APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1980, p. 518-5240099-2240/80/03-0518/07$02.00/0

Vol. 39, No. 3

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

S. B. MARTINSt* AND M. J. SELBY

Bactomatic, Inc., Palo Alto, California 94303

A 24-h instrumental procedure is described for the quantitative estimation ofcoliforms in ground meat. The method is simple and rapid, and it requires but a

single sample dilution and four replicates. The data are recorded automaticallyand 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 impedanceresponse of gram-negative organisms, and it is selective for coliforms. Seventysamples of ground beef were analyzed for coliforms by the IDT method and theconventional three-dilution, two-step most-probable-number test tube procedure.Seventy-nine percent of the impedimetric estimates fell within the 95% confidencelimits of the most-probable-number values. This corresponds to the criteria usedto evaluate other coliform tests, with the added advantage of a single dilution andmore rapid results.

Growth of microbial populations results inchanges in electrical impedance of the culturemedium. The Bactometer (Bactomatic, Inc.,Palo Alto, Calif.) microbial monitoring system isa sensitive instrument that monitors thesechanges during the course of microbial growth.Culture volumes from 1 to 100 ml can be testedin appropriately sized containers. Each con-tainer is monitored separately in reference tothe uninoculated medium, and the proportionalimpedance change is automatically recorded onstrip charts every 3 s; the change may also beanalyzed with a built-in computer (2, 4).

Typically, impedance changes become detect-able when the bacterial density reaches 105 to106 organisms/ml and then continue at a rateand for a period depending upon the bacterialspecies and the medium. The detection timeprovides an approximate measure of the numberof organisms in the initial inoculum (2, 4). Thisimpedimetric procedure offers a simple andrapid method for estimating total bacterial den-sities in frozen vegetables (16), urine (3), andmilk samples (5).We report here on the selective estimation of

coliforms in ground meat by impedimetric pro-cedures, using a medium especially developedfor this purpose.The subject of coliform standards for ground

meat is ofmuch current interest. Six states haveproposed guidelines for acceptable limits of col-iform densities in this food product (22). Otherstates are seriously considering such guidelines(7, 22). The proposed limits range from 50 to

t Present address: 777-58 San Antonio, Palo Alto, CA94303.

1,000 coliforms/g (22). Both the limits and thenecessity of any microbiological standard forground meat have been debated (6, 7, 9, 13, 14,20, 23). Standard methods in use today for esti-mating coliforns are tedious and time consum-ing. The availability of a simple and rapidmethod would greatly facilitate the acceptanceof, and compliance with, the coliform standardsby the industry (13, 23).

MATERIALS AND METHODSImpedance measurements. The impedance-mon-

itoring instrument used in this study was the Bacto-meter 32 microbial monitoring system described fullyelsewhere (2, 4). The system was operated at 2 KHz,gain 9, and the impedance changes were continuouslyrecorded on a strip chart recorder at 2.54 cm/h. Thetime at which the impedance change becomes detect-able is called the detection time (DT). For this study,DT was defined as the time when the rate of imped-ance change reaches two strip chart channel widthsper 2 h. This rate is equivalent to an impedance changeof 1.6% in 2 h. An impedance response was consideredto be positive for coliforms if detection by the abovecriterion was obtained within 20 h of inoculation.

h Impedimetric responses after 20 h were scored asnegative.Medium. The composition of the selective medium

developed for the detection of coliforms by the impe-dimetric method is as follows: tryptone, 20 g; lactose,5 g; L-asparagine, 1 g; Triton X-100, 4 ml; sodiumdihydrogen phosphate, 7 g; and distilled water, 1,000ml (pH 6.3).The medium was dispensed in 100-ml amounts in

bottles and sterilized by autoclaving at 121°C (15-lbsteam pressure) for 15 min.

Before use, freshly prepared potassium sulfite andsterile filtered novobiocin were added to the medium

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RAPID ESTIMATION OF COLIFORMS IN MEAT

to give a final concentration of 0.035% K2SO3 and 3jg of novobiocin per ml. The medium was distributedaseptically in 0.5-mnl amounts in the Bactometer mod-ule wells. Disposable modules with eight sample andeight reference wells, each provided with verticalstainless-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, ofwhich 114 were gram-negative and 17 were gram-pos-itive organisms, were used in this study. Members ofthe Enterobacteriaceae, other than Escherichia coli,were classified as coliforms only if they fermentedlactose with the production of gas. Thus, of the 13strains of Enterobacter agglomerans used in thisstudy, 4 were termed coliforms by the above criteria,whereas 9 were noncoliforms.

