1) Where the 45.5°C. air incubation is utilized comparative tests Fig. 1 Diagram for Coliform and Fecal Coliform Tests APPENDIX T INTERIM BACTERIOLOGICAL CRITERIA FOR OYSTERS By C. B. Kelly Shellfish Sanitation Branch The development of satisfactory criteria of quality for shellfish as received at the market has been continuously progressing since the early days of shellfish sanitation. The degree of interest in this problem can be measured by discussion at the last three Shellfish Sanitation Workshops where reports of collaborative studies and reports by individual workers have been presented and discussed. (1,2,3,4,5,6,7) In 1956, (1) after consideration of a large mass of data the Workshop adopted a bacteriological standard to be applied to shucked oysters as received at the market. The standards were regarded as for interim purposes. They set as upper limits of acceptability coliform MPN 16,000 per 100 grams and plate count of 1,000,000 per gram. At the same time, however, the Workshop recognized the gross deficiencies in attempting to work with coliform organisms as an index of quality and serious doubt was expressed as to the practicality of these coliform limits. The change in acceptable coliform limit from the former standard MPN 2400 to MPN 16,000 reflected the lack of confidence of the group on the significance of these coliform organisms. The change met with objection from northern interests and rightfully so because coliform levels of 16,000 per 100 gram in oysters harvested or processed in northern areas would quite likely indicate serious pollution or contamination. The data presented included evidence that high coliform densities were obtained in shell and shucked oysters in the face of evidence that these oysters were from safe approved areas and had been processed in approved plants in conformity with the requirements of the Shellfish Manuals. It was quite evident to the group that the coliform group failed in its prime purpose to reveal whether the shellfish had been harvested from polluted areas or had been exposed to dangerous contamination during handling and processing after removal from the water. A recommendation, therefore, was made that serious consideration be given to the validity of the coliform group as an index of quality and that simultaneous investigations be conducted to evaluate the significance of other bacterial indices. The fecal coliform group was suggested as a possible substitute for the coliform group. In partial fulfillment of this charge, a report on an interstate cooperative study to evaluate bacteriological criteria for market oysters was presented at the 1958 Shellfish Sanitation Workshop. (4) This study as in others that preceded and followed involved the bacteriological examination of oysters from key points in all commercial handling processes from shucking to delivery to the market. A feature of this study was the development and evaluation of a method for the estimation of fecal coliform organisms following procedure originally developed by Hajna and Perry. 18) Gross increases in coliform organisms were observed during normal, acceptable commercial practices. The magnitude of changes in coliform organisms was of the same order as those observed in plate counts. The results clearly demonstrated the inadequacy of the coliform group as an indicator of sanitary quality of shellfish. It was, therefore, concluded that the plate count was of equal significance in revealing chance contamination or violations of acceptable storage time and temperature. On the other hand, the results of the examinations for fecal coliform organisms revealed a much higher degree of stability as the shellfish proceeded through commercial channels and thus suggested the greater suitability of this parameter as an index of sanitary quality at the market level. The collaborative evaluation of the method for fecal coliforms revealed that it was reliable in the hands of all of the cooperating investigators provided that certain requirements, particularly temperature of incubation was rigidly adhered to. In the light of the findings of this report, the 1958 Workshop adopted (9) an interim bacteriological standard for fresh and frozen oysters at the market level. The standard regarded as satisfactory, shucked oysters with the fecal coliform density of 78 or less and a plate count of 100,000 or less. Oysters with fecal coliform MPN's between 79 and 230 and plate count between 100,000 and 500,000 were regarded with greater suspicion and considered as acceptable in occasional samples. Those with fecal coliform MPN's in excess of 230 and/or plate counts in excess of 500,000 were considered as unsatisfactory and subject to rejection. The Workshop recommended that the standard be adopted for a two year period. It was also recommended that the cooperative studies be extended to evaluate the applicability of these criteria. EFFECT OF COMMERCIAL HANDLING Studies continued in the interim between the 1958 and 1961 Workshop followed procedures established for the earlier investigations. It was wider in scope, however, since the City of Chicago accepted the interim standard and applied the test for fecal coliform organisms to oysters as received in the wholesale market. Data from this large receiving and distributing center was an important factor in the interstate cooperative study. The report on the study included data on more than 3,000 samples of shell and shucked oysters collected at various points in the commercial handling chain. Points of sampling included the shell stock on arrival at the shucking house, the product as shucked, as received and repacked in repacking establishments, and as received in the marketing areas. In general, results obtained were quite similar to those reported in 1958. (Tables 1 and 2) Gross changes in coliform density and plate counts again were observed. Changes in fecal coliform organisms again demonstrated the greater stability of this group of organisms. The coliform and fecal coliform densities of oysters as delivered to the shucking house during the winter