Rapid Measurement Of Meat Quality By Resazurin Reduction. I. Factors Affecting Test Validity*

Rapid Measurement Of Meat Quality By Resazurin Reduction. I. Factors Affecting Test Validity* R. A Holley', Susan M. Smith and A G. Kempton. .Department of Biology, University of Waterloo, Waterloo, Ontario. N2L3GI lpresent Address: Nucro- Tc(.-hntes Limited. 2.000 Ellesmere Road. Scarborough. Ontario.

Abstract The bacteriological quality of fresh meat can be determined rapidly by resazurin reduction. Test mixtures contained I ml of decanted supernatant from a Colworth Stomacher preparation of fresh or frozen meats added to 10 ml of a solution of 10% skim milk and 0.00055% (W IV) resazurin. Unsatisfactory meat reduced the dye within 3 h when tests were incubated at 30°C. The rate of dye reduction was affected by the number and type of organism. concentration of skim milk and by reagents affecting the redox potential or pH of the test medium. Of bacteria tested, Bacillus megalerium reduced resazuriil rapidly; Pseudomonas aereuginosa was slowest. This method is presently not applicable to ground meat because of reductive compounds in animal tissue released during grinding or blending. This problem is avoided in the analysis of meat cuts by the gentle action of the Stomacher.

Resume La qualite bacteriologique des viandes fraiches peut etre determinee rapidement iI ('aide de I'epreuve iI la resazurine. Les melanges etudies contenaient I ml de surnageant decante d'une preparation de viandes fraiches ou congelees dans un Stomacher Colworth, plus 10 ml d'une solution de lait ecreme i110% et de resazurine iI 0.00055% (P/V). Les viandes non acceptables reduisent la teinture en moins de 3h lorsque I'incubation est faite iI 30°C. La vitesse de reduction de la teinture a ete affectee par Ie nombre etle type d'organismes, la concentration du lait ecreme et par les reactifs qui influent sur Ie potentiel d'oxydo-reduction ou sur Ie pH du milieu. Des bacteries qui ont ete etudiees, c'est Ie Bacillus megalerium qui a reduit la resazurine Ie plus rapidement tandis que Ie Pseudomonas aereuginosa a ete Ie plus lent. Cette methode comme telle ne s'applique pas aux viandes hachees iI cause des substances reductrices qui sont liberees des tissus animaux lors du hachage et du melange. Ce probleme est evite dans I'analyse des coupes des viande grace iI I'action douce du Stomacher.

Introduction Meat packers routinely buy quantities of cut, boxed pork in either the frozen or refrigerated state. Usmg conventional methods, at least 48 h are required before the quality of the product can be established; but by then the meat has probably been processed and defective raw meat will already be causing economic loss in the finished product. An enzyme activity test capable of assessing the ~acteriological quality of meats within 3 h would essentIally eliminate such losses. Poor quality meats could then be termed "unacceptable" and returned to the sender under the terms of the Canadian Transport Tariff Bureau Association (1970). The most extensively studied enzymatic methods used to detect microbial activity include the methylene blue, the tetrazolium salts and the resazurin reduction assays; all of ~hich monitor dehydrogenase levels. The resazurin reductIon test has been used internationally for many years by the dairy industry as an indicator of hygienic and keeping qualities of raw milk (Thomas and Thomas, 1974a,b). Unfortunately, the conventional test-tube resazurin test does not produce results that can be confidently correlated with ~eef and