For screening the selectivity of the new medium forcoliforms, overnight Trypticase soy broth (TSB; BBLMicrobiology Systems, Cockeysville, Md.) cultureswere diluted 10-7 with Butterfield phosphate water(BPB) to give an approximate concentration of 200organisms/ml. The diluted cultures were inoculated in0.5-ml amounts into 0.5 ml of the medium containedin the module wells. Testing was done in duplicate at35°C for 24 h.Meat sampling. A total of 70 samples of ground

beef were obtained randomly from local supermarketshelves 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 intosterile polyethylene bags (18 by 30 cm), and 190 ml ofBPB containing 0.05% tryptone solution (BPB+) wasadded to give a final dilution of 1:20.The Colworth Stomacher 400 was used to prepare

the samples. Each sample portion was stomached for1 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 dilu-tions were inoculated in 0.5-ml amounts into modulewells containing an equal amount of the medium andincubated at 35°C for 24 h.The inoculated wells were scored as positive or

negative for coliforms in terms of the detection criteriadescribed earlier. From the data obtained, the IMPNper gram was calculated by using a four-tube MPNtable. The table was computer generated, using theequations developed by Halvorson and Ziegler (15).

Conventional MPN. Coliforms were also esti-mated by the standard two-step American PublicHealth Association (APHA) method (1) by using lau-ryl sulfate tryptose broth at 35°C for 48 h for thepresumptive test and brilliant green bile 2% at 35°Cfor 48 h for the confirmed test. Four tubes, eachcontaining 10 ml of medium, were used per dilution.The dilutions and the volume inoculated into laurylsulfate tryptose broth were the same as those used inthe IMPN test.VRBA counts. Coliform plate counts were made

on violet red bile agar (VRBA) by the standard APHApour plate technique (1) by using two or more serialdecimal dilutions of the meat sample. The plates wereoverlaid before incubation at 35°C for 24 h. All darkred colonies 0.5 mm or more in diameter were counted(1).

RESULTSSelective medium for impedimetric esti-

mation of coliforms. Conventional methodsfor enumerating coliforms are heavily dependenton the differential properties of the bacteriolog-ical medium used, for example, the productionof acid or gas. Neither of these differential char-acteristics provides a parameter than can bemeasured by the impedimetric procedure. Ap-plication of the impedimetric method for thequantitative estimation of coliforms was there-fore critically dependent upon the developmentof a medium which selectively amplifies theimpedimetric responses of coliforms.The medium described here achieves this, first

through the incorporation of asparagine andphosphates, which preferentially enhance theimpedimetric responses of gram-negative bacte-ria and inhibit the impedimetric responses ofgram-positive bacteria, and second through theuse of K2SO3 (0.035%) and novobiocin (3 jig/ml),which at pH 6.3 are inhibitory to most Proteus,pseudomonads, and other noncoliform orga-nisms.The specific amplifying effect of asparagine on

the impedimetric responses of gram-negativebacteria is observed in the new medium de-scribed above as well as in the more complexmedia suitable for the growth of the more fastid-ious gram-positive organisms. Figure 1 illus-trates the amplification of impedimetric re-sponses of selected gram-negative bacteria (E.coli and Pseudomonas aeruginosa) by incorpo-ration of asparagine in TSB and standard meth-ods broth (SMB); in contrast, asparagine hadlittle effect on the impedimetric responses of thegram-positive bacterium (Staphylococcus au-reus) and yeast (Candida albicans) tested. Fur-thermore, as shown in the above figure, theinclusion of phosphate salts in SMB-asparaginedepressed the impedance responses of S. aureusand C. albicans.A variety of antibiotics and other inhibitors

(e.g., vancomycin, methicillin, crystal violet, anddeoxycholate) were next tested to determine theoptimal conditions for the selective detection ofcoliforms. The impedimetric responses due topseudomonads were most satisfactorily con-trolled 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 byother investigators (11, 17-19), novobiocin elim-inated the responses of most Proteus strains.The productivity and selectivity of the me-

dium as finally defined by the above-mentionedpreliminary studies was determined first with ascreen of 131 pure cultures of various bacteria(Table 1).

