聽聽聽聽聽聽聽聽聽MICROBIAL聽LIMIT聽TESTS聽（微生物限度检查USP）
This聽chapter聽provides聽tests聽for聽the聽estimation聽of聽the聽number聽of聽viable聽
aerobic聽microorganisms聽present聽and聽for聽freedom聽from聽designated聽microbial聽
species聽in聽pharmaceutical聽articles聽of聽all聽kinds,聽from聽raw聽materials聽to聽the
聽finished聽forms.聽An聽automated聽method聽may聽be聽substituted聽for聽the聽tests聽
presented聽here,聽provided聽it聽has聽been聽properly聽validated聽as聽giving聽equivalent聽
or聽better聽results.聽In聽preparing聽for聽and聽in聽applying聽the聽tests,聽observe聽aseptic聽
precautions聽in聽handling聽the聽specimens.聽Unless聽otherwise聽directed,聽where聽the聽
procedure聽specifies聽simply聽“incubate,”聽hold聽the聽container聽in聽air聽that聽is聽
thermostatically聽controlled聽at聽a聽temperature聽between聽30聽and聽35,聽for聽a聽period
聽of聽24聽to聽48聽hours.聽The聽term聽“growth”聽is聽used聽in聽a聽special聽sense聽herein,
聽i.e.,聽to聽designate聽the聽presence聽and聽presumed聽proliferation聽of聽viable聽
microorganisms.

这一章规定，测试估计的数目可行的好氧微生物，从指定的微生物物种在制药文章的所有
品种，从原料到成品的形式。一个自动化的方法，可取代的考验就在这里，只要它已妥善
验证给予同等或更好的结果。在准备和在应用试验，观察无菌的预防措施，再处理标本。
除非另有指示，凡指定的程序是简单的“孵化”聽，举行容器中的空气是恒温的控制在温度
30至35聽，时间在24至48小时。相对而言，聽“增长”是用来在一个特殊的意义在这里，即
指定的存在和扩散的微生物。........
聽
Preparatory聽Testing聽（筹备测试）
The聽validity聽of聽the聽results聽of聽the聽tests聽set聽forth聽in聽this聽chapter聽rests聽
largely聽upon聽the聽adequacy聽of聽a聽demonstration聽that聽the聽test聽specimens聽to聽which聽
they聽are聽applied聽do聽not,聽of聽themselves,聽inhibit聽the聽multiplication,聽under聽the聽
test聽conditions,聽of聽microorganisms聽that聽may聽be聽present.聽Therefore,聽preparatory
聽to聽conducting聽the聽tests聽on聽a聽regular聽basis聽and聽as聽circumstances聽require
聽subsequently,聽inoculate聽diluted聽specimens聽of聽the聽material聽to聽be聽tested聽with聽
separate聽viable聽cultures聽of聽Staphylococcus聽aureus,聽Escherichia聽coli,聽
Pseudomonas聽aeruginosa,聽and聽Salmonella.聽This聽can聽be聽done聽by聽adding聽1聽mL聽of聽not聽
less聽than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Fluid聽Soybean–Casein聽Digest
聽Medium,聽or聽Fluid聽Lactose聽Medium)聽of聽the聽test聽material聽and聽following聽the聽test
聽procedure.聽Failure聽of聽the聽organism(s)聽to聽grow聽in聽the聽relevant聽medium聽
invalidates聽that聽portion聽of聽the聽examination聽and聽necessitates聽a聽modification聽of聽
the聽procedure聽by聽(1)聽an聽increase聽in聽the聽volume聽of聽diluent,聽the聽quantity聽of聽
test聽material聽remaining聽the聽same,聽or聽by聽(2)聽the聽incorporation聽of聽a聽sufficient聽
quantity聽of聽suitable聽inactivating聽agent(s)聽in聽the聽diluents,聽or聽by聽(3)聽an聽
appropriate聽combination聽of聽modifications聽(1)聽and聽(2)聽so聽as聽to聽permit聽growth聽of
聽the聽inocula.
聽
The聽following聽are聽examples聽of聽ingredients聽and聽their聽concentrations聽that聽may聽be
聽added聽to聽the聽culture聽medium聽to聽neutralize聽inhibitory聽substances聽present聽in聽
the聽sample:聽soy聽lecithin,聽0.5%;聽and聽polysorbate聽20,聽4.0%.聽Alternatively,聽
repeat聽the聽test聽as聽described聽in聽the聽preceding聽paragraph,聽using聽Fluid聽Casein聽
Digest–Soy聽Lecithin–Polysorbate聽20聽Medium聽to聽demonstrate聽neutralization聽of聽
preservatives聽or聽other聽antimicrobial聽agents聽in聽the聽test聽material.聽Where聽
inhibitory聽substances聽are聽contained聽in聽the聽product聽and聽the聽latter聽is聽soluble,
聽a聽suitable,聽validated聽adaptation聽of聽a聽procedure聽set聽forth聽in聽the聽section
聽Membrane聽Filtration聽under聽Test聽for聽Sterility聽of聽the聽Product聽to聽be聽Examined聽
under聽Sterility聽Tests聽71,聽may聽be聽used.
