not known prior to his lecture. Following his presentation, I gave my presentation on what I think is novel organism. I am sorry it I seemed a little nervous. I’ve never been too good at presenting in front of classes. We then headed to class to perform casein, oxidase and catalase tests on our strains. The positive result for oxidase is the appearance of blue pigment after application to a colony streaked on filter paper. The positive result of catalase would be the formation of bubbles after a loopful of specimen is placed in a droplet of 3% hydrogen peroxide. There seemed to be no correlation between the oxidase and catalase results. On Wednesday, we had our last class of the semester. We had a short review of what will be covered on the final and then we had a party to celebrate the successful semester. I really enjoyed this class this semester. I got to meet a lot of interesting people that have shown the dedication required to succeed. It’s kind of sad that it’s all over, now I got to find a job. I wish everybody the best of luck in pursuing their goals. It has truly been a pleasure this semester.

Semester at a Glance....

This semester was very short, at least it seemed that way. We started out doing simple tests for our research project, but as the semester went on, we did things like PCR and gamma radiation tolerance. My favorite part of this class was being able to gain research experience. Many students lack this important and now, I have it and it will set me apart from the rest. Another nice factor of the lab is that the work we did may eventually get published in an IJSEM publication. This surely will set me apart from other students, if I decide to go to med school one day. My least favorite part of the lab was being there from 130-5pm every Monday and Wednesday, although some days went by fast. Throughout this semester, Dr. Rainey was very helpful. I’m extremely happy that all of our media was pre-made and we didn’t have to do it ourselves. I think that this is an important component of the lab. If we had to make the media ourselves, we would’ve lost a lot of time and wouldn’t have been able to do as many tests. I must say, in doing the different tests, many our plates were contaminated. In the beginning we may not have used proper streaking technique, but once contamination became too frequent, we did. Even though we used proper techniques we were still plagued by contamination. Every time, it was the same strains, those that grew on 1/10 PCA. I’m convinced that those strains are pure at all.
This past week we did tests such as caseinase, catalase, and oxidase. If the organism is able to break down caseinase a clearing will be seen in the opaque, white media. If the organism is positive for catalase bubbles of oxygen will be produced in the hydrogen peroxide. If an organism produces oxidase it will turn blue once inoculated with hydrogen peroxide. We had strains that were positive for all of these tests. The last day we talked about the dreaded final (just kidding you guys) and had frozen Krispey Kreme and had delicious Papa Johns pizza. I also bought popsicles. We all had a BLAST (get it), even though we waited 10 minutes for Larry to show up to class.... I enjoyed this class and I know that Rainey can't wait for his next 4126 class so that they can continue with this project.

On Monday,

we turned in our masterpieces (IJSEM paper). However, there was still work to be done. Previously in the class, we made a list of all the tests left to be done, so today we were able to mark a few off the list. We streaked our 26 strains onto agar plates containing casein. We will check them in a couple of weeks. A clearing in the milky media we be a positive test. We also performed tests for catalase and oxidase. The catalase tests was familiar to most of us having taken general micro lab back in the day. Cells were inoculated into a small drop of hydrogen peroxide. The presence of bubbles denotes a positive test (the bubbles are oxygen). For the oxidase tests, cells were placed on filter paper. A drop of oxidase solution was placed onto of the cells. A color change, mostly a blue color change, denotes a positive test. The majority of our strains were positive for catalase and negative for oxidase. The results of these tests are on the spreadsheet. Unfortunately, the Luedemann (1968) paper did not perform catalase or oxidase tests to compare my strains to.
We also determined that our soil isolate, GB-20, grew the best on R2A and 1/10 PCA.
On Wednesday, we enjoyed pizza and donuts, courtesy of Dr. Rainey. Thank you!!! They were delicious. We also talked about the final, database, and lab notebook which are all due or take place on Monday. Yikes! Finals week got here so fast.

The End is Near......

