Week 8 (10/24/22- 10/28/22) Contamination Difficulties

 

Introduction: 

Minimum Inhibitory Concentration (MIC) is evaluated to determine the amount of antibiotic (kanamycin) to add to a bacterial sample to inhibit growth. This is important in the transformation experiment to determine if the cells are transformed or not. Plating transformed cells on media that contains an antibiotic at its MIC allows for only transformed cells to grow. This is due to the antibiotic of choice kanamycin, inhibiting growth in all cells that do not contain the kanamycin resistance gene. The kanamycin resistance gene will be added to all D. aquaticus cells during the transformation procedure, so growth leads to the conclusion that the bacterial cells are transformed and contain this gene.

Methods: 

OD600 of D. aquaticus was taken on the nanodrop prior to starting the MIC procedure.

Protocol for MIC is the same as the previous week:

Before running the experiment, the UV light was turned on in the hood for 15 minutes. The blower was also turned on and the glass was lifted slightly.  All equipment, gloves, and surfaces under the hood were wiped down using Kim wipes with alcohol.

 

A sterile 96-well plate was used. 6 reps of different concentrations were done, with 3 runs for each rep. Specific concentrations and amounts of TGY/kanamycin added are shown in figure 1. TGY was added to the wells first, using a P100, P20, and P2 pipette. Kanamycin was added next, using a P2 pipette. Each well was mixed 6 times using a P1000 pipette, then 52.5 ul of liquid was removed from each well and deposited in the waste beaker. 2.5 ul of D. aquaticus was added to each well using a P20 pipette, making the total volume in each well 250 ul. The 96-well plate was placed in the micro-plate reader for 48 hours. Everything under the hood was wiped down with Kim wipes and alcohol, and the UV light was turned back on for 15 minutes to sterilize the area. 

Rep	Volume of Media (ul)	Volume of Kanamycin (ul)	Concentration of Antibiotic (ug/ml)
1	299.4	0.6	2
2	299.48	0.525	1.75
3	299.55	0.45	1.5
4	299.63	0.375	1.25
5	299.7	0.3	1
6	299.78	0.225	0.75

Figure 1: Amount of antibiotic and TGY media added into each tube to get the final antibiotic concentration.

Figure 2: Outline of MIC procedure.

Figure 3: Concentrations of kanamycin in each well, in ug/ml. Rows A-C have the same concentrations. Row H contains the blanks (containing 250 ul of TGY),  and row F contains the positive controls (containing 2.5 ul of D. aquaticus and 247.5 ul of TGY).

On 10/25-10/26, test tubes were prepared for the microbiology courses at GCC. PR Sucrose media was personally made by me, by adding 21g of sucrose to 1 liter of deionized water and mixing on hot plate without heat. All test tubes contain a Durham tube inverted upside down prior to adding the media. 3 different types of test tubes were created with different types of media: PR Mannitol, PR Sucrose, and PR Lactose. 7 ml of the desired media was added into a test tube and the lid was screwed on loosely to allow for air exchange. All test tubes were placed in the autoclave.

A gram stain was done on the TGY that was used for our MIC procedure and the results were viewed under a microscope.

Results: 

OD600	Correction	Wavelength	A(wavelength)
0.97	0.10	600	0.97

Figure 4: OD600 results taken on 10/24 prior to starting the MIC procedure.

Figure 5: Minimum Inhibitory Concentration results of kanamycin with D. aquaticus after 48 hours, resulted on 10/26. Row A has antibiotic concentrations decreasing from 2 ug/ml to 0.75 ug/ml. Rows B and C are duplicates of row A, making there 3 runs total. All samples in row F are positive controls, containing TGY and D. aquaticus. All Samples in row H are blanks, containing only TGY.

Figure 6: Gram stain results taken from contaminated TGY. Both images are from the same gram stain from different angles of the slide, viewed under the 100x oil immersion lens.

Discussion: 

The OD600 values obtained from our D. aquaticus sample were consistent with the bacterial cells being in the exponential growth phase. This meant the bacterial cells are sufficient to test on to determine the MIC. 

After analyzing the microplate results after 48 hours, it was clear that something went wrong in the experiment. The negative controls indicate this; all of the negative controls containing only TGY seemed to have a large growth curve. This means that the TGY used in the experiment was contaminated, and invalidates all of the results from this MIC test. Gram staining was done on the TGY broth in attempt to visualize the organism that contaminated the media. Based on the stain, it looks like there may be multiple organisms in the media. It is unclear what the pink cells are because they are large and do not resemble a bacterial cell. In the second image in figure 6, there does appear to be cells that could be bacterial. These cells are purple and pink, and after analyzing the slide with Dr. Tuohy, it appears that these cells are gram negative. It is likely that this error was made while pipetting into the TGY, which was done about 50 times. When repeating this procedure next week, fresh TGY will be used and proper technique will be followed when pipetting. I have seen mistakes with pipettes where a student will accidentally drag their pipette on the bin while disposing their tip, and I will be more conscious of this during our next run to ensure there is not contamination. 

Going forward next week, we will change some of the procedure. The test will continue to be run on concentrations decreasing from 2ug/ml to 0.75 ug/ml Because mixing in the microplate is difficult and could lead to transfer of contents into other wells, we will change our technique. The TGY, kanamycin, and D. aquaticus will all be added into a test tube and mixed prior to adding it to the well. This will make the procedure easier to follow and increase the chances of an accurate result. 6 positive controls will be done, as well as 6 negative controls that will now contain TGY and kanamycin. There will also be 6 blanks that only contain TGY. With these changes implemented, I believe our results will be more conclusive in giving us the correct MIC for this bacterium.