聽
If聽in聽spite聽of聽the聽incorporation聽of聽suitable聽inactivating聽agents聽and聽a聽
substantial聽increase聽in聽the聽volume聽of聽diluent,聽it聽is聽still聽not聽possible聽to
聽recover聽the聽viable聽cultures聽described聽above聽and聽where聽the聽article聽is聽not聽
suitable聽for聽employment聽of聽membrane聽filtration,聽it聽can聽be聽assumed聽that聽the聽
failure聽to聽isolate聽the聽inoculated聽organism聽is聽attributable聽to聽the聽bactericidal聽
activity聽of聽the聽product.聽This聽information聽serves聽to聽indicate聽that聽the聽article
聽is聽not聽likely聽to聽be聽contaminated聽with聽the聽given聽species聽of聽microorganism.
聽Monitoring聽should聽be聽continued聽in聽order聽to聽establish聽the聽spectrum聽of聽
inhibition聽and聽bactericidal聽activity聽of聽the聽article.

Buffer聽Solution聽and聽Media聽（缓冲溶液和培养基）
Culture聽media聽may聽be聽prepared聽as聽follows,聽or聽dehydrated聽culture聽media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
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
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聽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
PH聽7.2聽Phosphate聽Buffer聽
Stock聽Solution—聽Dissolve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pH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聽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and聽
sterilize.聽

Media聽
Unless聽otherwise聽indicated,聽the聽media聽should聽be聽sterilized聽by聽heating聽in聽an聽
autoclave聽(see聽Steam聽Sterilization聽under聽Sterilization聽1211),聽the聽exposure聽
time聽depending聽on聽the聽volume聽to聽be聽sterilized.

I.聽Fluid聽Casein聽Digest–Soy聽Lecithin–Polysorbate聽20聽Medium聽
聽
Pancreatic聽Digest聽of聽Casein聽20聽g聽
Soy聽Lecithin聽5聽g聽
Polysorbate聽20聽40聽mL聽
Water聽960聽mL聽
Dissolve聽the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and聽dispense聽as聽desired.
聽
II.聽Soybean–Casein聽Digest聽Agar聽Medium聽
聽
Pancreatic聽Digest聽of聽Casein聽15.0聽g聽
Papaic聽Digest聽of聽Soybean聽Meal聽5.0聽g聽
Sodium聽Chloride聽5.0聽g聽
Agar聽15.0聽g聽
Water聽1000聽mL聽
pH聽after聽sterilization:聽7.3聽±聽0.2.聽
III.聽Fluid聽Soybean–Casein聽Digest聽Medium聽
聽
Prepare聽as聽directed聽for聽Soybean–Casein聽Digest聽Medium聽under聽Sterility聽Tests聽
聽
IV.聽Mannitol–Salt聽Agar聽Medium聽
聽
Pancreatic聽Digest聽of聽Casein聽5.0聽g聽
Peptic聽Digest聽of聽Animal聽Tissue聽5.0聽g聽
Beef聽Extract聽1.0聽g聽
D-Mannitol聽10.0聽g聽
Sodium聽Chloride聽75.0聽g聽
Agar聽15.0聽g聽
Phenol聽Red聽0.025聽g聽
Water聽1000聽mL聽
Mix,聽then聽heat聽with聽frequent聽agitation,聽and聽boil聽for聽1聽minute聽to聽effect
聽solution.聽
pH聽after聽sterilization:聽7.4聽±聽0.2.聽
V.聽Baird–Parker聽Agar聽Medium聽
聽
