U . , a: a — “E“ § I' I” I l I I ”I” l I I —'i_s_x -'I\Ir\> H @900 THE INFLUENCE. OF ORGANIC ACIDS; SUGARS, AND SODIUM CHLORIDE ON TYPICAL STRAINS OP POISOMNG STAPHYLOCOCC! .‘K/ \ka/ Thesis for the Degree of M. S. MICHIGAN STATE COLLEGE Tom D. Nunhtiimer 1938 Q ‘l \U . . .é uVYM.:to'x§cfixI!- aduimfiafiuI xi THE INFLUENCE OF ORGAFIC ACIDS, SUGARS, AND SODIUM CHLORIDE ON TYPICAL STRAINS OF FOOD POISONING STAPHYLOCOCCI THE EFLUEHCE OF ORGAEIC ACIDS, SUGARS, ND SODIUM CHLORIDE OH TYPICAL STRAINS OF FOOD POISONING STAPHYLOCOCCI Thesis for degree of M.S. Michigan State College Tom Duvall‘gynheimer 1938 ““5973 Acknowledgments The writer wishes to express his sincere appreciation to Dr. F. W. Fabian, Associate Professor of Bacteriology, under whose able guidance this work was done, for his never failing interest throughout the course of the work and for his assistance and criticisms during the preparation of this manuscript. The writer also wishes to eXpress his sincere gratitude for the other members of the Department of Bacteriology for many helpful suggestions made throughout the course of the experiment. 1159 -13 Table of Contents Introduction;-;;§;;;_;§_-_;_-;-;_;;_;]_ Review of Literature acids;;;;-_;;-_--§_-______-_;-;L;_ 2 saits;;-_--;----------_____-;;;;-; 7 sugars--4---—----—_-___--_---_;-.;10 Present Work Verification of strains-—-—-—-44-¥ll Method—44------—-4 ------------------- 16 Results Effect of acids; ................... 17 discussion --------------------- 21 Effect of sugars and salt --------- 24 discussion ..................... 26 Summary ______________________________ 29 Tables 8 4 20; ....................... 31 Figures 1 ; lO—---4 .................. 41 Bibliography? ........................ 52 Introduction The importance of Staphylococci in food poisoning has not been fully appreciated until comparatively re; cently. In many cases of food poisoning staphylococci were isolated but no particular importance was attached to their presence-~all the emphasis being placed on bac- teria of the Salmonella and botulinum groups. 0wen(22) first called attention to the possibility of staphyloe cocci being a cause of food poisoning when he isolated a staphylococcus from dried beef which had caused acute gastroenteritis in persons consuming the beef. Eight years later Barber (1) isolated a white staphylococcus from milk which had caused gastrointestinal disturbances to the consumers of the milk. Later Dach, et al (8) isolated a yellow hemolytic staphylococcus which was present in considerable numbers in sponge cake which had caused illness in ll persons who had consumed the cake. A 40 hour broth filtrate of the staphylococci swallowed by a human volunteer produced a typical at; tack of the same character as that occuring among the original consumers of the cake. Further investigations have shown beyond question that staphylococci are capable of causing food poisoning and should be one of a group of bacteria suspected in any outbreak. In view of this fact and also in view of the fact that staphylococci as a group are more resistant to physical and chemical agents than are some of the asporo; genic bacteria suspected in outbreaks of food poisoning, a study was made of the resistance of typical strains to some of the chemical agents commonly found in food such as acids, sugar and salt. Literature Review Influence of Acids. As early as 1898 Kahlenberg and True (17) observed that many of the weaker acids exerted antiseptic powers disprOportionate to the Héion concentration which they produced and are antiseptic and bactericidal at pH values far higher than the highly dissociated acids. They sugé gested that the undissociated molecules and the anions may exert a toxic effect in the case of the weaker acids. Clark (7) observed that acetic acid at a dilution which was only 2 per cent ionized, inhibited the germina; tion of the spores of a group of filamentous fungi, while highly dissociated acids failed to show so high a retard; ing effect. He also attributed the activity of the weekly dissociated acids to the undissociated molecule. Kronig and Paul (19) carried out experiments upon the disinfectant action of various salts, bases and acids upon Staphylococcus aureus and the spores of Bacillus anthracis. They found that the number of organisms, or spores, which developed after treatment for a given time varied inversly with the amount of dissociation. Solutions of mercuric chloride, silver nitrate etc. in alcohol, where no dissociation occurs, showed almost no disinfec- tant action. Furthermore, they found the toxic action of salts having poisonous metallic ions was markedly diminished by the presence of a nonetoxic salt of the same acid. The investigators concluded that there is a general relation between the action of the acids and the amount of dissociated hydrogen present; but there appear many exceptions to a strict parallelism. The authors, however, attribute these exceptional effects to the anion or the undissociated molecule. In 1902 Bial (2) made a study of the antiseptic action of the hydrogen ion of dilute acids upon yeasts. The yeast was cultivated in fermentation tubes filled with grape-sugar solution to which various amounts of acid had been added and the antiseptic action was in; versely registered by the amount of gas produced. Bial did not make exact calculations of the amount of dissocie ated hydrogen necessary to inhibit the yeast, but he found that a general relation existed between the ionization and the antiseptic action. The highly dissociated acids- hydrochloric, sulfuric, nitric and trichloracetic- entire? 