Post on 19-Dec-2015
Objective of Experiment To mTo masteraster basicbasic principleprinciple and and
methodmethod,which use to isolate and identify ,which use to isolate and identify pyogenic cocci pyogenic cocci from clinical from clinical sspecimens.pecimens.
To To diagnosediagnose clinical clinical disease and guide disease and guide doctors to select proper medicines .doctors to select proper medicines .
Specimens
(Gram-stain) Morphological characteristics (Microscopic examination)
Isolation culture
Smears
(blood agar plate)
Colonial characteristics
Hemolytic reaction
Pigmentation
PROCEDURE
morphology observation
colonial
typical colonies
stained and observe
Pure culture Antibiotic
susceptibility test
Direct identification
Identified method for Staphylococci
Gram-stainGram-stain Isolation Isolation and cultureand culture Pure culturePure culture
DirectDirect identifidentificationication
Direct identification
The mannitol fermentation test.The mannitol fermentation test. The Coagulase TestThe Coagulase Test The Dnase TestThe Dnase Test The phage typing test The phage typing test Animal ExperimentAnimal Experiment
The mannitol fermentation test.
Inoculate the bacteria into a mannitol Inoculate the bacteria into a mannitol micro-tube,incubate at 37micro-tube,incubate at 3700C for 18h.SC for 18h.S.aureus will ferment mannitol to prod.aureus will ferment mannitol to produce acid,which causes the medium tuce acid,which causes the medium to turn yellowo turn yellow. .
Test of Microbiological experiment While a patient is infected by pathogenic enterobacteria (While a patient is infected by pathogenic enterobacteria (Sal
monella),how to diagnose with microbiological methods in la,how to diagnose with microbiological methods in lab?b?
(simple procedure)(simple procedure)
While a patient is infected by pyogenic cocci,how to diagnosWhile a patient is infected by pyogenic cocci,how to diagnose with microbiological methods in lab?(simple procedure)e with microbiological methods in lab?(simple procedure)
While a patient is infected by influenza virus,how to diagnosWhile a patient is infected by influenza virus,how to diagnose with microbiological methods in lab?(simple procedure)e with microbiological methods in lab?(simple procedure)
The Coagulase Test
Coagulase is an enzyme converting fibrinogen inCoagulase is an enzyme converting fibrinogen into fibrin promoting blood clotting. to fibrin promoting blood clotting.
It might be a virulence factor with the coagulateIt might be a virulence factor with the coagulated blood around the bacteria protecting them frod blood around the bacteria protecting them from the immune system. m the immune system.
Coagulase-negative strains are often as pathogeCoagulase-negative strains are often as pathogenic as coagulase-positive strains.nic as coagulase-positive strains.
The Dnase Test Inoculate Dnase agar plates with a loop so Inoculate Dnase agar plates with a loop so
that the growth is in plaques about 1 cm in that the growth is in plaques about 1 cm in diameter.Incubate at 37diameter.Incubate at 3700C overnight.Flood C overnight.Flood the plate with 1 N hydrochloric acid.Clearithe plate with 1 N hydrochloric acid.Clearing around the colonies indicates Dnase acng around the colonies indicates Dnase activity.The hydrochloric acid reacts with untivity.The hydrochloric acid reacts with unchanged deoxyribonucleic acid to give a clchanged deoxyribonucleic acid to give a cloudy precipitate.A few other bacteria,e.g. oudy precipitate.A few other bacteria,e.g. Serratia,may give a positive reaction. Serratia,may give a positive reaction.
The phage typing test
This test is used to trace the infective This test is used to trace the infective agent in epidemiology if necessary.agent in epidemiology if necessary.
It is usually not done for routine cliniIt is usually not done for routine clinical purposecal purpose. .
Animal Experimentvomit excrement remaindered food
Isolation and identification of bacteria
Meat soup media
filter injection
6—8w cat
observationFood poisoning
The antibiotic susceptibility test This test is helpful for the treatment This test is helpful for the treatment
of S.aureus infectionof S.aureus infection.. materialsmaterials
StaphaureusStaphaureus (( isolated from the pus of isolated from the pus of a patienta patient )) . .
Several kinds of filter paper Several kinds of filter paper (( each coneach contains different kinds of antibioticstains different kinds of antibiotics ))
Nutrient agar plateNutrient agar plate
The antibiotic susceptibility test
Streaking the staphaureus on agar plate
(thoroughly covered the plate)
Put 4 kinds of paper contained different antibiotics on the plate (each paper are far away about 2 cm)
Incubate at 370C for 18-24 hours.
Observe the results the plates The sensitivity of the organism is indicated by the diameter
of the zone of growth inhibition.
Dimidiation of the enterobacteria according to the fermentation of lactose
Lactase fermenters:Lactase fermenters: saprophytic and commensalsaprophytic and commensal
Escherichia Klebsiella Citrobater Escherichia Klebsiella Citrobater
EnterobacterEnterobacter
Non lactase fermentersNon lactase fermenters: : pathogens pathogens
Salmonella Shigella some Citrobacter Salmonella Shigella some Citrobacter
Proteus SerratiaProteus Serratia
Colonial characteristic observation
Specimens isolation Gram Staining
(SS/EMB plate) Serological identification
TSI
Biochemical reaction
Procedure
Specimens
Different specimens should be taken depeDifferent specimens should be taken depending on the kind and the process of the nding on the kind and the process of the disease.disease.
blood blood
bone marrowbone marrow
Urine Urine
stoolstool
Isolation
Culture medium: S.S agar Culture medium: S.S agar Method: streak plateMethod: streak plate Result: Result:
Pathogenetic colonies: middle size, redPathogenetic colonies: middle size, red
Suspect colonies: colorless, small, opSuspect colonies: colorless, small, opaque aque
Biochemical reactions of Salmonella, etc
Species bottom slope H2S motilitySpecies bottom slope H2S motility E.coli AG AG - +E.coli AG AG - + Salmonella A - +/- + Salmonella A - +/- + Other Salmonella AG - +/- +Other Salmonella AG - +/- + Shigella A - +/- -Shigella A - +/- -
bottom: ferment dextrose slope: lactosebottom: ferment dextrose slope: lactose A:acid AG: acid and gasA:acid AG: acid and gas
Antigens of salmonella O antigen polysaccharide of LPS somatic antigen
used to divide Salmonella to 42 groups A-Z groups are pathogenic stable to heat (remains activity at 100 )℃H antigen flagella antigen divide Salmonella tow phase: special no special
Vi antigen related to the virulence of Salmonella sensitive to heat ( lose activity at 60 )℃
Serological Identification of Salmonella
Select the specimenSelect the specimen
React with A-Z polyvalent antiserumReact with A-Z polyvalent antiserum
React with several individual React with several individual types of anti-O and anti-H types of anti-O and anti-H
antiserum antiserum
React with anti-Vi React with anti-Vi antiserumantiserum
Agglutination reaction Agglutination reaction
(( ++ ) () ( -- ) )
Identify its group and phage