Can. tnsl. Food Sci. Technol. J. Vol. 10. No.3, July 1977

bacterial numbers in milk. In the Standard Methods for the Examination of Dairy Products (AP.H.A, 1967) it is stated explicitly that results must not be reported in terms of bacterial numbers under any circumstances. Otsuka and Nakae (1969) have shown that strips of resazurin-impregnated filter paper could be used to obtain good correlation between reduction time and bacterial numbers; but in 1974 the Government of Ontario replaced the resazurin reduction test with the Plate Loop method of obtaining direct counts of bacteria as the official test for milk quality (pers. comm. E. H. Smith, Director, Milk Industry Board, Ontario Ministry of Agriculture and Food). A wider application of the resazurin reduction method in the food industry has not won general acceptance. As early as 1939, Proctor and Greenlie found that resazurin reduction could be used to detect bacterial spoilage rapidly in ground meat, eggs, crab and lobster meat. Results with various vegetables, however, were not promising, possibly because the redox potential of the test medium was not stabilized. Ferguson et at. (1958) confirmed that resazurin dye reduction could not be used to detect bacterial spoilage of frozen vegetables because reductones present in the food generated inconsistent results. They also found that a preincubation period was necessary to stimulate resazurin reduction by bacteria in frozen foods. Wells (1959) developed a surface sampler for use in the resazurin test which in turn was used to estimate the shelf-life of poultry meat. He obtained a good correlation between bacterial numbers and reduction time; and, moreover, the reduction time for poultry meat nearing spoilage was 2 h. Wells (1959) also cited work carried out on precooked frozen foods, including meat pies, where resazurin reduction time was a good indicator of spoilage. P. Eckrich and Sons Inc., Fort Wayne, Indiana (Bull. Mt-9-21, 1971) have used cotton patches in a surface-contact resazurin reduction test to evaluate surface spoilage in meats. Unfortunately, a clear end point is not always obtained. Recently, very promising studies conducted in 2 different laboratories (Baumgart et at. 1975; Elmswiler et at. 1976) showed that resazurin reduction time did correlate with bacterial numbers in meat. Although Elmswiler et at. favoured the methylene-blue reduction test, both of these reports provide statistical evidence that the resazurin reduction test can be applied to meats and that bacterial numbers can be generated. In addition, spoiled meats reduced the dye significantly within 2 h. Unfortunately, these latter methods require the use of laboratory spectrophotometers and are not suitably portable. Purchasers of 153

large volumes of meat require not only an accurate and rapid method for the determination of meat quality expressed in terms of bacterial numbers; but also a portable method to use at any site in the plant and particularly on loading docks. The purpose of the present study was to develop the resazurin reduction test for use in accurately identifying grossly contaminated fresh meat within a 3 h period without recourse to complex laboratory equipment. This report contains the background information which had to be considered before the method could be evaluated within a meat-packing plant and implemented as a routine quality assurance procedure.

Materials and Methods Sample preparation. It is generally acknowledged that bacterial contamination of large pieces of meat is mainly on the surface; but the ICMSF (1974) has suggested microbiological specifications based on weight (number per g) without stipulating surface area. At the present time there is no industry-wide method for sampling that assures a consistent weight to surface area ratio. The development of such a method is beyond the scope of this work. Consequently, the irregular-shaped pieces of meat were sampled empirically. A 10 g sample of fresh or frozen meat, representing approximately 4 in 2 of outer surface area, was taken with a scalpel, weighed in a plastic petri dish and transferred to a sterile plastic bag, 18 cm x 30 cm, supplied with the' Colworth Stomacher (Model 400, A J. Seward, Blackfriars Road, London SEI 9UG). Sterile glass-distilled water (100 ml) was added and the meat homogenized in the Stomacher for 30 sec. The liquid was carefully decanted and stored on ice for use as the "meat homogenate". One-ml quantities were used directly in resazurin dye reduction tests. Appropriate dilutions were made in sterile glass-distilled water for use in bacteriological analysis. Resazurin reduction test. A standard solution of resazurin (Allied Chemicals, Morristown, N.J.) was made at double the concentration recommended in Standard Methods for the Examination of Dairy Products (AP.H.A., 1967); that is, one II mg dye tablet was dissolved in 100 ml of previously-boiled hot water. This solution was stored in a dark bottle at 4°C and used over a period of no more than 3 days. An indicator solution containing 0.00055% (W IV) resazurin and 10% (W IV) unsterile skim milk ("Vim" powdered skim milk, Silverwoods Limited, Kitchener, Ontario) was prepared immediately before use and brought to 30°C in a water bath. Ten ml of this solution was added at zero time to 1 ml of meat homogenate in a screw-capped test tube. The tube was inverted once, incubated at 30°C in a covered water bath and examined every 15 min for the first hour and at subsequent 30 min intervals thereafter. All tubes were inverted once every h. Resazurin reduction was measured visually in a box containing an 18 inch, 15 watt daylight type fluorescent lamp. The interior of the box was a neutral grey color. Resazurin was considered reduced when a purplepink color, equated to both the Lovibond disc number 3 and Munsell color standard 5P 7/4, was obtained (Thomas and Thomas, 1974a). Munsell color standards (Munsell

