4/23/24, 3:08 PM Appendix XIV A.

Microbiological Assay of Antibiotics - British Pharmacopoeia

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Appendix XIV A. Microbiological Assay of Antibiotics

(Ph. Eur. method 2.7.2)

The potency of an antibiotic is estimated by comparing the inhibition of growth of sensitive micro-
organisms produced by known concentrations of the antibiotic to be examined and a reference
substance.

The reference substances used in the assays are substances whose activity has been precisely
determined with reference to the corresponding international standard or international reference
preparation.

The assay must be designed in a way that will permit examination of the validity of the mathematical
model on which the potency equation is based. If a parallel-line model is chosen, the 2 log dose-
response (or transformed response) lines of the preparation to be examined and the reference
preparation must be parallel; they must be linear over the range of doses used in the calculation.
These conditions must be verified by validity tests for a given probability, usually P = 0.05. Other
mathematical models, such as the slope ratio model, may be used provided that proof of validity is
demonstrated.

Unless otherwise stated in the monograph, the confidence limits (P = 0.95) of the assay for potency
are not less than 95 per cent and not more than 105 per cent of the estimated potency.

Carry out the assay by method A or method B.

A. DIFFUSION METHOD

Liquefy a medium suitable for the conditions of the assay and inoculate it at a suitable temperature,
for example 48 °C to 50 °C for vegetative forms, with a known quantity of a suspension of micro-
organisms sensitive to the antibiotic to be examined, such that clearly defined zones of inhibition of
suitable diameter are produced with the concentrations of the antibiotic used for the assay.
Immediately pour into Petri dishes or large rectangular dishes a quantity of the inoculated medium to
form a uniform layer 2-5 mm thick. Alternatively, the medium may consist of 2 layers, only the upper
layer being inoculated.

Store the dishes so that no appreciable growth or death of the micro-organisms occurs before the
dishes are used and so that the surface of the medium is dry at the time of use.
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Using the solvent and the buffer solution indicated in Table 2.7.2.-1, prepare solutions of the
reference substance and of the antibiotic to be examined having known concentrations and
presumed to be of equal activity. Apply the solutions to the surface of the medium, for example, in
sterile cylinders of porcelain, stainless steel or other suitable material, or in cavities prepared in the
agar. The same volume of solution must be added to each cylinder or cavity. Alternatively, use sterile
absorbent paper discs of suitable quality; impregnate the discs with the solutions of the reference
substance or the solutions of the antibiotic to be examined and place on the surface of the agar.

In order to assess the validity of the assay, use not fewer than 3 doses of the reference substance
and 3 doses of the antibiotic to be examined having the same presumed activity as the doses of the
reference substance. It is preferable to use a series of doses in geometric progression. In routine
assays when the linearity of the system has been demonstrated over an adequate number of
experiments using a three-point assay, a two-point assay may be sufficient, subject to agreement by
the competent authority. However, in all cases of dispute, a three-point assay as described above
must be applied.

Arrange the solutions on each Petri dish or on each rectangular dish according to a statistically
suitable design, except for small Petri dishes that cannot accommodate more than 6 solutions,
arrange the solutions of the antibiotic to be examined and the solutions of the reference substance
in an alternate manner to avoid interaction of the more concentrated solutions.

Incubate at a suitable temperature for about 18 h. A period of diffusion prior to incubation, usually 1-
4 h, at room temperature or at about 4 °C, as appropriate, may be used to minimise the effects of
the variation in time between the application of the solutions and to improve the regression slope.

Measure the diameters to the nearest 0.1 mm or the areas of the circular inhibition zones to the
nearest 0.01 and calculate the potency using appropriate statistical methods.

Use in each assay the number of replications per dose sufficient to ensure the required accuracy
and precision. The assay may be repeated and the results combined statistically to obtain the
required accuracy and precision and to ascertain whether the potency of the antibiotic to be
examined is not less than the minimum required.

