R. B. TOMPKIN Swift & Company Research and Development Center Oak Brook, I l l i n o i s

Transcription

1 270 MICROBIOLQGICAL TECHNIQUES : SAMPLE TRANSPORT, SAMPLE PREPARATION, MEDIA, AND INCUBATION* R B TOMPKIN Swift & Company Research and Development Center Oak Brook, I l l i n o i s Many options e x i s t f o r t r a n s p o r t i n g samples, preparing samples f o r a n a l y s i s, media, and incubation temperatures Since t h e choice of options influences t h e a n a l y t i c a l r e s u l t s, t h i s information must be a v a i l a b l e when t h e r e s u l t s are i n t e r p r e t e d The reason f o r which t h e samples a r e collected determines t h e options s e l e c t e d A l i s t of reasons would include: 1 A r o u t i n e q u a l i t y assurance program 2 To determine t h e cause of a problem i n a product o r process 3 Samples submitted as part of a research program 4 Determining i f a product w i l l meet a customer s p e c i f i c a t i o n 5 Samples from a government s u r v e i l l a n c e or regulatory program 6 To develop d a t a t o s e t t l e a c o n f l i c t between a supplier and a customer 7 Samples of a food implicated i n an outbreak of foodborne illness Sample Transport The method of t r a n s p o r t i n g should be one which r e s u l t s i n a minimum of microbial growth o r death i n t h e sample The word m i n i m u m must be emphasized because some growth o r death w i l l l i k e l y occur The sample received in t h e laboratory must be r e p r e s e n t a t i v e of t h e product a t t h e time t h e sample was c o l l e c t e d Most perishable meat samples which must be shipped a long d i s t a n c e or where t h e r e i s t o be a lengthy time delay should be shipped frozen The most common method is t o pack t h e sample with d r y i c e i n an insulated o r Styrofoam container and s h i p by a i r freight It i s b e s t t o have a d e l i v e r y s e r v i c e prearranged s o t h e samples a r e transported d i r e c t l y f r o m t h e a i r p o r t t o t h e laboratory If t h i s is not done, t h e samples may be delayed a t t h e a i r p o r t and undergo spoilage When v i s i t i n g a p l a n t t o c o l l e c t samples, t h e most convenient way of t r a n s m i t t i n g samples t o t h e laboratory i s t o p a c b g e with dry i c e and c a r r y them back Problems might be encountered i f t h e samples a r e checked as personal baggage A i r l i n e s have had t o pay claims t o owners * Presented a t t h e 29th Annual Reciprocal Meat Conference of t h e American Meat Science Association, 1976

2 of p e t s which presumably died due t o suffocation from carbon dioxide leaking from packages This problem can be avoided by using one of t h e These a r e p l a s t i c containers containing commercial gel-type pcroducts a g e l which can be frozen and packaged with t h e samples Perishable samples which a r e t o be analyzed t h e same day they a r e c o l l e c t e d m y be r e f r i g e r a t e d Not a l l samples need t o be r e f r i g e r a t e d or frozen For example, swab samples may be taken from equipment a f t e r clean-up t o determine if t h e equipment has, indeed, been thoroughly cleaned and s a n i t i z e d These swab samples may be s e n t t o t h e laboratory through t h e regular m a i l a t ambient temperature Samples yielding low t o t a l p l a t e counts i n d i c a t e t h e equipment was thoroughly cleaned and s a n i t i z e d Conversely, samples yielding high t o t a l p l a t e counts i n d i c a t e t h e equipment was not thoroughly cleaned and s a n i t i z e d Another example may be i n sampling equipment o r meat products f o r salmonellae If t h e primary purpose i s t o determine t h e mere presence of salmonellae, shipping t h e samples a t ambient temperature should not a l t e r the end r e s u l t, although some d i s c r e t i o n should be applied It is b e s t t o avoid f r e e z i n g samples which w i l l be analyzed f o r Clostridium perfrinkens, since considerable d i e off can occur due t o f r e e z i n g Sample Preparation Once t h e samples a r e a t t h e Laboratory, t h e y must be prepared f o r a n a l y s i s Frozen samples a r e frequently allowed t o t h a w overnight i n a r e f r i g e r a t o r I n some instances, time is c r i t i c a l s o t h e samples m i g h t be placed under cold running water Care should be taken that t h e package does not leak and permit e n t r y of water Large boxes of frozen meat m y be sampled by d r i l l i n g i n t o t h e product using a p r e s t e r i l i z e d d r i l l The d r i l l shavings a r e then used f o r a n a l y s i s Several d i f f e r e n t methods a r e used t o prepare an i n i t i a l 1:lO suspension of t h e sample in a d i l u e n t Many l a b o r a t o r i e s use a "blender But, w h a t i s a blender? There a r e d i f f e r e n t manufacturers, blenders have d i f f c r e n t ranges of speed, and t h e jars come i n d i f f e r e n t shapes and s i z e s We use an Osterizer f o r blending samples This allows t h e use of an ordinary canning jar of t h e B a l l or Mason type The jars withstand autoclaving, a r e r e l a t i v e l y inexpensive, and r e q u i r e l e s s storage space The most frequent method we use is t o shake t h e sample with t h e d i l u e n t i n a small Jar having a t o t a l volume of approximately 165 ml When t e s t i n g meat products, an 11-gram sample is weighed i n t o a p r e s t e r i l i z e d jar containing a tablespoon of broken g l a s s chips The glass chips a r e obtained i n a 55-galloa drum from a glass manufacturer Ninety-nine m l of b u f f e r s o l u t i o n is then added t o t h e j a r and t h e cap is t i g h t l y sealed The jars a r e placed i n t o a s p e c i a l l y designed wooden block which can hold up t o 4 jars a t a time The samples a r e then shaken on a mechanical paint shaker f o r 3 t o 5 minutes The small "

