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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 ransport ing samples, preparing samples f o r analysis , media, and incubation temperatures. Since the choice of options influences the ana ly t i ca l r e s u l t s , t h i s information must be avai lable when t h e r e s u l t s are interpreted.

The reason f o r which the samples a r e collected determines the options selected. A l i s t of reasons would include:

1. A rout ine qua l i t y assurance program. 2. To determine the cause of a problem i n a product or process. 3 . Samples submitted as part of a research program. 4. Determining i f a product w i l l meet a customer spec i f ica t ion . 5 . Samples from a government survei l lance or regulatory program. 6 . To develop data t o s e t t l e a conf l i c t between a supplier and a

customer 7. Samples of a food implicated i n an outbreak of foodborne

i l l n e s s

Sample Transport

The method of t ransport ing should be one which r e s u l t s i n a minimum

The sample of microbial growth o r death i n the sample. emphasized because some growth or death w i l l l i k e l y occur. received in the laboratory must be representat ive of t he product a t the time t h e sample was col lected. Most perishable meat samples which must be shipped a long dis tance or where the re is t o be a lengthy time delay should be shipped frozen. The most common method is t o pack the sample with dry ice i n an insulated or Styrofoam container and sh ip by a i r freight. It is bes t t o have a del ivery service prearranged s o the samples a r e transported d i r e c t l y f romthe a i rpo r t t o the laboratory. If t h i s is not done, t he samples may be delayed a t t h e a i r p o r t and undergo spoilage.

The word m i n i m u m must be

When v i s i t i n g a plant t o c o l l e c t samples, t he most convenient way

Problems might be encountered i f the samples a re of t ransmit t ing samples t o t h e laboratory is t o pacbge with dry ice and car ry them back. checked as personal baggage. Air l ines have had t o pay claims t o owners

* Presented at the 29th Annual Reciprocal Meat Conference of the American Meat Science Association, 1976.

of pets which presumably died due t o suffocation from carbon dioxide leaking from packages. This problem can be avoided by using one of the commercial gel-type pcroducts . These a r e p l a s t i c containers containing a g e l which can be frozen and packaged with the samples. Perishable samples which a r e t o be analyzed the same day they a r e col lected m y be re f r igera ted .

Not a l l samples need t o be re f r igera ted or frozen. swab samples may be taken from equipment a f t e r clean-up t o determine if the equipment has, indeed, been thoroughly cleaned and san i t ized . These swab samples may be sen t t o the laboratory through the regular m a i l a t ambient temperature. Samples yielding low t o t a l p l a t e counts indicate the equipment was thoroughly cleaned and san i t ized . samples yielding high t o t a l p l a t e counts indicate the equipment was not thoroughly cleaned and san i t ized . Another example may be i n sampling equipment or meat products f o r salmonellae. If the primary purpose is t o determine t h e mere presence of salmonellae, shipping the samples a t ambient temperature should not a l t e r the end r e s u l t , although some discre t ion should be applied. which w i l l be analyzed f o r Clostridium perfrinKens, since considerable d i e off can occur due t o f reezing.

For example,

Conversely,

It is best t o avoid freezing samples

Sample Preparation

Once the samples a r e a t the Laboratory, they must be prepared fo r ana lys i s . Frozen samples a r e frequently allowed t o t h a w overnight i n a r e f r ige ra to r . I n some instances, time is c r i t i c a l s o the samples m i g h t be placed under cold running water. Care should be taken that the package does not leak and permit en t ry of water. Large boxes of frozen meat m y be sampled by d r i l l i n g in to the product using a pre- 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 ana lys i s .

Several d i f f e ren t methods a r e used t o prepare an in i t ia l 1:lO suspension of the sample in a d i luen t . But, w h a t i s a blender? There a r e d i f f e ren t manufacturers, blenders have d i f f c ren t ranges of speed, and the jars come i n d i f f e ren t shapes and s i zes . We use an Osterizer f o r blending samples. This allows the use of an ordinary canning jar of t he 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 require l e s s storage space.

Many laborator ies use a "blender ."

The most frequent method we use is t o shake the sample with the d i luent 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 in to a pre- s t e r i l i z e d jar containing a tablespoon of broken glass chips. The glass chips a r e obtained i n a 55-galloa drum from a glass manufacturer. Ninety-nine m l of buffer solut ion is then added t o the jar and the cap is t i g h t l y sealed. The jars a r e placed in to a spec ia l ly designed wooden block which can hold up t o 4 jars a t a time. then shaken on a mechanical paint shaker f o r 3 t o 5 minutes.