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520 MARTINS AND SELBY

c

z

u

5 10 15 20 2S S 10 15 20 25TIME 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 inoc-ulated into each Bactometer module well, and theexperiment was performed in duplicate. Similar re-

sponse curves were obtained with an inoculum levelof 102 organisms; the onset of impedance change was

2 to 4 h later.

Of the 17 gram-positive bacteria tested in thefinal medium, none gave a positive impedimetricresponse. In fact, none gave a positive impedi-metric response when tested in the same basalmedium in the absence ofK2SO3 and novobiocin.Given the potent antimicrobial effect of novo-

biocin on gram-positive bacteria (11), it is un-

likely that gram-positive bacteria will give false-positive responses in this medium.Among the 114 gram-negative bacteria

screened, 67 were coliforms and 47 were noncol-iforms. Eighty-eight percent (59/67) of the coli-forms gave positive impedimetric responses,whereas 21% (10/47) of the noncoliforms gave

positive responses. The positive responsesamong the noncoliforms were obtained mainlywith Pseudomonas aeruginosa and nonaero-

genic strains of Enterobacter agglomerans.The medium described thus appears to be

specific for gram-negative bacteria with selectiv-ity for the coliform group of bacteria. The selec-tivity of the medium could conceivably be af-fected by the binding of S02 (the presumed

APPL. ENVIRON. MICROBIOL.

inhibitor released from K2SO3) and novobiocinto food constituents. To test this possibility,roast beef was cut aseptically to obtain centralportions free of surface contamination. The meatwas then homogenized as described in Materialsand Methods and diluted in the medium to givea final concentration of 1:20. This homogenatewas then inoculated with 30 different strains ofbacteria. The selectivity of the medium wasunaffected by the presence of meat constituentsin the concentration tested (data not shown).Estimation by IMPN. The MPN method

was used on 70 meat samples to compare theestimation of coliforms by the impedimetric pro-cedure with estimation by the standard test tubeprocedure (Fig. 2). The coefficient of correlationbetween the MPN and the IMPN determina-tions was 0.68, with 77% of the IMPN valuesfalling within the 95% confidence limits of thecorresponding MPN values.

Fifty-two of the meat samples were examinedin duplicate by the impedimetric procedure. Thecoefficient of correlation between the duplicateIMPN values was 0.78.Estimation ofcoliforms by the impedance

detection time (IDT) method. In previous re-ports using the Bactometer microbial monitor-ing system, it had been shown that the DTprovides an approximate measure of the totalmicrobial burden in several types of food (5, 16).In the present study, the detection criteria werespecifically defined to achieve a high degree ofselectivity for the coliform group of organismsby excluding weak responses or late responsesdue to inadequately inhibited noncoliforms. Forexample, impedimetric responses occurring after20 h of incubation were treated as negative. Incomputing the average DT, where one or moreof the module wells was negative, a suitablyweighted detection time for the negative wellshad to be assigned. The DT assigned to thenegative wells was 23 h. The 23-h DT is abouttwo generation times away from the 20-h detec-tion limit for positive wells, and corresponds toa hypothetical bacterial load roughly one-fourththat required to give DT of 20 h. It can becalculated from the MPN tables that when onlyone-fourth of the inoculated wells are positive,the average bacterial concentration in the inoc-ulum is about one-fourth of the minimum re-quired to give a positive response.

Figure 3 gives the plot of DT (at 1:200 dilu-tion) against IMPN per gram. The coefficient ofcorrelation was -0.91. From the regression equa-tion derived from the above data, coliform den-sities were calculated from the IDT. The IDTestimates ranged from <100 to 21,000/g. Thecoefficient of correlation between the IDT esti-mates and the MPN per gram determined by

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RAPID ESTIMATION OF COLIFORMS IN MEAT 521

TABLE 1. Impedimetric responses of the organisms tested in the new coliform medium containingpotassiumsulfite and novobiocin as inhibitors

Impedimetric responsebOrganim No. of strains

tested Positive Negative

ColiformsEscherichia coli 31 28 3Enterobacter aerogenes 4 4 0Enterobacter agglomerans 4 2 2Enterobacter cloacae 16 13 3Klebsiella pneumoniae 10 10 0Serratia liquefaciens 1 1 0Serratia rubideae 1 1 0