Pancreatic聽Digest聽of聽Casein聽10.0聽g聽
Beef聽Extract聽5.0聽g聽
Yeast聽Extract聽1.0聽g聽
Lithium聽Chloride聽5.0聽g聽
Agar聽20.0聽g聽
Glycine聽12.0聽g聽
Sodium聽Pyruvate聽10.0聽g聽
Water聽950聽mL聽
Heat聽with聽frequent聽agitation,聽and聽boil聽for聽1聽minute.聽Sterilize,聽cool聽to聽
between聽45聽and聽50,聽and聽add聽10聽mL聽of聽sterile聽potassium聽tellurite聽solution聽(1聽in聽
100)聽and聽50聽mL聽of聽egg-yolk聽emulsion.聽Mix聽intimately聽but聽gently,聽and聽pour聽into聽
plates.聽(Prepare聽the聽egg-yolk聽emulsion聽by聽disinfecting聽the聽surface聽of聽whole聽
shell聽eggs,聽aseptically聽cracking聽the聽eggs,聽and聽separating聽out聽intact聽yolks聽
into聽a聽sterile聽graduated聽cylinder.聽Add聽sterile聽saline聽TS聽to聽obtain聽a聽3聽to聽7聽
ratio聽of聽egg聽yolk聽to聽saline.聽Add聽to聽a聽sterile聽blender聽cup,聽and聽mix聽at聽high聽
speed聽for聽5聽seconds.)聽
pH聽after聽sterilization:聽6.8聽±聽0.2.聽
VI.聽Vogel–Johnson聽Agar聽Medium聽
聽
Pancreatic聽Digest聽of聽Casein聽10.0聽g聽
Yeast聽Extract聽5.0聽g聽
Mannitol聽10.0聽g聽
Dibasic聽Potassium聽Phosphate聽5.0聽g聽
Lithium聽Chloride聽5.0聽g聽
Glycine聽10.0聽g聽
Agar聽16.0聽g聽
Phenol聽Red聽25.0聽mg聽
Water聽1000聽mL聽
Boil聽the聽solution聽of聽solids聽for聽1聽minute.聽Sterilize,聽cool聽to聽between聽45聽and聽
50,聽and聽add聽20聽mL聽of聽sterile聽potassium聽tellurite聽solution聽(1聽in聽100).聽

pH聽after聽sterilization:聽7.2聽±聽0.2.聽
VII.聽Cetrimide聽Agar聽Medium聽
聽
Pancreatic聽Digest聽of聽Gelatin聽20.0聽g聽
Magnesium聽Chloride聽1.4聽g聽
Potassium聽Sulfate聽10.0聽g聽
Agar聽13.6聽g聽
Cetyl聽Trimethylammonium聽Bromide聽(Cetrimide)聽0.3聽g聽
Glycerin聽10.0聽mL聽
Water聽1000聽mL聽
Dissolve聽all聽solid聽components聽in聽the聽water,聽and聽add聽the聽glycerin.聽Heat,聽with聽
frequent聽agitation,聽and聽boil聽for聽1聽minute聽to聽effect聽solution.聽
pH聽after聽sterilization:聽7.2聽±聽0.2.聽
VIII.聽Pseudomonas聽Agar聽Medium聽for聽Detection聽of聽Fluorescin聽
聽
Pancreatic聽Digest聽of聽Casein聽10.0聽g聽
Peptic聽Digest聽of聽Animal聽Tissue聽10.0聽g聽
Anhydrous聽Dibasic聽Potassium聽Phosphate聽1.5聽g聽
Magnesium聽Sulfate聽(MgSO4·7H2O)聽1.5聽g聽
Glycerin聽10.0聽mL聽
Agar聽15.0聽g聽
Water聽1000聽mL聽
Dissolve聽the聽solid聽components聽in聽the聽water聽before聽adding聽the聽glycerin.聽Heat,
聽with聽frequent聽agitation,聽and聽boil聽for聽1聽minute聽to聽effect聽solution.聽

pH聽after聽sterilization:聽7.2聽±聽0.2.聽
IX.聽Pseudomonas聽Agar聽Medium聽for聽Detection聽of聽Pyocyanin聽
聽
Pancreatic聽Digest聽of聽Gelatin聽20.0聽g聽
Anhydrous聽Magnesium聽Chloride聽1.4聽g聽
Anhydrous聽Potassium聽Sulfate聽10.0聽g聽
Agar聽15.0聽g聽
Glycerin聽10.0聽mL聽
Water聽1000聽mL聽
Dissolve聽the聽solid聽components聽in聽the聽water聽before聽adding聽the聽glycerin.聽Heat,
聽with聽frequent聽agitation,聽and聽boil聽for聽1聽minute聽to聽effect聽solution.