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Color Co., Inc., 2441 North Calvert St., Baltimore, Mdl 21218) 5PB 7/4, blue; IOPB 7/5.5, mauve; 5P 7/4, purpld pink and lOP 7/8, pink were sealed in glass tubes for USt as reference standards. To facilitate reading, the Lovibond comparitor with numerical scale and Munsell color stand. ard system were combined as shown in Table 1. Transi, tional colors between those noted above were recorded as half color units, eg., a color between blue and lilac was reo corded as 5.5. Any further numerical division was deemed to be inaccurate. Table I. Comparison of visual color with establish~d standards. Lovibond disc number

M unsell color standard

visual color grading

6 5 4 3 2 I 0

5PB 7/4

blue lilac mauve purple-pink pink vivid pink white

IOPB 7/5.5 5P 7/4 lOP 7/8

Bacterial numbers. Total counts were obtained from pour plates using all purpose trypticase agar (APT agar, Difco). Violet red bile agar (VRB), KF Streptococcus agar and lactobacillus selection agar (LBS) were obtained from Bioquest division of Baltimore Biological Laboratories to enumerate coliforms, Group D streptococci and lactobacilli respectively. Baird-Parker medium (BP, Difco) was surface spread to enumerate staphylococci. Drake's medium (Drake, 1966) was used to count pseudomonads. APT and LBS plates were incubated at 30°C for 48 h. The other media were incubated at 37°C for 48 h. Drake's medium was then incubated for a further 48 h at room temperature and the number of colonies growing on this medium were recorded after the dual incubation. Meat samples. Six different meat samples were obtained from J. M. Schneider Ltd (Kitchener). These were: fresh ham imported from the United States; fresh ham slaughtered by Schneider's Ltd.; fresh beef, bull; fresh beef, cow; frozen beef, bull, intended for use in Thuringer sausage; and frozen boneless pork. All meat samples were stored at 4°C in unsealed Nasco "Whirl-pak" plastic bags for 15 days. At intervals during this 15 day storage period, subsamples were analysed bacteriologically and by the resazurin reduction test in order to determine whether a correlation existed between bacterial numbers and reduction time.

Results and Discussion A) Standard Test System: Preliminary experiments wi~h resazurin-impregnated cotton and water-based medIa failed. The development of a test-tube resazurin reduction system in which milk was included: (a) permitted the use of accepted color standards for visual comparison of resazurin reduction (AP.H.A, 1967); (b) added stability to the test system since resazurin reduction to the proposed endpoint (Lovibond # 3, purple-pink color) is irreversible in milk; (c) permitted the use of a homogeneous test system; (d) satisfied the requirement that equipment be portable on a laboratory cart. Incubation and visual measure1. lnst. Can. Sci. Technol. Aliment. Vol. 10. No.3. July 1977

ritents could be completed without returning to the laboratory, while delivery vehicles were still at the plant. Milk sterilized by autoclave treatment was unsatisfactory. Resazurin was completely reduced within a few moments of its addition to autoclaved skim milk. Since membrane filtration of reconstituted powdered skim milk was also unsuccessful, freshly reconstituted skim milk powder was incorporated into the test system. Powdered milk containing less than 500 organisms per g was found to cause no color change in the test system after 3 h and could therefore be used routinely at a concentration of 10% (W I V). The use of "Vim" powdered skim milk does not constitute endorsement of this particular brand name. The pertinent specifications are that the brand used should not contain more than a few hundred bacteria per g, and should not contain inhibitors of dehydrogenase activity. It is suggested that reduction time by a known number of cells of a specific organism be used to detect inhibitory substances, should this be a matter of concern. A number of tests were made to determine the optimum amount of sample which could be added to the test system. The guideline used was that a satisfactory system should yield a purple-pink endpoint (Lovibond disc number 3) within 3 h when 10" or more organisms per g of sample were present. Meat is generally considered to be bacteriologically spoiled at this level (pers. comm. D. S. Clark, National Research Council, Ottawa). As shown in Table 2, the optimal amount of meat to be added to the system was 0.1 gm; or, more precisely, 1 Table 2. Selection of optimum amount of meat required to determine quality within the 3 h time period.

