人人文库网 > 专业文献 > 学术论文 > MFLP-46 Isolation of Thermophilic Campylobacter from Foods_其它国外
MFLP-46 Isolation of Thermophilic Campylobacter from Foods_其它国外
收藏
资源目录
编号：62235444
类型：共享资源
大小：108.64KB
格式：RAR
上传时间：2020-03-26
上传人：hon****an
认证信息
个人认证
丁**（实名认证）
江苏
IP属地：江苏
3.6
积分
- 关 键 词：
-
MFLP-46
Isolation
of
Thermophilic
Campylobacter
from
Foods_其它国外
MFLP
46
Foods_
其它
国外
- 资源描述：
-
MFLP-46 Isolation of Thermophilic Campylobacter from Foods_其它国外,MFLP-46,Isolation,of,Thermophilic,Campylobacter,from,Foods_其它国外,MFLP,46,Foods_,其它,国外
- 内容简介：
-
Published on the Food Directorates (Health Canadas) website at http:/www.hc-sc.gc.ca/food-aliment. Government of Canada Gouvernement du Canada Laboratory ProcedureMFLP-46March 2002 HEALTH PRODUCTS AND FOOD BRANCH OTTAWAISOLATION OF THERMOPHILIC CAMPYLOBACTER FROM FOODD. Medeiros and L. HofmannMicrobiology Research DivisionBureau of Microbial Hazards, Food DirectoratePostal locator: 2204A2Ottawa, K1A 0L2E-mail: Lisa_Hofmannhc-sc.gc.ca 1.APPLICATIONThis method is applicable to the determination of the thermotolerant and microaerophilic bacteria of the genusCampylobacter in foods to determine compliance with the requirements of Section 4 and 7 of the Food andDrugs Act. This revised method replaces MFLP-46, dated July 1998.2.PRINCIPLEThe procedure consists of four stages. The initial handling of the food at the enrichment stage variesaccording to the type of food under investigation.2.1Selective EnrichmentThe food is initially inoculated into a selective enrichment medium designed to slow or inhibit thegrowth of competing microorganisms while favouring the growth of campylobacters.2.2Colony Formation on Selective AgarsSelectively enriched cultures are streaked onto selective agars specifically developed for the isolationand identification of campylobacters. Presumptive colonies are further identified by examining forcharacteristic morphology and motility using phase contrast microscopy.2.3PurificationThe isolates are purified on an appropriate selective blood or charcoal agar plate.2.4IdentificationMFLP-46March 2002-2-Presumptive campylobacters are identified by biochemical reactions, tolerance to some chemicals andantibiotics, and by growth temperatures. Several biotypes are determined by biochemical reactions.Serotyping schemes for identifying heat-labile antigens (Liors scheme) and heat-stable antigens(Penners scheme) have been developed, however the antisera required are not readily available. Ifserotyping is required cultures may be sent to Health Canadas Laboratory Center for Disease Controlin Winnipeg, Manitoba. Various nucleic acid-based and antibody-based rapid methods for theidentification of Campylobacter are commercially available (9.1, 9.2, 9.4) 3.DEFINITIONSee Appendix A of Volume 3.4.COLLECTION OF SAMPLES4.1 Sampling See Appendix B of Volume 3. 4.1.1Holding and Shipping For holding and shipping, place the sample units in sealed containers at 4oC until analysis. With raw milk samples, supplement with (a) 5% cysteine or (b) 0.01% sodium bisulfite andeither 0.15% sodium thioglycolate or an atmosphere of 100% nitrogen. Sample units of frozenproducts shall be kept frozen. 