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Antibiotic HiVeg
TM
Assay Media
Antibiotic HiVeg Assay Media are used in the microbiological
assay of different antibiotics in pharmaceutical products and
also in foods, etc.
General Introduction
Anitibiotic HiVeg Assay Media are prepared by using vegetable
peptones in place of animal peptones making the media
free of BSE/TSE risk. Antibiotic Assay Media are used in
the performance of antibiotic assays. Grove and Randall
have elucidated those antibiotic assays and media in their
comprehensive treatise on antibiotic assays (1). Schmidt and
Moyer have reported the use of antibiotic assay medium for the
liquid formulation used in the performance of antibiotic assay
(2). These media are prepared according to the specifications
detailed in the USP (3, 4, 5) and by the FDA (6). Antibiotic HiVeg
Assay Media are the modifications of these assay media. The
use of these media like the conventional media, assures well
defined inhibition zones of the test organism. All conditions in
the microbiological assay must be controlled carefully. The use
of standard culture media in the test is one of the important
step for good results.
Nutrients and growth factors are supplied by the ingredients
like HiVeg peptone, HiVeg hydrolysate and/ or Papaic digest of
soyabean meal, yeast extract, HiVeg extract etc. Sodium chloride
maintains the osmotic equilibrium. Phosphates are included in
most of these media for good buffering action. In some media,
dextrose as a sugar is also supplemented as a carbon and energy
source. Ingredients like magnesium sulphate, sodium sulphite,
L-cystine, polysorbate 80 (tween 80) are also added in some
media depending on the requirement of the test organism.
General Assay Methods :
1. Cylinder plate method : This method was first devised
by Abraham et al (7) and later modified by Schmidt and Moyer
(2) and it depends upon diffusion of the antibiotic from vertical
steel cylinders placed on the surface of inoculated agar
medium. This produces zones of inhibition around the cylinder
containing antibiotic solution depending upon the concentration
of the antibiotic. This method is commonly employed in the
assay of pharmaceutical preparations of Penicillin and other
antibiotics. For assay, use of petri plates with 20 X 100 mm
dimension and stainless steel or porcelain cylinders with the
outside diameter 8 mm, inside diameter 6 mm and length 10
mm is recommended. All dimensions should have a tolerance
of 0.1 mm. The cylinders should be carefully cleaned to remove
all the impurities. For assays requiring base and seed layer, the
base layer is allowed to solidify first and then overlaid with the
seed agar containing the appropriate concentration of the test
organism. Most assays require base layer of 21 ml and seed
layer of 4 ml. Generally 6 cylinders are used per plate. The
cylinders are placed on inoculated plates at equal distance.
2. Punched-hole method : Holes are punched out of the
inoculated culture medium and the antibiotic solutions are
then loaded into them. Rest of the procedure is similar to the
cylinder plate method.
3. Paper-disc method : Paper discs with a diameter of 9
mm are impregnated with the antibiotic solution and placed
on the culture medium. Antibiotic can also be applied to the
disc after it has been placed on the medium. Plates containing
a single layer of medium with 2 mm thickness may be used
for these tests. All other steps are similar to the cylinder plate
method. Antibiotic HiVeg Medium No. 2 (MV005) or 5 (MV006)
can be employed depending on the required pH.
4. Serial dilution method : Minimum inhibitory concentration
(MIC) of an antibiotic can be expressed by determining the
antibiotic activity quantitatively. It can be done by using the
known sensitivity of a test organism towards a particular
antibiotic. Serial dilutions of an antibiotic to be tested are
pipetted into the antibiotic broth which is then inoculated
with a defined quantity of the relevant test organism. The last
tube which does not show any turbidity due to suppression of
microbial growth indicates the presence of active antibiotic at
a concentration corresponding to MIC.
