【分享】USP30 ＮＦ２５　61 MICROBIAL LIMIT TESTS微生物限度检查法

BUFFER SOLUTION AND MEDIA缓冲溶液和培养基
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.
可以用下列方法配制培养基，或者使用脱水培养基，只要根据其制造商或者分销商说明进行重组之后，该脱水培养基与那些使用在此给出的配方所得到的培养基有近似的成分和/或生产介质。

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 .
在使用在此列出的配方配制培养基的过程中，将可溶性固体溶于水，如果需要，加热以达到彻底溶解，并且，在使用时加入足够量的盐酸或者氢氧化钠，使培养基的PH达到要求。在25 ± 2 测定该pH值。

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.
当配方中要求使用琼脂时，使用水分含量不超过15%的琼脂。当配方中要求使用水时，使用纯净水。

PH 7.2 Phosphate Buffer
PH 7.2磷酸盐缓冲液
Stock Solution— Dissolve 34 g of monobasic potassium phosphate in about 500 mL of water contained in a 1000-mL volumetric flask. Adjust to pH 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.
贮备液：将34g磷酸二氢钾在1000mL容量瓶中溶解于约500mL水中。用氢氧化钠试液（约175mL）调节pH值至7.2±0.1，加水至容量瓶刻度，混匀。分装溶液并灭菌。冰箱保存。

For use, dilute the Stock Solution with water in the ratio of 1 to 800, and sterilize.
使用时，将贮备液以1:800的比例加水稀释，并灭菌。

Media 培养基
Unless otherwise indicated, the media should be sterilized by heating in an autoclave (see Steam Sterilization under Sterilization 1211 ), the exposure time depending on the volume to be sterilized.
除另外指出，培养基应在高压灭菌器中加热灭菌。（见灭菌<1211>下蒸汽灭菌）。灭菌时间取决于需要灭菌的培养基体积。

I. Fluid Casein Digest–Soy Lecithin–Polysorbate 20 Medium
液体酪蛋白消化物－大豆卵磷脂－聚山梨酯20培养基
Pancreatic Digest of Casein酪蛋白胰酶消化物    20 g
Soy Lecithin大豆卵磷脂    5 g
Polysorbate 20聚山梨酯20    40 mL
Water水    960 mL
Dissolve the 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, and dispense as desired.
将酪蛋白胰酶消化物和大豆卵磷脂溶于960mL水中，48 至50 水浴加热30分钟使其溶解。加入40mL聚山梨酯20。混匀，并按需要分装。

II. Soybean–Casein Digest Agar Medium 大豆酪蛋白消化物琼脂培养基
Pancreatic Digest of Casein酪蛋白胰酶消化物    15.0 g
Papaic Digest of Soybean Meal大豆粉木瓜蛋白酶消化物    5.0 g
Sodium Chloride氯化钠    5.0 g
Agar琼脂    15.0 g
Water水    1000 mL
pH after sterilization: 7.3 ± 0.2.
灭菌后pH值：7.3 ± 0.2

III. Fluid Soybean–Casein Digest Medium液体大豆酪蛋白消化物培养基
Prepare as directed for Soybean–Casein Digest Medium under Sterility Tests 71 .按照无菌检查<71>项下规定的大豆酪蛋白消化物培养基的方法进行配制。

IV. Mannitol–Salt Agar Medium甘露醇-氯化钠琼脂培养基
Pancreatic Digest of Casein酪蛋白胰酶消化物    5.0 g
Peptic Digest of Animal Tissue动物组织胃蛋白酶消化物    5.0 g
Beef Extract牛肉浸膏    1.0 g
D-Mannitol D-甘露醇    10.0 g
Sodium Chloride氯化钠    75.0 g
Agar琼脂    15.0 g
Phenol Red酚磺酞    0.025 g
Water水    1000 mL
Mix, then heat with frequent agitation, and boil for 1 minute to effect solution.
混匀，加热伴以频繁搅拌，煮沸1分钟得均匀溶液。
pH after sterilization: 7.4 ± 0.2.
灭菌后pH值：7.4 ± 0.2。

V. Baird–Parker Agar Medium
Baird-Parker琼脂培养基
Pancreatic Digest of Casein酪蛋白胰酶消化物    10.0 g
Beef Extract牛肉浸膏    5.0 g
Yeast Extract酵母提取物    1.0 g
Lithium Chloride氯化锂    5.0 g
Agar琼脂    20.0 g
Glycine 甘氨酸    12.0 g
Sodium Pyruvate丙酮酸钠    10.0 g
Water水    950 mL
Heat with frequent agitation, and boil for 1 minute. Sterilize, cool to between 45 and 50 , and add 10 mL of sterile potassium tellurite solution (1 in 100) and 50 mL of egg-yolk emulsion. Mix intimately but gently, and pour into plates. (Prepare the egg-yolk emulsion by disinfecting the surface of whole shell eggs, aseptically cracking the eggs, and separating out intact yolks into a sterile graduated cylinder. Add sterile saline TS to obtain a 3 to 7 ratio of egg yolk to saline. Add to a sterile blender cup, and mix at high speed for 5 seconds.)
加热伴以频繁搅拌，并煮沸1分钟。灭菌，放凉至45 与50 之间，加入10mL无菌亚碲酸钾溶液（1:100）和50mL乳状蛋黄。轻轻混合至均匀，倒入平皿。（制作乳状蛋黄时，消毒带壳蛋的表面，以无菌方式打开鸡蛋，并将完整无缺的蛋黄分离至无菌量桶。加入无菌盐试液，以使蛋黄与盐水比例为3比7。加入至无菌搅拌器，高速混合5秒。）
pH after sterilization: 6.8 ± 0.2.
灭菌后pH值：6.8 ± 0.2。

