‘lrl 1! llltl MI! I I I I 1| ) 0.. \JN H 04:. THS_ THE INFLUENCE OF CARBON DIOXIDE ON FOOD-POISONING ORGANISMS Thesis for the Degree of M. S. MICHIGAN STATE COLLEGE Marvin H. Ruster 1940 The Influence of Carbon Dioxide on Food-Poisoning Organisms THE INFLUENCE OF CARBON DIOXIDE ON FOOD-POISONING ORGANISMS by Marvin H. Buster A THESIS Submitted to the Graduate School of Michigan ’ State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Hygiene 1940 THESIS ACKNOWLEDGHMENT The writer wishes to express his sincere appreciation to Dr. W. L. Hallmann for his ad- vice and guidance throughout this work. He al- so desires to thank the following manufacturers for providing the equipment and materials that have made this work possible: General Motors Corp., Frigidaire Div., Dayton, Ohio; Harrisburg Steel Corp., Harrisburg, Pa; White Mfg. Co., St. Paul, Minn; Michigan Alkali 00., wyandotte, Mich. 199118 iv TABLE or comrahrs Title Acknowledgement I. Introduction II. Historical A. The Bactericidal.mffect of Carbon Dioxide B. Carbonated waters and Beverages C. The Carbonatibn of Dairy Products D. Carbon Dioxide as a Meat Preservative E. Carbon Dioxide as a mycostatic Agent E. The Nature of the Inhibitory Action of Carbon Dioxide ‘c. The Stimulating Effect of Carbon Diaxide ;H. The Influence of Carbon Dioxide on Toxin hProduotion and Preservation I. Carbon Dioxide as an Essential Growth Eactor J. The Role of Carbon Dioxide in Bacterial Metabolism III. History of Cultures IV. .Experimental V. Discussion and Conclusions VI. Citation of the Literature I. INTRODUCTION With the exception of the disease known as botulism, most cases of food poisoning result from eating meat in- fected with certain members of the Salmonella group or an enterotoxic substance elaborated by staphylococci. Such meat may come from an animal infected during life, or from a healthy animal, and be contaminated by contact with rats or mice or through handling by human carriers during the course of processing or preparation for eating. If living organisms are present, infection may result and cause a slow after-development of symptoms. Even though these organisms may be destroyed during the process of cooking, their toxins are frequently thermostable. Therefore, meat which has been the site of bacterial de- veIOpment, may give rise to sharp outbreaks of food poi- soning characterized by acute gastrointestinal symptoms. From the standpoint of prevention, rigid government inspection has been advocated as the only means whereby such outbreaks may be prevented. It must be remembered, however, that since these organisms are found even in healthy animals, it is highly questionable whether any a- mount of inspection would entirely eliminate the danger. How great the danger is it is rather difficult to determine. Since the great majority of food poisoning cases are gen- erally of a mild character, and of short duration, they are never heard from beyond the immediate family circle. vi Only when the attack is more serious than the average or when a large number of persons are affected simultaneously does knowledge of its occurrence become more widespread. .A very small proportion of food poisoning cases receives notice in the public press, a still smaller proportion is reported in the scientific Journals, while very few are thoroughly investigated as to their origin. 36 this as it may, the safeguarding of the healthful-- ness of the public food supply constitutes one of the most difficult yet one of the most fundamental problems of public health. So, in Spite of the great advances in scientific methods of food preservation, the food technologist is still confronted with one very important question, namely, what is the effect of the particular method of preservation on the viability of pathogenic organisms which may be present in or upon the food? ' During recent years, considerable work has been_done by English investigators at the Low Temperature Research Station at Cambridge, England, on the application of carbon dioxide gas as a supplement to cold storage in the preserva- tion of meat and meat products. In this country, most of the work thusfar has been carried out with fruits and veg- etables, but at present the method is also being applied to meat, poultry, fish, eggs and dairy producha According to the literature, it has been conclusively demonstrated that carbon dioxide is a vital factor in the growth of practically all bacteria. In addition, a 10 per vfl. cent concentration of this gas is generally recognized as having a stimulating effect on the growth of such disease- producing organisms as the meningococcus, gonococcus, staph- ylococcus, the tubercle bacillus, Brucella abortus and the _Salmonella group. Moreover, it has been well established that the toxi- city of Staphylococcus aureus is increased by growing the organism in the presence of 10 to 25 per cent carbon di- oxide. The presence of 10 per cent carbon dioxide has also been known to stimulate the growth and toxin production of Salmonella aertrycke. Finally,-carbon dioxide has been re- ported to have a preservative effect upon the toxins of path- ogenic organisms after they have once been formed thus pre- venting detoxification through oxidation by molecular oxygen. From the standpoint of public health, all this is very significant, for it would seem to indicate that the appli- cation of carbon dioxide to the preservation of foodstuffs would be an exceedingly dangerous practice inasmuch as it would not only tend to promote the growth of pathogenic or- ganisms but increase the toxin production of staphylococci as well. It should be remembered, however, that all of the studies which have been cited relative to this point were conducted at 57°C. Nevertheless, there has been a fear on the part of those concerned with the processing of meat and meat products that the introduction of carbon dioxide into the meat storage re- frigerator would tend to stimulate the growth and toxin viii production of food-poisoning organisms commonly found on meat and thus create a serious health hazard. Therefore, a series of tests was made, using various members of the Salmonella group and staphylococci as test organisms, to determine the effect of carbon dioxide at both high and low temperatures on the viability of these organisms. Special attention was directed to moderate concentrations of carbon dioxide at low temperatures. -1- II. HISTORICAL The influence of carbon dioxide on the growth of mi- cro-organisms has been exhaustively studied by a great num- ber of investigators, and in 1928, Valley'(l4) compiled a most excellent and comprehensive, chronological review of the work which had been done on the subject up to that time. During the 12 years which have elapsed, many valuable ad- ditions to our knowledge of the subject have been made es; pecially with respect to the practical application of car- bon dioxide in the preservation of foodstuffs.. .At present, studies relative to the preserving and tenderizing action of this gas on meat are in progress in this laboratory. As the result of constant research in this field, the existent literature has assumed vast