months in general reflect the low levels found in shell oysters as harvested. Plate counts were proportionately low. Shell stock as received at the shucking houses during the summer showed quite higher coliform MPN's and plate counts than in the winter. These higher bacterial levels in shell stock during the warmer seasons are due mainly to bacterial multiplication. It is inconceivable that shellfish taken from approved areas showing an average coliform MPN of 70 would show bacterial counts of the order of magnitude demonstrated in the examination of shell oysters on the shucking bench. That multiplication does take place in shell oysters after removal from the water was demonstrated in the Gulf Coast studies presented by Presnell and Kelly. (10) The shucking process did not contribute materially to the fecal coliform content. In fact, in many instances the values for shucked oysters are lower than for shell stock. The picture for coliform organisms and plate count is not clear. On some occasions significantly higher values are found, on other occasions there was not much change. The effect of time and temperature again becomes evident in the data for the product as received at repacking houses or as received at market. The coliform and plate count levels attained at these points are very much higher than that of the product of the shucking houses as collected at the shucking houses. Again the relative stability of the fecal coliform organisms becomes apparent although the higher densities, particularly in the upper percentile levels of the shucked oyster samples suggests adverse time-temperature situations. SIGNIFICANCE OF INDICATOR ORGANISMS During the past three years a number of investigations have produced scientific evidence quite pertinent to the establishment of bacteriological criteria. The vast amount of work (11, 12, 13, 14, 15, 16, 17) done in the collaborative study to evaluate indicator organisms for shellfish growing areas presented at the 1961 Workshop, but unavailable in advance of that meeting, has contributed much toward an understanding of the significance of these organisms. Continuation of these studies and investigations in the ecological aspects of indicators of pollution conducted by the Shellfish Sanitation Laboratory and the Sanitary Engineering Center are pertinent and finally a series of studies of the time-temperature effect of storage of shucked shellfish on the bacterial quality are of particular importance in interpreting the results of the past cooperative studies. It is the purpose of this report to discuss these recent findings and to apply this knowledge to a better interpretation of the technological data collected through the years. Recent work including the many reports presented at the 1961 Shellfish Sanitation Workshop has resulted in a new look at the real sanitary significance of the traditional coliform group of organisms as indicators of pollution. The 1961 Workshop reports reviewed and summarized by Mr. Beck (18) and presented at this Workshop present evidence that fecal coliform organisms are a more meaningful indicator of pollution than the coliform group. This contention is supported by an extensive ecological study in an estuarine area in the State of Washington (17,19) and investigations of Kabler, Clark, Geldrich (20) and others in assessing the sanitary significance of coliform and fecal coliform organisms in surface waters. These latter two works come to the similar conclusion that fecal coliform test results are more specific in revealing the presence of fecal pollution than the coliform tests in assessing water quality. Since shellfish are reflective of the marine environment in which they grow, the greater suitability of fecal coliforms as an index of pollution would also apply to shellfish. The past three years has also seen technological studies on the effect of commercial handling on the coliform, fecal coliform and plate count densities of shellfish. Studies conducted by the Shellfish Sanitation Laboratory at Pensacola, Florida (10) included the first of the recent series of studies on the comparative behavior of the three parameters under controlled time-temperature situations. Of particular significance is the series that compared the behavior of indicator organisms with a selected group of Salmonella. At 4 to 7oC. coliforms and plate counts showed identical behavior, a rapid rise throughout a six day period while fecal coliforms and Salmonella both showed little or no change. At 20°C. (68°F.) parallel multiplication of all organisms occurred including the Salmonella and the fecal coliform organisms. These investigations prompted more closely controlled studies at the Gulf Coast Research Center and at the Northwest Shellfish Laboratory at Purdy, Washington. The results, reported in another presentation at the current Workshop (21) showed multiplication of coliform and plate count organisms in oysters packed in ice and stored under dry refrigeration at 3°C. (380F.) throughout a twenty-five day period of storage. Fecal coliforms show a low rate of decline throughout the same period of time. Of particular importance is the extremely rapid rise in both coliform and plate count in oysters stored at 10°C. (50°F.). The rate of increase at this temperature far exceeds that of the oysters stored in ice and at 3°C. In fact, the bacterial densities attained at five days storage at 10°C. exceeded by at least tenfold those attained during the entire twenty-five days storage at the lower temperatures. It is also important that at 50°F. multiplication of fecal coliforms was observed in many of the lots of both Pacific and Eastern oysters studied. OTHER SPECIES OF SHELLFISH The applicability of the fecal coliform and plate count criteria to Pacific oysters was investigated by the State of Washington over a complete oyster season. (22) Results obtained on 333 samples from various wholesale establishments in the Seattle area indicate a 90% conformity with fecal coliform MPN 230. Conformity with plate count 500,000 was also 90% in samples collected during