5/16/2020 USP-NF

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© 2020 USPC

〈81〉 ANTIBIOTICS—MICROBIAL ASSAYS

Change to read:

INTRODUCTION AND GENERAL INFORMATION

The activity (potency) of antibiotics can be demonstrated by their inhibitory effect on microorganisms under suitable conditions. A
reduction in antimicrobial activity may not be adequately demonstrated by chemical methods. This chapter summarizes procedures
for the antibiotics recognized in the United States Pharmacopeia (USP) for which the microbiological assay is the standard analytical
method.
Two general techniques are employed: the cylinder-plate (or plate) assay and the turbidimetric (or tube) assay. Table 1 lists all the
antibiotics that contain microbial assays and speci es the type of assay (cylinder-plate or turbidimetric).

Table 1

Antibiotic Type of Assay

▲Amoxicillin Cylinder-plate▲ (USP 1-Dec-2019)

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Amphotericin B Cylinder-plate
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Bacitracin Cylinder-plate

Bleomycin Cylinder-plate

Capreomycin Turbidimetric
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▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

Chloramphenicol Turbidimetric
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Chlortetracycline Turbidimetric

Cloxacillin Cylinder-plate

Colistemethate Cylinder-plate
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Colistin Cylinder-plate

Cylinder-plate

Dihydrostreptomycin Turbidimetric

Erythromycin Cylinder-plate

Gentamicin Cylinder-plate

Gramicidin Turbidimetric

Nafcillin Cylinder-plate

▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

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Antibiotic Type of Assay

Cylinder-plate

Neomycin Turbidimetric

Novobiocin Cylinder-plate

Nystatin Cylinder-plate

Oxytetracycline Turbidimetric

Paromomycin Cylinder-plate

Penicillin G Cylinder-plate

Polymyxin B Cylinder-plate

▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

Tetracycline Turbidimetric

Thiostrepton Turbidimetric

▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

Tylosin

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Turbidimetric
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Vancomycin Cylinder-plate

[NOTE—Perform all procedures under conditions designed to avoid extrinsic microbial contamination. Take adequate safety precautions
while performing these assays because of possible allergies to drugs and because live cultures of organisms are used in the
procedures.]
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Cylinder-Plate Assay
The cylinder-plate assay depends on diffusion of the antibiotic from a vertical cylinder through a solidi ed agar layer in a Petri dish
or plate. The growth of the speci c microorganisms inoculated into the agar is prevented in a circular area or “zone” around the
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cylinder containing the solution of the antibiotic .

Turbidimetric Assay
The turbidimetric assay depends on the inhibition of growth of a microorganism in a uniform solution of the antibiotic in a uid
medium that is favorable to the growth of the microorganism in the absence of the antibiotic .

Units and Reference Standards

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▲For substances like the antibiotics quanti ed using the method in this chapter which are not easily characterized by chemical or
physical means, it is still necessary to express quantities of biological activity in units of biological potency, each de ned by an
authoritative reference standard. The potency of antibiotics is designated in either units (U) or µg of activity. In each case, the unit or
µg of antibiotic activity was originally established against a US Federal Master Standard for that antibiotic . This practice started
because originally the antibiotic selected as a reference standard was thought to consist entirely of a single chemical entity and was
therefore assigned a potency of 1000 µg/mg. In several such instances, as the manufacturing and puri cation methods for particular
antibiotics became more advanced, antibiotics containing more than 1000 µg of activity/mg became possible. Since relative potency
was used to assign this value and not the mass of the antibiotics themselves, such antibiotics had an “activity” equivalent to a given
number of µg of the original reference standard (rather than a µg of mass as would be expected). Over time, less complex antibiotics
or those with a single primary active substance were demonstrated to have µg of activity equivalent numerically to the µg (weight) of
the pure substance. In these cases, the antibiotics have moved away from the microbial assay methods and are assigned by a mass
balance approach; until that exercise is completed for a given antibiotic , manufacturers cannot assume that the µg of activity
corresponds to the µg (weight) of the antibiotic substance. Since the US Federal Master antibiotic standards are no longer available,
USP Reference Standards for antibiotics that are assigned relative potency by the methods in this chapter are calibrated either

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against the World Health Organization (WHO) International Standard for Antibiotics (ISA), if it exists, or the previous USP lot when a
WHO ISA or another authoritative reference standard calibrated against an ISA does not exist. Once assigned, the International Units
(IUs), when used, are converted to USP units by the previously agreed upon bridging value between the two when the respective
Reference Standards were originally assigned. Over time, it may be possible to convert the assay methods of more antibiotics from
the microbial bioassay to a physicochemical method much like that suggested in Validation of Alternative Methods to Antibiotic
Microbial Assays 〈1223.1〉 or other suitable strategies that include validated methods with associated bridging data.▲ (USP 1-Dec-2019)

Apparatus
Labware used for the storage and transfer of test dilutions and microorganisms must be sterile and free of interfering residues (see
Cleaning Glass Apparatus 〈1051〉). Use a validated sterilization method, such as dry heat, steam, or radiation; or use sterile, disposable
labware.

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. Refer to speci c temperature requirements below for each type of assay.

Test Organisms
The test organism for each antibiotic is listed in Table 3 for the cylinder-plate assay and Table 8 for the turbidimetric assay. The
test organisms are speci ed by the American Type Culture Collection (ATCC) number.
In order to ensure acceptable performance of test organisms, store and maintain them properly. Establish the speci c storage
conditions during method validation or veri cation. Discard cultures if a change in the organism’s characteristics is observed.

Prolonged Storage
For prolonged storage, maintain test organisms in a suitable storage solution such as 50% fetal calf serum in broth, 10%–15%
glycerol in tryptic soy broth, de brinated sheep blood, or skim milk. Prolonged-storage cultures are best stored in the freeze-dried

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state; temperatures of −60° or below are preferred; temperatures below −20° are acceptable.

Primary Cultures
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Prepare primary cultures by transferring test organisms from prolonged-storage vials onto appropriate media, and incubate under
appropriate growth conditions. Store primary cultures at the appropriate temperature, usually 2°–8°, and discard after 3 weeks. A
single primary culture can be used to prepare working cultures only for as many as 7 days.

Working Cultures
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Prepare working cultures by transferring the primary culture onto appropriate solid media to obtain isolated colonies. Incubate
working cultures under appropriate conditions to obtain satisfactory growth for preparation of test inocula. Prepare fresh working
cultures for each test day.

Uncharacteristic Growth or Performance of a Test Organism

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Use new stock cultures, primary cultures, or working cultures when a test organism shows uncharacteristic growth or performance.

Assay Designs
Suitable experimental designs are key to increasing precision and minimizing bias. Control of the incubation parameters,
temperature distribution and time, is critical for minimizing bias; it can be accomplished by staging the plates and racks as described
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for each assay.

CYLINDER-PLATE ASSAY
The comparisons are restricted to relationships between zone diameter measurements within plates, excluding the variation
between plates. Individual plate responses are normalized on the basis of the relative zone size of the standard compared to the
mean zone size of the standard across all plates.

TURBIDIMETRIC ASSAY
To avoid systematic bias, place replicate tubes randomly in separate racks so that each rack contains one complete set of
treatments. The purpose of this con guration is to minimize the in uence of temperature distribution on the replicate samples. The
turbidimetric assay, because of the con guration of the samples in test tube racks, is sensitive to slight variations in temperature.
The in uence of temperature variation can also be decreased by ensuring proper air ow or heat convection during incubation. At
least 3 tubes for each sample and Standard concentration (one complete set of samples) should be placed in a single rack. The
comparisons are restricted to relationships between the observed turbidities within racks.