VI. Vogel–Johnson Agar Medium
Vogel-Johnson 琼脂培养基
Pancreatic Digest of Casein酪蛋白胰酶消化物    10.0 g
Yeast Extract酵母提取物    5.0 g
Mannitol甘露醇    10.0 g
Dibasic Potassium Phosphate磷酸氢二钾    5.0 g
Lithium Chloride氯化锂    5.0 g
Glycine 甘氨酸    10.0 g
Agar琼脂    16.0 g
Phenol Red酚磺酞    25.0 mg
Water水    1000 mL
Boil the solution of solids for 1 minute. Sterilize, cool to between 45 and 50 , and add 20 mL of sterile potassium tellurite solution (1 in 100).
将这些固体的溶液煮沸1分钟。灭菌，放凉至45 与50 之间，加入20mL无菌亚碲酸钾溶液（1:100）。
pH after sterilization: 7.2 ± 0.2.
灭菌后pH值：7.2 ± 0.2。

VII. Cetrimide Agar Medium 十六烷三甲基溴化铵琼脂培养基
Pancreatic Digest of Gelatin白明胶胰酶消化物    20.0 g
Magnesium Chloride氯化镁    1.4 g
Potassium Sulfate硫酸钾    10.0 g
Agar琼脂    13.6 g
Cetyl Trimethylammonium Bromide (Cetrimide)
十六烷三甲基溴化铵    0.3 g
Glycerin甘油    10.0 mL
Water水    1000 mL
Dissolve all solid components in the water, and add the glycerin. Heat, with frequent agitation, and boil for 1 minute to effect solution.
将所有固体组分溶解于水中，并加入甘油。加热，伴以频繁搅拌，并煮沸1分钟以使其溶解。
pH after sterilization: 7.2 ± 0.2.
灭菌后pH值：7.2 ± 0.2。

VIII. Pseudomonas Agar Medium for Detection of Fluorescin
用于检测二氢荧光素的假单胞菌琼脂培养基
Pancreatic Digest of Casein酪蛋白胰酶消化物    10.0 g
Peptic Digest of Animal Tissue动物组织胃蛋白酶消化物    10.0 g
Anhydrous Dibasic Potassium Phosphate无水磷酸氢二钾    1.5 g
Magnesium Sulfate (MgSO4•7H2O)硫酸镁    1.5 g
Glycerin甘油    10.0 mL
Agar琼脂    15.0 g
Water水    1000 mL
Dissolve the solid components in the water before adding the glycerin. Heat, with frequent agitation, and boil for 1 minute to effect solution.
在加入甘油之前，先将固体组分溶解于水中。加入，加热，伴以频繁搅拌，并煮沸1分钟以使其溶解。
pH after sterilization: 7.2 ± 0.2.
灭菌后pH值：7.2 ± 0.2。

IX. Pseudomonas Agar Medium for Detection of Pyocyanin
用于检测绿脓菌素的假单胞菌琼脂培养基
Pancreatic Digest of Gelatin白明胶胰酶消化物    20.0 g
Anhydrous Magnesium Chloride无水氯化镁    1.4 g
Anhydrous Potassium Sulfate无水硫酸钾    10.0 g
Agar琼脂    15.0 g
Glycerin甘油    10.0 mL
Water水    1000 mL
Dissolve the solid components in the water before adding the glycerin. Heat, with frequent agitation, and boil for 1 minute to effect solution.
在加入甘油之前，先将固体组分溶解于水中。加入，加热，伴以频繁搅拌，并煮沸1分钟以使其溶解。
pH after sterilization: 7.2 ± 0.2.
灭菌后pH值：7.2 ± 0.2。

X. Fluid Lactose Medium 液体乳糖培养基
Beef Extract牛肉提取物    3.0 g
Pancreatic Digest of Gelatin白明胶胰酶消化物    5.0 g
Lactose乳糖    5.0 g
Water水    1000 mL
Cool as quickly as possible after sterilization.
灭菌后尽快放凉。
pH after sterilization: 6.9 ± 0.2.
灭菌后pH值：6.9 ± 0.2。

XI. Fluid Selenite–Cystine Medium液体亚硒酸-双硫丙氨酸（胱氨酸）培养基
Pancreatic Digest of Casein酪蛋白胰酶消化物    5.0 g
Lactose乳糖    4.0 g
Sodium Phosphate磷酸钠    10.0 g
Sodium Acid Selenite亚硒酸钠    4.0 g
L-Cystine L-胱氨酸    10.0 mg
Water水    1000 mL
Final pH: 7.0 ± 0.2.
最终pH值：7.0 ± 0.2。
Mix, and heat to effect solution. Heat in flowing steam for 15 minutes. Do not sterilize.
混匀，并加热以使其溶解。在流动蒸汽中加热15分钟，不得灭菌。