B. TURBIDIMETRIC METHOD

Inoculate a suitable medium with a suspension of the chosen micro-organism having a sensitivity to
the antibiotic to be examined such that a sufficiently large inhibition of microbial growth occurs in the
conditions of the test. Use a known quantity of the suspension chosen so as to obtain a readily
measurable opacity after an incubation period of about 4 h.

Use the inoculated medium immediately after its preparation.

Using the solvent and the buffer solution indicated in Table 2.7.2.-2 prepare solutions of the
reference substance and of the antibiotic to be examined having known concentrations presumed to
be of equal activity.

In order that the validity of the assay may be assessed, use not fewer than 3 doses of the reference
substance and 3 doses of the antibiotic to be examined having the same presumed activity as the
doses of the reference substance. It is preferable to use a series of doses in geometric progression.
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In order to obtain the required linearity, it may be necessary to select from a large number
3 consecutive doses, using corresponding doses for the reference substance and the antibiotic to be
examined.

Distribute an equal volume of each of the solutions into identical test-tubes and add to each tube an
equal volume of inoculated medium (for example, 1 mL of the solution and 9 mL of the medium). For
the assay of tyrothricin add 0.1 mL of the solution to 9.9 mL of inoculated medium.

Prepare at the same time 2 control tubes without antibiotic, both containing the inoculated medium
and to one of which is added immediately 0.5 mL of formaldehyde R. These tubes are used to set
the optical apparatus used to measure the growth.

Place all the tubes, randomly distributed or in a Latin square or randomised block arrangement, in a
water-bath or other suitable apparatus fitted with a means of bringing all the tubes rapidly to the
appropriate incubation temperature and maintain them at that temperature for 3-4 h, taking
precautions to ensure uniformity of temperature and identical incubation time.

After incubation, stop the growth of the micro-organisms by adding 0.5 mL of formaldehyde R to
each tube or by heat treatment and measure the opacity to 3 significant figures using suitable optical
apparatus. Alternatively use a method which allows the opacity of each tube to be measured after
exactly the same period of incubation.

Table 2.7.2.-1. – Diffusion assay

Solvent to be used
Reference Micro-
Antibiotic in preparing the Buffer solution (pH)
substance organism
stock solution

Saccharomyces
Amphotericin B for
Amphotericin cerevisiae
microbiological Dimethyl sulfoxide R pH 10.5 (0.2 M)
B ATCC 9763
assay CRS
IP 1432-83
Micrococcus
luteus
Bacitracin Bacitracin 0.01 M hydrochloric
pH 7.0 (0.05 M) NCTC 7743
zinc zinc CRS acid
CIP 53.160
ATCC 10240
Mycobacterium
Bleomycin Bleomycin
Water R pH 6.8 (0.1 M) smegmatis
sulfate sulfate CRS
ATCC 607
Colistimethate Colistimethate Water R pH 6.0 (0.05 M) Bordetella
sodium sodium CRS bronchiseptica
NCTC 8344
CIP 53.157
ATCC 4617

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Solvent to be used
Reference Micro-
Antibiotic in preparing the Buffer solution (pH)
substance organism
stock solution

Escherichia coli
NCIMB 8879
CIP 54.127
ATCC 10536
Bordetella
bronchiseptica
NCTC 8344
Colistin sulfate for CIP 53.157
Colistin ATCC 4617
microbiological Water R pH 6.0 (0.05 M)
sulfate
assay CRS Escherichia coli
NCIMB 8879
CIP 54.127
ATCC 10536
Bacillus subtilis
NCTC 10400
CIP 52.62
Framycetin Framycetin ATCC 6633
Water R pH 8.0 (0.05 M)
sulfate sulfate CRS Bacillus
pumilus NCTC
8241
CIP 76.18
Bacillus
pumilus NCTC
8241
CIP 76.18
Gentamicin Gentamicin
Water R pH 8.0 (0.05 M) Staphylococcus
sulfate sulfate CRS
epidermidis
NCIMB 8853
CIP 68.21
ATCC 12228
Bacillus subtilis
Methanol R (see the CIP 52.62
Josamycin Josamycin CRS pH 5.6
monograph) ATCC 6633
NCTC 10400
Bacillus subtilis
Josamycin Josamycin Methanol R (see the CIP 52.62
pH 5.6
propionate propionate CRS monograph) ATCC 6633
NCTC 10400

