The activity (potency)of antibiotics may be demonstrated under suitable conditions by their inhibitory effect on microorganisms.Areduction 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 and the turbidimetric or “tube”assay.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 for holding and transferring test organisms is sterilized by dry heat or by steam.
Temperature Control
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 ±0.5of the temperature selected.Closer control of the temperature (±0.1of 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 inocula.
Cylinder-Plate Assay Receptacles
For assay plates,use glass or plastic petri dishes (approximately 20×100mm)having covers of suitable material.For assay cylinders,use stainless steel or porcelain cylinders with the following dimensions,each dimension having a tolerance of ±0.1mm:outside diameter 8mm;inside diameter 6mm;and length 10mm.Carefully clean cylinders to remove all residues.An occasional acid bath,e.g.,with about 2Nnitric acid or with chromic acid (see Cleaning Glass Apparatus á1051ñ)is needed.
Turbidimetric Assay Receptacles
For assay tubes,use glass or plastic test tubes,e.g.,16×125mm or 18×150mm 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 1L,and adjust the solutions with either 1Nsodium hydroxide or 1Nhydrochloric acid as required,so that after steam sterilization the pHis as specified.
Peptone 6.0g
Pancreatic Digest of Casein 4.0g
Yeast Extract 3.0g
Beef Extract 1.5g
Dextrose 1.0g
Agar 15.0g
Water 1000mL
pHafter sterilization:6.6±0.1.
Peptone 6.0g
Yeast Extract 3.0g
Beef Extract 1.5g
Agar 15.0g
Water 1000mL
pHafter sterilization:6.6±0.1.
Peptone 5.0g
Yeast Extract 1.5g
Beef Extract 1.5g
Sodium Chloride 3.5g
Dextrose 1.0g
Dibasic Potassium Phosphate 3.68g
Monobasic Potassium Phosphate 1.32g
Water 1000mL
pHafter sterilization:7.0±0.05.
Same as Medium 2,except for the additional ingredient 1.0g of Dextrose.
Same as Medium 2,except that the final pHafter sterilization is 7.9±0.1.
Same as Medium 2,except that the final pHafter sterilization is 5.9±0.1.
Pancreatic Digest of Casein 17.0g
Papaic Digest of Soybean 3.0g
Sodium Chloride 5.0g
Dibasic Potassium Phosphate 2.5g
Dextrose 2.5g
Agar 20.0g
Water 1000mL
pHafter sterilization:7.2±0.1.
Same as Medium 9,except to use 12.0g of Agar instead of 20.0g,and to add 10mLof Polysorbate 80after boiling the medium to dissolve the agar.
pHafter sterilization:7.2±0.1.
Same as Medium 1,except that the final pHafter sterilization is 8.3±0.1.
Dextrose 20.0g
Peptone 10.0g
Water 1000mL
pHafter sterilization:5.6±0.1.
Peptone 9.4g
Yeast Extract 4.7g
Beef Extract 2.4g
Sodium Chloride 10.0g
Dextrose 10.0g
Agar 23.5g
Water 1000mL
pHafter sterilization:6.1±0.1.
Same as Medium 1,except for the additional ingredient 0.3g of Manganese Sulfate.
Glycerol 10.0g
Peptone 10.0g
Beef Extract 10.0g
Sodium Chloride 3.0g
Water 1000mL
pHafter sterilization:7.0±0.1.
Same as Medium 34,except for the additional ingredient 17.0g of Agar.
Pancreatic Digest of Casein 15.0g
Papaic Digest of Soybean 5.0g
Sodium Chloride 5.0g
Agar 15.0g
Water 1000mL
pHafter sterilization:7.3±0.1.
Same as Medium 3,except that the final pHafter sterilization is 7.9±0.1.
Yeast Extract 20.0g
Polypeptone 5.0g
Dextrose 10.0g
Monobasic Potassium Phosphate 2.0g
Polysorbate 80 0.1g
Agar 10.0g
Water 1000mL
pHafter sterilization:6.7±0.2.
Pancreatic Digest of Casein 9.0g
Dextrose 20.0g
Yeast Extract 5.0g
Sodium Citrate 10.0g
Monobasic Potassium Phosphate 1.0g
Dibasic Potassium Phosphate 1.0g
Water 1000mL
pHafter sterilization:6.8±0.1.
