Comparison of Dry Culture Medium and Conventional Plating Techniques for Enumeration of Bacteria in Pasteurized Fluid Milk

Comparison of Dry Culture Medium and Conventional Plating Techniques for Enumeration of Bacteria in Pasteurized Fluid Milk G. F. S E N Y K , S. M. KOZLOWSKI, P. S. NOAR, W. F.SH|PE, and D. K. BANDLER Cornell University Ithaca, NY 14853 ABSTRACT

INTRODUCTION

Standard plate counts, psychrotrophic bacterial counts, and coliforms were determined by conventional plating techniques and by Petrifilm TM plates, a dry culture medium, for 48 commercially processed milk samples (24 whole milk and 24 skim milk). The Petrifilm SM plate counts were compared with counts on standard methods agar for the standard plate count, psychrotrophic bacterial count, and rapid psychrotrophic bacterial count. The Petfifitm violet red bile plate counts were compared with counts on violet red bile agar for coliform test with a solid medium and the preliminary incubation m e t h o d for detection of coliforms. Standard plate counts were determined within 24 h of packaging aond after 7, 10, and 14 d of storage at 6.1 C. Psychrotrophic bacterial counts and colifrom counts were determined with 24 h of packaging and after 7 d storage. There was a strong linear relationship between Petrifilm SM and standard methods agar plates (excluding counts on samples plated within 24 h of packaging) and for the psychrotrophic bacterial count method. Petrifilm SM had a weak linear relationship with Standard Methods Agar plates for the rapid psychrotrophic bacterial count. Coliform counts determined on Petrifilm violet red bile plates were generally within the same range as counts on violet red bile agar plates. The positive predictive values for the Petrifitm violet red bile plates and violet red bile agar plates were essentially the same for samples plated within 24 h of packaging.

Agar plating methods for enumeration of bacteria date back to the turn of the century. Recently, several other techniques to enumerate bacteria have been developed~ In 1984, Petrifilm SM and violet red bile (VRB) (dry media film) were evaluated for determining total bacterial (3) and coliform (8) counts, respectively, in raw milk. Total bacteria count and coliform count were compared with those obtained by conventional methodology in a collaborative study. These earlier studies indicated that the Petrifilm Plate methods had potential as an alternative to conventional agar plating methods for raw milk analyses. Petrifilm plates consist of nutrients or selective and differential agents coated on films, along with a cold-water-soluble gelling agent and triphenyl tetrazolium chloride. Colony counts were accomplished by 1.0 ml of appropriate dilutions plated directly onto the Petrifilm Plates, spread of inoculum by pressure to an overlay film with a plastic spreader, and incubation of inoculated plates under the same conditions used for conventional plating techniques. The primary objective of this research was to evaluate Petrifilm plates as a suitable alternative to conventional plating techniques for enumerating total and psychrotrophic bacteria, and coliforms in commercially pasteurized skim and whole milk. This study incorporated various methods available to the dairy industry today. The standard plate count ( S P C ) i s considered the reference method for estimation of bacterial populations in most dairy products (10). Psychrotrophic bacteria are usually considered responsible for most spoilage problems in fluid milk and therefore are of particular interest to the dairy industry. The SPC and both the psychrotrophic and rapid psychrotrophic bacterial count methods employ standard methods agar (SMA). However, incubation times and temperatures are modified in the latter methods to promote the growth of psychrotrophs (9, 10). Coliform detection is

Received August 18, 1986. Accepted January 23, 1987. 1987 J Dairy Sci 70:1152-1158

1152

BACTERIAL COUNT BY DRY CULTURE MEDIUM used in the dairy industry to indicate postpasteurization contamination. The coliform test with a solid medium is a standard m e t h o d in the dairy industry t o d a y (10). Additionally, Ledford et al., (5, 6) have shown that the preliminary incubation method detects coilforms that will grow over the shelf-life of fluid milk and thus provide a good index of storage quality. Incubation parameters of the latter method have been modified to enhance coliform growth. Both methods employ VRB agar as the plating medium. MATERIALS A N D METHODS Experimental Design

