The microbial assays have been effectively extended to a plethora of pharmaceutical
preparations i.e., the secondary pharmaceutical products. However, this particular section will deal
with only the following three types of such products, namely :
(a) Antibiotics, (b) Vitamins, and (c) Amino Acids.
Antibiotics Assays
The microbial assays of ‘antibiotics’ are usually carried out by two standard methods as per
Indian Pharmacopoea* (1996), namely :
Method A i.e., the ‘Cylinder-Plate Method’ as discussed in Section 10.1.3.1 and Section 10.3.1.
Method B i.e., the ‘Turbidimetric Method’ as described in Section 10.1.3.2.
A comprehensive account of the ‘Antibiotic Assays’ shall now be dealt with under the following
sub-heads :
Standard Preparation and Units of Activity
Standard preparation may be defined as— ‘the authentic sample of the appropriate antibiotic
for which the potency has been precisely determined with reference to the appropriate international
standard’
However, the potency of the standard preparation may be duly expressed either in International
Units (IU) or in μg.mg–1 with respect to the ‘pure antibiotic’.
Important Points
(1) Standard Preparation for India are adequately maintained at the Central Drugs Laboratory,
Kolkata. A unit referred to in the ‘official assays’ and ‘tests’ refers to the specific activity contained
in such an amount of the respective standard preparation as is duly indicated by the Ministry of
Health and Family Welfare, Government of India from time to time.
(2) Standard Preparation may be suitably replaced by a ‘working standard’ prepared by any
laboratory that must be compared at definite intervals under varying conditions with the ‘standard’.
[A] Media. The media necessarily required for the preparation of ‘test organism inocula’ are
duly made from the various ingredients as listed specifically. However, one may make
minor modifications of the individual ingredients as and when required or ‘reconstituted dehydrated
media’ may be employed provided the resulting media have either almost equal or definitely better
growth-promoting characteristic features, and ultimately give a similar standard curve-response.
Method : Dissolve the various prescribed ingredients in sufficient distilled water (DW) to produce
1L, and add sufficient 1M sodium hydroxide or 1M hydrochloric acid, as required so that after
sterilization the pH must be as stated
[B] Buffer Solutions : Prepare the buffer solutions by dissolving the quantities
of K2HPO4 and KH2PO4 in sufficient distilled water to produce 1L after adjusting the pH with 8 M .
H3PO4 or 10 M.KOH. The buffer solutions are duly sterilized after prepares and the final pH specified in
each case must be the one that is obtained after sterilization.
Preparation of Standard Solution
In order to prepare a ‘Stock Solution’, dissolve a quantity of the Standard Preparation of a
given antibiotic, weighed accurately and precisely, and dried previously as duly indicated
in the solvent specified in the said Table ; and subsequently dilute to the required concentration as
indicated specifically. It is advisable to store the ‘Stock Solution’ duly in a refrigerator (+ 1–5°C), and
use within the stipulated period indicated.
On the particular day intended for carrying out the assay, prepare from the ‘Stock Solution’ at
least five or even more test dilutions whereby the successive solutions increase stepwise in concentration,
invariably in the ratio 1 : 1.25 for method A or smaller for method B. Use the final diluent
specified and a sequence in a such a manner that the middle or median should have the concentration as
specified duly
Preparation of Sample Solution
Based on the available information for the ‘drug substance’ under investigation (i.e., the ‘unknown’)
assign to it an assumed potency per unit weight or volume, and on this assumption prepare on
the day of the assay a ‘Stock Solution’ and test dilution(s) as duly specified for each individual antibiotic
taking particular care to use the same final diluent as employed for the Standard
Preparation. The assay with 5 levels of the Standard necessarily requires only one level of the ‘unknown’
at a concentration assumed very much equal to the ‘median level’ of the ‘Standard’.
For Amphotericin B, further dilute the stock solution with dimethylformamide to give concentrations of
12.8, 16, 20, 25 and 31.2 μg per ml prior to making the test solutions. The test dilution of the sample
prepared from the solution of the substance being examined should contain the same amount of
dimethylformamide as the test dilutions of the Standard Preparation.
For Bacitracin, each of the standard test dilutions should contain the same amount of hydrochloric acid as the
test dilution of the sample.
For Nystatin, further dilute the stock solution with dimenthylformamide to give concentrations of 64.0, 80.0,
100.0, 125.0 and 156.0 μg per ml prior to making the test dilutions. Prepare the standard response line
solutions simultaneously with dilutions of the sample being examined. The test dilution of the sample prepared
from the solution of the substance being examined should contain the same amount of dimethylformamide
as the test dilutions of the Standard Preparation. Protect the soultions from light.
When making the stock solution of Polymyxin B, add 2 ml of water for each 5 mg of the weighed Standard
Preparation material.
Where indicated, dry about 100 mg of the Standard Preparation before use in an oven at a pressure not
exceeding 0.7 kPa at 60° for 3 hours, except in the fine of Bleomycin (dry at 25° for 4 hours), Novobiocin (dry
at 100° for 4 hours), Gentamicin (dry at 110° for 3 hours) and Nystatin (dry at 40° for 2 hours).
Where two-level factorial assays are performed use the following test doses per ml; Amphotericin B, 1.0 to 4.0
μg; Bacteracin, 1.0 to 4.0 Units; Kanamycin Sulphate, 5.0 to 20.0 units; Streptomycin, 5.0 to 20.0 μg.
Test Organisms
The various test organisms for each antibiotic is duly listed in Table 10.4, along with its properly
documented identification number in the following recognized and approved compendia as :
• American Type Culture Collection (ATCC)
• National Collection of Type Cultures (NCTC)
• National Collection of Industrial Bacteria (NCIB).
Usually maintain a ‘culture’ on the slants of the medium, and under the specified incubation
conditions as mentioned duly in Table 10.5, and transfer weekly to fresh slants.
1. Use Medium A containing 300 mg of manganese sulphate per litre.
2. For Pseudomonas aeruginosa in the assay of Carbenicillin, use the dilution yielding 25% light transmission,
rather than the stock suspension, for preparing the inoculum suspension.
Methods of preparation of test organism suspension
1. Maintain the test organism on slants of Medium A and transfer to a fresh slant once a week. Incubate the
slants at the temperature indicated above for 24 hours. Using 3 ml of saline solution, wash the organism from
the agar slant onto a large agar surface of Medium A such as a Roux bottle containing 250 ml of agar.
Incubate for 24 hours at the appropriate temperature. Wash the growth from the nutrient surface using 50 ml
of saline solution. Store the test organism under refrigeration. Determine the dilution factor which will give
25% light transmission at about 530 nm. Determine the amount of suspensions to be added to each 100 ml of
agar of nutrient broth by use of test plates or test broth. Store the suspension under refrigeration.
2. Proceed as described in Method 1 but incubate the Roux bottle for 5 days. Centrifuge and decant the
supernatant liquid. Resuspend the sediment with 50 to 70 ml of saline solution and heat the suspension for
30 minutes at 70°. Wash the spore suspension three times with 50 to 70 ml of saline solution. Resuspend in
50 to 70 ml of saline solution and heat-shock again for 30 minutes. Use test plates to determine the amount
of the suspension required for 100 ml of agar. Store the suspension under refrigeration.
3. Maintain the test organism on 10 ml agar slants of Medium G. Incubate at 32° to 35° for 24 hours. Inoculate
100 ml of nutrient broth. Incubate for 16 to 18 hours at 37° and proceed as described in Method 1.
4. Proceed as described in Method 1 but wash the growth from the nutrient surface using 50 ml of Medium 1
(prepared without agar) in place of saline solution