This week we had our final classes of the semester. On Monday, we had a presentation by Jun a Post Doc in Dr. Moe’s lab he told us about quantitative PCR. We had an idea about how it works from the Salazaar paper that we read it talked about qPCR with a set of primers specific for the family Geodermatophilacea. After the presentation we had a few test to run. Milk agar plates were streaked these plates are used to test caseinase activity and should be graded in 2 wks. Also tested were oxidase and catalase which give almost instantaneous results. To test catalase we took a look of our isolates and placed it in a drop of 3% H2O2 if oxygen bubbles began to form then it was positive if no bubbles negative. To test oxidase we took a loopful of isolate and smeared it onto a filter paper made of cellulose. Then we added the oxidase reagent which was at 1% concentration to the smear if it turns dark purple to black it was positive. Light blue is the natural reaction between the paper and the reagent and is a negative test result. Interestingly about our results is that we had because of the variability within each genus for the oxidase test result that some of our isolates would be positive and some negative however all of our isolates were catalase positive but oxidase negative.
Wednesday was a discussion about what is going to be on the final and what is expected of us in our database/ excel sheet and our lab journals which are all due on the day of the final. After the discussion we had an end of the semester party with pizza and donuts.

LAST BLOG!!!

This week was the most exciting of the semester!!! On Monday, we performed oxidase, catalase, and casein tests on each of our 26 strains. Most of the strains tested positive for catalase (meaning they produced oxygen bubbles when mixed with hydrogen peroxide) and negative for oxidase (did not turn a dark blue/purple when mixed with solution on filter paper). We will not know the results of the casein tests because these will be recorded during the summer. We also took another look at our dessication plates and re-scored them, if necessary. My group found that not much had changed since the last time we viewed these plates. On Wednesday, we talked about our final exam and what we needed for our database. We also had a party which was wonderful (thank you, Dr. Rainey!!). This week was extra exciting since we received our presentation grades and I also got into medical school. I can't believe summer break is almost here!

all good things must come to an END!!!

So this week was rather bitter sweet. On Monday there were two presentations. First there was the qPCR presentation, presented by Jun. Last, but definitely not least came Larry's presentation on his his new found species, Geodermatophilus geauxtigeris. I learned a few things about qPCR (Quantitative PCR). First, qPCR cannot combine with single stranded DNA. However, it can combine with double stranded DNA. When attached to double stranded DNA it absorbs light at 488 nanometers, and emits light at 520 nanometers. The advantage of qPCR is it can recognize the presence of and quantify the target gene. In addition, I learned a few new things about an unknown species from the family of Geodermatophilaceae thanks to Mr. Roach.
In addition to presentations we streaked our strains unto plates which contained casein and performed catalase and oxidase test. Catalase test was performed by adding a loop full of the strain to hydrogen peroxide, and checking for bubbles. The oxidase test was performed by adding an solution to and checking for the formation of a color.
Wednesday brought on the tears. We got good news as well as bad. The good news was that Lauren had been accepted into med school (YAY LAUREN)...however, the bad news was that we may never be able to work alongside each other again :(
All in all this has been a great semester and I love all of you!!!!♥

Blog

On Monday, after turning in the final drafts to our IJSEM papers, we attended a presentation on Real-Time PCR. Real-Time PCR was used in the Salazar 2006 paper in determining the GEO primers, which we used when performing PCR throughout the class. Then we streaked casein plates and tested for oxidase and catalase. The oxidase experiment was preformed by spreading the culture onto filter paper. Then we added a drop of oxidase solution to the culture, and observed for a color change. The catalase experiment was performed by placing a loop with culture into a drop of hydrogen peroxide and observing for the formation of bubbles. The results from catalase and oxidase were put into the spread sheet. Before leaving, we looked at the progress of our Gobi isolate and noted on which media the organism was growing best on. On Friday, we attended our final class. We received our grades from our presentations and had a lab party with donuts and pizza thanks to Dr. Rainey. Then before we left we had a discussion about the final, lab books, and the database.

Final Blog

This was our last week of class. On Monday we had a little lecture on qPCR which is used to not only detect fragments of DNA but quantify the amount of DNA present. We also streaked our strains on casein plates and preformed oxidase and catalase tests. All twenty-six of our strains were catalase positive and oxidase negative. Weird. Wednesday was our class party. It was awesome!! We also discussed the final coming up on Monday and our databases and notebooks that are due next week. I have really enjoyed this class and working with everyone! I feel as if I have learned a lot and gained knowledge that will be extremely useful in the future. Thanks to Eugene for making all the media, Cristi for the study guide and of course Dr. Rainey! See yall at the final!

