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Stool Culture. Dr. Kiarash Ghazvini Department for bacteriology and virology, Mashhad University of medical Sciences. مقدمه. مهمترين باكتري هاي بيماريزاي روده اي كه قابل گزارش است شامل Salmonella Shigella sp Campylobacter 0157:H7 , E. Coli Yersinia enterocolitica Clostridium difficile
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Stool Culture Dr. Kiarash Ghazvini Department for bacteriology and virology, Mashhad University of medical Sciences
مقدمه • مهمترين باكتري هاي بيماريزاي روده اي كه قابل گزارش استشامل Salmonella • Shigella sp • Campylobacter • 0157:H7,E. Coli • Yersinia enterocolitica • Clostridium difficile • Vibrios cholera • Aeromonas • Plesiomonas. • مي باشد.
مقدمه • S. aureus و Candida albicans را مي توان به تعداد زياد و بصورت خالص از مدفوع كساني كه آنتي بيوتيك گسترده مصرف مي كنند جدا نمود و ارزش تشخيصي دارد.
When a stool specimen is sent to the microbiology laboratory for analysis, the physician mustcorrectly request the appropriate examination andunderstand the limitations of that test. • In theUnited States, stool specimens are most commonlysent for "bacterial culture." For most laboratories,this means culturing for C jejuni and Salmonellaand Shigella organisms. If other organisms are suspected, the laboratory must be notified.
مقدمه • در بيماران بستري كه بعد از سه روز بستري در بيمارستان دچار اسهال شده اند بايد به C. difficile شك نمود و مدفوع از نظر حضور توكسين باكتري بررسي شود.
جمع آوري و انتقال نمونه مدفوع • نمونه تازه مدفوع بايستي حداكثر ظرف 30 دقيقه به آزمايشگاه منتقل و در عرض 2 ساعت آماده سازي و كشت شود. اگر زمان انتقال و كشت بيش از اين مقدار باشد بايستي نمونه مدفع را در محيط انتقالي مناسب (Cary-Blair medium) اخذ و انتقال داد.
Refrigeration mustbe avoided as much as possible. • No preservativescan be added to stool samples for bacterial detection. Cary-Blair or other suitable transport • Stool specimens should be transported to the laboratory soon after collection.
جمع آوري و انتقال نمونه مدفوع • سواب ركتال براي تشخيص و شناسايي باكتري هاي بيماريزا درموارد اسهال حاد كافي است ولي نمونه مناسبتر و انتخابي نمونه مدفوع تازه است. ازمايشگاه مي تواند روزانه يك نمونه مدفوع از هر بيمار را پذيرش نمايد و تكرار آزمايش بيش از دوبار در روز براي يك بيمار توجيهي ندارد. نمونه مدفوعي كه براي رديابي سمC. difficileارسال مي گردد بايد فاقد محيط انتقالي باشد.
نمونه هاي غير قابل قبول كه نبايستي پذيرش شود • مدفوع جمع آوري شده كه داراي آلودگي خارجي است • نمونه مدفوع آلوده شده با ادرار • نمونه هاي بدون مشخصات و يا داراي مشخصات نا مناسب در تمام موارد فوق بايستي بخش و پزشك معالج را از عدم پذيرش و غير قابل قبول بودن نمونه مطلع نمود. تا در صورت نياز نمونه مناسب ارسال شود
روش كشت و محيطهاي كشت • نمونه رابر روي محيط كشت مناسب بصورت سه فاز كشت نماييد و پليتها را بمدت پيشنهاد شده در جدول زير در انكوباتور قرار دهيد..
Campylobacter jejuni • C. jejuni grows best at 42° C in an atmosphere containing 5% to 10% oxygen. Campylobacter species are therefore considered microaerophilic. • Many laboratories purchase tanks of gas with an appropriate mixture to provide an atmosphere for culturing this organism.
Campylobacter jejuni • In settings where this is not practical (e.g., when evaluating an outbreak in an area without immediate access to a laboratory), an acceptable atmosphere can be created with little expense by using a candle jar.
Campylobacter jejuni • C jejuni has characteristic colonial morphology, described as "running" and "wet-looking"because the colonies seem to run together. Microscopic morphology shows the typical gram-negative, curved rods that look like a seagull'swings. • This characteristic morphology of the genus Campylobacter differentiates it from P. aeruginosa, which also grows at 42° C and is oxidase positive.
Salmonellae • Salmonella infections are confirmed by culture • Organisms are most likely to be recovered from blood cultures of patients suspected of typhoid fever if the specimens are obtained during the first week of the infection.
Salmonellae • Stool cultures yield the organisms during the third and fourth week of the infection. • Routine microbiologic media such as sheep blood agar and MacConkey agar, and highly selective enteric media, such as Hektoen enteric (HE) agar and xylose-lysine-deoxycholate (XLD) agar are used for recovery. • Serotyping should be performed whenever possible.
Shigellae • Shigellae are fragile organisms that do not survive well outside the host for a long period. These organisms are particularly susceptible to acid pH; therefore stool samples should be processed as soon as they are received in the laboratory .
Shigellae • Diarrheic stools from patients with suspected shigellosis contain pus and blood, a presentation typical of an invasive agent. • Bloody stools must be plated as soon as possible on appropriate enteric media.
Escherichia coli • Diarrheogenic E. coli do not look different on a growth plate from E. coli that do not cause diarrheal disease. • Diagnosis of diarrheal disease caused by E. coli requires a high index of suspicion from the clinician on the basis of history and physical findings.
Escherichia coli • In addition, because E. coli are part of the normal fecal flora, special tests are needed to differentiate these pathogens from the routinely isolated nonpathogenic E. coli. • For example, • many enterohemorrhagic E. coli do not ferment sorbitol. Sorbitol-negative E. coli can be selected in the laboratory by using a sorbitol plate, such as sorbitol MacConkey agar • Antisera are also used to screen for specific serotypes. • Enteroinvasive E. coli produce colonies and biochemical reactions similar to those of Shigella species. Tests to differentiate between these pathogens should be performed.
Yersinia species • Yersinia sp. grow well at 25° C. • This characteristic may be used in the laboratory. • Plating and incubation at this temperature and the use of selective media (e.g., cefsulodin-irgasan-novobiocin [CIN] agar) permit ready isolation of Yersinia organisms.
Yersinia species • Cold enrichment procedures, such as placing fecal samples on isotonic saline and keeping them at 4° C before the inoculation of selective medium, have increased recovery of the organism.
Vibrio species • Vibrio sp. requires highly selective medium for maximum recovery. Thiosulfate-citrate-bile salts-sucrose (TCBS) agar inhibits the usual colon flora. • In addition, TCBS agar differentiates sucrose-fermenting from non-sucrose-fermenting vibrios
كلني باكتري هايي كه احتمال مي رود بيماريزا باشد را بايستي با كمك تستهاي بيوشيميايي شناسايي و تعيين جنس و گونه نمود. براي باكتري هاي بيماريزا انجام تست آنتي بيوگرام بر اساس روش استاندارد ضروري است.
گزارش نتايج جداسازي و شناسايي باكتري هاي • Salmonella • Shigella sp • Campylobacter • 0157:H7E. Coli • Yersinia enterocolitica • Vibrios cholera • Aeromonas • Plesiomonas. با اهميت مي باشد و بايستي گزارش گردد. • لازم به ذكر است كه گزارش عدم جداسازي هر يك از باكتري هاي فوق نيز بايد اعلام گردد.
