Sporulation Test and Starting work on the Poster

     This week we started work on the poster as well as were able to finish the sporulation tests. For the sporulation tests we used the two sets of plates that had sat out through spring break in order to truly starve the cells in order to force them to produce spores if they are able. The procedure for a sporulation test is very fundamentally similar to gram staining, using malachite green to distinct vegetative cells from spores. 


Sporulation Procedure

1. Bring 250mL of water in a beaker to a slight boil using a hot plate.

2. Use a wax crayon to draw a circle onto six slides. 

3. Label two slides each of the positive control , negative control, and tested bacteria (+B. subtilis, - S. epi, ? D. sonorensis.) as well as the date in order to separate the two sets

4. Spread each sample within notated circle and use Bunsen burner to heat fix onto slide. 

5. Attach clothing pins on either end of the slide and rest upon the top of the beaker, allowing the steam to hit the sample.

6. Flood slide with malachite green. 

7. Place a damp paper towel over slide to prevent malachite green from evaporating.

8. Allow steam to permeate sample for 5 minutes.

9. After waiting allotted duration, relocate slide to gram stain tray. Thoroughly rinse slide with DI water.

10. Flood with the counterstain safranin and allow it to sit for 50 seconds.

11. Thoroughly rinse with DI water and blot slide dry. 

12. Repeat procedure with each bacteria being tested. 

13. View stain under microscope to analyze and distinguish vegetative cells from sporulated cells.

One of the sonorensis sporulation images

    Unfortunately, while we could see the distinction in the positive and negative controls, we couldn't conclusively tell whether or not sonorensis had produced spores. While one of the sono plates looked very similar to the negative control, the other had a number of green spots very similar to the positive control. While I felt that it was negative along with other lab members, other in the lab felt as if it was positive, so we decided to label it inconclusive.  There were also more failed attempts on working with the casein media but we did eventually get it working and inoculate those plates. 

    We also started to work on another RNA isolation and plot out our time to get the D. caeni done intime however the sequencing for D. caeni wouldn't even be done until a week before the conference at the soonest, so it was decided that that project would be put on hold until after the conference and possibly until the summer. Because of this, a majority of the week was spent planning and working on the poster, mainly gathering images and personally working on the abstract.


First edition of the Abstract- 

Deinococcus Sonorensis was discovered in the Sonora desert in Arizona in 2005. The genus Deinococcus is known for its extremophile properties regarding oxidation, radiation and desiccation. Within all of the deinococcus genus, Deinococcus Sonorensis has been found to have a unique plaque forming structure. Because of its plaque forming structure, it's not is known about it beyond what was learned during its initial discovery. This study has analyzed it through various biochemical tests; enzyme testing, substrate utilization, and various staining methods. Beyond that it also tested colony morphology differences through various nutrients. As it is a UV radiation extremophile within the deinococcus genus this study also did a UV analysis in comparison to another extremophiles within the same genus (Deinococcus radiodurans) another non-extremophile bacteria in the same genus (Deinococcus aquaticus) and a non-extremophile outside of the genus (E. coli)




Second edition of the Abstract-


Deinococcus sonorensis was discovered in the Sonora desert in Arizona in 2005. Within the Deinococcus genus, D. sonorensis has been found to have a unique plaque forming structure. Due to this plaque forming structure, not much is known about this species beyond what was characterized during its initial discovery. This study has expanded upon the current knowledge from past research by conducting various biochemical tests including enzymatic, substrate, imaging, and staining as well as UV radiation tolerance. 


D. sonorensis is a gram-positive rod, grown on TGY, R2A and NB media with an optimal growth temperature of 30°C. It is a nonmotile, non sporulating aerobe that forms a plaque. It was found to have the enzymes Amylase, Oxidase, Catalase and Protease but lacks Urease and Tryptophanase. It can utilize the substrate Lactose but not Citrate, and does not have any mixed acid fermentation pathways. It cannot produce hydrogen sulfide gas as a byproduct of enzyme hydrolysis of sulfur containing molecules or ions. The tests for both sucrose substrate utilization and the butanediol pathways turned out to be inconclusive. 


Colony morphology differences were also tested through various nutrients. D. sonorensis was grown on both TGY and R2A and the morphology differences were initially observed, then further tested. Plaque stability was found to weaken when grown on less nutrient dense media, and the plaque structure was imaged using fluorescent microscopy. 




Final Version of Abstract-


Deinococcus sonorensis was discovered in the Sonoran Desert in Arizona in 2005. However, characteristics of the bacteria have remained largely unknown, obscured by the challenging plaque structure in which its cells are embedded. This project looked to explore the basic biology of D. sonorensis – the morphological, biochemical and genomic characteristics, as a preliminary study for investigating the plaque itself. D. sonorensis was found to be a gram-positive, aerobic, non-motile, non-sporulating rod with an optimal growth temperature of 30°C. It proved positive for Amylase, Oxidase, Catalase and Protease but negative for Urease and Tryptophanase. It utilizes the substrate Lactose but not Citrate and does not have mixed acid fermentation pathways. It does not produce hydrogen sulfide. The tests for both sucrose substrate utilization and the butanediol pathways proved inconclusive. Colony morphology differences were also observed and imaged under conditions of varying nutrient density. Plaque stability was clearly diminished as nutrient availability was reduced. The genus Deinococcus is well known for its radiation tolerance but D. sonorensis was capable of withstanding a significantly higher level of UV exposure under conditions of elevated nutrient density. Finally, a preliminary genomic analysis was performed. The organism had a genome size of 4.8 Mbp and a coding DNA sequence (CDS) count of approximately 4700. The GC-content was in the characteristically high range for Deinococcus at 69%. In sum, this work provides a valid foundation on which to pursue studies of the nature of the sonorensis plaque.


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