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?61? MICROBIAL LIMIT TESTS
This chapter provides tests for the estimation of the number of viable aerobic microorganisms present and for freedom from designated microbial species in pharmaceutical articles of all kinds, from raw materials to the finished forms. An automated method may be substituted for the tests presented here, provided it has been properly validated as giving equivalent or better results. In preparing for and in applying the tests, observe aseptic precautions in handling the specimens. Unless otherwise directed, where the procedure specifies simply ―incubate,‖ hold the container in air that is thermostatically controlled at a temperature between 30 and 35, for a period of 24 to 48 hours. The term ―growth‖ is used in a special sense herein, i.e., to designate the presence and presumed proliferation of viable microorganisms.
Preparatory Testing
The validity of the results of the tests set forth in this chapter rests largely upon the adequacy of a demonstration that the test specimens to which they are applied do not, of themselves, inhibit the multiplication, under the test conditions, of microorganisms that may be present. Therefore, preparatory
to conducting the tests on a regular basis and as circumstances require subsequently, inoculate diluted specimens of the material to be tested with separate viable cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella. This can be done by adding 1 mL of not less than 10-3 dilution of a 24-hour broth culture of the microorganism to the first dilution (in pH 7.2 Phosphate Buffer, Fluid Soybean–Casein Digest Medium, or Fluid Lactose Medium) of the test material and following the test procedure. Failure of the organism(s) to grow in the relevant medium invalidates that portion of the examination and necessitates a modification of the procedure by (1) an increase in the volume of diluent, the quantity of test material remaining the same, or by (2) the incorporation of a sufficient quantity of suitable inactivating agent(s) in the diluents, or by (3) an appropriate combination of modifications (1) and (2) so as to permit growth of the inocula.
The following are examples of ingredients and their concentrations that may be added to the culture medium to neutralize inhibitory substances present in the sample: soy lecithin, 0.5%; and polysorbate 20, 4.0%. Alternatively, repeat the test as described in the preceding paragraph, usingFluid Casein Digest–Soy Lecithin–Polysorbate 20 Medium to demonstrate neutralization of preservatives or other antimicrobial agents in the test material. Where inhibitory substances are contained in the product and the latter is soluble, a
suitable, validated adaptation of a procedure set forth in the
section Membrane Filtration under Test for Sterility of the Product to be Examined under Sterility Tests ?71?, may be used.
If in spite of the incorporation of suitable inactivating agents and a substantial increase in the volume of diluent, it is still not possible to recover the viable cultures described above and where the article is not suitable for employment of membrane filtration, it can be assumed that the failure to isolate the inoculated organism is attributable to the bactericidal activity of the product. This information serves to indicate that the article is not likely to be contaminated with the given species of microorganism. Monitoring should be continued in order to establish the spectrum of inhibition and bactericidal activity of the article.
Buffer Solution and Media
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
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.
For use, dilute the Stock Solution with water in the ratio of 1 to 800, and sterilize.
Media
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.
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.
solution.
pH after sterilization: 7.4 ± 0.2.
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.)
pH after sterilization: 6.8 ± 0.2.
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).
pH after sterilization: 7.2 ± 0.2.
frequent agitation, and boil for 1 minute to effect solution.
pH after sterilization: 7.2 ± 0.2.
VIII. Pseudomonas Agar Medium for Detection of Fluorescin
with frequent agitation, and boil for 1 minute to effect solution. pH after sterilization: 7.2 ± 0.2.
with frequent agitation, and boil for 1 minute to effect solution. pH after sterilization: 7.2 ± 0.2.
pH after sterilization: 6.9 ± 0.2.
Final pH: 7.0 ±
Mix, and heat to effect solution. Heat in flowing steam for 15 minutes. Do not sterilize.
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.
medium, pour into petri dishes, and allow to cool.
pH after sterilization: 6.9 ± 0.2.
Final pH: 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.
Final pH: 7.6 ±
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.
pH after sterilization: 7.1 ± 0.2.
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.
pH after sterilization: 7.1 ± 0.2.
pH after sterilization: 5.6 ± 0.2.
pH after sterilization: 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.
Sampling
Provide separate 10-mL or 10-g specimens for each of the tests called for in the individual monograph.
Procedure
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.
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.
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.
For a fluid specimen in aerosol form, chill the container in an alcohol-dry ice 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.
Total Aerobic Microbial Count
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.
PLATE METHOD
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.‖
MULTIPLE-TUBE METHOD
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.
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.
Table 2. Morphologic Characteristics of Staphylococcus aureus on Selective
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.
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.
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 ofPseudomonas aeruginosa. The presence of Pseudomonas aeruginosa may be confirmed by other suitable cultural and biochemical tests, if necessary.
Test for Salmonella species and Escherichia coli
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.)
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.
Table 4. Morphologic Characteristics of Salmonella Species on Selective
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.*
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.
Table 5. Morphologic Characteristics of Escherichia coli on MacConkey
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.
Total Combined Molds and Yeasts Count
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.
Retest
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.
* Additional, confirmatory evidence may be obtained by use of procedures set forth in Official Methods of Analysis of the AOAC, 12th ed. (1975), sections 46.013-46.026.
