88

Microbial Limit Test / General Tests

temperature variation greater than 0.29

C between the two
successive temperature readings or rabbits having an initial
temperature higher than 39.89C are withdrawn from the test.
Warm the test solution to a temperature of 3729C before
injection, and inject the solution slowly into the marginal
vein of the ear of each rabbit over a period not exceeding 10
min. Hypotonic test sample may be made isotonic by the addition of pyrogen-free sodium chloride. Record the temperature of each rabbit during a period of 3 hours after the injection, taking the measurements at intervals of not more than
30 min. The dierence between the control temperature and
the maximum temperature of each rabbit is taken to be the
rise in body temperature. Consider any temperature
decreases as zero rise.
Interpretation of results
The test is carried out on a group of three rabbits and the
result is judged on the basis of the sum of the three temperature rises. Repeat if necessary on further groups of three rabbits to a total of three groups, depending on the results
obtained. If the summed response of the rst group does not
exceed 1.39
C, the sample is judged to be pyrogen-negative. If
the summed response exceeds 2.59
C, the sample is judged to
be pyrogen-positive. If the summed response exceed 1.39C
C, repeat the test on another group
but does not exceed 2.59
of three rabbits. If the summed response of the rst and second group does not exceed 3.09C, the sample is judged to be
pyrogen-negative. If the summed response of the 6 rabbits exceeds 4.29
C, the sample is judged to be pyrogen-positive. If
the summed response exceeds 3.09
C but does not exceed 4.29
C, repeat the test on one more group of three rabbits. If the
summed response of the 9 rabbits does not exceed 5.09
C, the
sample is judged to be pyrogen-negative. If the summed
response exceeds 5.09
C, the sample is judged to be pyrogenpositive.
When the test sample is judged to be pyrogen-negative, the
sample passes the pyrogen test.

4.05 Microbial Limit Test

This chapter provides tests for the qualitative and quantitative estimation of viable microorganisms present in pharmaceutical articles. It includes tests for total viable count
(bacteria and fungi) and specied microbial species (Escherichia coli, Salmonella, Pseudomonas aeruginosa and
Staphylococcus aureus). Microbial limit test must be carried
out under conditions designed to avoid accidental microbial
contamination of the preparation during the test. When test
specimens have antimicrobial activity, or contain antimicrobial substances, any such antimicrobial properties
must be eliminated by means of procedures such as dilution,
ltration, neutralization or inactivation. For the test, use a
mixture of several portions selected at random from the bulk
or from the contents of a sucient number of containers. If
test specimens are diluted with uid medium, the test should
be performed quickly. In performing the test, precautions
must be taken to prevent biohazard.
1.

Total viable aerobic count

This test determines the mesophilic bacteria and fungi
which grow under aerobic conditions. Psychrophilic, thermophilic, basophilic and anaerobic bacteria, and microor-

