大肠杆菌的检测：大肠菌群测定的操作细则
大肠菌群系指一群能发酵乳糖、产酸产气、需氧和兼性厌氧的革兰氏阴性无芽胞杆菌。该菌主要来于人畜粪便,故以此作为粪便污染指标来评价食品的卫生质量,推断食品中有否污染肠道致病菌的可能。
食品中大肠菌群数系以100mL(g)检样内大肠菌群最可能数(MPN)表示。
1 设备和材料
1.1 温箱：36±1℃。 1.2 冰箱:0～4℃。 1.3 恒温水浴 :44.5±0.5℃。 1.4 天平。 1.5 显微镜。1.6 均质器或乳钵。 1.7 平皿:直径为90mm。 1.8 试管。 1.9 吸管。 1.10 广口瓶或三角烧瓶:容量为500mL。 1.11 玻璃珠:直径约5mm。 1.12 载玻片。 1.13 酒精灯。
1.14 试管架。
2 培养基和试剂
2.1 乳糖胆盐发酵管:按GB 4789.28中4.9规定。
2.2 伊红美蓝琼脂平板:按GB 4789.28中4.25规定。
2.3 乳糖发酵管:按GB 4789.28中4.10规定。
2.4 EC 肉汤:按GB 4789.28中4.11规定。
2.5 磷酸盐缓冲稀释液:按GB 4789.28中3.22规定。
2.6 生理盐水。
2.7 革兰氏染色液:按GB 4789.28中2.2规定。
3.1 检样稀释
3.1.1 以无菌操作将检样25mL(或g)放于有225mL灭菌生理盐水或其他稀释液的灭菌玻璃瓶内(瓶内予置适当数量的玻璃珠)或灭菌乳钵内,经充分振摇或研磨做成1:10的均匀稀释液。固体检样最好用均质器,以8 000-10 000 r/min的速度处理1min,做成1:10的均匀稀释液。
3.1.2 用1mL灭菌吸管吸取1:10稀释液1mL,注入含有9mL灭菌生理盐水或其他稀释液的试管内,振摇试管混匀,做成1:100的稀释液。
3.1.3 另取1mL灭菌吸管,按上条操作依次做10倍递增稀释液,每递增稀释一次,换用1支1mL灭菌吸管。
3.1.4 根据食品卫生标准要求或对检样污染情况的估计,选择三个稀释度,每个稀释度,接种3管。
3.2 乳糖发酵试验
将待检样品接种于乳糖 胆盐发酵管内,接种量在1mL以上者,用双料乳糖胆盐发酵管,1mL及1mL以下者,用单料乳糖胆盐发酵管。每一稀释度接种3管,置36±1℃ 温箱内,培养24±2h,如所有乳糖胆盐发酵管都不产气,则可报告为大肠菌群阴性,如有产气者,则按下列程序进行。
3.3 分离培养
将产气的发酵管分别转种在伊红美蓝琼脂平板上,置36±1℃ 温箱内,培养18-24h,然后取出,观察菌落形态,并做革兰氏染色和证实试验。
3.4 证实试验
在上述平板上,挑取可疑大肠菌群菌落1-2个进行革兰氏染色,同时接种乳糖发酵管,置 36±1℃温箱内培养24±2h,观察产气情况。凡乳糖管产气、革兰氏染色为阴性的无芽胞杆菌,即可报告为大肠菌群阳性。
3.5 报告
根据证实为大肠菌群阳性的管数,查MPN检索表,报告每100mL(g)大肠菌群的MPN值。 4 粪大肠菌群(faecal coliform)
4.1 用接种环将所有产气的乳糖胆盐发酵管培养物(见3.2条)转种于EC肉汤管内,置
44.5±0.2℃水浴箱内(水浴箱内的水面应高于EC肉汤液面),培养24±2h,经培养后,如所有EC肉汤管均不产气,则可报告为阴性;如有产气者,则将所有产气的EC肉汤管分别转种于伊红美蓝琼脂平板上,置 培养18-24h,凡平板上有典型菌落者,则证实为粪大肠菌群阳性。
4.2 结果报告
根据证实为粪大肠菌群的阳性管数,查MPN检索表,报告每100mL(g)粪大肠菌群的MPN值 乳酸菌的检测：
一 概述
乳酸菌是指一群能分解葡萄糖或乳糖产生乳酸，需氧和兼性厌氧，多数无动力，过氧化氢酶阴性，革兰氏阳性的无芽胞杆菌和球菌。这类细菌在自然界分布广泛，可栖居在人和各种动物的口腔、肠道等器官内，在土壤、食品、饲料、水及一些临床标本中都有乳酸菌的存在。乳酸菌在工业、农业和医药等与人类生活密切相关的领域应用价值很高，相当多的乳酸菌对人、畜的健康起着有益的作用，但个别菌种能对人畜致病，乳酸菌主要包括23个属的细菌。
二 样本采集
