July, 1919
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TABLEVI Grams per 100 c c . -DISTRIBUTION OF ACIDCalc. as Acetic Acetic -DISTRIBUTIONOF ALKALINaOH a t ApproxiAcid in Acid Alkali Alkali A!kali Time mate Acetic Mother in Mother in in in Vol. a t of Temp. a t Vol. of Volume Acid in Alkali Liquor and CrysAcetic Liquor Crys- Mother Wood Time of Crys- Crystalli- Mother of Washings Mother Acid in Washings and tals Liquor in Washings Washings t als Used in Crystalli- talli- zation Liquor I Cc. Liquor c Per cent Washings Per Per --Per centPer Per RUNNo. Fusion zation zation Dee. C. Cc. 1 2 3 Per cent 1 2 3 cent cent 1 2 3 Per cent cent cent 10 1 Oak 660 28.8 258 212 186 202 65.6 22.4 7.4 2.3 97.7 2.3 21.8 14.2 10.9 10.1 57.0 43.0 Elm 1150 24.9 352 52.4 2 376 278 330 13 55.1 30.9 1 1 . 1 3 . 6 98.1 1.9 14.3 12.1 8 . 5 10.0 44.9 9.50 30.1 298 60.5 25 3 242 179 177 27.8 6.1 2.5 96.9 3.1 17.8 15.4 12.2 10.3 55.5 Elm 44.5 1250 30.0 415 61.4 15.5 41 340 298 250 22.8 9.0 2.7 Elm 55.1 95.9 11.6 1 0 . 5 4.1 14.9 7.6 44.9 1050 35.0 292 20.0 386 252 401 Elm 5’ 56.6 2 9 . 0 10.4 2 . 8 1.2 11.5 1 7 . 6 18.4 21.0 68.4 31.6 98.8 25.0 925 30.9 290 62 258 216 225 76.5 16.6 2.9 2 . 6 34.0 98.9 1.0 22.9 1 7 . 4 1 3 . 3 12.4 66.0 Elm 950 27.4 295 17.0 MaDle 58.1 7 255 256 236 25.4 11.8 3 . 0 98.3 1.7 16.7 15.1 13.9 11.7 57.5 42.5 1 Selectivelwashing employed. Centrifuged instead of filtered.
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*
i n maintaining the reduced temperatures employed in some of the runs. Results obtained under varying conditions are given in Table VI. From 96 t o 99 per cent of the acetic acid is separated from the crystals by three washings, while 4 5 t o 68 per cent of the alkali is also carried into solution, the amounts depending upon the temperature a t which t h e process of separation is carried out and the volume of wash mater used. To make the operation successful with respect t o the recovery of alkali it would be necessary t o remove a’ much larger percentage of the alkali from the mother liquor and washings. This could be done by selective washing as carried out in Runs 4 and j on elm. I n these runs the following scheme was followed: I-First washings from previous runs added t o original solution, t h e solution treated with carbon dioxide, concentrated, and the carbonate “seeded” out. ;.--Second washings from previous runs used for first washing. 3-Third washings from previous runs used for second washing. 4-Fresh water used for third washing. Run 4 resulted in 95.9 per cent of the acetic acid going into the mother liquor and washings, and represents the amount of acetic acid which would be recovered unless one or more additional washings with pure water proved feasible. Since the washings obtained would be used for washing in subsequent runs, t h e only alkali lost would be t h a t contained in the mother liquor, which in this case amounts t o 14.8 per cent of the total alkali contained in the original ,solution and wash water used, or 2 0 . 1 per cent of the alkali in t h e original solution. This loss of alkali could be further reduced by cooling the mother liquor. I n the case under consideration, if the 415 g. of mother liquor were cooled t o oo, only 2 2 . 2 g. of alkali, calculated as sodium hydroxide, would remain in solution, representing a loss of 7.77 per cent of the alkali used. A similar result could be obtained by concentrating t h e mother liquor t o approximately I O O cc. and allowing the carbonate in solution t o crystallize a t room temperature. To check the above conclusions, the mother liquor in Run 5 was cooled t o o o C. and the carbonate crystals formed filtered off. I t was found t h a t the loss of alkali could be reduced in this way t o 9 per cent of the original amount used, 5.4 per cent of which is in combination with the acetic acid present in the mother liquor. I t seems probable, therefore, t h a t a
recovery of alkali of 8; t o 90 per cent could be ob: tained without difficulty . The runs on the different species were made primarily t o determine what effect, if any, the organic matter in solation or suspension might have in carrying out the separation of acid and alkali. Any effect this material has is negligible, and since i t is concentrated in the mother liquor the crystals are probably sufficiently free from organic matter t o make calcining unnecessary before causticizing. SUMMARY
