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The direct epifluorescent filter technique, cytochrome c oxidase test and plate count method for predicting the keeping quality of pasteurized cream
Food Microbiology, 1986, 3, 185-194
The direct epifluorescent filter technique, cytochrome c oxidase test and plate count method for predicting the keeping quality of pasteurized cream R. G. Kroll* and U. M. Rodrigues D e p a r t m e n t o f F o o d Microbiology, A F R C I n s t i t u t e o f F o o d Research, R e a d i n g Laboratory, S h i n f i e l d , R e a d i n g R G 2 9 A T , U K R e c e i v e d 20 F e b r u a r y 1986 The ability of the direct epifluorescent filter technique (DEFT) to predict the keeping quality (KQ) of pasteurized cream, after an 18-h preincubation stage, was poor unless (particularly with samples that had a corresponding long KQ) undiluted cream was filtered. The DEFT then gave as good, if not better, prediction of the KQ as the plate count. The cytochrome c oxidase test (Kroll 1985, J. Appl. Bacteriol. 59, 137-141) could also be used successfully to predict the KQ of pasteurized cream but its predictive value was not as good as the DEFT or plate count method. However, because of its simplicity, rapidity and low cost, the method could be of use to milk processors to monitor the potential KQ of their products.
Introduction Milk processors need rapid, simple and cheap methods to determine the potential shelf lives of their products. Traditional plate counts provide reliable and sensitive methods to monitor the hygienic status of foods but require at least several days for results to be obtained. Furthermore, the initial microbial content of pasteurized milk and cream, as determined by plate count, has little value in predicting the keeping quality (KQ) of the product (for references see Bishop et al. 1984). This is because such counts include the thermoduric organisms that survive pasteurization and become significant spoilage organisms only if they are psychrotrophic or the product is not properly refrigerated (Cox 1975, McKinnon and Pettipher 1983). Post-pasteurization contamination by psychrotrophic Gramnegative bacteria is the most significant factor in reducing the KQ of refrigerated pasteurized milk and cream (Phillips et 0740--0020/86/020185 + 10 $02.00/0
al. 1981, Schroder et al. 1982) but the numbers of these contaminants after pasteurization are low. Several methods have been described to predict more reliably the KQ of pasteurized milk and cream. Modifications of the plate count to obtain direct counts of these post-pasteurization contaminants can be obtained by incubation of plates at lower temperatures (Anon 1972) or by the inclusion of specific inhibitors (Langeveld et al. 1976). In the last decade new, more rapid methods of assessing bacterial numbers have been developed and the potential advantages of using the measurement of impedance, assay of bacterial ATP or the direct epifluorescent filter technique (DEFT) have been evaluated. (Waes and Bossuyt 1982, Bossuyt and Waes 1983, Bishop et al. 1984, Griffiths et al. 1984, Rodrigues and Pettipher 1984). The last two methods require an 18-h preincubation stage but results are available within a day. Alternatively, the bac© 1986 Academic Press Inc. (London) Limited
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R.G. Kroll and U. M. Rodrigues
terial n u m b e r s can be assessed by plate count, after preincubation, but this requires at least a f u r t h e r day for results to be obtained. Impedance methods take 1 to 2 days but have the a d v a n t a g e of being less labour intensive. All four methods can give reasonable prediction of the KQ of pasteurized milk or cream but can be expensive in t e r m s of capital, r u n n i n g or labour costs and a l t e r n a t i v e simple, cheap, rapid and reliable methods are needed. Kroll (1985) used a simple, rapid and cheap ( < l p per sample) dye oxidation method to detect the G r a m - n e g a t i v e psychrotrophic bacteria in milk. The degree of dye colouration was proportional to the n u m b e r s of psychrotrophs when cell n u m b e r s exceeded 104 m1-1. Although too insensitive to detect these bacteria in freshly pasteurized milk, the test could predict the KQ of >89% of samples of pasteurized milk after an 18-h preincubation stage (Kroll and Rodrigues 1986). The selective basis of the test is that, in general, the r e l e v a n t psychrotrophic G r a m - n e g a t i v e bacteria possess the enzyme cytochrome c oxidase whereas the r e l e v a n t non-psychrotrophic G r a m - n e g a t i v e and most Grampositive bacteria do not. Cytochrome c oxidase is a t e r m i n a l enzyme of the electron t r a n s p o r t chain of some bacteria which catalyses the reduction of 02 to H20. Many bacteria have branched respiratory chains with t e r m i n a l oxidases other t h a n cytochrome c oxidase (Poole 1983). This oxidase can be detected using the dye N N N ' N ' t e t r a m e t h y l p-phenylene-diamine (TMPD, Kovac 1956, Jones 1980). Electrons are donated from TMPD (Eo' = 0.26 mV) to the t e r m i n a l portion (cytochrome c-cytochrome c oxidase) of the respiratory chain. Reduced TMPD is colourless whereas w h e n oxidized a stable blue compound (Wursters blue) is formed. This paper compares, using preincu-
bated samples, this m e t h o d and the D E F T with the plate count, for predicting the KQ of pasteurized cream.
Materials and Methods Cream samples Samples of commercially pasteurized single cream were obtained from a local dairy and used either immediately on arrival in the laboratory or after storage for up to 7 d at 5°C.
Determination of KQ Samples (c. 100 ml) were stored at 5 +_ 0.5°C or 10 + 0.5°C (to represent temperature abuse) and viable counts were made at regular intervals by serial dilutions of cream samples in sterile quarter-strength Ringers solution, plating in Yeastrel Milk Agar (Oxoid Ltd) and counting colonies after 3 d incubation at 30°C. The KQ was taken as the time for the bacterial count of the cream samples to reach 5 × 106 cfu ml -I (Rodrigues and Pettipher 1984). Estimations of this arbitrary value of KQ were accurate to only +0.5 d due to errors in the construction of growth curves.
Preincubation of cream samples Aqueous solutions of 1.0% benzalkonium chloride (Sigma) or 0-1% crystal violet (Gurr) were sterilized by membrane filtration (0.22 ~m pore size, Millipore Ltd). In initial experiments when the DEFT and plate count were being compared, these inhibitors were added to preincubated samples to selectively inhibit the growth of gram-positive bacteria at concentrations of 0-03% w/v and 0.002% w/v respectively (Rodrigues and Pettipher 1984). When the cytochrome c oxidase test was compared with the DEFT and plate count, crystal violet was omitted from all samples for preincubation as this would have interfered with the reading of the oxidase test, and the concentration of benzalkonium chloride was increased to 0.05% w/v. Experiments on pure cultures inoculated into nutrient broth and UHT cream showed that this concentration completely inhibited the growth of Gram-positive bacteria but did not inhibit the Gram-negative bacteria. Two 20 ml aliquots of each cream sample were incubated at 20°C or 25°C for 18-h and then examined by the plate count, DEFT or cytochrome c oxidase test.
