Sulfide Spoilage Anaerobes ROBERT V

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Thermophilic Organisms in Food Spoilage: Sulfide Spoilage Anaerobes ROBERT V. SPECK Campbell Institute for Research and Technology, Campbell Place, Camden, New Jersey 08101

(Received for publication May 28, 1980) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/2/149/1666072/0362-028x-44_2_149.pdf by guest on 27 September 2021 ABSTRACT germination and subsequent vegetative multiplication of thermophiles. The history of sulfide spoilage in canned foods is traced from Although in the 1920s and 1930s large portions of its earliest reported occurrence in an Iowa cannery in 1919 seasonal packs were lost because of sulfide spoilage, through several outbreaks in the midwest and east in 1945. The subsequent to 1940 the spoilage rate was drastically taxonomy of the causative organism, beginning with the name, reduced. Wilson and Tanner (18), in their 1944 study of Clostridium nigrificans, proposed by Werkman and Weaver in 1927, and ending with Desulfotomaculum nigrificans intro 72 corn-canning plants located in Illinois and Wisconsin duced by Campbell and Postgate in 1965, is discussed. Mention and their 1945 study of 200 corn canneries in Illinois, is made of the kinds of canned foods that have been involved in Wisconsin, Minnesota, Maryland, Delaware and Penn sulfide spoilage, and the spoilage characteristics, such as sylvania, reported that only 0.31 o/o of the unprocessed appearance, odor, pH, etc. exhibited by spoiled product. The can samples of cream style corn contained hydrogen morphology, staining characteristics, and culturing methods sulfide producing anaerobes. This is extremely low when are discussed. Methods of analysis of common canned food compared to the fact that 24.5% of the samples ingredients such as sugar, flour, starch and nonfat dry milk are contained flat-sour spoilage producing spores. presented. A brief account of the thermal resistance of the spores of D. nigrificans is also given. Ingredient and equipment CLASSIFICATION contamination and spoilage prevention are discussed in detail. The name Clostridium nigri.ficans, proposed by Werkman and Weaver (17) in 1927 has been in common usage up to the present time. However, in 1938 Starkey Probably the earliest documented study on sulfide (15) proposed the new genus Sporovibrio for the spoilage of canned foods was that of W erkman and anaerobic vibrio-shaped cells which produced endo Weaver (17) in 1927. Working in the Bacteriology Section spores. The organism Sporovibrio desulfuricans was later of the Iowa Agricultural Experiment Station, these two shown by Campbell et al. (6) to be identical to C. investigators conducted many detailed experiments and nigri.ficans. Since the latter had taxonomic priority, the collected voluminous data for their monumental and thermophilic sporeforming, sulfate reducer was consid classic publication on "sulphur stinker" spoilage of ered to be properly named, C. nigri.ficans. In 1965 canned sweet corn. This paper, published in the Iowa Campbell and Postgate (7) proposed the name Desulfoto State College Journal of Science in 1927, traced this type maculum nigri.ficans. It was their feeling that the of spoilage back to 1919 when an Iowa cannery lost classification of W erkman and Weaver (I 7) for the practically its entire season pack of sweet corn. A similar original nigri.ficans in the genus Clostridium was a outbreak occurred in another Iowa cannery in 1925. mistake for several reasons. (a) Most clostridia are gram Reported outbreaks of sulfide spoilage in corn and positive and nigri.ficans is gram negative. (b) The DNA peas have been usually restricted to the midwestern base composition of C. nigri.ficans is remote from that of states, although at least one unreported outbreak the typical clostridia. (c) The presence of cytochrome in occurred in the state of Maryland. Although relatively the three sporulating types that they studied is not typical rare, four outbreaks reported before 1928, resulted in of clostridia. For these reasons they proposed that C. substantial financial losses. Studies by W erkman and nigri.ficans be removed from the genus Clostridium and Weaver (17) involved one pack in which the