Week 19: March 6, 2023 - March 10, 2023

 This week we were able to begin working with D. roseus to try to find its minimum inhibitory concentration. In order to move through our list of species quicker, we had to begin working on several different bacteria at a time rather than waiting to wrap up a species before moving on to the next. D. roseus was the bacteria with an appropriate amount of growth that would allow us to work with it. 

Performing MIC Protocol on D. roseus 

1. Pipette 5 ml of TGY into each test tube

1. 13 x 100 mm test tubes with metal caps 

1. 7 for each of the three antibiotics that will be used 

1. 21 test tubes total

2. Autoclave to sterilize tubes and TGY

2. Take the OD600 value of bacteria 

1. Want it between 0.80 - 1.00

2. If the value is higher, dilute it with TGY until it is within our desired range 

1. Our sample of D. roseus was at 1.76 so we diluted and the new value was 0.9

3. Once TGY has been autoclaved and cooled, remove appropriate amount of TGY per tube 

1. Amounts are shown in the table below 

4. Add in appropriate amount of antibiotic to each tube 

1. Amounts are shown in the table below 

1. The amounts for methicillin and kanamycin are the same because our stock solution of them is both 2 mg/mL

2. Ofloxacin has a stock solution of 1 mg/mL therefore the amounts added are different 

5. Vortex the tubes to mix the TGY and antibiotic 

1. Level 6 for 6 seconds

6. Remove 50 μL of TGY + antibiotic solution from each tube 

7. Add 50 μL of bacteria into each tube

1. In this case, we were working with D. roseus 

8. Vortex each tube on the vortexer 

1. Level 6 for 6 seconds 

9. Place tubes in shaking incubator for 30°C

1. Should be left in there for at least 48 hours 

*NOTE: The amount of TGY removed in step 3 is the same amount as the amount of antibiotic that will be added in step 4. The amount of TGY + Antibiotic removed in step 6 will be the same amount of bacteria added in step 7. This is to ensure that the total volume of 5 ml does not change.* 

Concentration	TGY Removed/Antibiotic Added  for Kanamycin/Methicillin	TGY Removed/Antibiotic Addedfor Ofloxacin	TGY + Antibiotic Solution Removed/Bacteria Added
1.56μg/mL	3.91 μL	7.8 μL	50 μL
3.125 μg/mL	7.8125 μL	15.625 μL	50 μL
6.25 μg/mL	15.625 μL	31.25 μL	50 μL
12.5 μg/mL	31.25 μL	62.5 μL	50 μL
25 μg/mL	62.5 μL	125 μL	50 μL
50 μg/mL	125 μL	250 μL	50 μL
100 μg/mL	250 μL	500 μL	50 μL

Table showing the amounts that will be pipetted out/in during the MIC protocol

Absorbance Readings of D. roseus on Kanamycin, Methicillin, and Ofloxacin 

We took a spectrophotometer reading after 72 hours for D. roseus on Kanamycin, Methicillin, and Ofloxacin. Since they were put in the incubator before the weekend, we had to wait until Monday to be able to take the absorbance values.  

Spectrophotometer Reading of D. roseus After 3 Days

Concentration	Kanamycin	Methicillin	Ofloxacin
1.56 μg/ml	0.41	0.21	0.08
3.125 μg/ml	0.01	0.05	1.76
6.25 μg/ml	0.03	0.11	0.05
12.5 μg/ml	0.02	0.00	0.03
25 μg/ml	0.01	0.03	0.06
50 μg/ml	0.02	0.07	0.09
100 μg/ml	0.11	0.00	0.02

The results of the spectrophotometer reading after three days seemed promising. It was still too early for us to draw any conclusions but by simply looking at the values, I predicted that D. roseus might be sensitive to antibiotics. In other words, this species of bacteria might have a low concentration MIC. 

We did, however, have an error with the Ofloxacin tubes at the concentration of 1.56 μg/ml. The bacteria was not added into the tube during the protocol therefore we cannot rely on the results that we obtained from that tube. Next week, we will need to redo the protocol with concentrations of 1.56 μg/ml, 3.125 μg/ml, and 6.25 μg/ml. This will give us the ability to properly test the lowest concentrations and compare them against each other.   

OD600 Values 3 Days Post-Inoculation in Broth 

It is important for us to consistently check up on how the new species are growing because we are unfamiliar with them. We need to have an idea of how quickly or slowly each species grows in order for us to plan out our project and see which species is ready to be worked with. In order to check the growth, we took the OD600 values of each species we have growing. 

Species	OD600 Value
D. caeni	0.22
D. indicus	0.32
D. Gobiensis (D. GoB)	0.39
D. aquatilis	0.28
D. grandis	1.52

Table showing the OD600 values of the new species we are growing 

So far, we are able to see that D. grandis has the most growth but it has also been growing for longer than the other four species. D. GoB is second for the most growth followed by D. indicus. D. aquatilis and D. caeni are not too far behind. It is too soon to say which ones will be the fastest growing.