Potency Considerations

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Within the restrictions listed above, the recommended assay design employs a ve-concentration standard curve and a single
concentration of each sample preparation.
For the cylinder-plate assay, each plate includes only 2 treatments, the reference treatment (median level standard, i.e., S3) and one
of the other 4 concentrations of the standard (S1, S2, S4, and S5) or the sample (U3). The concentration of the sample is an estimate
based on the target concentration. The sample should be diluted to give a nominal concentration that is estimated to be equivalent to
the median reference concentration (S3) of the standard. The purpose of diluting to the median reference concentration is to ensure
that the sample result will fall within the linear portion of the standard curve. The test determines the relative potency of U3 against
the standard curve. The sample (U3) should have a relative potency of about 100%. The nal potency of the sample is obtained by
multiplying the U3 result by the dilution factor.
An assay should be considered preliminary if the computed potency value of the sample is less than 80% or more than 125%. In this
case, the results suggest that the sample concentration assumed during preparation of the sample stock solution was not correct. In
such a case, one can adjust the assumed potency of the sample on the basis of the preliminary potency value and repeat the assay.
Otherwise, the potency will be derived from a portion of the curve where the Standard and sample responses will likely not be parallel.
Microbial determinations of potency are subject to inter-assay as well as intra-assay variables; therefore ▲three▲ (USP 1-Dec-2019) or
more independent assays are required for a reliable estimate of the potency of a given sample. Starting with separately prepared
stock solutions and test dilutions of both the Standard and the sample, perform additional assays of a given sample ▲▲ (USP 1-Dec-
▲
2019). The mean potency should include the results from all the valid independent assays. ▲ (USP 1-Dec-2019) The latter is assessed
by the width of the con dence interval (refer to Con dence Limits and Combination of Assays Calculations). The combined result of a
series of smaller, independent assays ▲▲ (USP 1-Dec-2019) is a more reliable estimate of potency than one from a single large assay
with the same total number of plates or tubes. Note that additional assays or lower variability allows the product to meet tighter
speci cation ranges. Reducing assay variability achieves the required con dence limit with fewer assays.

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Change to read:

CYLINDER-PLATE METHOD
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Temperature Control
Use appropriately quali ed and calibrated equipment to obtain the temperature ranges speci ed in Table 3.

Apparatus
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PLATES
Use glass or disposable plastic Petri dishes (approximately 20 mm × 100 mm or other appropriate dimensions) with lids.

CYLINDERS
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Use stainless steel or porcelain cylinders; 8 ± 0.1-mm o.d.; 6 ± 0.1-mm i.d.; 10 ± 0.1-mm high. [NOTE—Carefully clean cylinders to
remove all residues; occasional cleaning in an acid bath, e.g., with about 2 N nitric acid or with chromic acid (see 〈1051〉) is
required.]

Standard Solutions
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To prepare a stock solution, dissolve a suitable quantity of the USP Reference Standard of a given antibiotic , or the entire contents
of a vial of USP Reference Standard, where appropriate, in the solvent speci ed in Table 2; and dilute to the speci ed concentration.
Store at 2°–8°, and use within the period indicated. On the day of the assay, prepare from the stock solution ve or more test dilutions,
in which the successive solutions increase stepwise in concentration, usually in the ratio of 1:1.25. Use the nal diluent speci ed such
that the median has the concentration suggested in Table 2.

Sample Solutions
Assign an assumed potency per unit weight or volume to the sample. On the day of the assay, prepare a stock solution in the same
manner speci ed for the USP Reference Standard (see Table 2). Dilute the sample stock solution in the speci ed nal diluent to obtain
a nominal concentration equal to the median concentration of the standard (S3).

Table 2

Antibiotic Stock Solution Test Dilution

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Antibiotic Initial Stock Solution Final Test Dilution

Median
Initial Concentratio Further Concentratio Use Within Concentratio
Solvent n Diluent n (days) Final Diluent n (S3)a,b

Initial Final Median

Initial Concentratio Further Concentratio Use Within Concentratio
Solvent n Diluent n (days) Final Diluent n (S3)a,b

— — 0.1 µg/mL▲

(USP 1-Dec-
▲Amoxicillinc Water 100 µg/mL 7 B.3d 2019)

Amphotericin Dimethyl — —
Bc,e sulfoxide 1 mg/mL Same day B.10d 1 µg/mL

0.01 N — —
hydrochloric
Bacitracinf acid 100 U/mL Same day B.1d 1 U/mL

Bleomycin B.16d — — 2 U/mL 14 B.16d 0.04 U/mL

▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1-Dec-

Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) 2019)

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Cloxacillin B.1d — — 1 mg/mL 7 B.1d 5 µg/mL

Colistimethat 10 mg/mL
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ec Water B.6d 1 mg/mL Same day B.6d 1 µg/mL

Colistin Water 10 mg/mL B.6d 1 mg/mL 14 B.6d 1 µg/mL

Dihydrostrept — —
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omycing B.3d 1 mg/mL 30 B.3d 1 µg/mL

Erythromycin Methanol 10 mg/mL B.3d 1 mg/mL 14 B.3d 1 µg/mL

Gentamicin B.3d — — 1 mg/mL 30 B.3d 0.1 µg/mL

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Nafcillin B.1d — — 1 mg/mL 2 B.1d 2 µg/mL

▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1-Dec-
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Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) 2019)

Neomycing B.3d — — 1 mg/mL 14 B.3d 1 µg/mL

Novobiocin alcohol 10 mg/mL B.3d 1 mg/mL 5 B.6d 0.5 µg/mL

Dimethylfor — —
Nystatinc,h mamide 1000 U/mL Same day B.6d 20 U/mL

Paromomyci — —
n B.3d 1 mg/mL 21 B.3d 1 µg/mL

Penicillin G B.1d — — 1000 U/mL 4 B.1d 1 U/mL

Polymyxin Bi Water — B.6d 10,000 U/mL 14 B.6d 10 U/mL

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Antibiotic Stock Solution Test Dilution

Initial Final Median

Initial Concentratio Further Concentratio Use Within Concentratio
Solvent n Diluent n (days) Final Diluent n (S3)a,b

▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1-Dec-

Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) 2019)

Vancomycin Water — — 1 mg/mL 7 B.4d 10 µg/mL

a
  It is acceptable to adjust the median concentration to optimize zone sizes if the data remain in the linear range.
b
  µg in this column refers to µg of activity.
c
  Prepare the USP Reference Standard and sample test dilutions simultaneously.
d  The letter B refers to buffer. See Buffers for a description of each buffer listed in this table.
e  Further dilute the stock solution with dimethyl sulfoxide to give concentrations of 12.8, 16, 20, 25, and 31.2 µg/mL before making
the test dilutions. The test dilution of the sample should contain the same amount of dimethyl sulfoxide as the test dilutions of the
USP Reference Standard.
f
  Each of the standard test dilutions should contain the same amount of hydrochloric acid as the test dilution of the sample.
g
  The turbidimetric assay can be used as an alternative procedure.
h
  Further dilute the stock solution with dimethylformamide to give concentrations of 256, 320, 400, 500, and 624 U/mL before
making the test dilutions. Prepare the standard test dilutions simultaneously with test dilutions of the sample to be tested. The test

