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.

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 1051) is needed.

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 subsequent use.

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.

MEDIUM 1

pH after sterilization: 6.6 ± 0.1.

MEDIUM 2

pH after sterilization: 6.6 ± 0.1.

MEDIUM 3

pH after sterilization: 7.0 ± 0.05.

MEDIUM 4

MEDIUM 8

Pancreatic Digest of Casein	17.0 g
Papaic Digest of Soybean	3.0 g
Sodium Chloride	5.0 g
Dibasic Potassium Phosphate	2.5 g
Dextrose	2.5 g
Agar	20.0 g
Water	1000 mL

MEDIUM 10

MEDIUM 11

Dextrose	20.0 g
Peptone	10.0 g
Water	1000 mL

Peptone	9.4 g
Yeast Extract	4.7 g
Beef Extract	2.4 g
Sodium Chloride	10.0 g
Dextrose	10.0 g
Agar	23.5 g
Water	1000 mL

MEDIUM 32

Glycerol	10.0 g
Peptone	10.0 g
Beef Extract	10.0 g
Sodium Chloride	3.0 g
Water	1000 mL

MEDIUM 35

Pancreatic Digest of Casein	15.0 g
Papaic Digest of Soybean	5.0 g
Sodium Chloride	5.0 g
Agar	15.0 g
Water	1000 mL

MEDIUM 39

Yeast Extract	20.0 g
Polypeptone	5.0 g
Dextrose	10.0 g
Monobasic Potassium Phosphate	2.0 g
Polysorbate 80	0.1 g
Agar	10.0 g
Water	1000 mL

Pancreatic Digest of Casein	9.0 g
Dextrose	20.0 g
Yeast Extract	5.0 g
Sodium Citrate	10.0 g
Monobasic Potassium Phosphate	1.0 g
Dibasic Potassium Phosphate	1.0 g
Water	1000 mL

Prepare as follows, or by other suitable means, the potassium phosphate buffers required for the antibiotic under assay. The buffers are sterilized after preparation, and the pH specified in each case is the pH after sterilization.

The test organism for each antibiotic is listed in Table 2, together with its identification number in the American Type Culture Collection. The method of assay is given for each in Table 1. Maintain a culture on slants of the medium and under the incubation conditions specified in Table 3, and transfer weekly to fresh slants. For K. pneumoniae use a noncapsulated culture. For Enterococcus hirae, stab cultures may be used.

Table 1. Preparation of Stock Solutions and Test Dilutions of Reference Standards

Table 2. Test Organisms for Antibiotics Assayed by the Procedure Indicated in Table 1

Table 3. Preparation of Inoculum

Preparatory to an assay, remove the growth from a recently grown slant or culture of the organism, with 3 mL of sterile saline TS and sterile glass beads. Inoculate the surface of 250 mL of the agar medium specified for that organism in Table 3 and contained on the flat side of a Roux bottle except in the case of Enterococcus hirae and Staphylococcus aureus (ATCC 9144), which are grown in a liquid medium. Spread the suspension evenly over the surface of the agar with the aid of sterile glass beads, and incubate at the temperature shown for approximately the indicated length of time. At the end of this period, prepare the stock suspension by collecting the surface growth in 50 mL of sterile saline TS, except for Bleomycin (use 50 mL of Medium 34).

Determine by trial the quantity of stock suspension to be used as the Inoculum, starting with the volume suggested in Table 3. The trial tests should be incubated for the times indicated in the section Turbidimetric Method for Procedure. Adjust the quantity of Inoculum on a daily basis, if necessary, to obtain the optimum dose-response relationship from the amount of growth of the test organism in the assay tubes and the length of the time of incubation. At the completion of the incubation periods described in the section Turbidimetric Method for Procedure, tubes containing the median dose of the Standard should have absorbances of at least 0.3 absorbance unit, except for Amikacin, Chlortetracycline, Gramicidin, and Tetracycline (0.35 absorbance unit), and Capreomycin, Methacycline, and Tobramycin (0.4 absorbance unit).

For the cylinder-plate assay, determine by trial the proportions of stock suspension to be incorporated in the Inoculum, starting with the volumes indicated in Table 3, that result in satisfactory demarcation of the zones of inhibition of about 14 to 16 mm in diameter and giving a reproducible dose relationship. Prepare the inoculum by adding a portion of stock suspension to a sufficient amount of agar medium that has been melted and cooled to 45 to 50, and swirling to attain a homogeneous suspension.

Microbial assays gain markedly in precision by the segregation of relatively large sources of potential error and bias through suitable experimental designs. In a cylinder-plate assay, the essential comparisons are restricted to relationships between zone diameter measurements within plates, exclusive of the variation between plates in their preparation and subsequent handling. To conduct a turbidimetric assay so that the differences in observed turbidity will reflect the differences in the antibiotic concentration requires both greater uniformity in the environment created for the tubes through closer thermostatic control of the incubator and the avoidance of systematic bias by use of a random placement of replicate tubes in separate tube racks, each rack containing one complete set of treatments. The essential comparisons are then restricted to relationships between the observed turbidities within racks.

Within these restrictions, the assay design recommended is a 1-level assay with a standard curve. For this assay with a standard curve, prepare solutions of 5, 6, or more test dilutions, provided they include one corresponding to the reference concentration ( S 3), of the Standard and a solution of a single median test level of the Unknown as described under Preparation of Standard and Preparation of the Sample. Consider an assay as preliminary if its computed potency with either design is less than 80% or more than 125% of that assumed in preparing the stock solution of the Unknown. In such a case, adjust its assumed potency accordingly and repeat the assay.