preportions. Yet no systematic endeavour has been made to present an all-inclu- sive and up-to-date resume of this material. Therefore, for the sake of completeness, and somewhat for the feeling of making this information more readily anessible to future investigators, the whole subject has been unified and brought up to date. With this thought in mind, both the theoretical and practical sepects of the subject are presented in the following historical review. A. The Bactericidal Effect of Carbon Dioxide The first attempt to make a practical use of the anti- septic preperties of carbon dioxide was made by Barthell, (1) in 1848, when he patented a process for preserving milk -2- by means of carbonation. Many years later, Pasteur and his cc-worker Joubert (9) (1877) claimed that carbon dioxide had a lethal effect on Bacillus anthracis. This was con- trary, however, to the observation of Szpilman 05) (1880) who reported that 5 to 8 hours' exposure to pure carbon di- oxide did not kill this organism nayalter its pathogeni- city. .Likewise, Grossmann and mayerhausen (5) (1881) main- tained that carbon dioxide does not kill bacteria but merely “inhibits their motility while minimal amounts accelerate it. Frankle (5) (1889) studied the influence of carbon di- oxide on the growth of various organisms in liquid and solid media. He observed that the true beer yeasts were able to grow as well in 100 per cent carbon dioxide as in air but the majority of the parasitic and sapr0phytic forms failed to sur~ vive. Since these organisms again resumed their normal growth upon subsequent exposure to ordinary air, Frankle concluded the effect was not of a lethal nature but merely inhibitory. Like Frankland, (4) (1889) be emphasized the fact that even members in the same culture (aside from Spore forms) may vary in their susceptibility to the action of carbon dioxide. According to Schaffer and Freudenrich, (12) (1891) broth cultures of typhoid and anthrax bacilli were unaffected by 7 atmOSpheres of carbon dioxide. (Moreover, Sabrazes and Ba- zin (11) (1895) demonstrated that a concentration of 95 to 97 per cent carbon dioxide at 90 atmospheres pressure and be- yond did not prove injurious to Staphylococcus aureus, Agra- bacter aerogenes and B. anthracis inasmuch as these organisms -5- grew unhindered in both plain agar and that containing ad- ded buffer. In a study of the effect of carbon dioxide on members of the colon-typhoid group, Hoffman (7) (1906) obtained steri- lization of water suspensions of Escherichia 221i and the dys- sentery bacillus with 50 atmospheres of carbon dioxide in from 2 to 5 hours. Berghaus (2) (1907) considered the effect of carbon dioxide upon various organisms which he subjected to carbon dioxide treatment on freshly poured agar plates at 57°C. He found that Vibric 19.12.”; was killed in 24 hours at atmOSpheric pressure, B. anthracis was inhibited but not killed, while Egg}: ggli and Salmonella enteritidis were able to develop under a pressure of 2 atmOSpheres of carbon di- oxide. Plummer do) (1913 observed that the ammonifying bac- teria were very insensitive to the action of this gas since a concentration of 60 per cent had no appreciable effect on the rate of ammonification. .Althcugh carbon dioxide of less than 40 atmospheres had no effect whatsoever on the organisms studied, Larson et a1. (8) (1918) found that when they in- creased the pressure to 50 atmospheres, they were able to de- stroy Eberthella typhosa, High. gq]_._i_, mcobacterium M- ouldsis, staphylococci, streptococci and pneumococci in 1% to 2% hours. Yeast cells, however, remained unaffected. after 48hours. According to Valley and Rettger, (15) (1927) such or- ganisms as §3222° aureus, Salmonella paratzphi and Salaa- nella schottmuelleri were able to grow equally well both in plain agar and phosphate-buffered media in 95 to 97 -4- per cent carbon dioxide. ~ In~buffered media, such delicate organisms as X} ggmma could develop in atmospheres of car- bon dioxide as high as 70 to 75 per cent. More recently, in their experiments with Eggh,‘ggli and Eberth. typhosa Guillerd and Lieffrig (6) (1955) have shown that carbon dicx-_ ide has no appreciable bactericidal action on water-borne bacteria. B. Carbonated waters a Beverages much of the earlier knowledge concerning the germici- dal value of carbon dioxide was derived from a study of the behaviour of micro-organisms in carbonated waters and bever- ages. Leone (6) (1886) succeeded in reducing bacterial de- ve10pment in the city water supply of Munich by means of car- bonation. Likewise, Hochsetter (5) (1887) reported that,1. gggga was unable to survive in carbonated waters but Eberth. typhosa remained viable after 5 to 7 days. Scalla and San- felice, (7) (1891) on the other hand, demonstrated that path- ogers like 1. m, B. anthracis, mg. aureus and mg. glpgg and Eberth. typhosa were unaffected by quantities of carbon dioxide naturally dissolved in water at 15°C. Acid- sensitive organisms like 1. ggmma were injured when the water was saturated with carbon dioxide but.the others remained un- affected. Carbon dioxide introduced into soda water under pressure proved injurious to Bacillus subtilus and the spores of this organism as well as those of B, anthracis failed to germinate, but Proteus remained unharmed. In his study of -5- artificial carbonated waters, Slater (8) (1895) found car- bon dioxide had a germicidal or inhibitory effect on @ggh. 'ggli,ijerth. typhosa,.1.'ggmma and.§£§22° aureus but the effect varied with the organism in question. , Colin (1) (1915) performed a number of experiments with water suspensions of organisms which he subjected to varying pressures of carbon dioxide. Eberth. typhosa was killed in 20 hours at 10 kilos. pressure, while Eggh..ggli remained vi- able after 5 days under 25 kilos. pressure and sterilization of Eggynebacterium diphtheriae was obtained in 5 hours at 20 atmOSpheres. In their studies on the behaviour of the colon- .typhcid group in carbonated waters and beverages, Kbser and ‘ Skinner (5) (1922) found that gggh..ggli remained viable in non-acid beverages after 7 days under 28 pounds pressure at 24°C., but agar plates were sterile after 5 days. .At room temperature, Eberth. typhosa was destroyed in 24 hours under 24 pounds pressure, but at 1°C. it persisted for 4 days. §§;. schottmuelleri was viable after 48 hours at 24°C. and 25 pounds pressure, and was still alive after 10 days at 1°C. Bacillus mesentericus and B. anthracis spores were unaffected in carbonated water even after 1 month. Donald et a1. (2) (1924) have pointed out that the decline in bacterial numbers in carbonated beverages is directly proportional to high pres- sure and storage. .More recently, Kliewe and Kindhauser (4) (1955) have shown that the germicidal action of carbonic acid on water vibrio, colon and typhoid bacilli, and on Breslau, suipestifer and Schottmueller types of paratyphoid Organisms, p -0- was enhanced by raising the temperature to 57°C. but was al- most entirely absent at 18°to 20°C. C. The Carbonation of Dairy Products On the whole,the reduction of microflora by the carbo- nation of such food products as milk, ice cream and butter has been attended with very little success. Still Hoffman (1) (1906) maintained that he was able to obtain a material reduction of members of the colon-typhoid group after 48 hours by employing 5O atmospheres of carbon dioxide. But this is contrary to the observations of Van Slyke and Bos- worth (6) (1907) who claimed that the carbonation of milk without added pressure had no preservative value although lactic acid fermentation was slightly delayed. Likewise, Prucha and his associates (3) (1922) found that carbon di- oxide had no germicidal action on Eggh.,ggli or Eberth. 