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Solvent to be used
Reference Micro-
Antibiotic in preparing the Buffer solution (pH)
substance organism
stock solution

Bacillus subtilis
Kanamycin NCTC 10400
monosulfate CIP 52.62
ATCC 6633
Kanamycin
Water R pH 8.0 (0.05 M) Staphylococcus
monosulfate CRS
aureus
Kanamycin
NCTC 7447
acid sulfate
CIP 53.156
ATCC 6538 P
Bacillus
pumilus
NCTC 8241
Neomycin sulfate CIP 76.18
Neomycin
for microbiological Water R pH 8.0 (0.05 M)
sulfate Bacillus subtilis
assay CRS
NCTC 10400
CIP 52.62
ATCC 6633
Staphylococcus
Netilmicin Netilmicin aureus
Water R pH 8.0 ± 0.1
sulfate sulfate CRS ATCC 6538 P
CIP 53.156
Candida
tropicalis
CIP 1433-83
pH 6.0 (0.05 M) NCYC 1393
containing 5 per
Nystatin Nystatin CRS Dimethylformamide R Saccharomyces
cent V/V of
dimethylformamide R cerevisiae
NCYC 87
CIP 1432-83
ATCC 9763
Bordetella
Polymyxin B
bronchiseptica
Polymyxin B sulfate for
Water R pH 6.0 (0.05 M) NCTC 8344
sulfate microbiological
CIP 53.157
assay CRS
ATCC 4617
Rifamycin Rifamycin Methanol R pH 7.0 (0.05 M) Micrococcus
sodium sodium CRS luteus
NCTC 8340

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Solvent to be used
Reference Micro-
Antibiotic in preparing the Buffer solution (pH)
substance organism
stock solution

CIP 53.45
ATCC 9341
Bacillus subtilis
NCTC 10400
Spiramycin Spiramycin CRS Methanol R pH 8.0 (0.05 M)
CIP 52.62
ATCC 6633
Bacillus subtilis
NCTC 8236
CIP 1.83
Streptomycin Streptomycin
Water R pH 8.0 (0.05 M) Bacillus subtilis
sulfate sulfate CRS
NCTC 10400
CIP 52.62
ATCC 6633
Bacillus subtilis
NCTC 10400
Teicoplanin Teicoplanin CRS pH 6.0 (0.05 M) pH 6.0 (0.05 M)
CIP 52.62
ATCC 6633
Tylosin for
veterinary use A mixture of
2.5 per cent V/V Micrococcus
Tylosin 40 volumes of
solution of luteus
phosphate for methanol R and
Tylosin CRS methanol R in 0.1 M NCTC 8340
veterinary use 60 volumes of 0.1 M
phosphate buffer CIP 53.45
Tylosin phosphate buffer
solution pH 7.0 R ATCC 9341
tartrate for solution pH 8.0 R
veterinary use
Bacillus subtilis
Vancomycin Vancomycin NCTC 10400
Water R pH 8.0
hydrochloride hydrochloride CRS CIP 52.62
ATCC 6633

Calculate the potency using appropriate statistical methods.

Linearity of the dose-response relationship, transformed or untransformed, is often obtained only

over a very limited range. It is this range which must be used in calculating the activity and it must
include at least 3 consecutive doses in order to permit linearity to be verified. In routine assays when
the linearity of the system has been demonstrated over an adequate number of experiments using a
three-point assay, a two-point assay may be sufficient, subject to agreement by the competent
authority. However, in all cases of dispute, a three-point assay must be applied.

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Use in each assay the number of replications per dose sufficient to ensure the required accuracy
and precision. The assay may be repeated and the results combined statistically to obtain the
required accuracy and precision and to ascertain whether the potency of the antibiotic to be
examined is not less than the minimum required.