Phosphate Buffers and Other Solutions
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 pHspecified in each case is the pHafter sterilization.
BUFFER NO.1,1PERCENT,pH6.0— Dissolve 2.0g of dibasic potassium phosphate and 8.0g of monobasic potassium phosphate in 1000mLof water.Adjust with 18Nphosphoric acid or 10Npotassium hydroxide to 6.0±0.05.
BUFFERNO.3,0.1M,pH8.0— Dissolve 16.73g of dibasic potassium phosphate and 0.523g of monobasic potassium phosphate in 1000mLof water.Adjust the pHwith 18Nphosphoric acid or 10Npotassium hydroxide to 8.0±0.1.
BUFFER NO.4,0.1M,pH4.5— Dissolve 13.61g of monobasic potassium phosphate in 1000mLof water.Adjust the pHwith 18Nphosphoric acid or 10Npotassium hydroxide to 4.5±0.05.
BUFFERNO.6,10PERCENT,pH6.0— Dissolve 20.0g of dibasic potassium phosphate and 80.0g of monobasic potassium phosphate in 1000mLof water.Adjust the pHwith 18Nphosphoric acid or 10Npotassium hydroxide to 6.0±0.05.
BUFFER NO.10,0.2M,pH10.5— Dissolve 35.0g of dibasic potassium phosphate in 1000mLof water,and add 2mLof 10Npotassium hydroxide.Adjust the pHwith 18Nphosphoric acid or 10Npotassium hydroxide to 10.5±0.1.
BUFFER NO.16,0.1M,pH7.0— Dissolve 13.6g of dibasic potassium phosphate and 4.0g of monobasic potassium phosphate in 1000mLof water.Adjust with 18Nphosphoric acid or 10Npotassium hydroxide to a pHof 7.0±0.2.
OTHER SOLUTIONS— Use the substances specified under Reagents,Indicators,and Solutions.For water,use Purified Water.For saline,use Sodium Chloride Injection.Dilute formaldehyde is Formaldehyde Solutiondiluted with water 1:3.

The potency of antibiotics is designated in either “Units”or “µg”of activity.In each case the “Unit”or “µg”of antibiotic activity is established and defined by the designated federal master standard for that antibiotic.The corresponding USP Reference Standard is calibrated in terms of the master standard.USP Reference Standards for antibiotic substances are held and distributed by the U.S.Pharmacopeial Convention,Inc.
The concept of “µg”of activity originated from the situation where the antibiotic preparation selected as the reference standard was thought to consist entirely of a single chemical entity and was therefore assigned a potency of 1000“µg”per mg.In several such instances,as a result of the development of manufacturing and purification methods for particular antibiotics,preparations became available that contained more than 1000“µg”of activity per mg.It was then understood that such preparations had an activity equivalent to a given number of “µg”of the original reference standard.In most instances,however,the “µg”of activity is exactly equivalent numerically to the µg (weight)of the pure substance.Complications arise in some situations,e.g.,where an antibiotic exists as the free base and in salt form,and the “µg”of activity has been defined in terms of one such form;where the antibiotic substance consists of a number of components having close chemical similarity but differing antibiotic activity;or where the potencies of a family of antibiotics are expressed in terms of a reference standard consisting of a single member which,however,might itself be heterogeneous.In such cases the “µg”of activity defined in terms of a “Master Standard”is tantamount to a “Unit.”The “µg”of activity should therefore not be assumed necessarily to correspond to the µg (weight)of the antibiotic substance.

To prepare a stock solution,dissolve a quantity of the USP Reference Standard of a given antibiotic,accurately weighed,or the entire contents of a vial of USP Reference Standard,where appropriate,in the solvent specified in that table,and then dilute to the required concentration as indicated.Store in a refrigerator,and use within the period indicated.On the day of the assay,prepare from the stock solution 5or more test dilutions,the successive solutions increasing stepwise in concentration,usually in the ratio of 1:1.25for a cylinder-plate assay or smaller for a turbidimetric assay.Use the final diluent specified and a sequence such that the middle or median has the concentration designated.