The keeping quality of cream Bacteriological examinations of preincubated samples Plate counts. Viable counts were performed on preincubated samples as described and colonies were counted after incubation of plates for 2 d at 30°C.
The DEFT. The apparatus and reagents for analysis by the DEFT were as described by Pettipher et al. (1980} using the modified filter support bases and staining procedure described by Rodrigues and Kroll (1985). The enzyme/detergent pretreatment described by Pettipher et al. (1980} enabled only 2 ml of a 10-fold dilution of cream to filter. Replacement of the 2 ml 0.5% Triton X-100 with 2 ml 1% Tween 80 or 20 ml 0.1% Triton X-100 (Pettipher et al. 1981} enabled 2 ml of undiluted cream to be completely and reliably filtered. These alternative pretreatments produced DEFT slides of good quality and did not appear to affect the staining properties of the bacteria. In the experiments reported here the latter modification was used. With a few samples, corresponding to short KQ samples, it was still necessary to dilute the cream samples 10-fold before examination by the DEFT. This was not a problem as the counts in these samples were always well above the sensitivity of the DEFT.
Cytochrome c oxidase test. This was performed as described (Kroll 1985}, except that before addition of TMPD, 1 ml of 0.1% w/v Tween 80 made up in 0-1 M citrate/NaOH buffer, pH 6-6, was added. This assisted the mixing of the dye with the cream and did not affect the degree of dye oxidation in control experiments. The blue colouration al~er 5 min incubation at 25°C was assessed by eye with reference to a set of 10PB Munsell Colour Standards (Tintometer Ltd, Salisbury, England) on a scale of 1 Ccolour standard 9/1), 2 (8/2), 3 (8/4), 4 (7/6), 5 (7/8}, 6 (6/10) and 7 (5/10). Half scores were easily discernible. A reference score of 0 was obtained by addition of 1 ml of distilled water (without TMPD) to a sample of cream and incubating the sample in parallel with the test samples.
Results Use of the D E F T and plate count to predict the KQ of pasteurized cream In initial e x p e r i m e n t s , the ability of the D E F T to predict the K Q (range 3.0-25 d)
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of 23 s a m p l e s of p a s t e u r i z e d c r e a m stored a t 5°C or 10°C, a f t e r p r e i n c u b a t i o n of s a m p l e s a t 15, 20 or 30°C w a s poor (r range; - 0 . 0 6 to - 0 . 8 2 ) . T h e s e low correlations w e r e m o s t serious in c r e a m s of long K Q as, a t t h e s e low cell densities after p r e i n c u b a t i o n , the D E F T tended to o v e r e s t i m a t e the count. The 10-fold dilution of c r e a m u s i n g n o r m a l D E F T p r e t r e a t m e n t reduced the s e n s i t i v i t y of the DEFT. A v a r i e t y of d e t e r g e n t a n d e n z y m e p r e t r e a t m e n t s were investig a t e d and the use of 1% T w e e n 80 or 20 ml 0.1% Triton X-100 as described by P e t t i p h e r et al. (1981) enabled 2 ml of u n d i l u t e d c r e a m to be filtered. This a l t e r a t i o n improved the correlation of the D E F T of s a m p l e s p r e i n c u b a t e d at 15°C or 20°C with K Q (r range; - 0 . 7 3 to - 0 - 8 3 ) as c o m p a r e d with the plate count m e t h o d (r range; - 0 . 8 1 to - 0 . 9 2 ) (16 samples, K Q range, 1.4 to 18.8 days).
Comparison of the DEFT, plate count and oxidase test In the e x p e r i m e n t s j u s t described crystal violet was added to s a m p l e s for preincubation. As crystal violet would h a v e interfered with the r e a d i n g of the oxidase test, this inhibitor was o m i t t e d a n d the concentration of b e n z a l k o n i u m chloride was increased to 0-05% w/v. The K Q of c r e a m s stored a t 5°C r a n g e d from 0-0 to 16.0 d (63 samples) and a t 10°C from 0.0 to 7.2 d (50 samples). T h e p r e d o m i n a n t spoilage o r g a n i s m s a t both storage temperatures were G r a m n e g a t i v e psychrotrophic rods b u t w i t h c r e a m s of long K Q stored a t 10°C, G r a m positive sporeformers, as judged by colony morphology, b e c a m e m o r e significant. Both the D E F T a n d p l a t e count of s a m p l e s p r e i n c u b a t e d at 25°C correlated well w i t h K Q (results not shown). T h e cytochrome c oxidase t e s t on such p r e i n c u b a t e d s a m p l e s did not correlate as well, due to a n u m b e r of false n e g a t i v e results as p r e v i o u s l y observed a f t e r
188
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Fig. I. Relationship between log1• plate count and KQ at 10°C (upper figure; regression line data; c = 7.24, m = -0.58, r = -0-86) and 5°C (lower figure; regression line data; c = 6.95, m - -0.25, r = -0-87) after preincubation of pasteurized cream samples at 20°C for 18 h. preincubation of pasteurized milk samples at 30°C (Kroll and Rodrigues 1986), and analysis of results was restricted to samples that had been preincubated at 20°C. The relationship between plate count, DEFT count and cytochrome c oxidase activity of samples preincubated at 20°C, and KQ at 5°C or 10°C was good (Figs 1-3) and the correlation coefficients of the regression lines for all three methods were reasonable (Figs 1-3), the DEFT and plate count giving similar values and the oxidase test slightly lower correlation coefficients.
The degree of correct classification of KQ Using the analysis described by Rodrigues and Pettipher (1984) the three methods correctly classified the KQ of more than 81% (DEFT), 83% (plate count) and 88% (oxidase test) of samples (Table 1).