Table 2.7.2.-2. – Turbidimetric assay

Solvent to Medium
be used in Buffer and
Reference Micro- Incubation
Antibiotic preparing solution final pH
substance organism temperatu
the stock (pH) (± 0.1
solution pH unit)
Escherichia coli
Colistimethate Colistimethate NCIMB 8666 C-
Water R pH 7.0 35-37 °C
sodium sodium CRS CIP 2.83 pH 7.0
ATCC 9637
Escherichia coli
Colistin sulfate for
Colistin NCIMB 8666 C-
microbiological Water R pH 7.0 35-37 °C
sulfate CIP 2.83 pH 7.0
assay CRS
ATCC 9637
Staphylococcus
aureus
Framycetin Framycetin C-
Water R pH 8.0 NCTC 7447 35-37 °C
sulfate sulfate CRS pH 7.0
CIP 53.156
ATCC 6538 P
Staphylococcus
aureus
Gentamicin Gentamicin C-
Water R pH 7.0 NCTC 7447 35-37 °C
sulfate sulfate CRS pH 7.0
CIP 53.156
ATCC 6538 P
Enterococcus
hirae
CIP 58.55
C-
Gramicidin CRS Methanol R pH 7.0* ATCC 10541 35-37 °C
pH 7.0
Gramicidin Staphylococcus
aureus
ATCC 6538 P
*Addition of a detergent may be necessary to avoid adsorption on the material during
the dilutions, for example 0.1 mg/mL of polysorbate 80 R
Staphylococcus
Methanol R aureus
C-
Josamycin Josamycin CRS (see the pH 5.6 CIP 53.156 35-37 °C
pH 8.0
monograph) ATCC 6538 P
NCTC 7447

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Solvent to Medium
be used in Buffer and
Reference Micro- Incubation
Antibiotic preparing solution final pH
substance organism temperatu
the stock (pH) (± 0.1
solution pH unit)
Staphylococcus
Methanol R aureus
Josamycin Josamycin C-
(see the pH 5.6 CIP 53.156 35-37 °C
propionate propionate CRS pH 8.0
monograph) ATCC 6538 P
NCTC 7447
Kanamycin Staphylococcus
monosulfate aureus
Kanamycin C-
Kanamycin Water R pH 8.0 NCTC 7447 35-37 °C
monosulfate CRS pH 7.0
acid sulfate CIP 53.156
ATCC 6538 P
Staphylococcus
Neomycin sulfate aureus
Neomycin C-
for microbiological Water R pH 8.0 NCTC 7447 35-37 °C
sulfate pH 7.0
assay CRS CIP 53.156
ATCC 6538 P
Escherichia coli
Rifamycin Rifamycin NCIMB 8879 C-
Methanol R pH 7.0 35-37 °C
sodium sodium CRS CIP 54.127 pH 7.0
ATCC 10536
Staphylococcus
aureus
C-
Spiramycin Spiramycin CRS Methanol R pH 7.0 NCTC 7447 35-37 °C
pH 7.0
CIP 53.156
ATCC 6538 P
Klebsiella
pneumoniae
Streptomycin Streptomycin C-
Water R pH 8.0 NCTC 7427 35-37 °C
sulfate sulfate CRS pH 7.0
CIP 53.153
ATCC 10031
2.5 per
Tylosin for cent V/V
veterinary use solution of Staphylococcus
methanol R aureus
C-
Tylosin CRS in 0.1 M pH 7.0 NCTC 6571 37 °C
pH 7.0
Tylosin phosphate ATCC 9144
tartrate for buffer CIP 53.154
veterinary use solution
pH 7.0 R

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Solvent to Medium
be used in Buffer and
Reference Micro- Incubation
Antibiotic preparing solution final pH
substance organism temperatu
the stock (pH) (± 0.1
solution pH unit)
Enterococcus
C-
Tyrothricin Gramicidin CRS Alcohol R Alcohol R hirae 37 °C
pH 7.0
ATCC 10541
Staphylococcus
Vancomycin Vancomycin aureus C-
Water R pH 8.0 37-39 °C
hydrochloride hydrochloride CRS CIP 53.156 pH 7.0
ATCC 6538 P

The following section is published for information.