Sample frozen beef(bull) frozen pork fresh pork fresh beef (bull) fresh beef (cow) fresh bork fresh eef

Count per gram 1.7 x 10" 1.2 x 10" 5.9 x 10' 2.7 x 10 7 3.3 x 10' 2.0x 10"' 7.9 x 10'

Amount of meat per test 0.01 g 4.0* 3.0 4.0 > 6.0 > 6.0 > 6.0 >6.0

0.1 g

0.3 g

0.5 0.25 0.75 4.0 >6.0 >6.0 6.0

< 0.25 < 0.25 < 0.25 0.25 0.25

*hours to end point of Lovibond disc number 3.

ml of the 1: 10 meat homogenate from the Stomacher. At this concentration 108 organisms per g reduced resazurin to the desired endpoint within the critical 3 h time limit, whereas the sample containing 10 7 organisms per g required 4 h. When the equivalent of 0.3 g of meat was added to the test system, samples with as few as 10 5 bacteria per g reduced resazurin to Lovibond disc number 3 within 0.25 h; and 0.01 g samples of spoiled meats failed to reduce the dye within 3 h. Determination of the extent of non-enzymatic reductive capacity of meats was carried out using several types of steam-sterilized samples, including Kam luncheon meat (Canada Packers Ltd.) combined with either 0.2% K 2 HP04 or 0.2% KN0 3 in the test system (Table 3). Sterilized meat at the optimal level of 0.1 g per tube produced a minimal change in the color of the dye. The prospect that the nonenzymatic reduction of resazurin would yield a steady background of Lovibond disc number 5 throughout the test period was encouraging. Can. Inst. Food Sci. Techno!. J. Vol. 10. No.3, July 1977

Table 3. Resazurin reduction by sterile meats. Grams of meat per test

Lovibond Compari tor Reading 2h

0.05 g autoclaved Kam 0.1 g autoclaved Kam 0.1 gautoclaved Kam + 0.2% K,HPO, 0.1 gautoclaved Kam + 0.2% KNO" 0.1 gautoclaved pork 0.1 gautoclaved beef

6.0 5.0 5.0 5.0 5.0 5.0

3h 6.0 5.0 5.0 5.0 5.0 5.0

Although a volume of the meat homogenate from the Stomacher equivalent to 0.1 g of meat per test was satisfactory for samples of both fresh and frozen beef and pork (Table 2), exploratory tests showed that this same concentration of ground meat or meats comminuted with a Waring Blendor containing as few as 10 5 organisms per g reduced the dye to Lovibond disc number 3 within 2 h. It was believed that this was due to the release of reductones from the meat tissue during grinding or comminution. Originally, the Stomacher was chosen for this work because it was portable and compact; and Sharpe and his coworkers (1972,1976) have shown repeatedly that there is no significant difference between bacterial counts obtained when the Stomacher is used in place of blending. Extraction of bacteria from meat by the Stomacher does not cause extensive disruption of the animal tissue, and hence the use of the Stomacher proved to be vital to this application of the resazurin test. Even sterile beef extract had a powerful reducing capacity as shown in Table 4. More than 0.00 1 g of sterile beef extract reduced resazurin (blue) to resorufin (pink) without changing the pH from its initial 6.9. Ascorbic acid at a level of 0.0 1 g also changed the color of the dye rapidly but this was the result of a change in pH from 6.9 to

