Antibiotic Assay Medium No.
3 (Assay Broth) M042
Intended Use:
Recommended for microbiological assay of antibiotics.
Composition**
Ingredients Gms / Litre
Peptone 5.000
HM peptone B # 1.500
Yeast extract 1.500
Dextrose (Glucose) 1.000
Sodium chloride 3.500
Dipotassium hydrogen phosphate 3.680
Potassium dihydrogen phosphate 1.320
Final pH ( at 25°C) 7.0±0.2
**Formula adjusted, standardized to suit performance parameters
# Equivalent to Beef extract
Directions
Suspend 17.5 grams in 1000 ml purified / distilled water. Heat if necessary to dissolve the medium completely. Sterilize by
autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C.
Advice: Recommended for the microbiological assay of Amikacin, Capreomycin, Chlortetracycline,Chloramphenicol,
Cycloserine,Demeclocycline,Dihydrostreptomycin, Doxycycline, Gentamicin, Gramicidin, Kanamycin, Methacycline,
Neomycin, Novobiocin, Oxytetracycline, Rolitetracycline, Streptomycin, Tetracycline, Tobramycin, Trolendomycin and
Tylosin according to official methods.
Principle And Interpretation
Antibiotic Assay Medium is 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). Antibiotic Assay Medium No. 3
(Assay Broth) is used in the microbiological assay of different antibiotics in pharmaceutical and food products by the
turbidimetric method. Ripperre et al reported that turbidimetric methods for determining the potency of antibiotics are
inherently more accurate and more precise than agar diffusion procedures (4).
Turbidimetric antibiotic assay is based on the change or inhibition of growth of a test microorganims in a liquid medium
containing a uniform concentration of an antibiotic. After incubation of the test organism in the working dilutions of the
antibiotics, the amount of growth is determined by measuring the light transmittance using spectrophotometer. The
concentration of antibiotic is determined by comparing amounts of growth obtained with that given by the reference
standard solutions. Use of this method is appropriate only when test samples are clear.
Peptone, HM peptone B and yeast extract provides essential nutrients and growth factors for enhanced microbial growth.
Sodium chloride maintains the osmotic equilibrium of the medium and retains the cell viability and cell integrity.
Phosphates in the medium provide good buffering action. Dextrose serves as the carbon and energy source. All conditions in
the microbiological assay must be controlled carefully. The use of standard culture media in the test is one of the important
steps for the good results.
Type of specimen
Pharmaceutical preparations
Please refer disclaimer Overleaf.
HiMedia Laboratories Technical Data
Specimen Collection and Handling:
For pharmaceutical samples follow appropriate techniques for handling specimens as per established guidelines (1).
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions :
Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection.
Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per
established guidelines should be followed while handling specimens. Safety guidelines may be referred in
individual safety data sheets.
Limitations :
1. Freshly prepared plates must be used or it may result in erroneous results.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at
recommended temperature.
Quality Control
Appearance
Cream to yellow homogeneous free flowing powder
Colour and Clarity of prepared medium
Light yellow coloured clear solution
Reaction
Reaction of 1.75% w/v aqueous solution at 25°C. pH : 7.0±0.2
pH
6.80-7.20
Cultural Response
Cultural characteristics observed after an incubation at 32-37°C for 18-24 hours.
Organism Inoculum Growth Serial dilution
(CFU) with
Escherichia coli ATCC 50-100 luxuriant Chloramphenicol
10536
Klebsiella pneumoniae 50-100 luxuriant Capreomycin, Dihydrostreptomycin,
ATCC 10031 Streptomycin, Troleandomycin
Staphylococcus aureus 50-100 luxuriant Amikacin,
ATCC 29737 Chlortetracycline, Cycloserine,
Demeclocycline, Doxycycline,
Kanamycin,Kanamycin sulphate,
Methacycline, Oxytetracycline,
Rolitetracycline, Tetracycline,
Tobramycin, Tylosin, Gentamicin,
Gramicidin, Neomycin, Novobiocin
Enterococcus hirae ATCC 50-100 luxuriant
10541
Staphylococcus aureus 50-100 luxuriant Tylosin
ATCC 9144
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and use freshly prepared medium. Use before expiry date on the
label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump
formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation.
Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly
after use. Use before expiry date on the label.
Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow
established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must
be decontaminated and disposed of in accordance with current laboratory techniques (2,3).
Please refer disclaimer Overleaf.
HiMedia Laboratories Technical Data
Reference
1. Grove and Randall, 1955, Assay Methods of Antibiotics Medical Encyclopedia, Inc. New York.
2. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
3. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015)
Manual of Clinical Microbiology, 11th Edition. Vol. 1.
4. Rippere R. A.. Some principles of microbiological turbidimetric assays of antibiotics. J. Assoc. off. Anal. Chem. 1979
62(4):951-6.
Revision :02 / 2019
Disclaimer :
User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in
this and other related HiMedia™ publications. The information contained in this publication is based on our research and development
work and is to the best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to
specifications and information related to the products at any time. Products are not intended for human or animal or therapeutic use but
for laboratory,diagnostic, research or further manufacturing use only, unless otherwise specified. Statements contained herein should not
be considered as a warranty of any kind, expressed or implied, and no liability is accepted for infringement of any patents.
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