RECOMMENDED MICRO-ORGANISMS

The following text details the recommended micro-organisms and the conditions of use. Other
micro-organisms may be used provided that they are shown to be sensitive to the antibiotic to be
examined and are used in appropriate media and appropriate conditions of temperature and pH.
The concentrations of the solutions used should be chosen so as to ensure that a linear relationship
exists between the logarithm of the dose and the response in the conditions of the test.

Preparation of inocula

Bacillus cereus var. mycoides; Bacillus subtilis; Bacillus pumilus. Spore suspensions of the
organisms to be used as inocula are prepared as follows.

Grow the organism at 35-37 °C for 7 days on the surface of a suitable medium to which has been
added 0.001 g/L of manganese sulfate R. Using sterile water R, wash off the growth, which consists
mainly of spores. Heat the suspension at 70 °C for 30 min and dilute to give an appropriate
concentration of spores, usually 10 × 10 6 to 100 × 10 6 per millilitre. The spore suspensions may be
stored for long periods at a temperature not exceeding 4 °C.

Alternatively, spore suspensions may be prepared by cultivating the organisms in medium C at

26 °C for 4-6 days, then adding, aseptically, sufficient manganese sulfate R to give a concentration
of 0.001 g/L and incubating for a further 48 h. Examine the suspension microscopically to ensure
that adequate spore formation has taken place (about 80 per cent) and centrifuge. Re-suspend the
sediment in sterile water R to give a concentration of 10 × 10 6 to 100 × 10 6 spores per millilitre, and
then heat to 70 °C for 30 min. Store the suspension at a temperature not exceeding 4 °C.

Bordetella bronchiseptica. Grow the test organism on medium B at 35-37 °C for 16-18 h. Wash off
the bacterial growth with sterile water R and dilute to a suitable opacity.

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Staphylococcus aureus; Klebsiella pneumoniae; Escherichia coli; Micrococcus luteus;

Staphylococcus epidermidis. Prepare as described above for B. bronchiseptica but using medium A
and adjusting the opacity to one which has been shown to produce a satisfactory dose-response
relationship in the turbidimetric assay, or to produce clearly defined zones of inhibition of convenient
diameter in the diffusion assay, as appropriate.

Saccharomyces cerevisiae; Candida tropicalis. Grow the test organism on medium F at 30-37 °C for
24 h. Wash off the growth with a sterile 9 g/L solution of sodium chloride R. Dilute to a suitable
opacity with the same solution.

Buffer solutions

Buffer solutions having a pH between 5.8 and 8.0 are prepared by mixing 50.0 mL of 0.2 M
potassium dihydrogen phosphate R with the quantity of 0.2 M sodium hydroxide indicated in
Table 2.7.2.-3. Dilute with freshly prepared distilled water R to produce 200.0 mL.

Table 2.7.2.-3

pH 0.2 M Sodium hydroxide (mL)

5.8 3.72
6.0 5.70
6.2 8.60
6.4 12.60
6.6 17.80
6.8 23.65
7.0 29.63
7.2 35.00
7.4 39.50
7.6 42.80
7.8 45.20
8.0 46.80

These buffer solutions are used for all microbiological assays shown in Table 2.7.2.-1 with the
exception of bleomycin sulfate and amphotericin B.

For bleomycin sulfate, prepare the buffer solution pH 6.8 as follows: dissolve 6.4 g of potassium
dihydrogen phosphate R and 18.9 g of disodium hydrogen phosphate dodecahydrate R in water R
and dilute to 1000 mL with water R.

For amphotericin B, prepare the 0.2 M phosphate buffer solution pH 10.5 as follows: dissolve 35 g of
dipotassium hydrogen phosphate R in 900 mL of water R, add 20 mL of 1 M sodium hydroxide and
dilute to 1000.0 mL with water R.

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Culture media

The following media or equivalent media may be used.