1y stOpped the action of yeast in concentrations between .005 and .008 normal. Acids of intermediate dissociation- phosphoric, formic, oxalic- accomplished the same effect .01 normal; while acids still less dissociated; acetic, benzoic, and butyric4 stopped all fermentation only when .04 to .07 normal. The most striking feature of Bial's work was a series of experiments showing the diminution of the antiseptic action of acids by the addition of neutral salts whose action is to decrease the dissociation of the acidic hydrogen. A solution of .01 normal formic acid and 0.3 normal sodium formate showed active fermentation. The same action was noticed with salts of the other acids used. Bial studied also the effect of hydrochloric acid and sodium chloride in the presence of peptone and found that the peptone increased the resistance of the yeast to a mixture of these compounds. In 1906 Winslow and Lochridge (34) found that the mineral acids such as hydrochloric and sulfuric were toxic for Escherichia coli and fiberthella typhosa in prOportion to the hydrogen ion concentration. With both acids equal concentrations of the dissociated hydrogen produced the same percentage reduction in numbers. Since at the low dilutions used the hydrochloric acid was about 96 per cent dissociated. its effect must have been entire- ly ionic, and since the sulfuric at 75 per cent dissocia; tion showed only the toxicity which would have been ex; pected from its dissociated hydrogen, the unionized mole; cule appears to exert no appreciable influence. Extending their study to the effect of organic acids (acetic and benzoic) upon the same organisms they found that these acids exerted the same toxic action in dilue tions in which they are only about one per cent dissociated. They concluded then, that the toxicity of these organic acids is due not mainly to hydrogen ions, but to the action of the undissociated molecule, varying widely, as might be expected, with the acid employed. Having determined the toxic dilutions of the acids when acting in tap water, they determined the effect of organic matter by using a one per cent peptone solution in place of the tap water. It was found that the peptone greatly decreased the toxic action of the acids, hydro; chloric being affected the most, the organic acids the least. This was found to be due to the fact that the hydrogen was largely bound by the peptone. This effect would naturally be more noticeable with the more highly ionized acid. Paus (23) noted that fatty acids were more strongly inhibiting toward E. coli and Eb. typhosa than were the dibasic organic acids tested (with the exception of oxalic and malonic). He concluded that there was little relation between the hydrogen ion concentration and growth, but that the kind of acid as well as the acidity was respon- sible for the germicidal value. In 1912 Jonannessohn (l6) conducted experiments similar to those of Bial with fatty acids and came to the concluse ion that with acids the main part of the acid action is played by the undissociated molecules, not by the ions. In 1917 Wolf and Harris (35), after investigating the characteristics of Clostridium welchii, were of the Opinion that the degree of acidity rather than the nature of the acid was the controlling factor in the germicidal and antiseptic actions of the acids. Norton and Hen (21) found the disinfectant action of acids to correspond closely to the hydrogen ion con; centration produced. Even with formic acid, the toxicity for bacteria was decreased when the number of undissociated molecules was increased, and the number of free hydrogen ions decreased. The germicidal action of the organic acids was studied in detail by Reid (26) in 1932 who concluded that the resistance of Bacillus pyocyaneous to acids is not con; stant, but varies with the kind of acid. The monoebasic acids, the least dissociated of the acids used, inhibited growth at a much lower Réion concentration than the highly dissociated acids such as oxalic. A wide difference was found to exist between the ability of an acid to exert a bactericidal effect and to inhibit growth. Acids which are strongly bactericidal frequently exhibit weak inhibiting powers in liquid media. Oxalic, the most toxic acid in bactericidal dilutions, exhibited a comparatively weak inhibiting effect. Acetic, propionic, and butyric, which are weakly bactericidal, were among the most inhibiting of all the acids tested. It appears that the bactericidal action of the weaker organic acids is not alone dependent upon the cations but the undissociated molecules are also active in this respect. Influence of Salt Brown (4) in studying pickle brine fermentation found that the acids, principally lactic, are relied upon to suppress the growth of putrefactive bacteria. Many bacteria may enter the pickle tank, but only those which can tolerate 12 to 20 per cent salt are concerned in the normal fermentation. In 1910 Pettersson (25) studied the influence of salt upon the bacterial flora and the decomposition of fish. With low concentrations of salt (0-8 per cent) the flora is heterogenous, containing both rods and cocci. Between 12 and 15 per cent concentration of salt the rods are killed while the cocci were