XII. Fluid Tetrathionate Medium液体连四硫酸盐（四连磺酸钠）培养基
Pancreatic Digest of Casein酪蛋白胰酶消化物    2.5 g
Peptic Digest of Animal Tissue动物组织胃蛋白酶消化物    2.5 g
Bile Salts牛胆盐    1.0 g
Calcium Carbonate碳酸钙    10.0 g
Sodium Thiosulfate硫代硫酸钠    30.0 g
Water水    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 solution of brilliant green (1 in 1000), and mix. Do not heat the medium after adding the brilliant green solution.
加入这些固体的溶液至沸腾。在使用当日，加入以5g碘化钾和6g碘溶解于20mL水中配制成的溶液。然后，加入10mL亮绿（1:1000）溶液，混匀。加入亮绿溶液后，不可加热培养基。

XIII. Brilliant Green Agar Medium亮绿琼脂培养基
Yeast Extract酵母提取物    3.0 g
Peptic Digest of Animal Tissue动物组织胃蛋白酶消化物    5.0 g
Pancreatic Digest of Casein酪蛋白胰酶消化物    5.0 g
Lactose乳糖    10.0 g
Sodium Chloride氯化钠    5.0 g
Sucrose蔗糖    10.0 g
Phenol Red酚磺酞    80 mg
Agar琼脂    20.0 g
Brilliant Green亮绿    12.5 mg
Water水    1000 mL
Boil the solution of solids for 1 minute. Sterilize just prior to use, melt the medium, pour into petri dishes, and allow to cool.
将这些固体的溶液煮沸1煮沸1分钟。在马上要使用之前，灭菌，融化培养基，倒入培养皿中，静置放凉。
pH after sterilization: 6.9 ± 0.2.
灭菌后pH值：6.9 ± 0.2。

XIV. Xylose–Lysine–Desoxycholate Agar Medium
木糖-赖氨酸-脱氧胆酸钠琼脂培养基
Xylose木糖    3.5 g
L-Lysine L-赖氨酸    5.0 g
Lactose乳糖    7.5 g
Sucrose蔗糖    7.5 g
Sodium Chloride氯化钠    5.0 g
Yeast Extract酵母提取物    3.0 g
Phenol Red酚磺酞    80 mg
Agar琼脂    13.5 g
Sodium Desoxycholate脱氧胆酸钠    2.5 g
Sodium Thiosulfate硫代硫酸钠    6.8 g
Ferric Ammonium Citrate柠檬酸铁铵    800 mg
Water水    1000 mL
Final pH: 7.4 ± 0.2.
最终pH值：7.4 ± 0.2。
Heat the mixture of solids and water, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to a water bath maintained at about 50 , and pour into plates as soon as the medium has cooled.
加热这些固体与水的混合物，伴以旋转搅拌，至恰好到沸点。不得过度加热或灭菌。立刻转移至保持在约50 的水浴，并在培养基刚刚放凉时，立即倒入平皿。

XV. Bismuth Sulfite Agar Medium亚硫酸铋琼脂培养基
Beef Extract牛肉提取物    5.0 g
Pancreatic Digest of Casein酪蛋白胰酶消化物    5.0 g
Peptic Digest of Animal Tissue动物组织胃蛋白酶消化物    5.0 g
Dextrose葡萄糖    5.0 g
Sodium Phosphate磷酸钠    4.0 g
Ferrous Sulfate硫酸亚铁    300 mg
Bismuth Sulfite Indicator亚硫酸铋指示剂    8.0 g
Agar琼脂    20.0 g
Brilliant Green亮绿    25 mg
Water水    1000 mL
Final pH: 7.6 ± 0.2.
最终pH值：7.6 ± 0.2。
Heat the mixture of solids and water, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to a water bath maintained at about 50 , and pour into plates as soon as the medium has cooled.
加热这些固体与水的混合物，伴以旋转搅拌，至恰好到沸点。不得过度加热或灭菌。立刻转移至保持在约50 的水浴，并在培养基刚刚放凉时，立即倒入平皿。

XVI. Triple Sugar–Iron–Agar Medium三糖-铁-琼脂培养基
Pancreatic Digest of Casein酪蛋白胰酶消化物    10.0 g
Pancreatic Digest of Animal Tissue动物组织胰酶消化物    10.0 g
Lactose乳糖    10.0 g
Sucrose蔗糖    10.0 g
Dextrose葡萄糖    1.0 g
Ferrous Ammonium Sulfate硫酸亚铁铵    200 mg
Sodium Chloride氯化钠    5.0 g
Sodium Thiosulfate硫代硫酸钠    200 mg
Agar琼脂    13.0 g
Phenol Red酚磺酞    25 mg
Water水    1000 mL
pH after sterilization: 7.3 ± 0.2.
灭菌后pH值：7.3 ± 0.2。

XVII. MacConkey Agar Medium麦康凯（MacConkey）琼脂培养基
Pancreatic Digest of Gelatin白明胶胰酶消化物    17.0 g
Pancreatic Digest of Casein酪蛋白胰酶消化物    1.5 g
Peptic Digest of Animal Tissue动物组织胃酶消化物    1.5 g
Lactose乳糖    10.0 g
Bile Salts Mixture牛胆盐混合物    1.5 g
Sodium Chloride氯化钠    5.0 g
Agar琼脂    13.5 g
Neutral Red中性红    30 mg
Crystal Violet结晶紫    1.0 mg
Water水    1000 mL
Boil the mixture of solids and water for 1 minute to effect solution.
将这些固体与水的混合物煮沸1分钟以使其溶解。
pH after sterilization: 7.1 ± 0.2.
灭菌后pH值：7.1 ± 0.2。