The samples a r e The small

jar-mechanical paint shaker technique has been used a t Swift & Company f o r more than 30 years. The equipment is v i r t u a l l y t rouble-free, low in cost , and the system is su i ted t o analyzing a large nurriber of samples ef f i c i en t l y .

A r e l a t i v e l y new technique f o r emulsifying samples is the use of a Stomacher (Dynatech Laboratories, Inc ., Alexandria, Vi rg in ia ) . This system cons is t s of placing t h e sample and d i luent i n to a p l a s t i c bag which is then placed behind t h e f ron t door of the Stomacher. Two metal paddles inside the apparatus beat the sample against the 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 he meat sample is dis integrated 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 rna t ive t o mechanical devices is the standard hand shake. This consis ts of placing t h e sample and d i luent i n t o a jar and shaking t h e sample 25 times over an a rc of 1 foot i n 7 seconds. This is the same procedure used f o r shaking subsequent d i lu t ions prepared from the o r ig ina l 1:lO suspension. It is bes t sui ted f o r l iqu id samples such as ju ice from a packaged meat product or br ine solut ions. It can be used f o r analyzing swab samples; however, we prefer using the mechanical paint shaker technique. Industr ies Inc., Queens Village, N.Y.) is not normally used f o r t he i n i t i a l preparation of a 1 : l O d i lu t ion , it is convenient f o r preparing subsequent d i lu t ions . One last method f o r preparing the i n i t i a l sample is t o place the sample in to a s t e r i l e p l a s t i c bag such as a Whirlpack bag. mixed by shaking or squeezing. t o advantage such as determining a surface count of a f rankfur te r .

Although the vortex mixer (Sc ien t i f i c

After adding t h e d i luen t t o t h e bag, the sample can be There are times when t h i s can be used

Each of the foregoing methods has ce r t a in advantages and disad- vantages. skin and dr ied meats. Skin, espec ia l ly poul t ry skin, becomes wrapped around t h e blades of blenders. This is not a problem with the mechanical p a i n t shaker or Stomacher techniques. Hard, dry mater ia l tends t o be chased around t h e s ide of t h e blender jar. A t r i c k t o overcome t h i s is t o tilt the blender apparatus

Among the most d i f f i c u l t samples t o prepare f o r analysis are

D i l uen t s

There has been a considerable amaunt of research over t h e years on various types of d i luents f o r bac te r io logica l ana lys i s . For most meat products, t h i s is of minor importance, espec ia l ly f o r the i n i t i a l preparation of t h e 1:lO d i l u t i o n . While the d i luent m i g h t influence recovery of the maxinaun population, this is normally not s ign i f icant i n a cmmercial s i t ua t ion . 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 he analysis of meat products.

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Two types of media a re used f o r determining aerobic p l a t e counts i n meat products. general medium. However, most l a c t i c acid producing bac ter ia y ie ld colonies which a r e pinpoint i n s i z e on p l a t e count agar. a second type medium is used, especial ly f o r vacuum packaged cured meats and fermented products. purpose. We use APT agar. Although the same count i s obtained on APT and p la t e count agar, the colonies formed on APT agar a r e much larger in s i ze and eas i e r t o count.

P l a t e count agar is one type which serves as a good,

A s a r e s u l t ,

Several media are ava i lab le f o r t h i s

The bes t medixun cur ren t ly avai lable f o r detect ing Staphylococcus aureus is Baird-Parker agar. productive i n terms of recovering the highest l eve l of 2. aureus from foods. It is espec ia l ly usefu l f o r cured meats because it is possible t o de tec t 5. aureus from amng the background of normal f l o r a ex is t ing in the product. It is analagous t o having a spot l igh t on a fox in the middle of a f lock 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.

This medium has been shown t o be the most

Several media and methods e x i s t f o r aetermining coliforms i n meat products. Violet work. products, tubed l iqu id media a r e normally used. The media usual ly consis ts of lauryl s u l f a t e try-ptose broth f o r p r e l h i n a r y incubation followed by t r ans fe r r ing t o b r i l l i a n t green lactose b i l e broth f o r a coliform count and t o EC broth f o r a presumptive E. c o l i count. approach is m o s t commonly used by regulatory agency laborator ies .

red b i l e agar is s t i l l good f o r rout ine ana ly t i ca l If information is needed on the l eve l of E. c o l i i n meat

This

Some i n t e r e s t s t i l l e x i s t s i n quant i ta t ing the gruup D s t reptococci . The group D s t reptococci continues t o be a po ten t i a l spoilage organism i n m e a t products. t h i s group of bac ter ia i n t he United S ta t e s . Current AOAC, FDA and USDA mthodology do not include procedures f o r quant i ta t ing the group D s t reptococci i n foods. This is not t rue i n Canada where meats continue t o be monitored f o r ce r t a in group D s t reptococci .