常见的微生物检测 方法
摘要：微生物的检测，无论在理论研究还是在生产实践中都具有重要的意义，本文分生长量测定法，微生物计数法，生理指标法和商业化快速微生物检测简要介绍了利用微生物重量，体积，大小，生理代谢物等指标的二十余种常见的检测方法，简要介绍了这些方法的原理，应用范围和优缺点。 概述： 一个微生物细胞在合适的外界条件下，不断的吸收营养物质，并按自己的代谢方式进行新陈代谢。如果同化作用的速度超过了异化作用，则其原生质的总量（重量，体积，大小）就不断增加，于是出现了个体的生长现象。如果这是一种平衡生长，即各细胞组分是按恰当的比例增长时，则达到一定程度后就会发生繁殖，从而引起个体数目的增加，这时，原有的个体已经发展成一个群体。随着群体中各个个体的进一步生长，就引起了这一群体的生长，这可从其体积、重量、密度或浓度作指标来衡量。微生物的生长不同于其它生物的生长，微生物的个体生长在科研上有一定困难，一般情况下也没有实际意义。微生物是以量取胜的，因此，微生物的生长一般指群体的扩增。微生物的生长繁殖是其在内外各种环境因素相互作用下的综合反映。因此生长繁殖情况就可作为研究各种生理生化和遗传等问题的重要指标，同
时，微生物在生产实践上的各种应用或是对致病，霉腐微生物的防治都和她们的生长抑制紧密相关。因此有必要介绍一下微生物生长情况的检测方法。既然生长意味着原生质含量的增加，因此测定的方法也都直接或间接的以次为根据，而测定繁殖则都要建立在计数这一基础上。微生物生长的衡量，能够从其重量，体积，密度，浓度，做指标来进行衡量。 生长量测定法 体积测量法：又称测菌丝浓度法。 经过测定一定体积培养液中所含菌丝的量来反映微生物的生长状况。方法是，取一定量的待测培养液（如10毫升）放在有刻度的离心管中，设定一定的离心时间（如5分钟）和转速（如5000 rpm），离心后，倒出上清夜，测出上清夜体积为v，则菌丝浓度为（10-v）/10。菌丝浓度测定法是大规模工业发酵生产上微生物生长的一个重要监测指标。这种方法比较粗放，简便，快速，但需要设定一致的处理条件，否则偏差很大，由于离心沉淀物中夹杂有一些固体营养物，结果会有一定偏差。 称干重法：
Microscope 显微镜 Antony van leeuwenhoek 吕文虎克 Louis Pasteur 巴斯德 Joseph Lister 李斯德 Robert Koch 郭霍 Koch postulate 郭霍法则 molecular Koch postulate 分子郭霍法则Bacterium 细菌 Coccus 球菌 Diplococcus 双球菌 Bacillus 杆菌 Spiral bacterium 螺型菌 Vibrio 弧菌 Spirillum 螺菌Polymorphism 多形性 Cell wall 细胞壁Peptidoglycan 肽聚糖Mucopeptide 粘肽 Glycopeptide 糖肽 Murein 胞壁质 N-acetylmuramic acid ，NAM N-乙酰胞壁酸N-acetylglucosamine, NAG N-乙酰葡糖胺Diaminopimelic acid, DAP 二氨基庚二酸Teichoic acid 磷壁酸 Ribitol 核糖醇 Lipoteichoic acid, LTA 脂磷壁酸 Outer membrane 外膜Lipopolysaccharide LPS 脂多糖 Lipid A 脂质A Lysozyme 溶菌酶 Protoplast 原生质体Spheroplast 圆球体Bactrrial L form 细菌L形 Cytoplasmic membrane 细胞膜 Mesosome 中介体 Cytoplasm 细胞质 Ribosome 核糖体 Plasmid 质粒 Inclusion 内含物 Metachromatic granule 异染颗粒 Volutin 纡回体 Nuclear material 核质 Nucleoid 拟核 Capsule 荚膜 Microcapsule 微荚膜 Slime layer 黏液层 Smooth colony 光滑型菌落 Mucoid colony 黏液型菌落 Rough colony 粗糙型菌落 Flagellum 鞭毛 Pilus(fimbria) 菌毛 Common pilus 普通菌毛 Sex pilus 性菌毛 Fertility 致育性 Spore 芽孢 Endospore 内芽孢 Vegetative form 繁殖体 Spore wall 芽孢壁 Cortex 皮质层 Coat 芽孢壳 Exosporium 芽孢外衣 Light microscope 光学显微镜 Electron microscope 电子显微镜 Dark microscope 暗视野显微镜 Gram stain 革兰染色法 Bacterial metabolism 细菌代谢 Obligate aerobe 专性需氧菌 Microaerophilic bacterium 微需氧菌 Facultative anaerobe 兼性厌氧菌 Obligate anaerobe 专性厌氧菌 Superoxide dismutase SOD 超氧化物歧化酶 Catalase 触酶 Peroxidase 郭氧化物酶 Binary fission 二分裂法 Growth curve 生长曲线 Lag phase 迟晚期 Logarithmic phase 对数生长期 Stationary phase 稳定期 Decline phase 衰亡期 Autotroph 自养菌 Heterotroph 异养菌 Saprophyte 腐生菌 Permease 通透酶 Fermentation 发酵 Pyrogen 热原质 Antibiotic 抗生素 Exotoxin 外毒素 Endotoxin 内毒素 Bacteriocin 细菌素 Culture medium 培养基 Basal medium 基础培养基 Nutrient medium 营养培养基 Selective medium 选择性培养基 Different medium 鉴别培养基 Anaerobic medium 厌养培养基 Cooked meat medium 庖肉培养基 1