4.1.2 For environmental samples, follow MFLP-41.5. MATERIALS AND SPECIAL EQUIPMENTThe following media and reagents (1-10) are commercially available and are to be prepared and sterilizedaccording to the manufacturers instructions. See also Appendix G of Volume 3 and reference 9.1 for theformula of individual media.1)Brucella Broth2)Brucella Semi-solid Agar3)Preston Agar (Also known as Campylobacter Agar Base)4) Mueller Hinton Agar5)Mueller Hinton Agar with Blood6)Wilkins Chalgren Anaerobic Blood Agar7)Wilkins Chalgren Anaerobic Broth8)Campylobacter Agar with Charcoal and Deoxycholate plus supplement (CCDA) (Oxoid)9)SIM media10)Nitrate reagents N(1-naphthyl)-ethylene diamine2 HCl and Sulfanilic Acid11)Each of the following biochemicals prepared in a base of Brucella Semi-solid Media: 1% glucose, 1%nitrite, 1% glycine and 3.5% sodium chloride MFLP-46March 2002-3-12)Park and Sanders Enrichment Broth13)Rapid H2S Test Medium (for biotyping)14)Oxidase Test strips or Tetramethylparaphenylenediamine2 HCl reagent15)Gas Mixture (10% CO2, 5% O2, 85% N2) or CampyPak 2 envelope (BBL) or Gas Generating Kit forcampylobacters (Oxoid) or Anaerocult C (Merck)16)Nalidixic Acid (30g) disks, Cephalothin (30g) disks or E-test strips (Oxoid) 17)Sodium Hippurate18)Liquid Nitrogen19)Dimethyl Sulfoxide 20)Hydrogen peroxide 21) Lead acetate strips 22)Vacuum Leak Detector (Electro-Technic Products)23)Cryotubes24)Positive control (ATCC or equivalent) 25)Shaking Incubator (37oC and 42oC) 26)CO2 Incubator (37oC and 42oC) (optional) NOTE: It is the responsibility of each laboratory to ensure that the temperature of the incubators or waterbaths are maintained at the recommended temperatures. Where 35C is recommended in thetext of the method, the incubator may be 35 +/-1.0C. Similarly, lower temperatures of 30 or 25/C maybe +/- 1.0C. However, where higher temperatures are recommended, such as 43 or 45.5C, it isimperative that the incubators or waterbaths be maintained within 0.5C due to the potential lethality ofhigher temperatures on the microorganism being isolated. 27)Glass slides and coverslips28)Blender, stomacher or equivalent 29)Refrigerated centrifuge capable of 16,000 X g.30)Deep Freezer, -700C31)Phase Contrast Microscope6. PROCEDUREAnalyse each sample unit individually.The test shall be carried out in accordance with the following instructions:MFLP-46March 2002-4-6.1Handling of Samples6.1.1See 4.1.1 and .1.2Analyse samples as soon as possible after receipt in the laboratory because campylobactersare extremely sensitive to oxygen, particularly at room temperature. Campylobacters are alsosensitive to freezing, thawing and drying conditions.6.2EnrichmentNOTE: Always include a pure culture and an uninoculated broth as positive and negative controls, respectively,to ensure that the proper incubation conditions are met, and that the broth is not contaminated.6.2.1Fresh raw meat, pork and poultry samples: Cut sample into pieces (0.3-0.5 cm3) and add 25g into a 250 mL flask containing 100 mL of Park and Sanders Enrichment Broth. Incubateunder microaerobic conditions (6.4) at 37oC for 3-4 h. Transfer flask to a 42oC incubator undermicroaerobic atmosphere for 24 and 48 hours. Alternately, increase the incubator temperatureor use a programmable incubator which automatically adjusts temperature after 4h. Shake thecontents of the flask through all steps at 100-120 cycles/min. using a shaking incubator(required for gas exchange during enrichment).6.2.2Milk: Pour 100 mL of milk into a centrifuge bottle. Spin at 16,000 x g for 20 min. at 4oC. Remove the fat layer with a sterile spatula and discard the fat and liquid. Suspend the pellet in100 mL of Park and Sanders selective enrichment broth and follow the procedures forincubation and plating described in 6.2.1 and 6.3. 