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dilution of the sample should contain the same amount of dimethylformamide as the test dilutions of the standard. Use low-actinic
glassware.
i  Prepare the stock solution by adding 2 mL of water for each 5 mg of the USP Reference Standard.
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Inocula
Suspend the test organism from a freshly grown slant or culture in 3 mL of sterile saline TS. Glass beads can be used to facilitate
the suspension. Spread the saline suspension onto the surface of two or more agar plates (covering the entire surface) or onto the
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surface of a Roux bottle containing 250 mL of the speci ed medium (see Table 3).
Incubate for the speci ed time and at the temperature as speci ed in Table 3, or until growth is apparent.
After incubation, harvest the organism from the plates or Roux bottle with approximately 50 mL of sterile saline TS (except use
Medium 34 for bleomycin; see Media and Solutions), using a sterile bent glass rod or sterile glass beads. Pipet the suspension into a
sterile glass container. This is the harvest suspension.
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▲To make the stock suspension,

▲ (USP 1-Dec-2019) dilute an appropriate amount of the harvest suspension with sterile saline TS.
Using the UV-Vis spectrophotometer, measure the percentage transmittance at 580 nm. The target value is approximately 25%
transmittance at 580 nm. This value is used to standardize the harvest suspension volume added to the seed layer agar.
Starting with the suggested volumes indicated in Table 3, determine during method veri cation the proportions of stock suspension
to be added to the inoculum medium that result in satisfactory zones of inhibition of approximately 14–16 mm in diameter for the
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median concentration of the Standard (S3).

[NOTE—Zone sizes that are outside the 11–19-mm range are not desirable, because these contribute to assay variability.]
If the dilution percentage transmittance is above 25%, a ratio may be used to normalize the addition of organism to the seed layer.
The normalization factor can be determined by dividing the percentage transmittance obtained from the dilution by 25. This ratio can
then be multiplied by the suggested inoculum amount to obtain the volume (mL) of harvest suspension that needs to be added to the
seed layer. Adjust the quantity of inoculum on a daily basis, if necessary, to obtain an optimum concentration–response relationship.
Alternatively, determine during method veri cation the proportion of harvest suspension to be incorporated into the inoculum,
starting with the volumes indicated in Table 3, that result in satisfactory demarcation of the zones of inhibition of about 14–16 mm in
diameter for the median concentration of the Standard (S3) and giving a reproducible concentration–response relationship. Prepare
the inoculum by adding a portion of stock suspension to a su cient amount of agar medium that has been melted and cooled to
45°–50°. Swirl the mixture without creating bubbles in order to obtain a homogeneous suspension.

Table 3

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Suggested Inoculum
Incubation Conditions Composition

Test ATCCa Temperature Amount

Antibiotic Organism Number Mediumb (°) Time Mediumb (mL/100 mL)

0.5▲ (USP 1-
Kocuria
▲Amoxicillin rhizophila 9341 1 32–35 24 h 11 Dec-2019)

Amphotericin Saccharomyc
B es cerevisiae 9763 19 29–31 48 h 19 1.0

Bacitracin Micrococcus
luteus 10240 1 32–35 24 h 1 0.3

Bleomycin Mycobacteriu
m smegmatis 607 36 36–37.5 48 h 35 1.0

▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
▲ (USP 1- ▲ (USP 1-Dec- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1-Dec-

Dec-2019) 2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) 2019)

Cloxacillin Staphylococc
us aureus 29737 1 32–35 24 h 1 0.1

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Colistimethat Bordetella
e bronchiseptic
a 4617 1 32–35 24 h 10 0.1
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Colistin Bordetella
bronchiseptic
a 4617 1 32–35 24 h 10 0.1
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Dihydrostrept Bacillus
omycin subtilis 6633 32 32–35 5 days 5 As required

Erythromycin ▲Kocuria
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rhizophila▲

(USP 1-Dec-

2019) 9341 1 32–35 24 h 11 1.5

Gentamicin Staphylococc
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us
epidermidis 12228 1 32–35 24 h 11 0.03

Nafcillin Staphylococc
us aureus 29737 1 32–35 24 h 1 0.3

Neomycin Staphylococc
us
epidermidis 12228 1 32–35 24 h 11 0.4

Novobiocin Staphylococc
us
epidermidis 12228 1 32–35 24 h 1 4.0

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Suggested Inoculum
Incubation Conditions Composition

Test ATCCa Temperature Amount

Antibiotic Organism Number Mediumb (°) Time Mediumb (mL/100 mL)

Nystatin Saccharomyc
es
▲Kudriavzevii

▲ (USP 1-Dec-

2019) 2601 19 29–31 48 h 19 1.0

Paromomyci Staphylococc
n us
epidermidis 12228 1 32–35 24 h 11 2.0

Penicillin G Staphylococc
us aureus 29737 1 32–35 24 h 1 1.0

Polymyxin B Bordetella
bronchiseptic
a 4617 1 32–35 24 h 10 0.1

▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
▲ (USP 1- ▲ (USP 1-Dec- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1-Dec-

L
Dec-2019) 2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) 2019)
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Vancomycin Bacillus
subtilis 6633 32 32–35 5 days 8 As required

▲ 
▲ (USP 1-Dec-2019)
a  American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 (http://www.atcc.org).
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b
  See Media.

Analysis
Prepare the base layer for the required number of assay Petri plates, using the medium and volume shown in Table 4. Allow it to
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harden into a smooth base layer of uniform depth. Prepare the appropriate amount of seed layer inoculum (see Table 5) as directed
for the given antibiotic (see Table 3) with any adjustments made based on the preparatory trial analysis. Tilt the plate back and forth
to spread the inoculum evenly over the base layer surface, and allow it to harden.

Table 4. Base Layer

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Antibiotic Mediuma Target Volume (mL)

▲Amoxicillin 11 21▲ (USP 1-Dec-2019)

Amphotericin Bb — —

Bleomycin 35 10

▲ ▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

Colistimethate 9 21

Colistin 9 21

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Antibiotic Mediuma Target Volume (mL)

Dihydrostreptomycin 5 21

Erythromycin 11 21

Gentamicin 11 21

Neomycin 11 21

Nystatinb — —

Paromomycin 11 21

Polymyxin B 9 21

▲ ▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

Vancomycin 8 10

All others 2 21

a
  See Media.
b  No base layer is used. [NOTE—The base layer may be warmed to facilitate a uniform seed layer.]

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Table 5. Seed Layer

Antibiotic Mediuma Target Volume (mL)

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Amphotericin B 8

Bleomycin 6
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Nystatin 8

All others Refer to Table 3 4

a  See Media.
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Drop 6 assay cylinders on the inoculated surface from a height of 12 mm, using a mechanical guide or other device to ensure even
spacing on a radius of 2.8 cm, and cover the plates to avoid contamination. Fill the 6 cylinders on each plate with ▲the same volume
of▲ (USP 1-Dec-2019) dilutions of antibiotic containing the test levels (S1–S5 and U3) speci ed in the following paragraph. Incubate the
plates as speci ed in Table 6 for 16–18 h, and remove the cylinders. Measure and record the diameter of each zone of growth
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inhibition to the nearest 0.1 mm.

Table 6

Antibiotic Incubation Temperature (°)

Amphotericin B 29–31

▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

Colistimethate 36–37.5

Colistin 36–37.5

Dihydrostreptomycin 36–37.5

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Antibiotic Incubation Temperature (°)

Gentamicin 36–37.5

Neomycin 36–37.5

Novobiocin 34–36

Nystatin 29–31

Paromomycin 36–37.5

Polymyxin B 36–37.5

▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

Vancomycin 36–37.5

All others 32–35

The Standards (S1–S5) and a single test level of the sample (U3) corresponding to S3 of the standard curve, as de ned in Standard
Solutions and Sample Solutions will be used in the assay. For deriving the standard curve, ll alternate cylinders on each of 3 plates
with the median test dilution (S3) of the Standard and each of the remaining 9 cylinders with one of the other four test dilutions of the
standard. Repeat the process for the three test dilutions of the Standard. For the sample, ll alternate cylinders on each of 3 plates
with the median test dilution of the Standard (S3), and ll the remaining 9 cylinders with the corresponding test dilution (U3) of the
sample.