spoilage rate be reclassified with the mesophilic sporeformers under a was estimated to be 25o/o. More detailed evaluation new generic name. The proposed name, Desulfoto showed that at least 55% of the pack was spoiled. To the macululm, was chosen to indicate reduction of sulfur best of my knowledge there have been no reported compounds (desulfo) and sausage (tomaclum) (toma incidences of sulfide spoilage in home-canned foods. culum). More than likely this is due to the fairly rapid cooling of The authors went on further and described the type the processed jars or cans usually afforded by the species, D. nigri.ficans as "rods, 0.3 to 0.5 by 3 to 6 JJ• housewife. The rapid cooling would prevent spore rounded ends, sometimes lentictilate and swollen,

JOURNAL OF FOOD PROTECTION, VOL. 44, FEBRUARY i981 150 SPECK sometimes paired; motile with 'twisting and tumbling' To the best of my knowledge, there has never been an motility, peritrichous flagella; spores oval, terminal or occurrence of mixed spoilage due to D. nigrificans and subterminal, slightly swelling the cells; gram negative!' Bacillus stearothermophilus in the same can of product. D. nigrificam is a gram-negative obligate anaerobe I'm sure that the growth rate of the latter is significantly which reduces sulfate to sulfide. It does not reduce more rapid than the former. The acid produced by B. nitrate, does not ferment glucose and other carbohy stearothermophilus would certainly reduce the product drates, and does not liquefy gelatin, coagulated albumin, pH sufficiently to inhibit germination and growth of D. or blood serum. It produces H 2S from cystine, is nigrificans. non-pathogenic to man, guinea pig, mouse, rat or rabbit, MICROSCOPY and has been isolated from soils, compost heaps, Morphology manure, thermal spring water and "sulphur stinker" Normally smears of spoiled product show a minimum spoiled foods. number of organisms per field, not the abundance of PRODUCT SPOILAGE AND organisms found in product exhibiting thermophilic flat SPOILAGE CHARACTERISTICS sour-type spoilage. Vegetative cells are either straight or Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/2/149/1666072/0362-028x-44_2_149.pdf by guest on 27 September 2021 The term "sulphur stinker" is a commonlv used term slightly curved rods usually with rounded ends. They for a can of product exhibiting sulfide sp~ilage. The range from 2 to 6 !Jlll in length and 0.3 to 0.5 J.lll1 in dia appearance of such a can is normal as there is no meter. They occur singly, in pairs and in short chains. evidence of swelling or bulging. This is attributed to the The cells have peritrichous flagella which are responsible solubility of hydrogen sulfide in the liquid portion of the for the "twisting and tumbling" motility. Spores are oval, pack. Rarely is there any loss of vacuum of the can. terminal and subterminal, and cause only a slight Spoilage detection, therefore, depends on opening the swelling of the cell. can and examining it~; contents. Although now considered a rather rare type of spoilage, sulfide spoilage Staining characteristics has been reported in canned sweet corn, peas, In their original paper, Werkman and Weaver (17} mushrooms and mushroom-containing non-acid foods described the organism as a gram-positive rod. They and canned baby clams. Products most likely involved in noted 1 to 6 gram-positive spherical granules in many of sulfide spoilage are those with a pH of- 6.8 to 7.3. the cells. Sealey (13) described the organism as being so Although scanty growth occasionally occurs at pH values weakly gram-positive that for all practical taxonomic as low as 5.6, 6.2 is considered the lower limit. The purposes, it may be regarded as gram-negative. maximum pH for growth has been recorded as 7.8 Twenty-four-hour-old cultures and older cultures are Although several investigators (6, 7,15) have reported almost uniformly gram-negative and show practically no that D. nigrificans can be "trained" to grow at 30 to granular staining. Campbell and Postgate (7} described 37 C, to the best of my knowledge, spoilage in canned the organism as being gram-negative. product has never occurred at the mesophilic tempera CULTURING ture range. There has been one isolated case where Many of the