Evaluation of a Rapid Method for the Quantitative Estimation of Coliforms in Meat by Impedimetric Procedures

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1980, p /80/ /07$02.00/0 Vol. 39, No. 3 Evaluation of a Rapid Method for the Quantitative Estimation of Coliforms in Meat by Impedimetric Procedures S. B. MARTINSt* AND M. J. SELBY Bactomatic, Inc., Palo Alto, California A 24-h instrumental procedure is described for the quantitative estimation of coliforms in ground meat. The method is simple and rapid, and it requires but a single sample dilution and four replicates. The data are recorded automatically and can be used to estimate coliforms in the range of 100 to 10,000 organisms per g. The procedure is an impedance detection time (IDT) method using a new medium, tested against 131 stock cultures, that markedly enhances the impedance response of gram-negative organisms, and it is selective for coliforms. Seventy samples of ground beef were analyzed for coliforms by the IDT method and the conventional three-dilution, two-step most-probable-number test tube procedure. Seventy-nine percent of the impedimetric estimates fell within the 95% confidence limits of the most-probable-number values. This corresponds to the criteria used to evaluate other coliform tests, with the added advantage of a single dilution and more rapid results. Growth of microbial populations results in changes in electrical impedance of the culture medium. The Bactometer (Bactomatic, Inc., Palo Alto, Calif.) microbial monitoring system is a sensitive instrument that monitors these changes during the course of microbial growth. Culture volumes from 1 to 100 ml can be tested in appropriately sized containers. Each container is monitored separately in reference to the uninoculated medium, and the proportional impedance change is automatically recorded on strip charts every 3 s; the change may also be analyzed with a built-in computer (2, 4). Typically, impedance changes become detectable when the bacterial density reaches 105 to 106 organisms/ml and then continue at a rate and for a period depending upon the bacterial species and the medium. The detection time provides an approximate measure of the number of organisms in the initial inoculum (2, 4). This impedimetric procedure offers a simple and rapid method for estimating total bacterial densities in frozen vegetables (16), urine (3), and milk samples (5). We report here on the selective estimation of coliforms in ground meat by impedimetric procedures, using a medium especially developed for this purpose. The subject of coliform standards for ground meat is of much current interest. Six states have proposed guidelines for acceptable limits of coliform densities in this food product (22). Other states are seriously considering such guidelines (7, 22). The proposed limits range from 50 to t Present address: San Antonio, Palo Alto, CA ,000 coliforms/g (22). Both the limits and the necessity of any microbiological standard for ground meat have been debated (6, 7, 9, 13, 14, 20, 23). Standard methods in use today for estimating coliforns are tedious and time consuming. The availability of a simple and rapid method would greatly facilitate the acceptance of, and compliance with, the coliform standards by the industry (13, 23). MATERIALS AND METHODS Impedance measurements. The impedance-monitoring instrument used in this study was the Bactometer 32 microbial monitoring system described fully elsewhere (2, 4). The system was operated at 2 KHz, gain 9, and the impedance changes were continuously recorded on a strip chart recorder at 2.54 cm/h. The time at which the impedance change becomes detectable is called the detection time (DT). For this study, DT was defined as the time when the rate of impedance change reaches two strip chart channel widths per 2 h. This rate is equivalent to an impedance change of 1.6% in 2 h. An impedance response was considered to be positive for coliforms if detection by the above criterion was obtained within 20 h of inoculation. h Impedimetric responses after 20 h were scored as negative. Medium. The composition of the selective medium developed for the detection of coliforms by the impedimetric method is as follows: tryptone, 20 g; lactose, 5 g; L-asparagine, 1 g; Triton X-100, 4 ml; sodium dihydrogen phosphate, 7 g; and distilled water, 1,000 ml (ph 6.3). The medium was dispensed in 100-ml amounts in bottles and sterilized by autoclaving at 121 C (15-lb steam pressure) for 15 min. Before use, freshly prepared potassium sulfite and sterile filtered novobiocin were added to the medium 518