Change to read:
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TURBIDIMETRIC METHOD
Temperature Control
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Use appropriately quali ed and calibrated equipment to obtain the temperature ranges speci ed in Table 8. [NOTE—Temperature
control can be achieved using either circulating air or water. The greater heat capacity of water lends it some advantage over
circulating air.]

Spectrophotometer
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Measuring absorbance or transmittance within a fairly narrow frequency band requires a suitable spectrophotometer in which the
wavelength can be varied or restricted by the use of 580- or 530-nm lters. Alternatively, a variable-wavelength spectrophotometer
can be used and set to a wavelength of 580 or 530 nm.
The instrument may be modi ed as follows:
1. To accept the tube in which incubation takes place (see Apparatus below)
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2. To accept a modi ed cell tted with a drain that facilitates rapid change of contents
3. To contain a ow cell for a continuous owthrough analysis
Autozero the instrument with clear, uninoculated broth prepared as speci ed for the particular antibiotic , including the same
amount of test dilution (including formaldehyde if speci ed) as found in each sample.
Either absorbance or transmittance can be measured while preparing inocula.

Apparatus
Use glass or plastic test tubes, e.g., 16 mm × 125 mm or 18 mm × 150 mm. [NOTE—Use tubes that are relatively uniform in length,
diameter, and thickness and substantially free from surface blemishes and scratches. In the spectrophotometer, use matched tubes
that are free from scratches or blemishes. Clean tubes thoroughly to remove all antibiotic residues and traces of cleaning solution.
Sterilize tubes before use.]

Standard Solutions
To prepare a stock solution, dissolve a quantity of the USP Reference Standard of a given antibiotic or the entire contents of a vial
of USP Reference Standard, where appropriate, in the solvent speci ed in Table 7, and dilute to the required concentration. Store at
2°–8°, and use within the period indicated. On the day of the assay, prepare from the stock solution ve or more test dilutions, the

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successive solutions increasing stepwise in concentration, usually in the ratio of 1:1.25. [NOTE—It may be necessary to use smaller
ratios for the successive dilutions from the stock solution for the turbidimetric assay.] Use the nal diluent speci ed such that the
median level of the Standard (S3) has the concentration suggested in Table 7.

Sample Solutions
Assign an assumed potency per unit weight or volume to the unknown, and on the day of the assay prepare a stock solution in the
same manner speci ed for the USP Reference Standard (see Table 7). Dilute the sample stock solution in the speci ed nal diluent at
a nominal concentration equal to the median concentration of the Standard (S3) as speci ed in Table 7.

Table 7

Stock Solution Test Dilution

Initial Final Stock Median

Initial Concentratio Further Concentratio Use Within Concentratio
Antibiotic Solvent n Diluent n (days) Final Diluent n (S3)a

Capreomycin Water — — 1 mg/mL 7 Water 100 µg/mL

Chloramphen
icol Alcohol 10 mg/mL Water 1 mg/mL 30 Water 2.5 µg/mL

0.1 N — —
Chlortetracyc hydrochloric

L
line acid 1 mg/mL 4 Water 0.06 µg/mL

Dihydrostrept — —
IA
omycinb Water 1 mg/mL 30 Water 30 µg/mL

Gramicidin Alcohol — — 1 mg/mL 30 Alcohol 0.04 µg/mL

Neomycinb,c B.3d — — 100 µg/mL 14 B.3d 1.0 µg/mL

IC

0.1 N — —
Oxytetracycli hydrochloric
ne acid 1 mg/mL 4 Water 0.24 µg/mL
FF

0.1 N — —
hydrochloric
Tetracycline acid 1 mg/mL 1 Water 0.24 µg/mL

— — ▲100 U/mL
▲
O

Dimethyl (USP 1-Dec- Dimethyl

Thiostrepton sulfoxide 2019) Same day sulfoxide 0.80 U/mL

▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1-Dec-

Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) 2019)

Methanol
and B.3d
d
Tylosin Methanol 10 mg/mL B.16 1 mg/mL 30 (1:1) 4 µg/mL

a  µg in this column refers to µg of activity.

b
  The cylinder-plate assay can be used as an alternative procedure.
c  Dilute the 100-µg/mL stock solution with Buffer B.3 to obtain a solution having a concentration equivalent to 25 µg/mL of
neomycin. To separate 50-mL volumetric asks add 1.39, 1.67, 2.00, 2.40, and 2.88 mL of this solution. Add 5.0 mL of 0.1 N

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hydrochloric acid to each ask, dilute with Buffer B.3 to volume, and mix to obtain solutions having concentrations of 0.69, 0.83, 1.0,
1.2, and 1.44 µg/mL of neomycin. Use these solutions to prepare the standard response line.
d  The letter B refers to buffer. See Buffers for a description of each buffer listed in this table.

Inocula
Suspend the test organism from a freshly grown slant or culture in 3 mL of sterile saline TS. Glass beads can be used to facilitate
the suspension. Enterococcus hirae (ATCC 10541) ▲(when used for the gramicidin assay),▲ (USP 1-Dec-2019) and Staphylococcus
aureus (ATCC 9144) are grown in a liquid medium, not on agar. Spread the saline suspension onto the surface of two or more agar
plates (covering the entire surface) or onto the surface of a Roux bottle containing 250 mL of the speci ed medium (see Table 8).
Incubate at the time and temperature speci ed in Table 8, or until growth is apparent.
After incubation, harvest the organism from the plates or Roux bottle with approximately 50 mL of sterile saline TS, using a sterile
bent glass rod or sterile glass beads. Pipet the suspension into a sterile glass bottle. This is the harvest suspension.
Determine during method veri cation the quantity of harvest suspension that will be used as the inoculum, starting with the volume
suggested in Table 8. Prepare also an extra Standard (S3) as a test of growth. Incubate the trial tests for the times indicated in Table
11. Adjust the quantity of inoculum daily, if necessary, to obtain the optimum concentration–response relationship from the amount
of growth of the test organism in the assay tubes. At the completion of the speci ed incubation periods, tubes containing the median
concentration of the Standard should have absorbance values as speci ed in Table 9. Determine the exact duration of incubation by
observing the growth in the reference concentration (median concentration) of the standard (S3).

Table 8

Suggested Inoculum
Incubation Conditions Composition

Antibiotic
Test
Organism
ATCCa
Number Mediumb

L
Temperature
(°) Time (h) Mediumb
Amount
(mL/100 mL)
IA
Klebsiella
Capreomycin pneumoniae 10031 1 36–37.5 16–24 3 0.05

Chloramphen Escherichia
IC

icol coli 10536 1 32–35 24 3 0.7

Chlortetracyc Staphylococc
line us aureus 29737 1 32–35 24 3 0.1
FF

Dihydrostrept Klebsiella
omycin pneumoniae 10031 1 36–37.5 16–24 3 0.1

Enterococcus
Gramicidin hirae 10541 3 36–37.5 16–18 3 1.0
O

Klebsiella
Neomycin pneumoniae 10031 1 36–37.5 16–24 39 2

Oxytetracycli Staphylococc
ne us aureus 29737 1 32–35 24 3 0.1

Staphylococc
Tetracycline us aureus 29737 1 32–35 24 3 0.1

Enterococcus
Thiostrepton hirae 10541 40 36–37.5 18–24 41 0.2

▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1- ▲ (USP 1-Dec-

Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) Dec-2019) 2019)

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Suggested Inoculum
Incubation Conditions Composition

Test ATCCa Temperature Amount

Antibiotic Organism Number Mediumb (°) Time (h) Mediumb (mL/100 mL)

Staphylococc
Tylosin us aureus 9144 3 35–39 16–18 39 2–3

a  American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 (http://www.atcc.org).
b  See Media.