XVIII. Levine Eosin–Methylene Blue Agar Medium
Levine曙红亚甲基蓝琼脂培养基
Pancreatic Digest of Gelatin白明胶胰酶消化物    10.0 g
Dibasic Potassium Phosphate磷酸氢二钾    2.0 g
Agar琼脂    15.0 g
Lactose乳糖    10.0 g
Eosin Y黄色曙红    400 mg
Methylene Blue亚甲基蓝    65 mg
Water水    1000 mL
Dissolve the pancreatic digest of gelatin, the dibasic potassium phosphate, and the agar in the water, with warming, and allow to cool. Just prior to use, liquefy the gelled agar solution, add the remaining ingredients, as solutions, in the following amounts, and mix: for each 100 mL of the liquefied agar solution—5 mL of lactose solution (1 in 5), 2 mL of the eosin Y solution (1 in 50), and 2 mL of methylene blue solution (1 in 300). The finished medium may not be clear.
将白明胶胰酶消化物、磷酸氢二钾、琼脂溶解于水中，伴以加温，并静置放凉。在马上要使用之前，使凝胶状的琼脂液化，按如下数量以溶液形式加入剩余的成分，并混匀：对应于每100mL液化的琼脂溶液——5mL乳糖溶液（1:5）、2mL黄色曙红溶液（1:50）、2mL亚甲基蓝溶液（1:300）。完成后的培养基可能不够澄清。
pH after sterilization: 7.1 ± 0.2.
灭菌后pH值：7.1 ± 0.2。

XIX. Sabouraud Dextrose Agar Medium
Sabouraud（沙氏）葡萄糖琼脂培养基
Dextrose葡萄糖    40 g
Mixture of equal parts of Peptic Digest of Animal Tissue and Pancreatic Digest of Casein
同比配制的动物组织胃酶消化物和酪蛋白胰酶消化物    10 g
Agar琼脂    15 g
Water水    1000 mL
Mix, and boil to effect solution.
混匀，并煮沸以使其溶解。
pH after sterilization: 5.6 ± 0.2.
灭菌后pH值：5.6 ± 0.2。

XX. Potato Dextrose Agar Medium马铃薯葡萄糖琼脂培养基
Cook 300 g of peeled and diced potatoes in 500 mL of water prepared by distillation, filter through cheesecloth, add water prepared by distillation to make 1000 mL, and add the following:
取300g剥皮的马铃薯，放入500mL蒸馏水中煮溶，通过粗棉布过滤，加入蒸馏水至1000mL，并加入以下物质：
Agar琼脂    15 g
Glucose葡萄糖    20 g
Dissolve by heating, and sterilize.
加热熔解，并灭菌。
pH after sterilization: 5.6 ± 0.2.
灭菌后pH值：5.6 ± 0.2。
For use, just prior to pouring the plates, adjust the melted and cooled to 45 medium with sterile tartaric acid solution (1 in 10) to a pH of 3.5 ± 0.1. Do not reheat the pH 3.5 medium.
使用时，在马上要倒入平皿之前，以酒石酸溶液（1:10）将溶化后又放凉至45 的培养基调节到pH值3.5±0.1。调节pH后不得再加热。

SAMPLING取样
Provide separate 10-mL or 10-g specimens for each of the tests called for in the individual monograph.
为每一个在具体的各论中所要求的检验，提供单独的10-mL或10-g样品。

PROCEDURE步骤
Prepare the specimen to be tested by treatment that is appropriate to its physical characteristics and that does not alter the number and kind of microorganisms originally present, in order to obtain a solution or suspension of all or part of it in a form suitable for the test procedure(s) to be carried out.
For a solid that dissolves to an appreciable extent but not completely, reduce the substance to a moderately fine powder, suspend it in the vehicle specified, and proceed as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli.
配制供试样品时，使用适合其物理特性，并且不会改变其原本已有的微生物数量与种类的处理方式，以便以适合将要进行的试验步骤的状态获得全部或部分样品的溶液或者悬浮液。对于能够溶解到相当的程度，但无法彻底溶解的固体，将该物质研磨成大小适中的细粉，将其悬浮于指定的载体中，并如好氧微生物总数检查法、金黄色葡萄球菌和绿脓杆菌检查法、沙门氏菌和大肠杆菌检查法项下的规定继续进行。

For a fluid specimen that consists of a true solution, or a suspension in water or a hydroalcoholic vehicle containing less than 30 percent of alcohol, and for a solid that dissolves readily and practically completely in 90 mL of pH 7.2 Phosphate Buffer or the media specified, proceed as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli.
对于液体样品，其中包括了能在水中或在不低于30%乙醇的醇水载体中形成的真溶液或者悬浮液，以及比较容易且彻底地溶解在90mLpH值7.2磷酸盐缓冲液或指定介质中的固体样品，如好氧微生物总数检查法、金黄色葡萄球菌和绿脓杆菌检查法、沙门氏菌和大肠杆菌检查法项下的规定继续进行。

