Thermophilic Organisms in Food Spoilage: Sulfide Spoilage Anaerobes

Transcription

1 149 Journal of Food Protection, Vol. 44, No.2. Pages (February 1981) Copyright 1981, International Association of Milk, Food, and Environmental Sanitarians Thermophilic Organisms in Food Spoilage: Sulfide Spoilage Anaerobes ROBERT V. SPECK Campbell Institute for Research and Technology, Campbell Place, Camden, New Jersey (Received for publication May 28, 1980) ABSTRACT The history of sulfide spoilage in canned foods is traced from its earliest reported occurrence in an Iowa cannery in 1919 through several outbreaks in the midwest and east in The taxonomy of the causative organism, beginning with the name, Clostridium nigrificans, proposed by Werkman and Weaver in 1927, and ending with Desulfotomaculum nigrificans introduced by Campbell and Postgate in 1965, is discussed. Mention is made of the kinds of canned foods that have been involved in sulfide spoilage, and the spoilage characteristics, such as appearance, odor, ph, etc. exhibited by spoiled product. The morphology, staining characteristics, and culturing methods are discussed. Methods of analysis of common canned food ingredients such as sugar, flour, starch and nonfat dry milk are presented. A brief account of the thermal resistance of the spores of D. nigrificans is also given. Ingredient and equipment contamination and spoilage prevention are discussed in detail. Probably the earliest documented study on sulfide spoilage of canned foods was that of W erkman and Weaver (17) in Working in the Bacteriology Section of the Iowa Agricultural Experiment Station, these two investigators conducted many detailed experiments and collected voluminous data for their monumental and classic publication on "sulphur stinker" spoilage of canned sweet corn. This paper, published in the Iowa State College Journal of Science in 1927, traced this type of spoilage back to 1919 when an Iowa cannery lost practically its entire season pack of sweet corn. A similar outbreak occurred in another Iowa cannery in Reported outbreaks of sulfide spoilage in corn and peas have been usually restricted to the midwestern states, although at least one unreported outbreak occurred in the state of Maryland. Although relatively rare, four outbreaks reported before 1928, resulted in substantial financial losses. Studies by W erkman and Weaver (17) involved one pack in which the spoilage rate was estimated to be 25o/o. More detailed evaluation showed that at least 55% of the pack was spoiled. To the best of my knowledge there have been no reported incidences of sulfide spoilage in home-canned foods. More than likely this is due to the fairly rapid cooling of the processed jars or cans usually afforded by the housewife. The rapid cooling would prevent spore germination and subsequent vegetative multiplication of thermophiles. Although in the 1920s and 1930s large portions of seasonal packs were lost because of sulfide spoilage, subsequent to 1940 the spoilage rate was drastically reduced. Wilson and Tanner (18), in their 1944 study of 72 corn-canning plants located in Illinois and Wisconsin and their 1945 study of 200 corn canneries in Illinois, Wisconsin, Minnesota, Maryland, Delaware and Pennsylvania, reported that only 0.31 o/o of the unprocessed can samples of cream style corn contained hydrogen sulfide producing anaerobes. This is extremely low when compared to the fact that 24.5% of the samples contained flat-sour spoilage producing spores. CLASSIFICATION 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 (15) proposed the new genus Sporovibrio for the anaerobic vibrio-shaped cells which produced endospores. The organism Sporovibrio desulfuricans was later shown by Campbell et al. (6) to be identical to C. nigri.ficans. Since the latter had taxonomic priority, the thermophilic sporeforming, sulfate reducer was considered to be properly named, C. nigri.ficans. In 1965 Campbell and Postgate (7) proposed the name Desulfotomaculum nigri.ficans. It was their feeling that the classification of W erkman and Weaver (I 7) for the original nigri.ficans in the genus Clostridium was a mistake for several reasons. (a) Most clostridia are gram positive and nigri.ficans is gram negative. (b) The DNA base composition of C. nigri.ficans is remote from that of the typical clostridia. (c) The presence of cytochrome in the three sporulating types that they studied is not typical of clostridia. For these reasons they proposed that C. nigri.ficans be removed from the genus Clostridium and be reclassified with the mesophilic sporeformers under a new generic name. The proposed name, Desulfotomacululm, was chosen to indicate reduction of sulfur compounds (desulfo) and sausage (tomaclum) (tomaculum). The authors went on further and described the type species, D. nigri.ficans as "rods, 0.3 to 0.5 by 3 to 6 JJ rounded ends, sometimes lentictilate and swollen, JOURNAL OF FOOD PROTECTION, VOL. 44, FEBRUARY i981