JP XV
ganisms which require specic ingredients for growth, may
give a negative result, even if a signicant number exists in
the test specimens. The test may be carried out using one of
the following 4 methods, i.e., membrane ltration method,
pour plate method, spread plate method or serial dilution
method (most probable number method). Use an appropriate
method depending on the purpose. An automated method
may be used for the test presented here, provided it has been
properly validated as giving equivalent or better results.
Dierent culture media and temperature are required for the
growth of bacteria and fungi (molds and yeasts). Serial
dilution method is applicable only to the enumeration of bacteria.
Preparation of the test solution
Phosphate Buer (pH 7.2), Buered Sodium ChloridePeptone Solution or uid medium used for the test is used to
dissolve or dilute the test specimen. Unless otherwise specied, 10 g or 10 mL of the test specimen is used for the test,
but another quantity or volume may be used according to the
nature of the test specimen. The pH of the solution is adjusted to between 6 and 8. The test solution must be used within
an hour after preparation.
Fluid specimens or soluble solids: Take 10 g or 10 mL of
the test specimen, mix with the buer or uid medium specied to make 100 mL, and use this as the test uid. A uid
specimen containing insoluble materials must be shaken well,
just prior to mixing, to eect ne suspension.
Insoluble solids: Take 10 g or 10 mL of the test specimen,
reduce the substance to a ne powder, suspend it in the buer
or uid medium specied to make 100 mL, and use this as the
test uid. A larger volume of the buer or uid medium than
indicated may be used for the suspension, depending on the
nature of the test specimen. The suspension may be dispersed
well using, if necessary, a mechanical blender. A suitable surface-active agent (such as 0.1 wW
vz polysorbate 80) may be
added to aid dissolution.
Fatty products: For water-immiscible uids, ointments,
creams, waxes, and lotions which consist mainly of lipid,
take 10 g or 10 mL of the test specimen, emulsify it in the
buer or uid medium specied with the aid of a suitable surface-active agent such as polysorbate 20 or 80 to make 100
mL, and use this as the test uid. An emulsion may be made
by warming to a temperature not exceeding 459
C, but do not
maintain this temperature for more than 30 minutes.
Test procedures
(1) Membrane Filtration Method
This method is applicable especially to specimens which
contain antimicrobial substances. Use membrane lters of
appropriate materials, having a normal pore size not greater
than 0.45 mm. Filter discs about 50 mm in diameter are
recommended, but lters of a dierent diameter may also be
used. Filters, the ltration apparatus, media, etc., should be
sterilized well. Usually, take 20 mL of the test uid (containing 2 g of test specimen), transfer 10 mL of the solution to
each of two membrane lters, and lter. If necessary, dilute
the pretreated preparation so that a colony count of 10 to 100
may be expected. After the ltration of the test uid, wash
each membrane by ltering through it three or more times
with a suitable liquid such as Buered Sodium Chloride-Peptone Solution, Phosphate Buer, or the uid medium to be
used. The volume of the washings to be used is approximately
100 mL each, but in case lter disc is signicantly dierent

JP XV
from 50 mm in diameter, the volume may be adjusted according to the size of the lter. For fatty substances, the washings
may contain a suitable surface-active agent such as polysorbate 80. Put one of the membrane lters, intended primarily
for the enumeration of bacteria, on the surface of a plate of
Soybean-Casein Digest Agar and the other, intended primarily for the enumeration of fungi, on the surface of a plate of
one of Sabouraud Glucose Agar, Potato Dextrose Agar, or
GP Agar Medium (each contains antibiotics). After incubation of the plates at least for 5 days, between 309
C and 359
C
in the test for the detection of bacteria and between 209C and
259C in the test for fungi, count the number of colonies that
are formed. If a reliable count is obtained in a shorter incubation time than 5 days, this may be adopted.
(2) Pour Plate Method
Use petri dishes 9 to 10 cm in diameter. Use at least two
petri dishes for each dilution. Pipet 1 mL of the test uid or
its diluted solution onto each petri dish aseptically. Promptly
add to each dish 15 to 20 mL of sterilized agar medium that
has previously been melted and kept below 459C, and mix.
Primarily for the detection of bacteria, use Soybean-Casein
Digest Agar Medium, and, primarily for the detection of
fungi, use one of Sabouraud Glucose Agar, Potato Dextrose
Agar, and GP Agar Medium (each contains antibiotics).
After the agar solidies, incubate the plates for at least 5
days, between 309
C and 359
C for bacteria and between 209C
and 259
C for fungi. If too many colonies are observed, dilute
the uid as described above so that a colony count of not
more than 300 per plate may be expected in the case of bacteria, and not more than 100 per plate in the case of fungi. If
a reliable count is obtained in a shorter incubation time than
5 days, this may be adopted.
(3) Spread Plate Method
On the solidied and dried surface of the agar medium,
pipet 0.05 to 0.2 mL of the test uid and spread it on the surface with a spreader. The diameter of petri dishes, the kind
and volume of the medium to be used, the temperature and
time of incubation, and the method for calculation of total
viable count are the same as described in the Pour Plate
Method section.
(4) Serial Dilution Method (Most Probable Number
Method)
Prepare a series of 12 tubes each containing 9 to 10 mL of
Fluid Soybean-Casein Digest Medium. Use three tubes for
each dilution. To each of the rst three tubes add 1 mL of the
test uid (containing 0.1 g or 0.1 mL of specimen), resulting
in 1 in 10 dilution. To the next three tubes add 1 mL of a 1 in
10 dilution of the uid, resulting in 1 in 100 dilution. To the
next three tubes add 1 mL of a 1 in 100 dilution of the uid,
resulting in 1 in 1000 dilution. To the last three tubes add 1
mL of the diluent as a control. Incubate the tubes between
309C and 359
C for not less than 5 days. The control tubes
should show no microbial growth. If the reading of the
results is dicult or uncertain, transfer about 0.1 mL to a liquid or solid medium and read the results after a further
period of incubation between 309
C and 359
C for 24 to 72
hours. Determine the most probable number of microorganisms per g or mL of the specimen from Table 4.05-1.