For water-immiscible fluids, ointments, creams, and waxes, prepare a suspension with the aid of a minimal quantity of a suitable, sterile emulsifying agent (such as one of the polysorbates), using a mechanical blender and warming to a temperature not exceeding 45 , if necessary, and proceed with the suspension as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli.
对于与水不混溶的液体、油膏、乳膏、蜡状物，以最小剂量的适合的无菌乳化剂，使用机械混合器并在需要时加温到不超过45 ，来制备悬浮液，并如好氧微生物总数检查法、金黄色葡萄球菌和绿脓杆菌检查法、沙门氏菌和大肠杆菌检查法项下的规定使用该悬浮液继续进行。

For a fluid specimen in aerosol form, chill the container in an alcohol- mixture for approximately 1 hour, cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible, and transfer the quantity of test material required for the procedures specified in one of the two preceding paragraphs, as appropriate. Where 10.0 g or 10.0 mL of the specimen, whichever is applicable, cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residues. If the results of the test are inconclusive or doubtful, repeat the test with a specimen from 20 more containers.
对于以气体中悬浮颗粒状态存在的液体样品，在乙醇-干冰混合物中冷却约1小时，打开该容器，令其达到室温，使推进剂散发掉，或者加温（如果可行）以驱推进剂，并且如果适合则按照下面两个段落中一段所指定的此步骤所必需的数量转移供试品。如果10.0g或10.0mL（不论那种单位适合此种情况）供试品无法从10个气体悬浮颗粒状态的容器中获得，将10个冷却后的容器的全部内容物转入培养基，使推进剂散发掉，并使用剩余物继续进行该试验。如果试验的结果是不确定的或者可疑的，使用来自20个容器的供试品重复该试验。

Total Aerobic Microbial Count好氧微生物总数检查法
For specimens that are sufficiently soluble or translucent to permit use of the Plate Method, use that method; otherwise, use the Multiple-Tube Method. With either method, first dissolve or suspend 10.0 g of the specimen if it is a solid, or 10 mL, accurately measured, if the specimen is a liquid, in pH 7.2 Phosphate Buffer, Fluid Soybean–Casein Digest Medium, or Fluid Casein Digest–Soy Lecithin-Polysorbate 20 Medium to make 100 mL. For viscous specimens that cannot be pipeted at this initial 1:10 dilution, dilute the specimen until a suspension is obtained, i.e., 1:50 or 1:100, etc., that can be pipeted. Perform the test for absence of inhibitory (antimicrobial) properties as described under Preparatory Testing before the determination of Total Aerobic Microbial Count. Add the specimen to the medium not more than 1 hour after preparing the appropriate dilutions for inoculation.
对于能够充分溶解或透明、可以采用平皿法的样品，需使用该方法；否则，使用多管法。不管使用哪一种方法，首先将10.0g固体样品或精密量取的10mL液体样品，放入pH 7.2的磷酸盐缓冲液，或液体大豆酪蛋白消化物培养基，或液体酪蛋白消化物-大豆卵磷脂-聚山梨酯20中，制成100mL的溶液或悬浮液。对于在此初始的1:10溶液中无法移取的黏稠样品，稀释该样品直到得到可以移取的悬浮液，例如1:50或1:100等。如好氧微生物总数检查法之前的预备试验项下的描述，进行证实样品不具备抑制（抗微生物）特性的试验。在用于接种的适当稀释液制备之后1小时内，将样品加入培养基。

PLATE METHOD 平皿法
Dilute further, if necessary, the fluid so that 1 mL will be expected to yield between 30 and 300 colonies. Pipet 1 mL of the final dilution onto each of two sterile petri dishes. Promptly add to each dish 15 to 20 mL of Soybean–Casein Digest Agar Medium that previously has been melted and cooled to approximately 45 . Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and allow the contents to solidify at room temperature. Invert the petri dishes, and incubate for 48 to 72 hours. Following incubation, examine the plates for growth, count the number of colonies, and express the average for the two plates in terms of the number of microorganisms per g or per mL of specimen. If no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the specimen, express the results as “less than 10 microorganisms per g or per mL of specimen.”
如果需要，进一步稀释液体，以便每1mL液体预期能够生成30到300个菌落。移取1mL最终稀释液到1个无菌平皿，共做两个平皿的平行样。迅速地在每个平皿里加入15至20mL此前已经加热融化并放凉到约45 的大豆酪蛋白消化物琼脂培养基。盖上皿盖，倾斜或旋转平皿以使供试品和培养基混匀，然后静置于室温使内容物凝固。倒置平皿，培养48至72小时。培养完成后，观察平皿内微生物生长状况，进行菌落计数，将两个平皿的平均值以每g或mL供试品中微生物的数量来表达。如果在代表初始的1:10样品稀释度的平皿中未检出微生物，则结果表达为“每g或每mL样品中微生物数量少于10个”。

