The activity (potency) of antibiotics may be demonstrated under suitable
conditions by their inhibitory effect on microorganisms. A reduction in
antimicrobial activity also will reveal subtle changes not demonstrable by
chemical methods. Accordingly, microbial or biological assays remain generally
the standard for resolving doubt with respect to possible loss of activity. This
chapter summarizes these procedures for the antibiotics recognized in this
Pharmacopeia for which microbiological assay remains the definitive method.
Two general methods are employed, the cylinder-plate or “plate” assay（“板”
法）and the turbidimetric or “tube” assay（“管”法）. The first depends upon
diffusion of the antibiotic from a vertical cylinder through a solidified agar
layer in a petri dish or plate to an extent such that growth of the added
microorganism is prevented entirely in a circular area or “zone” around the
cylinder containing a solution of the antibiotic. The turbidimetric method
depends upon the inhibition of growth of a microbial culture in a uniform
solution of the antibiotic in a fluid medium that is favorable to its rapid
growth in the absence of the antibiotic.
All equipment is to be thoroughly cleaned before and after each use.
Glassware（玻璃仪器）for holding and transferring test organisms is sterilized
by dry heat or by steam.
Temperature Control （温度控制）
Thermostatic control is required in several stages of a microbial assay, when
culturing a microorganism and preparing its inoculum, and during incubation in
plate and tube assays. Maintain the temperature of assay plates at ±0.5 of the
temperature selected. Closer control of the temperature (±0.1 of the selected
temperature) is imperative during incubation in a tube assay, and may be
achieved in either circulated air or water, the greater heat capacity of water
lending it some advantage over circulating air.
Measuring transmittance within a fairly narrow frequency band requires a
suitable spectrophotometer in which the wavelength of the light source can be
varied or restricted by the use of a 580-nm filter or a 530-nm filter for
reading the absorbance in a tube assay. For the latter purpose, the instrument
may be arranged to accept the tube in which incubation takes place (see
Turbidimetric Assay Receptacles), to accept a modified cell fitted with a drain
that facilitates rapid change of content, or preferably, fixed with a
flow-through cell for a continuous flow-through analysis; set the instrument at
zero absorbance with clear, uninoculated broth prepared as specified for the
particular antibiotic, including the same amount of test solution and
formaldehyde as found in each sample.
NOTE—Either absorbance or transmittance measurement may be used for preparing
Cylinder-Plate Assay Receptacles（板法）
For assay plates, use glass or plastic petri dishes (approximately 20 × 100 mm)
having covers of suitable material. For assay cylinders, use stainless steel or
porcelain cylinders with the following dimensions, each dimension having a
tolerance of ±0.1 mm: outside diameter 8 mm; inside diameter 6 mm; and length
10 mm. Carefully clean cylinders to remove all residues. An occasional acid
bath, e.g., with about 2 N nitric acid or with chromic acid (see Cleaning Glass
Apparatus á1051ñ) is needed.
Turbidimetric Assay Receptacles（管法）
For assay tubes, use glass or plastic test tubes, e.g., 16 × 125 mm or 18 ×
150 mm that are relatively uniform in length, diameter, and thickness and
substantially free from surface blemishes and scratches. Tubes that are to be
placed in the spectrophotometer are matched and are without scratches or
blemishes. Cleanse thoroughly to remove all antibiotic residues and traces of
cleaning solution, and sterilize tubes that have been used previously, before
MEDIA（培养基） AND DILUENTS（稀释剂）
The media required for the preparation of test organism inocula are made from
the ingredients listed herein. Minor modifications of the individual
ingredients, or reconstituted dehydrated media, may be substituted, provided the
resulting media possess equal or better growth-promoting properties and give a
similar standard curve response.
Dissolve the ingredients in water to make 1 L, and adjust the solutions with
either 1 N sodium hydroxide or 1 N hydrochloric acid as required, so that after
steam sterilization the pH is as specified.