Applications of Bioluminescence in the Dairy Industry

M. W. GRlFFlTHS Deparbnent of Food Science University of Guelph Guelph, ON, Canada N1G 2W1

publicity given to foodborne disease in the media. In the past decade, headlines have been Several applications of ATP biorelated to Listeria monocytogenes in soft luminescence of relevance to the dairy cheese, Salmonella enteritidis in eggs, Esindustry have been proposed. This paper cherichia coli 0157 in hamburgers, bovine reviews some of the major benefits of spongiform encephalopathy in beef, and other the technology. New developments in problems. It sometimes appears that the food bioluminescence research have made the industry is lurching from one crisis to another. simple, rapid, sensitive detection of However, most foodborne illnesses were due foodborne pathogens a distinct possibilto mishandling in food service establishments ity. (46.8%) or at home (20.5%) (Table 1) (40, 1 . 4) (Key words: bioluminescence, food Nevertheless, a significant proportion of illness safety, evaluation, dairy products) was the result of mishandling by food processors (2.9%) and retailers (16.0%). This necessiINTRODUCTION tates, in any food manufacturing operation, the Current trends in nutrition and food tech- provision for adequate microbiological testing nology are increasing the demands on food to verify quality of raw materials, to instigate microbiologists to ensure a safe food supply. process controls such as Hazard Analysis CritiThe trend toward foods that are low in fat and cal Control Point (HACCP) procedures, and to high in fiber creates unknown pressures on monitor hygiene. For these measures to be food as an ecosystem. For example, increases effective, results must be obtained as quickly in the unsaturated fatty acid concentration of as possible. foods can change the growth rates of bacteria New techniques have had greatest applica(15, 49). The consumer is also demanding tion in the dairy industry, but their usefulness more variety, which means that the food transcends commodities. Of the emerging techprocessor is developing novel products con- nologies for rapid microbiological analysis, artinuously. These involve, in some instances, guably the technique giving results in the bringing together ingredients with different as- shortest time is bioluminescence. Two distinct sociated microbiological problems, the result areas of bioluminescence are of use to the food of which is uncertain. The concern over food industry: ATP bioluminescence and bacterial additives has led to greater reliance on low bioluminescence. temperatures to effect preservation and the emergence of minimally processed and cookATP BIOLUMINESCENCE chill products; thus, the need for strict microbiological control during processing becomes All living cells contain the molecule ATP. evident. In addition, processors, retailers, and This molecule may be assayed simply using an consumers are constantly making new de- enzyme and coenzyme complex (luciferasemands for shelf-life of products and expect luciferin) found in the tail of the firefly, Photithese to be met. To achieve this, microbial nus pyralis. The reaction is essentially the loads in foods must be minimal. stoichiometric conversion of ATP to photons The consumer has become much more of light (Figure 1). If the level of ATP in aware of food safety issues as a result of bacterial cells is assumed to be relatively constant, the amount of light emitted during the reaction is proportional to the numbers of bacteria present. The ATP bioluminescence assay Received August 19, 1993. has been adapted for a variety of applications Accepted January 8, 1993.
ABSTRACT 1993 J Dairy Sci 76:3118-3125

3118

SYMPOSIUM: EVALUATION OF MILK AND DAIRY PRODUCTS

TABLE 1. Places of acquisition of incidents of foodborne illness of known microbiological origin in Canada between 1975 and 1986.1

3119

Place
Food service establishment Home Food processing plant Retail outlet Farms and dairies Delicatessens Other Unknown 'Data of Todd (40, 41).

Incidents
46.8 20.5 2.9 16.0
5.0

(W

.6 2.2 6.0

in the dairy industry (Table 2), which have been reviewed by Griffiths (11).
Raw Mllk Quallty