MULTIPLE-TUBE METHOD 多管法
Into each of fourteen test tubes of similar size place 9.0 mL of sterile Fluid Soybean–Casein Digest Medium. Arrange twelve of the tubes in four sets of three tubes each. Put aside one set of three tubes to serve as the controls. Into each of three tubes of one set (“100”) and into a fourth tube (A) pipet 1 mL of the solution or suspension of the specimen, and mix. From tube A, pipet 1 mL of its contents into the one remaining tube (B) not included in a set, and mix. These two tubes contain 100 mg (or 100 µL) and 10 mg (or 10 µL) of the specimen, respectively. Into each of the second set (“10”) of three tubes pipet 1 mL from tube A, and into each tube of the third set (“1”) pipet 1 mL from tube B. Discard the unused contents of tubes A and B. Close well, and incubate all of the tubes. Following the incubation period, examine the tubes for growth: the three control tubes remain clear and the observations in the tubes containing the specimen, when interpreted by reference to Table 1, indicate the most probable number of microorganisms per g or per mL of specimen.
向14支大小相同的试验试管的每一支中，放置9mL无菌液体大豆酪蛋白消化物培养基。将其中12支试管分为4组，每组3支。将其中1组的3支试管放在一边作为对照。向1组（标记为“100”）3支试管中的每一支和第4支试管（标记为A）中，移取1mL供试品的溶液或悬浮液，并混匀。从试管A中，移取1mL其内容物至剩下的一个没有包括在任何一组中的试管（标记为B）中，并混匀。这两个试管分别含有100mg（或100µL）和10mg（或10µL）供试品。向第2组（标记“10”）3支试管的每一支中，移取1mL来自试管A的液体，并向第3组（标记“1”）3支试管的每一支中，移取1mL来自试管B的液体。丢弃试管A和B的内容物。封好并培养所有的试管。在培养期结束之后，观察试管内微生物生长状况：3支对照试管应保持澄清；样品试管的观察数据，根据表1的计数规则，指出每g或每mL样品中最可能含有的微生物数量。

Table 1. Most Probable Total Count by Multiple-Tube Method
表1：以多管法测定的最可能细菌总数
Observed Combinations of Numbers of
Tubes Showing Growth in Each Set
所观察到的每一组中显示微生物生长的试管数的组合    Most Probable
Number of
Microorgan-
isms per g or per mL
每g或每mL中
最可能微生物数量
No. of mg (or mL) of Specimen per Tube
每个试管中供试品的克（毫升）数   
100
(100 µL)    10
(10 µL)    1
(1 µL)   
3    3    3    >1100
3    3    2    1100
3    3    1    500
3    3    0    200
3    2    3    290
3    2    2    210
3    2    1    150
3    2    0    90
3    1    3    160
3    1    2    120
3    1    1    70
3    1    0    40
3    0    3    95
3    0    2    60
3    0    1    40
3    0    0    23

Test for Staphylococcus aureus and Pseudomonas aeruginosa
金黄色葡萄球菌和绿脓杆菌检查法
To the specimen add Fluid Soybean–Casein Digest Medium to make 100 mL, mix, and incubate. Examine the medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of Vogel–Johnson Agar Medium (or Baird–Parker Agar Medium, or Mannitol–Salt Agar Medium) and of Cetrimide Agar Medium, each plated on petri dishes. Cover and invert the dishes, and incubate. If, upon examination, none of the plates contains colonies having the characteristics listed in Tables 2 and 3 for the media used, the test specimen meets the requirements for freedom from Staphylococcus aureus and Pseudomonas aeruginosa.
向供试样品中加入液体大豆酪蛋白消化物培养基，制成100mL，混匀，并培养。观察试管内微生物生长状况，如果有微生物生长，使用接种环挑取少许培养物在Vogel-Johnson琼脂培养基（或Baire-parker琼脂培养基，或甘露醇氯化钠琼脂培养基）和十六烷三甲基溴化铵琼脂培养基表面上划线接种，每一种培养基都倾倒在平皿上。盖上皿盖倒置培养。培养后，如果观察的时候，没有一个平皿出现的菌落具有表2和表3所列出的在特定培养基上的生长特征，则可判断此样品符合要求，不含有金黄色葡萄球菌和绿脓杆菌。

Table 2. Morphologic Characteristics of Staphylococcus aureus on Selective Agar Media表2：金黄色葡萄球菌在选择性琼脂培养基上的形态学特征
Selective Medium
可选择的培养基    Characteristic Colonial Morphology
菌落的形态学特征    Gram Stain
革兰氏染色
属性
Vogel-Johnson
Agar Medium
Vogel-Johnson
琼脂培养基    Black Surrounded by yellow zone
黄色圈围绕的黑色菌落    Positive cocci
(in clusters)
阳性球菌
（群生）
Mannital-Salt
Agar Medium
甘露醇-氯化钠
琼脂培养基    Yellow colonies with yellow zones
黄色圈围绕的黄色菌落    Positive cocci
(in clusters)
阳性球菌
（群生）
Baird-Parker
Agar Medium
Baire-parker
琼脂培养基    Black, shiny, surrounded by clear zones 2 to 5 mm
黑色，有光泽，周围有2至5mm透明圈    Positive cocci
(in clusters)
阳性球菌
（群生）