2 150 SPECK sometimes paired; motile with 'twisting and tumbling' motility, peritrichous flagella; spores oval, terminal or subterminal, slightly swelling the cells; gram negative!' D. nigrificam is a gram-negative obligate anaerobe which reduces sulfate to sulfide. It does not reduce nitrate, does not ferment glucose and other carbohydrates, and does not liquefy gelatin, coagulated albumin, or blood serum. It produces H 2 S from cystine, is non-pathogenic to man, guinea pig, mouse, rat or rabbit, and has been isolated from soils, compost heaps, manure, thermal spring water and "sulphur stinker" spoiled foods. PRODUCT SPOILAGE AND SPOILAGE CHARACTERISTICS The term "sulphur stinker" is a commonlv used term for a can of product exhibiting sulfide sp~ilage. The appearance of such a can is normal as there is no evidence of swelling or bulging. This is attributed to the solubility of hydrogen sulfide in the liquid portion of the pack. Rarely is there any loss of vacuum of the can. Spoilage detection, therefore, depends on opening the can and examining it~; contents. Although now considered a rather rare type of spoilage, sulfide spoilage has been reported in canned sweet corn, peas, mushrooms and mushroom-containing non-acid foods and canned baby clams. Products most likely involved in sulfide spoilage are those with a ph of- 6.8 to 7.3. Although scanty growth occasionally occurs at ph values as low as 5.6, 6.2 is considered the lower limit. The maximum ph for growth has been recorded as 7.8 Although several investigators (6, 7,15) have reported that D. nigrificans can be "trained" to grow at 30 to 37 C, to the best of my knowledge, spoilage in canned product has never occurred at the mesophilic temperature range. There has been one isolated case where sulfide spoilage would not develop at the optimal thermophilic temperature of 55 C; however, after increasing the temperature to 65 C, spoilage was detected. There is also sound evidence that dormant spores in canned product may remain viable for extended periods. Our laboratory has shown that naturally occurring spores which survive a commercial process can remain dormant for up to 5 years, providing the product is held at a mesophilic temperature. After increasing the temperature to C, the product will spoil within 72 h. Upon opening a can of spoiled product, the odor of hydrogen sulfide is immediately discernible. Spoiled sweet corn usually has a bluish gray colored liquor with many blackened grains, particularly the separated germs, throughout the can. Spoiled peas sometimes show little or no discoloration. However, more frequently, peas show blackening, especially those that are broken or crushed. A black colored brine is also typical. In many instances, spangling ofthe inside ofthe can occurs and is the results of the interaction of the dissolved hydrogen sulfide with the iron of the container. This is evident through the semi-transparent enamel system. To the best of my knowledge, there has never been an occurrence of mixed spoilage due to D. nigrificans and Bacillus stearothermophilus in the same can of product. I'm sure that the growth rate of the latter is significantly more rapid than the former. The acid produced by B. stearothermophilus would certainly reduce the product ph sufficiently to inhibit germination and growth of D. nigrificans. MICROSCOPY Morphology Normally smears of spoiled product show a minimum number of organisms per field, not the abundance of organisms found in product exhibiting thermophilic flat sour-type spoilage. Vegetative cells are either straight or slightly curved rods usually with rounded ends. They range from 2 to 6!Jlll in length and 0.3 to 0.5 J.lll1 in diameter. They occur singly, in pairs and in short chains. The cells have peritrichous flagella which are responsible for the "twisting and tumbling" motility. Spores are oval, terminal and subterminal, and cause only a slight swelling of the cell. Staining characteristics In their original paper, Werkman and Weaver (17} described the organism as a gram-positive rod. They noted 1 to 6 gram-positive spherical granules in many of the cells. Sealey (13) described the organism as being so weakly gram-positive that for all practical taxonomic purposes, it may be regarded as gram-negative. Twenty-four-hour-old cultures and older cultures are almost uniformly gram-negative and show practically no granular staining. Campbell and Postgate (7} described the organism as being gram-negative. CULTURING Many of the earlier workers reported difficulty