The study was designed as a paired comparison; all tests were performed in duplicate. The SMA (BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD) pour plate was the reference in evaluation of the Petrifim SM plate (PSM; Medical-Surgical Division/3M, St. Paul, MN). The VRB agar (BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD) pour plate, with an overlay, was the reference in evaluation of the Petrifilm VRBA plate (PVRB; Medical-Surgical Division/3M, St. Paul, MN). Sample Selection and Preparation

On the day of processing, whole and skim milk samples from 24 different commercial processing plants were collected and transported to the laboratory on ice. Each sample was aseptically split and labeled so that aliquots could be tested. Stored samples were held at 6.1°C representing the mean temperature in supermarket display cases (7). Appropriate samples were serially diluted in phosphate and magnesium chloride dilution water (10). The primary dilution was prepared by addition of 11.0 ml of milk to 99.0 ml of diluent and so forth. Up to three dilutions were assessed for each sample and test. Tests Performed

The SMA pour plates and (PSM) plates were employed for the following tests: 1) SPC (10) on the samples within 24 h of and 7, 10, and 14 d after packaging; 2) psychrotrophic bacterial count (PBC) (10) and 3) rapid psychrotrophic bacterial count (RPBC) (9) on samples within

115 3

24 h of and 7 d after packaging. The VRBA pour plates with an overlay and PVRB plates were employed for coliform enumeration according to the following tests: 1) Coliform test with a solid medium (COLI) (10) on samples within 24 h of and 7 d after packaging and the preliminary incubation m e t h o d for detection of coliforms (PI) (5) on samples within 24 h of packaging. For each coliform enumeration test, five colonies (when available) were selected randomly from each of the plating media and confirmed in 2% brilliant green lactose bile broth (BGLB; BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD). Figure 1 gives a complete schematic description of the testing program. Statistical Analyses

Bacterial counts were first converted to logz0 to match more nearly the underlying statistical assumptions. Standard regression methods (11) were used to construct least squares regression lines and 95% confidence limits. Repeatability was calculated as coefficient of variation between replicates (standard deviation expressed as percentage of mean). F o r analysis o f coliform data, a positive predictive value was calculated as the proportion of the number of colonies identified as coliforms by a confirmatory test compared with the number of colonies considered to be coliforms by the presumptive test. RESULTS AND DISCUSSION

Statistical analysis indicated that the t y p e of milk (whole or skim) had no effect on the relationship between dry culture medium and conventional plating techniques. Therefore, combined data for whole and skim milk are provided. The SMA mean log counts for the SPC were significantly higher than PSM tested on d 0, 7, and 14. Differences between SMA and PSM were less on d 7 and 14 (significantly different at P<.05 but not P<.01). The slope and intercept of the regression line of the PSM and SMA for d 0 were significantly different from 1 and 0, respectively, indicating a lack of equivalence of the two methods when samples were plated within 24 h of packaging. No differences were significant for slopes or intercepts for d 7 Journal of Dairy Science Vol. 70, No. 6, 1987

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SENYK ET AL.

Each Milk Sample Three Dilutions (vhen appropriate) Plated in Duplicate (Whole and Skim from Each of 24 Plants)

I

I

Petrifil m VRB Violet and

Petrifilm SM and Standard Methods Agar SPC (d o, 7, I o, 14)

I

Plate and Incubate at 52+_. 1°C for 48

PBC (d o, 7)

I

Plate end Incubate at7 + I°C for lOd

Red Bile Agar

I

RPBC (d o, 7)

Coli (d O, 7)

Pl (d O)