plentiful in 18 per cent concentration after 15 days. Stadler (29) found that Escherichia coli communior, Salmonella enteritidis, proteus vulgaris and Cl. botulinus were inhibited by a salt concentration of between 7 and 10 per cent. In 1910 Weichel (32) working with meat inoculated before pickling found that the inoculated meat showed complete reduction in bacteria in 12419 per cent salt only after 75 days. In this connection Serkowski and Tomczak (28) stated that 15420 per cent of salt was reé quired to be of prOphylactic value with respectin bacteria which cause meat poisoning. KaraffaeKarbutt (18) found salt to possess weak bactericidal power. SaprOphytic bacteria required a higher concentration than the pathogenic bacteria. Eight to nine per cent inhibited the colon group while the pyogenic bacteria required 10 to 12 per cent. On the other hand, Falk (12) found that micrococci and sarcinae, because of their form of growth, could withstand about 15 per cent of salt. In 1915 Giltner and Baker (13) studying the effect of salt upon the flora of butter found that even 12 per cend does not retard growth in all cases and that some bacteria can withstand 20 per cent. Streptococci were found to be susceptible while staphylococci and micrococci were not. About 8 per cent of salt was believed to limit the physiological activities of most bacteria. Since it has been found that the bacteria are more resistant to changes in osmotic pressure than other forms of life it is necessary to look to other causes to explain the toxic action of salts upon them. Thus Eisenberg (9), Winslow and Hotchkiss (33), Fabian and Winslow (11) and others, have shown that the potency of a salt is a funce tion of the potency of both ions as well as the osmotic pressure involved. In 1919 Le Fevre (20) studied the ability of 50 different organisms to attack vegetables. Host of these failed to grow in a solution containing above 1 per cent salt. The only ones capable of growing in nigh concen- trations were Bacillus vulgatus which grew in a concen- tration as high as 6 per cent, and Bacillus mesentericus fuscus which grew in a 4 per cent solution. ’Of the organisms studied only 16 were more or less capable of softening cucumbers. A study of these led to the con; clusion that in the preservation of cucumbers, the critical point was between seven and eight per cent. On the other hand, Fabian and Johnson (10) isolated an organism corresponding in all essential details to B. mesentericus fuscus which was capable forming "slips”, and "mushy" pickles within 24 hours. The salt tolerance of this organism was higher than that reported by the other investigators. It grew readily in concentrations of salt up to and including 9 per cent, and could be induced to grow in salt concentrations up to and including 11 per cent. It was also shown that the type of medium had an important bearing on the salt tolerance of not only this organism but also upon other organisms of the same group, such as B. vulqatusg B. mesentericus, and L A 3. ruminatus which grew in higher concentrations of salt (11 per cent) in a special beet molasses medium than has been reported by other workers for nutrient broth (6 per cent). 10 Influence of Sugars Not much information is available upon the effect of concentrated sugar solutions upon bacteria. In 1909 Bitting (3) studied the effect of sugar on both the mold and yeast on tomato juice. No effect was noted until the concentration of sugar had reached 25 grams per 100 ml, where the growth occured as readily but less abundantly. In the 25 to 40 gram concentrations there was less development as the sugar was increased. The yeast was completely inhibited in concentrations above 80 grams per 100 ml. In the same year, Pederson and Breed (24) concluded sugar to be ineffective as a preservative in ketchup. Even 35 per cent concentrations inhibited certain types only. It was found, however, that 15 per cent of sugar and 3.5 per cent of salt inhibited growth of all organisms used except the yeast. Sackett (27) studied the longevity of members of the colon typhoid group in pure alfalfa honey. Eberthella typhosa remained alive for 48 hours in the pure honey, but was dead after 24 hours in dilutions above 50 per cent. The 10 per cent dilution was sterile after 4 days. These results are somewhat different from what would be expected, for with pure honey we are dealing with a very concentrated sugar solution. Sackett believed the failure of the organism 11 to die out as readily in concentrated honey as in the diluted solutions to be due to the fact that the former is a saturated colloidal solution, therefore, having a_ low osmotic pressure. In such a solution the plasmolysis would take place relatively slowly. When water was added, some of the sugar would form a molecular solution increase ing the osmotic pressure and hence the rate of plasmolysis. I. Verification of Strains In the past few years much work has been done in an effort to test the strains of staphylococci for produc; tion of the enterotoxic substance. In 1935 Stritar and Jordan’ (31) concluded there are no criteria for the differentiation of various types of staphylococci. The food poisoning strains agreed with other members of the group in not constituting a clearly marked division. They stated that, "the power to invoke food poisoning is not limited to any recognizable variety of staphylococci.