Aside frcnn t h i s , there is a d d n d l h g i n t e r e s t i n

Several media a r e used f o r quant i ta t ing Clostridium perfringens in meat products. One such medium is SFP agar. Another incorporates cycloserine as discussed by Dr. H. W . Walker a t the 1972 Reciprocal Meats Conference .

The best procedure f o r detect ing salmonellae i n meat products appears t o cons is t of a lactose broth pre-enrichment followed by elevated temperature enrichment i n s e l en i t e cyst ine and t e t r a th iona te broths . Twenty-five gram samples a r e commonly used. The method and s i z e of sample determine the success of t h e salmonellae hunter. The harder one t r i e s , the =re l i k e l y salmonellae w i l l be found in f r e s h meat.

Incubation Temperatures for Aerobic P la t e Count

Considerable var ia t ion ex i s t s i n t h e temperatures used f o r de te r - mining aerobic p l a t e counts. The th ree most common temperature ranges consis t of 5-7W, 20-30%, and 35-37W. Each temperature range r e su l t s i n a s l i g h t l y d i f f e ren t answer. counted after 10 days. C a t s resu l t ing from t h i s procedure a re usefu l i n determining t h e levels of bac te r ia which can multiply i n re f r igera ted meats. Usually, 10 days is too long t o w a i t f o r an answer and, f o r t h i s reason, temperatures i n the range of 2O-3O0C have been adopted. bation times f o r p la tes held a t 20-30W range from 3 t o 2 days as the temperature is increased from 20 t o 3OoC. t h e highest count. The t h i r d temperature range of 35-37% has been used as an index of ex terna l contamination t o meats. There i s some logic and data t o support t h i s be l i e f . t o f ind higher leve ls of mesophiles on f resh ly slaughtered carcass meat as opposed t o the psychrotrophs.

P la tes held a t 5-7OC a r e usual ly

Incu-

This range usual ly yields

For example, one m i g h t expect

Suggestions fo 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 , bu t they have not gone beyond the research s tage. One reason f o r t h i s is that the methods normally are not s u f f i c i e n t l y sens i t ive f o r detect ing microbial levels below 1 million per g i n meat products. method which is sens i t ive i n t h e range of 50,000 t o 500,000 per g would be very helpful , pa r t i cu la r ly in screening r a w mater ia ls .

A

A recent paper by Oliver ia and Parmelee (1) showed excel lent corre- l a t ion f o r t o t a l counts determined after 25 hours a t 21°C versus 10 days a t 7OC. Their da t a are based upon 322 samples of r a w and pasteurized m i l k . Would t h i s same re la t ionship hold f o r meat products? The goal would be t o obtain an 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 io r t o analyzing meat products. This concept has been used successful ly f o r assessing t h e qua l i t y of milk. example of where t h i s might be used w o u l d be in predict ing the shelf l i f e of pre-packaged luncheon meats or frankmters. Would. a preliminary incubation a t 1 3 O C (55%) fo r 18 t o 20 hours pred ic t t he re f r igera ted shelf l i f e of these products i n terms of 30-45 days?

An

Surmnar y

There have been many attempts over t he years t o establish standard methods f o r t he 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 the sense that they w i l l with- stand t h e r igo 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 ac t ion . Such methods a r e necessary in a time of need. However, there w i l l always be a need f o r a va r i e ty of methods.

No s jngle method w i l l s a t i s f y the va r i e ty of reasons f o r which the samples a r e analyzed and t h e d i f f e ren t types of meat products. analyst has t h e option of se lec t ing t h e conditions f o r ana lys i s . Knowing how t h e samples were handled and analyzed is e s sen t i a l fo r va l id in t e rp re t a t ion of the da ta .

Each

REFERENCES

(1) Oliveria, J. S . and C . E. Parmalee. 1976. Rapid enumeration of psychrotrophic bac ter ia 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 the stolnacher : e f f e c t of fa t content, surfactant concentration, and blending t i m e . Can. Ins t . Food Sc i . Technol. J. 9:30.

( 3 ) Sharpe, A . N . and A . K. Jackson. 1972. Stomaching: a new concept Appl. Microbiol. 24 : 175. i n bac ter io logica l sample preparation.

( 4 ) Tuttlebee, J. W, 1975. The stomacher--its use f o r homogenization in food microbiology. J . Fd . Technol. 10: 113.