Microbial Q a i y Sharpe et al. (36) were ult. the first to determine ATP concentrations in milk contaminated by bacteria. Since then,

luciferin

+ ATP + M#+
Q, luciferase

several workers (11) have used ATP bioluminescence to determine the microbial content of milk. In general, a bacterial concentration of 1 x 106 cfulml was required for detection, but results were obtained in about 5 min. This lack of sensitivity w s mainly due to a high concentrations of nonmicrobial ATP present in milk as f e ATP associated with casein re micelles (33) and ATP present in somatic cells (6). This nonmicrobial ATP w s removed by a hydrolysis with apyrase, an ATP-hydrolyzing enzyme, after addition of chelating agents to disrupt the casein micelle and detergent to lyse somatic cells. A second detergent was added to release the microbial ATP for assay. Recent research has focused on ways to improve the sensitivity of the assay. The incorporation of a filtration step or other procedures to concentrate the bacteria following selective lysis of somatic cells with nonionic detergents has improved assay sensitivity (Figure 2). The ATP can then be extracted from the entrapped bacteria with a cationic detergent and assayed. Using this procedure, workers (4, 12, 14, 21, 26, 35,45) have claimed that ATP bioluminescence can be used to detect as few as 1 x 104 cWml of bacteria in milk within 5 to 10 min. This result makes the technique useful as a rejection test for incoming tanker milks at milk processing plants. These tests are available commercialiy and are marketed by Biotrace (Bridgend, Wales) and Lumac (Laandgraaf, The Netherlands). Bacteria can also be concentrated from milk by centrifugation following addition of a ''con-

oxyluciferin

Figure 1. Schematic representation of the firefly luciferase reaction.

0
light

+ AMP + C q +

TABLE 2. Applications of ATP bioluminescence curm t l y available to the dairy industry.

Raw Inilk quality somatic cell count Microbial count Rodua quality Pasteurized m l ik Pasteurized cream Milk powder UHT sterility testing Starter culture activity Hygiene monitoring Enzyme assay
Rotease

Auraline phosphatase
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GRIFFITHS
Pasteurized Milk Quality

centrating agent, EnlitenTM(Promega, Madison, WI). The concentration of ATP in the bacterial pellet can be determined with luciferase-luciferin. The manufacturers claim that the technique can detect as few as 2 x 104 cfdml, and up to 24 samples can be processed in 1 to 2 h without culturing (27). Somatic Cell Concentrations. An indication of somatic cell concentrations in milk can be obtained from the concentration of ATP in milk following treatment with a nonionic detergent such as Triton X-100(6, 9, 10). Those researchers claim that the technique can be used as an index of mastitic infection. An alternative method for detecting mastitis by bioluminescence has been proposed (22). When bacterial growth in milk was monitored by measuring ATP concentration, bacteria grew more rapidly in mastitic milk, and the increase in bacterial ATP correlated well with inflammatory markers for mastitis.

The prime factor controlling the shelf-life of pasteurized dairy products is the reintroduction of Gram-negative, psychrotrophic bacteria into the milk after heat treatment (29). In addition to limiting shelf-life, postpasteurization contamination of milk may have important public health significance. A number of large outbreaks of foodborne illness attributed to the consumption of pasteurized milk were subsequently shown to be due to postprocess contamination (29). These outbreaks included listeriosis and salmonellosis; of particular concern was an outbreak of salmonellosis in 11linois involving over 16,000 cases (29). A number of methods for detection of postpasteurization contamination of dairy products has been described (3. These methods invariably involve a preincubation procedure in the presence of inhibitors for Gram-positive organisms, which is designed to select for the

Raw milk

m = a + Somatic cell lysing agent

Incubate at 3 f ~ for 5 min

ATP ATP
ATP

IATP(
A b

ATP
ATP ATP

bacteria retaining filter

Syringa through

B r a c t bacterial ATP with bacterial lysing agent

)rTp

Measure light emission after addition of luciferaseluciferin

Figure 2. Schematic representation of the ATP bioluminescence assay for enumration of the microbial content of milk. Journal of Dairy Science Vol. 76, No. 10, 1993

growth of Gram-negative psychrotrophs. W e as and Bossuyt (46, preincubated pasteurized 47) milks at 3' for 24 h in the presence of 0C benzalkon A 50% (.06%) and crystal violet (.002%) to select for the growth of p s y c h trophic, postpasteurization contaminants. Following this preincubation, bacterial numbers were assessed using ATP bioluminescence. Those authors (46) claimed that they were able to detect initial contamination at 1 L of milk using this technique. The test could be made more quantitative by employing a most probable number approach. Phillips and Griffiths (28) claimed that growth of Gram-negative psychrotrophs was better when pasteurized milks were preincubated at 21'C for 25 h in the presence of crystal violet (20 pg/ml), penicillin (200 U/ml), and nisin (400 U/ml), added as inhibitors for Gram-positive bacterial growth. The shelf-life of pasteurized milk and cream could be accurately predicted when ATP counts were performed on the preincubated products. This test also gave good agreement with the recently introduced European Community keeping quality test. An ATP bioluminescent method for prediction of the shelf-life of pasteurized milk has recently been described using the Charm II system (42). Such tests can be useful during milk processing to detect sources of contamination within a reasonable time (25 to 26 h), especially when used in conjunction with on-line sampling techniques (13).
Sterility Testing of UHT and Other Dairy Producta