31? phgga in milk subjected to 10 to 50 pounds extra pressure, while at 20 pounds pressure the bacterial count actually rose from.47 to 155 million organisms per cc. They further showed that carbon dioxide does not cause a reduction of bacteria found in ice cream.‘ Moreover, in their experimental work on carbonated raw milk and ice cream, valley and Rettger (5) (1927) found that atmospheres of 95 to 97 per cent car- bon dioxide under ordinary atmOSpheric pressure had little or no inhibitive or bactericidal effect. Hunziker (2) (1924) has shown that butter cannot be suc- cessfully preserved by carbonation, while Prucha et a1. (4) (1925) state that if the process is to be of any value, the -7- the product should be stored in an air-tight container. D. Carbon Dioxide as a Heat Preservative Kblbe (15) (1882) first observed the preservative action of carbon dioxide on meat; he successfully preserved beef for 18 days at 52°C. in an atmOSphere of carbon dioxide, and even after 4 or 5 weeks, there was no evidence of putrefaction, but the meat was unpalatable when cooked. Killefer (12) (1950) studied the effect of carbon dioxide on meat and fish and presented bacteriological data on the reduction of bac- terial infection by the use of this gas. His work showed that meat and fish could be kept longer in an atmOSphere of carbon dioxide, even at relatively high temperatures, than in air. Although pork and lamb kept at 4.5'tc 7.2°C. Spoiled after 10 days in air, no deterioration occurred after_5 weeks in carbon dioxide. Similarly, Callow (3) (1952) preserved pork in perfect condition for over 2 months at 0°C. in an atmosphere of carbon dioxide. Even after 70 days, it was more palatable than the fresh product while pork kept in air spoiled in 17 days. He more than doubled the storage-life of mild-cured green bacon at 5°C. by employing an atmosphere of carbon dioxide. Culture medium heavily seeded with pork- Spoilage organisms showed no growth at -l°C. in the presence of carbon dioxide after 50 days, whereas in nitrogen and air, there were signs of growth after 4 days. Lea (l4, 15) (1955) demonstrated that the development of rancidity in beef fat stored at 0°C. was greatly retarded by 10 percent carbon -8- dioxide, the time required in a saturated atmosphere for the production of a perceptible off flavor being approxi- mately doubled in the presence of 10 per cent carbon di- oxide. This preservative effect was more marked when the meat was placed in an atmosphere of carbon dioxide shortly after slaugmer and the humidity reduced to 90 per cent. Em- pey and Vickery (9,10) (1955, 1954) succeeded in extending the storage-life of chilled beef 40 per cent by employing an atmOSphere of 10 to 12 per cent carbon dioxide. These workers found that with but slight initial contamination, chilled beef could be held for 55 days in an atmosphere of 12 per cent carbon dioxide, while beef stored in an atmos- phere of 11 per cent carbon dioxide showed no deterioration 44 days after slaugher and the bloom remained unimpaired. Likewise, Coyne ( 6, 7 ) (1952, 1955) found that various types of fresh fish could be kept in 20 per cent carbon di- oxide as long as 28 days without serious spoilage, while con- trols stored in air were inedible after 12 to 14 days. He also studied the influence of various concentrations of car- bon dioxide (5 to 100 per cent) at 03 lo: 253 and 57°C. on micro-organisms responsible for slime formation on chilled beef. Carbon dioxide suppressed the growth of Achromobacter, Flavobacterium, Micrococcus, Bacillus and Pseudomonas, but had no effect on Aerobacter and Proteus. This is in accord with the findings of Chistyakov (5) (1955) who reported that practically all putnfactive bacteria responsible for the spoilage of meat, fish and other food products were inhibited -9- by carbon dioxide. Moreover, these observations have been confirmed by Haines (11) (1955) and more recently, by Mall- mann and Zaikowski (17) (1940) in this laboratory. Callow (4) (1958), however, reports that pork stored in 10 to 15 per cent carbon dioxide at -10°C. for various periods up to 22 weeks showed considerable loss in weight and appreciable bacterial spoilage; I According to Brooks, (1) (1955) the color and bloom of fresh beef is not affected by concentrations of carbon di- oxide below 20 per cent. Nevertheless, Brooks and Moran (2) (1955) have pointed out that the gaseous storage of meat may be limited by the rate of absorption of carbon dioxide. _They claim that the commercial use of carbon dioxide requires equilibration for at least 5 days with 100 per cent carbon dioxide before storage in the final 20 per cent mixture. This, however, results in a marked discoloration of the pro- duct. Although gaseous storage at 0°C. practically eliminates mold and bacterial growth in chickens, Lea (15) (1954) has shown that autolysis of the tissues by enzymatic action pre- vents the extension of the storage-life of undrawn birds. Ox- idative rancidity also may be a contributing factor towards spoilage over long periods unless the concentration of carbon dioxide closely approaches 100 per cent. Smith (18) (1954) likewise states that at least 70 per cent carbon dioxide is necessary to retard microbial decomposition in chickens during the Minimum storage period of 4 months. In one extensive test -10- he actually obtained better results by storage in air than in carbon dioxide. E. Carbon Dioxide as a Mycostatic Agent Carbon dioxide not only serves as a bactericidal or bac- teriostatic agent but it may also function as a mycostat as well. Lopriore (3) (1889) has demonstrated that a 10 per cent concentration of carbon dioxide retarded epore germination of ,ggggg’mucedc, and although pure carbon dioxide caused total -inhibition, it had no antibiotic effect even after 5 months. Likewise, Brown (2) (1922) found that carbon dioxide retarded the development of molds responsible for fruit-rot. He ob- served that the concentration necessary to suppress growth varied with the particular Species, that the inhibitive effect was accentuated by lowering the temperature, and with regard to concentrations of carbon dioxide which would be commercial- ly practical, temperature is a more important factor in reduc- ing mold growth than carbon dioxide. At 15° a moderate con- centration of carbon dioxide (10 to 20 per cent), although it does produce an initial retardation, eventually causes an ac- celeration of fungal growth. According to Moran, Smith and Tompkins, (6) (1952) carbon dioxide in any concentration will suppress the growth of meat-attacking