Microbial determinations of potency are subject to inter-assay as well as intra-assay variables, so that two or more independent assays are required for a reliable estimate of the potency of a given assay preparation or Unknown. Starting with separately prepared stock solutions and test dilutions of both the Standard and the Unknown, repeat the assay of a given Unknown on a different day. If the estimated potency of the second assay differs significantly, as indicated by the calculated standard error, from that of the first, conduct one or more additional assays. The combined result of a series of smaller, independent assays spread over a number of days is a more reliable estimate of potency than that from a single large assay with the same total number of plates or tubes.

To prepare assay plates using Petri dishes, place 21 mL of Medium 2 in each of the required number of plates, and allow it to harden into a smooth base layer of uniform depth, except for Amphotericin B and Nystatin, where no separate base layer is used. For Erythromycin, Gentamicin, Neomycin B, Paromomycin, and Sisomicin, use Medium 11. For Bleomycin, use 10 mL of Medium 35. For Dihydrostreptomycin use Medium 5. For Vancomycin, use 10 mL of Medium 8. For Carbenicillin, Colistimethate Sodium, Colistin, and Polymyxin B, use Medium 9. For Netilmicin, use 20 mL of Medium 11. Add 4 mL of seed layer inoculum (see Preparation of Inoculum and Table 3), prepared as directed for the given antibiotic, except for Bleomycin (use 6 mL), for Netilmicin (use 5 mL), and for Nystatin and Amphotericin B (use 8 mL), tilting the plate back and forth to spread the inoculum evenly over the surface, and allow it to harden. Drop six assay cylinders on the inoculated surface from a height of 12 mm, using a mechanical guide or other device to insure even spacing on a radius of 2.8 cm, and cover the plates to avoid contamination. After filling the six cylinders on each plate with dilutions of antibiotic containing the test levels specified below, incubate the plates at 32 to 35, or at the temperature specified below for the individual case, for 16 to 18 hours, remove the cylinders, and measure and record the diameter of each zone of growth inhibition to the nearest 0.1 mm. Incubate the plates at 29 to 31 for Amphotericin B and Nystatin. Incubate at 34 to 36 for Novobiocin. Incubate at 36 to 37.5 for Carbenicillin, Colistimethate Sodium, Colistin, Dihydrostreptomycin, Gentamicin, Neomycin, Netilmicin, Paromomycin, Polymyxin B, Sisomicin, and Vancomycin.

For the 1-level assay with a standard curve, prepare dilutions representing five test levels of the Standard ( S1 to S5) and a single test level of the Unknown U3 corresponding to S3 of the standard curve, as defined under Preparation of the Standard and Preparation of the Sample. For deriving the standard curve, fill alternate cylinders on each of three plates with the median test dilution ( S3) of the Standard and each of the remaining nine cylinders with one of the other four dilutions of the Standard. Repeat the process for the three dilutions of the Standard. For each Unknown, fill alternate cylinders on each of three plates with the median test dilution of the Standard ( S3), and the remaining nine cylinders with the corresponding test dilution ( U3) of the Unknown.

On the day of the assay, prepare the necessary doses by dilution of stock solutions of the Standard and of each Unknown as defined under Preparation of the Standard and Preparation of the Sample. Add 1.0 mL of each dose, except for Gramicidin, Thiostrepton, and Tylosin (use 0.10 mL) to each of 3 prepared test tubes, and place the 3 replicate tubes in a position, selected at random, in a test tube rack or other carrier. Include similarly in each rack 1 or 2 control tubes containing 1 mL of the test diluent (see Table 1) but no antibiotic. Upon completion of the rack of test solutions (with Candicidin, within 30 minutes of the time when water is added to the dimethyl sulfoxide stock solution), add 9.0 mL of inoculum to each tube in the rack in turn, and place the completed rack immediately in an incubator or a water bath maintained at 36 to 37.5, except for Candicidin (incubate at 27 to 29). Incubate the tubes for 4 to 5 hours, except for Capreomycin, Chloramphenicol, Cycloserine, Dihydrostreptomycin, Spectinomycin, Streptomycin, and Troleandomycin (incubate these for 3 to 4 hours), Tylosin (incubate for 3 to 5 hours), and Candicidin (incubate for 16 to 18 hours). After incubation add 0.5 mL of dilute formaldehyde to each tube, except for Tylosin (heat the rack in a water bath at 80 to 90 for 2 to 6 minutes or in a steam bath for 5 to 10 minutes, and bring to room temperature), taking one rack at a time, and read its transmittance or absorbance in a suitable spectrophotometer fitted with a 530-nm or 580-nm filter (see Spectrophotometer under Apparatus).

For the 1-level assay with a standard curve, prepare dilutions representing 5 test levels of the Standard (S1 to S5) and a single test level (U3) of each of up to 20 Unknowns corresponding to S3 of the Standard. Prepare also an extra S3 as a test of growth. Add 1 mL of each test dilution, except for Gramicidin, Thiostrepton, and Tylosin (use 0.10 mL) to 3 tubes and 1 mL of antibiotic-free diluent to 6 tubes as controls. Distribute one complete set, including 2 tubes of controls, to a tube rack, intermingling them at random. Add 9.0 mL of inoculum, except for Thiostrepton (use 10.0 mL of inoculum), incubate, add 0.5 mL of dilute formaldehyde, and complete the assay as directed above. Determine the exact duration of incubation by observation of growth in the reference concentration (median dose) of the dilutions of the Standard (S3).