paratyphi. Second, a systematic comparison of media developed in recent years. Among investigations in which this

Transcription

1 A COMPARISON OF COBALT AND NICKEL SALTS WITH OTHER AGENTS FOR THE DETECTION OF HY- DROGEN SULFIDE IN BACTERIAL CULTURES WILLIAM P. UTERMOHLEN, JR.1 AND CARL E. GEORGI Departments of Chemistry and Bacteriology, University of Nebraska Received for publication March 20, 1940 INTRODUCTION Numerous methods have been proposed for the detection of hydrogen sulfide production by bacteria, several having been developed in recent years. Among investigations in which this reaction has been used as a means of differentiating bacteria are the contributions of Jordan and Victorson (1917) on the paratyphoid-enteritidis group, Kligler (1917) concerning the typhoidparatyphoid group and Levine and co-workers (1932, 1934 and 1936) involving the colon-aerogenes group. Comparisons of various methods with larger groups of bacteria have likewise been made by ZoBell and Feltham (1934) and Tittsler and Sandholzer (1937) who compared lead and iron salts. Hunter and Creselius (1938) introduced bismuth sulfite for the purpose of comparing it with iron. A survey of the literature revealed two facts. First, with one exception, the use of cobalt and nickel salts as detecting agents for hydrogen sulfide has not been reported. The exception is found in a report by Sacquepee and Chevrel (1905) who found that nickel sulfate gave positive hydrogen sulfide tests with cultures of Salmonella schottmilleri but not with those of Salmonella paratyphi. Second, a systematic comparison of media containing these various detecting ions has never been attempted with a representative group of bacteria. 1 Present address, Kingsport, Tenn. Research Laboratory, Tennessee Eastman Corporation, 449

2 450 WILLIAM P. UTERMOHLEN, JR. AND CARL E. GEORGI Certain disadvantages exist with all of the indicators now in use for the detection of hydrogen sulfide. Iron salts tend to hydrolyze and precipitate from solution. Neither are they as sensitive in neutral or acid media as other detecting ions whose sulfides have a lower solubility product. Lead and bismuth are limited in their use because of their toxicity. Compounds of bismuth are difficult to keep in solution. Theoretically cobalt and nickel are somewhat more sensitive to hydrogen sulfide than iron and nearly as sensitive as bismuth. In addition, they possess the added advantage that their salts are quite soluble and do not hydrolyze in alkaline or neutral media as do iron and bismuth salts. Thus, cobalt and nickel ions possess the requisite qualities of solubility, sensitivity and distinct color change desirable for hydrogen sulfide detection. However, all metallic ions are subject to the limitation that they attain maximal sensitivity for sulfide ion only at higher ph levels. Since previous investigations have indicated that hydrogen sulfide detection may vary with the method used, the object of the following studies has been to compare some of the more recent methods with one employing cobalt and nickel as detectors. EXPERIMENTAL A. Toxicities of cobalt and nickel ions for bacteria Before developing a medium containing cobalt and nickel, it was necessary to determine the tolerance of the test organisms to various concentrations of salts of these metals. The bacteria selected represented a number of genera predominantly of the Enterobacteriaceae group and were species capable of growing aerobically upon nutrient agar. A number of the pathogenic species used were obtained from the stock culture collection of the Kansas State Board of Health Laboratories. Hotchkiss (1923) studied the effects of varying concentrations of solutions of a number of metallic salts, including cobalt and nickel, on Escherichia coli (communis). With these and other metals, the medium (metal salt in Bacto peptone) could not be heat-sterilized and still yield consistent results, for the salts either

3 DETECTION OP HYDROGEN SULFIDE IN BACTERIA hydrolyzed or formed precipitates with the peptone. A procedure was therefore devised which eliminated these undesirable conditions. Nutrient broth and M solutions of cobalt and nickel nitrate hexahydrates were prepared separately. Measured quantities of the broth were then placed in filter flasks on which were mounted Berkefeld filters. After the assembly (flasks, broth and filters) had been sterilized by autoclaving at 15 pounds for 20 minutes, measured quantities of the salt solutions were filtered into the broth until the desired metal ion concentrations had been attained. The filter candles were then replaced by sterile tubing bells (Riker and Riker, 1936), the flasks inverted (the side-arm tubes then served as a means of admitting air to the flasks, thus maintaining atmospheric pressure within) and the contents aseptically distributed in sterile test tubes. The final concentrations of both nickel and cobalt salts in nutrient broth were M, M, M, and M. These were inoculated with single loop transfers of twenty-four hour broth cultures. Turbidity in the broth tubes was used as a criterion of growth. Incubation was at the optimum growth temperature for the individual organisms concerned. After twenty-four hours it was found that every test organism except Bacillus kaustophilus had grown to a visible extent in the two weaker solutions. In some instances, with further incubation, growth was apparent in the concentrated solutions, but for the majority of the organisms M cobalt remained the highest level tolerated. Identical tests employing nickel demonstrated that, in general, the bacteria could tolerate nickel solutions of M and lower concentration. Thus, the tolerated concentration of nickel nitrate was about five times that of cobalt nitrate. In subsequent work, therefore, M cobalt and M nickel solutions were employed. Only five of the test species would not grow in the M nickel solution; these were Mycobacterium phlei, Sarcina lutea, Rhodospirillum rubrum, Bacillus kaustophilus, and Klebsiella friedlanderii. These organisms, for which hydrogen sulfide production has never been observed, were omitted from further tests. 451