I

Incubat! Sample at 37 °C for 6h

Plate and Incubate et 21°C for 25h

/

I

I

Plate and Incubate at 32 + I°C for 24 +__2h Record All Counts

[

I

Confirm 5 Colonies

1

Record Count

in BGLB

1

Incubate 81 32 +--l°C I0r 48 2_ 3h

t

Record Results Figure 1. Schematic of Testing Program. Abbreviations: SPC, standard plate count; PBC, psychrotrophic bacterial count; RPBC, rapid psychrotrophic bacterial count; VRB, violet red bile; COLI, colifrom count; PI, preliminary incubation coliform count, and BGLB, briliant green lactose broth.

to 14 (Table 1). These data are presented in Figure 2, 3, 4, and 5 with regression lines and 95% confidence limits. The difference between PSM and SMA counts on d 0 may be related to the construction of the PSM plate as well as the flora of the samples. Lower counts on PSM are likely related to the comparative repair and growth rate of injured organisms on SMA and PSM. Small changes in medium have had significant effects on counts (1, 4, 12). The changes in growth medium from an agar plate to a thin gel contained between two films may be sufficient alteration in the microenvironment to change the repair rate of some microorganisms. To use PSM plates to test pasteurized milk additional time for the repair and growth of injured organisms may be necessary. In a trial with 10 Journal of Dairy Science Vol. 70, No. 6, 1987

fresh milk samples, increase of incubation time from 48 to 72 h resulted in no significant increase in SMA, but PSM count increased so as not to be significantly different from SMA incubated for either 48 or 72 h (P<.01). Incubation time and temperature are also critical factors, as indicated by the similiarity of the PBC results for PSM and SMA on samples within 24 h of packaging. Although PBC counts on PSM tended to be slightly lower than SMA, there was a strong linear relationship. Although the results for SPC in this study differ from the those in the collaborative study (2), the samples in the collaborative study were entirely different in their preparation and make-up than the commercially pasteurized samples used in this study. Milks in the collaborative study were sterilized and then spiked

BACTERIAL COUNT BY DRY CULTURE MEDIUM

1155

TABLE 1. Comparison of standard plate counts for whole and skim milks by standard methods agar (SMA) and Petrifilm SM (PSM) plates. Days of storage at 6.1°C

No. of samples Mean log SMA count/ml Mean log PSM count/ml Correlation coefficient Slope Intercept

0

7

10

14

47 2.82 (.57)' 2.38 (.41) .78 .56 .79

47 5.34 (2.22) 5.04 (2.24) .98 .99 -.23

47

44 7.06 (2.40) 6.96 (2.37) .99 .99 .01

6.29 (2.51) 6.19 (2.47) .99 .98 .05

a Standard deviations are in parentheses.

with viable m i x e d cultures o f Escbericbia coli and Staphylococcus aureus or E. coli and Streptococcus lactis. These organisms are s o m e w h a t different f r o m t h e p s y c h r o t r o p h i c organisms ( n o t a b l y Pseudomonas and Enterobacter species) c o m m o n l y f o u n d in commercially processed fluid milk supplies. Samples for t h e collaborative study w o u l d n o t likely have c o n t a i n e d injured organisms. If t h e commercially processed samples in the latest study were sterilized and spiked with s i m i l a r organisms, the results seemingly w o u l d be comparable. Duplicate SMA and PSM plates with actual numerical counts within the 30 t o 300 c o u n t

range were e x a m i n e d for repeatability. No significant difference was f o u n d b e t w e e n t h e overall repeatability of PSM (CV = 3.4%) and SMA plate (CV = 2.6%). Comparisons of PBC and RPBC are p r o v i d e d in Table 2. F o r the PBC, SMA m e a n log counts were significantly higher than PSM at d 0, but counts at this t i m e were very low. A t d 7, no significant difference was found. Slopes and intercepts f r o m t h e regressions for b o t h days were n o t significantly d i f f e r e n t f r o m 1 and 0, respectively. A strong linear relationship b e t w e e n SMA and PSM was evident for the PBC m e t h o d . The SMA counts t e n d e d to be slightly higher, b u t slopes close to 1 indicated t h a t the

0ay 7 Day 0

.