5. Turbidimetric Assay : The turbidimetric method depends
upon the inhibition of test organism in a medium containing
uniform solution of an antibiotic. This method has an advantage
over the Cylinder plate method in that it requires shorter
incubation period of 3-4 hours. Use of 18x150 mm test
tubes that are free from impurities is recommended. In this
method, working dilutions of the antibiotic reference standards
are prepared in specific concentrations. 9 ml of inoculated
broth is added to 1 ml quantities of these solutions in test
tubes. Similarly solutions of sample under test containing
approximately the same antibiotic activity are simultaneously
tested. The tubes are then incubated for 3-4 hours at the
specified temperature in a water bath. After the incubation
period, the growth is terminated by addition of one drop of
formalin and the growth is determined by measuring the light
transmittance using a suitable calibrated Spectrophotometer.
The concentration of the antibiotic is determined by
comparing density of growth obtained with that given by the
reference standard solutions.
MV004 Antibiotic HiVeg Assay Medium No. 11
M. luteus
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Selection of Media and Test Organism for Assay of Antibiotics
A N T I B I O T I C M E D I A F O R
CYLINDER PLATE Turbidi-
Antibiotic Assay Method Organism and (ATCC No.) Maintenance Inoculum Base Seed metric
Layer Layer Assay
Amikacin Turbidimetric Staphylococcus aureus (29737) 1 1 3
Amphotericin B Cylinder plate Saccharomyces cerevisiae (9763) 19 19 19 19
Ampicillin Cylinder plate Micrococcus luteus (9341) 1 1 11 11
Bacitracin Cylinder plate Micrococcus luteus (10240) 1 1 2 1
Bleomycin Cylinder plate Mycobacterium smegmatis (607) 36 35 35 36
Candicidin Turbidimetric Saccharomyces cerevisiae (9763) 19 13 13
Capreomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 3
Carbenicillin Cylinder plate Pseudomonas aeruginosa (25619) 1 1 9 10
Cephalexin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Cephalothin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Cephapirin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Cephradine Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Chloramphenicol Turbidimetric Escherichia coli (10536) 1 1 3
Chlortetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 3
Clindamycin Cylinder plate Micrococcus luteus (9341) 1 1 11 11
Cloxacillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Colistimethate sodium Cylinder plate Bordetella bronchiseptica (4617) 1 1 9 10
Colistin Cylinder plate Bordetella bronchiseptica (4617) 1 1 9 10
Cycloserine Turbidimetric Staphylococcus aureus (29737) 1 1 3
Dactinomycin Cylinder plate Bacillus subtilis (6633) 1 1 5 5
Demeclocycline Turbidimetric Staphylococcus aureus (29737) 1 1 3
Demethylchlortetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 3
Dicloxacillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Dihydrostreptomycin Cylinder plate Bacillus subtilis (6633) 1 1 5 5
Dihydrostreptomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 3
Doxycycline Turbidimetric Staphylococcus aureus (29737) 1 1 3
TABLE 1
Antibiotic HiVeg
TM
Assay Media
MV1667 Antibiotic HiVeg Assay Medium No. 37
1. Control
2. Escherichia coli
3. Straphylococcus aureus
4. Streptococcus pyogens
4 3 2 1
MV1666 Antibiotic HiVeg Assay Medium No. 36
Growth without antibiotic
MV003 Antibiotic HiVeg Assay Medium No. 1 Seed Layer
MV005 Antibiotic HiVeg Assay Medium No. 2 Base Layer
Inhibition of Staphylococcus aureus ATCC 29737 by different concentrations of Cephalexin
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Selection of Media and Test Organism for Assay of Antibiotics
A N T I B I O T I C M E D I A F O R
CYLINDER PLATE Turbidi-
Antibiotic Assay Method Organism and (ATCC No.) Maintenance Inoculum Base Seed metric
Layer Layer Assay
Erythromycin Cylinder plate Micrococcus luteus (9341) 1 1 11 11
Gentamicin Cylinder plate Staphylococcus epidermidis (12228) 1 1 11 11