4 452 WILLIAM P. UTERMOHLEN, JR. AND CARL E. GEORGI B. Preparation of the cobalt-nickel medium Factors influencing the detection of hydrogen sulfide in a medium incorporating cobalt and nickel ions were systematically studied. A number of media were prepared, each differing in one variable and the effect upon the test was noted. These experiments indicated the following optimum conditions. Sulfur sources. The naturally-occurring sulfur compounds found in the peptone constituents of the media were not sufficient for the detectable production of hydrogen sulfide; hence an additional sulfur source was necessary. Among those investigated were one organic and two inorganic compounds. A combination was also employed. Sodium sulfite proved unsatisfactory. Cysteine yielded much better tests than did sodium thiosulfate, the third sulfur source tested. All organisms producing hydrogen sulfide at all were always able to yield that product from cysteine. The latter was more desirable than a cysteine-sodium thiosulfate mixture. Glucose. The addition of one gram of glucose per liter of medium resulted in better tests for hydrogen sulfide than when it was omitted. Hydrogen ion concentration. Two ph levels, both slightly on the alkaline side, were selected because metal ions are theoretically more sensitive at higher ph values. A ph of 7.1 was the preferred one for the detection of hydrogen sulfide for all microorganisms except Proteus vulgaris, which produced it in amounts detectable by this test at ph 7.8 but not at the former ph level although it may be formed at the lower level also. Tarr (1934) reported similar behavior of this organism. Scant or negligible hydrogen sulfide production, apparent in most instances at the higher ph, is perhaps due to the inhibitory action of that hydrogen ion concentration upon the metabolism of the bacteria. Protein source. Bacto-proteose peptone, one of the two nitrogen sources used, produced somewhat better results than did Bacto-tryptone. Hydrogen sulfide indicator. Cobalt was more sensitive than

5 DETECTION OF HYDROGEN SULFIDE IN BACTERIA 4 nickel, as an indicator of hydrogen sulfide, in that it yielded positive tests in a larger number of cases. This is to be expected in view of the lower solubility product. However, nickel gave better tests than did cobalt (as evidenced by more intense darkening of the media) in the instances when it did give positive tests. Despite the fact that the concentrations of the two individual metals were near the maximum tolerated values, it was found that when those two concentrations of nickel and cobalt nitrates were employed in one medium, no cumulative inhibitory action took place. The combination gave tests much superior to those obtained when either of the two metals was used singly. The medium regarded as the best combination of the different variable factors investigated, and discussed above, has the following composition: Proteose peptone (Difco) grams K2HPO gram Glucose gram Cysteine hydrochloride (Merck) grams Agar (Bacto) grams M Co(NO3)2 solution* ml M Ni (NOs)2 solution ml. Water ml. * grams Co(NO3)2.6H20 in 500 ml. water. t grams Ni(NOs)2-6H2O in 500 ml. water. 453 The medium was adjusted to a ph of 7.2 i 0.1 with N/1 NaOH, then tubed and sterilized at 15 pounds for 20 minutes. The nickel and cobalt solutions can be made up as one solution if desired. COMPARISON OF VARIOUS MEDIA FOR THE ABILITY TO DETECT THE PRESENCE OF HYDROGEN SULFIDE The cobalt-nickel medium discussed in the preceding paragraph (designated as medium E in the table of results) was compared with four other media used for hydrogen sulfide detection. They were Bacto-Lead Acetate Agar (A), Bacto Iron Peptone Agar (B), Bacto-Kligler Iron Agar (C), and Hunter's bismuth sulfite tryptone agar with skim milk (D) (Medium III, Hunter

6 454 WILLIAM P. UTERMOHI N, JR. AN CARL E. GEORGI and Crecelius, 1938). These media were prepared, tubed, and sterilized simultaneously. In these tests, as in all preceding ones, TABLE 1 ORGAMNI A B C D E Aerobacter aerogenes Aerobacter cloacae Aerobacter oxytocum... 1 _ Aerobacter viscosum...i ++ + _ ++ Alcaligenea viscous Escherichia coli Eberthella typhosa Shigella dysenteriae... 2 { (2)- - + Skigella parady-senteriae 12)+ (Flexner)...4. (1,2,43) Shigella sonnei... 3 (2) + - _ + + (3)- _ - Shigella sp. (Newcastle) Salmonella gallinarum i Salmonella paratyphi i Salmonella schottmuellen i i Salmonella pullorum } +1 + Salmonella enteritidis Salmonella typhimurium i Proteus vulgaris... 4 (1,2) i ++ if ph is 7.8 Slow lactose fermenter weak +++ Proteus morgani Serratia marcescens weak Streptococcus lactis Citrobacter sulfidogenes } i +++ single stab inoculations were made from twenty-four hour nutrient broth cultures. Observations were made at the end of 1, 2, 3 and 5 day intervals, following incubation at 370C.

