微生物学检查

Experiment

IV Microbiological examination of bacteria

Pan Tingting, PhD

Key Lab of Medical Molecular Virology,

Shanghai Medical College, Fudan University

Contents

1. Video Lab diagnosis of bacterial infection

2.Discussion a)

Diagnostic principle of bacterial infection b)How to write a recorder of an experiment

3. Operation a)isolate bacterium from stool(1st ): inoculation onto

MacConkey agar

b)

Acid-fast stain 4.Design of widal test

5.Demonstration Mycobacterium and its

colonies

：

：

：：

Diagnosis of Bacterial Infection Patient Clinical

diagnosis

Non-microbiological

investigations

Sample Take the correct specimen

Take the specimen correctly

Label & package the

specimen up correctly

Appropriate transport &

storage of specimen

A proper clinical assessment is essential for optimal use of

laboratory services!

Haematology

Biochemistry

Radiology

Principle of collection

１．２．　３．．asepsis

avoid contamination by normal flora or bacteria from environment.

take the correct specimen

specimens include blood, urine, pus, stool, sputum, CSF, or other material, as indicated by the

localization of the disease process.

early before using antibiotics

4fresh

test ASAP （）

As Soon As Possible

septicemia

epidemic meningitis enteric fever Blood specimen

Disease or symptoms specimens

respiratory tract infection Sputum specimen urinary infection mid-stream urine digestive tract infection vomitus or stool purulent infection pus

STD

secreta form Urogenital Tract CSF or blood Blood (1-2weeks) Stool (2-3weeks)

０

１０

２０

３０

４０

５０

６０

７０

８０

９０

１２３４５６

７８

ｗｅｅｋｓ　ｐｏｓｔ　ｔｙｐｈｏｉｄ　ｆｅｖｅｒｐｏｓｉｔｉｖｅ　ｒａｔｅ（％）ｂｌｏｏｄｓｔｏｏｌｕｒｉｎｅPositive rate of specimens from patients suffering enteric fever

Agglutinins

specimen Common pathogenic bacteria

blood

stool CSF Bacteroides fragilis

Brucella

Burkholderia pseudomallei

Candida albicans and Cryptococcus neoformans

Haemophilus influenzae

Neisseria meningitidis

Non-fermenters other than Pseudomonas aeruginosa

Other Enterobacteriaceae

Pseudomonas aeruginosa

Salmonella typhi and non-typhi

Staphylococcus aureus

Streptococci(S.pyogenes,S.pneumoniae,viridans streptococci)

（脆弱类杆菌）

（布鲁氏菌）

（类鼻疽伯克氏菌）

（白色念珠菌和新型隐球菌）

（流感嗜血杆菌）

（脑膜炎奈瑟球菌）

（铜绿假单胞菌）

（肠杆菌）

（沙门氏菌）

（金黄色葡萄球菌）

（链球菌）

Aeromonas and Plesiomonas, campylobacter spp, salmonella typhi, shigella…

…

Cryptococcus neoformans

Enterobacteriaceae

Haemophilus influenzae

Listeria monocytogenes

（产气单胞菌、邻单胞菌、弯曲杆菌、伤寒沙门氏菌、志贺氏菌）

（新型隐球菌）

（肠杆菌）

（流感嗜血杆菌）

（单核细胞增多性李斯特氏菌）

Common pathogenic bacteria in clinical specimens

sputum

throat swab urine

Genital secretion wound stomach

Lab diagnosis of bacterial infection bacteria tranditional method——isolate and identify the bacteria

rapid diagnosis——1morphology

2antigen test (protein)

3nuclear acid (DNA/RNA)

antibodies Test the antibodies in patients using the known antigens

：

：

、、、

Test the antibodies in patients using the known antigens

q n

n q ａｎｔｉｇｅｎ：ｓｔｒｕｃｔｕｒｅｏｆｂａｃｔｅｒｉａ（Ｏ，Ｈ，Ｖｉ）ｔｏｘｉｎ（Ｏｓｔｒｅｐｔｏｌｙｓｉｎ）Ｓｐｅｃｉｆｉｃａｎｔｉｇｅｎ

ａｎｔｉｂｏｄｙ：ｅａｒｌｙＩｇＭ，ｌａｔｅｒＩｇＧ

n q

q

q method serum titer

：ＡｂｉｎｒｅｓｔｏｒａｔｉｏｎｓｔａｇｅＶｓＡｂｉｎａｃｕｔｅｓｔａｇｅ：≥４Ｔｙｐｈｏｉｄｆｅｖｅｒ、ｐａｒａｔｙｐｈｏｉｄｆｅｖｅｒＯ≥８０，Ｈ≥１６０，ＲｈｅｕｍａｔｉｃｆｅｖｅｒＯ≥５００

Procedure of identify the bacteria

Collect and transfer the specimen

Inoculate into suitable medium

streak staining morphology

biochemistry reaction

serology identify toxicity identify)

antibiotics resistance test

report

、、（

Isolate and identify bacteria from stool specimen (exp4-7)

n Purpose

asepsis

procedure for isolation of stool bacteria

Procedure

Collect,store and transfer the

specimens

Separation culture(streaking

on plate)

Pick up predominant isolates Gram staining

Inoculation in

cooked meal

medium

Inoculate on biochemistry slip, Antibiotic susceptibility tests

Judgment on the results

inoculation

ｃｏｔｔｏｎ　ｓｗａｂ　ｆｏｒ　ａｒｅａⅠ，　ｓｔｒｅａｋｉｎｇ　ｂｙ　

、、

ｉｎｏｃｕｌａｔｉｎｇ　ｌｏｏｐ　ｆｏｒ　ａｒｅａ　ⅡⅢⅣ．

MacConkey agar

ｔｒｙｐｔｏｎ ２％ＮａＣｌ ０．５％

ｌａｃｔｏｓｅ １％ｂｉｌｅ　ｓａｌｔ ０．５％　

ｐｈｅｎｏｌ　ｒｅｄ　（　ｐＨ　６．８～８．０，）

１．５ａｇａｒ １．５－－２％

Bile salt inhibit the growth of gram positive bacteria

phenol red indicate the pH of the medium

Selective & differential medium

：：

（）

E coli E coli lactose -

Proteus Salmonella typhimurium

Experiment recorder: Requirements

1date of experiments, number or source of samples, name of culture medium, lot nomber of medium and other products

procedure and results in detail

3To achieve a final report, you should summarize all the recorder, and give your analysis of the results, then draw a conclusion .

、、２、

Experiment recorder: model

Contents should be involved as following

：

1. Name of exp

2. Purpose

3. Design or procedure

4. Time of exp

5. Material in detail samples: source, number

instruments: name, type

reagents: name, company, batch number animal: strain: name, source

（）

……