CONGRATULATIONS TO LAUREN

One of our esteemed classmates got some very good news this past week:

LAUREN GOT ACCEPTED TO MEDICAL SCHOOL

Here Lauren is giving her 4126 presentation on her research findings

WE WISH LAUREN ALL THE BEST FOR THE FUTURE

WE ARE EXPECTING MORE GREAT THINGS FROM HER...........

the new 4126 was a BLAST (not an EZTAXON)....

BIOL 4126 was only taught once before in Spring 2003 - then Rainey served as the Assoc Dean in BASC for 5 years before finding the light and coming back from the dark side. The first 4126 was a very different "beast" to that of this semester. In fact it was taught in a different world, a world with no BLOGs or AirSet, in fact Al Gore had just finished inventing the internet and was starting on his next invention.......

The new format and the components included seemed to work well for everyone and a real hands on research experience was had by all. On the basis of the comments on the BIOL 4126 survey (thanks to those who completed it) there is not too much to change in the format and components of the class. The next class will work on a different set of strains that belong to a different family of bacteria.

At the end of the semester we have made new discoveries (that strains are resistant to radiation and desiccation and that it differs between strains and genera) and collected a set of data that can be used to describe some new species of the family Geodermatotophilaceae. There is still some data to collect and this will be done in the summer and a paper(s) will be submitted to IJSEM in late Summer/early Fall. We will all be coauthors on the paper(s) along with Keren (Keren Ray - first student from RaineyLab who went to Med School in 1999 and is now a Peds Doc in Baton Rouge) and Danielle (who you all met and is finishing her PA training) who isolated the strains in the first place.

The class party was fun and Rainey was impressed with these GOLD shoes some of our classmates had on - he wanted a pair. That was until today - now he wants a pair of these:
but he needs a pay raise as these cost $540 not $5.40 or $54.00 but $540 and guess who is wearing them? Click on the picture above to see the whole story :-)

My EXTRA LATE blog...

Writing an IJSEM paper is very different. It is unlike any paper that I have ever written. Throughout this process I was lost and confused and did not know where to start. However, with the help of my classmates i managed to make it through. I am, however, still working on it and finishing up minor details. Overall, I must say, this was one hard paper to write.
Like writing any paper there are pros and cons. For this IJSEM paper the pros would be that this paper is for a grade, it is very informative, it describes a new species (one that we discovered), and it can be used as a reference in future studies. The cons are that IJSEM papers have to follow a strict format. I am used to writing english papers, ones that don't require much research or study. In addition, writing IJSEM papers takes a lot of time. We have had all semester to start writing this paper and all of us only started a week ago. Writing these papers are very time consuming and the result of the paper is shown by how much time you put into it. I can write an english paper in one day, however, I can't say the same for an IJSEM paper.

p.s. I don't think the First dog, Bo, is the source of the swine flu...i think Rainey brought it back from Costa Rica. So everyone PLEASE wear face mask tomorrow

PROs and Cons of Writing an IJSEM Paper

While writing my paper it has certainly been a unique experience unlike any papers I have wrote in past semesters. Prior scientific papers I have completed were certainly more open in format and materials covered. Taking Biology 3116 Advanced Microbiology Lab with Dr. Sullivan provided a good background to fall back on to, but the amount of information covered in those papers pales in comparison to the information an IJSEM paper covers.
The CONs of an IJSEM paper are hard to say with such limited experience in authoring one. The format of the paper was certainly a difficult task to grasp. The information provided on the IJSEM website helped some, but it is difficult to deviate from prior experience and habits gained from writing other papers in previous biology lectures. The placement and alignment of figures, graphs, and other various aspects of the paper was difficult to accomplish. Another CON is the way to annotate the cited literature; this has been a common problem in other classes because there are multiple forms of citation. From the first semester at LSU on, I have encountered multiple ways to cite literature used in my essays and after awhile it becomes confusing on what and which information goes where when citing. My lack of experience in writing an IJSEM paper made it difficult to understand what needs to be in a paper pertaining to characterization of a new species. It was pretty difficult to decide what data goes where and what data I needed to include. Another con would be the chemotaxonomy section, since we preformed none of these tests; I found it very difficult to pick and choose what should go in it. It shear task of “going out on a limb” on deciding what to write about also made this a difficult paper to author.
The PROs of an IJSEM paper are also hard to say with minor experience in authoring one. After observing the IJSEM literature provided by Dr. Rainey it becomes clear that the data is presented in a format that is easy to read through. The format of the papers makes it easy to flip through and makes the data easy to understand. Another PRO would be that this paper is giving me experience in what it takes to write an article for a journal submission. If I was to pursue a research career, experience in journal acquisition/ submission would be a necessary requirement. I also gained a better insight into the amount of time and effort that each author puts into his/her work to be published.
Overall, this paper has made me a better researcher in my opinion. The experience gained by this task is invaluable to the process of seeking out my future profession.

