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English to Chinese: Microbial Limit Tests General field: Science Detailed field: Chemistry; Chem Sci/Eng
Source text - English The Microbial Limit Tests are designed to perform the qualitative and quantitative estimations of specific viable microorganisms present in samples. It includes tests for total viable count (bacteria and fungi) and Escherichia coli. The most care must be taken in performing the tests, so that microbial contamination from the outside can be avoided. When test samples have antimicrobial activity or when they include antimicrobial substances, these antimicrobial properties must be eliminated by dilution, filtration, neutralization, inactivation, or other appropriate means. The tests should be conducted for samples prepared by mixing multiple portions randomly chosen from individual ingredients or products. When samples are diluted with fluid medium, the tests must be conducted quickly. Due attention must be paid to the effective quality control and the prevention of biohazard.
Total viable aerobic count
This test is to determine mesophilic bacteria and fungi which grow under aerobic conditions. Psychrophilic, thermophilic, basophilic, and anaerobic bacteria, and microorganisms which require specific ingredients for growth may give a negative result, even if they exist in a significant number. There are four methods for this test: membrane filtration method, pour plate method, spread plate method, and serial dilution method (most probable number method). An appropriate method should be taken from among these four, depending on purposes. If automated methods are comparable or superior in sensitivity and accuracy to the methods given here, they may be used. Different media and incubation temperature are required for the growth of bacteria and fungi (molds and yeasts). The serial dilution method is applicable only to bacteria.
Preparation of test fluids
To dissolve or dilute the sample, use phosphate buffer (pH 7.2), sodium chloride-peptone buffer solution, or fluid medium used for the test. Unless otherwise specified, use 10 g or 10 ml of the sample. However, a different weight or volume of the sample should be used, depending on the nature of the sample. Adjust the test fluid to pH 6－8. Use the test fluid within one hour after preparation.
Fluid samples or soluble solid samples
Take 10 g or 10 ml of the sample and mix with the buffer or fluid medium given above to make 100 ml. Use this mixture as the test fluid. For a fluid sample including insoluble substances, shake well just before mixing make a homogeneous suspension.
Insoluble solid samples
Take 10 g of the sample, grind to a fine powder, and suspend it in the buffer or fluid medium given above to make 100 ml. Use this suspension as the sample fluid. A larger volume of the buffer or fluid medium than specified here may be used to make a suspension, depending on the nature of the sample. If necessary, a blender may be used to disperse the insoluble particles well in the suspension. Also, an appropriate surfactant (e.g., 0.1% w/v polysorbate 80) may be added to help dissolve the sample.
For semisolid samples and liquids consisting mainly of lipid, take 10 g or 10 ml of the sample, emulsify the sample in the buffer or fluid medium given above using a surfactant such as polysorbate 20 or polysorbate 80, and make to 100 ml. Use this emulsified sample as the sample fluid. If necessary, warm at a temperature not exceeding 45℃ to emulsify the sample. Avoid warming for not longer than 30 minutes.
(1) Membrane Filtration Method
This method is applied to the sample which contains antimicrobial substances. Use membrane filters with a pore size of 0.45 µm or less. Filters about 50 mm across are recommended, but other sizes may be used. Sterilize the filters, filtration apparatus, media, and other apparatus used. Usually, measure two test fluids of 10 ml each, pass each sample through a separate filter. Dilute the pretreated test fluid if the bacteria concentration is high, so that 10－100 colonies can develop per filter. After filtration, wash each filter three times or more with an appropriate liquid such as phosphate buffer, sodium chloride-peptone buffer, or fluid medium. If the filter used is not about 50 mm in diameter, use an appropriate volume of washing, depending on the size of the filter. If the sample includes lipid, polysorbate 80 or an appropriate emulsifier may be added to the washings. After filtration, for bacteria detection, place the two filters on a plate of soybean-casein digest agar medium, and for fungi detection, add an antibiotic to the medium and place them on a plate of one of Sabouraud glucose agar, potato-dextrose agar, or GP agar media. Incubate the plates at least for 5 days at 30-35℃ for bacteria detection and at 20-25℃ for fungi detection, and count the number of colonies. If counts obtained are considered to be reliable in shorter incubation time than 5 days, these counts may be adopted for calculation of the viable count.
(2) Pour Plate Method
Use petri dishes 9-10 cm in diameter. Use at least 2 agar media for each dilution. Take 1 ml of the test fluid or its dilution into each petri dish aseptically, add to each dish 15－20 ml of sterilized agar medium, previously melted and kept below 45℃, and mix. For bacteria detection, use soybean-casein digest agar medium and for fungi detection, use one of Sabouraud glucose agar, potato-dextrose agar, and GP agar media, to which antibiotic has previously been added. After the agar solidifies, incubate at least for 5 days at 30－35℃ for bacteria detection and at 20－25℃ for fungi detection. If a large number of colonies develop, calculate viable counts based on counts obtained from plates with not more than 300 colonies per plate for bacteria detection and from plates with not more than 100 colonies per plate for fungi detection. If counts are considered to be reliable in a shorter incubation time than 5 days, these counts may be adopted.
(3) Spread Plate Method
Place 0.05-0.2 ml of the test fluid on the solidified and dried surface of the agar medium and spread it uniformly using a spreader. Proceed under the same conditions
as for the Pour Plate Method, especially about petri dishes, agar media, incubation temperature and time, and calculation method.
(4) Serial Dilution Method (Most Probable Number Method)
Use 12 test tubes: 9 containing 9 ml of soybean-casein digest medium each and 3 containing 10 ml of the same medium each for control. Prepare dilutions using the 9 tubes. First, add 1 ml of the test fluid to each of three test tubes and mix to make 10 times dilutions. Second, add 1 ml of each of the 10-times dilutions to each of another three test tubes and mix to make 100-times dilutions. Third, add 1 ml of each of the 100-times dilutions to each of the remaining three test tubes and mix to make 1,000-times dilutions. Incubate all 12 test tubes for at least 5 days at 30 - 35℃. No microbial growth should be observed for the control test tubes. If the determination of the result is difficult or if the result is not reliable, take a 0.1ml fluid from each of the
9 test tubes and place it to an agar medium or fluid medium, incubate all media for
24－72 hours at 30－35℃, and check them for the absence or presence of microbial growth. Calculate the most probable number of microorganisms per ml or gram of the sample, using the table given below.
Translation - Chinese 微生物限度检测旨在对样品中存在的特定活动微生物进行定性和定量估计。它包括对总活菌数（细菌和真菌）和大肠杆菌的测定。在进行测试的过程中必须非常小心，以避免来自外界微生物的污染。 当测试样品具有抗微生物活性或含有抑菌物质时，必须通过稀释，过滤，中和，失活或其他适当方法来消除这些抗菌性能。测试所用的样品必须是从某成分或产品中随机挑选的多个部分的混合体。 在样品用液体培养基稀释的情况下，测试必须快速进行。有效的质量控制和生物危害预防必须给予重视。