* * *

Bruce Langlois: Are there any questions f o r Dr. Tompkin?

F. F . Busta, University of Minnesota: How do you san i t i ze the casings on the outside of that fermented sausage before opening the bag?

Bruce Tompkin: Well, ac tua l ly we do not f o r a b ig piece of bologna We a r e able t o e s sen t i a l ly cut out a window, p u l l or fermented sausage.

t he casing back i n such a way t h a t we have a large a rea that has not been touched by the knife a t a l l . So, we don't s an i t i ze .

Jim Chris t ian, University of Georgia: Did you ever use Calgon swabs and 1 percent sodium c i t r a t e f o r general qua l i ty cont ro 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 he most pressing things we need today i n microbiological sampling or evaluations is a r a t i o n a l sense of value. procurement, one of my biggest problems is tha t when we take samples f o r microbiological analysis , 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 the ch i l led sample. Is there anything being done these days t o t r y and co r re l a t e counts between f r e sh unfrozen samples and frozen

In mi l i ta ry

samples? frozen sample and state t h a t the count similar t o t h e f resh sample.

This would permit you t o go back and take a count on the

Bruce Tompkin: Well, we've dabbled with t h a t . There might ac tua l ly be something published, but I don't r e c a l l offhand. There is no e f f o r t underway right now, that I know of, t o develop that data.

John Secr i s t : Well, I think contractors buying from other contractors would have the same problem, t ry ing t o ju s t i fy the finding of t h e i r f irst sample.

Tony Kotula, USDA: I just wanted t o mention, John, that w e are working .on sample transport , and are considering t h i s problem.

Melvin Hunt, Kansas State: Bruce, in view of the l i g h t of the e a r l i e r paper talking about interact ion surface forces and how the bacter ia are loosely bounded or tightly bounded, have you had any experience w i t h using surfactant in rinse solutions that would a l ter the surface interact ion counts?

Bruce Tompkin: Well, no, not really. We did once t r y t o use a Tween 80, I believe it is, t o enhance the core c h i l l process on a laboratory basis , thinking that this would f a c i l i t a t e the destruction or t he removal of b a c t e r h . It didn ' t work f o r us. I 'd l i ke t o see what Carl is imagining would be employed not only f o r enhancing sampling, of course, but t o f a c i l i t a t e the removal or destruction of bacter ia . Now there is another thing you have t o consider, that when you use a surfactant be careful how and when you use it, because some surfactants are effect ive against some bacter ia . analyzing a sample fo r Salmonella and you want t o get a t o t a l count, you r e a l l y should not use the normal surfactant that you'd use f o r Sa h o n e lla

So when you're

Tony Kotula, USDA: I just wanted t o add that if you use a sample area that is f a t , then you do use Tween 80.

Berry, Colorado State: In working w i t h a product l i k e frankfurters, or perhaps ones w i t h low levels of micro-organisms, how do you prevent or get around some of the problems i n d i lu t ions with t h e fa t and meat t i s sue?

Bruce Tompkin: Well, i t ' s a nuisance, but w e jus t don't bother with it. We just go right down through the fat and j u s t take the l iqu id portion and use that. N o w i n the i n i t i a l plating, say of a one t o ten d i lu t ion , t h e person reading the plates the next day or a f e w days Later m u s t be able t o dis t inguish between fat (blood) and bac te r i a l colonies

Bruce Ianglois, Kentucky: Have you compared the recovery of bacter ia using hand shaking, your paint shaker method, and the bag method?

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Bruce Tompkin: No. We have done t h i s i n the past, a long time ago. In f ac t , if I r e c a l l , our method says t h a t If you do use hand shaking, you have t o shake a f an ta s t i c number of times i n order t o equal the mechanical paint shaker. it rea l ly is. I ' m not sure that it is r e a l l y perfect , whether you're using a blender or t h i s mechanical painter shaker. you use, you should understand the system and know w h a t it is capable of doing. On the basis of experience we know w h a t t o expect from our product. So we're basing our interpretat ions on experience. So, whatever you become familiar with is w h a t you use.

But I don't know how meaningful

Whichever approach

Bruce L a g l o i s : Our next speaker, D r , J. E. Campbell, was born He got his B.S . Degree a t Rollins College and h i s Ph.D. in Georgia.

from the University of Wisconsin. went t o work f o r the U.S. Government where he is presently Director of the Cinc ina t t i Food Research kbora to r i e s discussing "Automtion i n Plat ing and Counting."

After a short s t ay in industry, he

D r Campbell w i l l be Dr. Campbell.