Week 14 (12/05/22 - 12/09/22) Contamination and Identification


Introduction:

 

This week, major focus was placed on developing an outline for procedures and the purity of the D. aquaticus samples. There are many opportunities for error while working in the lab. Contamination and improper planning were some difficulties we experienced in the lab this week. Developing a thorough plan will minimize the risk of error because every aspect of the experiment has already been planned out. This also leaves less opportunity for contamination, because we have more confidence going into the procedure. Contamination has affected our project before while working with kanamycin, due to improper pipetting or contaminated TGY. Although we have reformed our technique since then, contamination has become an issue again. Though the use of gram stains, we will hope to identify the organisms that contaminated the bacterial sample, and therefore determine the cause of contamination.  This will lead to improvements in preparation and technique going forward.

 

Methods: 

 

D. aquaticus was inoculated from a liquid sample from 11/10. Gram staining was performed to confirm that the sample was not contaminated. The bacteria was inoculated into 25 ml of TGY and stored in the 30o C incubator. OD values were taken from this sample on 12/6, 12/7, and 12/8. Gram staining was performed on 12/6, 12/7, and 12/8 to look for contamination. Gram staining was performed on the TGY, the bacterial sample from 11/10, and the bacterial sample from 12/05. 

 

An additional 25 ml of TGY was added to 2 125 ml flasks and autoclaved for 30 minutes. After the flasks were removed from the autoclave and cooled down, D. aquaticus was inoculated into both flasks from the liquid sample from 11/10. 

 

 

Results: 

 

Date

A600

Correction

λ

A(λ)

12/06

0.22

0.10

600

0.22

12/07

0.56

0.10

600

0.56

12/08

1.63

0.10

600

1.63

Figure 1: OD600 values on D. aquaticus sample inoculated on 12/05. The OD600 was taken on the nanodrop each day after inoculation. 


Figure 2: Gram staining done on two separate samples of D. aquaticus. The sample on the left was inoculated on 11/10, containing a lot of cells. The sample on the right was inoculated 12/05, with an OD value of 0.22. Both gram stains were made on 12/06 and viewed on the oil immersion lens at 100X. 


Figure 3: Gram stain done on D. aquaticus sample that was inoculated on 12/05. Contamination is shown on each slide, with the red arrows pointing to the contaminating cells. The cells could potentially be Staphylococcus epidermidis, as this type of bacteria is normal flora on human skin.  

 

Discussion: 

 

Not much progress was made this week. The first error I made was not inoculating D. aquaticus on 12/01. This would have prepared us to finish our soft agar experiment at the beginning of the week. Because we want to work with a newer sample of bacteria in our minimum inhibitory concentration (MIC) testing, we inoculated on 12/05. 

 

We had prepped our work to finish our MIC inoculations on 12/08, but we ran into another issue. The D. aquaticussample we inoculated on 12/05 seemed to be contaminated. We found a black “spot” floating in the liquid media. The gram stain confirmed that there were other cells in the sample. Because of this contamination, we were unable to move on with our MIC testing. I believe this contamination occurred while we were taking a gram stain on 12/06. I did see part of the inoculation loop touch the inside of the flask when the inoculation was occurring. This would definitely cause contamination, and in that moment I realized that next time I need to make multiple samples of D. aquaticus to have just in case another contamination occurs. Another mistake I made was not autoclaving the TGY before I put it in the flask. The TGY that I used was autoclaved after it was made, and I assumed that this was autoclaved enough to use for our inoculation. It turns out that after the TGY is added to the flask, it needs to be autoclaved again prior to inoculation. Although I was careful with transferring TGY into the flask, there was still room for contamination because it was not autoclaved again.

 

This week was a challenge because it seemed like nothing was going right. I believe errors were made due to the increased stress we are feeling during finals week. The errors we made were small, however they impacted the flow of our project. Next week I will be more intentional; we have multiple bacterial samples made and all of our TGY has been autoclaved. On 12/12 we will take the OD value of our samples, then finish up with our soft agar (including TGY, tetracycline, and D. aquaticus). We should have a result on 12/14, completing our MIC project just in time for winter break. Next semester we will decide if we want to move on with MIC testing for all of the Deinococcus species in the lab, or if we will continue working on transformation.

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