聽
pH聽after聽sterilization:聽7.2聽±聽0.2.聽
X.聽Fluid聽Lactose聽Medium聽
聽
Beef聽Extract聽3.0聽g聽
Pancreatic聽Digest聽of聽Gelatin聽5.0聽g聽
Lactose聽5.0聽g聽
Water聽1000聽mL聽
Cool聽as聽quickly聽as聽possible聽after聽sterilization.聽
pH聽after聽sterilization:聽6.9聽±聽0.2.聽
XI.聽Fluid聽Selenite–Cystine聽Medium聽
聽
Pancreatic聽Digest聽of聽Casein聽5.0聽g聽
Lactose聽4.0聽g聽
Sodium聽Phosphate聽10.0聽g聽
Sodium聽Acid聽Selenite聽4.0聽g聽
L-Cystine聽10.0聽mg聽
Water聽1000聽mL聽
Final聽pH:聽7.0聽±聽0.2.聽
Mix,聽and聽heat聽to聽effect聽solution.聽Heat聽in聽flowing聽steam聽for聽15聽minutes.聽Do聽not聽
sterilize.聽
XII.聽Fluid聽Tetrathionate聽Medium聽
聽
Pancreatic聽Digest聽of聽Casein聽2.5聽g聽
Peptic聽Digest聽of聽Animal聽Tissue聽2.5聽g聽
Bile聽Salts聽1.0聽g聽
Calcium聽Carbonate聽10.0聽g聽
Sodium聽Thiosulfate聽30.0聽g聽
Water聽1000聽mL聽
Heat聽the聽solution聽of聽solids聽to聽boiling.聽On聽the聽day聽of聽use,聽add聽a聽solution聽
prepared聽by聽dissolving聽5聽g聽of聽potassium聽iodide聽and聽6聽g聽of聽iodine聽in聽20聽mL聽of聽
water.聽Then聽add聽10聽mL聽of聽a聽solution聽of聽brilliant聽green聽(1聽in聽1000),聽and聽mix.
聽Do聽not聽heat聽the聽medium聽after聽adding聽the聽brilliant聽green聽solution.聽
XIII.聽Brilliant聽Green聽Agar聽Medium聽
聽
Yeast聽Extract聽3.0聽g聽
Peptic聽Digest聽of聽Animal聽Tissue聽5.0聽g聽
Pancreatic聽Digest聽of聽Casein聽5.0聽g聽
Lactose聽10.0聽g聽
Sodium聽Chloride聽5.0聽g聽
Sucrose聽10.0聽g聽
Phenol聽Red聽80聽mg聽
Agar聽20.0聽g聽
Brilliant聽Green聽12.5聽mg聽
Water聽1000聽mL聽
Boil聽the聽solution聽of聽solids聽for聽1聽minute.聽Sterilize聽just聽prior聽to聽use,聽melt聽
the聽medium,聽pour聽into聽petri聽dishes,聽and聽allow聽to聽cool.聽
pH聽after聽sterilization:聽6.9聽±聽0.2.聽
XIV.聽Xylose–Lysine–Desoxycholate聽Agar聽Medium聽
聽
Xylose聽3.5聽g聽
L-Lysine聽5.0聽g聽
Lactose聽7.5聽g聽
Sucrose聽7.5聽g聽
Sodium聽Chloride聽5.0聽g聽
Yeast聽Extract聽3.0聽g聽
Phenol聽Red聽80聽mg聽
Agar聽13.5聽g聽
Sodium聽Desoxycholate聽2.5聽g聽
Sodium聽Thiosulfate聽6.8聽g聽
Ferric聽Ammonium聽Citrate聽800聽mg聽
Water聽1000聽mL聽
Final聽pH:聽7.4聽±聽0.2.聽
Heat聽the聽mixture聽of聽solids聽and聽water,聽with聽swirling,聽just聽to聽the聽boiling聽
point.聽Do聽not聽overheat聽or聽sterilize.聽Transfer聽at聽once聽to聽a聽water聽bath聽
maintained聽at聽about聽50,聽and聽pour聽into聽plates聽as聽soon聽as聽the聽medium聽has聽cooled.