Gramicidin Turbidimetric Enterococcus faecium (19434) 3 3 3
Kanamycin Turbidimetric Staphylococcus aureus (29737) 1 1 3
Kanamycin B Cylinder plate Bacillus subtilis (6633) 1 1 5 5
Lincomycin Turbidimetric Staphylococcus aureus (29737) 1 1 3
Methacycline Turbidimetric Staphylococcus aureus (29737) 1 1 3
Methicillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Minocycline Turbidimetric Staphylococcus aureus (29737) 1 1 3
Mitomycin Cylinder plate Bacillus subtilis (6633) 1 1 8 8
Nafcillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Natamycin Cylinder plate Saccharomyces cerevisiae (9763) 19 19 19 19
Neomycin Cylinder plate Staphylococcus epidermidis (12228) 1 1 11 11
Neomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 39
Netilmycin Cylinder plate Staphylococcus epidermidis (12228) 1 1 11 11
Novobiocin Cylinder plate Staphylococcus epidermidis (12228) 1 1 2 1
Nystatin Cylinder plate Saccharomyces cerevisiae (2601) 19 19 19 19
Oxacillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Oxytetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 3
Paromomycin Cylinder plate Staphylococcus epidermidis (12228) 1 1 11 11
Penicillin-G Cylinder plate Staphylococcus aureus (29737) 1 1 2 1
Plicamycin Cylinder plate Staphylococcus aureus (29737) 1 1 8 8
Polymyxin-B Cylinder plate Bordetella bronchiseptica (4617) 1 1 9 10
Rifampicin Cylinder plate Bacillus subtilis (6633) 1 1 2 5
Rolitetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 3
Sisomycin Cylinder plate Staphylococcus epidermidis (12228) 1 1 11 11
Spectinomycin Turbidimetric Escherichia coli (10536) 1 1 3
Streptomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 3
Streptomycin Cylinder plate Bacillus subtilis (6633) 1 1 5 5
Tetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 3
Thiostreptone Turbidimetric Enterococcus faecium (19434) 40 3 41
Ticarcillin Cylinder plate Pseudomonas aeruginosa (25619) 36 38 38 38
Tobramycin Turbidimetric Staphylococcus aureus (29737) 1 1 3
Troleandomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 3
Tylosin Turbidimetric Staphylococcus aureus (29737) 1 1 39
Tyrothricin Turbidimetric Enterococcus faecium (19434) 3 3 3
Vancomycin Cylinder plate Bacillus subtilis (6633) 1 1 8 8
Viomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 3
Modification of animal peptone based Antibiotic Assay Medium as per United States Pharmacopeia / National Formulary, 2006/1985, U.S. Pharmaco-
peial Convention, Inc., Rockville, MD 20852.
Preparation of Inoculum : The test organisms are maintained on agar slants
and transferred at 2 weeks interval. On the day of the assay, a stock suspension
of the test organism is prepared in sterile saline. This stock suspension is then
diluted to the desired concentration of the test organism. Enterococcus faecium
is grown in a liquid medium.
When Bacillus subtilis is used as a test orgranism, the organism may be grown
on Antibiotic HiVeg Medium No. 1 (MV003) for 1 week or HiVeg Medium No. 6
(MV223) for 4 - 6 days, until 80% or more cells are in the sporulation as revealed
on microscopic examination.
References :
1. Grove and Randall, 1955, Assay Methods of Antibiotics Medical Encyclopedia, Inc. New
York.
2. Schmidt and Moyer, 1944, J. Bacteriol., 47:199.
3. United States Pharmacopeia / National Formulary (USP29/NF24) 2006, US Pharmacopoeial
Convention, Inc., Rockville, MD.

4. Indian Pharmacopoeia 2010, Minstry of Health and Family Welfare, Govt. of India, Delhi.
5. United States Pharmacopeia / National Formulary (USP34/NF29) 2011, US Pharmacopoeial
Convention, Inc., Rockville, MD.
6. Kramer, J., G.G. Carter, B. Arret, J. Wilner, W.W. Wright, and A. Kirshbaum. 1968. Antibiotic
residues in milk, dairy products and animal tissues: methods, reports and protocols. Food
and Drug Administration,Washington, DC.
7. Abraham et al, 1941, Lancet, 2:177.
Antibiotic HiVeg
TM
Assay Media
Refer individual technical specication for respective Antibiotic HiVeg Assay Media.