in culturing the organism. W erkman and Weaver (17}, after testing numerous media formulations, chose Beef Heart Infusion Agar with 0.1 o/o added ferric chloride and adjusted to ph 7.2 as a suitable stock medium. Use of shake tubes of this medium gave good colony growth; however, its use in plates incubated at 55 C in an atmosphere of hydrogen failed to produce growth. The pyrogallic acid - sodium hydroxide method was likewise unsuccessful. Studies by Bufton (2) and again by Postgate (12) concluded that all available media are unsatisfactory for providing accurate counts and recoveries of D. nigrificans. Lin and Lin {10) compared outgrowth and recovery of D. nigrificans in seven liquid and 10 solid media, and concluded that BETI (Beef extract, tryptone, iron) broth and BETI Agar showed a slight improvement in recovery and provided more rapid growth than other media. The use of Iron Sulfite Agar recommended by Cameron (3) has provided better recovery from spoiled product and ingredients than media proposed by earlier investigators. This medium, also recommended by Speck (14), contains Tryptone or Trypticase 10.0 g, sodium sulfite (anhydrous) 1.0 g, agar 20.0 g and distilled water JOURNAL OF FOOD PROTECTION, VOL. 44, FEBRUARY 1981

3 SULFIDE SPOILAGE ANAEROBES L. The medium has been used by the food industry for detection of H 2 S-producing anaerobes in starch and sugar for more than 40 years. Herson and Hullard (9), however, recommended using only 0.5 g of sodium sulfite in the same medium since it had been demonstrated that 0.1 o/o is inhibitory to certain bacteria. In our laboratory, best recoveries have been obtained with the use of Fluid Thioglycollate Medium plus freshly pickled iron strips or nails. The 15 to 20-ml quantities of the medium are sterilized in 20 x 150 screw-capped tubes. When ready for use, freshly pickled iron strips are rinsed in distilled water, flamed, and plunged hot into the tubes of sterile broth. We have found that medium without the iron strips can be stored longer than when the strips are added before sterilizing. We prefer iron strips to ferric citrate, which is frequently used in conjunction with sulfite agar. To analyze ingredients for sulfide spoilage organisms, the method originally described by National Canners Association (JJ) and later by Military Standard - Bacterial Standards for Starches etc., MIL-STD-900, 8 September 1958 is still recommended. The methods for sugar, starch, flour, nonfat dry milk and cream were detailed in Chapter 23 of the Compendium of Methods for the Microbiological Examination of Foods (14). Details of the methods follow. SUGAR, STARCH AND FLOUR [SUGAR: AOAC METHOD] For dry sugar, place 20 g of the sample into a dry sterile 2SO-ml Erlenmeyer flask with sterile rubber stopper. Add sterile water to the 100 ml mark and shake to dissolve. Replace the stopper with a sterile cotton plug and bring the solution rapidly to a boil, and continue boiling for 5 min. Replace the evaporated liquid with sterile water. Cool immediately in cold water. Prepare samples of liquid sugar the same way, except the amount added to the sterile flask should be determined, depending upon the Brix, to be equivalent to 20 g of dry sugar. In examining starch or flour, 20 g ofthe dry ingredients are placed in a dry sterile 2SO-ml Erlenmeyer flask, and sterile cold water added to the 100 ml mark, with intermittent swirling. Close the flask with the sterile rubber stopper and shake well to obtain a uniform, lump-free suspension of the sample in water. Sterile glass beads added to the sample water mixture will facilitate thorough mixing during shaking. Cultural methods When examining sugar, divide 20 ml of the heated solution among six 20 x ISO mm screw capped tubes containing approximately 10 ml of sulfite agar and a nail. Make the inoculations into freshly exhausted medium and solidify rapidly by placing the tubes in cold water. Preheat the tubes to SO to 55 C and incubate at that temperature for 24 and 48h. For starch or flour, divide 20 ml of the cold suspension among six 20 x lso mm screw capped tubes containing approximately 10 ml of sulfite agar and a nail. The tubes should be swirled manually and gently inverted several times before heating and during the 15-min heating period in a boiling water bath. The periodic swirling and inversion of the tubes will assure an even dispersion of the starch and flour in the tubes of medium. Following heating, cool the tubes immediately in cold water. Preheat the tubes to SO to 55 C, and incubate at that temperature for 24 to48 h. D. nigri:ficans will appear as jet black spherical areas, the color due to formation of iron sulfide. No gas is produced. Certain thermophilic anaerobes not producing H 2 S give rise to