7 DETECTION OF HYDROGEN SULFIDE IN BACTERIA 455 RESULTS A positive reaction for hydrogen sulfide consisted of a general darkening of the medium, or of a definite localized browning or blackening along the line of puncture. The organisms which yielded positive tests are listed in Table 1, together with estimations of the relative response in the various media. The following organisms did not give evidence of hydrogen sulfide production under any of the experimental conditions: Alcaligenes fecalis, Neisseria catarrhalis, Leuconostoc mesenteroides, Brucella abortus, Staphylococcus albus, Staphylococcus aureus, Micrococcus tetragenus, Flavobacterium vitarumen, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus megatherium, Alcaligenes radiobacter, and Corynebacterium xerosis. DISCUSSION The following facts, gathered from the above table, are apparent. The lead acetate, iron peptone, and Kligler iron media gave many positive tests; however, they were not quite as sensitive as the bismuth and the cobalt-nickel media. It should be noted that a number of bacteria usually considered hydrogensulfide-negative, including Salmonella paratyphi, several Aerobacter species, Escherichia coli, and several Shigella species, do produce hydrogen sulfide as demonstrated by the use of bismuth and cobalt-nickel indicators. The bismuth and the cobaltnickel media were both superior to the other media in respect to the total number of positive tests obtained. Where several media yielded positive tests, the cobalt-nickel medium usually gave the most intense positive test, or at least a test as strong as any others obtained. One noticeable exception occurs in the case of Eberthella typhosa, which causes intense blackening of the Kligler iron medium, but which produces no visible reaction in the cobalt-nickel medium. Ferric ammonium citrate tryptone agar was also prepared, according to directions in the Difco Manual (1939), and compared with the above media. However, the results obtained with it were not satisfactory. This may be due to the fact that the sulfur source in the commercially prepared material is better sensitized

8 456 WILLIAM P. UTERMOHLEN, JR. AND CARL E. GEORGI to the action of bacteria than that in the medium made and used in these investigations. (Private communication from Dr. H. G. Dunham of the Difco Laboratories.)2 These results indicate that certain of the media devised for the determination of hydrogen sulfide production by bacteria fail to agree among themselves. Shigella species and Streptococcus lactis give positive tests only with the bismuth indicator; Eberthella typhosa does not give a positive response with cobalt and nickel, but does with lead, iron, and bismuth; the strains of coliform and Salmonella species investigated give better tests with the cobalt-nickel medium than with any other; Aerobacter cloacae gives a positive test with cobalt-nickel indicator only. Some of the media compared in this work have been successfully used to distinguish various bacterial species in certain genera; this shows that the ability of an organism to produce detectable hydrogen sulfide is a function of the medium and of the indicator used to make the test. It is not enough to state merely that an organism does or does not produce hydrogen sulfide without stating the conditions under which it was investigated. Hunter (1938) was led to a similar conclusion. Several explanations probably are involved in the lack of agreement in these experiments. One is that certain detecting ions are more sensitive to hydrogen sulfide, and hence yield positive tests while others can not. This would explain why bismuth shows hydrogen sulfide production from Shigella species while no other detector ion does so. In other cases some of the metal ions used may poison the enzyme system essential to hydrogen sulfide production. For example, Eberthella typhosa gives positive tests in lead, iron, and bismuth media, and a negative test in the cobalt-nickel medium; possibly the latter ions inhibit some essential enzyme system of that particular organism. Similarly, the lead ion may poison the enzyme system of Sal- 2Dehydrated cobalt-nickel media of the above composition (with the sulfur source omitted) was prepared through the kindness of the Difco Laboratories. When 0.2 per cent cysteine was added, results obtained were identical with those from the media prepared according to the above formula. The Difco experimental medium is known as %S-34010B.