SYMPOSILTM: EVALUATION OF MILK AND DAIRY PRODUCTS

3121

luciferin. All of the contamination caused by understerilization of the milk could be detected in 3 d by this ATP method. Significant detection of contamination of UHT milks by bacterial ATP determination after a I-d preincubation at 30'C has been documented (8). Testing of UHT products for sterility by bioluminescence may be particularly useful for chocolate milks for which the color precludes the use of dye reduction (31). Bioluminescence techniques have been used to determine the bacterial content of milk powder with varying success (17, 23).
Monitoring Starter Culture Activity

Because ATP is an integral part of the metabolism of bacterial cells, the concentrations of ATP may provide a better indication of the activity of lactic acid bacteria than measurement of pH changes. A strong correlation between acid production and ATP concentrations exists for a number of lactic acid bacteria, including Lactococcus lactis and Lactobacillus acidophilus, during growth in milk (7, 25). Monitoring of the changes in ATP during growth in milk rapidly indicated the presence of antibiotic residues or phage (32. 50.51). Concentrations as low as ,005 U/ml of penicillin could be detected in about 90 min
(51).
Hygiene Monitoring

The sterility of UHT products is generally determined by assessment of plate count or pH changes after incubation of the product at 30'C for 3 d. Incubation followed by plate count is time-consuming, yielding results after 5 to 8 d. Although the monitoring of pH changes gives more rapid results, those results are not always reliable, particularly when contamination is due to sporeformers (48). Waes et al. (48) described a bioluminescence method of monitoring growth after storage of the product at 30'C that involved treatment with apyrase (.01 U) to remove free ATP prior to extraction and assay of bacterial ATP with luciferase-

Probably the most widely used current a p plication of ATP bioluminescence in the food industry is the estimation of surface cleanliness. The total ATP present on a swabbed surface can be extracted and assayed extremely rapidly (i.e., within 5 min) with no less accuracy than that obtained using traditional techniques (16). The result indicates the overall contamination of the surface because ATP from food residues and from microbial sources will be detected (4, 20). The amount of contamination determined by ATP and plate count methods correlated in about 80% of samples. More surfaces were identified as being soiled by the ATP test than by plate count (Table 3). A number of bioluminescence-based hygiene monitoring kits are now commercially available and are used routinely to monitor critical control points in milk processing operations worldwide.
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Enzyme Assays

GRIFFITHS

An interesting application of luminescence is emerging that has implications for the dauy industry in the future: the development of bioluminescent or chemiluminescent assays for a variety of enzymes of importance in the dajl Instrumentation industry. Profease. Rowe et al. (34)described a bioStanley (37)has documented over 90 comluminescent assay for proteases produced by mercially available luminometers with varying psychrotrophic bacteria during growth in milk. degrees of sophistication, ranging from fully These enzymes are heat-resistant and can sur- automated instruments to small, portable invive pasteurization and UHT processing, thus struments. The latter have been particularly reducing the shelf-life of these products (29). convenient for use in conjunction with hygiene The assay relies on rate of inactivation of the monitoring kits; they are easily transported protein luciferase, because of proteolysis, be- throughout the factory and are relatively inexing directly proportional to the protease con- pensive. centration at a constant initial luciferase conAn automated instrument, BactoFoss (Foss centration. In 5 min, protease can be detected Electric, H i l l e d Denmark), with a built-in in milk at concentrations as low as .01 U/ml, filtration sample preparation unit and dedicated which is well below concentrations found to for food use, has been developed (26). This limit shelf-life of UHT products. luminometer gave good correlations with plate Lipase. Thermostable lipases produced by counts for raw milks in the range 1 x 104 to 1 psychrotrophic bacteria also limit the shelf-life x 108 cfu/ml (21, 26). of dairy products (29). Possibilities exist for Luminometers are also available that are development of bioluminescent lipase assays designed to accept a microtiter plate format, (ll), but, so far, these have not been widely which is particularly useful when large numapplied to detection of the enzyme in dairy bers of assays are performed. products. Alkaline Phosphatase. A chemiluminescent BACTERIAL BIOLUMINESCENCE substrate, AMPDD (4-methoxy-4-(3-phosphaphenyl)spiro(1,2-deoxetane-3,2yl)phenylphosAlthough ATP bioluminescent techniques phate), for alkaline phosphatase is available, offer fast enumeration of bacterial populations and its application for detection of the enzyme in a variety of products, these methods lack following heat treatment of milk has been specificity. The need for rapid detection systems that are specific for certain bacterial spedescribed (52). fl-Galacrosidase. &Galactosidase is used to cies is highlighted by recent concerns over the hydrolyze the lactose in milk so that it may be presence of potential pathogens in dairy