earlier workers reported difficulty in sulfide spoilage would not develop at the optimal culturing the organism. W erkman and Weaver (17}, after thermophilic temperature of 55 C; however, after testing numerous media formulations, chose Beef Heart increasing the temperature to 65 C, spoilage was detected. Infusion Agar with 0.1 o/o added ferric chloride and There is also sound evidence that dormant spores in adjusted to pH 7.2 as a suitable stock medium. Use of canned product may remain viable for extended periods. shake tubes of this medium gave good colony growth; Our laboratory has shown that naturally occurring however, its use in plates incubated at 55 C in an spores which survive a commercial process can remain atmosphere of hydrogen failed to produce growth. The dormant for up to 5 years, providing the product is held pyrogallic acid - sodium hydroxide method was likewise at a mesophilic temperature. After increasing the unsuccessful. Studies by Bufton (2) and again by temperature to 55-65 C, the product will spoil within Postgate (12) concluded that all available media are 72 h. unsatisfactory for providing accurate counts and Upon opening a can of spoiled product, the odor of recoveries of D. nigrificans. Lin and Lin {10) compared hydrogen sulfide is immediately discernible. Spoiled outgrowth and recovery of D. nigrificans in seven liquid sweet corn usually has a bluish gray colored liquor with and 10 solid media, and concluded that BETI (Beef many blackened grains, particularly the separated extract, tryptone, iron) broth and BETI Agar showed a germs, throughout the can. Spoiled peas sometimes show slight improvement in recovery and provided more rapid little or no discoloration. However, more frequently, peas growth than other media. show blackening, especially those that are broken or The use of Iron Sulfite Agar recommended by crushed. A black colored brine is also typical. In many Cameron (3) has provided better recovery from spoiled instances, spangling ofthe inside ofthe can occurs and is product and ingredients than media proposed by earlier the results of the interaction of the dissolved hydrogen investigators. This medium, also recommended by Speck sulfide with the iron of the container. This is evident (14), contains Tryptone or Trypticase 10.0 g, sodium through the semi-transparent enamel system. sulfite (anhydrous) 1.0 g, agar 20.0 g and distilled water

JOURNAL OF FOOD PROTECTION, VOL. 44, FEBRUARY 1981 SULFIDE SPOILAGE ANAEROBES 151

1.0 L. The medium has been used by the food industry hydrogen which splits the agar and, with sulfite agar, reduces the sulfite for detection of H 2S-producing anaerobes in starch and thereby causing general blackening of the medium. Total the colonies sugar for more than 40 years. Herson and Hullard (9), in the six tubes. Calculate and report as number of spores per 10 g of ingredient. however, recommended using only 0.5 g of sodium sulfite An alternative method of analysis, using thioglycollate agar in lieu of in the same medium since it had been demonstrated that sulfite agar, has been used by some laboratories. 0.1 o/o is inhibitory to certain bacteria. NONFAT DRY MILK In our laboratory, best recoveries have been obtained Sample preparation with the use of Fluid Thioglycollate Medium plus freshly Weigh 10 g of the sample into a sterile 2SO-ml Erlenmeyer flask pickled iron strips or nails. The 15 to 20-ml quantities of marked to 100 mi. Add N/SO sodium hydroxide to the 100 ml mark and the medium are sterilized in 20 x 150 screw-capped shake to completely dissolve the sample. Heat lO min at 5 lb steam tubes. When ready for use, freshly pickled iron strips are pressure, then cool immediately. rinsed in distilled water, flamed, and plunged hot into Culturing methods the tubes of sterile broth. We have found that medium Transfer 2 ml of the heated solution to each of ten 20 x 1SO mm without the iron strips can be stored longer than when screw capped tubes of freshly exhausted sulftte agar and nail. Gently the strips are added before sterilizing. We prefer iron invert several times and solidify rapidly by placing the tubes in cold Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/2/149/1666072/0362-028x-44_2_149.pdf by guest on 27 September 2021 