Table 9

Antibiotic Absorbance, NLT (a.u.)

Capreomycin 0.4

Chlortetracycline 0.35

Gramicidin 0.35

Tetracycline 0.35

All others 0.3

L
Analysis
On the day of the assay, prepare the necessary concentration of antibiotic by dilution of stock solutions of the Standard and of
IA
each sample as speci ed under Standard Solutions and Sample Solutions. Prepare 5 test levels, each in triplicate, of the Standard (S1–
S5) and a single test level (U3), also in triplicate, of up to 20 samples corresponding to S3 (median concentration) of the Standard.

Place the tubes in test tube racks or other carriers. Include in each rack 1–2 control tubes containing ▲0.1 mL of the test diluent for
gramicidin, thiostrepton, and tylosin, or▲ (USP 1-Dec-2019) 1 mL of the test diluent ▲for all others▲ (USP 1-Dec-2019) (see Table 7), but no
IC

antibiotic . Add the volumes of the Standard and sample test dilutions as indicated in Table 10. Randomly distribute one complete
set, including the controls, in a tube rack. Add the volume of inoculum speci ed in Table 10 to each tube in the rack in turn, and place
the completed rack immediately in an incubator or a water bath maintained at 36.0°–37.5° for the time speci ed in Table 11.
FF

Table 10

Antibiotic Volume of Test Dilution (mL) Volume of Inoculum (mL)

Gramicidin 0.10 9.0

O

Thiostrepton 0.10 10.0

Tylosin 0.10 9.0

All others 1.0 9.0

Table 11

Antibiotic Incubation Time (h)

Capreomycin 3–4

Chloramphenicol 3–4

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Antibiotic Incubation Time (h)

Cycloserine 3–4

Dihydrostreptomycin 3–4

▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

▲ ▲
▲ (USP 1-Dec-2019) ▲ (USP 1-Dec-2019)

Tylosin 3–5

All others 4–5

After incubation, immediately inhibit the growth of the organism by adding 0.5 mL of dilute formaldehyde to each tube, except for
tylosin. For tylosin, heat the rack in a water bath at 80°–90° for 2–6 min or in a steam bath for 5–10 min, and bring to room
temperature. Read absorbance or transmittance at 530 or 580 nm, analyzing one rack at a time.

MEDIA AND SOLUTIONS

The media required for the preparation of test organism inocula are made from the ingredients listed herein. Minor modi cations of
the individual ingredients are acceptable; and reconstituted dehydrated media can be substituted, provided that the resulting media
possess equal or better growth-promoting properties and give a similar standard curve response.

Media
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

L
required, so that after steam sterilization the pH is as speci ed.

Medium 1
IA
Peptone 6.0 g

Pancreatic digest of casein 4.0 g

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Yeast extract 3.0 g

Beef extract 1.5 g

FF

Dextrose 1.0 g

Agar 15.0 g

Water 1000 mL
O

pH after sterilization 6.6 ± 0.1

Medium 2

Peptone 6.0 g

Yeast extract 3.0 g

Beef extract 1.5 g

Agar 15.0 g

Water 1000 mL

pH after sterilization 6.6 ± 0.1

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Medium 3

Peptone 5.0 g

Yeast extract 1.5 g

Beef extract 1.5 g

Sodium chloride 3.5 g

Dextrose 1.0 g

Dibasic potassium phosphate 3.68 g

Monobasic potassium phosphate 1.32 g

Water 1000 mL

pH after sterilization 7.0 ± 0.05

Medium 4

Peptone 6.0 g

Yeast extract 3.0 g

Beef extract

L 1.5 g
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Dextrose 1.0 g

Agar 15.0 g

Water 1000 mL
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pH after sterilization 6.6 ± 0.1

Medium 5
FF

Peptone 6.0 g

Yeast extract 3.0 g

Beef extract 1.5 g

O

Agar 15.0 g

Water 1000 mL

pH after sterilization 7.9 ± 0.1

Medium 8

Peptone 6.0 g

Yeast extract 3.0 g

Beef extract 1.5 g

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Agar 15.0 g

Water 1000 mL

pH after sterilization 5.9 ± 0.1

Medium 9

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

pH after sterilization 7.2 ± 0.1

Medium 10

Pancreatic digest of casein

L 17.0 g
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Papaic digest of soybean 3.0 g

Sodium chloride 5.0 g

Dibasic potassium phosphate 2.5 g

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Dextrose 2.5 g

Agar 12.0 g
FF

Water 1000 mL

Polysorbate 80 (added after boiling the medium to dissolve the

agar) 10 mL
O

pH after sterilization 7.2 ± 0.1

Medium 11

Peptone 6.0 g

Pancreatic digest of casein 4.0 g

Yeast extract 3.0 g

Beef extract 1.5 g

Dextrose 1.0 g

Agar 15.0 g

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Water 1000 mL

pH after sterilization 8.3 ± 0.1

Medium 13

Peptone 10.0 g

Dextrose 20.0 g

Water 1000 mL

pH after sterilization 5.6 ± 0.1

Medium 19

Peptone 9.4 g

Yeast extract 4.7 g

Beef extract 2.4 g

Sodium chloride 10.0 g

Dextrose 10.0 g

Agar

L 23.5 g
IA
Water 1000 mL

pH after sterilization 6.1 ± 0.1

Medium 32
IC

Peptone 6.0 g

Pancreatic digest of casein 4.0 g

FF

Yeast extract 3.0 g

Beef extract 1.5 g

Manganese sulfate 0.3 g

O

Dextrose 1.0 g

Agar 15.0 g

Water 1000 mL

pH after sterilization 6.6 ± 0.1

Medium 34

Glycerol 10.0 g

Peptone 10.0 g

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Beef extract 10.0 g

Sodium chloride 3.0 g

Water 1000 mL

pH after sterilization 7.0 ± 0.1

Medium 35

Glycerol 10.0 g

Peptone 10.0 g

Beef extract 10.0 g

Sodium chloride 3.0 g

Agar 17.0 g

Water 1000 mL

pH after sterilization 7.0 ± 0.1

Medium 36

Pancreatic digest of casein

L 15.0 g
IA
Papaic digest of soybean 5.0 g

Sodium chloride 5.0 g

Agar 15.0 g
IC

Water 1000 mL

pH after sterilization 7.3 ± 0.1

FF

Medium 39

Peptone 5.0 g

Yeast extract 1.5 g

O

Beef extract 1.5 g

Sodium chloride 3.5 g

Dextrose 1.0 g

Dibasic potassium phosphate 3.68 g

Monobasic potassium phosphate 1.32 g

Water 1000 mL

pH after sterilization 7.9 ± 0.1

Medium 40

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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

pH after sterilization 6.7 ± 0.2

Medium 41

Pancreatic digest of casein 9.0 g

Dextrose 20.0 g

Yeast extract 5.0 g

Sodium citrate 10.0 g

Monobasic potassium phosphate

L 1.0 g
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Dibasic potassium phosphate 1.0 g

Water 1000 mL

pH after sterilization 6.8 ± 0.1

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Solutions

BUFFERS
Prepare as directed in Table 12, or by other suitable means. The buffers are sterilized after preparation; the pH speci ed in each
FF

case is the pH after sterilization.