From the information available for the preparation to be assayed (the “Unknown”),assign to it an assumed potency per unit weight or volume,and on this assumption prepare on the day of the assay a stock solution and test dilution as specified for each antibiotic but with the same final diluent as used for the USP Reference Standard.The assay with 5levels of the Standard requires only one level of the Unknown at a concentration assumed equal to the median level of the Standard.

Test Organisms
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.pneumoniaeuse a noncapsulated culture.For Enterococcus hirae,stab cultures may be used.
Table 1.Preparation of Stock Solutions and Test Dilutions of Reference Standards
Stock Solution Test Dilution
Antibiotic and Type of
Assay [Cylinder-plate
(CP)or Turbidimetric (T)]
Initial Solvent (and initial concentration where specified);Further Diluent,if different Final Stock
per mL
Use Within Final Diluent Median Dose (µg of activity or Units per mL)
Amikacin (T) Water 1mg 14days Water 10µg
Amphotericin B(CP) Dimethyl sulfoxide 1mg Same day B.10 1.0µg
Bacitracin Zinc (CP) 0.01Nhydrochloric acid 100U Same day B.1 1.0U
Bleomycin (CP) B.16 2U 14days B.16 0.04U
Candicidin (T) Dimethyl sulfoxide 1mg Same day Water 0.06µg
Capreomycin (T) Water 1mg 7days Water 100µg
Carbenicillin (CP) B.1 1mg 14days B.1 20µg
Cephalothin (CP) B.1 1mg 5days B.1 1.0µg
Cephapirin (CP) B.1 1mg 3days B.1 1.0µg
Chloramphenicol (T) Alcohol (10mg/mL);[Water] 1mg 30days Water 2.5µg
Chlortetracycline (T) 0.01Nhydrochloric acid 1mg 4days Water 0.06µg
Cloxacillin (CP) B.1 1mg 7days B.1 5.0µg
Colistimethate Sodium (CP) Water (10mg/mL);
1mg Same day B.6 1.0µg
Colistin (CP) Water (10mg/mL);
1mg 14days B.6 1.0µg
Cycloserine (T) Water 1mg 30days Water 50µg
Demeclocycline (T) 0.1Nhydrochloric acid 1mg 4days Water 0.1µg
Dihydrostreptomycin (CP) B.3 1mg 30days B.3 1.0µg
Dihydrostreptomycin (T) Water 1mg 30days Water 30µg
Doxycycline (T) 0.1Nhydrochloric acid 1mg 5days Water 0.1µg
Erythromycin (CP) Methanol (10mg/mL);[B.3] 1mg 14days B.3 1.0µg
Gentamicin (CP) B.3 1mg 30days B.3 0.1µg
Gramicidin (T) Alcohol 95% 1mg 30days Alcohol 95% 0.04µg
Kanamycin (T) Water 1mg 30days Water 10µg
Methacycline (T) Water 1mg 7days Water 0.06µg
Nafcillin (CP) B.1 1mg 2days B.1 2.0µg
Natamycin (CP) Dimethyl sulfoxide 1mg Same day B.10 5.00µg
Neomycin (CP) B.3 1mg 14days B.3 1.0µg
Neomycin (T) B.3 100µg 14days B.3 1.0µg
Netilmicin (CP) B.3 1mg 7days B.3 0.1µg
Novobiocin (CP) Alcohol (10mg/mL);[B.3] 1mg 5days B.6 0.5µg
Nystatin (CP) Dimethylformamide 1,000U Same day B.6 20U
Oxytetracycline (T) 0.1Nhydrochloric acid 1mg 4days Water 0.24µg
Paromomycin (CP) B.3 1mg 21days B.3 1.0µg
Penicillin G(CP) B.1 1,000U 4days B.1 1.0U
Polymyxin B(CP) Water;[B.6] 10,000U 14days B.6 10U
Rolitetracycline (T) Water 1mg 1day Water 0.24µg
Sisomicin (CP) B.3 1mg 14days B.3 0.1µg
Streptomycin (T) Water 1mg 30days Water 30µg
Tetracycline (T) 0.1Nhydrochloric acid 1mg 1day Water 0.24µg
Thiostrepton (T) Dimethyl sulfoxide 1U Same day Dimethyl
Ticarcillin (CP) B.1 1mg 1day B.1 5.0µg
Tobramycin (T) Water 1mg 14days Water 2.5µg
Troleandomycin (T) Isopropyl alcohol-water (4:1) 1mg Same day Water 25µg
Tylosin (T) Methanol (10mg/mL);[B.16] 1mg 30days B.3:methanol (1:1) 4µg
Vancomycin (CP) Water 1mg 7days B.4 10µg
NOTES—B”denotes “buffer,”and the number following refers to the potassium phosphate buffers defined in this chapter.