fungi, the rate of growth in 20 per cent carbon dioxide being only 1/2 to 1/5 that in air, inhibition being more marked the lower the temperature. Similar observations were made by Tompkins (7) (1952) who found that the growth rate of various meat-attacking fungi on -11- artificial media is reduced to approximately 50 per cent of its value in air in 10 per cent carbon dioxide at 0°C. and 100 per cent relative humidity. Andreicha (1) (1956) in studying the combined mycostatic effect of carbon dioxide and high sugar concentrations, found that neither the high- est sugar concentrations used in commercial jams and jellies nor an atmosphere of carbon dioxide approaching 100 per cent, alone, can suppress the development of common molds. The combined action of these two preservative agents, however, gave satisfactory results. Gn60 per cent glucose media, an atmoSphere of 10 per cent carbon dioxide prevented the devel- Opment of Aspergillus repens and Penicillium glaucum, but lower sugar concentrations required higher carbon dioxide con- centrations to arrest growth. More recently, Tompkins (7) (1958) has pointed out that in_certain instances, the storing of food in concentrations of carbon dioxide up to 20 per cent may accelerate instead of retard the development of certain molds. (Moran (4, 5) (1957, 1959) found that carbon dioxide would retard mold growth on eggs and therefore permit the use of higher humidities in the storage room. At 0°C. and a rela- tive humidity of 85 per cent, 2.5 per cent carbon dioxide elim- inates fungal growth, prevents the appearance of a metallic "storage" taste and results in the retention of a firmer yolk, whereas in a saturated atmosphere, the carbon dioxide concen- tration must not be less than 60 per cent. E. The Nature of the Inhibitory Action of Carbon Dioxide Although experimental evidence concerning the inhibitory -12- effect of carbon dioxide on the growth of micro-organisms has been accumulating for over 90 years, the exact manner in which this gas retards microbial growth still remains unexplained. In this connection valley and Rettger (7) (1927) observed that bacteria become resistant to the action of carbon dioxide and grow normally if the medium is strongly buffered with phOSphates, whereas on ordinary media, growth is suppressed. Therefore, these authors concluded that the antibiotic effect. of carbon dioxide must be due primarily to the increased hy- drogen-ion concentration of the medium which is deleterious to bacterial growth. Killefer (5) (1950) also concurs with this view and explains the preservative action of this gas on meat on this basis. This hymnhesis has been adversely criticized by Callow (l) (1952) and refuted by Coyne (2) (1952) who found that if the pH of the medium is altered by means other than carbon di- oxide to the same amount, bacterial growth is not retarded to the same extent as with carbon dioxide. This is also in a- greement with the findings of Tompkins (6) (1952) who ob- tained a similar result quantitatively with certain fungi. Moreover, Haines (5) (1955) concluded that the change in pH which occurred in nutrient broth sown with.Achromobacter com- parable with that obtained by using 100 per cent carbon di- oxide does not account for the retardation of growth obtained with as low as 10 per cent concentration of this gas. According to these workers, the inhibitory action of car- bon dioxide may possibly be due to the fact that this gas ac- tually penetrates the cell wall thereby causing an alteration -15- in intracellular pH and consequently has a deleterious effect on the dehydrogenase systems of the cell. This conforms with the view of Jacobs (4) (1920), namely,'that since the ability of carbon dioxide to penetrate cell membranes is so great, it owes its physiological effect to hydrogen-ions which eaten the interior of the cell. Tompkins (6) (1952) has pointed out that the action of carbon dioxide is like unto that of ammo- nia which upon dissolving in water produces phys1010gically active ions. Hence, the presence of this gas affects a liv- ing system in two ways: "it influences the uptake of ions and depresses respiration." G. The Stimulating Effect of Carbon Dioxide In their natural environment, bacteria are constantly being exposed to carbon dioxide. Even so, ordinary air con- tains but .05 per cent of this gas and some bacteria require an excess of carbon dioxide above that found in the normal atmOSphere for their preper develOpment. So, it has been found that the growth of many fastidious organisms is greatly facilitated by small increments of added carbon dioxide. Thus, Nowak 00) in 1908, observed that the primary iso- lation of B332. abortus was greatly enhanced when he resorted to.the Bacillus subtilis tandem method of cultivation. Al- though Nowak attributed the success of his technique to the reduced oxygen tension in the cultural environment, subsequent work has shown that it was due to the additional carbon diox- ide given off by B, subtilis in the closed system. The partial- tension method was also successfully applied to the primary -14- cultivation of the gonococcus by Wherry and Oliver, 1916 (17); Reudiger, 1919 (12) and Swartz and Davis, 1920 (16); and to the meningococcus by Cohen and Harkle, 1916 (5) and Cohen and Fleming, 1918 (4). Subsequently, it was found that when the accompanying culture of B, subtilis was replaced by additional carbon di- oxide in air the cultures grew quite as well. Therefore,the tandem technique was superseded by direct application of the gas to the cultural environment. Investigators found that the growth of the gonococcus, Chapin, 1918 (2); the meningococcus, Cohen and Fleming, 1918 (4); Kohman, 1919 (7); and B532, 2222? Lug, Huddleson, 1920, 1921 5.6); T. Smith, 1924, 1926 as) and McAlpine and Slanetz, 1926 (9) was stimulated by an atmos- pheric concentration of 10 per cent carbon dioxide. Likewise, Kulp (8) (1926) observed that the same was true of Lactobacillus acidophilus. Rockwell 0.5,]4H1921, 1924) pointed out that carbon dioxide facilitates the growth of the gonococcus, the aerobic bacteria and the facultative group as well. Moreover, Nye and Lamb . (11) (1956) observed that 5 per cent carbon dioxide produced by burning a candle in a sealed container, enhanced the growth of the pneumococcus type VIII and therefore advised the grow- ing of all routine cultures in this manner. More recently, Auger (l) (1959) found that .5 to 25 per cent carbon dioxide not only stimulated the growth of all types of pneumococci, but other respiratory pathogens such as HemOphilus influenzae and Streptococcus hemolyticus as well. -15- H. The Influence of Carbon Dioxide on .Toxin Production & Preservation As early as 1918, Larson and his colleagues (7) report- ed that BBEB.,QQBB as well as other Gram negative bacilli be- came far more toxic after having been killed by carbon diox- ide than the living cultures. Parker et a1. (9) (1925) also observed that staphylococci grown in the presence of 10 per cent carbon dioxide produced much more skin toxin than in its absence. This in accord with the findings of Burnet (l, 2) (1929, 1950) who attributed the favorable