6.2.3Shellfish: Weigh a 25 g sample into a blender or 400 mL stomacher bag, and stomach or blendat low speed for 30 sec. Transfer the contents of the blender jar or stomacher bag into a 250mL flask containing 100 mL of Park and Sanders Enrichment Broth, and follow the proceduresfor incubation and plating described in 6.2.1 and .4Frozen foods: After thawing, cut sample into pieces (0.3-0.5 cm3)and add 25 g into a 250 mLflask containing 100 mL of Park and Sanders Enrichment Broth and follow the procedures forincubation and plating described in 6.2.1 and .5Swab samples: Transfer each swab into a 250 mL flask containing 100 mL of Park andSanders Enrichment Broth and follow the procedures for incubation and plating described in6.2.1 and PlatingAfter 24 + 2 h and 48 + 2 h incubation, streak two loopfuls/plate of the culture onto CCDA agar andPreston agar. Incubate the agar plates at 37oC for up to 72h under microaerobic conditions.6.4Procedures for Microaerobic AtmosphereMFLP-46March 2002-5-Thermophilic campylobacters are strict microaerophiles and this requirement must be taken intoaccount in enrichment and plating procedures. A commercial gas mixture of 5% O2, 10% CO2, and85% N2 is generally used. One of the following procedures may be used depending upon availabilityof the equipment and materials for generating microaerobic conditions.6.4.1Replacement of air with a gas mixture of 5% O2, 10% CO2, and 85% N2 or use of Gas generator ion envelope kits, such as GasPak (BBL) kits (commercially available)1)Place flasks (up to three) or stomacher bags containing enrichment broth or agarplates into an anaerobic jar or an air-tight cabinet without catalyst. Evacuate the airfrom the jar or cabinet up to about 610-635 mm or 24-25 inches Hg vacuum and refillthe jar with the gas mixture. Repeat the entire procedure at least once. Jars orcabinets can subsequently be placed in a shaking incubator, in order to allow formaximal gas exchange.2)If anaerobic jars with gas generator envelopes are being used follow manufacturesinstructions for use.3)Plates can be incubated in a CO2 incubator with a CO2 level at 10%. 6.5IdentificationUse Brucella Broth supplemented with 0.15 to 0.2% agar and 0.002% neutral red(Brucella Semi-solidmedium) in tubes for maintaining cultures or for the basal medium for biochemical tests. Tubes ofthese semi-solid media can be incubated aerobically. Do not tighten the screw caps of the tubesduring aerobic incubation. Since campylobacters grow near the surface of the medium (10 mm zone),inoculate the culture into this zone.6.5.1From each selective agar medium, select a portion of 3 suspect colonies (smooth, convex,translucent, colorless to cream-colored and pin-point to 2-4 mm in diameter or often spreadcolonies). 6.5.2Examine a wet mount preparation of each colony using a phase contrast microscope. Youngcells of Campylobacter are Gram-negative, small (0.2-0.8 :m wide by 1.5-5 :m long) and S-shaped They possess a characteristic corkscrew-like motility. Old cells of C. jejuni grown formore than 72 hours or cells exposed to air often change to coccoid forms. Carbol-fuchsin ismore effective than safranin as a counter stain. If safranin is used, the exposure time may beextended to 3 minutes or longer. However, Gram staining may be omitted. NOTE: The plate, cell morphology and motility patterns are most critical in the identification of the organism. 