聽
XV.聽Bismuth聽Sulfite聽Agar聽Medium聽
聽
Beef聽Extract聽5.0聽g聽
Pancreatic聽Digest聽of聽Casein聽5.0聽g聽
Peptic聽Digest聽of聽Animal聽Tissue聽5.0聽g聽
Dextrose聽5.0聽g聽
Sodium聽Phosphate聽4.0聽g聽
Ferrous聽Sulfate聽300聽mg聽
Bismuth聽Sulfite聽Indicator聽8.0聽g聽
Agar聽20.0聽g聽
Brilliant聽Green聽25聽mg聽
Water聽1000聽mL聽
Final聽pH:聽7.6聽±聽0.2.聽
Heat聽the聽mixture聽of聽solids聽and聽water,聽with聽swirling,聽just聽to聽the聽boiling聽
point.聽Do聽not聽overheat聽or聽sterilize.聽Transfer聽at聽once聽to聽a聽water聽bath聽
maintained聽at聽about聽50,聽and聽pour聽into聽plates聽as聽soon聽as聽the聽medium聽has聽cooled.聽
XVI.聽Triple聽Sugar–Iron–Agar聽Medium聽
聽
Pancreatic聽Digest聽of聽Casein聽10.0聽g聽
Pancreatic聽Digest聽of聽Animal聽Tissue聽10.0聽g聽
Lactose聽10.0聽g聽
Sucrose聽10.0聽g聽
Dextrose聽1.0聽g聽
Ferrous聽Ammonium聽Sulfate聽200聽mg聽
Sodium聽Chloride聽5.0聽g聽
Sodium聽Thiosulfate聽200聽mg聽
Agar聽13.0聽g聽
Phenol聽Red聽25聽mg聽
Water聽1000聽mL聽
pH聽after聽sterilization:聽7.3聽±聽0.2.聽
XVII.聽MacConkey聽Agar聽Medium聽
聽
Pancreatic聽Digest聽of聽Gelatin聽17.0聽g聽
Pancreatic聽Digest聽of聽Casein聽1.5聽g聽
Peptic聽Digest聽of聽Animal聽Tissue聽1.5聽g聽
Lactose聽10.0聽g聽
Bile聽Salts聽Mixture聽1.5聽g聽
Sodium聽Chloride聽5.0聽g聽
Agar聽13.5聽g聽
Neutral聽Red聽30聽mg聽
Crystal聽Violet聽1.0聽mg聽
Water聽1000聽mL聽
Boil聽the聽mixture聽of聽solids聽and聽water聽for聽1聽minute聽to聽effect聽solution.聽
pH聽after聽sterilization:聽7.1聽±聽0.2.聽
XVIII.聽Levine聽Eosin–Methylene聽Blue聽Agar聽Medium聽
聽
Pancreatic聽Digest聽of聽Gelatin聽10.0聽g聽
Dibasic聽Potassium聽Phosphate聽2.0聽g聽
Agar聽15.0聽g聽
Lactose聽10.0聽g聽
Eosin聽Y聽400聽mg聽
Methylene聽Blue聽65聽mg聽
Water聽1000聽mL聽
Dissolve聽the聽pancreatic聽digest聽of聽gelatin,聽the聽dibasic聽potassium聽phosphate,
聽and聽the聽agar聽in聽the聽water,聽with聽warming,聽and聽allow聽to聽cool.聽Just聽prior聽to聽
use,聽liquefy聽the聽gelled聽agar聽solution,聽add聽the聽remaining聽ingredients,聽as聽
solutions,聽in聽the聽following聽amounts,聽and聽mix:聽for聽each聽100聽mL聽of聽the聽liquefied聽
agar聽solution—5聽mL聽of聽lactose聽solution聽(1聽in聽5),聽2聽mL聽of聽the聽eosin聽Y聽solution聽
(1聽in聽50),聽and聽2聽mL聽of聽methylene聽blue聽solution聽(1聽in聽300).聽The聽finished聽medium聽
may聽not聽be聽clear.聽
pH聽after聽sterilization:聽7.1聽±聽0.2.聽
XIX.聽Sabouraud聽Dextrose聽Agar聽Medium聽
聽
Dextrose聽40聽g聽
Mixture聽of聽equal聽parts聽of聽Peptic聽Digest聽of聽Animal聽Tissue聽and聽Pancreatic聽Digest
聽of聽Casein聽10聽g聽
Agar聽15聽g聽
Water聽1000聽mL聽
Mix,聽and聽boil聽to聽effect聽solution.聽
pH聽after聽sterilization:聽5.6聽±聽0.2.聽
XX.聽Potato聽Dextrose聽Agar聽Medium聽
聽
Cook聽300聽g聽of聽peeled聽and聽diced聽potatoes聽in聽500聽mL聽of聽water聽prepared聽by聽
distillation,聽filter聽through聽cheesecloth,聽add聽water聽prepared聽by聽distillation聽
to聽make聽1000聽mL,聽and聽add聽the聽following:
聽
Agar聽15聽g聽
Glucose聽20聽g聽
Dissolve聽by聽heating,聽and聽sterilize.聽
pH聽after聽sterilization:聽5.6聽±聽0.2.聽
For聽use,聽just聽prior聽to聽pouring聽the聽plates,聽adjust聽the聽melted聽and聽cooled聽to聽45
聽medium聽with聽sterile聽tartaric聽acid聽solution聽(1聽in聽10)聽to聽a聽pH聽of聽3.5聽±聽0.1.聽
Do聽not聽reheat聽the聽pH聽3.5聽medium.聽

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