For amphotericin B,colistimethate sodium,and nystatin,prepare the USP Reference Standard solutions and the sample test solution simultaneously.
For amphotericin B,further dilute the stock solution with dimethyl sulfoxide to give concentrations of 12.8,16,20,25,and 31.2µg per mLprior to making the test dilutions.The Test Dilutionof the sample should contain the same amount of dimethyl sulfoxide as the test dilutions of the USP Reference Standard.
For bacitracin zinc,each of the Standard test dilutions should contain the same amount of hydrochloric acid as the Test Dilutionof the sample.
For neomycin turbidimetric assay,dilute the 100-µg-per-mLstock solution quantitatively with Buffer No.3to obtain a solution having a concentration equivalent to 25.0µg of neomycin per mL.To separate 50-mLvolumetric flasks add 1.39,1.67,2.00,2.40,and 2.88mLof this solution,add 5.0mLof 0.01Nhydrochloric acid to each flask,dilute with Buffer No.3to volume,and mix to obtain solutions having concentrations of 0.69,0.83,1.0,1.2,and 1.44µg of neomycin per mL.Use these solutions to prepare the standard response line.
For nystatin,further dilute the stock solution with dimethylformamide to give concentrations of 256,320,400,500,and 624Units per mLprior to making the test dilutions.Prepare the standard response line solutions simultaneously with dilutions of the sample to be tested.The Test Dilutionof the sample should contain the same amount of dimethylformamide as the test dilutions of the Standard.Use red low-actinic glassware.
For Polymyxin B,prepare the stock solution by adding 2mLof water for each 5mg of the weighed USP Reference Standard material.
Table 2.Test Organisms for Antibiotics Assayed by the Procedure Indicated in Table 1
Antibiotic Test Organism ATCC*Number
Amikacin Staphylococcus aureus 29737
Amphotericin B Saccharomyces cerevisiae 9763
Bacitracin Micrococcus luteus 10240
Bleomycin Mycobacterium smegmatis 607
Candicidin Saccharomyces cerevisiae 9763
Capreomycin Klebsiella pneumoniae 10031
Carbenicillin Pseudomonas aeruginosa 25619
Cephalothin Staphylococcus aureus 29737
Cephapirin Staphylococcus aureus 29737
Escherichia coli 10536
Staphylococcus aureus 29737
Cloxacillin Staphylococcus aureus 29737
Bordetella bronchiseptica 4617
Colistin Bordetella bronchiseptica 4617
Cycloserine Staphylococcus aureus 29737
Demeclocycline Staphylococcus aureus 29737
mycin (CP)
Bacillus subtilis 6633
mycin (T)
Klebsiella pneumoniae 10031
Doxycycline Staphylococcus aureus 29737
Erythromycin Micrococcus luteus 9341
Gentamicin Staphylococcus epidermidis 12228
Gramicidin Enterococcus hirae 10541
Kanamycin Staphylococcus aureus 29737
Methacycline Staphylococcus aureus 29737
Nafcillin Staphylococcus aureus 29737
Neomycin (CP) Staphylococcus epidermidis 12228
Neomycin (T) Klebsiella pneumoniae 10031
Netilmicin Staphylococcus epidermidis 12228
Novobiocin Staphylococcus epidermidis 12228
Nystatin Saccharomyces cerevisiae 2601
Oxytetracycline Staphylococcus aureus 29737
Paromomycin Staphylococcus epidermidis 12228
Penicillin G Staphylococcus aureus 29737
Polymyxin B Bordetella bronchiseptica 4617
Rolitetracycline Staphylococcus aureus 29737
Sisomicin Staphylococcus epidermidis 12228
Spectinomycin Escherichia coli 10536
Klebsiella pneumoniae 10031
Tetracycline Staphylococcus aureus 29737
Thiostrepton (T) Enterococcus hirae 10541
Tobramycin Staphylococcus aureus 29737
Troleandomycin Klebsiella pneumoniae 10031
Tylosin Staphylococcus aureus 9144
Vancomycin Bacillus subtilis 6633
*  American Type Culture Collection,10801University Boulevard,Manassas VA20110-2209.(http://www.atcc.org).
