The basic need for quality control in veterinary antimicrobials is to check whether it efficiently treat the disease and to reduce antimicrobial resistance (AMR). We can do quality control of antimicrobial by microbial assay to check AMR and also check the antimicrobial activity of the veterinary antimicrobial drugs.
By: Haseeb Asif, Arfa Tehreem
Microbial assay:
Microbial assay is defined as the qualitative or quantitative determination of chemical compounds with the use of microorganisms. The microbial assay of antibiotics is based upon a comparison of the inhibition of microorganisms by measured concentration of antibiotic under examination with that produced by known concentration of standard preparation of antibiotic having known activity
Comparison b/w gram-positive and gram-negative bacteria:
comparison | Gram-positive cells | Gram-negative cells |
Lipopolysaccharide (LPS) | Absent | present |
lipoprotein | Absent | present |
peptidoglycan | 60-80% | 10-20% |
Lipoteichoic acid | Present | absent |
Protein | 15% | 60% |
Useful bacteria | Actinomyces, bacillus subtilis, bacillus pumilus, clostridium tetani, staphylococcus aureus | Pseudomonas aeruginosa, Bordetella pertussis, salmonella enterica serovar typhi |
Isolation identification and purification of Bacillus subtilis for microbial assay:
Bacillus subtilis:
Bacillus subtilis are rod-shaped, Gram-positive bacteria that are naturally found in soil and vegetation. They grow in the mesophilic temperature range with the optimal temperature between 25-35oC. They form stress-resistant endospores that enable them to survive under harsh environmental conditions
Material and method:
- Collect at least five samples of 10 gram of soil from different location each sample is packed in sterile bottle and labelled appropriately
Isolation:
- 10gm soil sample is suspended in 90ml of sterile distilled water the soil sample is heat shocked at 60oC for one hour in water bath to kill non-spore-forming bacteria
- A loopful each of the soil suspensions was inoculated by streaking on nutrient agar medium. The inoculated plates were incubated aerobically at 37oC for 24hrs and examined for the appearance of colonies.
Identification:
The colonies that exhibited cultural characteristics typical of Bacillus species morphological characterization of the organism were determined by gram staining and endospore staining method
And the biochemical characterization is determined by biochemical tests
Basic characteristics | Properties (bacillus subtilis) |
Flagella | Flagellated |
Gram staining | Gram Positive (+ve) |
Motility | Positive (+ve) |
Pigment | Negative (-ve) |
Spore | Positive (+ve) |
Catalase | Positive (+ve) |
Citrate | Positive (+ve) |
Indole | Negative (-ve) |
Methyl red | Negative (-ve) |
Oxidase | Variable |
Urease | Negative (-ve) |
Voges Proskauer | Positive (+ve) |
- Microbiological assay for veterinary antimicrobial:
To perform microbiological assay on veterinary antimicrobial drugs we can use cylinder or cup plate method (diffusion phenomenon)
Requirement for microbial assay:
- Standard solution
- Test solution
- Medium
- Test organism (inoculum)
Standard preparation:
To prepare standard dissolve the quantity of standard preparation of antibiotics in the solvent given in the table. Dilute the preparation to get the required concentration as stated and store in refrigerator. From the stock solution prepare 5 or more test dilutions
Test solution:
For the substance under examination assumed potency per unit weight and volume and on this assumption prepare on the day of assay a stock solution and test dilution as specified for each antibiotic
Medium:
Media specified for bacillus subtilis is nutrient agar which is
prepared according to manufacturer instructions
Test organism (inoculum preparation):
- The test organism for each antibiotic is
listed along with its ATCC identification number
- Inoculum is prepared by subculturing the colonies of
bacillus subtilis on 10 ml nutrient agar slant and
incubate at 37oC
Procedure:
- Inoculate the specified organism (bacillus subtilis) in a
liquid medium (nutrient broth) and pour into a petri dish
so that depth of 3-4 mm is reached
- Cups or cavities are made by using a sterile borer
- Prepare standard and test solution as mentioned above
- Apply these solutions into cylinder or agar cavities and incubate at 37oC for 24 hours
- If antimicrobial has any antibacterial activity it will show the zone of inhibition
Minimum inhibitory concentration:
The minimum inhibitory concentration of an antimicrobial agent is the lowest concentration of the antimicrobial agent that inhibits a given bacterial isolate from multiplying and producing visible growth in the test system.
MIC test can be performed by dilution method. In the dilution method, a sequence of decreasing concentration of a drug is made in a broth which is then inoculated with test microorganisms and then MIC is determined by examining the tubes or wells which can find the lowest drug concentration that inhibit the visible growth of test organism