IJSEM

I believe that writing an IJSEM style paper is a great learning experience. For some of us writing this style of research paper will be becoming a major part of our Futures. I am also appreciative that for my first paper that I had someone there with a vast amount of experience in the area to be hard on me so that I can avoid similar mistakes in the future. This experience should be part of many more classes throughout our educational program at the university. The actual writing of the paper is straight forward some might even say it is cookie cutter in nature. Personally I was most worried about making a mistake in the sections that we smudge a little than I was in putting in any of my data that was retrieved from the experiments. Actual writing of the paper did take longer than expected trying to fine tune certain sections. After my consultation with Dr. Rainey much more tuning is in order before final drafts are to be turned in. Reading and understanding many different aspects of bacteria is needed to make a strong argument for ones case that they are correct in their conclusions. I can see how something as little as not being consistent with lettering for carbohydrates can cast doubt on the reliability of the conclusions of an author.

How to write an IJSEM paper?

The title page should include a short statement about what the paper is about. Provide any new names that are included in the text. It should also include the names of the authors, name of the laboratory, address of the laboratory, address and e-mail address of the corresponding author, a short title, and the GenBank accessions number. The abstract should be a concise summation of the results of the paper and the names of any new taxa proposed (if applicable). The introduction should be brief and give background information for the study. The Methods section should include in detail novel details of tests performed in the study. Standard techniques should not be included such as the plates were streaked using a loop in a clean bench. The methods section may include the names of the suppliers for the chemicals or tests used if it may impact the results. Details that should be included in the methods section include how many times tests were done. The results and discussion sections should include clear subheadings to organize the categories of the studies. It should be noted as to whether results are mean values or not and how many times tests were duplicated. Tables, figures, and the descriptions should be included in the end of this section. The type strain and other strain should be included in this section if new taxa are being described. Tables should only show differential data. Tables and figures should be comprehensible without having to look at the text. DNA base composition should be written as G+C in mol% only to the nearest significant digit. References should be in the name/date style. The author’s last name is written first followed by first initial.
The pros of writing an IJSEM paper are that it presents data in a way that is universally understood by scientists. The format is clear and easy to follow and progressively moves through the study from the beginning to end. The research is presented in a clear and concise way which makes reading the paper less time consuming and more easily understood.
The cons of writing an IJSEM paper are that it is difficult to format data in the way that IJSEM demands. For new scientists, writing an IJSEM paper may be difficult due to its restriction on creative writing techniques. Extra information is emitted from the IJSEM paper in order to make it concise, but for a person who is used to writing paper’s for English classes this may be a difficult task.