Table 3.Preparation of Inoculum
Incubation Conditions Suggested Inoculum Composition
Test Organism &
(ATCC No.)
Medium Temp.() Time Medium Amount (mLper 100mL) Antibiotics Assayed
Bacillus subtilis
32 32to 35 5days 5 As required Dihydrostreptomycin
8 As required Vancomycin
Bordetella bronchiseptica(4617) 1 32to 35 24hr. 10 0.1 Colistimethate Sodium,Colistin,Polymyxin B
Escherichia coli 1 32to 35 24hr. 3 0.7 Chloramphenicol
Klebsiella pneumoniae
1 36to 37.5 16to 24hr. 3 0.05 Capreomycin
0.1 Streptomycin,Troleandomycin,
39 2 Neomycin
Micrococcus luteus 1 32to 35 24hr. 11 1.5 Erythromycin
Micrococcus luteus
1 32to 35 24hr. 1 0.3 Bacitracin
36 36to 37.5 48hr. 35 1.0 Bleomycin
Pseudomonas aeruginosa(25619) 1 36to 37.5 24hr. 10 0.5 Carbenicillin
Saccharomyces cerevisiae(9763) 19 29to 31 48hr. 13 0.2 Candicidin
19 1.0 Amphotericin B
Saccharomyces cerevisiae(2601) 19 29to 31 48hr. 19 1.0 Nystatin
Staphylococcus aureus
3 35to 39 16to 18hr. 39 2-3 Tylosin
Staphylococcus aureus 1 32to 35 24hr. 1 0.1 Cephalothin,Cephapirin,Cloxacillin
1 0.3 Nafcillin
1 1.0 Penicillin G
3 0.1 Amikacin,Chlortetracycline,
3 0.2 Kanamycin
3 0.4 Cycloserine
3 0.15 Tobramycin
1 32to 35 24hr. 11 0.25 Netilmicin
(12228) 1 4.0 Novobiocin
11 0.03 Gentamicin,Sisomicin
11 0.4 Neomycin
11 2.0 Paromomycin
Enterococcus hirae
3 36to 37.5 16to 18hr. 3 1.0 Gramicidin
40 36to 37.5 18to 24hr. 41 0.2 Thiostrepton
NOTE—For Pseudomonas aeruginosa(ATCC25619)in the assay of Carbenicillin,use 0.5mLof a 1:25dilution of the stock suspension per 100mLof Medium 10.
Preparation of Inoculum
Preparatory to an assay,remove the growth from a recently grown slant or culture of the organism,with 3mLof sterile saline TSand sterile glass beads.Inoculate the surface of 250mLof the agar medium specified for that organism in Table 3and contained on the flat side of a Roux bottle except in the case of Enterococcus hiraeand Staphylococcus aureus(ATCC9144),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 50mLof sterile saline TS,except for Bleomycin (use 50mLof 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 Methodfor Procedure.Adjust the quantity of Inoculumon 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 Methodfor Procedure,tubes containing the median dose of the Standard should have absorbances of at least 0.3absorbance unit,except for Amikacin,Chlortetracycline,Gramicidin,and Tetracycline (0.35absorbance unit),and Capreomycin,Methacycline,and Tobramycin (0.4absorbance 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 14to 16mm 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 45to 50,and swirling to attain a homogeneous suspension.

Assay Designs
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.
NOTE—For some purposes,the practice is to design the assay so that a set of treatments consists of not fewer than three tubes for each sample and standard concentration,and each set is placed in a single rack.
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 (S3),of the Standard and a solution of a single median test level of the Unknown as described under Preparation of Standardand 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.
