大肠菌群的检测课程

大肠菌群的检测大肠菌群的定义:

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孢杆菌。

大肠菌群不是细菌学上的分类命名，而是根据卫生学方面的要求，提出的与粪便污染有关的细菌，即作为食品、水体等是否受过人畜粪便污染的指示菌，这些细菌在生化及血清学方面并非完全一致。根据进一步的生化试验，可将这群细菌再分为大肠艾希氏菌（俗称大肠杆

菌）、弗氏柠檬酸杆菌、肺炎克雷伯氏菌和阴沟肠杆菌等。大肠菌群基本形态：此菌为两端钝圆的短小杆菌，一般约0.5-0.8μm*1.0-3.0

μm，多单独存在或成双，但不呈长链排列。约50%的菌株有周生鞭毛，但多数只有1-4根，一般不超过10根，故菌体动力弱。多数菌株有菌毛，有的有荚膜或微荚膜，不形成芽孢，对普通碱性染料着色良好，革兰氏染色阴性。实验原理大肠菌群指一群能发酵乳糖、产酸产气、需氧和兼性厌氧的革兰氏阴性无芽孢杆菌。该菌主要来源于人畜粪便，以此作为粪便污染指标来评价食品的卫生质量，推断食品有否被肠道致病菌污染。食品中大肠菌群以100ml检样内大肠菌群最可能数（MPN）表示。实验器材蒸汽灭菌物品：1.乳糖胆盐发酵培养基试管10支2.9mL蒸馏水试管5支干热灭菌物体：10mL移液管1支1mL移液管5支实验试剂乳糖胆盐发酵培养基蛋白胨

20.0g 0.04%溴甲酚指示液25mL乳糖

10.0g 牛胆盐5.0g 水1000mL除0.04%溴甲酚指示液外，取上述成分，混合，微温溶解，调节PH值为7.2-7.6，加入0.04%溴甲酚指示液，根据要求的用量分装于含倒管的试管中，灭菌。麦康凯琼脂培养基大肠菌群及大肠杆菌测定——MPN法检验流程实验内容与步骤1）检样稀释（无菌操作）①将25mL（或g）检样置于含225mL灭菌生理盐水的

灭菌玻璃瓶，充分振摇（固体检样用匀浆器，用8000－10000r/min的速度处理1

min）制成1：10的均匀稀释液。②用1mL灭菌吸管吸取1：10稀释液1mL，注入含有9mL灭菌生理盐水的试管内，混匀，制成1

：100的稀释液。③另取1mL灭菌试管，按②操作依次做10倍递增稀释液，每稀释一次，换用一支1

mL灭菌试管。2）乳糖发酵实验接种1mL待检样品于乳糖胆盐发酵管内。接种3个稀释度，每一稀释度接种3管，置（36土1)℃温箱内培养（24士2)h。如所有乳糖胆盐发酵管都不产气，则可报告为大肠杆菌群阴性，如有产气者，则按下列程序进行。3）分离培养将产气的发酵管分别转接在伊红美蓝琼脂平板上，置（36士1)℃恒温箱内培养18一24

h。取出，观察菌落形态，并做革兰氏染色和证实试验。4）证实试验在上述平板上，挑取可疑大肠菌群菌落1一2个进行革兰氏染色，同时接种乳糖发

酵管，置（36士1)℃恒温箱内培养（24士2)h，观察产气情况。凡乳糖产酸产气、革兰氏染色为阴性的无芽袍杆菌，即可报告为大肠菌群阳性。查MPN表实验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（或g）大肠菌群的MPN。革兰氏染色基本原理：

革兰氏染色的机理主要是利用两类细菌的细胞壁成分和结构的不同。革兰氏阴性菌的细胞壁中含有较多的类脂质，而肽聚糖的含量较少。当用酒精或丙酮酸脱色时，类脂质被溶解，增加了细胞壁的通透性，使初染后的结晶紫和碘的复合物易于渗出，结果细胞被脱色，经复染后，又染上复染液的颜色。而革兰氏阳性菌细胞壁中肽聚糖的含量多而且交联度大，类脂质含量少，经乙醇或丙酮洗脱后，肽聚糖层的孔径变小，通透性降低，因此细胞仍保留初染时的颜色。★★The

GramreactionThe

Gramreaction

is

named

after

the

Danishphysician,

Christian

Gram,

who

developed

this

stainingtechnique

in

1884.