6.5.3Pick a portion of the same suspect colonies using an inoculating loop or needle and streak ontoone CCDA and Mueller Hinton with 5% blood plate, with each colony occupying 1/5 of a plate.Incubate plates at 37/C microaerobically for 1 - 2 days and check for purity.6.5.4 Tests for identification a)Catalase test: With an inoculating needle, mix a portion of a colony from the recentlyincubated CCDA plate with a drop of 3% hydrogen peroxide (H202) on a clean glassslide. (Or drop H202 directly onto the culture, provided all other tests have been donefirst). Development of bubbles is indicative of a positive reaction.b)Oxidase test: With an inoculating needle, smear a portion of a colony from the recentlyincubated CCDA plate onto an oxidase strip. The oxidase test is positive if the cellmass turns dark purple in 5-10 seconds. MFLP-46March 2002-6- c)Nalidixic Acid / Cephalothin Resistance / Sensitivity: Streak a MHB blood agar plate asa bacterial lawn. Place one disc each of nalidixic acid (30 g) and cephalothin (30 ug)on the plate. Incubate the plates at 42oC for 48 h under microaerophilic conditions. After the incubation period, determine if there is a transparent (non-growth) zonearound either disc. Resistance to nalidixic acid and cepthalothin is characterized by azone of clearing of #13 and 14 mm, respectively.6.5.5 Additional tests for identification a)Using the MHB plate from 6.5.3, prepare a fresh culture in 50 mL of Brucella broth contained in a 250 mL flask. Incubate at 37oC for 24-48 h under microaerobicatmosphere. The culture is used for inoculation of the following 6 tubes for biochemicaland growth tests (6.5.6b). b)For each isolate, prepare one tube each of at least 6 different Brucella Semi-solidmedia (see sec. 10): glucose, nitrate, SIM, cysteine, glycine, and NaCl.c)Inoculate all tubes with a Pasteur pipette by transferring several drops of the freshbroth culture to the surface zone (10 mm layer) of the media. Do not inoculate theentire tube from top to bottom.d)Place a lead acetate strip into the cysteine tube; crimp the top of the strip over the lip ofthe tube and hold it in place with the cap.e)Incubate all tubes aerobically or microaerobically at 37oC for 3-5 days. The screw capsshould not be tightened during the incubation period. Reading of biochemical testsa)Glucose fermentation: A positive reaction gives a yellow color (acidwith phenol red indicator) in the Brucella Semi-solid Glucose medium.b)Nitrate reduction test: Add an equal volume (0.5-1.0 mL) of the 2nitrate reagents to a semi-solid nitrate tube, and mix. Development ofa red color in one minute indicates a positive test for nitrite production. If no red color develops, add a pinch of zinc dust. A red colordeveloped with zinc dust indicates a negative test. The absence ofcolor with zinc powder is indicative of a positive reaction which resultsfrom a complete reduction of nitrate to ammonia. Note: (a) Zincpowder may become inactive with long storage. Check the activityperiodically with uninoculated nitrate medium. Add a pinch of zinc tothe medium, mix, and add the 2 nitrate solutions. If the zinc powder isstill in an active form, a red color will develop. (b) Nitrate may bechemically reduced to nitrite in prereduced medium without microbialgrowth. Check each batch of medium by adding the 2 test reagents tothe uninoculated medium. Discard the medium if it gives a red color. c)H2S tests: 1) SIM medium: development of a black color in the mediumindicates strong H2S production. 