consumed by the lactose-intolerant population. A bioluminescent substrate, D-luciferin-0-@galactoside, for this enzyme has recently been 4) synthesized ( 3 and may find application in the dairy industry.

TABLE 3. Comparison of hygiene monitoring &ed

Q LE'<27 ATP3

put by ATP bioluminescence and conventional meth0ds.l

Counts

2-3 LPC 2 8 4 9 ATP 31 23

3 4 LPC 50-149 ATP

>4 LPC >150 ATP

121 101

(no. samples)

16 12

70
38

of this diluent). 3ATP Count (relative light units per assay).

lData o Bautista et al. (4). f 2Log plate count (swab was placed i 5 ml of broth, and counts were determined as colony-formingunits per milliliter n

Journal of Diy Science Vol. 76. No. 10, 1993 ar

SYMPOSIUM:EVALUATION OF MILK AND DAIRY PRODUCTS

3 123

products. Advances in molecular genetics have made bioluminescent detection systems for specific types of bacteria a reality. The applications of this technology to the food industry have been reviewed (3, 38).
Detection of Pathogens

The genes responsible for bacterial bioluminescence (lux genes) have been identified and cloned (24).The DNA carrying these genes can be introduced into host-specific 4) phages ( 4 . The phages do not possess the intracellular biochemistry necessary to express these genes, and, therefore, phages remain "dark". However, on transfer of the lux genes to the host bacterium during infection, transcription results in light emission that can be easily detected with a luminometer. The amount of light emitted is proportional to the numbers of bacteria infected, and the specificity of the assay is dependent upon the specific-

ity of the phage (Figure 3). The bacteriophage P22 is essentially specific for SulmoneIZu typhimun'um This phage has been engineered to contain lux genes, and, upon infection of a . culture of S typhimurium with this modified phage, as few as 1 x 102 bacteria could be detected in 60 min (39). Kricka (19) has claimed that as few as 10 E. coli cells could be detected in milk within 1 h using this technolow. Stewart (39) postulated that incorporation of lux genes into phages specific for enteric bacteria or even Pseudomonas spp. would result in an inexpensive, on-line hygiene test for dairy products that could be completed in 1 h or less. A near on-line detection of enteric bacteria using lux recombinant bacteriophage has been developed (18). Research is ongoing at University of Guelph to produce bioluminescence-based assays for the detection of mastitis-causing organisms and foodborne pathogens.

@
genesfrom luminescent bacteria

Lux

&tnfection of host

Host-specific phage

Phage transformation

by phage

Production of host with luminescent phenotype

Figure 3. Schematic representation of the lux gene-based bacteriophage assay for bacteria.
Journal of Dairy Science Vol. 76, No. 10, 1993

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Monltorlng Starter Culture Activity

GRIFFITHS

The lux genes have been introduced into strains of lactic acid bacteria, including Lc. lactis, Lactobacillus casei, and Lactobacillus pluntamm (1). In the presence of exogenous aldehyde, these gknetically modified organisms become luminescent. However, luminescence is curtailed in the presence of inhibitory agents such as antibiotics and bacteriophage. Lactic acid bacteria possessing a luminescent phenotype have been proposed for use to indicate the effective fermentation of dsllry products (39). Using a bioluminescent mutant of Lb. casei, Ahmad and Stewart (1) showed that concentrations of penicillin G as low as .03 pg/ml (.05 U/ml) could be detected in 30 min. The technique also enabled bacteriophage concentrations as low as 105/ml to be observed within 100 min. Bacterial bioluminescence techniques can also be applied to evaluate biocidal efficiency (3).
Other Applications of Luminescence