检样主要为含乳酸菌活菌的饮料和微生态制剂，样本应放入冰箱保存，心快检验。
三 检验方法（中华人民共和国国家标准 乳酸菌饮料中乳酸菌的微生物学检验GB／T 16347-1996）
乳酸菌菌总数的测定：乳酸菌菌落总数是指标样在一定条件下培养后，所得1ml检样中所含乳酸菌菌落的总数。
（一）检验程序
乳酸菌菌落总数检验程序如下：
（二）培养基和试剂
改良TJA培养基（改良番茄汁琼脂培养基）；改良MC培养基（modified Chalmers培养基）；0.1％亚甲蓝牛乳培养基；6.5％氯化钠肉汤参照；pH9.6葡萄糖肉汤；40％胆汁肉汤；淀粉水解培养基；精氨酸水解培养基；乳酸杆菌糖发酵管；七叶苷培养基；革兰氏染色液；3％过氧化氢溶液；蛋白胨水、靛基质试剂；明胶培养基；硝酸盐培养基、硝酸盐试剂；生理盐水：定量分装于三角瓶和试管内灭菌。
（三）操作步骤
1．以无菌操作将经过充分摇匀的检样25ml（或25g）放入含有225ml灭菌生理盐水的来菌广口瓶内做成1：10的均匀稀释液。
2．用1ml灭菌吸管吸取1：10稀释液1m，沿管壁徐徐注入含有9ml灭菌生理盐水的试管内（注意吸管尖端不要触及管内稀释液）。
3．另取1ml灭菌吸管，按上述操作顺序，作10倍增稀释液，如此每递增一次，即换用1支1ml灭菌吸管。
4．选择2～3个以上适宜稀释度，分别在作10倍递增稀释的同时，即以吸取该稀释度的吸管移1ml稀释液于灭菌平皿内，每个稀释度作两个平皿。
5．稀释液移入平皿后，应及时将冷至50℃的乳酸菌计数培养基（改良TJA或改良MC）注入平皿约15ml，并转动平皿使混合均匀。同时将乳酸菌计数培养基倾入加有1ml稀释液检样用的灭菌生理盐水的灭菌平皿内作空白对照，以上整个操作自培养物加入培养皿开始至接种结束须在20min内完成。
6．待琼脂凝固后，翻转平板，置36℃±1℃温箱内培养72h±3h取出，观察乳酸菌菌特征，选取菌落数在30～300之间的平板进行计数。计算后，随机挑取5个菌落数进行革兰氏染色，显微镜检查并做过氧氢酶试验。革兰氏阳性，过氧化氢酶阴性，无芽胞的球菌
或杆菌可定为乳酸菌。根据证实为乳酸菌菌落计算出皿内的乳酸菌数，然后乘其稀释倍数即得每亳升样品中乳酸菌数。例如，检样10－4的稀释液在改良TJA琼脂平板上，生成的可疑菌落为35个，取5个鉴定，证实的乳酸菌的4个，则1ml检样中乳酸菌数为：
35×4／5×104＝2.8×105
7．乳酸菌在改良TJA和改良MC培养基上菌落生长形态特征。
（四）乳酸菌的鉴定
对上述分离到的乳酸菌需进行菌种鉴定时，则作以下试验。
1．菌种制备 自平板上挑取菌落，接种于改良TJA或改良MC琼脂斜面，于36℃±1℃，24～48h培养，刮取菌苔，分别进行下列试验。
2．乳酸杆菌鉴定试验 极少见还原硝酸盐，不液化明胶，不产生靛基质和硫化氢。
3．常见乳杆菌属内种的碳水化合物反应。
4．产酸乳的链球菌的鉴别试验。
Detection of Escherichia coli: Determination of coliform bacteria in the operating rules Coliform group refers to the group of ferment lactose, acid gas, aerobic and facultatively anaerobic gram-negative non-sporing bacillus.The bacteria mainly comes from human and animal feces, therefore as the faecal pollution indicators to evaluate the hygienic quality of food, food is not infer pollution intestinal pathogens may. The number of coliform bacteria in food by 100mL (g) samples in coliforms most probable number (MPN) said.