the warmer months. Ninety percent of the winter samples showed plate counts of 100,000 or less. Studies on soft clams were conducted in a collaborative study (23) undertaken by the States of Maryland, New York, New York City, and the Public Health Service over a two year period. The study involved the examination of shell soft clams at key points from harvesting to delivery at the market. It was concluded from this investigation that soft clam shell stock harvested in Maryland waters, can conform to the current tentative bacteriological standards for market oysters provided the harvesting areas meet the 1962 Cooperative Program requirements and recommended harvesting to market handling procedures are followed as stated in Part II of the Cooperative Program Manual. In Europe, the fecal coliform group has been the accepted indicator of quality of shellfish since 1947 and before in some countries. In England, the tests described by Knott and applied by the Worshipful Fishmongers (24) and the method of Clegg and Sherwood (25) both involve incubation at 44°C. in a water bath, thus the organisms detected are somewhat the same as those enumerated by the modified Eijkman procedure. Modifications of tests for fecal coliform organisms are also used in France and in Holland. (26) DISCUSSION Technological studies conducted during the past few years and recently reported are helpful in the interpretation of the results of cooperative studies to evaluate the applicability of the tentative market standards for shucked oysters. Probably the most revealing have been the numerous investigations on the bacterial changes in shucked oysters during storage at refrigerated temperatures. These show that high rates of multiplication of coliform organisms and plate count occur at the currently accepted temperature of 50°F. and that the oysters will not remain in saleable condition for much more than five to seven days. Storage at this temperature does more than effect the aesthetic quality of the food product since in many of the tests multiplication of E. coli and fecal coliform organisms was also demonstrated. The significance of the multiplication of these organisms assumes some proportion when one considers that experimental evidence has demonstrated a parallel behavior of Salmonella in oysters. It is, therefore, no surprise that shucked oysters as sampled at the market showed a wide range of bacterial densities and that these densities were consistently much higher than those that prevailed in the product as sampled in shucking establishments. The occurrence of low bacterial densities is at least in part due to the fact that in many cases the oysters were adequately refrigerated. The practice of packing containers of shucked oysters in ice is not uncommon and could account for the relatively high percentage of low bacterial count oysters on the market. Because coliform multiplication does take place even in oysters packed in ice, the value of this group as an indicator of pollution is practically nil. It, therefore, becomes of use only to reflect the time-temperature history of shucked oysters and it is likely that the more accepted plate count indicator would be a more suitable test for this purpose. The stability of the fecal coliform group, particularly at temperatures near 40°F. confirm the premise that this group of organisms would offer a better indication of the quality of the product from the sanitary viewpoint. The greater suitability of fecal coliform organisms as an index of pollution in shellfish growing areas offers confirmation of the applicability of this group in assessing the sanitary quality of shellfish at the market. While similar studies are still lacking with regard to other species of shellfish the results of recent investigations certainly suggest the applicability of the criteria of quality to West Coast species of shucked oysters, particularly Pacific oysters. A high degree of conformity with the tentative market standards was demonstrated in studies of the quality of that product both in shucking houses and as on arrival at the market. Limited studies suggests that the standards may be applicable to soft clams. The data recently gathered offers evidence for the acceptability of the currently tentative market criteria for all shucked oysters at the market level and it would thus, appear that these criteria would serve as a useful tool to the consuming States in assessing the quality of the product on arrival at the market. Certain admonitions must be made however. The quality of the product will not be maintained unless the shellfish are held at adequate refrigeration temperatures and these are lower than the current Manual upper limit of 50°F. Storage at temperatures close to 40°F. will not result in serious deterioration within the time the product is usually in transit. The bacteriological criteria should be considered as an indicator of the quality of the product and as such the term conditional is properly applied to those samples showing fecal coliform MPN's more than 230 and/or plate counts more than 500,000. Therefore, rejection on the basis of a single result is not desirable although still within the authority of a State or other governmental agency. The preferable procedure is, as stated in the following proposal for the agency in the receiving State to take action based on operational reports requested from the shellfish regulatory authority at the point of origin. RECOMMENDATIONS The following standards are recommended to be applied to all shucked oysters at the wholesale level as received by interstate shipment. Except for four changes, the standards are the same as the current interim criteria for shucked Eastern oysters. The first two changes are suggested to accomplish uniformity with the recommended criteria for shellfish growing areas. The third change would more accurately define the technique for bacteria counts and the fourth would convert the numerical expressions of density to currently accepted terms. The changes are: 1. Substitute "fecal coliforms" for "E. coli". |