influence of 20 to 25 per cent carbon dioxide on the toxin production of certain strains of BBBEB. aureus to the increase in intra- cellular acidity which favors haemolysin production. Par- ish and Clark (8) (1952) obtained powerful Staphylococcus toxins from cultures grown in an atmosphere of 25 per cent carbon dioxide. Likewise, Woolpert and Deck (14) (1955) se- cured lethal, hemolytic and skin-necrosing toxins from sev- eral cultures of food-poisoning strains of staphylococci grown under the same conditions. Similarly, Dolman (4) (1954), working with more than 200 strains of Staphylococ- cus from various clinical sources, found that 85 per cent were highly toxigenic when ingested by human volunteers after having been grown for 40 hours in an atmOSphere of 50 to 40 per cent carbon dioxide at 57°C. 0n the other hand, Burnet (5) (1951) found that carbon dioxide had a diminishing effect on the toxicity of the ty- phoid bacillus. This is in agreement with the observations -16- of Hanks and Hettger (5) (1952) who found it impossible tow demonstrate that the growth and toxin production of EEiES‘ Eflllfi pullorum was materially enhanced by an atmOSphere of 10 per cent or 20 per cent carbon dioxide. Herter and Bett- ger (6) (1957), however, reported that the presence of 10 per cent carbon dioxide increased the potentcy of BBB. ESE“ trycke toxin for mice and rabbits. Plastridge and Hettger (10, ll, l2, 15) (1927, 1929) reported that the growth and toxin production of B. diphtheriae was increased and made more regular when the cultures were aerated with 5 to 10 per cent carbon dioxide. Deterioration of the toxin through ox- idation was also prevented. I. Carbon Dioxide as an Essential Growth Factor Most of the earlier work dealing with the influence of carbon dioxide on micro-organisms had to do with its antibi- otic or bacteriostatic effect on bacteria. But now it is very well known that although excessive amounts of this agent may prove injurious to microbial life, a certain minimal a~ mount is just as indiSpensable for the sustenance of life in bacteria as it is in plants, the higher animals and man. Thus, Winogradsky (25) (1890) demonstrated that carbon dioxide is essential for the growth of the autotrophic nitri- fying bacteria. This was later confirmed by Bonazzi (1) (1921); Godlewsky (4) (1892, 1895, 1896) and Gowda (5) (1924). Likewise, Wakesman and Starkey (21, 22) (1922) and Starkey (17) (1925) recOgnized the necessity of carbon dioxide for the sulphur-oxidizing bacteria. -17- Nherry and Ervin (25) (1918) concluded that carbon dioxide was prerequisite for the growth of the of the tuber- cle bacillus. Although this conclusion has been disputed by Novy and Soule (8) (1925), it was later sustained by Rockwell and Highberger 04) (1926). Huddleson «$5 (1920, 1921) and T. Smith (16) (1924) recognized that carbon dioxide was also an essential factor in the growth of B332. abortus which is in full accord with the findings of Wilson (24) (1951). More- over,the carbon dioxide studies of Rockwell and McKann, 1921 (10); Rockwell, 1921, 1925, 1924 (ll, 12, 15); Rockwell and Highberger, 1926, 1927 (l4, 15); Valley and Rettger,l925-27,(18,19, 203m Gladstone, Fildes and Richardson (5) (1955) have shown that carbon dioxide, in minimal amounts, is a vital factor in the growth of all bacteria. It appears that the presence of carbon dioxide not only plays an essential role in bacterial development, but also is an important factor in the growth of molds and yeasts. Ac- cordingly, Durrell (2) (1924) observed that BasiSporum 52$? nggg spores did not germinate in the absence of carbon di- oxide and were in turn stimulated by small amounts of this gas. Rippel and Bortels (9) (1927) likewise noted that.B§- pergillus EEEEE spores germinated very poorly in the absence of carbon dioxide, while Rockwell and Highberger (15) (1927) demonstrated that carbon dioxide was essential for the proper growth and deve10pment of Mucor and Saccharomyces. J. The Role of Carbon Dioxide in Bacterial Metabolism Although the experimental evidence has been too inadequate -18- to warrant the formulation of any conclusions warious hypoth- eses have been preposed in an endeavour to explain how carbon dioxide produces its essential effect. At first, it was thought that the favorable action of carbon dioxide merely influenced bacterial growth indirectly. Consequently, the stimulating action of carbon dioxide under partial-tension conditions was generally attributed to the displacement of oxygen from the environment resulting in a reduced oxygen tension, Nowak, 1908 (7); Cohen and Fleming, 1918 (l) and Reudiger, 1919 (8). St. John (11) (1919) and Torrey and Buckell (12) (1924), however, ascribed the beneficial results to the increased moisture content of the closed system. Others were of the opinion that carbon dioxide affected a favorable adjustment in the hydrogen-ion concentration or the medium by the es- tablishment of a buffer system which prevented extreme devi- ationinn pH due to excessive alkalization, Gates, 1919 (3); Kohman, 1919 (6); Sierkowski and Zajdel, 1924 (10). Later on, however, it became apparent that the function of carbon dioxide was not that of an added stimulus, but that it was directly concerned with the very existence of the bac- terial cell itself. Thus, Huddleson (5, 4) (1920, 1921) rec- ognized its importance in the cultivation of B523. abortus, while Valley and Rettger (15) (1927) regarded it as satisfy- ing a definite requirement which ordinary air is incapable of meeting. Rockwell and Highberger (9) (1927) concluded that carbon dioxide served as a source of carbon and was the only source which could be so utilized. More recently, Koebs -19- (5) (1937) has suggested that carbon dioxide probably acts as a reSpiratory catalyst (hydrogen carrier) in Esch. 0011. III. HISTORY OF CULTURES All the paratyphoid cultures were originally received from Dr. P. R. Edwards of the Kentucky Agricultural Experi- ment Station, Lexington, Kentucky. Three of the Staphylo- coccus cultures, namely, Nos. 169, 168 and 85 were obtained from Dr. G. M. Deck of the Department of Bacteriology, Uni- versity of Chicago. These strains were all of the hemolytic variety and proved toxin-producers. .A fourth culture, a non-toxic strain, was acquired from the department stock culture collection in this laboratory. Iv3wEXEERIMENTAL In order to make a quantitative study of the effect of carbon dioxide on the viability of the organisms under in- vestigation, the following technique was employed through- out all the experiments: Fifty milliliter quantities of sterile Bacto-tryptose, dextrose, phoSphate-buffered broth in an Erlenmeyer flask were planted with a known number of organisms. Duplicate cultures of each organism were pre- pared, and then stored in a Frigidaire ice cream cabinet at temperatures of 3: 5‘and 2070. respectively. One culture was placed in a compartment and subjects Mitreatment with carbon dioxide, while the other was held at the same temp perature in the absence of the gas to serve as a control. -20- At the time the cultures were placed in storage, the cotton plugs were carefully removed from the flasks