1 the equipment and materials
1.1: 36 ± 1 ℃ temperature box.1.2: 0 ~ 4 ℃ refrigerator.1.3: 44.5 ± 0.5 ℃ constant
temperature water bath.In 1.4 days.The 1.5 microscope.1.6 homogenizer or mortar.1.7 Petri dish: diameter of 90mm.1.8 tube.The 1.9 straw.1.10 jars or Erlenmeyer flask: capacity of 500mL.1.11 glass beads: diameter of about 5mm.1.12 slides.1.13 alcohol lamp.The 1.14 test tube rack.
2 culture media and reagents
2.1 bile salt lactose fermentation tube: according to GB 4789.28 4.9 provisions.
Eosin methylene blue agar plate: 2.2 in GB 4789.28. 4.25 provisions.
2.3 lactose fermentation tube: according to GB 4789.28 4.10 provisions.
2.4 EC broth: according to GB 4789.28 4.11 provisions.
2.5 phosphate buffer solution at GB 4789.28: 3.22 set.
2.6 saline.
2.7 Gram staining solution: according to GB 4789.28 2.2 provisions.
3.1 sample dilution 3.1.1 to aseptic sample 25mL (or G) on the 225mL of sterile saline or other dilution liquid sterilization glass bottles (bottles to the appropriate number of glass beads) or sterilization mortar, after full shaking or grinding to make 1:10 uniform dilution.Solid sample with
homogenizer, 8 000-10 000 r/min 1min 1:10 speed of processing, made of uniform dilution. 3.1.2 1mL sterilizing straw to draw 1:10 dilution of 1mL, injected with 9mL of sterile saline or other dilution liquid inside the tube, shaking the tube mixing, made of 1:100 dilution.
3.1.3 another 1mL sterilization straw, according to the operation in turn do 10 times the incremental dilution, each incremental dilution once, for 1 1mL sterilizing straw. 3.1.4 according to the standards of food hygiene requirements or to sample contamination estimation, choice of three dilutions, each dilution, inoculated with 3 tubes.
3.2 lactose fermentation test
The sample to be detected with bile salt lactose fermentation tube, inoculation quantity is in 1mL above, using double bile salt lactose fermentation tube, 1mL and 1mL below, with lactose bile salt fermentation tube.Each dilution inoculation 3, is 36 ± 1 ℃ temperature is 24 ± 2h, culture, such as all bile salt lactose fermentation tube without gas production, can be reported as coliforms negative, such as gas production, according to the following procedures.
3.3 isolated culture
The gas production of the fermentation pipe are respectively used in the eosin methylene blue agar plate, is 36 ± 1 ℃ temperature box, 18-24h culture, then removed, colony morphologies were observed, and Gram staining and proof test.
3.4 proof test
In the plate, were suspicious of coliforms colony 1-2 for Gram staining, simultaneous inoculation of lactose fermentation tube, is 36 ± 1 ℃ temperature inside culture of 24 ± 2h, observation of gases.Where lactose tube gas production, gram negative bacillus can report no, as coliforms.
3.5 Report
According to confirmed as coliforms tube number, check the MPN retrieval table, each
100mL (g) MPN value of coliform bacteria.
4 fecal coliform (faecal coliform) 4.1 inoculation loop by all the gas producing bile salt lactose fermentation tube cultures (see clause 3.2) transferred to EC broth tubes, is 44.5 ± 0.2 ℃ water bath tank (water bath tank water level should be higher than the EC broth culture), 24 ± 2h, after cultivation, such as all EC broth tubes are not gas production, can be reported as negative; such as gas, then all the gas EC broth tubes are respectively switched to the Iranian red
methylene blue agar plate, the culture of 18-24h, where the plate has typical colony, is confirmed for fecal coliform positive.
