D. Test Sam pling
17.6.01 AOAC Offi Official Method 972.44 Micro Mi crobi bio olog logiical Method First Action Action 1972 Final Action Final Action 1978
(Personnel (Person nel with beards, mustaches, mustaches, or side burns below be low ear lobe should not perform per form steril ste rility ity exam ex amii na nation tion unless unless these are com pletely cov covered ered with sterile ster ile caps and masks. Wear clean labo lab ora ratory tory coat for exam examiina nation.) tion.) A. Prin Princi ci ple ple
“Low acid foods’’ means means any food with finished fin ished equilib librium rium pH value >4.6. Method ap plies only to con contain tainers ers which show no disten dis tention tion of either either end. Incu Incu bate contain con tainers ers 10 days at 21 –35°C before be fore exam examiina nation. tion. Commercial steril ste rility ity is defined de fined as that condi con dition tion achieved by ap pli plica cation tion of heat which ren ders food free of vi viaa ble form s o f micro mi croor organ ganisms isms having having public public health signif sig nifii cance, as well as mi cro croo o r g a ni n i s m s n o t o f h e a l t h s i gnif g nif i cance ca pa ca pa bl bl e o f re p re p ro duc ducing ing in th e food un der nor mal nonrefrigerated con di tions of stor storage age and distribution. B. Media Media and Reagents Reagents
See also 966.23A 966.23A ( ( see 17.2.01). see 17.2.01). (a ) Tryptone broth. —(Ae —(Ae r o bic me medium.) dium.) Dissolve Dissolve 10.0 g tryptone or Trypticase, 5.0 g glucose, glu cose, 1.25 g K 2HPO4, 1.0 g yeast extract, ex tract, and 2.0 mL 2% alco al coholic holic solu lution tion of bromocresol pur ple ple in 1 L H2O with gentle gentle heat, if neces necessary. sary. Dis pense 10 mL por portions tions into 20 150 mm screw-cap test tubes and auto autoclave clave 20 min at 121°C. Do not exhaust exhaust before using. us ing. (b) Mod Modified ified PE-2 medium. medium. —(Anaer —(Anaero o bic me medium.) dium.) Dissolve Dissolve 20.0 g peptone, 3.0 g yeast ex tract, tract, and 2.0 mL 2% alco alcoholic holic solu solution tion of bromocresol pur ple in 1 L H 2O with gentle gentle heat, if neces nec essary. sary. Dispense 19 mL portions portions into 20 150 mm screw-cap test tubes contain con taining ing 8–10 un untreated treated Alaska seed peas (hardware (hardware store). Auto Au toclave clave 30 min at 121°C. If not freshly pre pared, heat to 100°C and cool to 55°C before before using. using. (c) Glu Glucose cose starch agar. —Aero —Aero bic me medium dium (BD Biosci Biosciences, ences, 2350 Qume Dr, San Jose, CA 95131, USA; Codified Cat. No. 212000, or equivalent). Dis Dissolve solve 15.0 g proteose peptone No. 3, 2.0 g glucose, glucose, 10.0 g sol u ble starch, 5.0 g NaCl, 3.0 g Na2HPO4, 20.0 g gelatin, and 10.0 g agar in 1 L H2O, heat to bp, and auto toclave clave 15 min at 121°C in Erlenmeyer. Aseptically Aseptically pour into sterile sterile Petri dishes and allow allow to solid solidify. ify. (d) Nu —Aero bic medium me dium for spore produc production; tion; BD Nutri trient ent agar. —Aero Biosci Bio sciences ences Codified Cat. No. 2 12000, or equiv alent. alent. Dis solve 3.0 g beef extract, 5.0 g peptone, and 15.0 g agar in 1 L H2O, heat to bp, and auto autoclave clave 30 min at 121°C. (e) De Deter ter gent sanitizer solu solution. tion. —pHisoDerm, —pHisoDerm, or equiva equivalent. C. Ap pa para ratus tus
(a) Can opener. —Bacti-Disc —Bacti-Disc Cut Cutter ter (Wilkens-Anderson Co., 4525 W. Divi Division sion St, Chicago, Chicago, IL 60651, USA; No. 10810-01), bacte bac teri riolog ologiical can opener (Marmora Ma chine Co., 1956 N. Latrobe Ave, Chicago, Chicago, IL 60639, USA), or equiv alent. (b) Caps. —Dis —Dis pos posable, able, oper operat ating ing room-type (Baxter Hos pi pital tal Sup ply Division, Division, 1450 Waukegan Waukegan Rd, McGaw McGaw Park, IL 60085, USA, or equivalent). (c) Pipets. —Straight —Straight wall, 200–250 mm long 7 mm id, 9 mm od (Scientific (Scien tific Prod ucts, Inc.; cut and fire polished, pol ished, o r equiv alent).
