Week 15: February 6, 2023 - February 10, 2023

 February 6, 2023 

We decided to do gram stain on our sample of D. aquaticus from 1/31/23 because the plates that we made the week before for our MIC testing all contained growth. This was a setback for us since even the highest concentration showed growth when we predicted that it wouldn’t. In order for us to continue and redo our MIC testing plates, we wanted ot be sure that the growth that was on the plates was actually D. aquaticus and not contamination. 

Steps 

  1. Rinse slide with water and dry off

  2. Draw a circle on slide with wax pencil 

  3. Turn on gas and bunsen burner 

  4. Heat inoculation loop with flame until red hot to sterilize 

  5. Wait about 30 seconds for  loop to cool down 

  6. Dip into sample

  7. Spread in the middle of the circle 

  8. Heat up inoculation loop again until red hot

    1. Kill of the bacteria and sterilize the loop again

  9. Pass slide over the top of the flam carefully

    1. When fixed, the slide should be dry

  10. Move over to gram stain sink

  11. Fill circle with crystal violet 

    1. Let sit for 1 minute 

  12. Rinse with D.I. water 

  13. Fill circle with grams iodine 

    1. Let sit for 1 minute 

  14. Rinse with D.I. water 

  15. Fill circle with 95% ethanol 

    1. Let sit for 7 seconds 

  16. Rinse with D.I. water 

  17. Fill circle with sufranin 

    1. Let sit for 45 seconds 

  18. Use blotting paper to dry the slide 

  19. Move over to microscope and work your way through the different magnifications

    1. Once you get to oil, put a drop of emersion oil

    2. Use lens cleaner and lens cleaner sheets to wipe the microscope when finished

Results of Gram Stain

The gram stain confirmed that the growth which was present on the plates was D.aquaticus. The slide showed bacteria that was gram negative and rod-shaped just like D. aquaticus. Since we were able to confirm that D. aquaticus growed on the plates despite the concentrations of antibiotic, we decided to redo our testing with a a broader range of antibiotic concentrations. 


                                                                            Gram stain of D. aquaticus under a microscope


February 10, 2023

Materials 

  • 5 flasks of of autoclaved TGY and agar 

    • 125 ml flasks

    • Each containing 25 ml of TGY + 1.5% agar

  • 0.5 mg/ml tetracycline 

  • Pipettes and pipette tips

  • Hot plate


Procedure

  1. Heat up the flasks on the hot plate until they are completely liquified 

    1. If the flasks come out of the autoclave and are already liquified, this step can be skipped

    2. It is best to work on 1-2 steps at a time to avoid the solidification of the agar

When working with 1-2 flasks at a time, the protocol should be completed before starting the next flasks

  1. Label the plates with the antibiotic concentration before pouring

    1. For these plates we used tetracycline

  2. Once the media passes the baby bottle test, mix the antibiotic on the vortexer

    1. At level 6 for 6 seconds 

  3. Add the antibiotic to the flask and swirl it around to mix 

  4. Pour the contents of the flask onto the plate

  5. Leave the plates out overnight to dry 



Antibiotic Concentration 

Amount of Antibiotic 

1000 ng/ml 

500 μL

500 ng/ml 

250 μL

100 ng/ml 

50 μL

10 ng/ml

5 μL

1 ng/ml 

0.5 μL







                                                                                    Poured plates in the setting process




References 


Bruckner, M. Z. (2021, January 14). Gram Staining. Microbial Life Educational Resources. https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html 


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