Table 12. Buffers

Concentration of Concentration of Volume of 10 N

O

Dibasic Potassium Monobasic Potassium Potassium Hydroxide

Buffer Phosphate (g/L) Phosphate (g/L) (mL) pH after Sterilizationa

Buffer B.1 (1%, pH 6.0) 2 8 — 6.0 ± 0.05

Buffer B.3 (0.1 M, pH

8.0) 16.73 0.523 — 8.0 ± 0.1

Buffer B.4 (0.1 M, pH

4.5) — 13.61 — 4.5 ± 0.05

Buffer B.6 (10%, pH

6.0) 20 80 — 6.0 ± 0.05

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Concentration of Concentration of Volume of 10 N

Dibasic Potassium Monobasic Potassium Potassium Hydroxide
Buffer Phosphate (g/L) Phosphate (g/L) (mL) pH after Sterilizationa

Buffer B.10 (0.2 M, pH —

10.5) 35 2 10.5 ± 0.1

Buffer B.16 (0.1 M, pH

7.0) 13.6 4 — 7.0 ± 0.2

a  Adjust the pH with 18 N phosphoric acid or 10 N potassium hydroxide.

Other solutions: See Reagents, Indicators, and Solutions.

Water: Use Puri ed Water.
Saline: Use saline TS.
Dilute formaldehyde: Formaldehyde solution and water (1:3)

Change to read:

CALCULATIONS
Introduction
Antibiotic potency is calculated by interpolation from a standard curve using a log-transformed straight-line method with a least-
squares tting procedure (see below for calculation details). The analyst must consider three essential concepts in interpreting
antibiotic potency results:
1. Biological concentration–response relationships generally are not linear. The antibiotic potency method allows tting the data

L
to a straight line by evaluating a narrow concentration range where the results approach linearity. The assay results can be
considered valid only if the computed potency is 80%–125% of that assumed in preparing the sample stock solution. When the
IA
calculated potency value falls outside 80%–125%, the result for the sample may fall outside the narrow concentration range
where linearity has been established. In such a case, adjust the assumed potency of the sample accordingly, and repeat the
assay to obtain a valid result.
2. The most effective means of reducing the variability of the reportable value (the geometric mean potency across runs and
replicates) is through independent runs of the assay procedure. The combined result of a series of smaller, independent
IC

assays ▲▲ (USP 1-Dec-2019) is a more reliable estimate of potency than that from a single large assay with the same total
number of plates or tubes. Three or more independent assays are required for antibiotic potency determinations.
3. The number of assays needed in order to obtain a reliable estimate of antibiotic potency depends on the required
speci cation range and the assay variability. The con dence limit calculation described below is determined from several
FF

estimated log potencies that are approximately equal in precision. If the value calculated for the ▲half-▲ (USP 1-Dec-2019)width
of the con dence interval, W, is too wide, no useful decision can be made about whether the potency meets its speci cation.
The laboratory should predetermine in its standard operating procedures a maximum acceptable value for the con dence interval
▲half-
▲ (USP 1-Dec-2019)width. This maximum value should be determined during development and con rmed during validation or
O

veri cation. If the calculated con dence interval ▲half-▲ (USP 1-Dec-2019)width exceeds this limit, the analyst must perform additional
independent potency determinations to meet the limit requirement. Note that the decision to perform additional determinations does
not depend on the estimated potency but only on the uncertainty in that estimate as determined by the con dence interval ▲half-▲

(USP 1-Dec-2019)width. Assay variability has a greater impact on the calculated con dence limit than does the number of independent
potency determinations. As a result, the analyst should rst consider decreasing variability to the extent possible before conducting
potency determinations.
The following sections describe the calculations for determining antibiotic potency as well as for performing the con dence limit
calculation. Methods for calculating standard error are also shown in order to allow estimates of assay variance. Where logarithms
are used, any base log is acceptable. Appendix 1: Formulas for Manual Calculations of Regression and Sample Concentration provides
formulas for hand calculations applicable when the concentrations are equally spaced in the log scale. Alternative statistical methods
may be used if appropriately validated.

Cylinder-Plate Assay
This section details analysis of the sample data and determination of the potency of an unknown, using the cylinder-plate assay.

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SAMPLE DATA

Table 13 shows the data from one assay that will be used as an example throughout this section. For each of the ▲15▲ (USP 1-Dec-
▲for ▲
2019) plates, zones 1, 3, and 5 are ▲ (USP 1-Dec-2019) the reference concentration, (S3),▲ (USP 1-Dec-2019) and the other three

zones are for one of the other four ▲standard▲ (USP 1-Dec-2019) concentrations, ▲(S1, S2, S4, and S5) or the sample (U3),▲ (USP 1-Dec-

2019) as shown. Other columns are needed for calculations and are explained below.
Step 1: Perform initial calculations and variability suitability check.
For each set of 3 plates, average the 9 reference values and average the 9 Standard values.
Example: See Table 13.

15.867 = X(16.1, 15.6, …, 15.8)

14.167 = X(14.6, 14.1, …, 14.8)

For each set of 3 plates, determine the standard deviation of the 9 reference values and the standard deviation of the 9 Standard
values. For each standard deviation, determine the corresponding relative standard deviation.
Example: See Table 13.

0.200 = σ(16.1, …, 15.8)

1.3% = (0.200/15.867) × 100

0.324 = σ(14.6, …, 14.1)

2.3% = (0.324/14.167) × 100

L
For a variability suitability criterion, each laboratory should determine a maximum acceptable value for the relative standard
deviation. If any of the eight relative standard deviations (four for the reference and four for the Standard) exceed this
IA
predetermined maximum, the assay data are not suitable and should be discarded. [NOTE—The suggested limit for relative standard
deviation is NMT 10%.]
Step 2: Perform a plate-to-plate variation correction.
This correction is applied to convert the average zone measurement obtained for each concentration to the value it would be if the
average reference concentration measurement for that set of 3 replicate plates were the same as the value of the correction point:
IC

XC = XS − (XR − P)

XC = corrected standard mean

FF

XS = original standard mean

XR = reference mean

P = correction point
O

Example: For the rst set of 3 plates in Table 13 (S1), the correction is:

14.022 = 14.167 − (15.867 − 15.722) = 14.167 − 0.145

Step 3: Determine the standard curve line.

Generate the standard curve line by plotting the corrected zone measurements versus the log of the standard concentration
values. Calculate the equation of the standard curve line by performing a standard unweighted linear regression on these values,
using appropriate software or the manual calculations of Appendix 1: Formulas for Manual Calculations of Regression and Sample
Concentration. [NOTE—Use either the natural log or the base 10 log to plot the standard curve and determine the regression equation;
both provide the same nal test result.] Each laboratory should determine a minimum value of the coe cient of determination (%R2)
for an acceptable regression. The regression is acceptable only if the obtained %R2 exceeds this predetermined value. [NOTE—The
suggested limit for the percentage coe cient of determination is NLT 95%.]