Medium A

Peptone 6g
Pancreatic digest of casein 4g
Beef extract 1.5 g
Yeast extract 3g
Glucose monohydrate 1g
Agar 15 g
Water to 1000 mL

Medium B

Pancreatic digest of casein 17 g

Papaic digest of soya bean 3g
Sodium chloride 5g
Dipotassium hydrogen phosphate 2.5 g
Glucose monohydrate 2.5 g
Agar 15 g
Polysorbate 80 10 g
Water to 1000 mL

The polysorbate 80 is added to the hot solution of the other ingredients after boiling, and
immediately before adjusting to volume.

Medium C

Peptone 6g
Beef extract 1.5 g
Yeast extract 3g
Sodium chloride 3.5 g
Glucose monohydrate 1g
Dipotassium hydrogen phosphate 3.68 g
Potassium dihydrogen phosphate 1.32 g
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Water to 1000 mL

Medium D

Heart extract 1.5 g

Yeast extract 1.5 g
Peptone-casein 5g
Glucose monohydrate 1g
Sodium chloride 3.5 g
Dipotassium hydrogen phosphate 3.68 g
Potassium dihydrogen phosphate 1.32 g
Potassium nitrate 2g
Water to 1000 mL

Medium E

Peptone 5g
Meat extract 3g
Disodium hydrogen phosphate,12H2O 26.9 g

Agar 10 g
Water to 1000 mL

The disodium hydrogen phosphate is added as a sterile solution after sterilisation of the medium.

Medium F

Peptone 9.4 g
Yeast extract 4.7 g
Beef extract 2.4 g
Sodium chloride 10.0 g
Glucose monohydrate 10.0 g
Agar 23.5 g
Water to 1000 mL

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Medium G

Glycerol 10 g
Peptone 10 g
Meat extract 10 g
Sodium chloride 3g
Agar 15 g
Water to 1000 mL

pH 7.0 ± 0.1 after sterilisation.

Medium H

Peptone 5.0 g
Agar 15.0 g
Beef extract powder 3.0 g
Water to 1000 mL

pH 7.8 - 8.0 adjusted with 0.1 M sodium hydroxide.

Monographs of the British Pharmacopoeia

The following additional information and guidance apply to monographs of the British
Pharmacopoeia.

The required minimum precision for an acceptable assay of any particular antibiotic or preparation is
defined in the appropriate monograph in the paragraph on the Assay. This degree of precision is the
minimum acceptable for determining that the final product complies with the official requirements. It
may be inadequate for a decision about the potency that should be stated on the label or used as
the basis for calculating the quantity of an antibiotic to be incorporated in a preparation. In such
circumstances, assays of greater precision may be desirable with, for instance, fiducial limits of error
of the order of 98 to 102%. With this degree of precision, the lower fiducial limit lies close to the
estimated potency. By using this limit, instead of the estimated potency, to assign a potency to the
antibiotic either for labelling or for calculating the quantity to be included in a preparation, there is
less likelihood of the final preparation subsequently failing to comply with the official requirements
for potency.

Culture Media The following media or equivalent media may be used.

Medium I
D-Glucose 10 g
Tryptone 6g
Yeast extract* 2g

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Water to produce 1000 mL

Adjust to pH 8.0 with 1M sodium hydroxide or 0.1M orthophosphoric acid.

(*Ardamine yeast extract supplied by Champlain Industries Inc., Clifton, NJ 07012, USA is suitable.)

Method A. Diffusion Method

Table 2.7.2.-1 –Diffusion Assay; Additional Section for Monographs of the British Pharmacopoeia

Method B. Turbidimetric Method

Table 2.7.2.-2 –Turbidimetric Assay; Additional Section for Monographs of the British
Pharmacopoeia

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* Solution prepared by dissolving 16.73 g of dipotassium hydrogen orthophosphate and 0.523 g of

potassium dihydrogen orthophosphate in about 750 ml of water, if necessary adjusting to pH 8.0
with 0.1M sodium hydroxide or 0.1M orthophosphoric acid, and diluting to 1000 ml with water.

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