(一)、概述 食用被微生物污染的食品而导致的疾病，称作食源性疾病。导致这类疾病的微生物叫食源性致病菌。随着人们居住和卫生条件的不断改善，以及抗生素的滥用，人类对病菌的抵抗能力却在不断下降，食源性疾病一直呈上升的趋势。因此，对食品中致病菌的监测和检验也就越显示其重要性，常规的检验大多依靠培养目标微生物的方法来确定食品是否受到此微生物的污染，这些方法需要一定的培养时间，少则2～3天，多至数周，才能确定。而现行有效的一些快速检测方法不仅可以大大缩短检测时间提高微生物检出率并可用于微生物计数、早期诊断、鉴定等方面，以做到快速、简便、准确。快速方法包括了微生物学、分子化学、生物化学、生物物理学、免疫学和血清学等领域。 （二）、常见、常用的快速、简便的检测微生物数量的方法如下： 1、活细胞计数的改进方法 （1）、旋转平皿计数方法 （2）、疏水性栅格滤膜法（HGMF）或等格法（isogrid method） （3）、血膜系统（Pertrifilm） （4）、酶底物技术（ColiComplete） （5）、直接外荧光滤过技术（DEFT） （6）、“即用胶”系统（SimPlate） 2、用于估计微生物数量的新方法 （1）、阻抗法 （2）、A TP生物发光技术 3、其他方法 （1）、微量量热法 （2）、接触酶测定仪 （3）、放射测定法 （三）、食品中沙门氏菌的快速筛检方法 1、沙门氏菌显色培养基法 2、免疫学方法 3、分子生物学方法 4、自动传导法 （四）、大肠杆菌O157：H7快速检测方法 大肠杆菌O157：H7肠出血性大肠杆菌的主要血清型，自1982年在美国被分离并命名以来，陆续发现本菌与轻度腹泻、溶血性尿毒综合症、出血性肠炎、婴儿猝死综合症等多种人类病症密切相关，是食源性疾病的一种重要致病菌。E.coli O157：H7属于肠杆菌科埃希氏菌属，为革兰氏阴性杆菌，有鞭毛。近年来作为食品卫生及流行病学的研究热点，E.coli O157：H7的分离和鉴定方法已取得了较大进展。利用其生化特征、免疫原性建立的方法以及现代分子生物学技术的应用，可以从多方面对E.coli O157：H7进行检测。 1、E.coli O157：H7鉴别培养基及显色培养基 2、免疫学检测方法 3、分子生物学方法 （五）、金黄色葡萄球菌的快速检测方法 金黄色葡萄球菌为革兰氏阳性球菌，呈普通串状排列无芽孢，无鞭毛，不能运动。该菌在自然界中分布广泛，如空气、水、土壤、饲料和一些物品上，是最常见的化脓性球菌之一，食品受其污染的机会很多。金黄色葡萄球菌食物中毒是其肠毒引起的，目前已确认的肠毒素至少有A,B,C1,C2,C3,D,E和F8个型。由金黄色葡萄球菌肠毒素引发的中毒爆发事件，近年来
active immunity（主动免疫）: Immunity acquired through direct stimulation of the immune system by antigen. active transport（主动运输）:Transport of molecules against a concentration gradient (from regions of low concentration to regions of high concentration) with the aid of proteins in the cell membrane and energy from ATP. Alcohol fermentation（乙醇发酵）：is the formation of alcohol from sugar. Yeast, when under anaerobic conditions, convert glucose to pyruvic acid via the glycolysis pathways, then go one step farther, converting pyruvic acid into ethanol, a C-2 compound. aerobe（好氧微生物）: A microorganism that lives and grows in the presence of free gaseous oxygen (O2). aflatoxin（黄曲霉毒素）: From Aspergillus flavus t, a mycotoxin that typically poisons moldy animal feed and can cause liver cancer in humans and other animals. AIDS（爱滋病）: Acquired Immune deficiency syndrome. The complex of signs and symptoms characteristic of the late phase of human immunodeficiency virus (HIV) infection. Ames test（艾姆氏实验）: A method for detecting mutagenic and potentially carcinogenic agents based upon the genetic alteration of nutritionally defective bacteria anabolism（合成代谢）: The energy consuming process of incorporating nutrients into protoplasm through biosynthesis. anaerobe（厌氧微生物）: A microorganism that grows best, or exclusively, in the absence of oxygen. antibiotic（抗生素）:A chemical substance from one microorganism that can inhibit or kill another microbe even in minute amounts. antibody（抗体）: A large protein molecule evoked