“ Stone (30), on the other hand, using a specially prepared gelatin medium, claimed that the food poisoning strains liquefied gelatin while the nonepoisonous strains did not. In 1933 Woolpert and Back (36) comparing the entero; toxic substance with other toxic products produced by staphylococci obtained evidence indicating the former to be distinct from the hemolysin, dermotoxin and killing toxin formed in the filtrates of broth cultures of these organisms. 12 In 1934 Chapman et a1 (6) indicated the importance of the coagulase and hemolysis tests as measures of pathoé genicity, and in 1936, the use of crystal violet agar. Later (5) the following outline was suggested for the isolation and identification of the food poisoning strains. A. For isolation. l. Mannitol fermentation. Colonies surrounded by a yellow zone after over; night incubation on Bacto Phenol Red Mannitol agar are considered as positive. 2. Brom Thymol Blue test. Strains producing luxuriant growth on brom thymol blue agar in 43 hours are regarded as positive. B. For confirmation. 3. Pigment production. Food poisoning strains produce orange to yellow growth on proteose lactose agar in 48 hours. 4. Hemolysis test. Hemolytic strains (preferably on rabbit blood) are regarded as positive. 5. Coagulase test. A clot, jellyilike mass, or an opaque disk with human and rabbit plasma after 3 and/or 24 hours constitutes a positive test. Since the introduction of the preceeding scheme, many strains have been found containing variants. This led to further work by Chapman, Berens, Hilson, and Curcio 13 (5) indicating the steps by which a highly pathogenic strain degenerates until it becomes devoid of pathogenic preperties. On isolation a pathogenic strain of staphylococcus reacts positively to hemolysis (H), pigment production (P) coagulase test (C), crystal violet test (V), Brom thymol blue test (B) and lactose fermentation (L). The degeneration of the strain may be diagramatically illusé trated as follows: P H C B M L , /III In OHHH 1_”%0¢ H; _ o o l # t # t t t o x t t # pathogenic \ / } O 0 # ¥ } non pathogenic 22 C) <3 C) C)&———%3+——fl‘*~+——-W~é——#~éu———-fixe——H~< ‘1‘. A detailed discussion of the degeneration and criticism of the involved tests may be found in the above reference (5). 14 Experimental In studying the factors affecting the viability of Staphylococci in the acids, sugars, and sodium chloride, those strains were used which have been isolated from food poisoning outbreaks. The history of the various strains is as follows: Strain number 85 isolated by N. Y. Dept. Health 1932 Strain number 86 isolated by N. Y. Dept. Health 1932 Strain number 87 isolated by N. Y. Dept. Health 1932 Strain number 100 isolated from cake 4423432 Strain number 141 isolated from chocolate eclair 6;6;33 Strain number 161 isolated from sandwiches (1935) Strain number 166 isolated from head cheese 149437 Strain number 168 isolated from custard eclair 4428437 Strain number 169 isolated from chocolate eclair 5428437 The writer is indented to Dr. E. R. Hitchner, University of Haine for Strains 85. 86 and 87 and to Dr. G. M. Dack, University of Chicago, for the strains 1004169. 15 These strains together with a strain of Staphylococcus albus were tested according to the procedure suggested by Chapman et a1 (5). Table 1 The reactions of the various strains as tested for pathogenicity according to the technique of Chapman et a1. Test Strain number 85 Pigment 6 7 100 141 161 166 168 169 S.a1b. ,1 :- -' ,1 ,1 ,z ,1 Coagulase,human f ‘ ' 1 .t ,1; Coagulase,rabbit % Hemolysis,rabbit % Crystal violet % 8 2 ,z 2 2 ,z ii 2 ; W-W\-‘kw w~ w~ ‘hw H» ‘n. w» ‘*~ n-‘$.~w~ w» n~.~xr n~.~x. w~ n~. I ‘1‘. ‘I\w *JX- ‘N ‘1‘.- f i Brom thymol blue } } 2 1 Mannitol ferm. } 1 1 1 Lactose ferm. } 1 1 1 A consideration of table 1 and the diagram on page indicates that all the strains are pathogenic with the exception of number 141 and the strain of Staphylococcus albus. However, since some of the pathogenic strains were found to have partially degenerated as shown by the several negative tests for coagulase. hemolysis, and pigment, the 16 typical strains chosen for use throughout the course of the following experiments were numbers 85, 86, 87, 168, 169 and for comparison, the non4pathogenic strain of Staphylococcus albus. 151813110 6. Many fruits, vegetables and fermented foods such as sauerkraut, contain considerable amounts of naturally occuring organic acids which play a role in food presere vation. In studying the effect of acids upon staphylococci those commonly found in foods and food product were used, namely, acetic, citric, lactic, malic, and tartaric. For comparison benzoic and the mineral acid, hydrochloric, were also used. The medium used was one per cent glucose broth-having a pH of 6.846.9. All the acids except benzoic were made up to 0.1 normal, sterilized by filtration, and added aseptically to the broth. In order to obtain the different concentrations, various amounts of the acids measured in ml. were added to 10 m1. of sterile glucose broth. Since benzoic acid was found to be insoluble to the extent of a 0.1 normal solution, a saturated solution was made of benzoic acid in one per cent glucose broth. The amount which went into solution in this medium was found to be 0.4 per cent. In terms of normality this would be a 0.0327 normal solution. 