Predictive power of the three methods Good straight line fits for all three methods were obtained using the least squares analysis for simple linear regression of log1• KQ + 1 (to avoid taking the log1• of zero) against the log~o
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Fig. 2. Relationship between loglo DEFT count and KQ at 10°C (upper figure; regression line data; C = 7-17, m = -0.53, r = -0-86) and 5°C (lower figure; regression line data; c = 7.04, m = -0.24, r = -0.88) after preincubation of pasteurized cream samples at 20°C for 18 h. v a l u e s of the D E F T count, p l a t e count and oxidase t e s t (Table 2). The predicted K Q for a n e w l y observed v a l u e (x) is o b t a i n e d from t h e s e d a t a by loglo (KQ + 1) = c + m (loglox) w h e r e c is the i n t e r c e p t on t h e y-axis and m is the slope of t h e line. F u r t h e r m o r e , a n i m p o r t a n t factor is the reliability of the prediction o f y (KQ) from a new v a l u e of x i.e. w h a t v a l u e of K Q is to be expected from a new r e a d i n g a n d how m u c h can it be expected to v a r y ? T h e predicted v a l u e of y will v a r y so t h a t a 100 (1 - a)% confidence i n t e r v a l for the
predicted v a l u e of y for each new x v a l u e is needed. This is given by the expected v a l u e of y + the s t a n d a r d e r r o r of t h e individual predicted v a l u e as defined by y = c + mx___
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Fig. 3. R e l a t i o n s h i p b e t w e e n cytochrome c. oxidase a c t i v i t y ( a r b i t r a r y units) and KQ at 10°C (upper figure; r e g r e s s i o n line data; c = 3-19, m = - 0 . 4 0 , r = - 0 . 8 3 ) and 5°C (lower figure; regression line data; c = 2.95, s -- - 0 - 1 7 , r = - 0 . 7 9 ) after p r e i n c u b a t i o n of p a s t e u r i z e d c r e a m samples at 20°C for 18 h.
confidence widths are measured on a logarithmic scale therefore the larger the value of KQ the wider the confidence belts for the corresponding values of KQ. This means that, for all three methods, the longer the KQ of the cream, the less reliable the prediction of KQ is. The DEFT gave the best prediction of KQ with the narrowest confidence belts closely followed by the plato count method (Table 2). The oxidase test had slightly more variability but in part this is to be expected statistically as its scale of measurement has only 13 possible values, whereas the DEFT and plate
counts have more continuous scales. All three methods had greater predictive power of the KQ at 10°C than at 5°C. Discussion The microbiology of commercially freshly pasteurized milk and cream is similar in that the thermoduric organisms that survive pasteurization are present in fairly high numbers (102-104 ml -~) but that, when properly refrigerated, it is the low level postpasteurization contamination by psychrotrophic Gram-negative rods that are primarily responsible for the spoilage of
The keeping quality of cream
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Table 1. Classification of pasteurized cream samples on the basis of KQ at 5°C or 10°C by the DEFT, plate count or c y t o c h r o m e c oxidase activity following preincubation of samples at 20°C for 18 h.
Corresponding KQ (days) DEFT count (ml- 1) at 5°C 104 12.65 105 8.55 l0 s 4.5 107 <1.00 Plate count (ml-D 104 11-75 105 7-75 l0 s 3-75 107 < 1-00 Cytochrome c oxidase activity 1.0 10.72 2.0 3.31 3.0 1.02 4-0 <1-00
at 10°C 6.05 4.12 2.25 <1.00
Percentage samples correctly classified at 5°C 92 86 90 87
at 10°C 84 92 81 94
5.55 3.83 2.10 < 1-00
97 86 87 83
96 92 84 98
8.71 2-88 <1-00 <1-00
100 93 92 96
90 92 88 97
Table 2. Regression line data and 95% confidence belts of logl0 KQ + 1 against logl0 DEFT count, plate count and cytochrome c. oxidase score of pasteurized cream samples after preincubation for 18 h at 20°C.
Method of analysis DEFT Plate count Cytochrome c. oxidase
Storage temperature (°C)
Intercept (c)
Slope (m)
95% confidence belts
5 10 5 10 5 10
1.96 1.46 1.83 1.38 0-94 0-72
-0-22 - 0.16 -0.21 -0.15 - 1.12 -0-80
0.63-0-64 0.42,--0.46 0-67-0-69 0.44-0.48 0.71-0.73 0.51-0.53
these products (Phillips et al. 1981, Schroder et al. 1982). Both the D E F T (Rodrigues a n d P e t t i p h e r 1984) and c y t o c h r o m e c oxidase t e s t (Kroll and Rodrigues 1986) can be used to predict the K Q of p a s t e u r i z e d m i l k a f t e r an 18-h p r e i n c u b a t i o n stage. We w a n t e d to comp a r e the ability of these two new m e t h o d s a n d the p l a t e count to predict the K Q of p a s t e u r i z e d cream. To get good prediction of the K Q of p a s t e u r i z e d c r e a m in the D E F T undi-
luted c r e a m had to be filtered w i t h s a m p l e s of correspondingly long KQ. T h e D E F T t h e n g a v e as good, if not slightly better, prediction of K Q c o m p a r e d w i t h the p l a t e count a R e r p r e i n c u b a t i o n a t 25°C (results not shown) or 20°C (Figs 1 and 2, Tables 1 and 2). T h e p r e i n c u b a t i o n t e m p e r a t u r e does not a p p e a r to be too critical w i t h t h e s e two methods; indeed Rodrigues and P e t t i p h e r (1984) found t h a t D E F T g a v e t h e best prediction of the K Q of p a s t e u r i z e d m i l k a f t e r pre-
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preincubation at 30°C. Similarly, in this incubation at 30°C. A distinct advantage study many false negatives occurred of the DEFT compared with the plate after preincubation at 25°C (results not count method is that the latter requires at least a further day for results to be shown) but none occurred after incubation at 20°C (Fig. 3). In all cases false obtained. Both methods rely on the efficient negatives were not due to lack of growth in these samples, as judged by the correselective inhibition of Gram-positive sponding DEFT and plate count values, organisms during the preincubation and so were probably not due to inhibistage. We have used benzalkonium tion of Gram-negative psychrotrophs by chloride, with or without crystal violet, the inhibition system. The false negabut a combination of nisin and penicillin can be used (Griffiths et al. 1984). None tives probably result from the predomiof these inhibition systems may be per- nant growth of oxidase negative Gramnegative organisms which could occur in fectly selective so that no Gram-negative three ways. First, the test is not wholly psychrotrophs and all Gram-positive organisms are inhibited. However, we selective and the predominent population in a preincubated sample could have encountered no overt false negaresult from the growth of a few cells of tives or positives with either the DEFT or plate count method. The cytochrome c Acinetobacter. Fortunately these oxidase negative Gram-negative psychrotrophs oxidase test also requires a reliable are not very common contaminants of selective inhibition system in the preincubation stage. Although the test is pasteurized milk and cream (Tolle et al. 1979). Second, some species of coliforms broadly specific for the Gram-negative are psychrotrophic and these too are psychrotrophs (Kroll 1985), false posioxidase negative (Cousins and Bramley tives could arise. Two important Gram1981). Third, some species of Pseudopositive thermoduric types of organisms m o n a s are oxidase negative because they that survive pasteurization are the are deficient in cytochrome c (Jones micrococci and bacilli. Some Micrococcus 1980). Why more false negatives should species can be weakly oxidase positive occur at the higher preincubation tembut this depends on the method used peratures is not known but large (Cowan 1974) and some species of Bacilchanges in respiratory components can lus can be strongly oxidase positive but, occur in response to environment (Sweet interestingly, most psychrotrophic and Peterson 1978, Jones 1980). strains are totally oxidase negative or The range of oxidase scores is not as only very weakly oxidase positive (Jurthigh as that observed for pasteurized shuk and Liu 1983). Selective inhibitors milks (Kroll and Rodrigues 1986) which must therefore be added, although preliminary results suggest that omission of possibly decreases the sensitivity and statistical value of the test. Recent inhibitors results in usually the same or results suggest that extending the prelower oxidase scores (A. F. Kelly and incubation to 24 h may well enhance the R. G. Kroll, unpublished results) perhaps sensitivity of the method slightly (A. F. due to the production of inhibitors or the Kelly and R. G. Kroll, unpublished data). competition for nutrients by the GramIn conclusion, both the DEFT and positive bacteria. plate count give good prediction of the Kroll and Rodrigues (1986) found two KQ of pasteurized cream, but the DEFT overt false negative results using the results are available in under 24 h. The cytochrome c oxidase method after preincytochrome c oxidase test requires a cubation at 20°C and many more after
The keeping quality of cream similar short length of time but does not give quite as good prediction of KQ. It should, however, be of value to milk processors because of its cheapness and simplicity. It should be emphasized t hat the test will not detect psychrotrophic coliforms and in those dairies t ha t tend to have problems with this type of contamination the oxidase test may well not give reliable prediction of KQ. Why all three methods give better prediction of KQ at 10°C storage t han at 5°C is not certain, but may simply reflect the
193
larger possible range of values of KQ at 5°C.