5.7. Table 4. Resazurin reduction by sterile beef extract. grams/II ml (final volume) .00 I g beef extract .005 g beef extract .01 g beef extract .02 g beef extract

Lovibond comparitor reading 2 hours 3 hours 6.0 3.0 2.5 1.0

6.0 3.0 2.5 1.0

Several attempts were made to buffer the redox capacity of the test system so that it could be used with ground meat. Potential reduction retardants were added to the basic medium and then the system was challenged with 0.001 to 0.020 g of beef extract. The following compounds did not affect the rate at which sterile beef extract caused resazurin reduction: dibasic potassium phosphate, 0.1% (W IV); casamino acids, 1% (W IV); sodium thiosulfate, 1% (W IV); cystine, 0.01% (W IV) and an unsaturated fatty acid (crotonic acid), 5% (W IV croton oil). On the other hand, potassium nitrite, 1-4% (W IV); glycerol 20-50% (WI V); and sodium nitrite 2-4% (W IV) did retard the inherent reductive capacity of beef extract. Sodium nitrite was the most effective, but at the necessary level it was lethal to Pseudomosas aeruginosa when this organism was included in the text mixtur,e. A modification of this technique that would permit its use for examining ground meats was not found.

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B) Variation in the rate of resazurin reduction: It was expected t~at different organisms would reduce resazurin at vastly dlffere~t rate~, and that the resazurin test might prove .to be lffip~actIcal be~ause of the heterogeneity of bactenal populatIOns on vanous meats. The ability of several pure cultures to reduce resazurin was examined and the results are presented in Table 5. B. megaterium reTable 5. Difference in resazurin reduction rate of bacterial species. cells per test 10" 10' time (hr) to dye reduction

Organism

0.25 3.0 0.5 0.5 0.5 1.5

Bacillus megaterium Pseudomonas aeruginosa Escherichia coli Staphylococcusaureus Lactobacillus p Ian tarum Streptococcusfaecalis

1.0

> 4.0

> >

3.0 1.5 4.0 4.0

duced the dye most rapidly whereas cells of P. aeruginosa were the .slowest. Simi[ar results were obtained by Greene and Jamison (1959) who used a similar test system. They a~so found that S. lactis reduced resazurin just as fast as did B. ~ubtilis, whereas Table 5 indicates that S. faecalis was qUite slow to reduce resazurin. The addition of 0.05% IV) peptone to the tests slighly accelerated dye reductIOn by the more. fastidious L. plantarum and S. faecalis cultures but had httle effect on the rate of resazurin reduction by the other cell types. When Greene and Jamison (1959) halved the cell concentrations and mixed either B. subtilis or S. lactis with P. aeruginosa, the mixed culture reduced resazurin as fast as the same total numbers of B. subtilis or S. lactis. It was found during the current study that a mixture of 5 x 10" cells of B. megaterium and 5 x 10" cells of P. aeruginosa red.uced resazurin almost as fast as did 10' cells of B. megaterIum. Although Greene and Jamison (1959) concluded that the~e was some ~ynergistic activity responsible for more rapI~ dy~ reductIOn by mixed cultures, it is more likely that m mIXed cultures the rate of resazurin reduction is determined. simply by the number of rapid dye reducers present. If thiS latter comment were true, then it could be concluded that the presence of more than double the number o.f r~pid resazurin ~educers would be necessary before any slgmficant change m the rate of resazurin reduction could be noted. .In view of t~e effe~t o~ peptone on the rate of dye reduction by ce~~am species, It was necessary to simulate ac~ual test conditIOns as closely as possible. The effect of addmg the eqUivalent of 0.1 g of meat prepared with the Sto~acher to pure bacterial cultures during resazurin reduction was exammed and these results are presented in Table 6. It was apparent that the addition of meat extract

(yv

Table 6. Effect of meat addition on resazurin reduction by pure cultures.