2 VOL. 39, 1980 to give a final concentration of 0.035% K2SO3 and 3 jg of novobiocin per ml. The medium was distributed aseptically in 0.5-mnl amounts in the Bactometer module wells. Disposable modules with eight sample and eight reference wells, each provided with vertical stainless-steel electrodes (4), were used in this study. Each well had a nominal capacity of 2.0 ml. Stock cultures. A total of 131 stock cultures, of which 114 were gram-negative and 17 were gram-positive organisms, were used in this study. Members of the Enterobacteriaceae, other than Escherichia coli, were classified as coliforms only if they fermented lactose with the production of gas. Thus, of the 13 strains of Enterobacter agglomerans used in this study, 4 were termed coliforms by the above criteria, whereas 9 were noncoliforms. For screening the selectivity of the new medium for coliforms, overnight Trypticase soy broth (TSB; BBL Microbiology Systems, Cockeysville, Md.) cultures were diluted 10-7 with Butterfield phosphate water (BPB) to give an approximate concentration of 200 organisms/ml. The diluted cultures were inoculated in 0.5-ml amounts into 0.5 ml of the medium contained in the module wells. Testing was done in duplicate at 35 C for 24 h. Meat sampling. A total of 70 samples of ground beef were obtained randomly from local supermarket shelves and butcher shops over a period of 3 months. The samples were examined within 1 h of collection. A 10-g portion of each meat sample was weighed into sterile polyethylene bags (18 by 30 cm), and 190 ml of BPB containing 0.05% tryptone solution (BPB+) was added to give a final dilution of 1:20. The Colworth Stomacher 400 was used to prepare the samples. Each sample portion was stomached for 1 min. Subsequent dilutions were made in BPB+. Impedimetric most probable number (IMPN). Three sample dilutions (1:200, 1:2,000, and 1:20,000) were tested, each in quadruplicate. The sample dilutions were inoculated in 0.5-ml amounts into module wells containing an equal amount of the medium and incubated at 35 C for 24 h. The inoculated wells were scored as positive or negative for coliforms in terms of the detection criteria described earlier. From the data obtained, the IMPN per gram was calculated by using a four-tube MPN table. The table was computer generated, using the equations developed by Halvorson and Ziegler (15). Conventional MPN. Coliforms were also estimated by the standard two-step American Public Health Association (APHA) method (1) by using lauryl sulfate tryptose broth at 35 C for 48 h for the presumptive test and brilliant green bile 2% at 35 C for 48 h for the confirmed test. Four tubes, each containing 10 ml of medium, were used per dilution. The dilutions and the volume inoculated into lauryl sulfate tryptose broth were the same as those used in the IMPN test. VRBA counts. Coliform plate counts were made on violet red bile agar (VRBA) by the standard APHA pour plate technique (1) by using two or more serial decimal dilutions of the meat sample. The plates were overlaid before incubation at 35 C for 24 h. All dark red colonies 0.5 mm or more in diameter were counted (1). RAPID ESTIMATION OF COLIFORMS IN MEAT 519 RESULTS Selective medium for impedimetric estimation of coliforms. Conventional methods for enumerating coliforms are heavily dependent on the differential properties of the bacteriological medium used, for example, the production of acid or gas. Neither of these differential characteristics provides a parameter than can be measured by the impedimetric procedure. Application of the impedimetric method for the quantitative estimation of coliforms was therefore critically dependent upon the development of a medium which selectively amplifies the impedimetric responses of coliforms. The medium described here achieves this, first through the incorporation of asparagine and phosphates, which preferentially enhance the impedimetric responses of gram-negative bacteria and inhibit the impedimetric responses of gram-positive bacteria, and second through the use of K2SO3 (0.035%) and novobiocin (3 jig/ml), which at ph 6.3 are inhibitory to most Proteus, pseudomonads, and other noncoliform organisms. The specific amplifying effect of asparagine on the impedimetric responses of gram-negative bacteria is observed in the new medium described above as well as in the more complex media suitable for the growth of the more fastidious gram-positive organisms. Figure 1 illustrates the amplification of impedimetric responses of selected gram-negative bacteria (E. coli and Pseudomonas aeruginosa) by incorporation of asparagine in TSB and standard methods broth (SMB); in contrast, asparagine had little effect on the impedimetric responses of the gram-positive bacterium (Staphylococcus aureus) and yeast (Candida albicans) tested. Furthermore, as shown in the above figure, the inclusion of phosphate salts in SMB-asparagine depressed the impedance responses of S. aureus and C. albicans. A variety of antibiotics and other inhibitors (e.g., vancomycin, methicillin, crystal violet, and deoxycholate) were next tested to determine the optimal conditions for the selective detection of coliforms. The impedimetric responses due to pseudomonads were most satisfactorily controlled by the use of K2SO3 at ph 6.3. At ph 6.0, K2SO3 was inhibitory to most of the organisms, including coliforms. As previously reported by other investigators (11, 17-19), novobiocin eliminated the responses of most Proteus strains. The productivity and selectivity of the medium as finally defined by the above-mentioned preliminary studies was determined first with a screen of 131 pure cultures of various bacteria (Table 1).