.....

~

%

o~

......

.... ...

¢-

g

o

LOG COUNT/ML - S t l A

Figure 2. Relationship between log count/ml Petrifilm SM and log count/ml standard methods agar (SMA) for d O.

LOG COUNT/ML - S M A

Figure 3. Relationship between log count/ml Petrifilm SM and log count/ml standard methods agar (SMA) for d 7. Journal of Dairy Science Vol. 70, No. 6, 1987

1156

SENYK ET AL. 10-

10

° gAi

DayI0

~

E

=

-""

..ii!!iiiiiii!i!iiiii .... . . . !

_E

=

/

g E

2

;

,0

Log Countlml - 5 M A

Log Coont/ml

-SM A

Figure 4. Relationship between log count/ml Petrifilm SM and log count/ml standard methods agar (SMA) for d 10.

Figure 5. Relationship between log count/ml Petrifilm SM and log count standard methods agar (SMA) for d 14.

d i f f e r e n c e b e t w e e n PSM a n d SMA was c o n s t a n t over t h e e n t i r e r a n g e o f c o u n t s . T h e regression a n d c o r r e l a t i o n s for t h e R P B C m e t h o d i n d i c a t e d a p o o r linear relat i o n s h i p b e t w e e n S M A a n d PSM. Slopes were s i g n i f i c a n t l y d i f f e r e n t f r o m 1 at .51 (d 0) a n d .64 (d 7). T h e S M A c o u n t s were significantly h i g h e r t h a n PSM at each day. T h e R P B C m e t h o d gives small p i n p o i n t colonies o n SMA, a n d such c o l o n i e s m a y b e t o o small t o b e d e t e c t e d o n PSM. A d d i t i o n a l research on a

l i m i t e d n u m b e r o f d e t e r m i n a t i o n s (n = 26) e m p l o y i n g a n i n c u b a t i o n t i m e o f 4 8 h at 21°C gave c o u n t s n o t statistically d i f f e r e n t f r o m t h e S M A R P B C m e t h o d . T h e r e is a n e e d for f u r t h e r r e s e a r c h t o establish t h e exact i n c u b a t i o n t i m e r e q u i r e d for a rapid p s y c h r o t r o p h i c c o u n t using PSM; PSM c a n n o t be c o n s i d e r e d a suitable a l t e r n a t i v e to SMA f o r t h e R P B C m e t h o d e m p l o y i n g 25 h o f i n c u b a t i o n . D u p l i c a t e SMA a n d PSM plates for PBC a n d R P B C w i t h actual n u m e r i c a l c o u n t s w i t h i n t h e

TABLE 2. Comparison of psychrotrophic bacterial counts for whole and skim milks by standard methods agar (SMA) and Petrifilm SM (PSM) plates. Days of storage at 6.1°C Psychrotrophic bacterial count

No. of samples Mean log SMA count/ml Mean log PSM count/ml Correlation coefficient Slope Intercept

Rapid psychrotrophic bacterial count

0

7

0

7

48 1.02 (1.13) ~ .90 (1.09) .97 .93 --.05

48 5.17 (2.64) 5.03 (2.71) .98 1.01 --.18

48 1.33 (1.09) .39 (.80) .69 .51 --.28

48 5.24 (2.49) 3.71 (2.07) .77 .64 .33

1 Standard deviations are in parentheses. Journal of Dairy Science Vol. 70, No. 6, 1987

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TABLE 3. Levels of presumptive and confirmed coliform counts for whole and skim milks by violet red bile agar (VRBA) and Petrifilm VRB (PVRB) plates. Ranges of coliform count per milliliter Method

Day

Medium

<1

1 to 10

0 0

VRBA PVRB

34 38

13 9

1 1

0 0

COLI

7 7

VRBA PVRB

26 27

3 5

4 4

14 11

PI 2

0 0

VRBA PVRB

21 22

3 7

8 9

16 10

0 0

VRBA PVRB

36 39

11 8

1 1

0 0

COLI

7 7

BRBA PVRB

32 29

3 3

1 5

11 10

PI

0 0

VRBA PVRB

23 23

4 6

6 10

15 9

Presumptive data COLI 1

Confirmed data COLI

11 to 150

>150

i Coliform test with a solid medium. 2 Preliminary incubation method for detection of coliforms in commercially processed milk samples.