Organism B. megaterium E. coli P. aeruginosa S. aureus L. plantarum S·faecalis

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concentration per test 10' 10' 10' 10'

10'

H»

Time (h) required to reduce resazurin to Lovibond # 3 pork beef no meat 0.5 0.5 0.5 0.5 0.5 0.5

0.5 0.5 0.5 0.5 0.5 0.5

0.5 0.5 0.5 0.5 1.5 0.75

f~om either fresh beef or pork stimulated resazurin redu~ tIon by L. plantarum and S. faecalis as did the addition oj ~.05% (W IV) peptone. Added meat did not have an inhib.i ItOry effect on the rate of dye reduction by any of the a~ove cu~tures. The pork used in these experiments COn. tamed 10' cells per g; the beef contained 10" cells per g: These background counts were not considered higJ\ e.nou.gh t? have signific~n~ly affected the rate of dye reductIon m ml~ tubes ~o~tammg 10 8 -10" of the test organisms. At thiS sta!?e 10 I~ d~vel?pment, !t was acknowledged that only a major vanatlOn 10 the mlCfoflora of different meat products could affect the validity of the proposed test. C) Bacterial growth in refrigerated meats: Analyzing 6 meats .stored at 4°C for 15 d.ays with 6 media produced 36 bactenal growth curves which revealed all the variables that were anticipated. They need not be presented in de. tail. The initial total counts on APT agar ranged from less than 50 per g (fresh beef, cow) to more than 10" per g (fro. zen pork for Thuringer sausage). The total counts after 15 days ranged from less than 10" per g (fresh beef, cow) to more. than 10" per g (fresh beef, bull, frozen beef, bull for Thun~~er sausage). Coliform counts on VRB agar, lac· tobactlli on ~BS agar, Group D streptococci on KF agar, staphylOCOCCI on B-P agar and pseudomonads as counted ?n Drake's .medium were present in various numbers but 10 no consistent pattern. The growth curves differed in shape, ~arel>, exhibiting a typical exponential curve. It IS Widely accepted that the predominant flora in meats that are stored at 4°C with access to air are non spore-forming.' gram-negative rods of the pseudomonad, Mora~ella-Acmetobactergroups (pers. comm. D. S. Clark). In thIS work, the sum of the numbers of coliforms, staphylococci, lactobacilli, Group D streptococci and pseudomonads capable of growing on Drake's medium never ex~eeded 3~ ?f the total count. These latter groups, in spite of vanatlOns on any meat sample, would not be expected to exert any influence on the resazurin test as a measure of the total bacterial load. D) Bacterial numbers and resazurin reduction time: The resazurin test was conducted on all meat samples after 2, 7 and 1.4 days storage at 4°C. Comparisons between the resazunn test and bacterial numbers were also collected from useful data obtained during the preparation of Tables 3 and 4. There was no relationship between the res~zurin reduction tim~ and .the nu.mber of any specific species; ~ut 30 comparISons mvolvmg the total number of bacten~ produced the data presented in Figure 1. With these preliminary results it was decided to assesS the resazurin test using a two-class attributes plan (Haccept'.' or "reject") as described by the International CommiSSIOn on Microbiological Specifications for Foods (lCMSF, 1974). The key coordinates in Figure 1 represent the 3 h m~ximum. ~ime permitted by law to make an accept or reject decISIOn, and 10" bacteria per g as m (the value repr~senting the dividing line between acceptable and defective sample units). Applying this analysis to Figure 1, 13 samples would be rejected by both resazurin reduction. in 3 h or less and the standard plate count. The lo~er nght quadrant of Figure 1 contains 14 samples which would have been accepted by both the resazurin test and a direct count. A single sample of fresh ham containJ. lnst. Can. Sci. Technol. Aliment. Vol. 10. No.3. July 1977

accepted by the resazurin test although they contained more than 10 6 bacteria per g (upper right quadrant). The aberrant result with the beef sample was not explained; but the two unsatisfactory results with fresh ham appeared to be laboratory artifacts. This product was decomposed by endogenous enzymes before the bacteria numbers increased significantly. Under commercial conditions, it would have been rejected without analysis.