IJSEM Blog

We wrote a paper for the International Journal of Systematic and Evolutionary Microbiology (IJSEM). In this paper we described a new species belonging to the genera Blastococcus, Modestobacter, or Geodermatophilus. An IJSEM paper is very structured and there are specific guidelines one must follow that can be found in the instructions to the author on the IJSEM website. A typical paper includes a title with the novel species name and any emendations written. Next is the abstract which should tell something about the organism and what differentiates it from known species, as well as include the type strain number and culture collection number. The introduction discusses the known species and genre. A section on isolation and cultivation should include where the strain was collected, describe the environment and coordinates, tell what media it was grown on as well as temperature, pH, and NaCl concentration. The next section is colony morphology which should use scientific descriptions and include color changes in particular circumstances. A section on chemotaxonomy includes fatty acid and phospholipid composition and is sometimes found in the supplemental material. Phylogenetic analysis describes the 16S sequence of the strains and results from EzTaxon and/or BLAST as well as a phylogenetic tree which can be constructed using the program MEGA. The similarity of the new strains to previously described species should be used as evidence that the strain is a new species. DNA-DNA hybridization can also be included in this section. A section on physiology includes results from tests such as catalase, oxidase, and carbon utilization. A table can be used to display the data. Taxonomic conclusions should explain why the author is putting the suspected new species into the particular genus and why they believe it is new. A table differentiating the new and type species should be included. One of the most important sections is the description of the new species which should define the type strain, which collection it was put into, where it was isolated from, characteristics, name, and derivation. Finally the references section should be in IJSEM format. The biggest pro of IJSEM format is that every paper is formatted similarly and therefore comparisons between multiple papers is easier than if they were all formatted based on the preference of the author. The only con is that writing an IJSEM paper takes a lot of work to collect the data and a lot of time and put together the complete results.

...PAPERS...test...FAMILY EVENTS...presentations...MY SO CALLED LIFE!

So this week our primary focus was the IJSEM paper and a powerpoint presentation. All in all, the task was extremely time consuming, yet nothing hard work could not conquer. This type of paper has its ups and downs. The good thing about the paper was that the information was readily available, and the information that was needed to make the paper successful was very clear (following Urzi paper). However, on the other hand I found the format a litle confusing. Following the Urzi paper, as directed, went against the format that the instructions for authors called for. For instance the instructions for author called for a break up of specific different sections; however, in the Urzi paper some of the sections were merged. Also, some of the things discussed in the paper were unfamiliar to me such as DNA-hybridization. In addition, the papers used for reference were different. Trying to follow the papers, in an attempt to make my own paper successful was very difficult.
My draft was not as good as I would have liked, but I am solely to blame. After reading the comments, I find it a little bit clearer what is asked of me. Also, I made some crazy mistakes; which were possibly due to working under such time constraints. The mistakes which were made were very simple so I do BELIEVE my final draft will be much better. So, I'm kind of confused about what this blog should be about also, so I am going to end on this note!

Blog

All in all, I think writing an IJSEM paper was a good project for our class. I enjoyed reviewing everything that we've done during the semester and I'm sure this will help when studying for the final. Re-reading all of this information also made me realize how much our class has accomplished this semester....we really have learned a lot! I also liked learning how to write a scientific paper....all other papers I have written for biology classes have been lab reports. I think that turning in a rough draft had good and bad parts to it....I'm glad that Dr. Rainey was able to give us feedback on our papers and a chance to fix our mistakes....but at the same time I would have liked to just turn it in one time and be done. I also wished that we could have actually published something...maybe by writing a paper collectively as a class on the most unique strains. But, like I said initially, this was a good end-of-the-semester project and I would recommend Dr. Rainey requiring this for the next Biol 4126 class he teaches.

IJSEM Paper

The paper begins with a title page that is to the point in stating the contents of the paper, and it includes any new names that are proposed in the paper. Next is the summary/abstract. It is a clear and comprehensive summary of the contents of the paper. All new names are included with some specifics, but not full detailed descriptions. Then there is a brief introduction of about three hundred and fifty words that does not contain any detailed summaries of the results. The methods section follows. In this section, only important novel details are described, not standard methods, and the supplies of the chemicals and equipments should be mentioned. Next is the results and discussion, where the proposed name and formal description should appear. The final section is the references in which they are given the IJSEM format: Author (if more than three authors write et al after the first one), then year in parenthesis, followed by the title of the paper, and then abbreviated title of the journal .To write this type of paper can be very time consuming, but helps in comparing information between other papers by keeping a specific format. This paper appears professional and gives specific guide lines to help authors remain consistent.

Almost Spring Break!!!.....

