聽聽聽
聽聽聽MICROBIAL聽ENUMERATION聽TESTS—NUTRITIONAL聽AND聽DIETARY聽SUPPLEMENTS
聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽(微生物计数试验——营养和添加剂)
聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽BUFFER聽SOLUTION聽AND聽MEDIA（缓冲液和培养基）
聽聽聽Culture聽media聽may聽be聽prepared聽as聽follows,聽or聽dehydrated聽culture聽media聽may
be聽used聽provided聽that,聽when聽reconstituted聽as聽directed聽by聽the聽manufacturer聽or聽
distributor,聽they聽have聽similar聽ingredients聽and/or聽yield聽media聽comparable聽to聽
those聽obtained聽from聽the聽formulas聽given聽herein.
聽聽聽In聽preparing聽media聽by聽the聽formulas聽set聽forth聽herein,聽dissolve聽the聽soluble聽
solids聽in聽the聽water,聽using聽heat聽if聽necessary聽to聽effect聽complete聽solution,聽and聽
add聽solutions聽of聽hydrochloric聽acid聽or聽sodium聽hydroxide聽in聽quantities聽
sufficient聽to聽yield聽the聽desired聽pH聽in聽the聽medium聽when聽it聽is聽ready聽for聽use.聽
Determine聽the聽pH聽at聽25聽±聽2.
聽聽聽Where聽agar聽is聽called聽for聽in聽a聽formula,聽use聽agar聽that聽has聽a聽moisture聽content
of聽not聽more聽than聽15%.聽Where聽water聽is聽called聽for聽in聽a聽formula,聽use聽Purified聽
Water.
聽聽聽聽聽聽聽聽聽聽pH聽7.2聽Phosphate聽Buffer（磷酸盐缓冲液）
聽聽聽Prepare聽a聽stock聽solution聽by聽dissolving聽34聽g聽of聽monobasic聽potassium聽
phosphate聽in聽about聽500聽mL聽of聽water聽contained聽in聽a聽1000-mL聽volumetric聽flask.聽
Adjust聽to聽a聽pH聽of聽7.2聽±聽0.1聽by聽the聽addition聽of聽sodium聽hydroxide聽TS聽(about聽175
mL),聽add聽water聽to聽volume,聽and聽mix.聽Dispense聽and聽sterilize.聽Store聽under聽
refrigeration.聽For聽use,聽dilute聽the聽stock聽solution聽with聽water聽in聽the聽ratio聽of聽1聽to聽800,聽dispense聽as聽desired,聽and聽sterilize.
聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽Media
聽聽聽聽Prepare聽media聽for聽the聽tests聽as聽described聽below.聽Alternatively,聽dehydrated聽
formulations聽may聽be聽used聽provided聽that,聽when聽reconstituted聽as聽directed聽by聽the聽
manufacturer聽or聽distributor,聽they聽meet聽the聽requirements聽of聽the聽Growth聽
Promotion聽Testing.Unless聽otherwise聽indicated聽elsewhere聽in聽this聽chapter,聽media聽
are聽sterilized聽in聽autoclaves聽using聽a聽validated聽process.聽The聽exposure聽time聽
within聽the聽autoclave聽at聽121聽will聽depend聽on聽the聽volume聽of聽media聽to聽be聽
sterilized.聽Thus,聽for聽example,聽a聽500-mL聽volume聽would聽need聽to聽be聽autoclaved聽
using聽a聽temperature聽and聽time聽relationship聽that聽will聽ensure聽that聽the聽medium聽has聽
attained聽at聽least聽an聽F0聽of聽12–15聽in聽the聽sterilization聽process.聽However,聽the聽
appropriate聽time聽and聽temperature聽duration聽for聽sterilizing聽prepared聽media聽at聽
any聽given聽volume聽should聽be聽confirmed聽by聽a聽thermal聽penetration聽study聽using聽a聽
thermocouple聽or聽thermoprobe聽placed聽within聽the聽liquid聽medium.
聽聽聽聽聽聽 FLUID聽CASEIN聽DIGEST–SOY聽LECITHIN–POLYSORBATE聽20聽MEDIUM

Pancreatic聽Digest聽of聽Casein 20聽g
Soy聽Lecithin 聽聽聽聽聽聽聽聽聽5聽g
Polysorbate聽20 聽聽聽聽聽聽聽聽聽40聽mL
Water 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽960聽mL
聽聽聽Dissolve聽pancreatic聽digest聽of聽casein聽and聽soy聽lecithin聽in聽960聽mL聽of聽water,聽
heating聽in聽a聽water聽bath聽at聽48聽to聽50聽for聽about聽30聽minutes聽to聽effect聽solution.聽
Add聽40聽mL聽of聽polysorbate聽20.聽Mix,聽dispense聽as聽desired,聽and聽sterilize.

聽聽聽聽聽聽聽聽聽聽聽聽聽SOYBEAN–CASEIN聽DIGEST–AGAR聽MEDIUM

Pancreatic聽Digest聽of聽Casein 15.0聽g
Papaic聽Digest聽of聽Soybean聽Meal 5.0聽g
Sodium聽Chloride 聽聽聽聽聽聽聽聽聽5.0聽g
Agar 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽15.0聽g
Water 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽1000聽mL
聽聽pH聽after聽sterilization:聽7.3聽±聽0.2.