relatively large amounts of hydrogen which splits the agar and, with sulfite agar, reduces the sulfite thereby causing general blackening of the medium. Total the colonies in the six tubes. Calculate and report as number of spores per 10 g of ingredient. An alternative method of analysis, using thioglycollate agar in lieu of sulfite agar, has been used by some laboratories. NONFAT DRY MILK Weigh 10 g of the sample into a sterile 2SO-ml Erlenmeyer flask marked to 100 mi. Add N/SO sodium hydroxide to the 100 ml mark and shake to completely dissolve the sample. Heat lo min at 5 lb steam pressure, then cool immediately. Culturing methods Transfer 2 ml of the heated solution to each of ten 20 x 1SO mm screw capped tubes of freshly exhausted sulftte agar and nail. Gently invert several times and solidify rapidly by placing the tubes in cold 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 and report on the basis of 10 g of sample. CREAM 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. Transfer 20 ml of sample to a sterile 2SO-ml Erlenmeyer flask marked for 100 mi. Add the sterilized gum mixture to the 100 ml mark and carefully shake, using a sterile rubber stopper. Loosen stopper and autoclave for 5 min at 5 psi pressure. Culturing methods 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 nails. Gently invert several times and solidify rapidly by placing the tubes in cold 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 and report on the basis of 1 ml of sample. PRECAUTIONS AND LIMITATIONS OF PROCEDURES When analyzing ingredients by the above methods, thorough dispersion of the sample solution or slurry in each tube of medium is essential More difficulty will be encountered in the analysis of starch or flour because of the thickening effect during heating. Frequent swirling or gentle inversion of the tubes during the first 10 min of heating will assure proper dispersion. Since tubes containing numerous colonies of D. nigrificans may become completely blackened after 48 h of incubation, a preliminary count should be made after 20 to 24 ± 3 h. INTERPRETATION OF RESULTS A standard for sulfide spoilage spores need only apply to ingredients (sugar, starch, flour, etc.) to be used in low acid, heat processed canned foods. Sulfide spoilage spores shall be present in not more than 2 (40%) of the 5 samples tested and in any 1 sample to the extent of not more than 5 spores per 10 g. This would be equivalent to 2 colonies in the inoculated tubes. THERMAL RESISTANCE Generally speaking, the thermal resistance of D. nigrificans is greater than that of the thermophilic anaerobe Clostridium thermosaccharolyticum and less than the flat sour organism, B. stearothermophilus. In their 1927 publication, Werkman and Weaver (17) reported that 8 x 10 6 spores of D. nigrificans in pea medium adjusted to ph 7.0, survived 450 but not 465 min at 100 C (212 F). At 118 C (245 F), the spores survived SO but not 55 min. At 121 C (250 F), the spores survived 25 but not 30 min. In plotting these survival and JOURNAL OF FOOD PROTECTION, VOL. 44, FEBRUARY 1981

4 152 SPECK destruction points on sernilogarithrnic paper, a slope of 30 F is obtained. This seems highly unlikely so I must question the validity of the original data. In his later publication, Werkrnan (}6) expressed considerable difficulty in obtaining readings at temperatures of 245 and 250 F. He endeavored to generate data which could be used to show agreement with exposures at 212 F when the results were judged by logarithmic curves. In an attempt to avoid the problem, Werkrnan contrifuged his pea medium sufficiently to throw down particulate matter without af(ecting the suspended spores. The procedure reduced the resistance; however, at the higher temperatures the resistance still seemed high. Unpublished data by the writer showed that 10,000 spores of the NCA strain SS-3750 of D. nigrificans in phosphate buffer, ph 7.0 survived 250, 60 and 15 min at 230, 240 and 250 F, respectively. They were destroyed at 300, 80 and 20 min at the same temperatures, respectively. In plotting survival and destruction points on sernilogarithrnic paper, an F value of with corresponding z values of were obtained. Using Stumbo's method, ad of 2.6 and corresponding z of 16.3 were obtained. Studies reported by Donnelly and Busta (8) indicated that spores of highest heat resistance were produced using a 20% infusion of spent mushroom compost. D-values of 25.8 to 55.4 min were reported for strains of D. nigrificans in soy formula. Most data reported confirm that D. nigrificans is capable of surviving the standard commercial processes of most canned foods. Processing per se, therefore, cannot be relied upon to prevent sulfide spoilage in those products which normally