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HOURS TORESAZURIN REDUCTION Fig. I. Preliminary evidence of a relationship between bacterial numbers on meat and resazurin reduction time.

ing less than 106 bacteria per g would have been rejected by the resazurin test (lower left quadrant). One sample each of fresh ham and fresh beef (bull) would have been

Can. Inst. Food Sci. Techno!. J. Vo!. 10. No.3. July 1977

Acknowledgments This work was supported by National Research Council of Canada grant number P-7504 (Project Research Applicable in Industry). The authors wish to express their appreciation to Dr. D. S. Clark for his advice and guidance.

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In spite of variation in the number and type of organisms found during the monitored spoilage of several meats, there appears to be a relationship between bacterial numbers and the rate of resazurin reduction using the test-tube resazurin test as developed. The method is performed by adding 1.0 ml from the 1: 10 dilution of meat as prepared in the Colworth Stomacher to 10 ml of an indicator solution containing 10% (W/V) skim milk and 0.00055% (WI V) resazurin. The resazurin is said to be reduced when it visually compares to Lovibond disc number 3. A correct decision to "accept" or "reject" in 27 of 30 determinations provided sufficient impetus to proceed with further evaluation of this test in the laboratories of J. M. Schneider Ltd., Kitchener, Ontario.

American Public Health Association 1967. Standard Methods for Analysis of Dairy Products. 12 Ed. A.P.H.A.. lnc.. 1740 Broadway. New York. N.Y. 10019. Baumgart. A. Portner and G. Lassak 1975. Schnellmethode lur Bestimmung dec aeroben Bamtkeimzahl (Lebendkeimzahl) auf Frischfteisch mit Hilfe photometrisch meBbarer Extinktionsanderungen beim Resazurintesl. Die Fleischwirtschaft 55:969. Canadian Transport Tariff Bureau Association 1970. Class Tariff No. I-C. rule 46. Canadian Transport Tariff Bureau Association. 555 Dixon Rd .. Rexdale. Ontario. Drake. C. H. 1966. Evaluation of culture media for the isolation and enumeration of Pseudomonas aeruginosa. Health Lab. Sci 3: 10. Elmswiler. B. S.• A. W. Kotula. C. M. Chesnut and E. P. Young 1976. Dye reduction for estimating bacterial counts in ground beef. Appl. and Environ. Microbiol. 31 :618 . Ferguson, W. E., A. R. Yates and A. H. Jones 1958. Resazurin reduction by microorganisms in fresh and frozen vegetables. Food Technol. 12:641. Greene. V. W. and R. M. Jamison 1959. Influence of bacterial interaction on resazurin reduction times. J. Dairv Sci. 42: 1099. lCMSF 1974. Microorganisms in Foods Vol. II. Sampling fo! Microbiological Analysis:Principles and Specific Applications. Univ. ofToronto Press. ToronlO and Buffalo. Otsuka, G. and T. Nakae 1969. Resazurin test paper method for determining the sanitary quality of raw milk. J. Dairy Sci.. 52:2041. Proctor. B. E. and D. C. Greenlie 1939. Redox potential indicators in quality control of foods. I. Correlation of resazurin reduction rates and bacterial plate counts as indices of the bacterial condition of fresh and frozen foods. Food Res.. 4:441. Sharpe. A. N. and A. K. Jackson 1972. SlOmaching: a new concept in bacteriological sample prepa· ration. Appl. Microbiol. 24: 175. Sharpe. A. N. and G. C. Harshman 1976. Recovery of Clostridium perfringens. Staphylococcus aureus and molds from foods by the Stomacher: effect of fat content, surfactant concentration. and blending lime on the count. Can. Inst. Food. Sci. Technol. J. 9:30. Thomas. S. B. and B. F. Thomas 1974a. The development of dye reduction tests for the bacteriological grading of raw milk-part 2. Resazurin test( I). Dairy Indus.• 39:31. Thomas. S. B. and B. F. Thomas 1974b. The development of dye reduction tests for the bacteriological grading of raw milk-part 2. Resazurin test(2). Diary Indus.. 39:59. Wells. F. £.1959. Resazurin reduction tests for shelf life estimations of poultry meats. Food Technol. 13:584. Received September 7. 1976

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