strips to ferric citrate, which is frequently used in water. Preheat the tubes to SO to 55 C and incubate at that temperature for 24 and 48 ± 3 h. Count colonies of D. nigrificans described earlier conjunction with sulfite agar. and report on the basis of 10 g of sample. To analyze ingredients for sulfide spoilage organisms, the method originally described by National Canners CREAM Association (JJ) and later by Military Standard - Sample preparation Bacterial Standards for Starches etc., MIL-STD-900, 8 Mix 2 g of gum tragacanth and 1 g of gum arabic in 100 ml of water in an Erlenmeyer flask. Sterilize in the autoclave for 20 min at 121 C. September 1958 is still recommended. The methods for Transfer 20 ml of sample to a sterile 2SO-ml Erlenmeyer flask marked sugar, starch, flour, nonfat dry milk and cream were for 100 mi. Add the sterilized gum mixture to the 100 ml mark and detailed in Chapter 23 of the Compendium of Methods carefully shake, using a sterile rubber stopper. Loosen stopper and for the Microbiological Examination of Foods (14). autoclave for 5 min at 5 psi pressure. Details of the methods follow. Culturing methods SUGAR, STARCH AND FLOUR Transfer 2 ml of the sample and gum mixture to each of ten 20 x 1SO mm screw capped tubes of freshly exhausted sulfite agar and [SUGAR: AOAC METHOD] nails. Gently invert several times and solidify rapidly by placing the Sample preparation tubes in cold water. Preheat the tubes to SO to 55 C and incubate at that For dry sugar, place 20 g of the sample into a dry sterile 2SO-ml temperature for 24 and 48 ± 3 h. Count colonies of D. nigrificans Erlenmeyer flask with sterile rubber stopper. Add sterile water to the described earlier and report on the basis of 1 ml of sample. 100 ml mark and shake to dissolve. Replace the stopper with a sterile PRECAUTIONS AND LIMITATIONS OF PROCEDURES cotton plug and bring the solution rapidly to a boil, and continue When analyzing ingredients by the above methods, thorough boiling for 5 min. Replace the evaporated liquid with sterile water. Cool dispersion of the sample solution or slurry in each tube of medium is immediately in cold water. essentiaL More difficulty will be encountered in the analysis of starch or Prepare samples of liquid sugar the same way, except the amount flour because of the thickening effect during heating. Frequent swirling added to the sterile flask should be determined, depending upon the or gentle inversion of the tubes during the first 10 min of heating will Brix, to be equivalent to 20 g of dry sugar. assure proper dispersion. In examining starch or flour, 20 g ofthe dry ingredients are placed in Since tubes containing numerous colonies of D. nigrificans may a dry sterile 2SO-ml Erlenmeyer flask, and sterile cold water added to become completely blackened after 48 h of incubation, a preliminary the 100 ml mark, with intermittent swirling. Close the flask with the count should be made after 20 to 24 ± 3 h. sterile rubber stopper and shake well to obtain a uniform, lump-free suspension of the sample in water. Sterile glass beads added to the INTERPRETATION OF RESULTS sample water mixture will facilitate thorough mixing during shaking. A standard for sulfide spoilage spores need only apply to ingredients Cultural methods (sugar, starch, flour, etc.) to be used in low acid, heat processed canned When examining sugar, divide 20 ml of the heated solution among foods. Sulfide spoilage spores shall be present in not more than 2 (40%) six 20 x ISO mm screw capped tubes containing approximately 10 ml of of the 5 samples tested and in any 1 sample to the extent of not more sulfite agar and a nail. Make the inoculations into freshly exhausted than 5 spores per 10 g. This would be equivalent to 2 colonies in the medium and solidify rapidly by placing the tubes in cold water. Preheat inoculated tubes. the tubes to SO to 55 C and incubate at that temperature for 24 and THERMAL RESISTANCE 48h. For starch or flour, divide 20 ml of the cold suspension among six Generally speaking, the thermal resistance of D. 20 x lSO mm screw capped tubes containing approximately 10 ml of nigrificans is greater than that of the thermophilic sulfite agar and a nail. The tubes should be swirled manually and anaerobe Clostridium