fall in the ideal ph range. INGREDIENT AND EQUIPMENT CONTAMINATION Barlow (1) in 1913 demonstrated some of the fundamental causes of thermophilic spoilage. His detailed experiments showed that thermophilic spoilage in corn did not result from holding the raw product. His findings and resultant conclusions were either unknown or largely disregarded until 1926 when they were confirmed by Cameron et al. (4). These latter investigators concluded that, "when thermophilic spoilage occurs, it is due to contamination within the cannery or to contamination conveyed by something other than the basic raw product." In addition to the contribution of equipment, contamination from spores of spoilage therrnophiles could be found to a limited extent in soil, while refined sugar was found to carry contamination with greater frequency and generally to a much greater extent. Following these and similar studies, granulated sugar carne to be regarded as an outstanding source of thermophilic contamination. Although, the flat sour-type spores were found in essentially all of the cane-sugar samples that were examined, the thermophilic anaerobes, including sulfide spoilage spores were found in many samples and sometimes in undesirable numbers. Early in 1931 tentative specifications for canners' sugar were prepared and announced. Shortly thereafter, refineries which formerly furnished sugar harboring considerable thermophilic spores, produced a product practically free of spoilage producing spores. The significance of starch contamination by thermophilic spoilage organism was emphasized by Cameron and Williams (5) in It was demonstrated that contamination provided in starch will cause spoilage in canned cream-style corn. Spoilage from this source has also been traced in spaghetti and dog food. Although sulfide spoilage organisms are not as prevalent in starch as are flat sour-type spores, the ingredient can be a contributor to "sulphur stinker" spoilage. Following a number of publications, the production of low thermophile spore count starch became a reality. Producers of canned foods today must still scrutinize sugar and starch for thermophilic spores. This is particularly true of imported starches which may not be given the hydrogen peroxide sterilizing treatment. Other ingredient sources of sulfide spoilage-producing spores would include cream, condensed milk and nonfat dry milk solids. In considering all three types of thermophilic spoilage of canned products, flat sour bacteria (JJ. stearothermo philus) are most readily established in the canning plant and develop with greatest ease. Consequently, flat sour spoilage appears to be the most common. Possibly the greater resistance of spores to heat may be added to other factors contributing to this predominance. Sulfide spoilage, on the other hand, is the rarest and the organism responsible for it appears to be the least readily estabished in the canning plant. This may be due to, at least in part, the more exacting requirements for growth on the part of the organisms. Earlier mention was made of the investigations of Wilson and Tanner (18) wherein the low incidence (0.31 o/o) of sulfide spoilage organisms was compared to the high incidence (24.5%) of flat sour organisms in unprocessed cream-style corn. In the same survey, the investigators found less than 9.0% of the raw corn samples contained flat sour spores. No spores of sulfide spoilage organisms was detected. Although some increase in flat sour spore counts was evident in subsequent samples along the processing line, no spores of H 2 S-producers were encountered in any of the survey samples. Similar data were also presented from their survey of the canning of whole kernel and vacuum-pack corn. Peas, on the other hand, showed higher incidences of flat sour spores in both washed raw samples and in unprocessed live samples than were noted from studies of corn canning plants. Spores of sulfide spoilage organisms were lacking in all samples except that one sample in 9,654 processed cans showed these organisms. In their summary of these pea canneries, the authors stated "as in corn canning. thermophilic anaerobes not producing hydrogen sulfide and those producing hydrogen sulfide were only rarely demonstrated.'' The investigation of Cameron et at. (4) of pea packers in 1927 showed that all samples of overflow water from JOURNAL OF FOOD PROTECTION. VOL 44. FEBRUARY 1981