This week did not start off so well. I got sick over the weekend and was not able to go to class on Monday...and I HATE missing class (I always seem to miss something really important if I do). However, I was able to copy notes from Cristi's lab notebook (thank you, Cristi!), so hopefully I know everything that happened on Monday. From what I understand, we talked about our paper and the format that it must be written in (IJSEM). We also plated out our dessication experiment which had been stored in an arid environment for 42 days. We also resuspended wells with soil liquid media and spotted this on the media it grew best on. Lastly, we performed PCR with Geo primers on DNA that had been extraced from soil. I think this protocol is posted on AirSet. I know I do not have a lot of detail written about this lab day...sorry! I'm hoping I can learn more about the exact steps we did on Monday from everyone else's blog!!!On Wednesday we spotted more of our liquid media samples onto 17 new carbon sources! I think I heard that some of the plates may have been mislabeled with the wrong carbon source....hopefully they were not so we don't have to re-do everything!!! After we spotted the plates containing the carbon sources, we incubated them at 25C. That incubator is getting very, very full! We also purified eight different PCR products using protocol from the internet called "UltraClean PCR Clean up Kit". I thought that this process was going to take a long time, like DNA extraction...but, it ended up being pretty simple. Dr. Rainey made some agarose gel and pipetted our samples into the wells for us....he said if we would've done this part, "we would've been there forever". Anyway, we must've done a fairly good job because we had nice, bright, pretty bands show up on the gel. The only problem is, only 6 bands showed up instead of 8. When I asked why this happened, Dr. Rainey said that maybe the DNA stayed attached to the filter instead of coming out into the tube like it was supposed to. Who knows. I hope everyone has a wonderful, sunny Spring Break!!!!!!

Spring Break….WOOT WOOT

Monday’s lab began with a detailed discussion into what constitutes an IJCM paper. Basically, the paper is a step-by-step process of building up your case of why a couple of your assigned strains are species based on comparison to previously described species. We will use this in an attempt to write an IJCM- worthy paper. Personally I knew they had rules on the format of their submissions, but I didn’t realize that they had so many of them.
Our first lab procedure of the day involved gathering our stock plates to be re-plated on to new media. After streaking the strains we parafilmed them and placed the plates in the incubator. The next task began with gathering the desiccation wells. We resuspended the cells by adding 500 microliters of liquid media (optimal media for each strain was used) and allowing them to sit for thirty minutes. We then swished the cells for thirty seconds each to mix up the wells contents. We then took ten microliters of each strain from the wells and spot plated each onto their optimal media. We spot plated six organisms per plate, parafilmed and placed them in the incubator.
After that we observed the results of the strains exposure to 9 kGy or radiation. Only strain twenty-seven grew, this strain is from the Sahara which makes it more likely to be a candidate for UV resistance due to possible link between desiccation resistance (which the organism obviously needs since it’s found in the desert). Our last task of the day was making PCR Master Mix and selecting our LRH-4 DNA extraction as our DNA (+) control.

PCR Master Mix (in order, 49.5 µl in each tube)
· X10 buffer 80 µl
· Geosp1 8.0 µl
· Geosp2 8.0 µl
· dNTPs 80 µl
· water 632 µl
· sample DNA 0.5 µl
· Taq pol. 0.5 µl
We hot-started the PCR mix this time in order to make sure the DNA is accessible to the polymerase. The Taq polymerase was added after the hot start because at those temperatures Taq polymerase becomes inactivated.
PCR Cycle: 40 cycles
· 98 C for 10 min (hot start)
· 90 C for 5 min (add Taq pol.)
· 93 C for 30 sec
· 51 C for 30 sec
· 72 C for 2 min
· 72 to 46 C for 10 min

The next lab day began with gathering contaminated carbon utilization plates to be plated onto new media. It is suspected that a contaminated tip contaminated the cell suspensions (as demonstrated by all of our strain 33 plates being contaminated) so we made new cell suspensions for each of our organisms by: adding 500 µl of liquid media, a loop full of your organism and vortexing the mixture multiple times. We spot plated 10 µl of each organism to new carbon source plates. After that we parafilmed them and placed them in the incubator.
The final task of the day was spot plating 10 µl of each of our strains on to new carbon utilization plates.
Carbon Sources:
· Control C
· Fructose FR
· Cellobiose CB
· Pyruvate PY
· Trehalose TR
· Sorbitol SB
· Myo-inositol MI
· Maltose MT
· Citrate CT
· Acetate AC
· Succinate SC
· Mannose MS
· Alanine AL
· Arginine AG
· Asparagine AS
· Histidine HS
· Lysine LY
· Threonine TH
We parafilmed and placed the new plates into the incubator. Sorry it took so long guys we had a lot of plates to do (labeling all of them made felt like it took forever) and making new cell suspensions took up quite a bit of time. Well I’ve be staying home for Spring Break, hopefully working on our paper. I hope everyone has a happy and safe break.