thermosaccharolyticum and less gently inverted several times before heating and during the 15-min heating period in a boiling water bath. The periodic swirling and than the flat sour organism, B. stearothermophilus. In inversion of the tubes will assure an even dispersion of the starch and their 1927 publication, Werkman and Weaver (17) flour in the tubes of medium. Following heating, cool the tubes reported that 8 x 106 spores of D. nigrificans in pea immediately in cold water. Preheat the tubes to SO to 55 C, and medium adjusted to pH 7.0, survived 450 but not incubate at that temperature for 24 to48 h. 465 min at 100 C (212 F). At 118 C (245 F), the spores D. nigri:ficans will appear as jet black spherical areas, the color due to formation of iron sulfide. No gas is produced. Certain thermophilic survived SO but not 55 min. At 121 C (250 F), the spores anaerobes not producing H2S give rise to relatively large amounts of survived 25 but not 30 min. In plotting these survival and

JOURNAL OF FOOD PROTECTION, VOL. 44, FEBRUARY 1981 152 SPECK destruction points on sernilogarithrnic paper, a slope of were prepared and announced. Shortly thereafter, 30 F is obtained. This seems highly unlikely so I must refineries which formerly furnished sugar harboring question the validity of the original data. considerable thermophilic spores, produced a product In his later publication, Werkrnan (}6) expressed practically free of spoilage producing spores. considerable difficulty in obtaining readings at temp The significance of starch contamination by thermo eratures of 245 and 250 F. He endeavored to generate philic spoilage organism was emphasized by Cameron data which could be used to show agreement with and Williams (5) in 193 7. It was demonstrated that exposures at 212 F when the results were judged by contamination provided in starch will cause spoilage in logarithmic curves. In an attempt to avoid the problem, canned cream-style corn. Spoilage from this source has Werkrnan contrifuged his pea medium sufficiently to also been traced in spaghetti and dog food. Although throw down particulate matter without af(ecting the sulfide spoilage organisms are not as prevalent in starch suspended spores. The procedure reduced the resistance; as are flat sour-type spores, the ingredient can be a however, at the higher temperatures the resistance still contributor to "sulphur stinker" spoilage. Following a

seemed high. number of publications, the production of low thermo Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/2/149/1666072/0362-028x-44_2_149.pdf by guest on 27 September 2021 Unpublished data by the writer showed that 10,000 phile spore count starch became a reality. spores of the NCA strain SS-3750 of D. nigrificans in Producers of canned foods today must still scrutinize phosphate buffer, pH 7.0 survived 250, 60 and 15 min at sugar and starch for thermophilic spores. This is 230, 240 and 250 F, respectively. They were destroyed at particularly true of imported starches which may not be 300, 80 and 20 min at the same temperatures, given the hydrogen peroxide sterilizing treatment. Other respectively. In plotting survival and destruction points ingredient sources of sulfide spoilage-producing spores on sernilogarithrnic paper, an F value of 16.0- 19.0 with would include cream, condensed milk and nonfat dry corresponding z values of 16.0 - 17.7 were obtained. milk solids. Using Stumbo's method, aD of 2.6 and corresponding z In considering all three types of thermophilic spoilage of 16.3 were obtained. of canned products, flat sour bacteria (JJ. stearothermo· Studies reported by Donnelly and Busta (8) indicated philus) are most readily established in the canning plant that spores of highest heat resistance were produced and develop with greatest ease. Consequently, flat sour using a 20% infusion of spent mushroom compost. spoilage appears to be the most common. Possibly the D-values of 25.8 to 55.4 min were reported for strains of greater resistance of spores to heat may be added to other D. nigrificans in soy formula. factors contributing to this predominance. Sulfide Most data reported confirm that D. nigrificans is spoilage, on the other hand, is the rarest and the capable of surviving the standard