Acknowledgements The authors would like to t hank Chris Davis, Stephen Catling and Alison Kelly for their excellent technical assistance; Cliffords Dairies, Bracknell for the samples of pasteurized cream and C. Thompson, Food Biometrics Department, FRI, Reading for help with the statistical analyses.
References Anon (1972) Standard Methods for the Examination for Dairy Purposes. Washington. American Public Health Association. Bishop, J. R., White, C. M. and Firstenberg-Eden, R. (1984} Rapid impedimetric method for determining the potential shelf-life of pasteurized whole milk. J. Food Protect. 47,471-475. Bossuyt, R. G. and Waes, G. M. (1983) Impedance measurements to detect post-pasteurization contamination of pasteurized milk. J. Food Protect. 46, 622-624. Cousins, C. M. and Bramley, A. J. (1981} The microbiology of raw milk. In Dairy Microbiology Volume 1 The Microbiology of milk (Ed. Robinson, R. K.) pp. 119-163, London, Applied Science Publishers. Cowan, S. T. (1974) Cowan and Steels manual for the identification of medical bacteria (2nd ed.) Cambridge, Cambridge University Press. Cox, W. A. (1975} Problems associated with bacterial spores in heat treated milk and dairy products. J. Soc. Dairy Tech. 28, 59-68. Griffiths, M. W., Phillips, J. D. and Muir, D. D. (1984) Methods for rapid detection of post-pasteurization contamination in cream. J. Soc. Dairy Tech. 37, 22-26. Jones, C. W. (1980) Cytochrome patterns in classification and identification including their relevance to the oxidase test. In Microbiological Classification and Identification (Eds Goodfellow, M. and Board, R. G.) Society for Applied Bacteriology Symposium No. 89, pp. 127-138. London, Academic Press. Jurtshuk, P. and Liu, J. K. (1983) Cytochrome oxidase analyses of Bacillus strains: existence of oxidase-positive species. Int. J. System. Bacteriol. 33, 887-891. Kovac, N. (1956) Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature 178, 703. Kroll, R. G. (1985) The cytochrome c oxidase test for the rapid detection of psychrotrophic bacteria in milk. J. Appl. Bacteriol. 59, 137-141. Kroll, R. G. and Rodrigues, U. M. (1986) Prediction of the keeping quality ofpasteurised milk by the detection of cytochrome c oxidase. J. Appl. Bacteriol. 60, 21-27. Langeveld, L. P. M., Cuperus, F., Van Breeman, P. and Dijkers, J. (1976) A rapid method for the detection of post-pasteurization contamination in HTST pasteurized milk. Neth. Milk Dairy J. 30, 157-173. McKinnon, C. H. and Pettipher, G. L. (1983} A survey of the sources of heat resistant bacteria in milk with particular reference to psychrotrophic spore forming bacteria. J. Dairy Res. 50, 163-170. Pettipher, G. L., Mansell, R., McKinnon, C. H. and Cousins, C. M. (1980) Rapid membrane filtration-epifluorescent microscopy techniques for the direct enumeration of bacteria in raw milk. Appl. Environ. Microbiol. 39, 423--429. Pettipher, G. L. and Rodrigues, U. M. (1981) Rapid enumeration of bacteria in heat-treated milk and milk products using a membrane filtration-epifluorescencemicroscopy technique. J. Appl. Bacteriol. 50, 157-166.
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Phi|iips, J. D., Griffiths, M. W. and Muir, D. D. (1981) Factors affecting the shelf-life of pasteurized double cream. J. Soc. Dairy Technol. 34, 109-112. Poole, R. K. (1983) Bacterial cytochrome oxidases. A structurally and functionally diverse group of electron-transfer proteins. Biochim. Biophys. Acta 726, 205--243. Rodrigues, U. M. and Kroll, R. G. (1985) Increased selectivity, sensitivity and rapidity in the Direct Epifluorescent Filter Technique (DEFT). J. Appl. Bacteriol. 59, 493-499. Rodrigues, U. M. and Pettipher, G. L. (1984) Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality ofpasteurised milk within 24 hours. J. Appl. Bacteriol. 57, 125-130. Schroder, M. J. A., Cousins, C. M. and McKinnon, C. H. (1982) Effects of psychrotrophic post pasteurization contamination on the keeping quality at 11° and 5°C of HTST-pasteurised milk in the UK. J. Dairy Res. 49, 619-630. Sweet, W. J. and Peterson, J. A. (1978) Changes in the cytochrome content and electron transport patterns in Pseudomonas putida as a function of growth phase. J. Bacteriol. 133, 217-224. Tolle, V. A., Suhren, G., Otte, I. and Heeschen, W. (1979) Zur bakteriologie und sensorik der pasteurisierten Trinkmilch. Milchwissenschaft 34, 406--410. Waes, G. and Bossuyt, R. (1982) Usefulness of the Benzalkon-crystal violet ATP method for predicting the keeping quality of pasteurised milk. J. Food Protect. 10, 928-931.