General Tests / Microbial Limit Test

Table 4.05-1.

89

Most probable number of microorganisms

Number of tubes in which microbial

growth is observed for each
quantity of the specimen

Most probable
number of microorganisms per
g or per mL

0.1 g or
0.1 mL
per tube

0.01 g or
0.01 mL
per tube

1 mg or
1 mL
per tube

3
3
3
3

3
3
3
3

3
2
1
0

1100
1100
500
200

3
3
3
3

2
2
2
2

3
2
1
0

290
210
150
90

3
3
3
3

1
1
1
1

3
2
1
0

160
120
70
40

3
3
3
3

0
0
0
0

3
2
1
0

95
60
40
23

If, for the rst column (containing 0.1 g or 0.1 mL of

specimen), the number of tubes showing microbial growth is
two or less, the most probable number of microorganisms per
g or per mL is likely to be less than 100.
Eectiveness of culture media and conrmation of antimicrobial substances
Use microorganisms of the following strains or their
equivalent. Grow them in Fluid Soybean-Casein Digest Medium between 309
C and 359
C for bacteria and between 209C
and 259C for Candida albicans.
Escherichia coli, such as ATCC 8739, NCIMB 8545, NBRC
3972
Bacillus subtilis, such as ATCC 6633, NCIMB 8054, NBRC
3134, JCM 2499
Staphylococcus aureus, such as ATCC 6538, NCIMB 8625,
NBRC 13276
Candida albicans, such as ATCC 2091, ATCC 10231, NBRC
1594, JCM 2085
Dilute portions of each of the cultures using Buered Sodium Chloride-Peptone Solution, or Phosphate Buer to prepare test suspensions containing about 50 to 200 CFU per
mL. Growth-promoting qualities are tested by inoculating 1
mL of each microorganism into each medium. The test media
are satisfactory if clear evidence of growth appears in all inoculated media after incubation at indicated temperature for
5 days. When a count of the test organisms with a test specimen diers by more than a factor of 5 from that without the
test specimen, any such eect must be eliminated by dilution,
ltration, neutralization or inactivation. To conrm the
sterility of the medium and of the diluent and the aseptic performance of the test, carry out the total viable count method
using sterile Buered Sodium Chloride-Peptone Solution or
Phosphate Buer as the control.
2.

Test for the detection of specied microorganisms

Escherichia coli, Salmonella, Pseudomonas aeruginosa
and Staphylococcus aureus are included as the target strains
of the test. These four species of microorganisms are im-