9 DETECTION OF HYDROGEN SULFIDE IN BACTERIA 457 monella paratyphi, while bismuth, cobalt, and nickel ions do not. Thirdly, hydrogen sulfide production undoubtedly depends upon the ability of bacteria to cleave the material serving as a sulfur source. Desnuelle and Fromageot (1939) have demonstrated the presence of an adaptive enzyme, "cysteinase," produced by Escherichia coli, which splits cysteine to form hydrogen sulfide. It may be that with the coliform bacteria the cobalt-nickel medium gives better tests than do the other media for hydrogen sulfide because only the former medium contains added cysteine as a sulfur source. This agrees with the findings of Levine (1936). Inhibition of growth by the metal detector ions is not an acceptable explanation, as every species grew in all of the media investigated. It is interesting to note the wide difference in toxicity displayed toward bacteria by salts of the three metals, iron, cobalt and nickel, considering that the three elements are so similar chemically. Cobalt, although immediately between iron and nickel in the periodic system, is appreciably more toxic to bacteria than are either of the latter metals. The general maximum tolerated concentrations of iron (ferric), cobalt, and nickel ions are in the approximate ratio , respectively. Since these three elements are in the same period and column in the periodic table, and have very similar physical and chemical properties, this difference in toxicity is of interest. An incidental observation in this investigation was that the presence of added cysteine in the cobalt-nickel medium prevented the usual fermentation of glucose with the formation of gas, which is characteristic of the coliform and other enteric bacteria. This is possibly due to the fact that' the free glucose in the medium was used up in chemical combination with the excess of cysteine present (Schubert, 1939), and, thus, was not subject to normal attack by bacteria. SUMMARY 1. The toxicities of nickel and cobalt ions for a number of bacterial species in broth cultures were determined. 2. A medium containing proteose peptone, cysteine, glucose,

10 458 WILLIAM P. UTERMOHLEN, JR. AND CARL E. GBORGI dipotassium phosphate, nickel and cobalt nitrates, agar, and water was developed which compares well with present established media for detection of hydrogen sulfide production by bacteria. 3. Organisms usually considered to be negative hydrogensulfide producers, such as Salmonella paratyphi, Streptococcus lactis, Escherichia coli, and a number of Aerobacter and Shigella species, give positive reactions with bismuth or cobalt-nickel indicators, or both. 4. It has been shown that detection (and perhaps production) of hydrogen sulfide in bacterial cultures varies greatly according to the medium and indicator employed. A statement of the ability of a microorganism to produce hydrogen sulfide is of little value unless all factors are specified. 5. The possible explanation of this variation in the detection of hydrogen sulfide production by bacteria has been discussed. REFERENCES DESNUELLE, P., AND FROMAGEOT, C La decomposition ana6robie de la cystine par Bacterium coli. I. Existence d'une cyst6inase ferment d'adaptation. Enzymologia, 6, Difco Laboratories, Inc Manual of Dehydrated Culture Media and Reagents. 6th ed., rev. HOTCHKISs, M Studies on salt action. VI. The stimulation and inhibitive effect of certain cations upon bacterial growth: J. Bact., 8, HUNTER, C. A., AND CRECELIUS, H. G Hydrogen sulphide studies. I. Detection of hydrogen sulphide in cultures. J. Bact., 35, JORDON, E. O., AND VICTORSON, R Differentiation of the paratyphoidenteritidis group. II. Lead acetate agar. J. Infectious Diseases, 21, KLIGLER, I. J A simple medium for the differentiation of members of the typhoid-paratyphoid group. Am. J. Pub. Health, 7, LEVINE, M., EPSTEIN, S. S., AND VAUGHN, R. H Differential reactions in the colon group of bacteria. Am. J. Pub. Health, 24, LEVINE, M., VAUGHN, R., EPSTEIN, S. S., AND ANDERSON, D. Q Some differential reactions in the coli-aerogenes group of bacteria. Proc. Soc. Exptl. Biol. Med., 29, RIKER, A. J., AND RIKER, R. S Introduction to Research on Plant Diseases. pp Swift. SACQUAPfE, E., AND CHEVREL, F Action des bacilles typhiques, paratyphiques et du colibacille sur quelques sels metalliques. Compt. rend. soc. biol., 59,

11 DETECTION OF HYDROGEN SULFIDE IN BACTERIA 459 SCHUBERT, M. P The combination of cysteine with sugars. J. Biol. Chem., 130, TARR, H. L. A The enzymic formation of hydrogen sulphide by certain heterotrophic bacteria. Biochem. J., 27, TARR, H. L. A The enzymic formation of hydrogen sulphide by certain heterotrophic bacteria. II. Biochem. J., 28, TITTSLER, R. P., AND SANDHOLZER, L. A Advantages of peptone iron agar for the routine detection of hydrogen sulphide production. Am. J. Pub. Health, 27, VAUGHN, REESE, AND LEVINE, M Hydrogen sulfide production as a differential test in the colon group. J. Bact., 32, ZOBELL, C. E., AND FELTHAM, C. B A comparison of lead, bismuth, and iron as detectors of hydrogen sulphide produced by bacteria. J. Bact., 28, Downloaded from on June 18, 2018 by guest