so that the car- bon dioxide might have free access to the surface of the me- dium. All counts were made on Bacto-tryptose agar after being incubated for 48 hours at 37‘C. The cultures were plated out at various intervals for 4 days to 3 weeks. . The carbon dioxide concentrations were automatically maintained and controlled by means of a Carbostat unit manu- factured by the White Mfg. Co., St. Paul, Minn. This device automatically checked the gas concentration every 45 seconds and maintained the cOncentration within a range of plus or minus .3 per cent. -21- TABLE I A Comparison of the affect of 5 Per Cent and 10 Per Cent Carbon Dioxide on the Growth of staphylococci at 5 to 5 C. Name of’ ‘Treat— Initial N0. Per Cent Decrease in Numbers Organism ment of Orgs. 5 Days 12 Days 21 Days 5% Carbon Dioxide Staph. au- C02 118,000 1.70 72.88 88.75 reus $85 None 118,000 40.68 79.66 95.75 10% Carbon Dioxide 002 122,000 54.26 92.17 Hone 122,000 77.05 91.64 97.62 5% Carbon Dioxide ’ Staph. au- C02 45,000 74.04 86.66 100.00 reus (Lab.) None 45,000 15.55 90.00 100.00 10% Carbon Dioxide 002 157,000 70.06 91.78 97.07 None 157,000 75.86 94.27 98.06 A Comparison of the Effect of an Atmosphere of 5 Per Cent and 10.Per Cent Carbon Dioxide on Members of the Salmonella Group at 5'to 5°C. -22- TABLE II Name of* Treat- InitiaI’No.‘Per Cent Decrease in Numbers Organism ment of Crgs. 5 Days, 12 Days 21 Days 5% Carbon Dioxide Sal. abor- 002 41,000 87.80 *85.57 19.51 tus None 41,000 00.00 4.59 51.22 10% Carbon Dioxide 002 29,000 *88.20 *107.00 *70.00 Hone 29,000 00.00 *80.00 *80.00 5% Carbon Dioxide Sal. brede- 003 255,000 , 20.85 65.96 91.92 nay None 255,000 10.64 *21.70 88.94 10% Carbon Dioxide None 540,000 50.00 15.70 *851.00 5% Carbon Dioxide dal. derbey C02 114,000 85.86 87.72 99.04 None 114,000 62.28 87.72 95.86 10% Carbon Dioxide 002 450,000 94.22 95.55 98.10 None 450,000 94.67 90.67 49.11 5% Carbon Dioxide sal. ente- C02 221,000 55.75 44.54 76.47 ritidis Hone 221,000 55.04 59.27 100.00 10% Carbon Dioxide C02 510,000 55.00 15.00 80.97 None 510,000 52.22 49.56 61.50 5% Carbon Dioxide . Sal. gal- C02 91,000 5.50 48.55 94.59 linarum None 91,000 17.58 72.55 80.68 10% Carbon Dioxide 002 99,000 77.77 81.82 79.80 None 99,000 59.59 77.98 97.58 -25... TABLE II Continued Name of {Treat- Initial No. Per Cent Decrease in Numbers Organism ment of Orgs. 5 Days 12 Days 21 Days 5% Carbon Dioxide dal. muen- C02 400,000 49.75 74.25 99.12 Chen None 400,000 40.00 71.50 98.70 10% Carbon Dioxide 003 1,170,000 60.00 84.62 90.00 None 1,170,000 67.52 *151.00 *508.00 5% Carbon Dioxide Sal. sui- 002 65,000 41.54 58.50 95.85 pestifer Hone 55,000 *42.45 49.25 75.85 10% Carbon Dioxide C02 550,000 89.40 91.82 95.76 None 550,000 66.66 48.50 65.45 5% Carbon Dioxide dal. typhi- 002 250,000 57.59 76.52 89.51 murium None 250,000 41.74 25.65 42.18 10% Carbon Dioxide C02 410,000 66.00 70.75 85.66 None 410,000 00.00 47.52 95.17 * Denotes per cent increase in numbers. TABLE Ila A.Comparison of the Effect.of an atmOSphere of 5 Per Cent & 10 Per Cent Carbon Dioxide on Salmonella pullorum at 5‘to 5'C. flame of’ “Treaté‘lnitial 80. Per Cent Decrease in Numbers Organism ment of Orgs. 7 Days 14 Days 21 Days 5% Carbon Dioxide Sal. pul- 002 97,000 54.64 91.96 100.00 lorum None 97,000 67.01 95.05 100.00 10% Carbon Dioxide 002 54,000 57.81 91.87 94.05 None 54,000 75.44 95.78 100.00 -24- TABLE III The Effect of 10 Per Cent Carbon Dioxide on the Growth of Toxin-Producing Staphylococci at‘5’C. ** Name éfflTreath Initial No. Per Cent Decrease in Numbers Organism .ment of Orgs. 7 Days 14 Days 21 Days Staph. au- 002 85,000 58.14 55.98 55.98 reus #159 None 85,000 41.86 85.72 85.72 Staph. au- 002 550,000 81.14 57.45 *717.00 reus #168 None 550,000 50.00 75.45 100.00 * Denotes per cent increase in numbers. ** The cultures listed were tested at 10% COgonly. The Effect of 10 Per Cent Carbon Dioxide on Members of the Salmonella Group at 5°C. -25- TABLE IV Name of:Treat- Initial No. Per Cent Decrease in Numbers Organism ment of Orgs. 7 Days 14 Days 21 Days Sal. give 002 550,000 59.12 55.55 54.24 None 550,000 *55.50 24.24 59.12 Sal. ken- 002 121,000 50.55 70.25 15.70 tucky 'None 121,000 42.15 52.81 55.71 851. lon- 002 190,000 ' 51.58 50.55 70.00 don None 190,000 71.05 79.47 90.00 Sal. min- 002 150,000 *20.00 *15.55 84.55 nesota None 150,000 ’"15.55 56.70 75.00 Sal. mon- 002 500,000 55.55 85.55 90.00 tevideo Hone 500,000 50.00 16.66 45.55 551. new- 002 280,000 14.29 *5.57 *252.00 brunswick Hone 280,000 7.14 *71.86 *1556.00 Sal. new- 002 590,000 *9.08 87.29 95.27 port None 590,000 51.00 80.51 91.87 Sal. pan- 002 140,000 54.29 75.75 75.75 ama None 140,000 *51.75 57.14 75.75 Sal. senf— 002 400,000 50.00 85.25 91.50 tenberg None 400,000 70.00 80.00 85.75 Sal. worth- 002 180,000 85.89 100.00 95.11 ington None 180,000 85.11 85.55 95.51 * Denotes per cent increase in numbers. **The cultures listed were tested at 10% Cq30nly. -25- TABLE IVE The Effect of 10 Per Cent . Members of the Salmonella Group at 5°C. ** Carbon Dioxide on ‘Treat-Initial No. Per Cent Decrease in Numbers ’Name of Organism ment of Orgs. 5 Days 12 Days 21 Dayg Sal. bareilly C02 270,000 55.55 48.14 96.00 None 270,000 55.00 74.82 84.08 881. 8811- 002 720,000 12.50 27.77 95.70 fornia None 720,000 50.00 56.00 50.00 Sal. cer- C02 540,000 57.41 78.52 92.22 Tatum Hone 540,000 76.00 76000 51085 Sal. chol- 002 140,000 61.45 80.72 97.72 eraesuis None 140,000 15.00 62.86 92.14 Sal. new- 002 450,000 64.44 80.44 51.11 ington None 450,000 57.78 80.21 84.22 Sal. orani- 002 520,000 25.00 95.95 90.94 enberg None 520,000 62.50 45.75 65.65 Sal. para- 002 510,000 25.80 58.71 86.15 typhi A. None 510,000 61.50 81.62 45.90 Sal. schott- 002 520,000 92.50 95.65 100.00 muelleri None 520,000 85.75 65.44 65.65 ** The cultures listed were tested at 10% Cngnly. -27- TABLE V The Effect of a 10 Per Cent Concentration of Carbon Dioxide on the Growth of Staphylococci and Members of the Salmonella Group at 18120°C. after 4 Days. Name of Organism ’Treat- Initial No. Fold Increase ment of Orgs. in Thousands Staph. aureus $169 002 22,000 56.4 None 22,000 5.9 Staph. aureus #168 002 17,000 15.1 None 17,000 5.9 Staph. aureus #85 002 11,000 28.6 None 11,000 65.6 Staph. aureus (Lab.) 002 1,500 66.2 ' None 1,500 461.5 Sal. aertrycke 002 98,000 9.0 None 98,000 5.9 Sal. enteritidis 002 291,000 ' 45.7 None 291,000 25.4 Sal. muenchen ' 002 155,000 51.5 None 155,000 5.6 Sal. oranienberg 002 94,000 45.8 None 94,000 57.2 Sal. paratyphi A 002 171,000 ‘ ' 21.1 None 171,000 24.6 Sal. schottmuelleri 002 22,000 86.4 None 22,000 295.5 Sal. senftenberg 002 9,000 211.1 None 9,000 68.9 Sal. suipestifer CO2 55,000 15.7 None 55,000 27.7 -28- V. DISCUSSION & CONCLUSIONS Table I shows the results obtained by exposing 2 strains of filapg. aureus to atmospheres of 5 per cent and 10 per cent carbon dioxide at 5'to 5°C. It will be observed that the bacterial count of both strains was ap- proximately the same in the presence and in the absence of carbon dioxide at both concentrations of this gas. At the end of 5 days' exposure there was a reduction of 2 to 77 per cent. After 12 days, the number of viable organ- isms had reduced 75 to 90 per cent, while