commercial processes organism responsible for it appears to be the least readily of most canned foods. Processing per se, therefore, estabished in the canning plant. This may be due to, at cannot be relied upon to prevent sulfide spoilage in those least in part, the more exacting requirements for growth products which normally fall in the ideal pH range. on the part of the organisms. Earlier mention was made of the investigations of INGREDIENT AND EQUIPMENT CONTAMINATION Wilson and Tanner (18) wherein the low incidence Barlow (1) in 1913 demonstrated some of the (0.31 o/o) of sulfide spoilage organisms was compared to fundamental causes of thermophilic spoilage. His the high incidence (24.5%) of flat sour organisms in detailed experiments showed that thermophilic spoilage unprocessed cream-style corn. In the same survey, the in corn did not result from holding the raw product. His investigators found less than 9.0% of the raw corn findings and resultant conclusions were either unknown samples contained flat sour spores. No spores of sulfide or largely disregarded until 1926 when they were spoilage organisms was detected. Although some confirmed by Cameron et al. (4). These latter increase in flat sour spore counts was evident in investigators concluded that, "when thermophilic spoil subsequent samples along the processing line, no spores

age occurs, it is due to contamination within the cannery of H2 S-producers were encountered in any of the survey or to contamination conveyed by something other than samples. Similar data were also presented from their the basic raw product." In addition to the contribution of survey of the canning of whole kernel and vacuum-pack equipment, contamination from spores of spoilage corn. Peas, on the other hand, showed higher incidences therrnophiles could be found to a limited extent in soil, of flat sour spores in both washed raw samples and in while refined sugar was found to carry contamination unprocessed live samples than were noted from studies of with greater frequency and generally to a much greater corn canning plants. Spores of sulfide spoilage organisms extent. Following these and similar studies, granulated were lacking in all samples except that one sample in sugar carne to be regarded as an outstanding source of 9,654 processed cans showed these organisms. In their thermophilic contamination. Although, the flat sour-type summary of these pea canneries, the authors stated "as spores were found in essentially all of the cane-sugar in corn canning. thermophilic anaerobes not producing samples that were examined, the thermophilic anae hydrogen sulfide and those producing hydrogen sulfide robes, including sulfide spoilage spores were found in were only rarely demonstrated.'' many samples and sometimes in undesirable numbers. The investigation of Cameron et at. (4) of pea packers Early in 1931 tentative specifications for canners' sugar in 1927 showed that all samples of overflow water from

JOURNAL OF FOOD PROTECTION. VOL 44. FEBRUARY 1981 SULFIDE SPOILAGE ANAEROBES 153 the outlets of two blanchers were heavily contaminated not detrimental to the finished product quality, provide with sulfide spoilage bacteria. One of the blanchers was additional processing over the standard commercial new and had not been in use during the previous season. process, (e) provide rapid and adequate cooling of Since it was assumed that the bacteria were not normal processed product before casing and {f) set aside and inhabitants of new blanchers, the blanch water was incubate thermophilically samples representative of the suspected of providing a source of the bacteria. A survey pack to apprise sulfide spoilage potential. of the blanch water system revealed that the deep well REFERENCES water eventually supplying the blanchers gave entirely 1. Barlow, B. 1913. Master of Science Thesis, University of Illinois. negative results for sulfide organisms. A large overhead 2. Bufton, A. W. J. 1959. A note on the enumeration of thermophilic wooden storage tank used to supply cold water for sulfate reducing bacteria {Clostridium nigri:ficans). J. Appl. washing requirements and also to supply another wooden Bacteriol. 22:278-280. tank directly below was also free of sulfide organisms. 3. Cameron, E. J. 1938. J. Assoc. Off. Agric. Chern. 21:452. 4. Cameron, E. J., C. C. Williams, R. J. Thompson. 1928. Bacteriolo The second wooden tank was equipped with a closed gical field studies in canning. Thermophilic contamination in the