3 520 MARTINS AND SELBY c z u S S TIME IN HOURS FIG. 1. Impedimetric response curves of E. coli, P. aeruginosa, S. aureus, and C. albicans in five media: (1) TSB; (2) TSB with 0.1% asparagine (TSB-ASN); (3) SMB; (4) SMB with 0.1% asparagine (SMB-ASN); and (5) SMB-ASN with 0.5% phosphate salts (SMB- ASN-PO4). Approximately 10i organisms were inoculated into each Bactometer module well, and the experiment was performed in duplicate. Similar response curves were obtained with an inoculum level of 102 organisms; the onset of impedance change was 2 to 4 h later. Of the 17 gram-positive bacteria tested in the final medium, none gave a positive impedimetric response. In fact, none gave a positive impedimetric response when tested in the same basal medium in the absence of K2SO3 and novobiocin. Given the potent antimicrobial effect of novobiocin on gram-positive bacteria (11), it is unlikely that gram-positive bacteria will give falsepositive responses in this medium. Among the 114 gram-negative bacteria screened, 67 were coliforms and 47 were noncoliforms. Eighty-eight percent (59/67) of the coliforms gave positive impedimetric responses, whereas 21% (10/47) of the noncoliforms gave positive responses. The positive responses among the noncoliforms were obtained mainly with Pseudomonas aeruginosa and nonaerogenic strains of Enterobacter agglomerans. The medium described thus appears to be specific for gram-negative bacteria with selectivity for the coliform group of bacteria. The selectivity of the medium could conceivably be affected by the binding of S02 (the presumed APPL. ENVIRON. MICROBIOL. inhibitor released from K2SO3) and novobiocin to food constituents. To test this possibility, roast beef was cut aseptically to obtain central portions free of surface contamination. The meat was then homogenized as described in Materials and Methods and diluted in the medium to give a final concentration of 1:20. This homogenate was then inoculated with 30 different strains of bacteria. The selectivity of the medium was unaffected by the presence of meat constituents in the concentration tested (data not shown). Estimation by IMPN. The MPN method was used on 70 meat samples to compare the estimation of coliforms by the impedimetric procedure with estimation by the standard test tube procedure (Fig. 2). The coefficient of correlation between the MPN and the IMPN determinations was 0.68, with 77% of the IMPN values falling within the 95% confidence limits of the corresponding MPN values. Fifty-two of the meat samples were examined in duplicate by the impedimetric procedure. The coefficient of correlation between the duplicate IMPN values was Estimation of coliforms by the impedance detection time (IDT) method. In previous reports using the Bactometer microbial monitoring system, it had been shown that the DT provides an approximate measure of the total microbial burden in several types of food (5, 16). In the present study, the detection criteria were specifically defined to achieve a high degree of selectivity for the coliform group of organisms by excluding weak responses or late responses due to inadequately inhibited noncoliforms. For example, impedimetric responses occurring after 20 h of incubation were treated as negative. In computing the average DT, where one or more of the module wells was negative, a suitably weighted detection time for the negative wells had to be assigned. The DT assigned to the negative wells was 23 h. The 23-h DT is about two generation times away from the 20-h detection limit for positive wells, and corresponds to a hypothetical bacterial load roughly one-fourth that required to give DT of 20 h. It can be calculated from the MPN tables that when only one-fourth of the inoculated wells are positive, the average bacterial concentration in the inoculum is about one-fourth of the minimum required to give a positive response. Figure 3 gives the plot of DT (at 1:200 dilution) against IMPN per gram. The coefficient of correlation was From the regression equation derived from the above data, coliform densities were calculated from the IDT. The IDT estimates ranged from to 21,000/g. The coefficient of correlation between the IDT estimates and the MPN per gram determined by

4 VOL. 39, 1980 TABLE 1. RAPID ESTIMATION OF COLIFORMS IN MEAT 521 Impedimetric responses of the organisms tested in the new coliform medium containing potassium sulfite and novobiocin as inhibitors Impedimetric responseb Organim No. of strains tested Positive Negative Coliforms Escherichia coli Enterobacter aerogenes Enterobacter agglomerans Enterobacter cloacae Klebsiella pneumoniae Serratia liquefaciens Serratia rubideae Total (88%) 8 (12%) Noncoliforms Shigella Proteus Pseudomonas aeruginosa Pseudomonas (others) Enterobacter agglomerans Serratia rubideae Citrobacter Arizona Aeromonas Gram positivesc Total (15%) 54.5 (85%) a Organisms other than E. coli are classified as coliforms only if they fermented lactose with the production of gas. In the coliform group, one strain of E. coli did not produce gas in brilliant green bile 2%. In the noncoliform group, one strain of P. aeruginosa produced gas in brilliant green bile 2%. beach organism was tested in duplicate and was characterized as positive or negative by the detection criteria described in Materials and Methods. An organism positive in only one of the two duplicate wells was given a value of 0.5. 