Week 8: Doing gram stains on aquaticus

Introduction: 

     Following our previous 96 wells results of contamination, we found a foreign bacteria in our TGY which resulted in an unusual growth in our blanks and negative controls. In this week, we wanted to ensure that our antibiotic (kanamycin) and our bacteria and cell (aquaticus) is not also contaminated. Therefore, it was my job to do a gram stain and check on our bacteria to see if it is contaminated or not. This gram stain took a total of 2 tries due to some errors and mistakes.

Day 1: October 31st 

    For the first day, I did a gram stain on both of our aquaticus which dated back to 8/16/22 and 9/30/22. The results came back quite unusual and unfortunate. 

8/16/22 Cells

Fig. 1: 40X Lens                                                              


From a 40X Lens standpoint, the cells look quite blue and rod shaped. Some seem to look more cluttered than others like the big clutter in the far right above. 







Fig. 2: 100X Lens


Now in a 100X Lens standpoint, the results get even more unusual when the rods seem to be cluttering up significantly in the far right. There is also a tint of blue and red in the results. The most noticeable for me when the blue that was blending into the purple cell's clutters. 







Dr. Tuohy suggest that the 8/16/22 is likely to be gram negative. 

9/30/22 Cells

Fig. 3: 40X Lens


As shown here, the alarming size of this cell was not a rod-shape as expected but rather a big pink clump. There were other visible blue rod cells surrounding it, but it was very tiny and almost unnoticeable. 








Fig. 4: 100X Lens 


Zoomed in, the cell shaped did not change and the tiny blue rods became less visible. There is not sign of pink or purple rod-shaped cells and only had the big clump. This was a sign of contamination. 








Day 2: November 1st

Even though Day 1's results turned into a failure and could conclude that our aquaticus is in fact contaminated like our TGY, I still redid the gram stains one more time just to make sure I avoid any errors this time. 

8/16/22 Cells

Fig. 5: 40X Lens


As shown here, the results look quite similar to Fig. 1 but except had less clumps and more individual rod cells. 








Fig. 6: 100X Lens


The results is better than Fig. 2 because the cells are not as cluttered and it is more pink than having multiple colors. 








I conclude that this is gram negative. 

9/30/22 Cells

Fig. 7: 40X Lens 


This shows a more substantial and better result than Fig. 4. I was relieved to see tons of rods. Sure there are clumps, but it is more substantial than the big clump. NOTE: The blue stain is from the blue wax pen and it is not a foreign substance. 







Fig. 8: 100X Lens 


As expected from Fig. 7, the cell is not clumpy and is actually pink single rod shapes which has little to no clumps. This was a big relief knowing that our aquaticus is not in fact contaminated and that it was just a gram staining mistake in Day 1. 







I concluded that this is gram negative.

Conclusion: 

    After spending two days retrying the gram stain, Jessica, Kaylee, and I was relief that it was just a gram staining mistake and error that caused the unusual and alarming result in Day 1. The gram staining errors would be that I did not vortex the cells enough or I accidentally burnt the cells when I was going in with the hot metal loop. Either way, I am relieved and glad that the cells were not contaminated. 

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