90

Microbial Limit Test / General Tests

portant for the evaluation of microbial contamination not

only in the nished products, but also in the bulk or intermediate of the production process, and are representative of
the microorganisms which should not exist in these materials.
Preparation of the test uid
If necessary, refer to the paragraph on Preparation of the
Test Solution in Total viable aerobic count. When test specimens are dissolved in or diluted with a uid medium, use the
medium designated in each test, unless otherwise specied.
Test procedure
(1) Escherichia coli
To 10 g or 10 mL of the test specimen add a volume of
Fluid Lactose Medium to make 100 mL, and incubate beC and 359
C for 24 to 72 hours. Examine the meditween 309
um for growth, and if growth is present, mix by gentle shaking, take a portion by means of an inoculating loop, and
streak it on the surface of MacConkey Agar Medium. IncuC and 359C for 18 to 24 hours. If brick-red
bate between 309
colonies of Gram-negative rods surrounded by a reddish
precipitation zone are not found, the specimen meets the requirements of the test for absence of Escherichia coli. If colonies matching the above description are found, transfer the
suspect colonies individually to the surface of EMB Agar
Medium, and incubate between 309
C and 359C for 18 to 24
hours. Upon examination, if none of the colonies exhibits
both a characteristic metallic sheen and a blue-black appearance under transmitted light, the specimen meets the requirements of the test for the absence of Escherichia coli. Conrm
any suspect colonies on the plate by means of the IMViC test
(Indole production test, Methyl red reaction test, and VogesProskauer test, and Citrate utilization test), and the colonies
which exhibit the pattern of either [] or []
are judged as Escherichia coli. Rapid detection kits for Escherichia coli may also be used.
(2) Salmonella
As in the case of the detection of Escherichia coli, to 10 g
or 10 mL of the test specimen add a volume of Fluid Lactose
Medium to make 100 mL, and incubate between 309C and
359C for 24 to 72 hours. Examine the medium for growth,
and if growth is present, mix by gentle shaking, pipet 1 mL
portions, respectively, into 10 mL of Fluid Selenite-Cystine
Medium and Fluid Tetrathionate Medium, and incubate for
12 to 24 hours. 10 mL of Fluid Selenite-Cystine Medium may
be replaced by the same volume of Fluid Rappaport Medium.
After the incubation, streak portions from both the uid media on the surface of at least two of Brilliant Green Agar
Medium, XLD Agar Medium, and Bismuth Sulte Agar
Medium, and incubate between 309
C and 359C for 24 to 48
hours. Upon examination, if none of the colonies conforms
to the description given in Table 4.05-2, the specimen meets
the requirements of the test for the absence of the genus
Salmonella. If colonies of Gram-negative rods matching the
description in Table 4.05-2 are found, transfer suspect colonies individually, by means of an inoculating wire, to a slant
of TSI Agar Medium using both surface and deep inoculation. Incubate between 359C and 379
C for 18 to 24 hours.
The presence of genus Salmonella is conrmed if, in the deep
culture but not in the surface culture, there is a change of
color from red to yellow and usually a formation of gas with
or without production of hydrogen sulde. Precise identication and typing of genus Salmonella may be carried out by
using appropriate biochemical and serological tests addition-

JP XV
ally including an identication kit.
Table 4.05-2. Morphologic characteristics of Salmonella
species on selective agar media
Medium

Description of colony

Brilliant Green Agar

Medium

Small, transparent and colorless, or

opaque, pink or white (often surrounded by a pink to red zone)

XLD Agar Medium

Red, with or without black centers

Bismuth Sulte Agar

Medium

Black or green

(3) Pseudomonas aeruginosa

To 10 g or 10 mL of the specimen add Fluid SoybeanCasein Digest Medium, or another suitable uid medium
without antimicrobial activity, to make 100 mL. Fluid Lactose Medium is not suitable. Incubate between 309C and
359C for 24 to 48 hours. If, upon examination, growth is
present, use an inoculating loop to streak a portion of the
medium on the surface of Cetrimide Agar Medium or NAC
Agar Medium and incubate between 309
C and 359
C for 24 to
48 hours. If no growth of microorganisms is detected, the
specimen passes the test. If growth of colonies of Gram-negative rods with a greenish uorescence occurs, streak suspect
colonies from the agar surface of Cetrimide Agar Medium on
the agar surfaces of Pseudomonas Agar Medium for Detection of Fluorescein and Pseudomonas Agar Medium for Detection of Pyocyanin, and incubate between 309
C and 359C
for 24 to 72 hours. The production of yellowish uorescence
on the surface of the former medium shows the production of
uorescein, and the production of blue uorescence on the
latter medium indicates the production of pyocyanin. Conrm any suspect colonies as Pseudomonas aeruginosa by
means of the Oxidase test. Transfer each of the suspect colonies to lter paper that has previously been impregnated with
N, N-dimethyl-p-phenylenediammonium dichloride. If the
colony changes to purple within 5 to 10 seconds, the oxidase
test is judged as positive. If the oxidase test is judged to be
negative, the specimen meets the requirements of the test for
the absence of Pseudomonas aeruginosa. The presence of
Pseudomonas aeruginosa may also be conrmed by using appropriate biochemical tests including an identication kit.
(4) Staphylococcus aureus
To 10 g or 10 mL of the specimen add Fluid SoybeanCasein Digest Medium, or another suitable uid medium
without antimicrobial activity to make 100 mL. Incubate the
uid containing the specimen between 309
C and 359C for 24
to 48 hours. If, upon examination, growth is present, use an
inoculating loop to streak a portion of the medium on the
surface of one of the Vogel-Johnson Agar Medium, BairdParker Agar Medium, or Mannitol-Salt Agar Medium, and
incubate between 309C and 359
C for 24 to 48 hours. Upon
examination, if no colonies of Gram-positive cocci having the
characteristics listed in Table 4.05-3 are found, the specimen
meets the requirements of the test for the absence of
Staphylococcus aureus. Conrm any suspect colonies as
Staphylococcus aureus by means of the coagulase test. With
the aid of an inoculating loop, transfer suspect colonies to individual tubes, each containing 0.5 mL of mammalian,
preferably rabbit or horse, plasma with or without suitable
additives. Incubate in a thermostat at 3719
C. Examine the
coagulation after 3 hours and subsequently at suitable inter-