The direct epifluorescent filter technique, cytochrome c oxidase test and plate count method for predicting the keeping quality of pasteurized cream R. G. Kroll* and U. M. Rodrigues D e p a r t m e n t o f F o o d Microbiology, A F R C I n s t i t u t e o f F o o d Research, R e a d i n g Laboratory, S h i n f i e l d , R e a d i n g R G 2 9 A T , U K R e c e i v e d 20 F e b r u a r y 1986 The ability of the direct epifluorescent filter technique (DEFT) to predict the keeping quality (KQ) of pasteurized cream, after an 18-h preincubation stage, was poor unless (particularly with samples that had a corresponding long KQ) undiluted cream was filtered. The DEFT then gave as good, if not better, prediction of the KQ as the plate count. The cytochrome c oxidase test (Kroll 1985, J. Appl. Bacteriol. 59, 137-141) could also be used successfully to predict the KQ of pasteurized cream but its predictive value was not as good as the DEFT or plate count method. However, because of its simplicity, rapidity and low cost, the method could be of use to milk processors to monitor the potential KQ of their products.
Introduction Milk processors need rapid, simple and cheap methods to determine the potential shelf lives of their products. Traditional plate counts provide reliable and sensitive methods to monitor the hygienic status of foods but require at least several days for results to be obtained. Furthermore, the initial microbial content of pasteurized milk and cream, as determined by plate count, has little value in predicting the keeping quality (KQ) of the product (for references see Bishop et al. 1984). This is because such counts include the thermoduric organisms that survive pasteurization and become significant spoilage organisms only if they are psychrotrophic or the product is not properly refrigerated (Cox 1975, McKinnon and Pettipher 1983). Post-pasteurization contamination by psychrotrophic Gramnegative bacteria is the most significant factor in reducing the KQ of refrigerated pasteurized milk and cream (Phillips et 0740--0020/86/020185 + 10 $02.00/0
al. 1981, Schroder et al. 1982) but the numbers of these contaminants after pasteurization are low. Several methods have been described to predict more reliably the KQ of pasteurized milk and cream. Modifications of the plate count to obtain direct counts of these post-pasteurization contaminants can be obtained by incubation of plates at lower temperatures (Anon 1972) or by the inclusion of specific inhibitors (Langeveld et al. 1976). In the last decade new, more rapid methods of assessing bacterial numbers have been developed and the potential advantages of using the measurement of impedance, assay of bacterial ATP or the direct epifluorescent filter technique (DEFT) have been evaluated. (Waes and Bossuyt 1982, Bossuyt and Waes 1983, Bishop et al. 1984, Griffiths et al. 1984, Rodrigues and Pettipher 1984). The last two methods require an 18-h preincubation stage but results are available within a day. Alternatively, the bac© 1986 Academic Press Inc. (London) Limited
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R.G. Kroll and U. M. Rodrigues
terial n u m b e r s can be assessed by plate count, after preincubation, but this requires at least a f u r t h e r day for results to be obtained. Impedance methods take 1 to 2 days but have the a d v a n t a g e of being less labour intensive. All four methods can give reasonable prediction of the KQ of pasteurized milk or cream but can be expensive in t e r m s of capital, r u n n i n g or labour costs and a l t e r n a t i v e simple, cheap, rapid and reliable methods are needed. Kroll (1985) used a simple, rapid and cheap ( < l p per sample) dye oxidation method to detect the G r a m - n e g a t i v e psychrotrophic bacteria in milk. The degree of dye colouration was proportional to the n u m b e r s of psychrotrophs when cell n u m b e r s exceeded 104 m1-1. Although too insensitive to detect these bacteria in freshly pasteurized milk, the test could predict the KQ of >89% of samples of pasteurized milk after an 18-h preincubation stage (Kroll and Rodrigues 1986). The selective basis of the test is that, in general, the r e l e v a n t psychrotrophic G r a m - n e g a t i v e bacteria possess the enzyme cytochrome c oxidase whereas the r e l e v a n t non-psychrotrophic G r a m - n e g a t i v e and most Grampositive bacteria do not. Cytochrome c oxidase is a t e r m i n a l enzyme of the electron t r a n s p o r t chain of some bacteria which catalyses the reduction of 02 to H20. Many bacteria have branched respiratory chains with t e r m i n a l oxidases other t h a n cytochrome c oxidase (Poole 1983). This oxidase can be detected using the dye N N N ' N ' t e t r a m e t h y l p-phenylene-diamine (TMPD, Kovac 1956, Jones 1980). Electrons are donated from TMPD (Eo' = 0.26 mV) to the t e r m i n a l portion (cytochrome c-cytochrome c oxidase) of the respiratory chain. Reduced TMPD is colourless whereas w h e n oxidized a stable blue compound (Wursters blue) is formed. This paper compares, using preincu-
bated samples, this m e t h o d and the D E F T with the plate count, for predicting the KQ of pasteurized cream.
Materials and Methods Cream samples Samples of commercially pasteurized single cream were obtained from a local dairy and used either immediately on arrival in the laboratory or after storage for up to 7 d at 5°C.
Determination of KQ Samples (c. 100 ml) were stored at 5 +_ 0.5°C or 10 + 0.5°C (to represent temperature abuse) and viable counts were made at regular intervals by serial dilutions of cream samples in sterile quarter-strength Ringers solution, plating in Yeastrel Milk Agar (Oxoid Ltd) and counting colonies after 3 d incubation at 30°C. The KQ was taken as the time for the bacterial count of the cream samples to reach 5 × 106 cfu ml -I (Rodrigues and Pettipher 1984). Estimations of this arbitrary value of KQ were accurate to only +0.5 d due to errors in the construction of growth curves.
Preincubation of cream samples Aqueous solutions of 1.0% benzalkonium chloride (Sigma) or 0-1% crystal violet (Gurr) were sterilized by membrane filtration (0.22 ~m pore size, Millipore Ltd). In initial experiments when the DEFT and plate count were being compared, these inhibitors were added to preincubated samples to selectively inhibit the growth of gram-positive bacteria at concentrations of 0-03% w/v and 0.002% w/v respectively (Rodrigues and Pettipher 1984). When the cytochrome c oxidase test was compared with the DEFT and plate count, crystal violet was omitted from all samples for preincubation as this would have interfered with the reading of the oxidase test, and the concentration of benzalkonium chloride was increased to 0.05% w/v. Experiments on pure cultures inoculated into nutrient broth and UHT cream showed that this concentration completely inhibited the growth of Gram-positive bacteria but did not inhibit the Gram-negative bacteria. Two 20 ml aliquots of each cream sample were incubated at 20°C or 25°C for 18-h and then examined by the plate count, DEFT or cytochrome c oxidase test.