30 t o 3 0 0 c o u n t r a n g e w e r e e x a m i n e d f o r repeatability. No significant difference was f o u n d b e t w e e n t h e overall PSM (CV = 3.8% a n d 2.8%) a n d S M A r e p e a t a b i l i t y (CV = 3.2% a n d 2.2%) f o r t h e PBC a n d RPBC. Coliform c o u n t s were very low for the samples tested; therefore, each sample was cross-classified a c c o r d i n g t o p r e s u m p t i v e a n d c o n f i r m e d c o u n t s p e r milliliter b y m e t h o d , d a y , a n d m e d i u m ( T a b l e 3). N o t r e n d s w e r e e v i d e n t

in t h e C O L I d 0 a n d 7 data. T h e PI d a t a ind i c a t e s a s l i g h t l y h i g h e r t r e n d t o classify s a m ples as h a v i n g > 1 5 0 c o l i f o r m s / m l c o u n t s o n VRBA. The positive predictive value (representing the proportion of confirmed and presumptive c o l o n i e s ) w a s also c a l c u l a t e d f o r e a c h m e t h o d , d a y , a n d m e d i u m ( T a b l e 4). F o r C O L I , d 0, v a l u e s w e r e n o t s i g n i f i c a n t l y d i f f e r e n t at .86 ( P V R B ) a n d .81 ( V R B A ) . T h e p o s i t i v e predic-

TABLE 4. Positive predictive values for coliforms in whole and skim milks confirmed from Petrifilm VRB (PVRB) and violet red bile agar (VRBA) plates. No. of colonies selected for confirmation (A)

No. of colonies selected and confirmed (B)

Positive predictive value (B/A)

Method

Day

Medium

COLI 1

0 0

PVRB VRBA

29 36

25 29

.86 .81

COLI

7 7

PVRB VRBA

88 98

78 51

.89 .52

PI 2

0 0

PVRB VRBA

135 137

113 111

.84 .81

1 Coliform test with a solid medium. 2 Preliminary incubation method for detection of coliforms in commercially processed milk samples. Journal of Dairy Science Vol. 70, No. 6, 1987

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SENYK ET AL.

live value for P V R B (.89) was significantly higher than V R B A (.52) for COLI d 7. T w o factors possibly attribute for the significant difference in positive predictive values. Alt h o u g h c o l i f o r m s and n o n c o l i f o r m s grow on PVRB, t h e o n l y colonies c o u n t e d and subject to c o n f i r m a t o r y tests for c o l i f o r m s are t h o s e producing gas. Thus, c o n f i r m a t i o n rates f r o m P V R B should be high. No such selection of colonies can be m a d e for c o n f i r m a t i o n f r o m V R B A plates. F u r t h e r m o r e , studies in our l a b o r a t o r y showed that 30% o f aged milk samples tested for coliforms gave presumptive colonies growing on V R B A plates, which isolation and identification showed were Pseudornonas species (5). Additionally, in unpublished data in c o n n e c t i o n with studies o f the preliminary incubation m e t h o d for coliforms, 51 of 292 isolates f r o m V R B A plates o f aged milk samples were either of the genera S e r r a t i a or H a f n i a , which w o u l d grow on V R B A b u t n o t p r o d u c e gas in BGLB. In view of these findings, the lower positive predictive value for d 7 V R B A plates is not surprising. The positive predictive value for P V R B and V R B A plates by the PI m e t h o d were not significantly different (PVRB = .84 and V R B A = .81). Thus, differences in positive predictive values were only observed in aged milk samples. No c o l i f o r m analyses were p e r f o r m e d after d 7. CONCLUSIONS