Table 3. Morphologic Characteristics of Pseudomonas aeruginosa on Selective and Diagnostic Agar Media
表3：绿脓杆菌在选择性和诊断性琼脂培养基上的形态学特征
Selective Medium
选择性培养基    Characteristic Colonial
Morphology
菌落的形态学特征    Fluorescence in
UV Light
紫外荧光反应    Oxidase Test
氧化酶
试验    Gram Stain
革兰氏
染色属性
Centrimide Agar Medium
十六烷三甲基溴化铵琼脂培养基    Generally greenish
通常为绿色    Greenish
绿色    Positive
阳性    Negative rods
阴性杆菌
Pseudomonas Agar Medium for
Detection of Fluorescin
用于检测二氢荧光素的假单胞菌琼脂培养基    Generally colorless to yellowish
通常为无色至微黄    Yellowish
微黄    Positive
阳性    Negative rods
阴性杆菌
Pseudomonas Agar Medium for
Detection of Pyocyanin
用于检测绿脓菌素的假单胞菌琼脂培养基    Generally greenish
通常为绿色    Blue
兰色    Positive
阳性    Negative rods
阴性杆菌

Coagulase Test (for Staphylococcus aureus)— With the aid of an inoculating loop, transfer representative suspect colonies from the agar surfaces of the Vogel–Johnson Agar Medium (or Baird–Parker Agar Medium, or Mannitol–Salt Agar Medium) to individual tubes, each containing 0.5 mL of mammalian, preferably rabbit or horse, plasma with or without suitable additives. Incubate in a water bath at 37 , examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. Test positive and negative controls simultaneously with the unknown specimens. If no coagulation in any degree is observed, the specimen meets the requirements of the test for absence of Staphylococcus aureus.
凝固酶试验（针对金黄色葡萄球菌）：用接种环从Vogel-Johnson琼脂培养基（或Baire-parker琼脂培养基，或Mannitol-Salt甘露醇-盐琼脂培养基）的琼脂表面挑取具代表性的可疑菌落，每个菌落分别接种到单独的试管，试管内含有0.5mL哺乳动物（最好是兔子或马）血浆，其中适当的添加剂可有可无。在37 水浴中培养，3小时开始观察直至24小时。同时做阳性对照管和阴性对照管。如果没有发现任何程度的凝结，判断样品符合不得检出金黄色葡萄球菌的检验要求。

Oxidase and Pigment Tests (for Pseudomonas aeruginosa)— With the aid of an inoculating loop, streak representative suspect colonies from the agar surface of Cetrimide Agar Medium on the agar surfaces of Pseudomonas Agar Medium for Detection of Fluorescin and Pseudomonas Agar Medium for Detection of Pyocyanin contained in petri dishes. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be inoculated from a separate colony. Cover and invert the inoculated media, and incubate at 35 ± 2 for not less than three days. Examine the streaked surfaces under UV light. Examine the plates to determine whether colonies having the characteristics listed in Table 3 are present.
氧化酶和色素试验（针对绿脓杆菌）：用接种环从十六烷三甲基溴化铵琼脂培养基表面挑取具代表性的可疑菌落，接种到用于检测二氢荧光素的假单胞菌琼脂培养基和用于检测绿脓菌素的假单胞菌琼脂培养基上，每个可疑菌落分别接种。若需要接种的菌落数太多，可将接种的平皿分成四等分，每个区域接种一个可疑菌落。将接种后的培养基，盖上皿盖并倒置，在35 ± 2 条件下培养至少3天。用紫外光下观察接种菌落的表面。观察平皿以确定是否有具备表3所列的特征的菌落存在。

Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with N,N-dimethyl-p-phenylenediamine dihydrochloride: if there is no development of a pink color, changing to purple, the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa. The presence of Pseudomonas aeruginosa may be confirmed by other suitable cultural and biochemical tests, if necessary.
可以通过氧化酶试验，确认某个可疑菌落是绿脓杆菌。在菌落培养物上放置，或将菌落培养物转移到，预先浸润了二盐酸二甲基对苯二胺的条状或圆形滤纸：如果没有出现粉色并逐渐变为紫色，则该样品符合不得检出绿脓杆菌的要求。必要时，还可以使用其他适宜的培养和生化试验来确认绿脓杆菌的存在。

Test for Salmonella species and Escherichia coli
沙门氏菌属和大肠杆菌检查法
To the specimen, contained in a suitable vessel, add a volume of Fluid Lactose Medium to make 100 mL, and incubate. Examine the medium for growth, and if growth is present, mix by gently shaking. Pipet 1-mL portions into vessels containing, respectively, 10 mL of Fluid Selenite–Cystine Medium and Fluid Tetrathionate Medium, mix, and incubate for 12 to 24 hours. (Retain the remainder of the Fluid Lactose Medium.)
向放在适宜容器中的样品，加入液体乳糖培养基制成100mL，培养。检查培养基中的生长情况，若发现生长物，轻轻振荡混合均匀。用移液器吸取分别1mL培养物，并加入到10mL液体亚硒酸-双硫丙氨酸（胱氨酸）培养基和液体连四硫酸盐（四连磺酸钠）培养基中，混匀并培养12至24小时。（同时需保留该液体乳糖培养基的剩余物）

Test for Salmonella Species— By means of an inoculating loop, streak portions from both the selenite-cystine and tetrathionate media on the surface of Brilliant Green Agar Medium, Xylose–Lysine–Desoxycholate Agar Medium, and Bismuth Sulfite Agar Medium contained in petri dishes. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 4, the specimen meets the requirements of the test for absence of the genus Salmonella.
沙门氏菌属试验：用接种环从液体亚硒酸-双硫丙氨酸（胱氨酸）培养基和液体连四硫酸盐（四连磺酸钠）培养基中挑取培养物，分别划线接种到亮绿琼脂培养基、木糖-赖氨酸-脱氧胆酸钠琼脂培养基、亚硫酸铋琼脂培养基的表面。盖上皿盖并倒置平皿，培养。在观察时，如果未发现符合表4所描述的菌落特征，则该样品符合不得检出沙门氏菌的要求。

