Antibiotic Assay Medium No.
2 (Base Agar) M005
Intended use
Antibiotic Medium No. 2 (Base Agar) is used as a basal medium for microbiological assay of antibiotics.
Composition**
Ingredients Gms / Litre
Peptone 6.000
HM Peptone B # 1.500
Yeast extract 3.000
Agar 15.000
Final pH ( at 25°C) 6.6±0.2
**Formula adjusted, standardized to suit performance parameters
#- Equivalent to Beef extract
Directions
Suspend 25.5 grams in 1000 ml purified/ distilled water. Heat to boiling to dissolve the medium completely. Sterilize by
autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile Petri plates.
Advice : Recommended for the microbiological assay of Spiramycin
Principle And Interpretation
This medium is commonly used as base agar for microbiological agar diffusion assays for wide variety of antibiotics. Agar
diffusion assays can be performed by cylinders, punched-hole or paper disc tests. This medium is identical numerically with
the name assigned by Grove and Randall (1) this medium is equivalent to Antibiotic Assay Medium No.B as per Indian
Pharmacopoeia (3). Peptone, yeast and HM peptone B provide the nitrogenous and carbonaceous compounds, long chain
amino acids, vitamins and mineral requirement for the growth of test organisms. This medium provides solidified substratum
for growth of organisms and supports the over layering of soft agar.
To perform the antibiotic assay the Antibiotic assay medium No.2 is used as Base Agar. This medium should be prepared on
the same day as the test. For the cylinder method, a base layer of 21 ml is required. Once the base medium has solidified,
Antibiotic assay medium No.1 as seed agar, inoculated with the standardized culture can be overlaid. Even distribution of the
layer is important.
Type of specimen
Pharmaceutical preparations.
Specimen Collection and Handling:
For pharmaceutical samples follow appropriate techniques for handling specimens as per established guidelines (3).
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.
Please refer disclaimer Overleaf.
HiMedia Laboratories Technical Data
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
Gelling
Firm,comparable with 1.5% Agar gel
Colour and Clarity of prepared medium
Amber coloured, clear to slightly opalescent gel forms in Petri plates
Reaction
Reaction of 2.55% w/v aqueous solution at 25°C. pH : 6.6±0.2
pH
6.40-6.80
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 18-48 hours.
Organism Inoculum Growth Recovery Antibiotics Basal layer
(CFU) assayed
Bacillus subtilis subsp. 50-100 luxuriant >=70% Spiramycin
spizizenii ATCC 6633
(00003*)
Micrococcus luteus ATCC 50-100 luxuriant >=70% Bacitracin
10240
Staphylococcus aureus 50-100 luxuriant >=70% Tylosin
ATCC 9144 (00035*)
Staphylococcus aureus 50-100 luxuriant >=70% Amikacin,Cephalothin,Cephapir
ATCC 29737 in, Chlortetracycline, Nafcillin,
Oxytetracycline,Rolitetracycline
Staphylococcus epidermidis 50-100 good-luxuriant >=70% Tetracycline
ATCC 12228 (00036*)
Klebsiella pneumoniae 50-100 luxuriant >=70% Capreomycin, Streptomycin,
ATCC 10031 Troleandomycin, Gramicidin,
Thiostrepton,Tobramycin
Enterococcus hirae ATCC 50-100 luxuriant >=70%
10541 (00011*)
Escherichia coli ATCC 50-100 luxuriant >=70% Chloramphenicol,
10536 Spectinomycin
*- Corresponding WDCM numbers
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 (3,4).
Please refer disclaimer Overleaf.
HiMedia Laboratories Technical Data
Reference
1. Grove and Randall, 1955, Assay Methods of Antibiotics Medical Encyclopedia, Inc. New York.1.
2. Indian Pharmacopoeia 2018, Ministry of Health and Family Welfare, Govt. of India, Delhi.
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
4. Jorgensen, J.H., Pfaller, M.A., Carrol , K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual
of Clinical Microbiology, 11th Edition. Vol. 1.
Revision : 03/ 2018
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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