in response to an antigen that interacts specifically with that antigen. antigen（抗原）: Any cell, particle, or chemical that induces a specific immune response by B cells or T cells and can stimulate resistance to an infection or a toxin. antigenic determinant（抗原决定基）:The precise molecular group of an antigen that defines its specificity and triggers the immune response. antimetabolite（抗代谢物）:A substance such as a drug that competes with, substitutes for, or interferes with a normal metabolite. antiseptic（防腐剂）：A growth-inhibiting agent used on tissues to prevent infection. antiserum（抗血清）：Antibody-rich serum derived from the blood of animals (deliberately immunized against infectious or toxic antigen) or from people who have recovered from specific nfections. antitoxin（抗毒素）：Globulin fraction of serum that neutralizesa specific toxin. Also refers to the specific antitoxin antibody itself. arthrospore（节孢子）：A fungal spore formed by the septation fragmentation of hyphae. ascospore（子囊）：A spore formed within a saclike cell (ascus) of Ascomycota following nuclear fusion and meiosis. asepsis（无菌）：A condition free of viable pathogenic microorganisms. autoantibody（自身抗体）：An "anti-self antibody having an ffinity for tissue antigens of the subject in which it is formed. autoantigen（自身抗原）：Molecules that are inherently part of self but are perceived by the
微生物英文名词解释 1.Mycoplasma：The mycoplasma are a group of the smallest organisms without cell wall that can be free-living in nature, can pass through bacterial filter and also grow on laboratory media. 2.Chlamydia：Chlamydia are small Gram-negative bacteria which are obligate intercellular parasites like virus, but differ from them in that they have both RNA and DNA, ribosome, cell wall, and divided by binary fission. 3.L forms of bacteria：In osmotically protective media, removal of the bacterial wall with lysozyme or penicillin liberate protoplasts from Gram-positive cells and spheroplasts from Gram-negative cells. If such wall-defective cells are able to grow and divide, they are called L forms. L forms are difficult to cultivate. They require a special media. Some L form can revert tothe normal bacillary form. L form in the host may produce chronic infection that are relatively resistant to antibiotic treatment. 4.Capsule：Many bacteria synthesize large amounts of extracellular polymer when growing in their natural environments. When the polymer forms a condensed, well-defined layer closely surrounding the cell, it is called the capsule. With one known exception (the polypeptide capsule), the polymer is polysaccharide. 