2)Brucella Semi-solid Cysteine Medium with lead acetate strip:blackening of the lead acetate strip indicates H2S production. MFLP-46March 2002-7-Since the reaction in the semi-solid Brucella Cysteine Mediumis more sensitive than in the SIM medium, weak H2Sproducers may give a positive test with the lead acetate stripbut not with the SIM medium. d) Test for growth in Semi-solid Brucella Medium with 1% Glycine and inSemi-solid Brucella Medium with 3.5% NaCl: Observe the growth atthe top zone (10 mm layer) of the medium. If the growth is notdistinctive, record as no growth.6.5.6Additional tests for differentiating C. jejuni from C. coli and C. lari a)Hippurate hydrolysis test: 1)Inoculate a loopful of a 24 h culture from Mueller Hinton Blood Agarinto 0.5 mL of sterile sodium hippurate solution in a small screw-capped or corked tube.2)Incubate tubes, including an uninoculated control, aerobically at 37oCfor a minimum of 3 h.3)Overlay 0.2 mL of ninhydrin solution. (Do not mix)4)Development of a deep purple color within 5 minutes indicates apositive result; development of a slight bluish color is regarded asnegative. b) Trimethylamine N-Oxide (TMAO) test: Test the nalidixic acid resistant strains (presumptive C. lari) in TMAO medium as follows: 1)Add a couple of drops of semi-solid medium culture to the top of aTMAO medium tube and incubate microaerobically at 37oC for 3-7days . 2)Observe for growth spreading below the top of the TMAO media. OnlyC.lari will grow in the presence of TMAO.6.5.7Identification criteriaPure cultures of suspected Campylobacter are Gram-negative, vibroid shaped rods that haveone curve or are S shaped (occasionally spiral). They are motile with a typical corkscrew-likemotion. They have a single polar flagellum at one or both ends of the cell. C. jejuni cells olderthan 72 h, or cells exposed to air often change to spheric forms. Campylobacter sp. are alloxidase positive, non-fermentative, and will also not oxidize carbohydrates.The following are major steps for identifying C. jejuni, C. coli and C. lari from other species ofCampylobacter (see Table 1).a)Campylobacters can be divided into 2 groups on the basis of the catalase test: Discardcatalase-negative cultures as they are C. sputorum (ss. sputorum, ss. bubulus, or ss.mucosalis).MFLP-46March 2002-8-b)Catalase-positive campylobacters can be further divided into 2 groups on the basis ofgrowth temperature, and sensitivity to antimicrobial compounds (nalidixic acid andcephalothin). (1) The heat-tolerant catalase-positive members (C. jejuni, C. coli and C.lari) grow at 42oC but not at 25oC. C. jejuni and C. coli are sensitive to nalidixic acid butresistant to cephalothin, and C. lari is resistant to both antibiotics. In TMAO medium,C. lari will grow but both C. jejuni and C. coli will not. (6.5.7). (2) The nonheat-tolerantcatalase-positive species (C. fetus ss. fetus and C. fetus ss. venerialis) grow at 25oCbut not at 42oC, and are resistant to nalidixic acid and sensitive to cephalothin.6.5.8Biotyping of C. jejuni, C. coli, and C. lari A provisional scheme is as follows:C. jejuniC. coliC. lariTestBiotypeIIIIIIIVIIIIIIHippurate hydrolysis+- - - - Rapid H2S test- - +- - +DNA hydrolysis- +- +- +- +a)Hippurate hydrolysis test:See 6.5.6b)Rapid H2S test:1)Gently suspend a large ball-like inoculum (5 mm loopful) from a 24h cultureinto the upper third of the Rapid H2S Test Medium.2)Do not mix.3)Incubate in a 370C waterbath for 2 h.4)A blackening around the bacterial mass represents a positive reaction andusually begins to appear within 30-45 min. NOTE: Use only 24-h cultures.c)DNA hydrolysis test:1)Inoculate a circular area (diameter 5 mm) on Toluidine Blue - DNA Agar with a3 mm loopful of a 24-48 h culture by pressing the loop with inoculum into theagar.2)Incubate at 42oC for 24-48 h