Week 14: January 30, 2023 - February 3, 2023

 Week 2: January 30, 2023 - February 3, 2023 

January 31, 2023

This week we resumed our work on MIC testing on Deinococcus aquaticus. MIC stands for minimum inhibitory concentration. This refers to resistance of specific bacterial strains to an applied antibiotic.  During the previous semester, we focused on D. aquaticus but this semester, we are going to be working with many other Deinococcus species using the protocols and techniques that we used on D. aquaticus. 

Before we can move onto other species, we needed to finalize our MIC testing on D. aquaticus. We began by taking out our D. aquaticus sample out of the 30°C incubator. It had mold in it so we inoculated a new sample into broth in order to continue.

Inoculation Steps:

  1. Wipe down surface and turn on gas

  2. Pipette 10 ml of TGY into clean flask. 

  3. Burn inoculation loop until red hot 

    1. Wait about 30 seconds for it to cool

  4. Dip sterile inoculation loop into D. aquaticus 

  5. Dip inoculation loop (with D. aquaticus) into flask of TGY

    1. Swirl the loop around a little bit

  6. Burn inoculation loop again until red hot 

    1. Sterilizes and kills bacteria on it

  7. Cover flask with aluminum foil and label

    1. TGY, D. aquaticus, date, initials of group members 

  8. Incubate at 30°C 

  9. Turn off gas, wipe down work surface, put materials away  


February 1, 2023

In order to finish our MIC testing for D. aquaticus, we did a serial dilution. A serial dilution is taking from a stock solution, or the highest concentration, and diluting it down into small concentrations that would be hard to achieve using a standard method such as pipettes. 

We began by heating up our four flasks of TGY and soft agar which had previously been made and autoclaved. Our concentrations would be 100 ng/ml, 10 ng/ml, 1 ng/ml, and 0 ng/ml. The 0 ng/ml would be used as our negative control. 

Steps: 

  1. Heat up flasks of TGY and soft agar until it is completely liquified

    1. Allow TGY to cool a little bit before continuing

  2. Remove appropriate amounts of TGY and soft agar from each flask

  3. Add in the appropriate amount of antibiotic 

    1. For this, we were using tetracycline

  4. Add in appropriate amount of bacteria to each flask and vortex

    1. We were working with D. aquaticus

    2. Vortex on level 6 for 6 seconds 

  5. Pour onto plates and spread around

  6. Let plates set then incubate in the 30°C incubator 



Concentration 

TGY + Agar Removed 

Antibiotic Added 

Bacteria Added 

100 ng/ml

5 ul

5 ul 

25.2 ul

10 ng/ml

250 ul 

250 ul 

25.2 ul

1 ng/nl

250 ul

250 ul

25.2 ul

0 ng/ml

None 

None 

25.2 ul








References 


Kowalska-Krochmal, B., & Dudek-Wicher, R. (2021). The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance. Pathogens, 10(2), 165. https://doi.org/10.3390/pathogens10020165

Sapkota, A. (2020, May 27). Serial dilution- definition, formula, calculator, procedure, uses. Microbe Notes. https://microbenotes.com/serial-dilution/

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