Total 67 59 (88%) 8 (12%)

NoncoliformsShigella 2 0 2Proteus 16 1.5 14.5Pseudomonas aeruginosa 7 3 4Pseudomonas (others) 6 2 4Enterobacter agglomerans 9 3 6Serratia rubideae 1 0 1Citrobacter 4 0 4Arizona 1 0 1Aeromonas 1 0 1Gram positivesc 17 0 17

Total 64 9.5 (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 thenoncoliform 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 criteriadescribed in Materials and Methods. An organism positive in only one of the two duplicate wells was given avalue 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).

* * 0 ** ~~~..*

* *0. m.

*0/

* .

0

LOG1 CONVENTIONAL M*N/G

FIG. 2. Scattergram of IMPN per gram as deter-mined by the impedimetric procedure versus MPNper gram as determined by the standard test tubemethod. The solid line is the least-squares linear fitto the IMPN data. Plotted data were obtained from70 samples of meat, 52 of which were tested in dupli-cate by the IMPN method.

2 16 20IMPEDANCE DETECTION TIME (HOURS)

FIG. 3. Scattergram of IMPN per gram as deter-mined by the impedimetric procedure versus the de-tection time in the impedimetric procedure at thesample dilution of 1:200. The solid line is the least-squares linear fit to the IMPN data. Plotted datawere obtained from 70 samples of meat, 52 of whichwere tested in duplicate by the IMPN method.

0

0-j

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522 MARTINS AND SELBY

the conventional method was 0.69, with 79% ofthe IDT estimates falling within the 95% confi-dence limits of the corresponding conventionalMPN values.Table 2 compares the data obtained by the

two methods. The data obtained by eachmethod are grouped into three classes corre-sponding to coliform counts of <100, 100 to 999,and 21,000/g. The tabulated data demonstratea remarkable uniformity in the distribution ofcoliform densities as determined by the conven-tional MPN test and the IDT method. Thisdistribution is very similar to that reported re-cently by Fowler et al. (10) from an analysis of810 samples of ground meat procured fromthroughout the United States.

DISCUSSIONApplication of impedimetric procedures for

the selective detection of bacteria has not beenpossible hitherto due to the lack of selective anddifferential media. The study described heresuggests that impedimetric responses of bacteriacan be selectively controlled by the use of en-hancers (e.g., asparagine) and inhibitors. Thus,the development of suitable media could greatlyextend the usefulness of impedimetric proce-dures.This study evaluated the impedance method

for the selective detection and estimation ofcoliforms. Geldreich (12) has proposed that themembrane filter procedure for estimating coli-forms in water and sewage be validated if80% ofthe membrane filter counts fall within 95% con-fidence limits of the corresponding MPN values.

In our study, 77% (IMPN) and 79% (IDT) ofthe impedimetric estimates fell within the 95%confidence limits ofthe corresponding MPN val-ues. This degree of correlation is particularlystriking in view of the inherent variability in theMPN techniques (24). The presence of coliformsin foods does not have quite the rigorous signif-icance 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) <100 100-999 >1,000 Total

<100 27 11 2 40(67.5)a (27.5) (5)

100-999 2 22 16 40(5) (55) (40)

>1,000 2 8 32 42(5) (19) (76)

Total 31 41 50 122

Numbers in parentheses are percentages.

tion of the impedimetric method as an alternateprocedure for estimating coliforms in foods.An analysis of all IDT estimates that fell

outside the 95% confidence limits of the MPNvalues is presented in Table 3. The results showthat in 13 of 15 instances when the impedimetricestimates gave coliform densities significantlyhigher than that obtained by the test tubemethod, the meat samples had excessive VRBAcounts. Further, the VRBA counts varied from1,400 to 160,000/g, and the ratio ofVRBA countsto MPN ranged from 12.5 to 1,500. These resultstherefore suggest that gram-negative, nonaero-genic, lactose-fermenting organisms, presumablymembers of the Enterobacteriaceae, were re-sponsible for the false-positive results obtainedwith the 70 meat samples analyzed in this study.The results reported in Table 3 also show that

the 15 false-positive estimates obtained with theIDT method were balanced almost exactly by 11false-negative results. Similarly, the data pre-sented in Table 2 show a remarkable uniformityin the distribution of coliform densities as deter-

