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.
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
."
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, N.Y.) 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.
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.
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 30-45 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.
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
samples? This would permit you t o go back and take a count on t h e
frozen sample and state t h a t t h e count similar t o t h e f r e s h sample.
Bruce Tompkin: Well, we've dabbled with t h a t . There might a c t u a l l y
be something published, but I don't r e c a l l offhand. There i s no e f f o r t
underway right now, that I know of, t o develop that d a t a .
John S e c r i s t : Well, I think contractors buying from other
contractors would have t h e same problem, t r y i n g t o j u s t i f y t h e finding
of t h e i r f i r s t sample.
Tony Kotula, USDA: I just wanted t o mention, John, that w e are
working .on sample t r a n s p o r t , and are considering t h i s problem.
Melvin Hunt, Kansas S t a t e : Bruce, i n view of t h e l i g h t of t h e
e a r l i e r paper talking about i n t e r a c t i o n surface forces and how t h e
b a c t e r i a are loosely bounded o r tightly bounded, have you had any
experience w i t h using s u r f a c t a n t in rinse s o l u t i o n s that would a l t e r
t h e s u r f a c e i n t e r a c t i o n counts?
Bruce Tompkin: Well, no, not really. We did once t r y t o use a
Tween 80, I b e l i e v e it is, t o enhance t h e core c h i l l process on a
laboratory b a s i s , thinking that t h i s would f a c i l i t a t e t h e d e s t r u c t i o n
or t h e removal of b a c t e r h . It d i d n ' t work f o r us. I ' d l i k e t o see
what Carl is imagining would be employed not only f o r enhancing
sampling, of course, b u t t o f a c i l i t a t e t h e removal or d e s t r u c t i o n of
b a c t e r i a . Now t h e r e is another t h i n g you have t o consider, that when
you use a s u r f a c t a n t be c a r e f u l how and when you use it, because some
s u r f a c t a n t s are e f f e c t i v e a g a i n s t some b a c t e r i a . So when you're
analyzing a sample f o r Salmonella and you want t o g e t a t o t a l count,
you r e a l l y should not use the normal s u r f a c t a n t that you'd use f o r
Sa h o n e lla
Tony Kotula, USDA: I j u s t wanted t o add that if you use a sample
a r e a that is f a t , then you do use Tween 80.
Berry, Colorado S t a t e : I n working w i t h a product l i k e frankfurters,
o r perhaps ones w i t h low l e v e l s of micro-organisms, how do you prevent
or g e t around some of t h e problems i n d i l u t i o n s with t h e f a t and meat
tissue?
Bruce Tompkin: Well, i t ' s a nuisance, but w e j u s t don't bother
with it. We just go right down through t h e f a t and j u s t t a k e t h e
l i q u i d portion and use that. N o w i n t h e i n i t i a l p l a t i n g , say of a one
t o t e n d i l u t i o n , t h e person reading the p l a t e s the next day o r a f e w
days Later m u s t be a b l e t o d i s t i n g u i s h between f a t (blood) and b a c t e r i a l
colonies
Bruce Ianglois, Kentucky: Have you compared t h e recovery of
b a c t e r i a using hand shaking, your paint shaker method, and t h e bag
method?
277
Bruce Tompkin: No. We have done t h i s i n t h e past, a long time
ago. In f a c 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 a n t a s t i c number of times i n order t o
equal t h e mechanical paint shaker. But I don't know how meaningful
it r e a l l y is. I ' m not sure that it i s r e a l l y p e r f e c t , whether you're
using a blender or t h i s mechanical painter shaker. Whichever approach
you use, you should understand t h e system and know w h a t it is capable
of doing. On t h e basis of experience we know w h a t t o expect from our
product. So we're basing our i n t e r p r e t a t i o n s on experience. So,
whatever you become familiar with is w h a t you use.
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.
from t h e University of Wisconsin. After a s h o r t s t a y i n industry, he
went t o work f o r t h e U.S. Government where he i s presently Director of
t h e C i n c i n a t t i Food Research k b o r a t o r i e s
D r Campbell w i l l be
discussing "Automtion i n P l a t i n g and Counting." Dr. Campbell.
in Georgia.
.