17 The test tubes thus implanted were incubated at room temperature, and at intervals the number of viable organisms was determined by the plate method using standard agar. The determination of the Héion concentration was performed according to the electrometric method using a quinhydrone electrode. Since foods are stored mainly at room temperature, the temperature of incubation used throughout the course of these experiments was 20 - 22°C. Antiseptic and Germicidal Action of Acids on Different Strains of Staphylococci In order to study the relative inhibiting effect of each of the acids on the various strains of staphylococci, a 0.1 normal solution of each acid was made and various amounts in m1. quantities were added to 10 ml. of one per cent glucose broth. The broth was then incubated for one week at room temperature, subcultures were made by transferring 0.1 m1. from each tube into 10 m1. of broth and incubating for 3 days at room temperature. The acids were considered to exert an inhibiting effect if there was no growth in the original tube but growth in the subcultured tubes. The results of this eXperi; ment are shown in Table 2. After determining the effect of the various concené trations of acids the work vas repeated and at intervals the number of viable organisms was determined by plate counts using standard agar. room temperature and counted after 3 days. Table 2 The number of mi. sary to exert an inhibiting effect upon the different strains of staphylococci. 18 The plates were incubated at of 0.1 normal acid neces— Ml. of 0.1 Normal Acid Added to 10 ml. Broth Strain Acetic Citric Lactic Malic Tartaric HCl 85 2 2.5 1 2 2 1.5 86 2 3 1 2 2 1.5 S.a1b. 1.5 2 2 2 2 0.75 168 2 4 1.5 3 1,5 1,5 169 2 4 1.5 3 1.5 1.5 19 Table 3 The number of ml. of each acid necessary to exert a germicidal effect upon the different strains of staphylococci. M1. of 0.1 Normal Acid Added to 10 ml. Broth Strain Acetic Citric Lactic Malic Tartaric HCl 85 3 3 3 3 3 2 86 3 5 3 3 3 2 87 3 2 2 3 3 2 s. alb 2 3 3 2 3 l 168 3 5 2 5 2 2 169 3 5 2 5 2 2 Due to the disagreement among several of the previous workers as to the relative importance of the hydrogen ion concentration in actions of this type, it was deemed advisable to determine the pH of each of the concentrations of acid which was found to exert an antiseptic or germicidal effect. This is shown in table 4. The results secured for benzoic acid (Fig.10 and Table 14) show that a one per cent glucose broth saturated with this acid had a pH of 3.47 and was definitely germi; cidal. The amount of benzoic acid present in the solution was found to be 0.4 per cent or a 0.0327 normal solution. Just what normality below this would be germicidal was not determined. 20 Table 4 The pH values of the various acids for the diferent strains of staphylococci at the point where there is no inhibition, and germicidal action. Acetic Acid Citric Acid Strain Killed Inh. No Inh. Killed Inh No lab. 85 4.37 4.57 4.91 4.15 4.25 4.40 86 4.37 4.57 4.91 3.70 4.15 4.40 87 4.37 4.57 4.91 4.15 4.40 4.76 S.alb 4.37 4.73 4.91 4.15 4.40 4.76 168 4.37 4.57 4.91 3.70 3.81 4.15 169 4.37 4.57 4.91 3.70 3.81 4.15 Strain Lactic Acid Malic Acid 85 3.72 4.32 4.66 3.89 4.06 4.49 86 3.72 4.32 4.66 3.89 4.06 4.49 87 3.90 4.32 4.66 3.89 3.91 4.06 S.a1b 3.72 4.32 4.66 3.89 4.06 4.49 168 3.90 4.17 4.32 3.55 3.89 4.06 169 3.90 4.17 4.32 3.55 3.89 4.06 Table 4 continued. Tartari Acid -ypro hlori, Acid Strain Killed Inh. To Inh. Killed Inh. Ho Inh. 85 3.61 3.85 4.32 2.37 2.93 3-55 86 3.61 3.85 4.32 2.37 2.93 3.55 87 3.61 3.85 4.32 2.37 2.93 3.55 S.a1b. 3.61 3.85 4.32 3.10 2.93 3.55 168 3.85 4.07 4.32 2.37 3.10 3.55 169 3.69 4.07 4.32 2.37 2.93 3.55 Table 5. Showing the decreasing order of antiseptic and germicidal value of the different acids together with their dissociation constants. Germicidal Kind of Inhibiting Dissociation Acid ppH acid ApH constant acetic 4.37 acetic 4.59 1.86 x 10"5 citric 3.8? lactic 4.27 1.38 x10 '4 lactic 3.81 citric 4.06 8.0 x 10-.4 malic 3.74 malic 3.98 4.0 x 10'-4 tartaric 3.65 tartaric 3.92 1.1 x 1053 301 2.43 H01 2.94 probably 90497fi The arrangement shown in Table 5 indicates that, in general, the antiseptic and germicidal power of the acids used parallel except in the case of lactic acid, which 22 has a stronger antiseptic action than a germicidal action. The reverse is true for citric acid. Thus, it is seen that a difference exists between the ability of an acid to exert a germicidal action and to inhibit growth. This supports the conclusion reached by Reid (26) The arrangement of activity is also especially significant in view of the fact that the dissociation constants of the acids used are practically the reverse. HCl and tartaric acids having the highest dissociation constants, (tartaric being 1.1x1013) exert the weakest germicidal and antiseptic action; while acetic with the lowest dissociation constant (1.86x1045) exerts the strongest action. This supports the findings of Clark, (7), Yaoi (37) and others who believe the unionized molecules are prominently concerned, since the toxic effect is not in direct proportion to the degree of dissociation. An inspection of Table 4 further indicates that the order of germicidal activity does not appear to be entirely consistent, but varies with the strain used. This might be explained as being due to the fact that since the union- ized molecules play a proment role it is natural that the negative radical may bring about a specificity of reaction to such an