at the end of 5 weeks, this reduction approached 100 per cent. The effect of concentrations of 5 per cent and 10 per cent carbon dioxide on various Species of Salmonella at 5‘ to 5°C. is presented in Tables 11 and Ila. In general, both the air and the carbon dioxide cultures showed a pro- gressive decrease in numbers throughout the period of ex- amination. The trend of development at the end of 5 weeks showed a reduction of approximately 80 to 90 per cent. The influence of a 10 per cent concentration of car- bon dioxide on 5 toxin-producing strains of gyapg. aureus at 5’0. is represented in Table 111. _These results show that the organisms decrease in numbers at the same rate re- gardless of the presence or absence of carbon dioxide. Tables IV and 17a show the trend of development of other representative members of the Salmonella group in an atmosphere of 10 per cent carbon dioxide at 5°C. Although on the whole, the trend shows a progressive decrease in é29- numbers both in the presence and absence of carbon dioxide, here again, the reduction in countshown by the carbon diox- ide cultures, parallels that of the controls. In analyzing these data, it should be remembered that each individual sample or strain cannot serve as an ulti- mate criterion as to the behaviour of a particular organ- ism under the conditions which obtain. Nevertheless, when these data are examined as a whole, it is evident that in general all the organisms investigated showed a marked re- duction in count irrespective of the presence or absence of carbon dioxide. Neither is this all, but this decrease becomes progressively greater the longer the period of ex- posure. From this it may be concluded that low temperatures not only have a bacteriostatic effect upon bacteria but produce an antibiotic effect as well. On the other hand, since there was no essential difference between the reduc- tion in numbers which occurred in the presence of carbon dioxide and that which took place in its absence, it may also be inferred that carbon dioxide has no supplementary effect on low temperatures in either reducing or increas- ing the deve10pment of food-poisoning organisms. Fumhermore, if the bacterial population of Salmonella and Staphylococcus tends to reduce in numbers during stor- age at 5‘to 5‘C., even though the added presence of carbon dioxide had a tendency to enhance toxin production or pre- vent detoxification of the toxin once it had been formed, it would have no significance as far as creating a health -30- hazard is concerned. For, the very essence of toxin pro- éufimn consists in an increase in the number of viable 0r- ganisms, so that toxin production and reproduction, roughly at least, parallel each other. Therefore, we may conclude that if food is kept at 5°C. or less the danger from food poisoning is relatively slight since the causative agents will not grow in food at such low temperatures. Finally, these data indicate that the application of carbon dioxide atmospheres as a supplement to refrigeration does not ere- ate any additional health hazard in foods stored under these conditions. Table V represents the effect of an atmosphere of 10 per cent carbon dioxide on various members of the Salmonella group and several strains of Staphylococcus at 18°to 20°C. In contrast to the results obtained at low temperatures, from this table it is evident that all species showed a marked increase in numbers both in the presence and in the absence of carbon dioxide. This was in accordance with an- ticipation based on the fact that the rapidity of microbial development is determined among other things by temperature. 0n the whole, it is also apparent that proliferation oc- curred more readily in an atmoSphere of 10 per cent carbon dioxide than in its absence. In this connection it is-interesting to recall the ob- servations of other workers already cited pertaining to the effect of carbon dioxide at higher temperatures on organ- isms commonly incriminated in food poisoning. As early as -01— 1895, Sabrazes and Bazin (1) demonstrated that 23222: aa- gggg grew well in the presence of carbon dioxide in both plain agar and that containing added buffer. This was later confirmed by valley and Rettger (2) (1927) who also showed that Sal, paratyphi and schottmuelleri were able to grow equally well in both plain agar and phosphate- buffered-media in an athSphere of 95 to 97 per cent car- bon dioxide. .Although these workers used solid media and employed a much higher gas concentration than was used in this investigation, the trend in either case was the same. If carbon dioxide has a general tendency to stimu- late the growth and reproduction of food-poisoning organ-' isms at higher temperatures, then it must follow that it enhances their toxicity as well. From this we may con- clude that it would be inexpedient to introduce carbon dioxide into food storage containers under conditions where high temperatures prevailed, for it would be bound to create a serious health hazard. The main conclusion arising from this phase of the investigation, however, is that it is wise to keep all food, especially animal food from the time of slaughter until its final preparation at a relatively low temperature. 1. 2. 5. 4. 6. 7. 8. 9. 10. -02- VI. CITATION ON THE LITERATURE A. The Bactericidal Effect of Carbon Dioxide Barthell (1848) Cited by Valley in Quart. Rev. BidL 5(2):209-24 (1928). Berghaus, Dr. H Ueber die Nirkung der Kohlensaure des Sauerstoffs und Wasaerstoffs auf Bakterien bei verschieden Druchfihen. ‘ Arch. f. Hyg. 62:172-99 (1907). Frankel, C. H 0 Die Einwirkung der Kohlensaure auf die Lebenstatig- keit der Mikroorganismen. zeitSChro f0 Hyg. 5:332-62 (1889). Frankland, P. F. . Ueber den Einfluss der Kohlensaure auf die Leben- statigkeit der Mikroorganismen. Zeitschr. f0 Hyg. 6:13-17 (1889). Grossmann, C. und Mayerhausen Ueber das Leben der Bakterien in Gasen. Pfluger's Arch. f. Physiol. 15:248 (1881). Guillerd,.A. et Lieffrig Action bactercide de l'acide carbonique sur les germes de 1'eau. Bull. Acad. Med. (Paris) ll5(25):864-66 (1955). Hoffman, W. ,, ' Uber den Einfluss hohen Kohlensaure drucks auf Bakterien im Nasser und in der Milch. AICho f0 Hyg. 57:579-400 (1906). Larson, W. P., Hartzell, T. B. and Diehl, H. S. The effect of high pressures on bacteria. Jour. Infect.Dis. 22:271-79 (1918). Pasteur, L. et Joubert N'tude sur la maladie charbonneuse. Compt. rend. Acad. Sci. 84:900 (1877). Plummer, J. K. Some effects of oxygen and carbon dioxide on ni- trification and ammonification in soils. ' Cornell Agr. Exp. Sta. Bull. 584:505-50 (1916). 11. 12. 15. 14. 15. 1. 2. 4. 