5.Pyrogen：This is a fever-producing substance synthesized by bacteria. In fact, it is the lipopolysaccharide of Gram-negative bacteria. For the injectable medicament, it is especially important to avoid the contamination of pyrogen in the course of pharmic production. 6.Exotoxin：Exotoxins are proteins produced inside Gram-positive bacteria cells and secreted into the environment. These toxins are some of the strongest poisons known to man and cause violent reactions in host organisms. 7.Endotoxin：Endotoxins are made up of lipids and carbohydrates associated with the outer membrane of gram-negative bacteria. These toxins usually produce fever, weakness, and capillary damage. 8.Disinfection: Reduce or eliminate pathogens病原体in or on inanimate无生命 的objects to a safe level, which are no longer health hazard危险. 9.Sterilization: A physical or chemical process that completely destroys or removes all microbial life，including bacteria spore and viruses. 10.Antisepsis: Use chemical agents to inhibit or destroy the growth of microorganisms on skin or other living tissue. 11.Plasmids：Plasmids are small genetic elements that replicate independently of the bacterial chromosome. Most plasmids are circular, double-stranded DNA molecules varying from 1,500 to 400,000 base pairs. Like the bacterial chromosomal DNA, they can autonomously replicate and as such are referred to as replicons. 12.Transformation：It is the process by which bacteria take up fragments of naked DNA and incorporate them into their genomes. During transformation, DNA
微生物检验（完整版） 名解 微生物：是一群个体微小结构简单肉眼不能看见的微小生物的总称 种：亲缘关系较近的微生物群体在进化发育阶段上有一定的共同形态和生理特征 微生物学：生物学的一个分支是研究微生物在一定条件下的形态结构生理生化遗传变异特性及微生物的进化分类生态等生命活动规律以及微生物之间与人类动植物自然界互相关系的一门科学 抗生素：是由某些微生物在代谢过程中产生的能抑制或杀灭某些其他微生物和肿瘤细胞的微量生物活性物质 细菌素：是某些细菌菌株产生的一类具有抗菌作用的蛋白质 条件致病菌：正常菌群在宿主体内具有相对稳定性一般不致病 消毒：指杀死物体上的病原微生物但不一定能杀死细菌芽孢的方法 灭菌：指杀灭物体上所有的微生物的方法 防腐：指防止或抑制微生物生长繁殖的方法 无菌：指没有货的微生物的存在 质粒：是细菌染色体外的遗传物质也是环状闭合的双联DNA分子比染色体小存在于细胞质中可自主复制 突变：是指细菌遗传物质的机构发生突然而稳定的改变所致的变异现象可遗传给后代 基因转移：外源性物质由供体菌转入受体细胞内的过程 基因重组：供体菌的基因进入受体菌细胞并在其中自行复制与表达或矛受体菌DNA整合在一起的过程 病毒：是一类个体微小结构简单只含一种核酸只能在活的易感细胞内以复制方式增殖的
非细胞型微生物 复制周期：病毒的增殖被人为分成吸附穿入脱壳生物合成装配成熟与释放七个步骤的完整过程 缺陷病毒：是指因病毒基因组不完整或因基因某一点改变而不能进行正常增殖的病毒 顿挫感染：病毒进入宿主细胞若细胞缺乏病毒复制所需的酶能量和必要成分等则病毒无法合成自身成分不能够装配和释放子代病毒的现象 干扰现象：两种病毒感染同一种细胞时可发生一种病毒抑制另一种病毒增殖的现象 人工自动免疫：是将疫苗等免疫原接种于人体刺激机体免疫系统产生特异性免疫应答使机体获得特异性免疫力 人工被动免疫：是指注射含某种病毒特异性中和抗体的免疫血清等一系列细胞因子是机体立即获得特异性免疫 干扰素：是由病毒或干扰素诱生剂作用于中性粒细胞成纤维细胞或免疫细胞产生的一种糖蛋白 致病性：一定种类的病原微生物在一定的条件下能在特殊的宿主体内引起特定疾病的能力半数致死量：在规定时间内通过一定途径能使一定体重或年龄的某种动物半数死亡或感染需要的最小病原体数量或毒素量 急性感染：发作突然病程较短一般是数天或数周 局部感染：病原体侵入机体后局限就在一定部位生长繁殖引起病变的一种感染类型 毒血症：致病菌侵入宿主体后只在机体局部生长繁殖病菌不进入血液循环但其产生的外毒素入血引起特殊的毒性症状 败血症：致病菌侵入血流后在其中大量繁殖并产生毒性产物引起全身性毒性症状 内毒素血症：革兰阴性菌侵入血流并在其中大量繁殖崩解后释放出大量毒素也可有病灶