The keeping quality of cream Bacteriological examinations of preincubated samples Plate counts. Viable counts were performed on preincubated samples as described and colonies were counted after incubation of plates for 2 d at 30°C.
The DEFT. The apparatus and reagents for analysis by the DEFT were as described by Pettipher et al. (1980} using the modified filter support bases and staining procedure described by Rodrigues and Kroll (1985). The enzyme/detergent pretreatment described by Pettipher et al. (1980} enabled only 2 ml of a 10-fold dilution of cream to filter. Replacement of the 2 ml 0.5% Triton X-100 with 2 ml 1% Tween 80 or 20 ml 0.1% Triton X-100 (Pettipher et al. 1981} enabled 2 ml of undiluted cream to be completely and reliably filtered. These alternative pretreatments produced DEFT slides of good quality and did not appear to affect the staining properties of the bacteria. In the experiments reported here the latter modification was used. With a few samples, corresponding to short KQ samples, it was still necessary to dilute the cream samples 10-fold before examination by the DEFT. This was not a problem as the counts in these samples were always well above the sensitivity of the DEFT.
Cytochrome c oxidase test. This was performed as described (Kroll 1985}, except that before addition of TMPD, 1 ml of 0.1% w/v Tween 80 made up in 0-1 M citrate/NaOH buffer, pH 6-6, was added. This assisted the mixing of the dye with the cream and did not affect the degree of dye oxidation in control experiments. The blue colouration al~er 5 min incubation at 25°C was assessed by eye with reference to a set of 10PB Munsell Colour Standards (Tintometer Ltd, Salisbury, England) on a scale of 1 Ccolour standard 9/1), 2 (8/2), 3 (8/4), 4 (7/6), 5 (7/8}, 6 (6/10) and 7 (5/10). Half scores were easily discernible. A reference score of 0 was obtained by addition of 1 ml of distilled water (without TMPD) to a sample of cream and incubating the sample in parallel with the test samples.
Results Use of the D E F T and plate count to predict the KQ of pasteurized cream In initial e x p e r i m e n t s , the ability of the D E F T to predict the K Q (range 3.0-25 d)
187
of 23 s a m p l e s of p a s t e u r i z e d c r e a m stored a t 5°C or 10°C, a f t e r p r e i n c u b a t i o n of s a m p l e s a t 15, 20 or 30°C w a s poor (r range; - 0 . 0 6 to - 0 . 8 2 ) . T h e s e low correlations w e r e m o s t serious in c r e a m s of long K Q as, a t t h e s e low cell densities after p r e i n c u b a t i o n , the D E F T tended to o v e r e s t i m a t e the count. The 10-fold dilution of c r e a m u s i n g n o r m a l D E F T p r e t r e a t m e n t reduced the s e n s i t i v i t y of the DEFT. A v a r i e t y of d e t e r g e n t a n d e n z y m e p r e t r e a t m e n t s were investig a t e d and the use of 1% T w e e n 80 or 20 ml 0.1% Triton X-100 as described by P e t t i p h e r et al. (1981) enabled 2 ml of u n d i l u t e d c r e a m to be filtered. This a l t e r a t i o n improved the correlation of the D E F T of s a m p l e s p r e i n c u b a t e d at 15°C or 20°C with K Q (r range; - 0 . 7 3 to - 0 - 8 3 ) as c o m p a r e d with the plate count m e t h o d (r range; - 0 . 8 1 to - 0 . 9 2 ) (16 samples, K Q range, 1.4 to 18.8 days).
Comparison of the DEFT, plate count and oxidase test In the e x p e r i m e n t s j u s t described crystal violet was added to s a m p l e s for preincubation. As crystal violet would h a v e interfered with the r e a d i n g of the oxidase test, this inhibitor was o m i t t e d a n d the concentration of b e n z a l k o n i u m chloride was increased to 0-05% w/v. The K Q of c r e a m s stored a t 5°C r a n g e d from 0-0 to 16.0 d (63 samples) and a t 10°C from 0.0 to 7.2 d (50 samples). T h e p r e d o m i n a n t spoilage o r g a n i s m s a t both storage temperatures were G r a m n e g a t i v e psychrotrophic rods b u t w i t h c r e a m s of long K Q stored a t 10°C, G r a m positive sporeformers, as judged by colony morphology, b e c a m e m o r e significant. Both the D E F T a n d p l a t e count of s a m p l e s p r e i n c u b a t e d at 25°C correlated well w i t h K Q (results not shown). T h e cytochrome c oxidase t e s t on such p r e i n c u b a t e d s a m p l e s did not correlate as well, due to a n u m b e r of false n e g a t i v e results as p r e v i o u s l y observed a f t e r
188
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Fig. I. Relationship between log1• plate count and KQ at 10°C (upper figure; regression line data; c = 7.24, m = -0.58, r = -0-86) and 5°C (lower figure; regression line data; c = 6.95, m - -0.25, r = -0-87) after preincubation of pasteurized cream samples at 20°C for 18 h. preincubation of pasteurized milk samples at 30°C (Kroll and Rodrigues 1986), and analysis of results was restricted to samples that had been preincubated at 20°C. The relationship between plate count, DEFT count and cytochrome c oxidase activity of samples preincubated at 20°C, and KQ at 5°C or 10°C was good (Figs 1-3) and the correlation coefficients of the regression lines for all three methods were reasonable (Figs 1-3), the DEFT and plate count giving similar values and the oxidase test slightly lower correlation coefficients.
The degree of correct classification of KQ Using the analysis described by Rodrigues and Pettipher (1984) the three methods correctly classified the KQ of more than 81% (DEFT), 83% (plate count) and 88% (oxidase test) of samples (Table 1).
