Week 3 - September 30, 2022

Continuation of Week 2: 

Friday, September 23, 2022 

This is a continuation of last week’s blog post. I was not able to be in the lab when the 48-hour reading was done but the results of the reading are listed in the table below. 

D. aquaticus on Kanamycin 48-Hour Absorbance Reading 

Blank With Control 1 (Media) 

Spectrophotometer on 200 with 600 wavelength

Run	Concentration of Antibiotic	A	B
1	3.595 μg/mL	-0.12	-0.12
1A	3.125 μg/mL	0.32	-0.12
2	2.733 μg/mL	-0.01	-0.04
3	2.34 μg/mL	-0.02	-0.045
4	1.95 μg/mL	0.17	-0.09
5	1.56 μg/mL	0.34	0.19

Concentration 5, our lowest concentration, seems to be the one that has the most amount of growth. Set A had an absorbance reading of 0.34 and Set B of the same concentration had an absorbance reading of 0.19. The readings, however, have a difference of 0.15. After this reading, it was decided that we would lower our concentrations to see how D.aquaticus reacts to Kanamycin in lower concentrations. 

Monday September 26, 2022

D. aquaticus on Kanamycin Absorbance Reading After 115 Hours 

Blank With Control 1 (Media)

Spectrophotometer on 200 with 600 wavelength 

Run	Concentration of Antibiotic	A	B
1A	3.595 μg/mL	0.81	0.06
1	3.125 μg/mL	0.50	1.24
2	2.733 μg/mL	2.21	1.27
3	2.34  μg/mL	1.61	1.88
4	1.95 μg/mL	1.31	1.63
5	1.56 μg/mL	1.51	1.91

This was another day that I was not in the lab. My lab partners did a reading after 115 hours out of curiosity and I have included the results here so that I can easily see them and compare them to each other. 

There was significant growth compared to the 48-hour reading. At the 48-hour mark, Concentration 5 had the most amount of growth. Here, we can see that Concentration 2 seems to have the most growth in Set A. in Set B, Concentration 5 still has the most amount of growth. In Set A, Concentrations 2-5 have absorbance values above 1.00. In Set B, Concentrations 1-5 have absorbance values above 1.00. Due to this, it was decided to lower the concentrations and have the highest concentration be 3.00 μg/mL. 

Tuesday, September 27, 2022 

I was out sick on Tuesday when my lab mates began working on the lower concentrations of Kanamycin to test D. aquaticus. Over the course of Monday-Wednesday, they set up the experiment and even took readings of the growth. Below are the lower concentrations that were decided upon. 

New Kanamycin Concentrations (Lower)

Run	Concentration of Antibiotic
1	3.00 μg/mL
2	2.75 μg/mL
3	2.50 μg/mL
4	2.25 μg/mL
5	2.00 μg/mL
6	1.75 μg/mL
7	1.50 μg/mL
8	1.25 μg/mL
9	1.00 μg/mL
10	0.75 μg/mL

There will be 10 different concentrations and three sets of each concentration. There will be three sets in case there is any contamination. This way, we can acquire more accurate results. Before doing any readings, each test tube of antibiotic, media, and D. aquaticus will be mixed via vortex on level 6 for 10 seconds. 

D. aquaticus on Kanamycin 20-Hour Absorbance Reading - Lower Concentration Range 

Blank With Control 1 (Media)

Spectrophotometer on 200 with 600 wavelength 

Run	Concentration of Antibiotic	Set A	Set B	Set C
1	3.00 μg/mL	0.05	-0.02	-0.01
2	2.75 μg/mL	0.02	0.01	-0.01
3	2.50 μg/mL	0.00	0.08	0.01
4	2.25 μg/mL	0.02	0.00	0.03
5	2.00 μg/mL	-0.01	0.09	0.03
6	1.75 μg/mL	0.06	-0.01	0.01
7	1.50 μg/mL	-0.01	0.04	0.04
8	1.25 μg/mL	-0.01	0.03	0.01
9	1.00 μg/mL	0.04	0.03	0.02
10	0.75 μg/mL	0.14	0.10	0.04

Control 2

0.68

The absorbance values of D. aquaticus on the lower concentrations of Kanamycin after 20 hours are included in the table above. There is not very much growth but Concentration 10A did have the highest absorbance value at 0.14.

Thursday, September 29, 2022 

D.  aquaticus on Kanamycin 48 Hour Absorbance Reading - Lower Concentration Range 

Blank With Control 1 (Media)

Spectrophotometer on 200 with 600 wavelength 

Run	Concentration of Antibiotic	Set A	Set B	Set C
1	3.00 μg/mL	0.02	-0.01	0.01
2	2.75 μg/mL	-0.02	-0.01	0.12
3	2.50 μg/mL	-0.02	-0.01	-0.03
4	2.25 μg/mL	0.00	-0.01	0.04
5	2.00 μg/mL	0.08	0.00	0.26
6	1.75 μg/mL	0.13	0.17	-0.02
7	1.50 μg/mL	0.34	0.13	0.27
8	1.25 μg/mL	0.44	0.07	0.11
9	1.00 μg/mL	0.64	0.70	0.72
10	0.75 μg/mL	1.00	1.27	1.11

Control 2

1.69

After the 48 hour mark, Concentration 10 continues to be the concentration with the most amount of growth. Concentration 10 only contains 0.75 μg/mL of Kanamycin and it has a mean absorbance value of about 1.127.

Friday, September 30, 2022

D. aquaticus on Kanamycin 72-Hour Absorbance Reading - Lower Concentrations

Blank With Control 1 (Media)

Spectrophotometer on 200 with 600 wavelength 

Run	Concentration of Antibiotic	Set A	Set B	Set C
1	3.00 μg/mL	0.05	0.02	0.01
2	2.75 μg/mL	0.02	0.02	0.02
3	2.50 μg/mL	0.46	0.09	0.03
4	2.25 μg/mL	0.16	0.24	0.12
5	2.00 μg/mL	0.29	0.46	0.16
6	1.75 μg/mL	0.69	0.63	0.01
7	1.50 μg/mL	1.17	1.02	1.49
8	1.25 μg/mL	1.24	1.02	0.79
9	1.00 μg/mL	1.65	1.63	1.57
10	0.75 μg/mL	1.90	1.97	1.78

Control 2

2.41

The lowest concentration continues to be the concentration with the most amount of growth. Concentration 9 was close behind it. In the search for the MIC, or the minimum inhibitory concentration, we are predicting that it will be close to either concentration 4 or 5. I believe this will be where bacteria growth can start to be inhibited by the antibiotic but there are still living cells.