A Abortive infection 顿挫感染 * Absorption 吸附 * Acid-fast bacilli 抗酸杆菌 Acquired immunity 获得性免疫 * Acquired immunodeficiency syndrome，AIDS 获得性免疫缺陷综合征* Actinomyces 放线菌属 Acute infection 急性感染 * Adenovirus 腺病毒 Adhesin 粘附素 A.flavus 黄曲霉 A. israelii 衣氏放线菌 * Anaerobic bacteria 厌氧性细菌 Anaerobic medium 厌氧培养基 Antibody dependent enhancement,ADE 抗体依赖的促进作用Antigenic drift 抗原漂移 Antigenic shift 抗原性转换 * Antisepsis 防腐 * Antistreptolysin O (ASO) test, 抗链球菌溶素O试验 Antiviral protein,AVP 抗病毒蛋白 Apoptosis 细胞凋亡 * Apparent infection 显性感染 Arbovirus 虫媒病毒 * Artificial active immunization 人工主动免疫 * Artificial passive immunization 人工被动免疫 * Asepsis 无菌 Aspergillus 曲霉 * Assembly and release 装备与释放 * Astrovirus 星状病毒 Autotroph 自养菌 * Attenuated vaccine 减毒疫苗 B * Bacillus 杆菌、芽胞杆菌属 * Bacillus anthracis 炭疽芽胞杆菌 Bacillus cereus 蜡样芽胞杆菌 * Bacteremia 菌血症 Bacterial infection 细菌感染 * Bacterial L form 细菌L型 * Bacteriocin 细菌素 * Bacteriophage 噬菌体 * Bacterium 细菌 Bartonella 巴尔通体属 * Bacteriodes fragilis 脆弱类杆菌
microorganism微生物；bacterium细菌；Gram stain革兰染色；coccus球菌；bacillus 杆菌；vibro弧菌；peptidoglycan肽聚糖；teichoic acid磷壁酸；lipopolysaccharide(LPS) 脂蛋白；lipid A 脂质A；L formed bacteria L型细菌；mesosome中介体；plasmid质粒 formed bacteria L型细菌；mesosome中介体；plasmid质粒；capsule, 荚膜；flagellum,鞭毛；pilus, 菌毛；spore，芽胞 Pyrogen,热原质；toxin,毒素；growth curve, 生长曲线 Sterilization灭菌；disinfection消毒；autoclave压力蒸汽灭菌器；bacteriophage噬菌体；virulent phage毒性噬菌体；temperate phage 温和噬菌体 transposable element转座元件；transposon转座子;mutation突变；transformation 转化；conjugation接合；transduction转导；lysogenic conversion溶原性转换 pathogenicity 致病性；virulence毒力；exotoxin外毒素；endotoxin内毒素；normal flora正常菌群；conditioned pathogen机会致病菌；opportunistic infection机会性感染 specimens标本；isolation分离；identification鉴定；artificial active immunization 人工主动免疫；artificial passive immunization人工被动免疫；vaccine疫苗；toxoid 类毒素；antitoxin抗毒素 Staphlococcus葡萄球菌；Staphlococcus A protein葡萄球菌A蛋白；coagulase血浆凝固酶；enterotoxin肠毒素；Strptococcus链球菌；pyrogenic exotoxin致热外毒素；streptolysin链球菌溶素；hyaluronidase透明质酸酶 E.coli大肠埃希菌；ETEC肠产毒性大肠埃希菌；EIEC肠侵袭性大肠埃希菌；EPEC
A/O法活性污泥中氨氧化菌群落的动态与分布 摘要： 我们研究了在厌氧—好氧序批式反应器（SBR）中氨氧化菌群落（AOB）和亚硝酸盐氧化菌群落（NOB）的结构活性和分布。在研究过程中，分子生物技术和微型技术被用于识别和鉴定这些微生物。污泥微粒中的氨氧化菌群落结构大体上与初始的接种污泥中的结构不同。与颗粒形成一起，由于过程条件中生物选择的压力，AOB的多样性下降了。DGGE测序表明，亚硝化菌依然存在，这是因为它们能迅速的适应固定以对抗洗涤行为。DGGE更进一步的分析揭露了较大的微粒对更多的AOB种类在反应器中的生存有好处。在SBR反应器中有很多大小不一的微粒共存，颗粒的直径影响这AOB和NOB的分布。中小微粒（直径<0.6mm）不能限制氧在所有污泥空间的传输。大颗粒（直径>0.9mm）可以使含氧量降低从而限制NOB的生长。所有这些研究提供了未来对AOB微粒系统机制可能性研究的支持。 关键词：氨氧化菌（AOB），污泥微粒，菌落发展，微粒大小，硝化菌分布，发育多样性 1.简介 在浓度足够高的条件下，氨在水环境中对水生生物有毒，并且对富营养化有贡献。因此，废水中氨的生物降解和去除是废水处理工程的基本功能。硝化反应，将氨通过硝化转化为硝酸盐，是去除氨的一个重要途径。这是分两步组成的，由氨氧化和亚硝酸盐氧化细菌完成。好氧氨氧化一般是第一步，硝化反应的限制步骤：然而，这是废水中氨去除的本质。对16S rRNA的对比分析显示，大多数活性污泥里的氨氧化菌系统的跟?-变形菌有关联。然而，一系列的研究表明，在氨氧化菌的不同代和不同系有生理和生态区别，而且环境因素例如处理常量，溶解氧，盐度，pH，自由氨例子浓度会影响氨氧化菌的种类。因此，废水处理中氨氧化菌的生理活动和平衡对废水处理系统的设计和运行是至关重要的。由于这个原因，对氨氧化菌生态和微生物学更深一层的了解对加强处理效果是必须的。当今，有几个进阶技术在废水生物处理系统中被用作鉴别、刻画微生物种类的有价值的工具。目前，分子生物技术的应用能提供氨氧化菌群落的详细分类说明。
