Chemotherapeutic Agents Practical: Disc Diffusion and Broth Dilution Method

Chemotherapeutic Agents Practical
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Introduction
Pharmacologists must understand sensitivity and specificity investigations, as well as the fundamentals of synergy and antagonism while creating novel antimicrobial therapeutic regimens. In vitro susceptibility testing on a variety of antibiotics suited for treating these bacteria at that specific body site is used to recommend appropriate antibiotic therapy. A few procedures were used in our experiments: reading the Etest strip, demonstration of antimicrobial synergy, which tested if their combined activities were greater than the sum of their individual activities, and demonstration of antimicrobial antagonism, which tested if the presence of the other harmed one drug's activity.
 
Materials and Methods
Protocol 2 involved a demonstration of antimicrobial synergy and antagonism. For this experiment, both the broth dilution method and the disc diffusion were used to demonstrate antimicrobial synergy, enhanced inhibitory action or antimicrobial resistance, and reduced inhibitory activity (1). 
The first methodology used two primary techniques to exhibit antibacterial cohesiveness: The Disc Diffusion Method and the Broth Dilution Method. A sterilized swab was utilized for the Disc Diffusion Method. On a DST agar plate, some E. coli suspension from the previous experiment (step A.7) was added. Sulphamethoxazole and trimethoprim antibiotic discs were then inserted, separated by 1cm, and sterilely placed on the agar plate. They were then incubated for 24 hours at 37°C, after which zones of inhibition were seen and measured.
Penicillin and gentamicin were used to demonstrate synergy in the serial dilutions using broth. In rows 1 and 2 of a 96-well dish, pits 2-12, 50 µl of nutritional broth were introduced. Each row of wells 1 and 2 received 50 µl of penicillin solution (concentration 80 g/ml). Each well of raw two only received 5 µl of gentamicin solution (concentration 100 g/ml) after penicillin from 2-12 in each row had been doubly diluted. After that, three colonies from the plate given were emulsified in 3 ml of sterile distilled water to make an Enterococcus faecalis solution. In wells, 2-12 of each row, 50 µl of the bacterial rest were added. Finally, incubation of the 96-well plate at 37oC for 24 hours and later on, observation of the MIC of penicillin alone occurs and that of penicillin with gentamicin (2).
Making a Proteus mirabilis suspension by emulsifying three colonies in 3 mL normal saline and proving Antimicrobial Antagonism was the alternative option (3). Afterward, the portion of the culture was transferred to a DST agar medium swabbing with a sterilized swab. Antibiotic discs containing nalidixic acid and nitrofurantoin, isolated by 1 cm, were placed on the DST plate. The inhibitory zones were observed and recorded after a 24-hour incubation period at 37°C. 
The Etest strip was imbued with a calibrated range of antibiotic concentrations, allowing the MIC to be read at the point where the bacterial growth zone crossed the strip. The final MIC tetracycline findings for E. coli were read and recorded.
Results
For the Disc Diffusion procedure, it was observed that there was no bacterial growth as there was an area around the disc that was visible, which is the zone of inhibition (ZOI). For the Broth dilution method, the broth became cloudy, which insinuated bacterial growth had occurred from 4-12 on the first line and none in the second line. Finally, microscopic observations from the procedure, which aimed at demonstrating Antimicrobial Antagonism, showed that with the addition of nitrofurantoin, cellular elongation was inhibited since Nalidixic acid has an inhibiting impact on the genetic recombination of cells (4). After some time, bacterial growth was observed in the Etest technique, with an asymmetrically inhibiting ellipse positioned along the strip at 1.5ug/ml
Discussion 
The same purpose of the Disc Diffusion procedure is to assess the sensitivity of isolates obtained from some particular symptoms to therapeutic applications of antibiotics so that doctors can prescribe the most appropriate antibiotic therapy (5). As a region around the disc was visible, the results showed no bacterial growth. The zone of inhibition (ZOI) was around 20mm, which means the drug is susceptible. As a result, greater inhibition zones correspond with lower minimum doses of sulphamethoxazole and trimethoprim against E. coli (MICs).
The goal of the Broth Dilution Method is to find the lowest doses of the tested antimicrobial agent (minimal inhibitory concentration, MIC) that suppress the observable growth of the bacterium being studied under certain test circumstances. The lowest concentration of MIC suppressed bacterial growth from 4 to 12 on the first line. The absence of bacterial growth on the second line, on the other hand, indicated that the MIC did not support bacterial development (antagonism).
The last method, which was designed to demonstrate Antimicrobial Antagonism, revealed that adding nitrofurantoin to the cells decreased cellular elongation due to that restraining action on the binary fission of cells by nalidixic acid. The two compounds, nitrofurantoin and nalidixic, were shown to be antagonistic. However, the nalidixic acid and tetracycline combination was synergistic against E coli.
The Etest technique was to determine antimicrobial sensitivity by inserting an antibiotic-impregnated strip onto an agar plate. Tetracycline was observed to be resistant to E coli. If bacteria or fungus are susceptible to antibiotics or antifungals, they will not thrive when exposed to certain concentrations.
References
1. Bauer. Antibiotic susceptibility testing by a standardized single disk method. American journal of clinical pathology [Internet]. 2022;45(4). Available from: https://pubmed.ncbi.nlm.nih.gov/5325707/
2. Tanz RR, Shulman ST, Barthel MJ, Willert C, Yogev R. Penicillin plus rifampin eradicates pharyngeal carriage of group A streptococci. The Journal of Pediatrics [Internet]. 1985 Jun; 106(6):876–80. Available from: https://pubmed.ncbi.nlm.nih.gov/3889257/
3. Eliopoulos GM, Moellering RC. Antibiotic Synergism and Antimicrobial Combinations in Clinical Infections George. Clinical Infectious Diseases [Internet]. 1982 Mar;4(2):282–93. Available from: https://pubmed.ncbi.nlm.nih.gov/7051231/
4. Engelkirk PG. Laboratory diagnosis of infectious diseases : essentials of diagnostic microbiology [Internet]. Missouri.edu. 2022. Available from: http://link.library.missouri.edu/portal/Laboratory-diagnosis-of-infectious-diseases-/AKIazlvYS8s/
5. Biemer JJ. Antimicrobial susceptibility testing by the Kirby-Bauer disc diffusion method. Annals of clinical laboratory science [Internet]. 2021;3(2). Available from: https://pubmed.ncbi.nlm.nih.gov/4575155/
Part B
Q. no. 1 
HYPOTHETICAL RESULTS FROM BROTH DILUTION ASSAY AND DISC DIFFUSION ASSAY
The basics of broth and agar disc dilution are to know the minimum amount of the assayed antimicrobial agent, MIC. MIC values are used to know the susceptibilities of bacteria to drugs. However, growth is analyzed after incubation, and the MIC value is...