Table 13. Sample Data (Cylinder-Plate Assay)

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▲Standard Corr
Reference (S3) ▲ (USP 1-Dec-2019)
Con ecte
cent d
ratio Plat Zon Zon Zon Mea Zone Zone Zone Mea Mea
n e e1 e3 e5 n 2 4 6 n n
Stan (U/m Repl (mm (mm (mm (mm %RS (mm (mm (mm (mm %RS (mm
dard L) icate ) ) ) ) SD D ) ) ) ) SD D )

1 16.1 15.6 15.8 14.6 14.1 13.5

2 16.0 15.9 16.2 14.5 14.1 14.4

15.8 0.20 14.1 0.32 14.0
S1 3.20 3 15.7 15.7 15.8 67 0 1.3 14.0 14.2 14.1 67 4 2.3 22

1 15.8 15.6 15.5 14.7 15.1 14.8

2 15.7 15.5 15.6 14.7 14.9 15.2

15.5 0.15 14.8 0.26 14.9
S2 4.00 3 15.7 15.4 15.3 67 8 1.0 14.8 15.0 14.3 33 5 1.8 89

1 15.6 15.8 16.0 16.6 16.8 16.3

2 15.8 15.6 15.7 16.6 16.5 16.2

15.7 0.16 16.5 0.23 16.5
S4 6.25 3 16.1 15.7 15.8 89 9 1.1 16.9 16.5 16.8 78 3 1.4 11

1 15.6 15.6 15.5

L 17.3 17.0 17.0

IA
2 15.6 15.7 15.5 17.3 17.4 17.2
7.81 15.6 0.14 17.1 0.22 17.2
S5 25 3 15.9 15.8 15.8 67 1 0.9 17.3 17.3 16.7 67 4 1.3 22

15.7
IC

22a

▲Sa
Reference (S3)▲ (USP 1-Dec-2019) ▲Sample
mple ▲ (USP 1-Dec-2019)
FF

1 15.7 15.8 15.7 15.3 15.8 15.7

O

2 15.9 15.7 15.7 15.8 15.8 15.5

Unk
now 15.6 0.17 15.4 0.30 15.5
U3 n 3 15.5 15.8 15.3 78 9 1.1 15.2 15.1 15.1 78 7 2.0 22

a  This is the value of the overall reference mean, referred to as the “correction point” below.

Example: Table 14 summarizes the portion of Table 13 needed for this part of the calculation.

Table 14

Standard Set Corrected Zone Measurements (mm) Concentration (U/mL)

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Standard Set Corrected Zone Measurements (mm) Concentration (U/mL)

S1 14.022 3.2

S2 14.989 4.0

Reference (S3) 15.722 5

S4 16.511 6.25

S5 17.222 7.8125

LINEAR REGRESSION RESULTS

Standard curve line:

Z = [3.551 × ln(C)] + 9.978

Z = corrected zone measurement

C = concentration

%R2 = 99.7

L
SAMPLE POTENCY DETERMINATION
To estimate the potency of the unknown sample, average the zone measurements of the Standard and the zone measurements of
IA
the sample on the 3 plates used. Correct for plate-to-plate variation using the correction point determined above to obtain a
corrected average for the unknown, U. [NOTE—An acceptable alternative to using the correction point is to correct using the value on
the estimated regression line corresponding to the log concentration of S3.] Use the corrected average zone measurement in the
equation of the standard curve line to determine the log concentration of the sample, LU, by:
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LU = (U − a)/b

a = intercept of the regression line

b = slope of the regression line

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To obtain the potency of the unknown, take the antilog of LU and multiply the result by any applicable dilution factor. This value can
also be expressed as a percentage of the reference concentration value.
Example: Corrected sample zone measurement (see Table 13) = 15.522
Natural log of the sample concentration:
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LU = (15.522 − 9.978)/3.551 = 1.561

Sample concentration:

CU = e1.561 = 4.765
Percentage of reference concentration:

Result = (4.765/5.000) × 100 = 95.3%

Turbidimetric Assay
This section details analysis of the sample data and determination of the potency of an unknown using the turbidimetric assay. The
method assumes that the tubes are randomly distributed within the heat block or other temperature control device. If the device has a
temperature pro le that is not uniform, a randomized blocks design is preferred. In such a design, the rack is divided into areas
(“blocks”) of relatively uniform temperature and at least one tube of each Standard concentration and of each unknown is placed in
each area. The data analysis of a randomized block design is different from the following.

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SAMPLE DATA
Table 15 shows the data from one assay that will be used for an example throughout this section. Other columns are needed for
calculations and are explained below.

Table 15. Sample Data (Turbidimetric Assay)

Concentration Standard
Standard (µg/mL) Replicate Absorbance (a.u.) Average (a.u.) Deviation

1 0.8545

2 0.8422

S1 64 3 0.8495 0.8487 0.0062

1 0.8142

2 0.8273

S2 80 3 0.8392 0.8269 0.0125

1 0.6284

2 0.6947

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S3 100 3 0.7563 0.6931 0.0640

1 0.6933
IA
2 0.6850

S4 125 3 0.6699 0.6827 0.0119

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1 0.5299

2 0.5779

S5 156 3 0.5316 0.5465 0.0272

FF

1 0.7130

2 0.7960

U3 Unknown 3 0.7201 0.7430 0.0460

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Step 1: Perform initial calculations and variability suitability check.

For each concentration (including the sample), average the three absorbance values.
Example: See S1 in Table 15.

0.8487 = X(0.8545, 0.8422, 0.8495)

For each concentration, determine the standard deviation of the three readings and a combined standard deviation for all the
concentrations.
Example: See S1 in Table 15.

0.0062 = SD(0.8545, 0.8422, 0.8495)

The combined value is calculated by taking the square root of the average of the ve variances:

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0.0322 = {[(0.0062) + (0.0125) + (0.0640)2 + (0.0119)2 + (0.0272)2]/5}1/2

2 2

For a variability suitability criterion, each laboratory should determine a maximum acceptable combined standard deviation. If
the combined standard deviation exceeds this predetermined maximum, the assay data are not suitable and should be discarded.
[NOTE—The suggested limit for the combined standard deviation is NMT 10% of the average absorbance value across the ve
concentrations.] If the number of replicates per concentration is at least 5, then a relative standard deviation can be computed for
each concentration after checking for outliers and compared to a maximum acceptable relative standard deviation. [NOTE—The
suggested limit for the relative standard deviation is NMT 10%.]
Step 2: Determine the standard curve line.
Generate the standard curve line by plotting the average absorbance values versus the log of the standard concentration values.
Calculate the equation of the standard curve line by performing an unweighted linear regression on these values using appropriate
software or the manual calculations of Appendix 1: Formulas for Manual Calculations of Regression and Sample Concentration. [NOTE
—Use either the natural log or the base 10 log to plot the standard curve and determine the regression equation; both provide the
same nal test result.] Each laboratory should determine a minimum value of the percentage coe cient of determination (%R2) for
an acceptable regression. The regression is acceptable only if the %R2 value obtained exceeds this predetermined value. [NOTE—The
suggested limit for the percentage coe cient of determination is NLT 90%.]
Example: Table 16 summarizes the portion of Table 15 needed for this part of the calculation.