微生物检测手段及注意事项
微生物检测手段及注意事项 微生物的检测，无论在理论研究还是在生产实践中都具有重要的意义，本文对生长量测定法、微生物计数法、生理指标法和商业化快速微生物检测简要介绍了利用微生物重量，体积，大小，生理代谢物等指标的二十余种常用的检测方法，简要介绍了这些方法的原理，应用范围和优缺点。 一个微生物细胞在合适的外界条件下，不断的吸收营养物质，并按自己的代谢方式进行新陈代谢。如果同化作用的速度超过了异化作用，则其原生质的总量（重量，体积，大小）就不断增加，于是出现了个体的生长现象。如果这是一种平衡生长，即各细胞组分是按恰当的比例增长时，则达到一定程度后就会发生繁殖，从而引起个体数目的增加，这时，原有的个体已经发展成一个群体。随着群体中各个个体的进一步生长，就引起了这一群体的生长，这可从其体积、重量、密度或浓度作指标来衡量。微生物的生长不同于其他生物的生长，微生物的个体生长在科研上有一定困难，通常情况下也没有实际意义。微生物是以量取胜的，因此，微生物的生长通常指群体的扩增。微生物的生长繁殖是其在内外各种环境因素相互作用下的综合反映。因此生长繁殖情况就可作为研究各种生理生化和遗传等问题的重要指标，同时，微生物在生产实践上的各种应用或是对致病，霉腐微生物的防治都和他们的生长抑制紧密相关。所以有必要介绍一下微生物生长情况的检测方法。既然生长意味着原生质含量的增加，所以测定的方法也都直接或间接的以次为根据，而
测定繁殖则都要建立在计数这一基础上。微生物生长的衡量，可以从其重量，体积，密度，浓度，做指标来进行衡量。 1. 微生物计量法 1.1 体积测量法 又称测菌丝浓度法，通过测定一定体积培养液中所含菌丝的量来反映微生物的生长状况。方法是，取一定量的待测培养液（如10 mL）放在有刻度的离心管中，设定一定的离心时间（如5 min）和转速（如5000 rpm），离心后，倒出上清夜，测出上清夜体积为v，则菌丝浓度为（10-v）/10。菌丝浓度测定法是大规模工业发酵生产上微生物生长的一个重要监测指标。这种方法比较粗放，简便，快速，但需要设定一致的处理条件，否则偏差很大，由于离心沉淀物中夹杂有一些固体营养物，结果会有一定偏差。 称干重法 可用离心或过滤法测定。一般干重为湿重的10~20%。在离心法中，将一定体积待测培养液倒入离心管中，设定一定的离心时间和转速，进行离心，并用清水离心洗涤1~5次，进行干燥。干燥可用烘箱在105 ℃或100 ℃下烘干，或采用红外线烘干，也可在80 ℃或40 ℃下真空干燥，干燥后称重。如用过滤法，丝状真菌可用滤纸过滤，细菌可用醋酸纤维膜等滤膜过滤，过滤后用少量水洗涤，在40 ℃下进行真空干燥。称干重发法较为烦琐，通常获取的微生物产品为菌体时，常采用这种方法，如活性干酵母（Activity Dry Yeast, ADY）,一些以微生物菌体为活性物质的饲料和肥料。
1.Hyphae（菌丝的复数） 2.Polymorphism 多态性：一个基因座位上存在多个等位基因。 3.Generation time 代时：繁殖一代，细菌增殖一代所需要的时间。 4.Log phase 对数期：又称指数生长期，细菌在该期以最大的生长速率生长与分裂，活菌数目按几何级数增加。 5.Colony 菌落：在固体培养基表面由单个细菌大量繁殖所形成的细菌群体。 6.Sterilization 灭菌：杀灭物体上的所有微生物。 7.Disinfection 消毒：杀灭物体上的病原微生物的作用与方法。 8.Antisepsis 9.防腐：防止或抑制微生物生长繁殖的方法。 10.LD50 and ID50 半数致死量：在规定时间内，通过指定感染途径，使一定体重或年龄的某种动物半数死亡所需最小细菌数或毒素量。 12.Bacteremia 菌血症：病原菌由局部侵入血液，经血液到达某部位，但未在血中繁殖，无明显中毒症状。 11.Septicemia 败血症：细菌侵入血液并大量生长繁殖，同时产生毒素，引起严重的中毒症状。 12.Vertical Transmission 垂直传播：指感染母体的病毒经胎盘或产道由亲代直接传播给子代的传播方式。 13.Outer membrane 外膜：是G-细胞壁的主要结构，约占细胞壁干重的80%，主要由脂蛋白、脂质双层和脂多糖三部分组成。 13.Mesosome 中介体：又称间体是一种由细胞质膜内褶而形成的囊状结构，其中充满着层状或管状的囊泡。 14.Bacterial L form 细菌L型：在实验室或宿主体内通过自发突变而形成的遗传性稳定的细胞壁缺陷菌株。15.Capsule 荚膜：某些细菌在生长过程中，向其细胞壁外分泌一层疏松、透明、排列有序且不易被清除的黏液状物质。 16.Spore 芽孢：某些细菌在一定条件下，胞浆脱水浓缩，在菌体内部形成具有多层膜包裹的圆形或卵圆形小体。 孢子：是真菌的繁殖结构，由生殖菌丝产生。 18.Growth curve 生长曲线：是指将一定量的细菌接种于适宜的液体培养基中进行培养，连续定时检查活菌数，