4.2 findings report
According to the confirmed fecal coliform positive number, check the MPN retrieval table, each 100mL (g) MPN value of fecal coliforms
Detection of lactic acid bacteria:
An overview
Lactic acid bacteria are a group of decomposition of glucose or lactose to produce lactic acid, aerobic and facultative anaerobic, most unpowered, catalase negative, gram positive bacillus and coccus without.This class of bacteria widely distributed in nature, can live in the human and animal gut and other organs, the mouth, in the soil, food, feed, water and some clinical specimens are lactic acid bacteria in the presence of.Lactic acid bacteria in industry, agriculture and medicine and human life is closely related to the field of application of value is very high, quite a number of lactic acid bacteria on human, animal health plays a beneficial role, but individual species to human and animal pathogenic, lactic acid bacteria mainly includes 23 genera of bacteria.
Two sample collection
The sample is mainly contains live lactic acid bacteria beverage and preparation, samples should be stored in the fridge, heart fast inspection.
Three test methods (the people's Republic of China national standards of lactic acid bacteria beverage of lactic acid bacteria microbiological examination of the GB / T 16347-1996) Lactic acid bacteria: Determination of the total number of lactic acid bacteria colony is the index sample cultured under certain conditions, the 1ml sample contained in the total number of lactic acid bacteria.
(a) inspection procedure
Lactic acid bacteria colony count determination procedure is as follows:
(two) culture media and reagents Improved TJA medium (modified tomato juice agar); improved MC medium (modified Chalmers medium); 0.1% methylene blue milk medium; Sodium Chloride Broth 6.5% reference; pH9.6 glucose broth; 40% Bile Broth; starch hydrolysate medium; arginine hydrolysis medium; lactobacillus fermentation tube seven leaf glucoside; culture medium; Gram staining solution; 3% hydrogen peroxide solution; peptone water, indole reagent; culture medium; nitrate medium, nitrate reagent; saline: quantitative packaging in triangular bottle and tube sterilization.
(three) steps 1 with sterile operation will thoroughly shaken sample 25ml (or 25g) into a 225ml of sterile saline in the jar made of 1:10 uniform dilution.
2 with 1ml sterilizing straw to draw 1:10 dilution of 1m, along the pipe wall was injected with 9ml of sterile saline in vitro (Note: do not touch the tip of straw tube dilution). 3 another 1ml sterilization straw, according to the order of operations, 10 doubling dilution, so each incremental time, namely the change with 1 1ml sterilizing straw. 4 select 2 to 3 or more appropriate dilution, respectively in 10 times diluted while
increasing, namely to draw the dilution of the straw shift 1ml dilution in sterile Petri dish, each dilution of two Petri dish.
5 dilution liquid into the Petri dish, will be timely cooling to 50 ℃ medium for lactic acid bacteria counts (modified TJA or modified MC) into a Petri dish of about 15ml, and rotating the flat dish make uniform mixing.At the same time, medium for lactic acid bacteria counts pour with 1ml dilution of samples with sterile saline sterile Petri dish for the blank control, the whole operation from above culture into Petri dishes to inoculation end shall be completed within 20min.
6 agar solidified, turning plate, is 36 ± 1 ℃, the temperature inside cultured 72h ± 3H removed, observation of lactic acid bacteria characteristics, select the colony number in 30 ~ 300 between the plate count.After calculation, randomly selected 5 colonies were Gram staining, microscopic examination and hydrogen peroxide enzyme test.Gram positive, catalase-negative, spore of Bacillus aureus or may be set for lactic acid bacteria.According to confirmed for lactic acid bacteria colony calculates a dish of lactic acid bacteria, and then by the dilution factor that each ml sample of lactic acid bacteria.For example, the sample from 10 - 4 dilution in improved TJA agar plates, generating the
suspected colonies for 35, taking 5 identification, confirmed the presence of lactic acid bacteria in 4, then 1ml sample of lactic acid bacteria for:
35 x 4 / 5 x 104 = 2.8 x 105 7 of lactic acid bacteria in the modified TJA and modified MC medium colony growth morphology.
(four) the identification of lactic acid bacteria
The lactic acid bacteria isolated from need for strain identification, for the following test. 1 Preparation of bacteria from flat screen colony, inoculated in the modified TJA or
improved MC agar, at 36 ± 1 ℃, 24 ～ 48h culture, the scraping of the lawn, conducted the following experiment. 2 Lactobacillus identification test rare nitrate reduction, liquefaction of gelatin, does not produce indole and hydrogen sulfide.
3 common Lactobacillus species in genus carbohydrate response.
4 production of yoghurt Streptococcus identification test.