JP XV

General Tests / Microbial Limit Test

vals up to 24 hours. Test positive and negative controls simultaneously. If no coagulation is observed, the specimen meets
the requirements of the test for the absence of Staphylococcus aureus.
Table 4.05-3. Morphologic characteristics of Staphylococcus aureus on selective agar media
Medium

Colonial characteristics

Vogel-Johnson Agar
Medium

Black surrounded by yellow zone

Baird-Parker Agar
Medium

Black, shiny, surrounded by clear zones

Mannitol-Salt Agar
Medium

Yellow colonies with yellow zones

Eectiveness of culture media and conrmation of antimicrobial substances

Grow the test strains listed in Table 4.05-4 in the media inC and 359
C for 18 to 24 hours. Dilute
dicated between 309
portions of each of the cultures using Buered Sodium Chloride-Peptone Solution, Phosphate Buer, or medium indicated for each bacterial strain to make test suspensions containing about 1000 CFU per mL. As occasion demands, using a
mixture of 0.1 mL of each suspension of Escherichia coli,
Salmonella, Pseudomonas aeruginosa and Staphylococcus
aureus containing about 1000 CFU, the validity of the medium and the presence of antimicrobial substances are tested in
the presence or absence of the specimen.
Table 4.05-4. Bacterial strains and media used for conrmation of the eectiveness of culture medium and validity of
the test for specied microorganisms
Microorganisms

Strain number

Media

Escherichia coli

ATCC 8739, NCIMB

8545, NBRC 3972 or
equivalent strains

Fluid Lactose
Medium

Salmonella

No strain number is
recommended*

Fluid Lactose
Medium

Pseudomonas
aeruginosa

ATCC 9027, NCIMB

8626, NBRC 13275 or
equivalent strains

Fluid SoybeanCasein Digest

Medium

Staphylococcus
aureus

ATCC 6538, NCIMB

8625, NBRC 13276 or
equivalent strains

Fluid SoybeanCasein Digest

Medium

*Salmonella strains of weak or no pathogenicity may be used.

Salmonella Typhi may not be used.

Retest
For the purpose of conrming a doubtful result, a retest is
conducted using 25 g or 25 mL of test specimen. Proceed as
directed under Test procedure, but make allowance for the
larger specimen size, for example by adjusting the volume of
the medium.
3.

