USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 .g under refrigeration at 7-10,000 RPM For iv. t and add original slurry,mix and centrifuge a second time as above. v. Decan end the 8.5,and then adjust the volume to 2 ml with Tris buffer. vi. and vii.Cut 2 mm diameter wells into air dried Toluidine Blue DNA Agar. viii. 86a8g"aaeu2oep8t6otn8n8ecoerHoE8ee出 11 ix.Incubate these plates,agar side down,at 37oc for 4 to 24 h. X. 3-12
USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 3-12 iii. Centrifuge under refrigeration at 7-10,000 RPM for 15 min. iv. Decant and discard the supernatant and add 0.05 ml cold 3M trichloroacetic acid for each ml of the original slurry, mix and centrifuge a second time as above. v. Decant and discard the supernatant. Re-suspend the precipitate in 1 ml of Tris buffer, adjusted to pH 8.5, and then adjust the volume to 2 ml with Tris buffer. vi. Boil the solution for ³15 but £90 min, cool and store under refrigeration until needed. vii. Cut 2 mm diameter wells into air dried Toluidine Blue DNA Agar. viii. Dispense the food extract into one or more wells using a Pasteur pipette. Do not overfill the well. ix. Incubate these plates, agar side down, at 37°C for 4 to 24 h. x. Any pink halo, extending 1 mm beyond the well is considered positive for thermonuclease
USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 3.7 Selected References a2a6taa-i6t92aie86f1c1a1A68no2ae82nat38g16e0ac AOAC International Inc., Emswiler-Rose,B.S.,R.W.Johnston,M.E.Harris,and W.H. Lee.1980. on foronted auaagee sausages. ureup.533-550.andn datin Staphylococcus stoesse Amer ubl.th. Wash otoxin Nutrition Press,Inc.,Westport,CT. 3-13
USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 3-13 3.7 Selected References Cunniff, P. (ed.). 1995. Official Methods of Analysis of AOAC International, 16th Edition. AOAC International Inc., Gaithersburg, MD 20877. Emswiler-Rose, B. S., R. W. Johnston, M. E. Harris, and W. H. Lee. 1980. Rapid detection of staphylococcal thermonuclease on casings of naturally contaminated fermented sausages. Appl. Environ. Microbiol. 440:13-18. Lancette, G. A., and S. R. Tatini. 1992. Staphylococcus aureus, p. 533-550. In C. Vanderzant and D. F. Splittstoesser (ed.), Compendium of Methods for the Microbiological Examination of Foods. Amer. Publ. Hlth. Assoc., Washington, D.C. 20005. Tatini, S. R. 1981. Thermonuclease as an indicator of staphylococcal enterotoxins in food, p. 53-75. In R. L. Ory (ed.), Antinutrients and Natural Toxicants in Foods. Food and Nutrition Press, Inc., Westport, CT
USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 CHAPTER 6. ISOLATI PRODUCTS Gerri M.Ransom and Bonnie E.Rose 6.1 Introduction for the of foods varietng of foods Isolation Ca ylobacter jejuni and Campylobacter coli is achieved both with and without selective broth enri nt 1 ures outline most raw/cooked meat and poultry products Campylobacters are sensitive to freezing and die off at be rature ed a DE A at4°c d h oossible since cam oylobacters can overgrown ht bacteria. If freezin of samples d,cryoprotective agents sh ould be use Stern and stored'fr in 吧10% Blankenship et al 1983 found that brucella broth su with 10%polyvinyl pyrrolidine was suitable for transporting frozen freshly processed poultry carcasses)to a central 1y919 Campylobacters are microaerophilic and certain environmental stresses such as exposure to drying, and ●ah detri nd suppress c etitors,significantly improve Campy lobacter recovery 6.2 Equipment,Reagents,and Media 6.21 Equipment a. Phase-contrast microscope with 100x oil immersion objective b. Agit ati ng in ubator(s)/water bath(s)at37±1.0°C 42+ Balance sensitivity of o 1g e. Quart-size Qwik seal®bags(Reynolds Metals Co. or non-vented) Anaerob 6-1
USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 6-1 CHAPTER 6. ISOLATION, IDENTIFICATION, AND ENUMERATION OF CAMPYLOBACTER JEJUNI/COLI FROM MEAT AND POULTRY PRODUCTS Gerri M. Ransom and Bonnie E. Rose 6.1 Introduction Procedures for the recovery of Campylobacter spp. from foods are evolving and no single method can be recommended for testing a wide variety of foods. Isolation of Campylobacter jejuni and Campylobacter coli is achieved both with and without selective broth enrichment. The procedures outlined below are among the most promising for the isolation and enumeration of these bacteria from raw/cooked meat and poultry products. Campylobacters are sensitive to freezing and die off at room temperature. Samples intended for Campylobacter examination should be transported and held at 4o C. Sample analysis should begin as soon as possible since campylobacters can be overgrown by contaminating psychrotrophic bacteria. If freezing of samples cannot be avoided, cryoprotective agents should be used. Stern and Kotula, 1982, reported improved recovery of C. jejuni from ground beef stored frozen in 10% dimethyl sulfoxide or glycerol. Blankenship et al., 1983, found that brucella broth supplemented with 10% polyvinyl pyrrolidine was suitable for transporting frozen swab samples (from freshly processed poultry carcasses) to a central laboratory for analysis. Campylobacters are microaerophilic and certain environmental stresses such as exposure to air, drying, low pH, and prolonged storage can be detrimental to their survival. Use of oxygenquenching agents, a microaerobic atmosphere, and antibiotics that suppress competitors, significantly improve Campylobacter recovery. 6.2 Equipment, Reagents, and Media 6.21 Equipment a. Phase-contrast microscope with 100X oil immersion objective b. Agitating incubator(s)/water bath(s) at 37 ± 1.0°C and 42 ± 1.0o C c. 42 ± 1.0o C incubator (static) d. Balance, sensitivity of 0.1 g e. Quart-size Qwik Seal® bags (Reynolds Metals Co., Richmond, VA; # RS78) f. Anaerobic jars (vented or non-vented)
USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 CampyPak PlusTM (BBL 71045) or h. and g riate tubing ands) connegtora for evacuatented anaerpbic iars i. der containing a mixture of5:10 CO2,and Na with appropriate d o and connectors for gassing 3. Regulator for gas cylinder compatible with Compressed Gas Association (CGA)connection on cylinder k Bilter paper (for glycerol humectant and oxidase test) (1002 Microscope slides,cover slips,and immersion oil nd needles n 0.2 e filters 16x150 mm and 16x 125 mm screw-cap test tubes 250-ml screw-cap bottles Ster e swabs or nt glass rods ("hockey sticks") t sterile pir and u Large sterile plastic bags Centrifuge,rotor, and 250-ml sterile centrifuge bottles X. Sterile cheesecloth-lined funnels 6.22 Reagents a. Glycerol 36 Hydrogen peroxide solution c. Cephalothin antibiotic susceptibility discs (30 ug) d Nalidixic acid antibiotic susceptibility discs (30 ug) e. Oxidase reagent (1%Tetramethyl-p-phenyienediamine dihydro chloride solution) dentific kit (optional 6.23 Media 6 t Broth (HEB) 01g otone wate a Modified Campylobacter Charcoal Differential Agar (MCCDA d ( se Medium Brucella-FBP (BFBP)Agar g. Enriched Semisolid Brucella Medium (optional) 6.3 Isolation and Enumeration 6-2
USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 6-2 g. CampyPak PlusÔ (BBL 71045) or Gas Generating Kits for Campylobacter (Oxoid BR56 for 3.0-3.5 liter jars, or BR60 for 2.5-3.0 liter jars) h. Vacuum pump and gauge with appropriate tubing and connectors for evacuation of vented anaerobic jars i. Gas cylinder containing a mixture of 5% O2, 10% CO2, and 85% N2 with appropriate tubing and connectors for gassing vented anaerobic jars and Qwik Seal® bags j. Regulator for gas cylinder compatible with Compressed Gas Association (CGA) connection on cylinder k. Filter paper (for glycerol humectant and oxidase test) l. Petri dishes (100 x 15 mm disposable) m. Platinum or sterile plastic inoculating loops and needles n. Microscope slides, cover slips, and immersion oil o. 0.2 mm sterile membrane filters p. 16 x 150 mm and 16 x 125 mm screw-cap test tubes q. 250-ml screw-cap bottles r. Sterile swabs or bent glass rods ("hockey sticks") s. Sterile forceps and scissors t. Sterile pipettes u. Large sterile plastic bags v. StomacherÔ 400, and StamacherÔ 400 bags w. Centrifuge, rotor, and 250-ml sterile centrifuge bottles x. Sterile cheesecloth-lined funnels 6.22 Reagents a. Glycerol b. 3% Hydrogen peroxide solution c. Cephalothin antibiotic susceptibility discs (30 mg) d. Nalidixic acid antibiotic susceptibility discs (30 mg) e. Oxidase reagent (1% Tetramethyl-p-phenylenediamine dihydrochloride solution) f. Campylobacter latex test kit (optional presumptive identification) 6.23 Media a. Hunt Enrichment Broth (HEB) b. 0.1% peptone water c. Modified Campylobacter Charcoal Differential Agar (MCCDA) d. Brucella-FBP (BFBP) Broth e. Semisolid Brucella Glucose Medium f. Brucella-FBP (BFBP) Agar g. Enriched Semisolid Brucella Medium (optional) 6.3 Isolation and Enumeration
USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 a. Place 25 g @h oles into 100 ml of HEB in Reynolds quart-size Qwik Seal@bag.Place the Qwik Seal bag inside a stomacherT 400 bag for reinforcement and stomach for 2 minutes. Flatten the Qwik Seal bao against the lab bencili edge as much air ng insert the tip-end of opensterile (o equivalent)into the bag through this opening. Be sure that il mouth-end of the pipe tte a nect 85 Na)with sterile Fubber tubing equipped with a sterile filter a sterile filter can made out of n autocla ic pipette stuff with thes ca mixtu and to excess gas flows from the bag. Then allow a small amount gas escape t。 for seal. before securing the step d b. Place a raw whole chicken carcass or meat pieces (up to 3 Ib)in 3500 hag ange add 200 m Twist bag to seal and shake contents for minutes Tilt the bag the meat pieces a lowing the olution or 70g then Aseptically cut the corner of the bag and pour the rinse 七hr ough sterile che esec 16000 ni.ce ntrifuge supernatant and suar end the" pellet in 10 ml 0.18 pe water. For detection, inoculate 1 ml of rinse HEB ba ng as ith c. If enumeration is desired, prepare a three tube MPN HEB oose test lutions HEB ecie tested. Py- e rinse samples (prior to centrifuging)begin by adding three 10m1 rinse to three 90 ml bottles natively,Owi bags may be use sne。8 to each oth0 e 9 mi tu山es of HEB, of he Prepare serial dilutions of the rinse in 0.18 peptone water. Prepare subsequent MPN tubes by transferring 1 ml portions of the 6-3
USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998 6-3 a. Place 25 g meat or swab samples into 100 ml of HEB in a Reynolds quart-size Qwik Seal® bag. Place the Qwik Seal® bag inside a StomacherÔ 400 bag for reinforcement and stomach for 2 minutes. Flatten the Qwik Seal® bag against the lab bench edge to remove as much air as possible without spilling the contents, then seal the bag, leaving a 1/2 inch opening at one end. Aseptically insert the tip-end of a sterile 10 ml pipette (or equivalent) into the bag through this opening. Be sure that the mouth-end of the pipette contains a sterile cotton filter. Connect the mouth-end of the pipette to the microaerobic Campy gas mixture (5% O2, 10% CO2, and 85% N2) with sterile rubber tubing equipped with a sterile filter (a sterile filter can be made out of an autoclaved, shortened 25 ml volumetric pipette stuffed with glass wool). Slowly inflate the bag to capacity with the Campy gas mixture and continue to fill until excess gas flows from the bag. Then allow a small amount of gas to escape to provide for expansion, before securing the remainder of the seal. Proceed to step d. b. Place a raw whole chicken carcass or meat pieces (up to 3 lb) in a large sterile plastic bag such as a StomacherÔ 3500 bag, and add 200 ml 0.1% peptone water. Twist bag to seal and shake contents for 2 minutes. Tilt the bag and hold back the meat pieces, allowing the rinse liquid to flow to one corner. Sanitize bag corner with 1000 ppm hypochlorite solution or 70% ethanol, then rinse in sterile distilled water. Aseptically cut the corner of the bag and pour the rinse through a sterile cheeseclothlined funnel into a sterile 250 ml centrifuge bottle. Centrifuge at 16,000 x g for 15 minutes. Discard the supernatant and suspend the pellet in 10 ml 0.1% peptone water. For detection, inoculate 1 ml of rinse concentrate into 100 ml HEB in a Qwik Seal® bag. Then follow gassing steps as outlined, beginning with the third sentence of step a. above. c. If enumeration is desired, prepare a three tube MPN series using HEB. Choose test dilutions and HEB volumes based on the expected numbers of campylobacters in the meat species being tested. For example, for poultry rinse samples (prior to centrifuging) begin by adding three 10 ml portions of the rinse to three 90 ml bottles of HEB. (Alternatively, Qwik Seal® bags may be used here [see step a. above]). Then add 1 ml portions of the rinse to each of three 9 ml tubes of HEB. Prepare serial dilutions of the rinse in 0.1% peptone water. Prepare subsequent MPN tubes by transferring 1 ml portions of the