The following conclusions can be drawn f r o m this research: 1) a strong linear relationship exists b e t w e e n SMA and PSM plates e m p l o y e d for the SPC (d 7 to 14) and PBC m e t h o d s ; 2) PSM p e r f o r m a n c e is comparable on w h o l e and skim milk, indicating no fat c o n t e n t bias for normal fluid milk; 3) PSM plates, particularly for fresh (d 0) samples, gave l o w e r counts than agar p o u r plates; 4) PSM plates are n o t a suitable alternative to SMA for the RPBC m e t h o d e m p l o y i n g t h e suggested incubation time; 5) P V R B and V R B A c o u n t s are generally within t h e same range; 6) P V R B and V R B A positive predictive values are essentially t h e same for samples plated within 24 h o f packaging.

Journal of Dairy Science Vol. 70, No. 6, 1987

The PSM could be considered an alternative to conventional plating t e c h n i q u e s e m p l o y e d for SPC (d 7 t o 14) and p r o b a b l y d 0 by using a 72 h incubation, p s y c h r o t r o p h i c bacterial counts, c o l i f o r m tests with solid m e d i u m , and preliminary i n c u b a t i o n d e t e c t i o n of coliforms for evaluation o f fluid milks.

REFERENCES

1 De Valdez, G. F., G. S. De Giogi, A. P. De Ruiz Holgado, and G. Oliver. 1985. Influence of the recovery medium on the viability of injured freeze-dried lactic bacteria. Milchwissenshaft 40: 518. 2 Ginn, R. E., V. S. Packard, and T. L. Fox. 1986. Enumeration of total bacteria and coliforms in Milk by rehydrated film methods: Collaborative Study. J. Assoc. Offic. Anal. Chem. 69: 527. 3 Ginn, R. E., V. S. Packard, and T. L. Fox. 1984. Evaluation of the 3M dry culture plate (Petrifilm SM) method for determining numbers of bacteria in raw milk. J. Food Prot. 47:753. 4 Lancette, G. A., J. T. Peeler, and J. M. Lanier. 1986. Evaluation of an improved MPN medium for recovery of stressed and nonstressed Staphylococcus aureus. J. Assoc. Offic. Anal. Chem. 69:44. 5 Ledford, R. A., G. F. Senyk, E. Kotsides, and E. T. Wolff. 1983. Preliminary incubation method for detection of coliforms in commercially processed milk samples. J. Dairy Sci. 66:1607. 6 Ledford, R. A., G. F. Senyk, W. F. Shipe, E. Kotsides, and E. T. Wolff. Influence of growth of coliforms on flavor acceptability of commercially processed milk samples. J. Dairy Sci. 66:161I. 7 Milk Quality Improvement Project Progress Report. 1979/1980. Dep. Food Sci., Cornell Univ., Ithaca, NY. 8 Nelson, C. L., T. L. Fox, and F. F. Busta. 1984. Evaluation of dry medium film (Petrifilm VRB) for coliform enumeration. J. Food Prot. 47:520. 90liveria, J. S., and C. E. Parmelee. 1975. Rapid enumeration of psychrotrophic bacteria in raw and pasteurized milk. J. Milk Food Technol. 39:269. 10 Richardson, G. H., ed. 1985. Standard methods for the examination of dairy products. 15th ed. Am. Publ. Health Assoc., Washington, DC. 11 Snedecor, G. W., and W. G. Cochran. 1980. Statistical methods. 7th ed. Iowa State Univ. Press, Ames. 12 Snigh, R. S., V. K. Batish, H. Chandler, and B. Ranganathan. 1985. Reactivation of heat injured Esbericbia coli cells in milk. Milchwissenshaft 40:398.