Buer solution and media

Buer solution and media used for the microbial limit test
are described below. Other media may be used if they have
similar nutritive ingredients, and selective and growthpromoting properties for the microorganisms to be tested.
(1) Buer solution
(i) Phosphate Buer (pH 7.2)
Stock Solution: Dissolve 34 g of potassium dihydrogen-

91

phosphate in about 500 mL of water. Adjust to pH 7.1 to 7.3

by the addition of 175 mL of sodium hydroxide TS, add
water to make 1000 mL, and use this solution as the stock solution. After sterilization by heating in an autoclave, store
under refrigeration. For use, dilute the Stock Solution with
water in the ratio of 1 to 800, and sterilize at 1219
C for 15 to
20 minutes.
(ii) Buered Sodium Chloride-Peptone Solution (pH 7.0)
Potassium dihydrogen phosphate
3.56 g
Disodium hydrogen phosphate
dodecahydrate
18.23 g
Sodium chloride
4.30 g
Peptone
1.0 g
Water
1000 mL
Mix all the components, and sterilize by heating in an autoclave at 1219
C for 15 to 20 minutes. pH after sterilization:
vz) may be ad6.9 7.1. Polysorbate 20 or 80 (0.1 to 1.0 wW
ded.
(2) Media
(i) Soybean-Casein Digest Agar Medium
Casein peptone
15.0 g
Soybean peptone
5.0 g
Sodium chloride
5.0 g
Agar
15.0 g
Water
1000 mL
Mix all the components, and sterilize by heating in an autoclave at 1219
C for 15 to 20 minutes. pH after sterilization:
7.1 7.3.
(ii) Fluid Soybean-Casein Digest Medium
Casein peptone
17.0 g
Soybean peptone
3.0 g
Sodium chloride
5.0 g
Dipotassium hydrogen phosphate
2.5 g
Glucose
2.5 g
Water
1000 mL
Mix all the components, and sterilize by heating in an autoclave at 1219
C for 15 to 20 minutes. pH after sterilization:
7.1 7.5.
(iii) Sabouraud Glucose Agar Medium with Antibiotics
Peptones (animal tissue and casein) 10.0 g
Glucose
40.0 g
Agar
15.0 g
Water
1000 mL
Mix all the components, and sterilize by heating in an autoclave at 1219
C for 15 to 20 minutes. pH after sterilization:
5.4 5.8. Just prior to use, add 0.10 g of benzylpenicillin
potassium and 0.10 g of tetracycline per liter of medium as
sterile solutions or, alternatively, add 50 mg of chloramphenicol per liter of medium.
(iv) Potato Dextrose Agar Medium with Antibiotics
Potato extract
4.0 g
Glucose
20.0 g
Agar
15.0 g
Water
1000 mL
Mix all the components, and sterilize by heating in an autoclave at 1219
C for 15 to 20 minutes. pH after sterilization:
5.4 5.8. Just prior to use, add 0.10 g of benzylpenicillin
potassium and 0.10 g of tetracycline per liter of medium as
sterile solutions or, alternatively, add 50 mg of chloramphenicol per liter of medium.
(v) GP (Glucose-peptone) Agar Medium with Antibiotics
Glucose
20.0 g
Yeast extract
2.0 g

92

Microbial Limit Test / General Tests

Magnesium sulfate heptahydrate

0.5 g
Peptone
5.0 g
Potassium dihydrogen phosphate
1.0 g
Agar
15.0 g
Water
1000 mL
Mix all the components, and sterilize by heating in an autoclave at 1219
C for 15 to 20 minutes. pH after sterilization:
5.6 5.8. Just prior to use, add 0.10 g of benzylpenicillin
potassium and 0.10 g of tetracycline per liter of medium as
sterile solutions or, alternatively, add 50 mg of chloramphenicol per liter of medium.
(vi) Fluid Lactose Medium
Meat extract
3.0 g
Gelatin peptone
5.0 g
Lactose monohydrate
5.0 g
Water
1000 mL
Mix all the components, and sterilize by heating in an autoclave at 1219
C for 15 to 20 minutes. pH after sterilization:
6.7 7.1. After sterilization, cool immediately.
(vii) MacConkey Agar Medium
Gelatin peptone
17.0 g
Casein peptone
1.5 g
Animal tissue peptone
1.5 g
Lactose monohydrate
10.0 g
Sodium desoxycholate
1.5 g
Sodium chloride
5.0 g
Agar
13.5 g
Neutral red
0.03 g
Crystal violet
1.0 mg
Water
1000 mL
Mix all the components, boil for 1 minute, mix, and sterilize by heating in an autoclave at 1219
C for 15 to 20 minutes.
pH after sterilization: 6.9 7.3.
(viii) EMB (Eosin-Methylene Blue) Agar Medium
Gelatin peptone
10.0 g
Dipotassium hydrogen phosphate
2.0 g
Lactose monohydrate
10.0 g
Agar
15.0 g
Eosin Y
0.40 g
Methylene blue
0.065 g
Water
1000 mL
Mix all the components, and sterilize by heating in an
autoclave at 1219C for 15 to 20 minutes. pH after sterilization: 6.9 7.3.
(ix) Fluid Selenite-Cystine Medium
Gelatin peptone
5.0 g
Lactose monohydrate
4.0 g
Trisodium phosphate dodecahydrate 10.0 g
Sodium selenite
4.0 g
L-Cystine
0.010 g
Water
1000 mL
Mix all the components, and heat to eect solution. Final
pH: 6.8 7.2. Do not sterilize.
(x) Fluid Tetrathionate Medium
Casein peptone
2.5 g
Animal tissue peptone
2.5 g
Sodium desoxycholate
1.0 g
Calcium carbonate
10.0 g
Sodium thiosulfate pentahydrate
30.0 g
Water
1000 mL
Heat the solution of solids to boiling. On the day of use,
add a solution prepared by dissolving 5 g of potassium iodide
and 6 g of iodine in 20 mL of water. Then add 10 mL of a so-

