The Antibacterial and Antifungal Activities

The antibacterial and antifungal activities were evaluated by standardized disc-agar diffusion method [29-31]. The efficacy of the novel antimicrobial compounds was determined against different strains of gram-positive and gram-negative bacteria. These compounds were also evaluated against different fungal strains. The gram-positive organisms that were used for culture sensitivity include Staphylococcus aureus (RCMB 010027), Streptococcus pneumoniae (RCMB 010010) and Bacillis subtilis (RCMB 010067). On the other hand, the gram-negative organisms that were used for culture sensitivity include Pseudomonas aeruginosa (RCMB 010043), Klebsiella pneumoniae (RCMB 0010093) and Escherichia coli (RCMB 010052) and Sallmonella typhimurium (RCMB 010315. The fungal strains that were used include Aspergillus fumigatus (RCMB 02568) and Candida albicans (RCMB 05036). Different antibiotics were used as reference for evaluating the antimicrobial activity of novel compounds. Ampicillin was used as the reference antibiotic for assessing the antimicrobial activity of the novel compounds against gram-positive bacteria. Gentamicin was used as the reference antibiotic for assessing the antimicrobial activity of the novel compounds against gram-negative bacteria. On the other hand, Amphotericin B was used as the reference antibiotic for assessing the antimicrobial activity of the novel compounds against for fungal strains.
Assay of Antimicrobial activity
M. tuberculosis (RCMB 010126) was used as the experimental microbe for the present study.

Pyrazinamide and Isoniazid were used as the reference antibiotic in comparison to the novel compounds. MABA (Microbial Alamar Blue Assay) was conducted to evaluate the antibacterial activity of the novel compounds. The assay was conducted in micro-titer plates. The micro-titer plates were round-bottomed and black in color. Such arrangements were used to prevent the background effects of light. The peripheral wells in the micro-titer plate were filed with sterile water. Sterile water was use to prevent dehydration in the wells. The wells were classified into two types; experimental and control. The experimental wells were filled with novel antibacterial compounds. However, the control wells consisted of bacterial colony only. The experimental wells were filled with different dilutions of the bacterial colony. Initial dilutions of the novel compounds were achieved with dimethyl sulfoxide. Subsequent dilutions were undertaken in the micro-titer plate. A two-fold dilution was achieved in the micro-titer plate. The experimental and control wells were inoculated with 0.1 ml (105 CFU//ml) Mycobacterium tuberculosis. The micro-titer plate was incubated at 37 degree centigrade. On the 4th day of experimentation, 20 micro liter of Alamar Blue Solution and 12.5 micro liters of 20% Tween-80 were added to the micro-titer plate. The micro-titer plate was further incubated for 24 hours at 37 degree centigrade. Spectrophtometric readings were recorded at 590 nm. The results were expressed as Percentage inhibition (% inhibition). Percentage inhibition was estimated as:
% inhibition = 1- (Mean O.D. of test well/ mean O.D. of B wells) × 100.
Results were also expressed as Visual Minimum Inhibitory Concentrations (VMIC).
VMIC is defined as the lowest concentration of the novel antimicrobial compounds that prevented color change.