Table 4. Morphologic Characteristics of Salmonella Species on Selective Agar Media表4：沙门氏菌属在选择性琼脂培养基上的形态学特征
Selective Medium
选择性培养基    Characteristic Colonial Morphology
菌落形态学特征
Brilliant Green
Agar Medium
亮绿琼脂培养基    Small, transparent, colorless or pink to white opaque (frequently surrounded by pink to red zone)
细小、透明、无色或粉色到白色不透明（常被粉色或红色环带环绕）
Xylose-Lysine-
Desoxycholate
Agar Medium
木糖-赖氨酸-脱氧胆酸钠琼脂培养基    Red, with or without black centers
红色，有或无黑色中心
Bismuth Sulfite
Agar Medium
亚硫酸铋琼脂培养基    Black or green
黑色或绿色
If colonies of Gram-negative rods matching the description in Table 4 are found, proceed with further identification by transferring representative suspect colonies individually, by means of an inoculating wire, to a butt-slant tube of Triple Sugar–Iron–Agar Medium by first streaking the surface of the slant and then stabbing the wire well beneath the surface. Incubate. If examination discloses no evidence of tubes having alkaline (red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from hydrogen sulfide production), the specimen meets the requirements of the test for the absence of the genus Salmonella.*
如果发现有符合表4所描述的革兰氏阴性杆菌菌落，继续进行进一步的鉴别。使用接种针，将单个可疑聚落挑起并转移到三糖-铁-琼脂培养基高层斜面上，方法如下：首先挑起斜面的表面，然后将接种针完全刺入表面之下。培养。如果通过观察没有发现试管呈现碱性（红色）底部呈现酸性（黄色）（有或没底部伴随氢化硫黑色产物），则该样品符合未检出沙门氏菌*属的要求。

Test for Escherichia coli— By means of an inoculating loop, streak a portion from the remaining Fluid Lactose Medium on the surface of MacConkey Agar Medium. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 5 for this medium, the specimen meets the requirements of the test for absence of Escherichia coli.
大肠杆菌检查：使用接种环，从剩余的液体乳糖培养基中液挑取一部分培养物接种到麦康凯琼脂培养基的表面上。盖上皿盖，倒置培养。观察结果，若没有菌落符合表5所列出的在此培养基上的形态学特征，则该样品符合未检出大肠杆菌的要求。

Table 5. Morphologic Characteristics of Escherichia coli on MacConkey Agar Medium表5：大肠杆菌在麦康凯琼脂培养基上的形态学特征
Gram Stain
革兰氏染色    Characteristic Colonial Morphology
菌落的形态学特征
Negative rods
(cocco-bacilli)
阴性杆菌
(球杆菌)    Brick-red; may have surrounding zone of precipitated bile
红色块状物；可能环绕着胆汁沉淀环
If colonies matching the description in Table 5 are found, proceed with further identification by transferring the suspect colonies individually, by means of an inoculating loop, to the surface of Levine Eosin–Methylene Blue Agar Medium, plated on petri dishes. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be seeded from a separate colony. Cover and invert the plates, and incubate. Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light, the specimen meets the requirements of the test for the absence of Escherichia coli. The presence of Escherichia coli may be confirmed by further suitable cultural and biochemical tests.
如果发现有符合表5所描述的菌落，继续进行进一步的鉴别。使用接种环，将单个可疑菌落分别挑取接种到Levine曙红亚甲基蓝琼脂培养基的表面上。若需要接种的菌落数太多，可将每个培养基等分四份，每个区域接种一个单独的菌落。盖上皿盖，倒置培养。观察，如没有菌落展现出在反射光下的特征性金属光泽和在透射光线下的蓝黑色外观，则该样品符合未检出大肠杆菌的要求。大肠杆菌的存在情况可以通过进一步的适宜的培养和生化试验来确认。

Total Combined Molds and Yeasts Count 霉菌与酵母菌联合总数检查
Proceed as for the Plate Method under Total Aerobic Microbial Count, except for using the same amount of Sabouraud Dextrose Agar Medium or Potato Dextrose Agar Medium, instead of Soybean Casein Digest Medium, and except for incubating the inverted petri dishes for 5 to 7 days at 20 to 25 .
采用好氧微生物总数检查法下的平皿法进行检测，但培养基需使用同样体积的Sabouraud葡萄糖琼脂培养基或马铃薯葡萄糖琼脂培养基，以代替大豆酪蛋白消化物培养基，并且应在倒置的平皿中于20 至25 培养5至7天。

Retest 复检
For the purpose of confirming a doubtful result by any of the procedures outlined in the foregoing tests following their application to a 10.0-g specimen, a retest on a 25-g specimen of the product may be conducted. Proceed as directed for Procedure, but make allowance for the larger specimen size.
为了确认按照前述试验中所描述的步骤每次使用10.0g供试品得出的不确定结果，可以使用25g供试品进行复检。按照步骤中的规定继续进行，但需改用此较大的样品量。

伙计，你这不是32版的，是30版的。