JP XV
lution of brilliant green (1 in 1000), and mix. Do not heat the
medium after adding the brilliant green solution.
(xi) Fluid Rappaport Medium
Soybean peptone
5.0 g
Sodium chloride
8.0 g
Potassium dihydrogen phosphate
1.6 g
Malachite green oxalate
0.12 g
Magnesium chloride hexahydrate
40.0 g
Water
1000 mL
Dissolve malachite green oxalate and magnesium chloride
hexahydrate, and the remaining solids separately in the
water, and sterilize by heating in an autoclave at 1219
C for 15
to 20 minutes. For the use, mix the both solutions after
sterilization. Final pH: 5.4 5.8.
(xii) Brilliant Green Agar Medium
Peptones (animal tissue and casein) 10.0 g
Yeast extract
3.0 g
Sodium chloride
5.0 g
Lactose monohydrate
10.0 g
Sucrose
10.0 g
Phenol red
0.080 g
Brilliant green
0.0125 g
Agar
20.0 g
Water
1000 mL
Mix all the components, and boil for 1 minute. Sterilize
just prior to use by heating in an autoclave at 1219C for 15 to
20 minutes. pH after sterilization: 6.7 7.1. Cool to about 50
9C and pour to petri dishes.
(xiii) XLD (Xylose-Lysine-Desoxycholate) Agar Medium
D-Xylose
3.5 g
L-Lysine monohydrochloride
5.0 g
Lactose monohydrate
7.5 g
Sucrose
7.5 g
Sodium chloride
5.0 g
Yeast extract
3.0 g
Phenol red
0.080 g
Sodium desoxycholate
2.5 g
Sodium thiosulfate pentahydrate
6.8 g
Ammonium iron (III) citrate
0.80 g
Agar
13.5 g
Water
1000 mL
Mix all the components, and boil to eect solution. pH
after boiling: 7.2 7.6. Do not sterilize in an autoclave or
overheat. Cool to about 509
C and pour to petri dishes.
(xiv) Bismuth Sulte Agar Medium
Meat extract
5.0 g
Casein peptone
5.0 g
Animal tissue peptone
5.0 g
Glucose
5.0 g
Trisodium phosphate dodecahydrate 4.0 g
Iron (II) sulfate heptahydrate
0.30 g
Bismuth sulte indicator
8.0 g
Brilliant green
0.025 g
Agar
20.0 g
Water
1000 mL
Mix all the components, and boil to eect solution. pH
after boiling: 7.4 7.8. Do not sterilize in an autoclave or
overheat. Cool to about 509
C and pour to petri dishes.
(xv) TSI (Triple Sugar Iron) Agar Medium
Casein peptone
10.0 g
Animal tissue peptone
10.0 g
Lactose monohydrate
10.0 g
Sucrose
10.0 g