E. Stanley and L. J. Kricka, ed. John Wiley & Sons,

1992. Bacterial bioluminescence: application in food microbiology. J. Food Rot. 55:62. 4Bautista. D. A., L. Mclntyre, L. Laleye, and M. W. Griffiths. 1992. The application of ATP bioluminescence for the assessment of milk quality and factory hygiene. J. Rapid Methods Automation Microbiol. 1: 179. 5Bishop. J. R. and C. H. White. 1986. Assessment of dauy product quality and potential shelf-life-a review. J. Food Rot. 49:739. 6Bossuyt, R. 1978. Usefulness of an ATP assay technique in evaluating the somatic cell content of milk. Milchwissenschaft 33:1 1. 7 Cardwell, J. T., and Y. Sasso. 1985. The relation of adenosine tri-phosphate activity to titratable activity in t r e dairy cultures. J. Dairy Sci. 68(Suppl. 1): he 66.(Abstr.) 8Dabaji, M. 1982. Bioluminescence entre dans les Ateliers. Usine Nouvelle 41:138. 9Emanuelson. U., T. Olsson, 0. Holmberg, M. Hageltom, T. Mattila, L. Nelson, and G. Astrom. 1987. Comparison of screening tests for detecting mastitis. J. D i y Sci. 70880. ar 10Emanuelson. U., T. Olsson, T. Mattila, G. Astrom, and 0. Holmberg. 1988. Effects of parity and stage of lactation on adenosine triphosphate, somatic cell count and antitrypsin in cows m l . J. Dairy Res. 5549. ik 11 Griffiths, M. W. 1991. Rapid estimation of microbial numbers in dairy products using ATP technology. Page 29 in Physical Methods for Microorganism Detection. w. H. Nelson, ed. CRC Press, Boca Raton, 12G~iffiths.M. W., L. McIntP, M. Sully. and I. Johnson. 1991. Enumeration of bacteria in raw milk. Page 479 in Bioluminescence and Chemiluminescence, Current Concepts. P. E. Stanley and L. J. Kricka, ed. John Wiley & Sons, Chichester, Engl. 13 Griffiths, M.W.. and J. D. Phillips. 1986. The application of the pre-incubation test in commercial dairies. Aust. J. Dairy Technol. 41:71. 14Griffiths, M. W., and I. D. Phillips. 1989. Rapid assessment of the bacterial content of milk by bioluminescent techniques. Page 13 in Rapid Methods for Foods,Beverages and Pharmaceuticals. SOC.Appl. B&ol. Tech. Ser. No. 25. C. J. Stannard, S. B. Pettit, and F. A. Skinner, ed. Blackwell Sci. Publ., oxford, Engl. 15 Griffiths,M. W.. J. D. Phillips. 1990. Strategies to and control the outgrowth of spores of psychrotrophic Bacillus in dairy products. I. Use of naturallyoccuning materials. Milchwissenschaft 45:621. 16Holah, J. T. 1991. Monitoring the hygienic status of surfaces. J. Food Rot. 54:819. (Abstr.) 17 Kahru. A., and R. Vilu. 1991. The ATP bioluminescence technique in evaluating the bacteriological quality of dried milk. Page 495 in Bioluminescence and Chemiluminescence, Current Concepts. P. E. Stanley and L. J. Kricka, ed. John Wiley & Sons, Chichester. End: 18Kodhra. C. P., H. H. Crew, M. K. Wmson, C.E.D. R e s , and G.S.A.B. Stewart. 1992. Near on-line detection of enteric bacteria using lux recombinant bacteriophage. Paper presented SOC. Appl. Bacteriol.

cence and Chemiluminescence, Current Concepts. P. Chichester, Engl.

3 Baker, J., M. W. Griffiths, and D. Collins-Thompson.

Chemiluminescent-based probes are available for use with immunological and nucleic acid hybridization assays (2, 30). The sensitivity of these labeling methods in conjunction with low light level image analyzing systems offers exciting possibilities for the detection of pathogens in food samples.
CONCLUSIONS

PL.