The data given show t h a t 17 t o 2 0 per cent of acetic acid can be obtained from hardwood sawdust by fusion with sodium hydroxide. A simultaneous production of oxalic acid amounting t o approximately 50 per cent of the dry weight of the wood is obtained. If the reaction is carried out in a closed vessel, a simultaneous production of methyl alcohol results amounting t o 2.4 per cent; but, as t h e temperature is increased beyond 200°, the yield of oxalic acid is considerably reduced. At lower temperatures both formic and acetic acids are produced, amounting t o approximately 1 5 per cent each. Higher yields of oxalic acid t h a n those obtained b y Thorn‘ with pine wood and caustic soda have been obtained. It appears that- the yields of this acid obtained with caustic soda will more nearly approach those obtained with caustic potash if the temperature is kept a t approximately zoo0, the heating prolonged, and the ratio of alkali t o sawdust maintained a t 3 :
I.
Somewhat lower yields of acetic acid than those obtained by Cross, Bevan, and Isaac2 with caustic potash have been obtained using caustic soda. It has been found possible t o recover as much as 91 per cent of the alkali used. FOREST PRODUCTS LABORATORY UNIVERSITY OF WISCONSIN MADISON,WISCONSIN
I N C 0 6 P B R A T I O N WITH THE
AN AEROBIC SPORE-FORMING BACILLUS TN CANNED SALMON By ALBERTC. HUNTERA N D CHARLESTHOM Received April 19, 1919
Failures in the sterilization of canned goods are well recognized from the occurrence of swelled cans, cans which give out foul odors when opened, or of glass jars in which there is turbidity or other visible 1
LOG
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evidence of decomposition. The. presence of living bacteria in such products without active spoilage is less well known, but accounts for some of the discordant bacteriological results which have been frequently attributed t o errors in manipulation. Such living bacteria may bear no relation t o the present condition of the product whether sound or spoiled. Bacteriological examination of about 500 cans of salmon has furnished a striking example of such survival. The salmon examined represented nine brands packed a t different factories and included salmon designated by the trade as red or sockeye salmon, coho or silver salmon, chum or dog salmon, and humpback or pink salmon. Routine examination designed t o test sterility was first applied. When living organisms were found, all species were isolated and studied t o ‘test their relationship t o the conditions met with. The large number of unsterile cans encountered, 44.7 per cent (Table I ) , forced a careful discrimination between unsterility and active spoilage. The cans were first examined for leaks or dents and as t o whether or not they were “swells” or “springers.” They were then opened aseptically by means of a flamed can opener. By means of a sterile pipette, from 5 t o I O mm. in diameter and not tapered a t the end, a small amount of the salmon was transferred t o each of two Petri dishes. Care was taken t o obtain some of the flesh together with the liquor. Small amounts were also carefully placed in each of two test tubes and about 5 g. in dextrose broth. Dextrose agar was poured over the fish in the Petri dishes and the test tubes and all cultures were incubated a t 37’ C. for 4 days. Notice was taken of the odor and general appearance of the fish in each can and a record of this was kept with the bacteriological data. Table I gives a summary of the cans examined and the number and per cent of cans found not sterile. TABLEI Number Examined BRAND 202 A... 66 B... c . .* . . . . . . . . . . . . . . . . . 72 32 D... 32 E.. . 24 F. 24 G. H. . . . . . . . . . . . . . . .?. 32 I.... 46
................. .................
................. ................. ................. .. ................. .. .. ................. TOTAL ......... 530
Number Not Sterile 110 38 31 19 19 131 7 0
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Per cent N o t Sterile 54.4 57.5 43.05 59.37 59.37 54.1 29.1 0 0
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237 44.7 1 These 13 cans were “swells” and did not show the characteristic organism discussed in this paper.