Predictive power of the three methods Good straight line fits for all three methods were obtained using the least squares analysis for simple linear regression of log1• KQ + 1 (to avoid taking the log1• of zero) against the log~o
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Fig. 2. Relationship between loglo DEFT count and KQ at 10°C (upper figure; regression line data; C = 7-17, m = -0.53, r = -0-86) and 5°C (lower figure; regression line data; c = 7.04, m = -0.24, r = -0.88) after preincubation of pasteurized cream samples at 20°C for 18 h. v a l u e s of the D E F T count, p l a t e count and oxidase t e s t (Table 2). The predicted K Q for a n e w l y observed v a l u e (x) is o b t a i n e d from t h e s e d a t a by loglo (KQ + 1) = c + m (loglox) w h e r e c is the i n t e r c e p t on t h e y-axis and m is the slope of t h e line. F u r t h e r m o r e , a n i m p o r t a n t factor is the reliability of the prediction o f y (KQ) from a new v a l u e of x i.e. w h a t v a l u e of K Q is to be expected from a new r e a d i n g a n d how m u c h can it be expected to v a r y ? T h e predicted v a l u e of y will v a r y so t h a t a 100 (1 - a)% confidence i n t e r v a l for the
predicted v a l u e of y for each new x v a l u e is needed. This is given by the expected v a l u e of y + the s t a n d a r d e r r o r of t h e individual predicted v a l u e as defined by y = c + mx___
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Fig. 3. R e l a t i o n s h i p b e t w e e n cytochrome c. oxidase a c t i v i t y ( a r b i t r a r y units) and KQ at 10°C (upper figure; r e g r e s s i o n line data; c = 3-19, m = - 0 . 4 0 , r = - 0 . 8 3 ) and 5°C (lower figure; regression line data; c = 2.95, s -- - 0 - 1 7 , r = - 0 . 7 9 ) after p r e i n c u b a t i o n of p a s t e u r i z e d c r e a m samples at 20°C for 18 h.
confidence widths are measured on a logarithmic scale therefore the larger the value of KQ the wider the confidence belts for the corresponding values of KQ. This means that, for all three methods, the longer the KQ of the cream, the less reliable the prediction of KQ is. The DEFT gave the best prediction of KQ with the narrowest confidence belts closely followed by the plato count method (Table 2). The oxidase test had slightly more variability but in part this is to be expected statistically as its scale of measurement has only 13 possible values, whereas the DEFT and plate
counts have more continuous scales. All three methods had greater predictive power of the KQ at 10°C than at 5°C. Discussion The microbiology of commercially freshly pasteurized milk and cream is similar in that the thermoduric organisms that survive pasteurization are present in fairly high numbers (102-104 ml -~) but that, when properly refrigerated, it is the low level postpasteurization contamination by psychrotrophic Gram-negative rods that are primarily responsible for the spoilage of
The keeping quality of cream
191
Table 1. Classification of pasteurized cream samples on the basis of KQ at 5°C or 10°C by the DEFT, plate count or c y t o c h r o m e c oxidase activity following preincubation of samples at 20°C for 18 h.
Corresponding KQ (days) DEFT count (ml- 1) at 5°C 104 12.65 105 8.55 l0 s 4.5 107 <1.00 Plate count (ml-D 104 11-75 105 7-75 l0 s 3-75 107 < 1-00 Cytochrome c oxidase activity 1.0 10.72 2.0 3.31 3.0 1.02 4-0 <1-00
at 10°C 6.05 4.12 2.25 <1.00
Percentage samples correctly classified at 5°C 92 86 90 87
at 10°C 84 92 81 94
5.55 3.83 2.10 < 1-00
97 86 87 83
96 92 84 98
8.71 2-88 <1-00 <1-00
100 93 92 96
90 92 88 97
Table 2. Regression line data and 95% confidence belts of logl0 KQ + 1 against logl0 DEFT count, plate count and cytochrome c. oxidase score of pasteurized cream samples after preincubation for 18 h at 20°C.
Method of analysis DEFT Plate count Cytochrome c. oxidase
Storage temperature (°C)
Intercept (c)
Slope (m)
95% confidence belts
5 10 5 10 5 10
1.96 1.46 1.83 1.38 0-94 0-72
-0-22 - 0.16 -0.21 -0.15 - 1.12 -0-80
0.63-0-64 0.42,--0.46 0-67-0-69 0.44-0.48 0.71-0.73 0.51-0.53
these products (Phillips et al. 1981, Schroder et al. 1982). Both the D E F T (Rodrigues a n d P e t t i p h e r 1984) and c y t o c h r o m e c oxidase t e s t (Kroll and Rodrigues 1986) can be used to predict the K Q of p a s t e u r i z e d m i l k a f t e r an 18-h p r e i n c u b a t i o n stage. We w a n t e d to comp a r e the ability of these two new m e t h o d s a n d the p l a t e count to predict the K Q of p a s t e u r i z e d cream. To get good prediction of the K Q of p a s t e u r i z e d c r e a m in the D E F T undi-
luted c r e a m had to be filtered w i t h s a m p l e s of correspondingly long KQ. T h e D E F T t h e n g a v e as good, if not slightly better, prediction of K Q c o m p a r e d w i t h the p l a t e count a R e r p r e i n c u b a t i o n a t 25°C (results not shown) or 20°C (Figs 1 and 2, Tables 1 and 2). T h e p r e i n c u b a t i o n t e m p e r a t u r e does not a p p e a r to be too critical w i t h t h e s e two methods; indeed Rodrigues and P e t t i p h e r (1984) found t h a t D E F T g a v e t h e best prediction of the K Q of p a s t e u r i z e d m i l k a f t e r pre-
192
R.G. Kroll and U. M. Rodrigues
preincubation at 30°C. Similarly, in this incubation at 30°C. A distinct advantage study many false negatives occurred of the DEFT compared with the plate after preincubation at 25°C (results not count method is that the latter requires at least a further day for results to be shown) but none occurred after incubation at 20°C (Fig. 3). In all cases false obtained. Both methods rely on the efficient negatives were not due to lack of growth in these samples, as judged by the correselective inhibition of Gram-positive sponding DEFT and plate count values, organisms during the preincubation and so were probably not due to inhibistage. We have used benzalkonium tion of Gram-negative psychrotrophs by chloride, with or without crystal violet, the inhibition system. The false negabut a combination of nisin and penicillin can be used (Griffiths et al. 1984). None tives probably result from the predomiof these inhibition systems may be per- nant growth of oxidase negative Gramnegative organisms which could occur in fectly selective so that no Gram-negative three ways. First, the test is not wholly psychrotrophs and all Gram-positive organisms are inhibited. However, we selective and the predominent population in a preincubated sample could have encountered no overt false negaresult from the growth of a few cells of tives or positives with either the DEFT or plate count method. The cytochrome c Acinetobacter. Fortunately these oxidase negative Gram-negative psychrotrophs oxidase test also requires a reliable are not very common contaminants of selective inhibition system in the preincubation stage. Although the test is pasteurized milk and cream (Tolle et al. 1979). Second, some species of coliforms broadly specific for the Gram-negative are psychrotrophic and these too are psychrotrophs (Kroll 1985), false posioxidase negative (Cousins and Bramley tives could arise. Two important Gram1981). Third, some species of Pseudopositive thermoduric types of organisms m o n a s are oxidase negative because they that survive pasteurization are the are deficient in cytochrome c (Jones micrococci and bacilli. Some Micrococcus 1980). Why more false negatives should species can be weakly oxidase positive occur at the higher preincubation tembut this depends on the method used peratures is not known but large (Cowan 1974) and some species of Bacilchanges in respiratory components can lus can be strongly oxidase positive but, occur in response to environment (Sweet interestingly, most psychrotrophic and Peterson 1978, Jones 1980). strains are totally oxidase negative or The range of oxidase scores is not as only very weakly oxidase positive (Jurthigh as that observed for pasteurized shuk and Liu 1983). Selective inhibitors milks (Kroll and Rodrigues 1986) which must therefore be added, although preliminary results suggest that omission of possibly decreases the sensitivity and statistical value of the test. Recent inhibitors results in usually the same or results suggest that extending the prelower oxidase scores (A. F. Kelly and incubation to 24 h may well enhance the R. G. Kroll, unpublished results) perhaps sensitivity of the method slightly (A. F. due to the production of inhibitors or the Kelly and R. G. Kroll, unpublished data). competition for nutrients by the GramIn conclusion, both the DEFT and positive bacteria. plate count give good prediction of the Kroll and Rodrigues (1986) found two KQ of pasteurized cream, but the DEFT overt false negative results using the results are available in under 24 h. The cytochrome c oxidase method after preincytochrome c oxidase test requires a cubation at 20°C and many more after
The keeping quality of cream similar short length of time but does not give quite as good prediction of KQ. It should, however, be of value to milk processors because of its cheapness and simplicity. It should be emphasized t hat the test will not detect psychrotrophic coliforms and in those dairies t ha t tend to have problems with this type of contamination the oxidase test may well not give reliable prediction of KQ. Why all three methods give better prediction of KQ at 10°C storage t han at 5°C is not certain, but may simply reflect the
193
larger possible range of values of KQ at 5°C.