On Monday we talked about......

the IJESEM paper and all that it entails. In the paper we will either have to write about a new species in one of the three genera we are working with or one species that we think represents a new genus. If we do have a new genus or strain WE can name it. I’m thinking something like Keithobacter, or something of that nature! A good example to look at when writing your paper would be the Ahrens and Moll, 1970 paper. It has the correct structure, although, the introduction needs to be longer. In the paper the required sections are: isolation and cultivation, colony and cell morophology, chemotaxonomy, phylogenetic analysis, physiology, and abstract. After, we plated out our dessication experiment, re-streaked our stock plates, and spotted the organisms used in the dessication experiment. Dr. Rainey made the PCR pre-mix which include x10 Buffer (80uL), Geosp1 (8uL), Geosp2 (8uL), dNTPs (80uL), and H2O (632 uL). We put 49.5uL into each tube along with 0.5uL of DNA. While in the PCR machine the first hot start was used to denature the DNA and a higher temperature. We put the taq in after because it also denatures at high temperatures.
On Wednesday nothing, and I mean NOTHING exciting happen. We were supposed to to re-streak our carbon source plates, plate more carbon sources, and run our DNA out on a gel. However, all of our time consisted of re-streaking our carbon. Then after that we had to streak a new batch of carbon sources. This took the entire 3.5 hours. My thumb started to go numb and I started to become nauseous after the first hour. Like I said Wednesday was unexciting and nothing happened.

My week for Biol 4126 got started rather late....

On Monday I was not able to attend class because of a malfunction within my body. Nonetheless, Rainey assured me that if I had passed away class would be canceled. I thought that was rather kind of him to cancel class just so my fellow classmates could mourn. Anyhow, I was brought up to speed and informed about EVERYTHING that had taken place in my absence. To my knowledge I missed the opportunity to perform the dessication experiment, and setup the PCR using the Arizona soils. In addition, our stock culture plates were restreaked. Most importantly, was the discussion about the IJESM paper which is due on April 20. Upon my return, I immediately got to work! First we got out our old carbon plates and analyzed them carefully. To my dismay, our plates were highly contaminated. To correct this we had to make liquid suspensions of our strains. This was done by combining 500 microliters of saline solution with a loopful of our strain. Following this we vortex the tubes vigourously. From this we spotted the strains on the carbon source from our previous experiment and the carbon sources intended for this lab. All in all, we had a lot of work to do. Oh...yea...and we also held up the entire class :)
On the bright side, spring break 2009 is this week! With that being said, I hope everyone has as much fun as I will ♥

Build up to Break.....

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

9kGy isolate 5 some cells survived most didn’t

9kGy isolate 25 did fairly well

happy spring break......

Monday we began by discussing our IJSEM, International Journal of Systematic and Evolutionary Microbiology, paper that we are turning in at the end of the month. We will be describing what we believe to be a new species belonging to one of the three genre we are focusing on: Geodermatophilus, Blastococcus, and Medestobacter. If we think we have strains with characteristics of a new genus we also have the option of describing those. We will describe their isolation and cultivation, colony morphology, chemotaxonomy, phylogenetic analysis, physiology, and taxonomic conclusions based on our research and data collected throughout the semester. In lab we plated out our desiccation spots by putting 500µl of liquid media into the wells and letting them sit for thirty minutes before mixing them with a pipette to resuspend the cells and spotting 10µl of the suspension onto a plate of its original media. We also made new stock plates of our 26 strains as well our soil samples, N97-6 and N97-19, that were positive when PCR was done with geo primers. We observed our 9kGy radiation plates. We scored strain 5 as ** and strain 25 and ****. The rest of the strains had no growth. The last thing we did was PCR with geo primers on our positive soil DNA that we extracted last week. Rainey made a premix with 80µl x10 buffer, 8µl geo primer 1, 8µl of geo primer 2, 80µl of dNTPs, and 632µl of water. 49.5µl of the premix was placed into each tube along with .5µl of DNA. We did a hot start to denature the DNA longer at 98C for 10 min. Next it was cooled to 90C and Rainey added .5µl of Taq since better results are achieved after adding the Taq later for these geo primers. Eugene did a wonderful job of running a gel of our results. Our soil DNA came out well. My groups 4126 isolate was positive, but not as bright as the others. On Wednesday we plated our second set of carbon sources:
C- control
F- fructose
CB- cellobiose
PY- pyruvate
TR- trehalose
SO/SB- sorbitol
MI- myo-inositol
MT- maltose
CT- citrate
AC- acetate
SC- succinate
MS- mannose
AL- alanine
AG- arginine
AS- asparagine
HS- histidine
LY- lysine
TH- threonine