extent that one and the same acid might act differently on different organisms. In this connection, Yaoi (37) found that some acids vary greatly in their accelerating or retarding effect upon bacterial growth 23 when applied to the medium in low concentrations. With the inorganic acids, HCl and HNO3 stimulated the growth. On the other hand, organic acids on the whole, have no stimulating effect except acetic and oxalic which, however, have much less stimulating power than the former two. This stimulating effect might explain the slightly weaker antiseptic action of acetic acid as compared to the others tested. The order of antiseptic potency was found to be about the same for all the organisms tested, that order being, acetic) lactic) citric) malic) tartaric) Hcl It was found that one and the same organism does not react the same to all of the acids. Figure l and table 8 show that during the first 24 hour phase, strain number 85 is rather resistant to the action of malic (3 ml) and tartaric acid (3 ml.), while during this same period it is very susceptable to the action of acetic and lactic acid. Figure 4 and Table 11 indicate that the strain of Staphylococcus albus is likewise more resistant to the action of acetic, citric, and tartaric acids during the first 24 hour period than it is to the other acids tested. The same resistance, though to a lesser extent, was found for strain 169 on acetic acid (Figure 6, Table 13). In order to determine whether the resistance was truly characteristic for the organism, the resistance of strain 85 on a higher concentration of malic acid (4 ml.) 24 was determined. Figure 1 shows that although the time required to kill all the organisms was shortened, the resistance during the first 24 hour phase was as great as in the lower concentration of acid. Thus it is in; dicated that the resistance or susceptibility of the organism in the presence of acid is specific for each acid and organism. Influence of sugar and Sodium Chloride on Different Strains of Staphylococci. In determining the effect of sugars upon the organ; isms, the sugars, glucose and sucrose, were added to sterile sugar free broth and autoclaved at 12 pounds. This was done to xeep the hydrolysis at a minimum. Both the sugar and the salt concentrations were made up by weight. The tubes were inoculated and then incubated at room temperature for one week. An inhibiting concentra4 tion was considered as one in which there was no growth in the original tube, but .rowth in the su cultured tube. 0 The results of this.experiment are found in Tables 6 and 7. 25 Table 6 Per cent of sucrose, glucose, and sodium chloride exerting an inhibiting effect upon the different strains of staphylococci. Per Cent Strain Sucrose Glucose Sodium chloride 85 so 35 17.5 86 50 4O 15 87 50 4O 15 S.a1b. 50 35 20 168 60 45 17.5 169 60 '45 17.5 Table 7 The per cent of sucrose, glucose and sodium chloride exerting a germicidal effect upon the different strains of staphylococci. Per cent ' Strain Sucrose (Glucose Sodium chloride 85 6O 4O 2O 86 6O 50 20 87 6O 5O 2O S.a1b. 6O 40 22.5 168 70 50 20 169 70 5O 20 26 From the data in Tables 6 and 7, it can be seen that the strains are comparatively resistant to the action of both sugars and salt. About 40 to 45 per cent of glucose was required to exert antiseptic action compared with 50 to 60 per cent sucrose, and 15 to 17.5 per cent of salt. The graphs shown in Figures 748 and the data in Tables 15420 indicate that there is a great difference between the action of glucose and sucrose. Vigorous growth took place even when the latter was present in large amounts. This phase was then followed by a period during which growth was materially checked although no decided reduction in numbers was apparent. Repeated observations showed that after five days the number of organisms rapidly decreased. At the end of seven days the organisms were all killed. This rapid decrease might be explained as being due to the combined effect of the sucrose and the acid pro; duced during the first period during which vigorous growth took place. Glucose, on the other hand, as shown by Figure 7 and Tables 15420 produced no such curve and the number of viable organisms was found to continually decrease. In this respect the salt and glucose acted alike. Sodium chloride exerted a germicidal effect when present in as low a concentration as 20 to 22.5 per cent . (See Fig. 9, Tables 15420). The order of activity, 27 sodium chloride> glucose) sucrose is to be explained by the fact that the action of each is, at least in part, due to plasmolysis of the cell. Since this effect is additive in nature, that is, it depends upon the number of particles or molecules present, it is natural that sodium chloride with a molecular weight of about 58.5 and glucose 180 would contain more molecules per unit weight than would sucrose whose molecular weight is 342. Therefore, the activity should tend to decrease as the molecular weight increases. The effect of sucrose upon several strains of food poisoning staphylococci was studied by Hucker and Haynes (15) who found the toxin producing micrococci to develOp in relatively high concentrations (20 to 50 per cent). At the end of 14 days it was found that 20 per cent sucrose broth was sufficient to reduce materially the numbers of organisms, while in a medium containing 35 per cent sucrose, or more, the organisms were practically all killed