'The 17 gram-positive organisms tested were: Staphylococcus aureus (five); Streptococcus faecalis (two); group D streptococci (four); Bacillus megaterium (three); B. polymyxa (one); B. pumilus (one); and B. subtilis (one) j * * 0 * * ~~~.* * *0. m. *0/ *. 0 LOG 1 CONVENTIONAL M*N/G FIG. 2. Scattergram of IMPN per gram as determined by the impedimetric procedure versus MPN per gram as determined by the standard test tube method. The solid line is the least-squares linear fit to the IMPN data. Plotted data were obtained from 70 samples of meat, 52 of which were tested in duplicate by the IMPN method IMPEDANCE DETECTION TIME (HOURS) FIG. 3. Scattergram of IMPN per gram as determined by the impedimetric procedure versus the detection time in the impedimetric procedure at the sample dilution of 1:200. The solid line is the leastsquares linear fit to the IMPN data. Plotted data were obtained from 70 samples of meat, 52 of which were tested in duplicate by the IMPN method.

5 522 MARTINS AND SELBY the conventional method was 0.69, with 79% of the IDT estimates falling within the 95% confidence limits of the corresponding conventional MPN values. Table 2 compares the data obtained by the two methods. The data obtained by each method are grouped into three classes corresponding to coliform counts of, 100 to 999, and 21,000/g. The tabulated data demonstrate a remarkable uniformity in the distribution of coliform densities as determined by the conventional MPN test and the IDT method. This distribution is very similar to that reported recently by Fowler et al. (10) from an analysis of 810 samples of ground meat procured from throughout the United States. DISCUSSION Application of impedimetric procedures for the selective detection of bacteria has not been possible hitherto due to the lack of selective and differential media. The study described here suggests that impedimetric responses of bacteria can be selectively controlled by the use of enhancers (e.g., asparagine) and inhibitors. Thus, the development of suitable media could greatly extend the usefulness of impedimetric procedures. This study evaluated the impedance method for the selective detection and estimation of coliforms. Geldreich (12) has proposed that the membrane filter procedure for estimating coliforms in water and sewage be validated if 80% of the membrane filter counts fall within 95% confidence limits of the corresponding MPN values. In our study, 77% (IMPN) and 79% (IDT) of the impedimetric estimates fell within the 95% confidence limits of the corresponding MPN values. This degree of correlation is particularly striking in view of the inherent variability in the MPN techniques (24). The presence of coliforms in foods does not have quite the rigorous significance attached to their presence in water (11, 20). These results therefore warrant considera- TABLE 2. Distribution of coliforms in meat: comparison ofmpn and IDT Conventional IDT (estimated organisms/g) (MPN/g) >1,000 Total (67.5)a (27.5) (5) (5) (55) (40) >1, (5) (19) (76) Total Numbers in parentheses are percentages. tion of the impedimetric method as an alternate procedure for estimating coliforms in foods. An analysis of all IDT estimates that fell outside the 95% confidence limits of the MPN values is presented in Table 3. The results show that in 13 of 15 instances when the impedimetric estimates gave coliform densities significantly higher than that obtained by the test tube method, the meat samples had excessive VRBA counts. Further, the VRBA counts varied from 1,400 to 160,000/g, and the ratio ofvrba counts to MPN ranged from 12.5 to 1,500. These results therefore suggest that gram-negative, nonaerogenic, lactose-fermenting organisms, presumably members of the Enterobacteriaceae, were responsible for the false-positive results obtained with the 70 meat samples analyzed in this study. The results reported in Table 3 also show that the 15 false-positive estimates obtained with the IDT method were balanced almost exactly by 11 false-negative results. Similarly, the data presented in Table 2 show a remarkable uniformity in the distribution of coliform densities as deter- TABLE 3. Comparison ofmpn with IDT and VRBA countsa Conventional IDT method Serial no. method (estimated VRBA (MPN/g) organisms/g) (CFUh/g) ,700 66, ,100 1, , , , , ,440 8,700 18, ,440 13,000 18, , , ,900 30, , ,500 9, , ,000 4, , , , , , , , , , , , ,200 APPL. ENVIRON. MICROBIOL ,800 7,000 4,200 20,000 1, ,000 11,000 58,000 2,000 a Data are drawn from IDT values that fell outside the 95% confidence limits of the corresponding MPN values. b Colony-forming units.