We spotted 10µl of each strain onto agar containing each of the above. Finally, Lauren, Christi and Sean Michael purified PCR that had been done earlier in the day on some of our 4136 strains we do not have 16S for. Happy spring break everyone, have fun and be safe!!!!!

Blog then beach...

On Monday, we plated our strains that were left in the dessicator for 42 days. We resuspended the cells in each well with 500 ul of liquid media. We left the liquid media in each well for 30 minutes and then repeatedly mixed the cells with the media for 30 secs by pipetting the solution up and down. We spotted the media each strain grew best on.
We Also ran a PCR on the DNA extracted from the three soil samples and the isolates from the 3 soil samples that showed up on the previous gel using the geo primers. Our group restreaked GB 20. The PCR instructions are:
98 C for 10 minutes to denature the DNA
90C for 5 minutes to cool and then add 0.5 ul of Taq polymerase
93C for 30 seconds to denature the DNA
51C for 30 seconds to anneal the primers
72 C for 2 minutes for elongation
-Repeat above steps 40 times
72 C for 10 minutes to extend elongation to make sure all fragments are the same size
4 C ??
The results of the gel showed that the three soil samples contain members of the Geodermatophilaceae family. The results were excellent with a single band at around 564. Also, the soil samples containing some of our strains showed a single band consistent with the Geodermatophilaceae family. Our GB-20 had two bands. I’m not quite sure why this would happen.
On Wednesday, we performed more carbon utilization tests using 16 different carbon sources. A control plate was also made for comparison. 10 ul of the strains were spotted on each of the carbon sources. Our previous carbon source plates were doing well.
We also purified the PCR products from our 26 strains.

Success with DNA

On Monday, we discussed our IJSEM paper. Individually, we will attempt to describe a new species from our 26 stains within the family Geodermatophilaceae. Then our desiccation experiment from February 9 was ready to be spotted onto media. 500µl of liquid media was added to each well on the desiccation tray. After thirty minutes, each well was gently mixed with a pipette for 30 seconds, then 10µl were spotted onto media. Then the PCR was set up with GEO primers for our DNA that was extracted from the dirt samples last week. PCR was set up a little different this week, by adding the Taq after the samples were heated at 98ºC for ten minutes. Also new stock plates were restreaked including our GB-20 strain that had positive PCR results. Then we recorded our 9 kGy results into the spread sheet. We found that five of our strains were able to tolerate this high irradiation.
The pictures show some of our stains that are tolerant of 9kGy.

Strain 67 and 72 both seem almost non-affected by the irradiation.
Strain 68 grew but showed less growth after the irradiation.
Tuesday, Eugene ran a gel with our PCR results from Monday and number of other dirt and bacteria samples. The results from out dirt samples showed a strong presence for bacteria from the family Geodermatophilaceae with bright bands around 565 bps.
Wednesday, we spotted of strains on 17 new carbon sources which included:
Fructose FR
Cellobiose CB
Pyruvate PY
Trehalose TR
Sorbitol SB
Myo-inositol MI
Maltose MT
Citrate CT
Acetate AC
Succinate SC
Mannose MS
Alanine AL
Arginine AG
Asparagine AS
Histidine HS
Lysine LY
Threonine TH
We also purified eight of Dr. Rainey’s PCR samples following the PCR purification protocol from Mo Bio. Six out of eight of these samples were also a success on a gel.

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.