聽聽聽聽聽聽聽聽聽聽聽聽聽聽FLUID聽SOYBEAN–CASEIN聽DIGEST聽MEDIUM

Pancreatic聽Digest聽of聽Casein 17.0聽g
Papaic聽Digest聽of聽Soybean聽Meal 3.0聽g
Sodium聽Chloride 聽聽聽聽聽聽聽聽聽5.0聽g
Dibasic聽Potassium聽Phosphate 2.5聽g
Dextrose 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽2.5聽g
Purified聽Water 聽聽聽聽聽聽聽聽聽1000聽mL
聽聽聽Dissolve聽the聽solids聽in聽the聽water,聽heating聽slightly聽to聽effect聽a聽solution.聽
Cool聽the聽solution聽to聽room聽temperature,聽and聽adjust聽the聽pH聽with聽1聽N聽sodium聽
hydroxide聽so聽that聽after聽sterilization聽it聽will聽have聽a聽pH聽of聽7.3聽±聽0.2.聽Filter,聽
if聽necessary,聽and聽dispense聽into聽suitable聽containers.聽Sterilize聽at聽a聽
temperature聽and聽time聽relationship聽that聽will聽ensure聽that聽the聽medium聽has聽
attained聽at聽least聽an聽F0聽of聽12–15聽in聽the聽sterilization聽process,聽or聽by聽a聽
validated聽filtration聽process.
聽聽 聽聽聽聽聽聽聽聽聽SABOURAUD聽DEXTROSE–AGAR聽MEDIUM

Dextrose 40.0聽g
聽聽聽Mixture聽of聽Peptic聽Digest聽of聽Animal聽Tissue聽andPancreatic聽Digest聽of聽Casein聽
(1:1) 聽聽聽聽聽聽聽聽聽聽10.0聽g
Agar 15.0聽g
Water 1000聽mL
Mix,聽and聽boil聽to聽effect聽solution.
pH聽after聽sterilization:聽5.6聽±聽0.2.
聽聽VIOLET-RED聽BILE聽AGAR聽WITH聽GLUCOSE聽AND聽LACTOSE

Yeast聽Extract 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽3.0聽g
Pancreatic聽Digest聽of聽Gelatin 7.0聽g
Bile聽Salts 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽1.5聽g
Lactose 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽10.0聽g
Sodium聽Chloride 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽5.0聽g
D-Glucose聽Monohydrate 聽聽聽聽聽聽聽聽聽10.0聽g
Agar 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽15.0聽g
Neutral聽Red 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽30聽mg
Crystal聽Violet 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽2聽mg
Water 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽1000聽mL
聽聽聽Adjust聽the聽pH聽so聽that聽it聽is聽7.4聽±聽0.2聽after聽heating.聽Heat聽to聽boiling,聽but
do聽not聽heat聽in聽an聽autoclave.聽Pour聽onto聽plates.

聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽MOSSEL–ENTEROBACTERIACEAE聽ENRICHMENT聽BROTH

Pancreatic聽Digest聽of聽Gelatin 10.0聽g
D-Glucose聽Monohydrate 聽聽聽聽聽聽聽聽聽5.0聽g
Dehydrated聽Ox聽Bile 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽20.0聽g
Monobasic聽Potassium聽Phosphate 2.0聽g
Dibasic聽Potassium聽Phosphate 聽聽聽聽聽聽聽聽聽8.0聽g
Brilliant聽Green 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽15聽mg
Water 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽1000聽mL
聽聽聽Suspend聽the聽solids聽in聽water,聽and聽heat聽to聽boiling聽for聽1聽to聽2聽minutes.聽
Transfer聽120-mL聽portions聽to聽250-mL聽volumetric聽flasks聽or聽9-mL聽portions聽to聽test聽
tubes,聽all聽being聽capped聽with聽cotton聽plugs聽or聽loose-fitting聽caps.聽Heat聽on聽a聽
steam聽bath聽for聽30聽minutes.聽Adjust聽the聽pH聽so聽that聽it聽is聽7.2聽±聽0.2聽after聽
heating.
聽聽聽聽聽聽聽聽聽聽聽聽聽GROWTH聽PROMOTION聽TESTING（增菌试验）
聽聽聽聽Each聽lot聽of聽dehydrated聽medium聽bearing聽the聽manufacturer’s聽identifying聽
number聽or聽each聽lot聽of聽medium聽prepared聽from聽basic聽ingredients聽must聽be聽tested聽
for聽its聽growth-promoting聽qualities.聽Cultures聽of聽Staphylococcus聽aureus(ATCC聽No.聽
6538),聽Escherichia聽coli(ATCC聽No.聽8739),聽Bacillus聽subtilis(ATCC聽No.聽6633),聽
Candida聽albicans(ATCC聽No.聽10231),聽and聽Aspergillus聽niger(ATCC聽No.聽16404)聽are聽
used.聽A聽10-3聽dilution聽of聽a聽24-hour聽broth聽culture聽of聽the聽microorganism聽to聽the聽
first聽dilution聽(in聽pH聽7.2聽Phosphate聽Buffer聽or聽Fluid聽Soybean–Casein聽Digest聽
Medium)聽may聽be聽used聽as聽the聽inocula.聽Serially聽streak聽plates聽of聽the聽media聽with聽
the聽appropriate聽inocula聽to聽obtain聽isolated聽colonies聽to聽demonstrate聽the聽growth-
promotion聽qualities聽of聽the聽Soybean–Casein聽Digestand聽Sabouraud聽Dextrose聽
Agarmedia.聽Inocula聽the聽Fluid聽Soybean–Casein聽Digest聽Mediumand聽Mossel–
Enterobacteriaceae聽Enrichment聽Brothwith聽10聽to聽100聽cfu聽of聽the聽appropriate聽
challenge聽organisms聽to聽demonstrate聽their聽growth-promotion聽qualities.

聽

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