Conduct Con duct test in clean room. (If neces nec essary, sary, open room may be used but outside out side windows windows must be closed and d irect drafts across work area must be elimi elim inated.) If available, avail able, use lami laminar flow cabi cabinet. Strip la bels from from cans, ex exam amine ine cans for exter external nal defects, defects, and record record descrip de scriptions. tions. Wash cans with soap (or deter detergent gent sanitizer solu solution) and H2O, and dry with clean pa per towels. tow els. Wipe counter counter top with 100 ppm (g/mL) Cl solu solution tion (e.g., Clorox or diluted diluted NaOCl solu so lution) tion) imme immedi diately ately before before placing placing washed and dried can on it. Place code end of can in down posi position tion and num ber cans in ink or with CuSO4 mark marking ing solu solution tion to right of side seam. Wash hands and face with soap, and rewash hands and face with deter de tergent gent sanitizer solu solution. tion. Com pletely cover hair with clean dis pos posable able oper operat ating ing room cap. Hold noncoded end of can over large Meker burner, just above blue portion portion of flame. Heat this end of can until until all conden condensa sation tion is evapo evap orated; then return re turn can to ta ble in for former mer posi position. tion. Clean handle handle and blade of spe cial can opener, C(a), with pa per towel moistened moistened with 70% alco cohol, hol, flame metal portion portion enough to destroy destroy all micro mi croor organ ganisms, isms, and use it to make 4 cm (1.5 in.) diam diameeter hole in noncoded, heated end of can. Imme Immedi diately ately and without without moving moving can, use straight-wall sterile sterile glass pipet, C(c), to trans fer ca 2 g food to sepaarate tubes, 2 each of aero sep aer o bic and 2 of anaer anaero o bic media media (4 total). total). (No other transfer ferring ring tool may be substi substituted.) tuted.) Preloosen screw cap and hold it between between little little and ring fingers fingers while transfer transfer is being being made. Flame lips of media media tubes both before before and after addi dition tion of food. When transfer transferring ring food to anaer anaero o bic tubes, food must be inoc in oculated ulated into lower por tion of me medium. dium. Tighten screw caps after af ter inoc in ocula ula tion, in cu bate tubes 72 h at 35°C, and and observe daily. daily. Record results re sults for each tube sepa separately. Remove Re move addi additional tional 10 g food test portion from each container container with sterile sterile pipet and place in sterile sterile 25 200 mm screw-cap test tube. Use pipet-like spatula, spatula, if nec es essary, sary, for this oper operaation (thermophilic contam contamiina nation tion unlikely). unlikely). Num ber tube to corre correspond spond to can and refrig refriger erate ate for later testing, test ing, if neces necessary. sary. E. Contam Contamiina nation tion Control Control
Use sterile sterile loop or glass rod to streak plate of glucose glucose starch agar, On ta ble, open pl ate of glu cose starch agar for time equal to longest lon gest dura duration that any medium me dium tube or plate is ex posed. Incu bate plates 72 h at 35°C, and ob serve daily. B(c).
F. Micro Microscopic scopic Exam Examiination nation
With pair of metal cutting cutting shears, enlarge hole in can and re cord odor. Micro Microscopically scopically (oil immer immersion) sion) exam examine ine heat-fixed thin smear of food, stained 10 s with 1% gentian gen tian (or crystal) crystal) vio violet and washed in running running tap water, or, alter alternatively, natively, ex am amine wet mounts with phase contrast contrast micro microscope. scope. If food con tains ap pre precia cia ble fat, xylol should be dripped across food smear while it is still hot from heat fixing. fixing. Com pare stained smear with one made from nor normal mal product, prod uct, if possi possi ble. ble. G. pH De Deter termi mina nation tion
Deter De termine mine pH with pH meter, meter, using using refer reference ence buffer near normal nor mal pH of food. Record Record both refer reference buffer pH and test sam ple pH. Com pare to normal normal can of food, if avail able. H. Confir Confirma mation tion of Results Results
If there is any abnor abnormal mal odor, abnor normal mal appearance, ab abnor normal mal pH, num bers of of bacte bacteria ria on micro microscopic scopic exam examiina nation, tion, and/or growth in media media from any can of food, subcul sub culture ture corre correspond sponding ing refrig friger erated ated tube as follows: follows: Flame lip of tube and, with straightwall sterile sterile glass
IN TERNA NATIONAL TIONAL 2005 AOAC INTER
pipet, C(c), transfer ca 2 g food to 2 tubes each of aero bic and anaero bic media (4 total). Flame lips of media tubes both before and after addition of food. Tighten caps after inoculation, incu bate tubes 72 h at 55°C, and observe daily. Record results for each tube separately. Any organisms isolated from normal cans hav ing ob vious vacuum which produce gas in anaero bic m edium at 35°C should immediately be sus pected as being from laboratory contamination. Aseptically inoculate growing organism into another normal can, close hole with solder, and incu bate 14 days at 35°C. Any swelling of container indicates that organism was not in original test sam ple. Record as laboratory contamination and review results of additional cans to verify finding of contamination. Growth in aero bic medium at 35°C from normal cans indicates either noncommercial sterility or laboratory contamination. Unless there is abnormal odor, abnormal ap pearance, abnormal pH, and/or
2005 AOAC IN TERNATIONAL
num bers of bacteria on microscopic examination from product in original can, record results as laboratory contamination and review results of additional cans to verify finding of contamination. Otherwise, observe subculture re sults at 55°C. Growth at 35°C and absence of growth at 55°C confirm nonsterility of original container. Check growth under aero bic conditions on nutrient agar plates, B(d), at 55°C and confirm for spores after 72 h. Confirmation indicates nonsterility due to flat sour spoilage. Record growth at 55°C under anaero bi c co ndi tions with gas production as commercially sterile. Growth is caused by dormant spores inca pa ble of growth at normal tem peratures of storage and distri bution. If only one of du plicate tubes is positive after incu bation and streaked glucose starch agar is also negative, record as laboratory contamination. Growth on air control plate of glucose starch agar also indicates potential laboratory contamination. Reference: JAOAC 55, 613(1972).