6 VOL. 39, 1980 mined by the MPN method and the IDT method. The results analyzed in Tables 2 and 3 therefore lead to the expectation that the MPN method and the IDT method would each reject roughly the same percentage of meat samples, irrespective of the coliform standard prescribed or the sampling plan adopted. These results also suggest that both methods detect primarily bacteria belonging to the Enterobacteriaceae and that the bacterial strains detected by either method have equal probability of occurrence within the meat samples examined. This notion is supported by the results of screening of the medium used in this study with various bacteria (Table 1). Of the 30 bacterial strains belonging to genera other than Enterobacteriaceae, only some strains of P. aeruginosa (three of seven) gave positive responses in the impedimetric test. On the other hand, of the 13 strains of E. agglomerans screened, the impedimetric method detected 5 and the test tube method detected 4, whereas only 2 were detected by both methods. It cannot be said that only aerogenic strains of E. agglomerans have sanitary significance. In the individual samples, the discrepancies between the standard test tube method and the impedimetric method can be quite large. For example, in four instances (no. 3, 4, 14, and 15) where the MPN values ranged from 104 to 240, the IDT estimates ranged from 3,500 to 21,000 and the VRBA counts were 140,000 to 160,000 (Table 3). Similarly, in three instances (no. 23, 24, and 25) where the MPN values were 10,400, the IDT estimates ranged from 100 to 330. There is room for concern when a newly proposed test can, however occasionally, seriously underestimate the coliform burden in a food sample. At the same time, it cannot be said that an MPN count of 10,000 is necessarily more significant than a VRBA count of 160,000. Certainly the standard APHA procedure (1) for determining the "confirmed" coliform count from the VRBA count operates on the assumption that the ratio of VRBA count to the confirmed coliform count will seldom exceed a factor of 10. Thus, the impedimetric method offers an alternative method for estimating coliforms. The rationale for accepting a measure of variability in the results obtained with different tests for estimating coliforms derives from two considerations. First, there is the uncertainty that aerogenic, lactose-fermenting members of the Enterobacteriaceae may not be exclusive indicators of the sanitary quality of foods (8). Second, it is founded on the expectation that if members of closely related group of bacterial species have RAPID ESTIMATION OF COLIFORMS IN MEAT 523 similar probabilities of distribution in a particular food or its environs, each member of that group will have equal sanitary significance (8, 21) Ċompared with other methods currently available for estimating coliforms in food, the impedimetric method is simple to execute, the data are recorded automatically, and the results are obtained within 24 h. In addition, the detection time method requires only one dilution of the food sample, thus resulting in considerable savings in labor and materials. ACKNOWLEDGMENTS Our thanks are due to Stuart Dufour for his continuous advice and help with the statistical analysis in the preparation of this manuscript; to Spring Kraeger for helpful suggestions during the course of this work; and to Paxton Cady for critically reviewing this manuscript. LITERATURE CrITD 1. American Public Health Association Compendium of methods for the microbiological examination of foods. American Public Health Association, Washington, D.C. 2. Cady, P Progress in impedance measurements in microbiology, p In A. N. Sharpe and D. S. Clark (ed.), Mechanizing microbiology. Charles C Thomas Publisher, Springfield, Ill. 3. Cady, P., S. W. Dufour, P. Lawless, B. Nunke, and S. J. Kraeger Impedimetric screening for bacteriuria. J. Clin. Microbiol. 7: Cady, P., S. W. Dufour, J. Shaw, and S. J. Kraeger Electrical impedance measurements: rapid method for detecting and monitoring microorganisms. J. Clin. Microbiol. 7: Cady, P., D. Hardy, S. Martins, S. W. Dufour, and S. J. Kraeger Automated impedance measurements for rapid screening of milk microbial content. J. Food Protect. 41: Carl, K. E Oregon's experience with microbiological standards for meat. J. Milk Food Technol. 38: Chambers, J. V., D. 0. Brechbill, and D. A. Hill A microbiological survey of raw ground beef in Ohio. J. 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