A study of this table shows t h a t nearly one-half of these cans of salmon were found t o contain living bacteria in contrast t o the findings of Weinzirll who reports very few unsterile cans of salmon. I n some brands t h e proportion of non-sterile cans is greater t h a n 5 0 per cent, while in other cases, for example, the fish from Brands H a n d I, all the cans examined were found t o be sterile. The presence of these living bacteria has little significance as t o the quality of the product a t t h e time the can is opened, since some of the sterile cans were found, on chemical examination, t o contain putrid and decomposed fish, while many of 1
“The Bacteriology of Canned Foods,” J . M e d . Res. 39 (1919),
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the cans from which bacteria were grown contained fish apparently sound. Comparative study of the organisms found in these cans showed t h a t a particular variety had been found in 2 2 4 out of 2 3 7 non-sterile cans, or 42.2 per cent of all cans examined. The 13 unsterile cans failing t o show this species were t h e “swells” belonging t o a single group of Brand F. Cans showing a mixed flora are not uncommon but in many cases this form alone survived the processing. This organism was first isolated from still another lot of salmon in this laboratory b y Mrs. Obst during the summer of 1918. The frequency of this species in salmon, together with its survival in commercially processed goods, makes a study of its characteristics especially important. The other organisms found were isolated €or s t u d y but thus far have furnished no significant information. DESCRIPTION OB THE ORGANISM
The morphological, cultural, and biochemical features of this organism as described here indicate t h a t it i s probably a member of the mesentericus group, although not clearly identified with any of the well-known species. Bacillus 3.2 p X 0.7 p Forms spores within 24 hrs. Grows on all media Produces red ring in dextrose agar Growth on nutrient agar slant along streak, wrinkled, gray N o gas in dextrose and lactose broth with and without fish
No in tryPtophan broth
MORPHOLOGY Spore-forms measure 1.6 p X 0.8 p
Motile Gram-positive
CULTURAL CHARACTERS Growth on dextrose Pellicle on dextrose agar spreading* broth, medium dark tough and wrinkled On dextrose agar plates brown growth entire Gelatin liquefied surface of plate Litmus milk peptonHeavy pellicle in plain ized turned brown iru broth, medium clear 48 hrs. BIOCHEMISTRY Acid produced in dextrose and lactose broths. Later turns alkaline 2.8 per cent normal acid produced in
dextrose broth plus.
fish. Much less i n lactose broth Spores of this organism very resistant to heat
The outstanding feature of this organism is t h e .production of a dark red ring when grown on solid carbohydrate medium. This ring is produced in t h e medium a t about a half-inch from the growth on t h e slant or from the growth on the surface of the column of agar. When sterile fish is present in the medium, this red color is often imparted t o it and a marked softening of the fish takes place. No such red ring is obtained when the organism is grown on plain agar. The action of this organism on sterilized salmon is. very marked. Approximately joo g. of fish were ground in a food chopper and sterilized for 50 min. a t 1 5 lbs. pressure. This fish was inoculated with I O cc. of a @-hour broth culture of the organism a n d incubated a t 37’ C. for one week. Within t h a t t i m e the fish became very soft, in fact, liquid, and gave off a very offensive odor. The color of the decomposed fish was a dark brown. Chemical examination1 of the decomposed salmon in this particular case failed t o show the presence of indol, skatol, or hydrogen 1 Determinations made by Dr H W. Houghton, of the Food Investigation Laboratory, Bureau of Chemistry.