or inhibited at the end of 18 days. During the first 24 hours growth was vigorous in concentrations as high as 50 per cent. During the second 24 hours growth was materially checked and after 5 days, the numbers of staphylococci were materially reduced. The results of this paper agree very closely with the conclusions reached by Hucker and Haynes (15). A survey of Figure 8, Tables 15420 shows a rapid growth rate during the first 24 hours, followed by a period of 28 about four days where the numbers of bacteria remain rather constant. At the fifth day a very rapid decrease in numbers is apparent, and at the end of seven days the organisms were all killed or inhibited. The action of sodium chloride, as shown by Fig. 9 and Tables 15420, bears a closer resemblance to the acid curves than to the curves showing the effect of sucrose upon the organisms. The action of the organic acids is attributed to the specific molecule and the specific potency of both ions, while the effect of sucrose is principally one of plasmolysis. Hence, the appearance of the curves indicates the importance of the potency of the ions in the action of sodium chloride. This conclusion supports those reached by Eisenberg (9), Fabian and Winslow (11), and others who have indicated the action of a salt is a function of the action of both ions as well as the osmotic pressure involved. 29 Summary A summary of the action of certain organic acids commonly occuring in food and of sucrose, glucose and sodium chloride upon typicd.strains of staphylococci which had been isolated previously from outbreaks of food poisoning shows: 1. That although the action of the highly dissociated mineral acid parallels the hydrogen ion concentration production; it was found that the organic acids exerted a germicidal and antiseptic effect disprOportionate to the hydrogen ion concentration produced. It is, there; fore, apparent that the observed effects are due to factors in addition to the hydrogen ion, presumably either the unionized molecule or the anion or both. 2. The differences in the action of the various organic acids upon one and the same strain of staphylococci is believed to be due to a specificity of reaction like; wise brought about by either the anion or undissociated molecule or both. 3. The decreasing order of germicidal action of the acids was found to be acetic) citric) lactic> maliC) tartaric) hydrochloric. 4. The decreasing order of antiseptic action of the acids was found to be acetiC) lactic) citriC) malic) tartaric) hydrochloric. In this connection it is interest; ing to note that citric and lactic acids change places in their antiseptic and germicidal prOperties. 5. Sodium chloride in a concentration of 15 to 20 per cent was found to exert an inhibiting efiect upon the staphylococci, while a 20425 per cent concentration exerted a definite germicidal action. 6. Glucose exerts an inhibiting effect in a concen; tration of 30 to 40 per cent and a germicidal effect at 40 to 60 per cent. 7. Sucrose is less active than either glucose or sodium chloride since a concentration of 50 to 60 per cent was required for inhibition and 60 to 70 per cent for germicidal action. 8. There is a great difference in the action of glucose and sucrose upon the staphylococci. During the first 24 hours vigorous growth took place in sucrose even when it was present in concentrations as high as 70 per cent. This phase was followed by a period of four days 1 during whicn the number of viable organisms remained nearly constant. On the fifth day the number of organisms rapidly decreased until the seventh day at which time all the organisms were killed. Whereas, with glucose the germicidal action was apparent at once and uniform throughout. 9. A saturated broth solution of benzoic acid having a normality of 0.0327 and a pH of 3.47 was found to be definitely germicidal to staphylococci. a o o o OOH o m 0 ON ooe.o o 0 cm a 0mm oHH ooe.mm o“ oem co m 00¢.m oms ooo.¢mfl 0mm oom.m ooo.m N ooo.o~a ooo.mea ooo.ooo.a ooo.oa ooo.OmH ooo.oe see H gases ooo.on ooo.ooo.m ooo.o~o.a ooo.m~m ooo.o~o.a ooo.mm¢ .Ha Hensmpoap a“ anadoon How assesses oases assess onusso. oapmoq .mm cashew .Hoooooamnmmpm mo hpwfiwpsfib can no mdwom oacsmho mo monmfiHMqH .w manna Hoooooahnmwpm mo thprsHb on» so mOHom quemno mo ooquHmnH a. O O O m OO OH O O O s OHm O OOO.N OH OH OH m OOO.sH OO OOa.ON OOO.N ONH OOO.OH m OOO.OO OOO.Oa OOO.OON OOO.OH OOO.OH OOO.Omm see H names OOO.OHm OOO.OOO.H OOO.OOH.N OOO.OON.H OOO.OOm.H OOO.OON.H .Hs Hannmposp GH ESHSoonH HOm oHnepsea oHHss ussomH oHssHO shamed . .mm dfidhvm .m manna O a 3 o O Oh O m OH O O OON OmH a OON O mam Om OO0.0 OOO.N m Oom.O Om OOO.HH OOO.OH OO0.0a OOO.OH m OO0.0m OOO.OOm OO0.0a OO0.00H OOO.OOm OO0.0~ ewe H sense OO0.00m OOO.OOH.H OO0.0HO.H OOO.mNm OOO.OOO.H OOO.OHa .Ha Hanmsosp aw BDHSUOQH HOm oHneshea oHHes capoeH cansao oHpooa .Hoooooahgmmpm mo thHHpmwb can so mOHom ownemno .am cashew mo mosodamsH .OH manna LO O a lOH ON O O O OmH OmO m ON OO O OOO OON.¢ e OeH OON.m O ONH OOO.NH OOO.mN m OOO.~ OOO.OHm OOH OO¢ OOO.HmH OOO.OOH m OO0.0mH OO0.0eO OOO.ON OOO.OH OOO.an OOO.OHO nae H seamen OO0.0mm OO0.000 OO0.0eO OOO.OOH.H OO0.0¢O OO0.000.H .Hs HsHpmsoap “Hi! qfi afiafioonH Hom oHssssse osHsz unposH caanO Ospoo< .Hoooooahgmmpw mo hpwawpswb map so mcwom oflsmmao mo monoaamnH amsQHm mdooooodfinpspm mo qwmnpm .HH manna o s OH O O OO O O O OH O O Owe Om ON Os OOa O OO OOO.m OOm.H OOO OON O¢O.N m OOO.N OOa.Om OOO.