The resurgence of interest in ATP bioluminescence and the dramatic improvements in reagent quality and instrument sensitivity have led to the availability of applications of direct relevance to the dairy industry (11). The most noteworthy are the hygiene monitoring assay and the new generation of milk bacteria enumeration kits. Developments in bacterial bioluminescence offer exciting possibilities for the detection of pathogens and spoilage organisms in foods (3, 38).
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39Stewart. G., T. Smith, and S. Denyer. 1988. Genetic engineering for bioluminescent bacteria. Food Sci. Technol. Today 3:19. 4OTodd E.C.D. 1990. Foodborne disease in Canada: a IO-year summary, 1975-1984. Health Prot. Branch, Health Welfare Canada, Ottawa, ON, Can. 41 Todd, E.C.D. 1991. Foodborne and waterborne disease in Canada, annual summaries 1985 and 1986. Health Rot. Branch, Health Welfare Canada, Ottawa, ON, Can. 42 Trivedi, S. H. Zarrin. E. Zomer, and S.Charm. 1992. . Shelf-life prediction of pasteurized fluid milk using the Charm I1 system. I. Food h t . 55:837.(Abs!r.) 43 Ugmva, N., Y.Vosny, G. K U ~ U Z O V ~E. &menand , tieva. 1991. Bioluminescent assay of &galactosidase using D-luciferin-0-&galactoside. Page 51 1 in Bioluminescence and Chemiluminescence. Current Concepts. P. E. Stanley and L. J. Kricka, ed. John Wiley & Sons, Chichester, Engl. . 44Ulitzer, S , and J. Kuhn. 1987. Introduction of lux genes into bacteria, a new approach for specific determination of bacteria and their antibiotic susceptibility. Page 463 in Bioluminescence and Chemiluminescence. New Perspectives. J. Scholmerich, R. Andreesen, A. Kapp, M. Emst, and W. G. Wood, ed. John Wiley & Sons, Toronto, ON, Can. 45Van Crombrugge, I., G. Waes, and W. Reybroek. 1989. The ATP-F test for estimation of the bacteriological quality of raw milk. Neth. Milk Dairy J. 43: 347. 46 Waes, G., and R. Bossuyt. 1981. A rapid method to detect postcontamination in pasteurized milk. Milchwissenschaft 36548. 47 Waes, G., and R. Bossuyt. 1982. Usefulness of the benzalkon-crystal violet-ATP method for predicting the keeping quality of pasteurized milk. J. Food Rot. 42928. 48Waes. G., R. Bossuyt, and J. Mottar. 1984. A rapid method for the detection of non-sterile UHT milk by the determination of the bacterial ATP. Milchwissenschaft 39:707. 49 Wang, L-L., and E. A. Johnson. 1992. Inhibition of Lisrcria monocyrogenes by fatly acids and monoglycerides. Appl. Envhn. Microbiol. 58:624. 5OWesthoff. D. C., and T. Engler. 1975. Detection of penicillin by bioluminescence. J. Milk Food Technol. 38537. 51 Williams, G. R. 1984. Use of bioluminescence in the determination of the antibiotic content of milk. J. Soc. Dairy Technol. 3740. 52 Zomer, E., S. Trivedi, and S. Charm. 1991. A rapid bioluminescence assay of alkaline phosphatase in m l ik and dairy products using the Charm II system. J. Food Prot. 54:813. Journal of Dairy Science Vol. 76, No. 10, 1993

Kricka, ed. John Wiley & Sons, Chichester. Engl. 36Sharpe, A. N., M. N. Woodrow, and A. K. Jackson. 1970. Adenosine tri-phosphate (ATP) levels in foods contaminated by bacteria. J. Appl. Bacteriol. 33:758. 37 Stanley, P. E. 1992. A survey of more than 90 commercially available luminometers and imaging devices for low-light measuxements of chemiluminescence and bioluminescence including instruments for manual, automatic and specialized operation for HPLC, LC, GLC and microtim plates. part 1: Descriptions. J . Biolumin. Chemilumin. 7:77. 38 Stewart, G.S.A.B. 1990. In vivo bioluminescence: new potentials for microbiology. Lett. Appl. Microbiol. 10:
1.