3 jar-mechanical paint shaker technique has been used a t Swift & Company f o r more t h a n 30 years The equipment i s v i r t u a l l y t r o u b l e - f r e e, low in c o s t, and t h e system is s u i t e d t o analyzing a large nurriber of samples ef f i c i e n t l y A r e l a t i v e l y new technique f o r emulsifying samples i s t h e use of a Stomacher (Dynatech Laboratories, Inc Alexandria, V i r g i n i a ) This system c o n s i s t s of placing t h e sample and d i l u e n t i n t o a p l a s t i c bag which i s then placed behind t h e f r o n t door of the Stomacher Two metal paddles i n s i d e the apparatus beat t h e sample a g a i n s t t h e metal door The paddles s t r i k e i n succession and, a f t e r 1 t o 3 minutes, t h e meat sample is d i s i n t e g r a t e d t o a workable suspension Research has shown t h i s system t o be equal t o blending in a mechanical blender (2-4), An a l t e r n a t i v e t o mechanical devices is t h e standard hand shake This c o n s i s t s of placing t h e sample and d i l u e n t i n t o a jar and shaking t h e sample 25 times over an a r c of 1 f o o t i n 7 seconds This i s t h e same procedure used f o r shaking subsequent d i l u t i o n s prepared from t h e o r i g i n a l 1:lO suspension It i s b e s t s u i t e d f o r l i q u i d samples such as j u i c e from a packaged meat product o r b r i n e s o l u t i o n s It can be used f o r analyzing swab samples; however, we p r e f e r using t h e mechanical p a i n t shaker technique Although t h e vortex mixer ( S c i e n t i f i c I n d u s t r i e s Inc, Queens Village, NY) i s not normally used f o r t h e i n i t i a l preparation of a 1 : l O d i l u t i o n, it is convenient f o r preparing subsequent d i l u t i o n s One last method f o r preparing t h e i n i t i a l sample i s t o place t h e sample i n t o a s t e r i l e p l a s t i c bag such as a Whirlpack bag After adding t h e d i l u e n t t o t h e bag, t h e sample can be mixed by shaking o r squeezing There are times when t h i s can be used t o advantage such as determining a s u r f a c e count of a f r a n k f u r t e r Each of t h e foregoing methods has c e r t a i n advantages and d i s a d vantages Among t h e most d i f f i c u l t samples t o prepare f o r a n a l y s i s are skin and d r i e d meats Skin, e s p e c i a l l y p o u l t r y skin, becomes wrapped around t h e blades of blenders This i s not a problem with t h e mechanical p a i n t shaker or Stomacher techniques Hard, d r y m a t e r i a l tends t o be chased around t h e s i d e of t h e blender jar A t r i c k t o overcome t h i s is t o tilt t h e blender apparatus D i l u e n ts There has been a considerable amaunt of research over t h e years on various types of d i l u e n t s f o r b a c t e r i o l o g i c a l a n a l y s i s For most meat products, t h i s i s of minor importance, e s p e c i a l l y f o r t h e i n i t i a l preparation of t h e 1:lO d i l u t i o n While t h e d i l u e n t m i g h t influence recovery of t h e maxinaun population, this i s normally n o t s i g n i f i c a n t i n a cmmercial s i t u a t i o n Simply stated, phosphate buffered d i s t i l l e d water, as described i n Standard Methods f o r the &amination of Dairy Products, is adequate f o r t h e a n a l y s i s of meat products

