Preparation of medium:
In 1000 mL distilled water, dissolve 28.0 grams of nutritional agar. Bring to a boil, then completely dissolve the medium. Autoclave at 15 Ibs pressure (121°C) for 15 minutes to sterilise. Pour into sterile Petri plates after thoroughly mixing.
Microorganisms
Gram – positive bacteria, Staphylococcus aureus (MTCC 3160), and Gram – negative bacteria, Escherichia coli, were used in the biological experiments (MTCC 732). Microbial type culture collection (MTCC) at Chandigarh, India's Institute of Microbial Technology (IMTECH).
Preparation of 24 hours pure culture
In a Roux bottle, a loop containing each of the bacteria was suspended in around 10ml of physiological saline. These were streaked onto the appropriate culture slants and incubated for 24 hours at 37°C. When growth was observed after the incubation period, the tubes were maintained at 2-8̊ C kept at room temperature.
Preparation of samples solutions for the experiment
Samples were prepared by dissolving 10 mg of chloramphenicol in 10 mL of distilled water and comparing them to a standard solution of chloramphenicol for bacteria (25 mg/mL pure water). Unless they were employed in the experiment, they were kept in the refrigerator.
Agar well – diffusion method
Antimicrobial activity was determined using the agar well diffusion method shown in Figure 15. With 24 hour culture and 48hour old broth culture of respective bacteria, nutrient agar (NA) plates were swabbed (sterilized cotton swabs). Using a sterile cork borer, agar wells of 5mm diameter were drilled onto each of these plates. Using sterilized dropping pipettes, 50l, 100l, and 150l of the sample, control 30l, and standard 30l were added to the wells, and plates were left for 1 hour to allow for pre-incubation diffusion.For bacterial strains, the plates were incubated in an upright position at 37 ̊C,2̊ C for 24 hours to reduce the effects of variation in time between the applications of different solutions. The existence or absence of an inhibitory zone was reported as a result. The lack of the tested organism was indicated by the inhibitory zone surrounding the well. The zones' sizes were measured on a diameter measurement scale. Antibacterial activity was measured in triplicates and the average values were recorded shown in Table 4.
Table: 4 Anti-bacterial activity of TPN Grown Crystal
Bacterial strains
|
Sample dose (µl)
|
Std. (30µl)
|
Control (30µl)
|
50µl
|
100µl
|
150µl
|
Gram positive
|
Staphylococcus aureus (mm)
|
3.10±0.21
|
5.40±0.37
|
9.25±0.64
|
11.15±0.78
|
0.00±0.00
|
Gram negative
|
Escherichia coli (mm)
|
3.50±0.24
|
6.75±0.47
|
9.60±0.67
|
11.80±0.82
|
0.00±0.00
|
Values expressed as Mean ± SD for triplicates,
Standard: Chloramphenicol; mm: Millimeter, Control: Distilled water