Quality control of veterinary antimicrobial by microbial assay

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