4 273 Two types of media a r e used f o r determining aerobic p l a t e counts i n meat products P l a t e count agar is one type which serves as a good, general medium However, most l a c t i c a c i d producing b a c t e r i a y i e l d colonies which a r e pinpoint i n s i z e on p l a t e count agar A s a r e s u l t, a second type medium is used, e s p e c i a l l y f o r vacuum packaged cured meats and fermented products Several media are a v a i l a b l e f o r t h i s purpose W e use APT agar Although the same count i s obtained on APT and p l a t e count agar, t h e colonies formed on APT agar a r e much l a r g e r i n s i z e and e a s i e r t o count The b e s t medixun c u r r e n t l y a v a i l a b l e f o r d e t e c t i n g Staphylococcus aureus i s Baird-Parker agar This medium has been shown t o be t h e most productive i n terms of recovering t h e highest l e v e l of 2 aureus from foods It i s e s p e c i a l l y u s e f u l f o r cured meats because it i s possible t o d e t e c t 5 aureus from amng t h e background of normal f l o r a e x i s t i n g i n t h e product It is analagous t o having a s p o t l i g h t on a fox in t h e middle of a f l o c k of chickens he high cost of t h i s mdium is j u s t i f i e d by i t s performance Several media and methods e x i s t f o r aetermining coliforms i n meat products Violet red b i l e agar i s s t i l l good f o r r o u t i n e a n a l y t i c a l work If information is needed on t h e l e v e l of c o l i i n meat products, tubed l i q u i d media a r e normally used The media u s u a l l y c o n s i s t s of lauryl s u l f a t e try-ptose b r o t h f o r p r e l h i n a r y incubation followed by t r a n s f e r r i n g t o b r i l l i a n t green l a c t o s e b i l e b r o t h f o r a coliform count and t o EC b r o t h f o r a presumptive E c o l i count This approach i s m o s t commonly used by regulatory agency l a b o r a t o r i e s E Some i n t e r e s t s t i l l e x i s t s i n q u a n t i t a t i n g t h e gruup D s t r e p t o c o c c i The group D s t r e p t o c o c c i continues t o be a p o t e n t i a l spoilage organism i n m e a t products Aside frcnn t h i s, t h e r e i s a d d n d l h g i n t e r e s t i n t h i s group of b a c t e r i a i n t h e United S t a t e s Current AOAC, FDA and USDA mthodology do not include procedures f o r q u a n t i t a t i n g t h e group D s t r e p t o c o c c i i n foods This is not t r u e i n Canada where meats continue t o be monitored f o r c e r t a i n group D s t r e p t o c o c c i Several media a r e used f o r q u a n t i t a t i n g Clostridium perfringens i n meat products One such medium is SFP agar Another incorporates cycloserine as discussed by Dr H W Walker a t t h e 1972 Reciprocal Meats Conference The b e s t procedure f o r d e t e c t i n g salmonellae i n meat products appears t o c o n s i s t of a l a c t o s e broth pre-enrichment followed by elevated temperature enrichment i n s e l e n i t e c y s t i n e and t e t r a t h i o n a t e b r o t h s Twenty-five gram samples a r e commonly used The method and s i z e of sample determine t h e success of t h e salmonellae hunter The harder one t r i e s, t h e =re l i k e l y salmonellae w i l l be found i n f r e s h meat