under microaerobic conditions.3) A clear-colorless or pinkish zone around the inoculum is considered a positivereaction.MFLP-46March 2002-9-6.5.9SerotypingSee section 2.4Rapid testsSome rapid immunological tests have been developed, including latex agglutination tests,enzyme linked fluorescent assay systems and DNA hybridization (9.1, 9.2, 9.4)7. MAINTENANCE OF CULTURESStock cultures may be prepared and stored as follows:7.1Semi-solid mediuma)Inoculate the culture (heavy inoculum) at the top (10 mm) portion of Semi-solid BrucellaMedium and incubate aerobically at 370C.b)Keep in incubator at 37oC, and transfer weekly or when most of the medium turns yellow.7.2Culture storage in freezerGlycerol peptone medium at -700C:a)Using a 24-h culture, prepare a heavy suspension of the bacteria in 1-2 mL of Glycerol-peptonemedium.b)Mix well and dispense 0.3-0.5 mL into cryogenic vials.c)Store the culture at -700C (Viability is excellent after several years of storage).7.3Liquid nitrogen method:a)Prepare a heavy cell suspension in Brucella Broth using a 24-48 h culture from a MuellerHinton Blood Agar plate; place 0.5 mL of the suspension into a (12.5 x 43 mm) tube designedfor immersion into liquid nitrogen, such as a Cryotube* containing 2 drops of dimethyl sulfoxide(DMSO). Mix and let stand for 5-10 min. before storage.b)Put the Cryotubes on metal racks and submerge the racks in liquid nitrogen.c)Do not seal the lid of the tank. Add vacliquid nitrogen regularly to fill the tank. NOTE: The major advantage of this method is eliminating accidents due to electrical power failure. 7.4Lyophilizationa)Make a heavy suspension of a 24 h culture in 1-2 mL of Serum-inositol medium and incubatemicroaerophilically at 42oC for 24 h.b)Distribute 0.2 mL into lyophilizing ampules and lyophilize.c)Store lyophilized ampules at 4oC.MFLP-46March 2002-10-NOTE: All ampules should be checked with a Vacumn Leak Detector (Model BD-10A available for 120V or 240V fromElectro-Technic Products, 4642 N. Ravenswood-Lo. 1-2349, Chicago, Illinois, 60640, U.S.A.). Absence of bluelight passing through the ampule denotes leakage of vacuum. Therefore, such ampules should be discarded.8.TRANSPORTING CULTURES THROUGH MAILSpecial attention should be paid when sending Campylobacter strains through the mail because the cells grownon agar plates or agar slopes are killed within 3 days in air at room temperature. The following is a procedurewhich will allow for survival of campylobacters for about 5 weeks at 4oC or 2 weeks at room temperature (100%recovery).a)Inoculate the culture onto a Mueller Hinton Blood Agar plate and incubate under microaerophilicconditions for 24 h at 42oC.b)Remove growth from the plate with a sterile swab, break off the handle of swab using sterileforceps. Place the swab (heavily inoculated) at the top layer of Wilkins Chalgren AnaerobicBlood Agar transport medium which has been dispensed in 6 mL quantities into sterile bottles.c)Leave the swab in the bottle,
- 温馨提示:
1: 本站所有资源如无特殊说明，都需要本地电脑安装OFFICE2007和PDF阅读器。图纸软件为CAD,CAXA,PROE,UG,SolidWorks等.压缩文件请下载最新的WinRAR软件解压。
2: 本站的文档不包含任何第三方提供的附件图纸等，如果需要附件，请联系上传者。文件的所有权益归上传用户所有。
3.本站RAR压缩包中若带图纸，网页内容里面会有图纸预览，若没有图纸预览就没有图纸。
4. 未经权益所有人同意不得将文件中的内容挪作商业或盈利用途。
5. 人人文库网仅提供信息存储空间，仅对用户上传内容的表现方式做保护处理，对用户上传分享的文档内容本身不做任何修改或编辑，并不能对任何下载内容负责。
6. 下载文件中如有侵权或不适当内容，请与我们联系，我们立即纠正。
7. 本站不保证下载资源的准确性、安全性和完整性, 同时也不承担用户因使用这些下载资源对自己和他人造成任何形式的伤害或损失。
人人文库网所有资源均是用户自行上传分享，仅供网友学习交流，未经上传用户书面授权，请勿作他用。
|
2:不支持迅雷下载,请使用浏览器下载
3:不支持QQ浏览器下载,请用其他浏览器
4:下载后的文档和图纸-无水印
5:文档经过压缩，下载后原文更清晰
|
川公网安备: 51019002004831号