Cylinder-Plate Method
To prepare assay plates using Petri dishes,place 21mLof Medium 2in each of the required number of plates,and allow it to harden into a smooth base layer of uniform depth,except for Amphotericin Band Nystatin,where no separate base layer is used.For Erythromycin,Gentamicin,Neomycin B,Paromomycin,and Sisomicin,use Medium 11.For Bleomycin,use 10mLof Medium 35.For Dihydrostreptomycin use Medium 5.For Vancomycin,use 10mLof Medium 8.For Carbenicillin,Colistimethate Sodium,Colistin,and Polymyxin B,use Medium 9.For Netilmicin,use 20mLof Medium 11.Add 4mLof seed layer inoculum (see Preparation of Inoculumand Table 3),prepared as directed for the given antibiotic,except for Bleomycin (use 6mL),for Netilmicin (use 5mL),and for Nystatin and Amphotericin B(use 8mL),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 12mm,using a mechanical guide or other device to insure even spacing on a radius of 2.8cm,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 32to 35,or at the temperature specified below for the individual case,for 16to 18hours,remove the cylinders,and measure and record the diameter of each zone of growth inhibition to the nearest 0.1mm.Incubate the plates at 29to 31for Amphotericin Band Nystatin.Incubate at 34to 36for Novobiocin.Incubate at 36to 37.5for Carbenicillin,Colistimethate Sodium,Colistin,Dihydrostreptomycin,Gentamicin,Neomycin,Netilmicin,Paromomycin,Polymyxin B,Sisomicin,and Vancomycin.
For the one-level assay with a standard curve,prepare dilutions representing five test levels of the Standard (S1to S5)and a single test level of the Unknown U3corresponding to S3of the standard curve,as defined under Preparation of the Standardand 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.
Turbidimetric Method
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 Standardand Preparation of the Sample.Add 1.0mLof each dose,except for Gramicidin,Thiostrepton and Tylosin (use 0.10mL)to each of three prepared test tubes,and place the three replicate tubes in a position,selected at random,in a test tube rack or other carrier.Include similarly in each rack one or two control tubes containing 1mLof the test diluent (see Table 1)but no antibiotic.Upon completion of the rack of test solutions (with candicidin within 30minutes of the time when water is added to the methyl sulfoxide stock solution),add 9.0mLof inoculum to each tube in the rack in turn,and place the completed rack immediately in an incubator or a water bath maintained at 36to 37.5,except for Candicidin (incubate at 27to 29).Incubate the tubes for 4to 5hours,except for Capreomycin,Chloramphenicol,Cycloserine,Dihydrostreptomycin,Spectinomycin,Streptomycin,and Troleandomycin (incubate these for 3to 4hours),Tylosin (incubate for 3to 5hours),and Candicidin (incubate for 16to 18hours).After incubation add 0.5mLof dilute formaldehyde to each tube,except for tylosin (heat the rack in a water bath at 80to 90for 2to 6minutes or in a steam bath for 5to 10minutes,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 Spectrophotometerunder Apparatus).
For the one-level assay with a standard curve,prepare dilutions representing five test levels of the Standard (S1to S5)and a single test level (U3)of each of up to 20Unknowns corresponding to S3of the Standard.Prepare also an extra S3as a test of growth.Add 1mLof each test dilution,except for Gramicidin,Thiostrepton,and Tylosin (use 0.10mL)to three tubes and 1mLof antibiotic-free diluent to six tubes as controls.Distribute one complete set,including two tubes of controls,to a tube rack,intermingling them at random.Add 9.0mLof inoculum,except for Thiostrepton (use 10.0mLof inoculum),incubate,add 0.5mLof 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).

To calculate the potency from the data obtained either by the cylinder-plate or by the turbidimetric method,proceed in each case as directed under Potencies Interpolated from a Standard Curve(see Design and Analysis of Biological Assays á111ñ),using a log transformation,straight-line method with a least squares fitting procedure,and a test for linearity.Where a number of assays of the same material are made with the same standard curve,calculate the coefficient of variation of results of all of the assays of the material.Where more than one assay is made of the same material with different standard curves,average the two or more values of the potency.