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.