It

involvesa

series

ofsimple

steps.1.

Bacterial

cells

are

dried

onto

a

glass

slide

and

stained

withcrystal

violet,

then

washed

brieflyin

water.2.

Iodine

solution

is

added

so

that

the

iodine

forms

acomplexwith

crystal

violet

in

the

cells.3.

Alcohol

or

acetone

is

added

to

solubilise

the

crystal

violet

-

iodine

complex.4.

The

cells

are

counterstained

withsafranin,

then

rinsed

and

dried

for

microscopy.The

method

separates

bacteria

into

two

types.

Gram-positive

cells

retain

the

crystal

violet-iodine

complex

and

thus

appear

purple

(shownforBacillus

cereus

in

the

left-hand

image

below).

Gram-negative

cells

are

decolourised

by

the

alcohol

or

acetone

treatment,

but

are

then

stained

withsafranin

so

they

appear

pink

(shownfor

Pseudomonas

aeruginosa

in

the

right-hand

image

below).

Thus,

the

essential

difference

between

Gram-positive

and

Gram-negative

cells

is

their

abilityto

retain

the

crystal

violet-iodine

complex

when

treated

with

a

solvent.★his

difference

instaining

reflects

a

fundamental

difference

in

the

organisation

of

the

bacterial

cell

wall

or

"cell

envelope",

shown

in

highlysimplifiedformbelow.C.Gram，1884年创立，是最重要的染色法。革革兰兰氏氏染染色色的的原原理理:从上图可知，甲、乙两种细菌经结晶紫初染后，分别染上了紫色，经碘液媒染，结晶紫与碘液形成了分子量较大的复合物，在乙醇脱色时，凡是染上的紫色容易被脱掉的细胞又成为无色菌体（如乙菌），反之则仍为紫色（如甲菌）。最后再用红色染料——沙黄复染，结果甲菌仍然维持最初染上的紫色，而乙菌则被复染而成红色，前者称为革兰氏阳性细菌，简称G+菌，后者则称为革兰氏阴性细菌，简称G-菌。革兰氏染色是细菌分类鉴定的重要指标，通过革兰氏染色，可以将几乎所有细菌分成革兰氏阳性细菌和革兰氏阴性细菌两大类。这两大类细菌在细胞结构、成分、形态、生理、生化、遗传、免疫、生态和药物敏感性等方面都呈现出明显的差异，因此任何细菌只要先通过很简单的革兰氏染色，即可提供不少其他重要的生物学特性方面的信息。Gram,

who

invented

the

gram-staining

method

in

1884.To

gramstain,

an

investigator

smears

a

sample

ofbacteria

on

a

slide,

soaks

itin

aviolet

dye

and

then

treats

it

with

iodine.

The

slideis

then

rinsedwith

alcohol

and

counterstained

witha

pink

dye

called

safranine.The

cell

walls

of

gram-negative

bacteria

have

a

very

lowaffinity

for

the

violet

stain,

whichis

rinsed

out

by

the

alcohol.

Oncecounterstained

with

safranine,

however,

the

gram-negative

bacteria

appear

bright

pink

to

red.

Gram-positive

cell

walls

have

a

highaffinity

for

the

violet

stain,

and

retain

iteven

through

the

alcohol

rinse.

When

the

process

is

complete,

they

appear

dark

purple

tobrown.The

difference

between

the

two

cell

types

appears

to

be

in

the

amount

of

peptidoglycan

in

the

cell

wall.

Grampositive

cell

walls

areabout

five

times

as

rich

in

peptidoglycan

as

gram-negative

cellwalls.

In

addition,

gram-negatives

have

a

second

membrane(chemically

different

fromthe

plasma

membrane)

external

to

the

cellwall,

and

may

also

have

a

gelatinous

sheath

external

to

thesecond

membrane.

Cozy.The

differences

in

the

cell

wall

are

more

than

simply

a

classification

tool.

Cell

wall

characteristics

are

intimately

related

to

the结果:阳性菌——紫色阴性菌——红色结晶紫初染碘液媒染乙醇脱色蕃红复染disease-causing

potentialof

the

bacterium.

In

fact,

medical

researchers

have

found

th