Acknowledgements The authors would like to t hank Chris Davis, Stephen Catling and Alison Kelly for their excellent technical assistance; Cliffords Dairies, Bracknell for the samples of pasteurized cream and C. Thompson, Food Biometrics Department, FRI, Reading for help with the statistical analyses.
References Anon (1972) Standard Methods for the Examination for Dairy Purposes. Washington. American Public Health Association. Bishop, J. R., White, C. M. and Firstenberg-Eden, R. (1984} Rapid impedimetric method for determining the potential shelf-life of pasteurized whole milk. J. Food Protect. 47,471-475. Bossuyt, R. G. and Waes, G. M. (1983) Impedance measurements to detect post-pasteurization contamination of pasteurized milk. J. Food Protect. 46, 622-624. Cousins, C. M. and Bramley, A. J. (1981} The microbiology of raw milk. In Dairy Microbiology Volume 1 The Microbiology of milk (Ed. Robinson, R. K.) pp. 119-163, London, Applied Science Publishers. Cowan, S. T. (1974) Cowan and Steels manual for the identification of medical bacteria (2nd ed.) Cambridge, Cambridge University Press. Cox, W. A. (1975} Problems associated with bacterial spores in heat treated milk and dairy products. J. Soc. Dairy Tech. 28, 59-68. Griffiths, M. W., Phillips, J. D. and Muir, D. D. (1984) Methods for rapid detection of post-pasteurization contamination in cream. J. Soc. Dairy Tech. 37, 22-26. Jones, C. W. (1980) Cytochrome patterns in classification and identification including their relevance to the oxidase test. In Microbiological Classification and Identification (Eds Goodfellow, M. and Board, R. G.) Society for Applied Bacteriology Symposium No. 89, pp. 127-138. London, Academic Press. Jurtshuk, P. and Liu, J. K. (1983) Cytochrome oxidase analyses of Bacillus strains: existence of oxidase-positive species. Int. J. System. Bacteriol. 33, 887-891. Kovac, N. (1956) Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature 178, 703. Kroll, R. G. (1985) The cytochrome c oxidase test for the rapid detection of psychrotrophic bacteria in milk. J. Appl. Bacteriol. 59, 137-141. Kroll, R. G. and Rodrigues, U. M. (1986) Prediction of the keeping quality ofpasteurised milk by the detection of cytochrome c oxidase. J. Appl. Bacteriol. 60, 21-27. Langeveld, L. P. M., Cuperus, F., Van Breeman, P. and Dijkers, J. (1976) A rapid method for the detection of post-pasteurization contamination in HTST pasteurized milk. Neth. Milk Dairy J. 30, 157-173. McKinnon, C. H. and Pettipher, G. L. (1983} A survey of the sources of heat resistant bacteria in milk with particular reference to psychrotrophic spore forming bacteria. J. Dairy Res. 50, 163-170. Pettipher, G. L., Mansell, R., McKinnon, C. H. and Cousins, C. M. (1980) Rapid membrane filtration-epifluorescent microscopy techniques for the direct enumeration of bacteria in raw milk. Appl. Environ. Microbiol. 39, 423--429. Pettipher, G. L. and Rodrigues, U. M. (1981) Rapid enumeration of bacteria in heat-treated milk and milk products using a membrane filtration-epifluorescencemicroscopy technique. J. Appl. Bacteriol. 50, 157-166.
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R. G, Kroll and U. M. Rodrigues
Phi|iips, J. D., Griffiths, M. W. and Muir, D. D. (1981) Factors affecting the shelf-life of pasteurized double cream. J. Soc. Dairy Technol. 34, 109-112. Poole, R. K. (1983) Bacterial cytochrome oxidases. A structurally and functionally diverse group of electron-transfer proteins. Biochim. Biophys. Acta 726, 205--243. Rodrigues, U. M. and Kroll, R. G. (1985) Increased selectivity, sensitivity and rapidity in the Direct Epifluorescent Filter Technique (DEFT). J. Appl. Bacteriol. 59, 493-499. Rodrigues, U. M. and Pettipher, G. L. (1984) Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality ofpasteurised milk within 24 hours. J. Appl. Bacteriol. 57, 125-130. Schroder, M. J. A., Cousins, C. M. and McKinnon, C. H. (1982) Effects of psychrotrophic post pasteurization contamination on the keeping quality at 11° and 5°C of HTST-pasteurised milk in the UK. J. Dairy Res. 49, 619-630. Sweet, W. J. and Peterson, J. A. (1978) Changes in the cytochrome content and electron transport patterns in Pseudomonas putida as a function of growth phase. J. Bacteriol. 133, 217-224. Tolle, V. A., Suhren, G., Otte, I. and Heeschen, W. (1979) Zur bakteriologie und sensorik der pasteurisierten Trinkmilch. Milchwissenschaft 34, 406--410. Waes, G. and Bossuyt, R. (1982) Usefulness of the Benzalkon-crystal violet ATP method for predicting the keeping quality of pasteurised milk. J. Food Protect. 10, 928-931.