steam coil and was used to preheat water supplying the canning of peas and com. Bulletin 25-L, National Canners Assoc .. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/2/149/1666072/0362-028x-44_2_149.pdf by guest on 27 September 2021 blanchers. The temperature of this water varied Washington. D.C. according to the amount used for the blanchers but 5. Cameron, E.J., and C. C. Williams. 1937. Significance of sugar and ranged between 105 and 150 F. A thorough bacteriologi starch contamination. Canner, Convention Number, February 27, 1937. cal survey established that this wooden hot water tank 6. Campbell, L. L., Jr., H. A. Frank, and E. R. Hall. 1957. Studies on was heavily contaminated with sulfide spoilage organ thermophilic sulfate reducing bacteria. I. Identification of Sporo· isms, as well as flat sour spores. In addition, the brine vibrio desulfaricans as Clostridium nigrificans. J. Bacteriol. tank supplied with water from the hot water tank was 73:516-521. also found to be heavily contaminated. The wooden brine 7. Campbell, L. L., and I. R. Postgate. 1965. Classification of the sporeforming sulfate-reducing bacteria. Bact. Rev. 29:359-363. tank was not only used to prepare and hold brine but also 8. Donnelly, L. S., and F. F. Busta. 1978. Abstracts of the annual sugar solution. The tank was most heavily contaminated meeting of the American Society for Microbiology. p. 190. at the startup in the morning. Counts of greater than 9. Hersom, A. C., and E. D. Hulland. 1964. Canned foods, an intro 1 ,000,000 per can equivalent were not uncommon. It duction to their microbiology. Chemical Publishing Co. p. 238. became apparent that the wooden tank acted like a 10. Lin, C. C., and K. C. Lin. 1970. Spoilage bacteria in canned foods. II. Sulfide spoilage bacteria in canned mushrooms and a versatile sponge, soaking up the sugar and salt solution and that medium for the enumerations of Clostridium nigrificans. AppL thermophilic spores could become established within the Microbiol. 19:283-286. pores of the wood. Such microenvirons were immune 11. National Canners Association Research Laboratories. 1968. from the usual cleaning process. Replacement of the two Laboratory manual for food cancers and processors, Vol. 1. The wooden tanks with galvanized iron tanks eliminated the AVI Publishing Co. Inc., Westport, Connecticut. 12. Postgate, J. R. 1966. Media for sulphur bacteria. Lab. Prac. sulfide spoilage problem. 15:1239-1244. SPOILAGE PREVENTION 13. Sealey, J. Q. 1951. The biology of Clostridium nigrificans, PhD. thesis, University of Texas. As with other types of thermophilic spoilage, 14. Speck, R. V. 1976. Sulfide spoilage sporeformers. pp. 270-274. In processing of canned foods cannot be relied upon to M. L. Speck {ed.) Compendium of methods for the microbiological eliminate "sulfide stinkers." Because of the unusually examination offoods. American Public Health Assoc .. Washington, high heat resistance of sulfide spoilage spores, a process D.C. 15. Starkey, R. L. 1938. A study of spore formation and other which would be adequate to insure their destruction, morphological characteristics of Vibrio desulfuricans. Arch. would render the product unpalatable. Control can only Microbiol. 9:268-304. be provided by: (a) ingredient (sugar, starch, nonfat dry 16. Werkman, C. H. 1929. Bacteriological studies on sulfide spoilage of milk, etc.) selection to assure freedom from or low canned vegetables. Iowa Agr. Exper. St. Res. Bull. 117:163-181. 17. Werkman, C. H., and H. J. Weaver. 1927. Studies in the bacteri· numbers of spoilage-producing spores, (b) whenever ology of sulphur stinker spoilage of canned sweet corn. Iowa State possible, provide thorough cleaning and washing of College J. Sci. 2:57-67. vegetables, (c) if practical, presterilize ingredients 18. Wilson, C. M., and F. W. Tanner. 1948. Microbiology of canned (stocks, mushrooms, clams, etc.) before blending, (d) if corn and peas. Food Res. 13:365-377.

JOURNAL OF FOOD PROTECTION, VOL. 44. FEBRUARY 1981