Overview:
Antibiotic Medium No. 8 is the official standardized seed agar used as an alternative to Medium No. 4 for the microbiological cylinder-plate assay of streptomycin and dihydrostreptomycin. It is designated as �Medium G� in USP <81>, European Pharmacopoeia 2.7.2, JP 17, and WHO guidelines. The higher pH (8.0�8.2) and slightly modified nutrient balance produce even larger and sharper inhibition zones with Bacillus subtilis ATCC 6633 than Medium No. 4 in many laboratories, making it the currently preferred seed medium for streptomycin assays in most modern compendial protocols.
Applications:
?? Compendial seed layer for streptomycin and dihydrostreptomycin potency assays
?? Cylinder-plate diffusion assay of streptomycin-containing pharmaceuticals
?? Regulatory QC testing (now the most commonly accepted seed medium for streptomycin)
Key Features:
?? Higher pH (8.1) � maximizes streptomycin diffusion and zone size
?? Internationally harmonized and currently preferred over Medium No. 4
?? Produces exceptionally large, clear zones with Bacillus subtilis
?? Reference seed agar in current USP/EP/JP/WHO streptomycin monographs
Composition (per liter):
Beef Extract � 1.5 g
Yeast Extract  � 6.0 g
Peptone (meat or casein)� 6.0 g
D-Glucose   � 1.0 g
Agar  � 15.0 g
Final pH: 8.1 � 0.1 at 25�C (after sterilization � strictly critical)
Preparation:
Suspend 29.5 g of the dehydrated medium in 1 liter of purified water. Heat with frequent agitation and boil for 1�2 minutes until completely dissolved. Sterilize by autoclaving at 121�C for 15 minutes. Cool to 48�50�C. Inoculate with standardized suspension of Bacillus subtilis ATCC 6633. Pour 4�6 mL seeded layer over a previously prepared base layer (usually Antibiotic Medium No. 5 or No. 2).
Test organism (mandatory):
Bacillus subtilis ATCC 6633
Storage:
Dehydrated medium: Store below 30�C in a cool, dry place.
Prepared seeded plates: Use within 24 hours; store at 2�8�C if necessary.
Note: In current editions of USP, Ph. Eur., and JP, Medium No. 8 + Bacillus subtilis has replaced Medium No. 4 + Klebsiella pneumoniae as the preferred system for streptomycin assay because of superior zone clarity and reproducibility.
For pharmaceutical quality-control and official compendial streptomycin assay only.