¢ OOO.N OO0.0 OOO.ON N OOO.ma OOO.OHm OOO.OO OOO.¢O OOO.OOH OOO.OHH see H “Opp OOO.OmO OO0.000.H OOO.ONO.H OOO.OOO.H OOO.OO¢.H OOO.OOO.H .Hs HOHsOOomp nH seafloqu HOm unnesuse oHHaa osOoaH enhpao oasmo< .OOH eHmnOO .Hoooooahgmmpm mo thHHpsfib on» so mOHos oHsmmao Mo mosmdfimsH .NH manna mmh 0 O O O O ON a OHN 0H 00H 0H 000.NvL 0 m OOO.Nm OON OOO.N OOH.H OOO.O OOH N OO0.00H OOO.H¢ OO0.0¢ OO0.0e OOO.mO OOm.eN see H HOOOO 000.00m 000.00m 000.00» 000.00m 000.00¢ 000.00¢ .HB Hmfinmpowp GO aaadoonH Hum oprpusa oHHME oHpoma owaawo caucus .mOH nHmhpm .HQUOOOthmmpm Mo hpwfiflnmfl> map so mmflom oHnmmpo mo mosmdamnH .MH manna b- O O O O O O O O OH ON Os OOH O OH m oom OHO OOO.N OOO.HH OOH OOH.m N OO0.00 OOO.HO OO0.0m OO0.00 OO0.0 OO0.00H see H HOOOO OOO.OOO OOO.Omm OO0.0NO OOO.OOO OOO.OHO OO0.0HO .Hs HOOHOOoOp :H aaHSOosH OOH OOH .pHe.O NO Ow mm .Hoooooflhnmmpm mo hpwawpmwb on» no A>¢.m mmv vHoe ofioanmn mo nodeHom spopp depmpdpmm a mo moqodamnH .¢H manna meOHHHHad.s. O. O O a OON O r OHH OH O sOON OO itO .zmaN ON O O . zOOH OaH OH sOON OOm s OH aeaH OOO.H qOm sOmH OO0.0 m . ”ii OON saNH OOO.Om OOO 2mm OOa.sH N OOH.O 2O.NN OOO.OOO OO0.0H .sOO OO0.0Nm awe H Hopes OO0.00~ OO0.000 OO0.0NO.H OOO.OOH.H OO0.0NO OO0.0aa .He Heanmposp . a“ afififioodH Hodz omoposm mmooaaw Homz omohofim omooaaw OO OHOHOO 1mm magnum OH OHOOO mH OHOOO .OO ens mm Owenpm .HOOOOOHngOOO mo sOHHapess on» no oOHpoano afifloom One omohosm .mmoofiam mo oosodHMmH .ma capes One ma manna mnOHHHHa » a. O O O 5 OO O OH OOH O O OOOH Om ONN OOON O OH OONH OHN OOO.¢ OFNH OOO a OOO zHOH OOH.O OO0.0N seNH OOO H O OOO.O~ ENOH OO¢.OOIEOOO.OOH OOO OON Hm N OOO.¢O 2O.» OOO.OOO OOO.OOO 2O.OH OOO. ONO see H amped OO0.0mO OOq+OOm, :m.H OOo.ON~ OO0.00w, .Ha HOHOOOoOp a“ SdafioonH Homz mmopodm omooSHw Homa mmonodm omooflaw fiHmmOm mspH< m NO cashew OH OHadH, OH OHpse .mfipam was um msHmnpm .Hoooooahnmmpm Mo hpflfiflpwflb map mm mcwhoaso ESHOOO Ode .omonosm .mmoodam mo mosodfimsH .mH manna dam NH manna OOOHHHHO w :. O O a O OOO OOH O OO =HNN O aHmN O O OO aOOH O ON aOOH OO O OOO :OHH OO OOO :OO OOO.O O OOH.H zOO OOO OON.N .aOO OOO.ON N OOO.ON 2N.O OOO.HH OOO.OO OOO.OOO OO0.00 see H “Opes OOO.OOH OOOlOOH OOO.OOH OOO.OHN OOO.OHN OOO.OH .Ha HOHHOOosO dfi afiaaoonH Hush mmonodm mwooflaw flown mmonodm mmooaflw \mOH nwmhpm mma!fia new ON OHOOO OH OHOOO .mOH was mOH wnwmhpmo.aoooooahsmmpm mo hpfififlnmwb can so ocfluoago adfidom use omoosm .mmoodam mo mononaqu .om manna was ma manna 31.1171} : - . . t/L'. 102 of 3. _‘¥ 4‘ ‘J ‘v ‘. . I 3 4 6" H» Pigire l. Inf] eice of acids on via1ility of at») y OCOCCi, cteria U . no) OJ. NUTS the n4; 1 40:0: 0 -. Figure 2. ‘..L COElC “' citric "‘- ttrzli. 1‘10 at“ t 1:213 in (1.71;; S biCC3Tifl \ - Oi f‘ " " r‘v .. .5 alt} . t} v A O" 10:: figure 5. D Influence of '11.} :0. {4‘7 (1) (t H '7 U acids 01 vi bilitx of st? | ‘ . EJJL lC _.—- tip” tiric-+n 1' 2 m1 ac-ztic -—— C» ;. citric ——- 1 1‘;CtiC + '1 lfif)‘. in ”o—- . .-A~—A- ~V 7'11 tart.- :‘ic -+H .151 -‘ a L Duct " 71' 1:110»)? 31' H 1') (Kw- #3 U" C-.(/ ti ie :11 df37s figure 4. Influ nee of zcids on viability of s: ohfilocic i. strain of.Staohvlococcze albis. bacteria the number of g of q .A Figure 5. 1 :2 7i time in dflr'rs Influence of acids on vixhility of stn strain £0. 16%. 3 :21 acetic _.— ' m1 Citric-- ’ ml lactic—4e- ml malic __u.. m1 t‘irt-sric H phrlococci, . 3 pl acetic ———- citric ----— lactic: -—¥-- rral ic —---- tartaric-4+? nber of bacteria 1... .. 3 11.4, T, Lt 10s of i Line in days Eigare 6. Influence of acids on viability of staohrlococci, strain 40. 163. 43] strain p r 4 9-“ ° 6d \ \\ 93‘ 8.1.5113] \ \\ “\ L3, strain \ 40” strain nimber of bacteria tre A-..’ of lo: E7 —+e- 0t 09.113715 ~ '_" 105 4+0 time in davs figure 7. ‘ \ y\ \ \f. \t. \ \ 1 3 5 6 Influence of glucose on the viability of staphylococci. bacteria I .7 L 2‘0 J the ninhe ‘1 L 10; o q figure 8. 3‘ fiOfi 1 1 7 0,; strain ' strain strain strain - strain strain r—H- 55 -*' 86-- 87-*- St. albus—n-o 1654++ 1694*- l 2 3 time in days (fir- Inf;uence of sucrose on viability of staphylococci. 3),- strain 55 —-- 20;: strain {6-- 21- strain {97 “*- . L” .52. strain r.‘3t. albus--~- ‘- 23 strain 168 4+. 23,-. strain 169 -o- 5 4 m "-1 H 0) +3 2 .o 4‘ 'M O E. ,O E3 3‘ a? \. p c” O 6 H 2 l o (p 0 b‘igzre 9. Influence of sodium chloride on viability- of st'mhylococci. str in 65 -* strain es—-"- strain F7-ak— strain ct. albus-—~—- strain loB 4** strain 163 -o- bacteria P Si 102 of the number Figure 10. Influence of benzoic acid on viability o: staohylococci. .‘JL strain 87-—&— strain it, gihus__u_. strain lee-uas ria strain 169-4»- ? bficte .a nnmher o ‘rc ()3 I F of t“, 10 tithd irl d?:]s Figure 11. Influence of hydrochloric wcid on viability of staphylococci. 10. Bibliography Barber M.A. ‘ Philippine Jour. Sc. 23515- (1914) Bial M. Ueber die antiseptiche Funktion des H ions verdunnter Sauren. Ztschr.f. phvs. Chemie. 49: 513 (1902) Bitting A.W. Experiments on the spoilage of tomato ketchup 'U.s. Dept. Agr. Bull. 119 (1909) Brown C. W. Notes on Brine Pickle Fermentation Abstracts Bact._1: 104;105 (1916) Chapman G., Berens C.. Nilson, E. and Curcio L. Differentiation of Pathogenic staphylococci from non; pathogenic staphylococci. J. Bact. ii: 311 (1938) Chapman G.. Berens C.. Peters A.. Curcio L. Coagulase and Hemolysis tests as measures of the pathogenicity of staphylococci. J. 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