Table 16

Set of Standards Average Absorbance Values (a.u.) Concentration (µg/mL)

S1 0.8487 64

S2 0.8269 80

S3 0.6931

L 100
IA
S4 0.6827 125

S5 0.5465 156
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LINEAR REGRESSION RESULTS

Standard curve line:
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Absorbance = 2.2665 − [0.7735 × log10 (concentration)]

%R2 = 93.0%

SAMPLE POTENCY DETERMINATION

To estimate the potency of the unknown sample, average the three absorbance measurements to obtain an average for the
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unknown, U. Use this average measurement in the equation of the standard curve line to determine the log concentration of the
unknown sample, LU, by:

LU = (U − a)/b

a = intercept of the regression line

b = slope of the regression line

To obtain the potency of the unknown, take the antilog of LU and multiply the result by any applicable dilution factor. This value can
also be expressed as a percentage of the reference concentration value.
Example: Average sample absorbance (see Table 15) = 0.7430.

log10(CU) = (0.7430 − 2.2665)/(−0.7735) = 1.9696

CU = 101.9696 = 93.2

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Percentage of reference concentration = (93.2/100.0) × 100 = 93.2%

CU = concentration of the sample

Con dence Limits and Combination of Assays Calculations

Because of interassay variability, three or more independent determinations are required for a reliable estimate of the sample
potency. For each independent determination, start with separately prepared stock solutions and test dilutions of both the Standard
and the sample, and repeat the assay of a given sample ▲▲ (USP 1-Dec-2019).
Given a set of at least three determinations of the unknown potency, use the method of Appendix 2: Procedure for Checking for
Outliers; Rejection of Outlying or Aberrant Measurements to check for any outlier values. This determination should be done in the log
scale.
To obtain a combined estimate of the unknown potency, calculate the average, M, and the standard deviation of the accepted log
potencies. [NOTE—Use either the natural log or the base 10 log.] Determine the con dence interval for the potency as follows:

antilog[M − t(0.05, N − 1) × SD/√N], antilog[M + t(0.05, N − 1) × SD/√N]

M = average

t(0.05, N−1) = the two-sided 5% point of a Student’s t-distribution with N−1 degrees of freedom
[NOTE—The t value is available in spreadsheets, statistics texts, and statistics software.]

SD = standard deviation

N = number of assays

L
W = antilog{[t(0.05, N − 1) × SD/√N]}

W = half-width of the con dence interval

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Compare the half-width of the con dence interval to a predetermined maximum acceptable value. If the half-width is larger than the
acceptance limit, continue with additional assays.

EXAMPLE
Suppose the sample is assayed four times, with potency results in the natural log scale of 1.561, 1.444, 1.517, and 1.535. Then:
IC

N=4

M = X(1.561, 1.444, 1.517, 1.535) = 1.514

FF

SD = σ(1.561, 1.444, 1.517, 1.535) = 0.050

t = 3.182
The con dence interval in the log scale is

1.514 ± (3.182 × 0.050/√4) = (1.434, 1.594)

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Taking antilogs, the estimated potency is

e1.514 = 4.546
1.434 1.594
with a 95% con dence interval for the potency of e ,e = (4.197, 4.924).
The half-width of the con dence interval to compare to an acceptance value is the ratio 4.924/4.546 = 1.083.

Change to read:

APPENDICES
Appendix 1: Formulas for Manual Calculations of Regression and Sample Concentration
If the concentrations are equally spaced in the logarithmic scale, the calculations can be performed using the following formula.
Let:

Sk = mean corrected zone measurement (cylinder-plate assay) or average absorbance value (turbidimetric assay) for standard
set k

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k = 1, 2, 3, 4, 5

S = mean of the ve Sk values

Lk = logarithm of the kth concentration. [NOTE—Use either the natural log or the base 10 log. Slope of the regression line is
calculated by:]

b = (Yhigh − Ylow)/(Xhigh − Xlow)

Yhigh = ⅕(3S5 + 2S4 + S3 − S1)

Ylow = ⅕(3S1 + 2S2 + S3 − S5)

Xhigh = L5

Xlow = L1

Combine and simplify to:

b = (4S5 + 2S4 − 2S2 − 4S1)/[5(L5 − L1)]

The log of the concentration of the sample is found using:

LU = Lreference + [(U − S)/b]

For example, using the data for the cylinder-plate assay in Table 13 and natural logarithms:

b = [(4 × 17.222) + (2 × 16.511) − (2 × 14.989) − (4 × 14.020)]/{5[ln(7.81) − ln(3.2)]} = 3.551

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S = (14.020 + 14.989 + 15.722 + 16.511 + 17.222)/5 = 15.693

Natural log of sample concentration = ln(5) + [(15.522 − 15.693)/3.551] = 1.561

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Sample concentration = e1.561 = 4.765

Appendix 2: Procedure for Checking for Outliers—Rejection of Outlying or Aberrant Measurements

A measurement that is clearly questionable because of a failure in the assay procedure should be rejected, whether it is discovered
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during the measuring or tabulation procedure. The arbitrary rejection or retention of an apparently aberrant measurement can be a
serious source of bias. In general, the rejection of measurements solely on the basis of their relative magnitudes is a procedure that
should be used sparingly.
Each suspected potency measurement, or outlier, may be tested against the following criterion. This criterion is based on the
variation within a single group of supposedly equivalent measurements from a normal distribution. ▲At a con dence level of 99%, a
FF

valid observation will be rejected once in 100 trials (when the suspected outlier can occur at only one end) or once in 50 trials (when
the suspected outlier can occur at either end), provided that relatively few, if any, responses within the group are identical. Arrange the
responses in order of magnitude from y1 to yN, where N is the number of observations in the group.▲ (USP 1-Dec-2019) Compute the
relative gap by using Table A2-1, and the formulas below:
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When N = 3–7:

G1 = (y2 − y1)/(yN − y1) ▲Candidate Outlier is Smallest (y1)

G1 = (yN − yN−1)/(yN − y1) Candidate Outlier is Largest (yN)▲ (USP 1-Dec-2019)

When N = 8–10:

G2 = (y2 − y1)/(yN − 1 − y1) ▲Candidate Outlier is Smallest (y1)

G2 = (yN − yN−1)/(yN − y2) Candidate Outlier is Largest (yN)▲ (USP 1-Dec-2019)

When N = 11–13:

G3 = (y3 − y1)/(yN −1 − y1) ▲Candidate Outlier is Smallest (y1)

G3 = (yN − yN−2)/(yN − y2) Candidate Outlier is Largest (yN)▲ (USP 1-Dec-2019)

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If G1, G2, or G3, as appropriate, exceeds the critical value in Table A2-1, for the observed N, there is a statistical basis ▲for identifying
the discordant measurement as an outlier and considering its removal.▲ (USP 1-Dec-2019)

Table A2-1. Test for Outlier Measurements

In samples from a normal population, gaps equal to or larger than the following values of G1, G2, and G3 occur with a probability P =
0.01, when outlier measurements can occur only at one end; or with P = 0.02, when they may occur at either end.

N 3 4 5 6 7

▲0.988 ▲0.780
▲ (USP 1- ▲ (USP 1-
G1 Dec-2019) 0.889 Dec-2019) 0.698 0.637

N 8 9 10 — —

0.635▲ (USP 1-Dec- — —

G2 ▲0.683 2019) 0.597

N 11 12 13 — —

0.615▲ (USP 1-Dec- — —

G3 ▲0.679 0.642 2019)

L
EXAMPLE
Estimated potencies of sample in log scale = 1.561, 1.444, 1.517, 1.535.
IA
Check the lowest potency for outlier:

G1 = (1.517 − 1.444)/(1.561 − 1.444) = 0.624 < 0.889

Therefore, 1.444 is not an outlier.
Check the highest potency for outlier:
IC

G1 = (1.561 − 1.535)/(1.561 − 1.444) = 0.222 < 0.889

Therefore, 1.561 is not an outlier.
Outlier potencies should be marked as outlier values and excluded from the assay calculations. NMT 1 potency can be excluded
as an outlier.
FF

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<81> ANTIBIOTICS--MICROBIAL ASSAYS Ying Han BIO42015 Biologics Monographs 4 -

Associate Science & Standards Liaison Antibiotics

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