TABLE 3. Comparison ofMPN with IDT andVRBA countsa

ConventionalSerial no. method

(MPN/g)

1 2402 6403 2404 2405 1,4406 1,4407 2408 1049 92010 <10011 <10012 64013 <10014 10415 104

16 2,48017 1,44018 92019 1,44020 1,44021 64022 64023 10,40024 10,40025 10,40026 2,200

IDT method(estimated

organisms/g)

1,7003,1003,5003,9008,70013,000

960430

3,900920

1,5003,6001,0005,000

21,000

290240200340

<100<100<100<100150330150

VRBA(CFUh/g)

66,0001,000

140,000140,00018,00018,00018,0001,400

30,0009,0009,000800

4,000160,000160,000

7,8007,0004,200

20,0001,400300300

11,00011,00058,0002,000

a Data are drawn from IDT values that fell outsidethe 95% confidence limits of the corresponding MPNvalues.

b Colony-forming units.

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RAPID ESTIMATION OF COLIFORMS IN MEAT 523

mined by the MPN method and the IDTmethod. The results analyzed in Tables 2 and 3therefore lead to the expectation that the MPNmethod and the IDT method would each rejectroughly the same percentage of meat samples,irrespective of the coliform standard prescribedor the sampling plan adopted.These results also suggest that both methods

detect primarily bacteria belonging to the En-terobacteriaceae and that the bacterial strainsdetected by either method have equal probabil-ity of occurrence within the meat samples ex-amined. This notion is supported by the resultsof screening of the medium used in this studywith various bacteria (Table 1). Of the 30 bac-terial strains belonging to genera other thanEnterobacteriaceae, only some strains of P.aeruginosa (three of seven) gave positive re-sponses in the impedimetric test. On the otherhand, of the 13 strains of E. agglomeransscreened, the impedimetric method detected 5and the test tube method detected 4, whereasonly 2 were detected by both methods. It cannotbe said that only aerogenic strains of E. agglom-erans have sanitary significance.

In the individual samples, the discrepanciesbetween the standard test tube method and theimpedimetric method can be quite large. Forexample, 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 21,000and 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 pro-

posed test can, however occasionally, seriouslyunderestimate the coliform burden in a foodsample. At the same time, it cannot be said thatan MPN count of 10,000 is necessarily moresignificant than a VRBA count of 160,000. Cer-tainly the standard APHA procedure (1) fordetermining the "confirmed" coliform countfrom the VRBA count operates on the assump-tion that the ratio of VRBA count to the con-firmed coliform count will seldom exceed a factorof 10.Thus, the impedimetric method offers an al-

ternative method for estimating coliforms. Therationale for accepting a measure of variabilityin the results obtained with different tests forestimating coliforms derives from two consider-ations. First, there is the uncertainty that aero-genic, lactose-fermenting members of the Enter-obacteriaceae may not be exclusive indicatorsof the sanitary quality of foods (8). Second, it isfounded on the expectation that if members ofclosely related group of bacterial species have

similar probabilities of distribution in a particu-lar food or its environs, each member of thatgroup will have equal sanitary significance (8,21).Compared with other methods currently

available for estimating coliforms in food, theimpedimetric method is simple to execute, thedata are recorded automatically, and the resultsare obtained within 24 h. In addition, the detec-tion time method requires only one dilution ofthe food sample, thus resulting in considerablesavings in labor and materials.

ACKNOWLEDGMENTSOur thanks are due to Stuart Dufour for his continuous

advice and help with the statistical analysis in the preparationof this manuscript; to Spring Kraeger for helpful suggestionsduring the course of this work; and to Paxton Cady forcritically reviewing this manuscript.

LITERATURE CrITD1. American Public Health Association. 1976. Compen-

dium of methods for the microbiological examination offoods. American Public Health Association, Washing-ton, D.C.

2. Cady, P. 1978. Progress in impedance measurements inmicrobiology, p. 199-239. In A. N. Sharpe and D. S.Clark (ed.), Mechanizing microbiology. Charles CThomas Publisher, Springfield, Ill.

