| The first part:GBS related Cj Colonization in guinea pigObjective:1Separating Campylobacter jejuni from the gastrointestinal tract ofguinea pigs.2To locate the Campylobacter jejuni in guinea pigs.3To study the morphological and physiological changes ofCampylobacter jejuni in different site of guinea pig gastrointestinal.Methods:1Cultivating Campylobacter jejuni: Campylobacter jejuni was isolatedfrom the Guillain-Barre syndrome patients, identified by the China Center forDisease Control and Prevention by the U.S. Center for Disease Control andPrevention as Campylobacter jejuni strains, serotype penner O:19type strains.Culture the campylobacter jejuni on Colombia blood agar plate undermicroaerobic condition at42℃,5%O2,10%CO2,85%N2. Then Identify thecampylobacter jejuni.2Identify the Campylobacter jejuni: Morphology inspection, oxidase test,sodium hippurate hydrolysis test, PCR and other methods were used toIdentify the Campylobacter jejuni.3Grouping experimental animal: there're all15Hartley guinea pigswhich were6weeks old, weight between250g and350g, and they wererandomly divided into two groups,12guinea pigs in the experimental groupand the others in the control group, the experimental group was also dividedinto two groups, the separated group and the colonized group.4The way and the concentration of bacterial suspension to feed theHartley guinea pigs: All the Hartley guinea pigs were starved for12hours, prepared3×10~8CFU/ml bacterial suspension, and then fed2ml to theexperimental group pigs,fed Brucella broth2ml to the control group.5Dissecting the animals: Keeping the separated group's guinea pigs for48hours, keeping the colonized group's guinea pigs for7days, and thendissecting the animals after weighing, all the animals were anesthetized by4%chloral hydrate (10ul/g), and then killed by heart bled. Removed thegastrointestinal tract by aseptic technique, selected four samples from stomachand cecum, selected one sample from small intestine and lager intestine every5-10cm length.6Isolating and culture: Took0.5cm of the intestinal tissue into5-8mlBrucella broth medium, in microaerophilic and42℃conditions cultured for24hours and subcultured to Columbia blood medium, the same conditions,cultured for24-48hours. Observe the colony morphology, and selected thesuspected colony, passage to the Colombian media, Then purified the strains.7Identified the strains: Morphology inspection, oxi-dase test, sodiumhippurate hydrolysis test, PCR and other methods were used to Identify thestrains.8Gram stain: Took0.5cm3gastrointestinal contents, used a sterile cottonswab to smear, after gram stain, used a1000times optical microscope toobserve whether there're some various like Campylobacter jejuni or theirmorphological changes in different parts of the gastrointestinal, while learningthe internal environment of the gastrointestinal tract.9Statistic the location and quantity of the gastrointestinal tract where canisolate the campylobacter jejuni.Results:1Irrigated the separated group with bacteria and dissected the animalsafter48hours, cultured of the organization and contents of the gastrointestinaltract, separation of the campylobacter jejuni from each of the intestinal tract.2Irrigated the colonization group with bacteria and dissected the animalsafter7days, cultured of the organization and contents of the gastrointestinaltract, separation of the campylobacter jejuni from each of the intestinal tract. 3Irrigated Brucella broth medium to the control group after48hours and7days, then dissected the animals, cultured of the organization and contents ofthe gastrointestinal tract, undetached campylobacter jejuni.4Gram stain of the gastrointestinal tract contents of the control group,used a1000times optical microscope to observe, didn't see some various likecampylobacter jejuni.5Dissected the animals after irrigating bacteria for48hours, Gram stainof the gastrointestinal tract contents, used a optical microscope to observethere're some various like Campylobacter jejuni; in different gastrointestinaltract, can not distinguish the form of Campylobacter jejuni changes, especiallyin the cecum and colon parts, there's too much bacteria to figure thecampylobacter jejuni out.Conclusions:1Campylobacter jejuni was not isolated from the gastrointestinal tract ofclean grade and healthy guinea pig.2Campylobacter jejuni was fed to guinea pigs,then it will survival in thegastrointestinal tract of guinea pig.3Directly Gram stain of the gastrointestinal tract contents, we can notdistinguish the Campylobacter jejuni because of the smaller form and could becovered by the other enteric bacteria. Objective:1To learn whether there's some Campylobacter jejuni in the intestinalfistula effluent.2To learn whether the isolation rate of Campylobacter jejuni from theintestinal fistula effluent is higher than that from non-intestinal fistula stool inthe non-diarrhea, non-Guillain-Barre syndrome population.3To learn whether the more close to the colostomies,the isolation rate ofCampylobacter jejuni is more higher.Methods:1Specimens Source: specimen were from three patients who wereclinical diagnosised of Hirschsprung patients. Three patients were fromPediatric Surgery of the Second Hospital of Hebei Medical University,September2011to October.2Collecting specimens: Collected the specimens from the intestinalfistula effluent and the stool,less in2hours we should put the specimens into5-8ml Brucella broth medium.3.Enrichment: culture the specimens under microaerobic condition at42℃,5%O2,10%CO2,85%N2for24hours.4Subculture: subcultured bacterial suspension to Columbia bloodagar,in microaerophilic environment at42℃,5%O2,10%CO2,85%N2for24to48hours5Purification: We observed the morphology of the Columbia blood agargrowth of bacteria, picked suspicious single colonies to Columbia blood agarpurified bacteria by using ring vaccination, and picked three colonies in everyplate to increasing the positive rate of selection of the purpose of strain.6Bacterial identification included bacterial colony morphology, Gramstaining and biochemical identification. Molecular identification wasperformance when necessary,7The isolated bacteria were saved at-80℃. Results:13cases of patients with fistula effluent bacteria were detachedCampylobacter jejuni by using this the experimental method;2The identified separation small colonies bacteria hippurate sodiumhydrolysis test was positive, and Gram staining was Gram-positive cocci.33cases were not detached Campylobacter jejuni by using thisexperimental method.Conclusion:The method is not suitable for isolating and cultivating of Campylobacterjejuni in patients with colostomy. |
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