5 Incubation Temperatures f o r Aerobic P l a t e Count Considerable v a r i a t i o n e x i s t s i n t h e temperatures used f o r d e t e r mining aerobic p l a t e counts The t h r e e most common temperature ranges c o n s i s t of 5-7W, 20-30%, and 35-37W Each temperature range r e s u l t s i n a s l i g h t l y d i f f e r e n t answer P l a t e s held a t 5-7OC a r e u s u a l l y counted a f t e r 10 days C a t s r e s u l t i n g from t h i s procedure a r e u s e f u l i n determining t h e l e v e l s of b a c t e r i a which can multiply i n r e f r i g e r a t e d meats Usually, 10 days i s t o o long t o w a i t f o r an answer and, f o r t h i s reason, temperatures i n t h e range of 2O-3O0C have been adopted Incubation times f o r p l a t e s held a t 20-30W range from 3 t o 2 days as t h e temperature is increased from 20 t o 3OoC This range u s u a l l y y i e l d s t h e highest count The t h i r d temperature range of 35-37% has been used as an index of e x t e r n a l contamination t o meats There i s some l o g i c and d a t a t o support t h i s b e l i e f For example, one m i g h t expect t o f i n d higher l e v e l s of mesophiles on f r e s h l y slaughtered carcass meat as opposed t o the psychrotrophs Suggestions f o r Research A need e x i s t s f o r a rapid method of estimating t o t a l count i n meat products Various approaches have been attempted, such as dye reduction t e s t s, b u t t h e y have not gone beyond t h e research s t a g e One reason f o r t h i s i s that the methods normally are not s u f f i c i e n t l y s e n s i t i v e f o r d e t e c t i n g microbial l e v e l s below 1 million per g i n meat products A method which is s e n s i t i v e i n t h e range of 50,000 t o 500,000 per g would be very h e l p f u l, p a r t i c u l a r l y in screening r a w m a t e r i a l s A recent paper by O l i v e r i a and Parmelee (1)showed e x c e l l e n t correl a t i o n f o r t o t a l counts determined a f t e r 25 hours a t 21 C versus 10 days a t 7OC Their d a t a are based upon 322 samples of r a w and pasteurized m i l k Would t h i s same r e l a t i o n s h i p hold f o r meat products? The goal would be t o obtain a n estimate of psychrotrophs i n meats a f t e r 25 hours Another concept which has been v i r t u a l l y untouched is t h e use of preliminary incubation p r i o r t o analyzing meat products This concept has been used s u c c e s s f u l l y f o r assessing t h e q u a l i t y of milk An example of where t h i s might be used w o u l d be i n p r e d i c t i n g t h e s h e l f l i f e of pre-packaged luncheon meats or f r a n k m t e r s Would a preliminary incubation a t 1 3 O C (55%) f o r 18 t o 20 hours p r e d i c t t h e r e f r i g e r a t e d s h e l f l i f e of t h e s e products i n terms of days? Surmnary There have been many attempts over t h e years t o establish standard methods f o r t h e examination of meat products To date t h e only methods which can be considered " o f f i c i a l, f f in t h e sense that they w i l l withstand t h e r i g o r s of a court case, a r e those methods published by the AOAC as o f f i c i a l f i r s t a c t i o n Such methods a r e necessary in a time of need However, t h e r e w i l l always be a need f o r a v a r i e t y of methods