5 SULFIDE SPOILAGE ANAEROBES 153 the outlets of two blanchers were heavily contaminated with sulfide spoilage bacteria. One of the blanchers was new and had not been in use during the previous season. Since it was assumed that the bacteria were not normal inhabitants of new blanchers, the blanch water was suspected of providing a source of the bacteria. A survey of the blanch water system revealed that the deep well water eventually supplying the blanchers gave entirely negative results for sulfide organisms. A large overhead wooden storage tank used to supply cold water for washing requirements and also to supply another wooden tank directly below was also free of sulfide organisms. The second wooden tank was equipped with a closed steam coil and was used to preheat water supplying the blanchers. The temperature of this water varied according to the amount used for the blanchers but ranged between 105 and 150 F. A thorough bacteriological survey established that this wooden hot water tank was heavily contaminated with sulfide spoilage organisms, as well as flat sour spores. In addition, the brine tank supplied with water from the hot water tank was also found to be heavily contaminated. The wooden brine tank was not only used to prepare and hold brine but also sugar solution. The tank was most heavily contaminated at the startup in the morning. Counts of greater than 1,000,000 per can equivalent were not uncommon. It became apparent that the wooden tank acted like a sponge, soaking up the sugar and salt solution and that thermophilic spores could become established within the pores of the wood. Such microenvirons were immune from the usual cleaning process. Replacement of the two wooden tanks with galvanized iron tanks eliminated the sulfide spoilage problem. SPOILAGE PREVENTION As with other types of thermophilic spoilage, processing of canned foods cannot be relied upon to eliminate "sulfide stinkers." Because of the unusually high heat resistance of sulfide spoilage spores, a process which would be adequate to insure their destruction, would render the product unpalatable. Control can only be provided by: (a) ingredient (sugar, starch, nonfat dry milk, etc.) selection to assure freedom from or low numbers of spoilage-producing spores, (b) whenever possible, provide thorough cleaning and washing of vegetables, (c) if practical, presterilize ingredients (stocks, mushrooms, clams, etc.) before blending, (d) if not detrimental to the finished product quality, provide additional processing over the standard commercial process, (e) provide rapid and adequate cooling of processed product before casing and {f) set aside and incubate thermophilically samples representative of the pack to apprise sulfide spoilage potential. REFERENCES 1. Barlow, B Master of Science Thesis, University of Illinois. 2. Bufton, A. W. J A note on the enumeration of thermophilic sulfate reducing bacteria {Clostridium nigri:ficans). J. Appl. Bacteriol. 22: Cameron, E. J J. Assoc. Off. Agric. Chern. 21: Cameron, E. J., C. C. Williams, R. J. Thompson Bacteriological field studies in canning. Thermophilic contamination in the canning of peas and com. Bulletin 25-L, National Canners Assoc.. Washington. D.C. 5. Cameron, E.J., and C. C. Williams Significance of sugar and starch contamination. Canner, Convention Number, February 27, Campbell, L. L., Jr., H. A. Frank, and E. R. Hall Studies on thermophilic sulfate reducing bacteria. I. Identification of Sporo vibrio desulfaricans as Clostridium nigrificans. J. Bacteriol. 73: Campbell, L. L., and I. R. Postgate Classification of the sporeforming sulfate-reducing bacteria. Bact. Rev. 29: Donnelly, L. S., and F. F. Busta Abstracts of the annual meeting of the American Society for Microbiology. p Hersom, A. C., and E. D. Hulland Canned foods, an introduction to their microbiology. Chemical Publishing Co. p Lin, C. C., and K. C. Lin Spoilage bacteria in canned foods. II. Sulfide spoilage bacteria in canned mushrooms and a versatile medium for the enumerations of Clostridium nigrificans. AppL Microbiol. 19: National Canners Association Research Laboratories Laboratory manual for food cancers and processors, Vol. 1. The AVI Publishing Co. Inc., Westport, Connecticut. 12. Postgate, J. R Media for sulphur bacteria. Lab. Prac. 15: Sealey, J. Q The biology of Clostridium nigrificans, PhD. thesis, University of Texas. 14. Speck, R. V Sulfide spoilage sporeformers. pp In M. L. Speck {ed.) Compendium of methods for the microbiological examination offoods. American Public Health Assoc.. Washington, D.C. 15. Starkey, R. L A study of spore formation and other morphological characteristics of Vibrio desulfuricans. Arch. Microbiol. 9: Werkman, C. H Bacteriological studies on sulfide spoilage of canned vegetables. Iowa Agr. Exper. St. Res. Bull. 117: Werkman, C. H., and H. J. Weaver Studies in the bacteri ology of sulphur stinker spoilage of canned sweet corn. Iowa State College J. Sci. 2: Wilson, C. M., and F. W. Tanner Microbiology of canned corn and peas. Food Res. 13: JOURNAL OF FOOD PROTECTION, VOL. 44. FEBRUARY 1981