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sulfide, although a very pronounced test for methyl mercapean was obtained. Further work, however, is necessary t o establish the correlation between the action of this organism on the salmon and the decomposition products resulting therefrom. The spores of this organism are resistant t o heat and in broth culture have been found t o survive heating in the Arnold sterilizer a t 100’ C. for one hour. Broth cultures of the spores survived 1 5 lbs. pressure in the autoclave for I 5 min., but were killed a t I 5 lbs. pressure for 3 0 min. It is very evident t h a t these forms when embedded in the center of the salmon within the can may survive the temperature of processing in the cannery. Study of the detailed records summarized in Table I shows t h a t some cans of Brands H and I, in which all cans were found sterile, consisted of markedly decomposed fish. I n the other samples with part of the cans sterile there was no correlation between sterility and the soundness of the fish except in Brand F, in which active spoilage is reported. An obligate adrobe such as this may be found in viable condition in a can properly sealed and showing no apparent spoilage. I n fact, organisms of several species were found in cans of fish in which no physical or chemical evidences of spoilage were detected. Some cans containing soft and obviously putrid fish contained no living organisms. The presence of these organisms may result in quick decay when opening the can supplies the oxygen needed for activity. On the other hand, their presence may or may not supply information as t o the previous history of the material canned. Such correlation depends upon the establishment of a correlation between a particular microorganism and a condition or process. The salmon organism is a heat-resistant spore-former capable of decomposing salmon with great rapidity, but its exact source and significance in the canned products remains t o be demonstrated. The survival of such an organism shows t h a t the method of cooking or processing the commercial product has failed t o produce sterility. The temperature may not have reached the proper limit, the period may have been too short, or the heat may not have been evenly applied, hence some cans escaped the adequate cooking which sterilized the remainder. The large number of unsterile cans found among well-known brands of salmon points t o a widespread failure in factory operation. Sterility merely means t h a t whatever organisms may have been present in the material a t some time are now dead. The material itself may have been rotten or putrid from the activity of these organisms before canning, hence totally unfit for food. Some of this salmon was manifestly putrid but not in active spoilage as it came from the can. The evidences of decay were, however, manifest t o sight and smell. I n specially prepared foods these evidences are commonly masked or more or less completely destroyed. Jams, jellies, apple butter, canned pumpkin, mince meat, soup stock, and tomato products such as catsup, pulp, and paste, may be made u p in large part of
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decomposed material yet so comminuted and so flavored in cooking as t o leave neither visible evidence nor flavor t o guide the consumer who would unhesitatingly reject the raw materials if he could examine them. Yet these products are commonly sterile. Sterility is not t o be confused with fitness for food; i t does establish the presumption t h a t t h e material under examination is in the same condition as when packed. Similar observations have been made in this laboratory in a considerable variety of canned foods. Living bacteria rather frequently are found in canned foods which show no‘ signs of decomposition which are apparent t o the senses. The organism described in this paper is only one of many forms, both cocci and bacilli, isolated from canned products apparently sound. This leads t o the conclusion t h a t lack of fermentation or active spoilage of canned food is not a guarantee of sterility. This condition in sardines is discussed by 0bst.l The bacteria in the product may be in a dormant state while in the can and only grow when more favorable conditions are supplied. Many of the bacteria isolated, as the organism described above, are obligate aerobes and could not grow under the anaerobic conditions occurring in a properly exhausted and sealed can. Processing, t o use the commercial term, should then be distinguished from sterilization. SUMMARY
Bacteriological examination of 530 cans of salmon, representing g brands, showed 23 7 unsterile cans. 2 2 4 of these cans contained the same organism of the mesentericus group either in pure culture or in connection with other species. Only 1 3 of these cans showed active spoilage. The organism is a n obligate aerobic spore-former, gram positive, motile, and produces a dark red ring about a centimeter below the colony in carbohydrate media. I t decomposes fish rapidly. Such obligate aerobic spore-formers may be present in Tiiable condition in canned products without any appearance of spoilage. Actual sterility is very properly the aim of the packer. The survival of viable organisms in the final product may occasionally be unavoidable but calls for a careful survey of their source and significance with a view t o their complete destruction. BUREAUOF
CHEMISTRY
DGPARTMENT OF AGRICULTURE
WASHINGTON, D. C.
TEMPERATURE-TIMERELATIONS IN CANNED FOODS DURING STERILIZATION By GEO.E. THOMPSON Received June 26, 1918 I-INTRODUCTION
It is well known t h a t the death rates of the bacteria which caused spoilage in canned foods depend on the temperature and composition of the substrate. As these death rates, under varying conditions of temperature and substrate, become accurately established, a knowl1
“A Bacteriologic Study of Sardines,” J . Infect. Dis., 24 (1919), 158.
