MICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY SUPPLEMENTS
(微生物计数试验——营养和添加剂)
BUFFER SOLUTION AND MEDIA（缓冲液和培养基）
Culture media may be prepared as follows, or dehydrated culture media may
be used provided that, when reconstituted as directed by the manufacturer or
distributor, they have similar ingredients and/or yield media comparable to
those obtained from the formulas given herein.
In preparing media by the formulas set forth herein, dissolve the soluble
solids in the water, using heat if necessary to effect complete solution, and
add solutions of hydrochloric acid or sodium hydroxide in quantities
sufficient to yield the desired pH in the medium when it is ready for use.
Determine the pH at 25 ± 2.
Where agar is called for in a formula, use agar that has a moisture content
of not more than 15%. Where water is called for in a formula, use Purified
Water.
pH 7.2 Phosphate Buffer（磷酸盐缓冲液）
Prepare a stock solution by dissolving 34 g of monobasic potassium
phosphate in about 500 mL of water contained in a 1000-mL volumetric flask.
Adjust to a pH of 7.2 ± 0.1 by the addition of sodium hydroxide TS (about 175
mL), add water to volume, and mix. Dispense and sterilize. Store under
refrigeration. For use, dilute the stock solution with water in the ratio of 1 to 800, dispense as desired, and sterilize.
Media
Prepare media for the tests as described below. Alternatively, dehydrated
formulations may be used provided that, when reconstituted as directed by the
manufacturer or distributor, they meet the requirements of the Growth
Promotion Testing.Unless otherwise indicated elsewhere in this chapter, media
are sterilized in autoclaves using a validated process. The exposure time
within the autoclave at 121 will depend on the volume of media to be
sterilized. Thus, for example, a 500-mL volume would need to be autoclaved
using a temperature and time relationship that will ensure that the medium has
attained at least an F0 of 12–15 in the sterilization process. However, the
appropriate time and temperature duration for sterilizing prepared media at
any given volume should be confirmed by a thermal penetration study using a
thermocouple or thermoprobe placed within the liquid medium.
FLUID CASEIN DIGEST–SOY LECITHIN–POLYSORBATE 20 MEDIUM
Pancreatic Digest of Casein 20 g
Soy Lecithin 5 g
Polysorbate 20 40 mL
Water 960 mL
Dissolve pancreatic digest of casein and soy lecithin in 960 mL of water,
heating in a water bath at 48 to 50 for about 30 minutes to effect solution.
Add 40 mL of polysorbate 20. Mix, dispense as desired, and sterilize.
SOYBEAN–CASEIN DIGEST–AGAR MEDIUM
Pancreatic Digest of Casein 15.0 g
Papaic Digest of Soybean Meal 5.0 g
Sodium Chloride 5.0 g
Agar 15.0 g
Water 1000 mL
pH after sterilization: 7.3 ± 0.2.
FLUID SOYBEAN–CASEIN DIGEST MEDIUM
Pancreatic Digest of Casein 17.0 g
Papaic Digest of Soybean Meal 3.0 g
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose 2.5 g
Purified Water 1000 mL
Dissolve the solids in the water, heating slightly to effect a solution.
Cool the solution to room temperature, and adjust the pH with 1 N sodium
hydroxide so that after sterilization it will have a pH of 7.3 ± 0.2. Filter,
if necessary, and dispense into suitable containers. Sterilize at a
temperature and time relationship that will ensure that the medium has
attained at least an F0 of 12–15 in the sterilization process, or by a
validated filtration process.
SABOURAUD DEXTROSE–AGAR MEDIUM
Dextrose 40.0 g
Mixture of Peptic Digest of Animal Tissue andPancreatic Digest of Casein
(1:1) 10.0 g
Agar 15.0 g
Water 1000 mL
Mix, and boil to effect solution.
pH after sterilization: 5.6 ± 0.2.
VIOLET-RED BILE AGAR WITH GLUCOSE AND LACTOSE
Yeast Extract 3.0 g
Pancreatic Digest of Gelatin 7.0 g
Bile Salts 1.5 g
Lactose 10.0 g
Sodium Chloride 5.0 g
D-Glucose Monohydrate 10.0 g
Agar 15.0 g
Neutral Red 30 mg
Crystal Violet 2 mg
Water 1000 mL
Adjust the pH so that it is 7.4 ± 0.2 after heating. Heat to boiling, but
do not heat in an autoclave. Pour onto plates.
MOSSEL–ENTEROBACTERIACEAE ENRICHMENT BROTH
Pancreatic Digest of Gelatin 10.0 g
D-Glucose Monohydrate 5.0 g
Dehydrated Ox Bile 20.0 g
Monobasic Potassium Phosphate 2.0 g
Dibasic Potassium Phosphate 8.0 g
Brilliant Green 15 mg
Water 1000 mL
Suspend the solids in water, and heat to boiling for 1 to 2 minutes.
Transfer 120-mL portions to 250-mL volumetric flasks or 9-mL portions to test
tubes, all being capped with cotton plugs or loose-fitting caps. Heat on a
steam bath for 30 minutes. Adjust the pH so that it is 7.2 ± 0.2 after
heating.
GROWTH PROMOTION TESTING（增菌试验）
Each lot of dehydrated medium bearing the manufacturer’s identifying
number or each lot of medium prepared from basic ingredients must be tested
for its growth-promoting qualities. Cultures of Staphylococcus aureus(ATCC No.
6538), Escherichia coli(ATCC No. 8739), Bacillus subtilis(ATCC No. 6633),
Candida albicans(ATCC No. 10231), and Aspergillus niger(ATCC No. 16404) are
used. A 10-3 dilution of a 24-hour broth culture of the microorganism to the
first dilution (in pH 7.2 Phosphate Buffer or Fluid Soybean–Casein Digest
Medium) may be used as the inocula. Serially streak plates of the media with
the appropriate inocula to obtain isolated colonies to demonstrate the growth-
promotion qualities of the Soybean–Casein Digestand Sabouraud Dextrose
Agarmedia. Inocula the Fluid Soybean–Casein Digest Mediumand Mossel–
Enterobacteriaceae Enrichment Brothwith 10 to 100 cfu of the appropriate
challenge organisms to demonstrate their growth-promotion qualities.
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