WHONET微生物中英文对照及其代码 WHONET微生物中英文对照及其代码 常见细菌中英文对照、菌组、菌属及代码（革兰阴性部分） 代码革兰英文名称中文名称细菌分类菌属 139-Vibrio cholerae O139霍乱弧菌O139血清型FERM弧菌属 157-Escherichia coli O157:H7大肠埃希菌O157:H7EBC埃希菌属 aba-Acinetobacter baumannii鲍曼不动杆菌NFR不动杆菌属 abu-Arcobacter butzleri比茨莱弓形菌GNCB弓形菌属 ac--Acinetobacter sp.不动杆菌属NFR不动杆菌属 aca-Acinetobacter calcoaceticus醋酸钙不动杆菌NFR不动杆菌属 acb-Actinobacillus sp.放线杆菌属GNCB放线杆菌属 ach-Achromobacter sp.无色杆菌属NFR无色杆菌属 acv-Aeromonas caviae豚鼠气单胞菌FERM气单胞菌属 acx-Acidovorax sp.食酸菌属NFR食酸菌属 acy-Arcobacter cryaerophilus嗜低温弓形菌GNCB弓形菌属 ade-Alcaligenes xylosoxidans ss. denitrificans反硝化无色杆菌NFR无色杆菌属adf-Acidovorax delafieldii德拉菲尔德食酸菌NFR食酸菌属 adf-Pseudomonas delafieldii德拉菲尔德食酸菌NFR 食酸菌属 aeh-Aeromonas hydrophila嗜水气单胞菌FERM气单胞菌属 aeq-Actinobacillus equuli马驹放线杆菌马驹亚种GNCB放线杆菌属 aer-Aeromonas sp.气单胞菌属FERM气单胞菌属 aeu-Aeromonas eucrenophila嗜矿泉气单胞菌FERM气单胞菌属 afa-Alcaligenes odorans香味类香味菌NFR产碱杆菌属 afc-Acidovorax facilis速生食酸菌NFR食酸菌属 afc-Pseudomonas facilis速生食酸菌NFR 食酸菌属 afd-Afipia clevelandensis克利夫兰阿菲波菌GNCB阿菲波菌属 afi-Afipia sp.阿菲波菌属GNCB阿菲波菌属 afo-Afipia broomeae布氏阿菲波菌GNCB阿菲波菌属 agr-Agrobacterium sp.根瘤菌属NFR根瘤菌属 aha-Acinetobacter haemolyticus溶血不动杆菌NFR不动杆菌属 aja-Aeromonas jandaei让达气单胞菌FERM气单胞菌属 ajo-Acinetobacter johnsonii约氏不动杆菌NFR不动杆菌属 aju-Acinetobacter junii琼氏不动杆菌NFR不动杆菌属 alc-Alcaligenes sp.产碱杆菌属NFR产碱杆菌属 ali-Actinobacillus lignieresii利尼埃放线杆菌GNCB放线杆菌属 alm-Alteromonas sp.交替单胞菌属check交替单胞菌属 alw-Acinetobacter lwoffii鲁氏不动杆菌NFR不动杆菌属 aly-Alysiella sp.小链球菌check小链球菌 amd-Aeromonas media中间气单胞菌FERM气单胞菌属 ant-Arcobacter nitrofigilis固氮弓形菌GNCB弓形菌属 apf-Afipia felis猫阿菲波菌GNCB阿菲波菌属 api-Alcaligenes piechaudii皮埃肖无色杆菌NFR产碱杆菌属
微生物快速检测方法及应用进展 随着人们生活水平不断提高，各种安全问题越来越受到人们的重视，微生物的污染问题也相应地备受关注。在食品和环境等各个方面都有微生物污染的可能，一旦污染，微生物将大量繁殖而导致食源性疾病或环境污染甚至医院内感染。特别是近年来随着环境污染的加剧和生态平衡的不断破坏，导致感染的致病菌的种类越来越多，病原微生物对人类的威胁越来越大。传统的检验方法，主要包括形态检查和生化方法，其准确性、灵敏性均较高，但涉及的实验较多、操作烦琐、需要时间较长、准备和收尾工作繁重，而且要有大量人员参与［1，2］。所以，迫切需要准确、省时、省力和省成本的快速检验方法。本文对微生物快速检测方法的进展情况及实际应用进行综述，以利于预防食源性疾病及公共卫生突发事件的发生。 1 即用型纸片法 3M公司的perrifilmTMPlate系列微生物测试片，可分别检测菌落总数、大肠菌群计数、霉菌和酵母计数［3］。由RCP Scientific Inc 公司开发上市的Regdigel系列，除上述项目外还有检测乳杆菌、沙门氏菌、葡萄球菌的产品［4］，这两个系列的产品与传统检测方法之间的相关性非常好。如用大肠菌群快检纸片检测餐具的表面，操作简便、快速、省料，特异性和敏感性与发酵法符合率高，已经被列为国标方法。使用时应正确掌握操作技术和判断标准，从而达到理想的检测效果［5］。美国3M公司生产的PF(Petrifilm)试纸还加入了染色剂、显色剂，增强了菌落的目视效果，而且避免了热琼脂法不适宜受损细菌恢复的缺陷。霉菌快速检验纸片，应用于食品检验中的霉菌具有操作简便，仅需36℃培养，不需要低温设备；快速，仅需2 d就可观察结果，比现在的国家标准检验方法缩短3～5 d，大大提高了工作效率。纸片法与国标法在霉菌检出率上差异无统计学意义，且菌落典型，易判定。纸片荧光法利用细菌产生某些代谢酶或代谢产物的特点而建立的一种酶—底物反应法。只需检测食品中大肠菌群、大肠杆菌的有关酶的活性，将荧光产物在365 nm紫外光下观察即可。同时纸片可高压灭菌处理，4℃保存，简化了实验准备、操作和判断［6］。但由于它们价格昂贵，限制了在基层单位的实际应用。 2 生物化学技术 2.1 PCR技术PCR技术采用体外酶促反应合成特异性DNA片段，再通过扩增产物来识别细菌。由于PCR灵敏度高，理论上可以检出一个细菌的拷贝基因，因此在细菌的检测中只需短时间增菌甚至不增菌，即可通过PCR进行筛选，节约了大量时间，但PCR技术也存在一些缺点：食物成分、增菌培养基成分和其他微生物DNA对Taq酶具有抑制作用，可能导致检验结果假阴性；操作过程要求严格，微量的外源性DNA进入PCR后可以引起无限放大产生假阳性结果，扩增过程中有一定的装配误差，会对结果产生影响。由于以上原因，PCR技术对操作者的自身素质要求很高，对于基层单位而言难以做到。短时间内也不会有经济效益和社会效益，因此影响了这项技术在基层的应用。 2.2 基因探针技术基因探针技术利用具有同源性序列的核酸单链在适当条件下互补形成稳 定的或链的原理，采用高度特异性基因片段制备基因探针来识别细菌。基 因探针的优点是减少了基因片段长度多态性所需要分析的条带数。如法国生物一梅里埃公司的 基因探针检测系统，对于分离到的单个菌落，30 min完成微生物的确证试验［7］， 基因探针的缺点是不能鉴定目标菌以外的其他菌。 3 选择、鉴定用培养基法