3. Cady, P., S. W. Dufour, P. Lawless, B. Nunke, and S.J. Kraeger. 1978. Impedimetric screening for bacteri-uria. J. Clin. Microbiol. 7:273-278.

4. Cady, P., S. W. Dufour, J. Shaw, and S. J. Kraeger.1978. Electrical impedance measurements: rapidmethod for detecting and monitoring microorganisms.J. Clin. Microbiol. 7:265-272.

5. Cady, P., D. Hardy, S. Martins, S. W. Dufour, and S.J. Kraeger. 1978. Automated impedance measure-ments for rapid screening of milk microbial content. J.Food Protect. 41:277-283.

6. Carl, K. E. 1975. Oregon's experience with microbiologicalstandards for meat. J. Milk Food Technol. 38:483-486.

7. Chambers, J. V., D. 0. Brechbill, and D. A. Hill. 1976.A microbiological survey of raw ground beef in Ohio. J.Milk Food Technol. 39:530-535.

8. Drion, E. F., and D. A. A. Mossel. 1977. The reliabilityof the examination of foods, processed for safety, forenteric pathogens and Enterobacteriaceae: a mathe-matical and ecological study. J. Hyg. 78:301-324.

9. Foster, J. F., J. L. Fowler, and W. C. Ladiges. 1977. Abacteriological survey of raw ground beef. J. Food Pro-tect. 40:790-794.

10. Fowler, J. L., D. L. Stutzman, J. F. Foster, and W. H.Langley, Jr. 1977. Selected food microbiological datacollected through a computerized program. J. FoodProtect. 40:166-169.

11. Garrod, L. P., H. P. Lambert, and F. O'Grady. 1973.Various anti-bacterial antibiotics, p. 214-215. In Anti-biotics and chemotherapy, 4th ed. Churchill Living-stone, London.

12. Geldreich, E. E. 1975. Membrane filter coliform proce-dures, p. 117-132. In Handbook for evaluating waterbacteriological laboratories, 2nd ed. U.S. Environmen-tal Protection Agency, Washington, D.C.

13. Goepfert, J. M. 1976. The aerobic plate count, coliformand Escherichia coli content of raw ground beef at theretail level. J. Milk Food Technol. 39:175-178.

4. Goepfert, J. M., and H. U. Kim. 1975. Behavior ofselected food-borne pathogens in raw ground beef. J.

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Milk Food Technol. 38:449-452.15. Halvorson, H. O., and N. R. Ziegler. 1933. Application

of statistics to problems in bacteriology. I. A means ofdetermining bacterial population by the dilutionmethod. J. Bacteriol. 25:101-121.

16. Hardy, D., S. J. Kraeger, S. W. Dufour, and P. Cady.1977. Rapid detection of microbial contamination infrozen vegetables by automated impedance measure-

ments. Appl. Environ. Microbiol. 34:14-17.17. Jeffries, L. 1959. Novobiocin-tetrathionate broth: a me-

dium of improved selectivity for the isolation of salmo-nellae from faeces. J. Clin. Pathol. 12:568-571.

18. Moats, W. A. 1978. Comparison offour agar plating mediawith and without added novobiocin for isolation ofsalmonellae from beef and deboned poultry meat. Appl.Environ. Microbiol. 36:747-751.

19. Restaino, L, G. S. Grauman, W. A. McCall, and W.M. Hill. 1977. Effects of varying concentrations of no-

vobiocin incorporated into two Salmonella plating me-

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dia on the recovery of Enterobacteriaceae. Appl. En-viron. Microbiol. 33:585-589.

20. Thatcher, F. S., and D. S. Clark. 1975. Microorganismsin foods. I. Their significance and methods of enumer-ation, p. 23-31. University of Toronto Press, Torontoand Buffalo.

21. Thomas, H. A., Jr., and R. L. Woodward. 1964. Esti-mation of coliform density by the membrane filter andthe fermentation tube methods. Am. J. Public Health45:1431-1437.

22. Westhoff, D., and F. Feldstein. 1976. Bacteriologicalanalysis of ground beef. J. Milk Food Technol. 39:401-404.

23. Winslow, R. L. 1975. A retailer's experience with theOregon bacterial standards for meat. J. Milk FoodTechnol. 38:487489.

24. Woodward, R. L. 1957. How probable is the most prob-able number? Am. Water Works Assoc. J. 49:1060-1068.

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