5. -53- Sabrazes, J. et Bazin, E. L'acide carbonique a haute pression peut-il etre considers comme un antiseptique pussiant? Compt. rend. Biol. Ser. 9 (5)3909-14 (1895). Schaffer and Nreudenrich Pressure and microbes. Ann. de Micrographie 4:105-19 (1891). bzpilman, Jo Ueber das Ierhalten der Milzbrand Bacillen in Gasen. Zeitschr. physiol. Chem. 4:550 (1880). Valley , G o The effect of carbon dioxide on bacteria. Quart. Rev. Biol. 5(2):209-24 (1928). Valley;G. and Rettger, L. F. The influence of carbon dioxide on bacteria. Jour. Bact. l4 (2):101~57 (1927). B. Carbonated Waters & Beverages Colin, H. Sterilization de l'eau par l'acide carbonique sous pression. Compt. rend. Aca. Sci. 161:652-55 (1915). Donald, J. 3., Jones, C. L.. and MacLeon,.A. R. M. The effect of carbonation on bacteria in bev- erages o Amer. Jour. Pub. Health 14:122-28 (1924). Hochsetter, M. Ueber Mikroorganismen im kunstlichen Selters- wasser nebst einigen vergleichenden Unter- suchungen uber ihr Verhalten im Berliner.Leitungs- wasser und im destillierten Nasser. Arb. aus dem Kaiserl. Gesundheitsamte 2:1-29 1887 . Kliewe, H. und Kindhauser, J. Uber die Keimtatende Kraft der Kohlensaure. Arch. f. Hyg. 110(4). 211-18 (1955). Koser, S. A. and Skinner, W. N. Viability of the colon-typhoid group in carbonated waters and carbonated beverages. Jour. Bact. 7:111-21 (1922). 6. 8. 1. 2. 6. l. Leone, C. " Untersuchungen uber die Mikroorganismen des Trink- wassers und ihr Verbalten in Kohlensauren Nassern. Arab. f0 Hyg. 4:168'75 (1886). 80818 e Sanfelice Azione dell'acido carbonico disciolto nelle acque potabile su alcuni microorganismi. Centr. f. Bakt. u. Parasitenk. 9:110-15 (1891). Slater, C. A bacteriological investigation of artificial mineral waters. Jour. Path. & Bact. 1:468-88 (1895). C. The Carbonation of Dairy Products Hoffman, w. ,, Ueber den Einfluss hohen Kohlensaure drucks auf Bak- terien im Nasser und in der Milch. Arch. f. Hyg. 57:579-400 (1906). Hunziker, O. E. Facts abbut carbonated butter. Jouro Dairy 5010 7:484-96 (1924). Prucha, M. J., Brannon, J. J. and Ambrose,.A. 8. Does carbon dioxide in carbonated milk and milk products destroy bacteria? Cir. 256, Ill. Agr. Exp. Sta., Urbana (1922). Prucha, M. J., Brannon, J. M. and Ruehe, H. A. Carbonation of’butter. Jour. Dairy Sci. 8:518-29 (1925). valley, G. and Rettger, L. F. The influence of carbon dioxide on bacteria. Jour. Bact. I4(2):101-57 (1927). van Slyke, L. L. and Bosworth, A. W. Effect of treating milk with carbon dioxide gas under pressure. N. Y. (Geneva) Agr. Exp. Sta. Bull. 292:571-84 (1907). D. Carbon Dioxide as a Meat Preservative Brocks, Jo The effect of carbon dioxide on the color and bloom of lean meat. Jour. Soc. Chem. & Ind. 52(4):17T-19T (1955). 2. 7. 9. 10. 11. Brooks, J. and Moran, T. The absorption of carbon dioxide by meat. Dept. Sci. & Ind. Res. Rept. Food Invest. Bd. (Great Britain) 44-45 (1955). 'Ca11ow, E. H. Gas storage of pork and bacon. Jouro Chemo & Ind. 51 (l5)3116T-19T (1932). Ida! The storage of frozen legs of pork for the subse- quent manufacture of hams. Dept. Sci. & Ind. 385., Rept. Food Invest. Bd. (Great Britain) 68 (1958). Chistyakov, F. M. _ . Action of carbonic acid on putrefactive organisms. Mikrobiologiya 2:192-210 (1955). Coyne, F0 P0 The effect of carbon dioxide on bacterial growth with reference to the preservation of fish. Jour. Soc. Chem. & Ind. 51 (l5):ll9T-21T (1952). Idem The effect of carbon dioxide on bacterial growth with special reference to fish. II. Gas storage 0f fiSho Jour. Soc. Chem. & Ind. 52:19T-24T (1955). Idem. The effect of carbon dioxide on bacterial growth. Proc. Roy. Soc. (London) B115 (782):l96-2l6 (1955). Nmpey, N. A. and Vickery, J. R. V The use of carbon dioxide in the storage of chilled beef. Jour. Counc. Sci. & Ind. Res., Australia 6 (4)255- 45 (1955). ' Idem The exPort of chilled beef-~The preparation of the "Iodmeneus" shipment at the Brisbane abattoir. Jour. Counc. Sci. & Ind. 888., nustralia 7 (2): 75-77 (1954). Haines, R. B. The influence of carbon dioxide on the rate and multiplication of certain bacteria as judged by viable counts. Jour. Soc. Chem. & Ind. 52 (4):15T-17T (1955). 12. 15. 14. 16. 17. 18. 1. 2. -55- Killefer, D. H. Carbon dioxide preservation of meat and fish. Ind. & Eng. Chem. 22(2);l40-45 (1950). Kolbe, H. Lea Idem. Idem. Antiseptische Nigenschaften der Kohlensaure. Jour. prakt. Chem. 26:249-55 (1882). C. H. Chemical changes in the fat of frozen and chilled meat. The protective influence of carbon dioxide on the fat of chilled beef stored at 0°C. . Jouro 5000 Chemo & Ind. 52(4):9T‘12T (1955). The protective influence of carbon dioxide on the fat of chilled beef. Dept. Sci. & Ind. Res., Rept. Food Invest. Bd. .(Great Britain) 59-45 (1955). 11. Chemical changes in the fat of gas-stored chicken. Jour. Soc. Chem. & Ind. 55 (Pt. 2):547T-49T (1954). Mallmann, W. L. and Zaikowski, L. J. Effect of carbon dioxide on the growth of meat Spoilage organisms. National Provisioner 105(7):l6-l7 (1940). Smith, F. C. 4 Cold storage of poultry. 1. Gas storage of chickens. Jour. Soc. Chem. & Ind. 55 (Pt. 2);545T-47T (1954). E. Carbon Dioxide as a mycostatic Agent Andreicha, H. I. The combined effect of carbon dioxide and high sugar concentrations on the development of molds. Microbiologia 5(5):709-l5 (1956). Brown, W. 0n the germination and growth of fungi at various temperatures and in various concentrations of carbon dioxide. Ann. Bot. 56:257-85 (1922). Loprigre, G. Uber die Einwirkung der Kohlenséure und anderer Gase auf die Entwicklungsffighigkeit der Mikro- organisms. Zeitohro f0 Hyg. 6:13-22 (1889). -37- 4. Moran, T. Gas storage of eggs. Jour. Soc. Chem. & Ind. 56:99T-101T (1957). 5. Idem. The cold storage and gas storage of eggs. r‘ood Invest. Bd. Leaflet f8 (Great Britain) (1959). 6. Moran, T., Smith and Tompkins The inhibition of mold growth on meat by carbon di- oxide. Jour. Soc. Chem. & Ind. 51(15): ll4T-16T (1952). 7. Tompkins R. G. The inhibition of the growth of meat-attacking fungi by carbon dioxide. Jour. Soc. Chem. & Ind. 51 (Pt. 2);262T-64T (1952). 80 Idem- Inhibitory effect of carbon dioxide on the growth 0f m01d30 Dept. Sci. Ind. Res., Rept. Food Invest. Bd. (Great Britain) 50-9 (1958). F. The Nature of the Inhibitory Action of Carbon Dioxide l. Callow, E. H. Gas storage of pork and bacon. Jouro SOC. Chemo & Ind. 51 (Pt. 2):116T“19T (1952). 2. Coyne, F. P. The effect of carbon dioxide on bacterial growth with reference to the preservation of fish. Jour. Soc. Chem. & Ind. 51 (Pt. 2):ll9T-21T (1952). 30 Hain33, Bo Bo The influence of carbon dioxide on the rate and multiplication of certain bacteria as judged by viable counts. Joun Soc. 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Jour. Bact. 14 (2):101-57 (1927). Discussion & Conclusions sabraZés, Jo at BaZin, E0 L'acide carbonique a haute pression peut-il etre con- sidere oomme un antiseptique pussiant? . Compt. rend. Biol. Ser. 9 (5):909-l4 (1895). valley G. and Rettger, L. E. The influence of carbon dioxide on bacteria. Jour. Bact. 14 (2):101-57 (1927). D . . \.. d C . .. . . .uv .. . I. ) P . v . u | v .. . _ .7 (I . . . . u I u t . ’ ‘ y I ’ - I I l _ n 4 I I 1 n H n- I v 1 ‘ I A I b \ r . . u u r \ ‘1... .. V...\..u . ... .. . s. .‘ I x ...\ . v .. . . . I n , s l . . , _ ,. . .. .. ..7 . . . . . a. . .. .\ . .. a . a . v ... ,. . . ... fl. . - . . . . .6 v . F . \ .v.‘ uwvc. . 21.7.. .K... .c s. . '1... ... u w. .«w \ 1. . . . . . .. A. .\u . . I . - a. a . e . ._ . y .. . . V. V... u._ .0 5 I ... {‘(J... chub-K... .' .4 A w. h _ _ ‘ 1:. V). v (I u . $ U i . ‘. 95. ml a. . ‘ .'. ‘ N \n’ \.I . Ala .. I . .1 O} H . .7 T“. u‘. r O , at I 4 o u « nu r L n r s . V. vA. . 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