6 No s j n g l e method w i l l s a t i s f y t h e v a r i e t y of reasons f o r which t h e samples a r e analyzed and t h e d i f f e r e n t types of meat products Each a n a l y s t has t h e option of s e l e c t i n g t h e conditions f o r a n a l y s i s Knowing how t h e samples were handled and analyzed is e s s e n t i a l f o r v a l i d i n t e r p r e t a t ion of t h e d a t a REFERENCES (1) Oliveria, J S and C E Parmalee 1976 Rapid enumeration of psychrotrophic b a c t e r i a i n raw and pasteurized milk J Milk Food Technol 39:269 ( 2 ) Sharpe, A N and G C Harshman 1976 Recovery of Clostridium perfringens, Staphylococcus aureus and molds from foods by t h e stolnacher : e f f e c t of f a t content, s u r f a c t a n t concentration, and blending t i m e Can I n s t Food S c i Technol J 9:30 ( 3 ) Sharpe, A N and A K Jackson 1972 Stomaching: a new concept i n b a c t e r i o l o g i c a l sample preparation Appl Microbiol 24 :175 ( 4 ) Tuttlebee, J W, 1975 The stomacher--its use f o r homogenization J Fd Technol 10:113 in food microbiology * * * Bruce Langlois: Are t h e r e any questions f o r Dr Tompkin? F F Busta, University of Minnesota: How do you s a n i t i z e t h e casings on t h e outside of that fermented sausage before opening t h e bag? Bruce Tompkin: Well, a c t u a l l y we do not f o r a b i g piece of bologna o r fermented sausage We a r e a b l e t o e s s e n t i a l l y c u t out a window, p u l l t h e casing back i n such a way t h a t we have a l a r g e a r e a that has not been touched by t h e knife a t a l l So, we don't s a n i t i z e Jim C h r i s t i a n, University of Georgia: Did you ever use Calgon swabs and 1 percent sodium c i t r a t e f o r general q u a l i t y c o n t r o l work i n swabbing? Bruce Tompkin: No, we've not used them John S e c r i s t : F i r s t of a l l, Bruce, I want t o agree with you t h a t one of t h e most pressing things we need today i n microbiological sampling o r evaluations is a r a t i o n a l sense of value I n m i l i t a r y procurement, one of my biggest problems is t h a t when we t a k e samples f o r microbiological a n a l y s i s, i n order f o r it t o stand up i n Court, we have t o have a stand-by sample, which has t o be frozen, while you're analyzing t h e c h i l l e d sample Is t h e r e anything being done t h e s e days t o t r y and c o r r e l a t e counts between f r e s h unfrozen samples and frozen

7 samples? This would permit you to go back and take a count on the frozen sample and state that the count similar to the fresh sample Bruce Tompkin: Well, we've dabbled with that There might actually be something published, but I don't recall offhand There is no effort underway right now, that I know of, to develop that data John Secrist: Well, I think contractors buying from other contractors would have the same problem, trying to justify the finding of their first sample Tony Kotula, USDA: I just wanted to mention, John, that we are working on sample transport, and are considering this problem Melvin Hunt, Kansas State: Bruce, in view of the light of the earlier paper talking about interaction surface forces and how the bacteria are loosely bounded or tightly bounded, have you had any experience with using surfactant in rinse solutions that would alter the surface interaction counts? Bruce Tompkin: Well, no, not really We did once try to use a Tween 80, I believe it is, to enhance the core chill process on a laboratory basis, thinking that this would facilitate the destruction or the removal of bacterh It didn't work for us I'd like to see what Carl is imagining would be employed not only for enhancing sampling, of course, but to facilitate the removal or destruction of bacteria Now there is another thing you have to consider, that when you use a surfactant be careful how and when you use it, because some surfactants are effective against some bacteria So when you're analyzing a sample for Salmonella and you want to get a total count, you really should not use the normal surfactant that you'd use for Sa hone lla Tony Kotula, USDA: I just wanted to add that if you use a sample area that is fat, then you do use Tween 80 Berry, Colorado State: In working with a product like frankfurters, or perhaps ones with low levels of micro-organisms, how do you prevent or get around some of the problems in dilutions with the fat and meat tissue? Bruce Tompkin: Well, it's a nuisance, but we just don't bother with it We just go right down through the fat and just take the liquid portion and use that Now in the initial plating, say of a one to ten dilution, the person reading the plates the next day or a few days Later must be able to distinguish between fat (blood) and bacterial colonies Bruce Ianglois, Kentucky: Have you compared the recovery of bacteria using hand shaking, your paint shaker method, and the bag method?

8 277 Bruce Tompkin: No We have done this in the past, a long time ago In fact, if I recall, our method says that If you do use hand shaking, you have to shake a fantastic number of times in order to equal the mechanical paint shaker But I don't know how meaningful it really is I'm not sure that it is really perfect, whether you're using a blender or this mechanical painter shaker Whichever approach you use, you should understand the system and know what it is capable of doing On the basis of experience we know what to expect from our product So we're basing our interpretations on experience So, whatever you become familiar with is what you use Bruce Laglois: Our next speaker, Dr, J E Campbell, was born in Georgia He got his BS Degree at Rollins College and his PhD from the University of Wisconsin After a short stay in industry, he went to work for the US Government where he is presently Director of the Cincinatti Food Research kboratories Dr Campbell will be discussing "Automtion in Plating and Counting" Dr Campbell