develop in a medium having considerable variation in reaction.

Transcription

1 INFLUENCE OF HYDROGEN-ION CONCENTRATION OF MEDIUM ON THE REPRODUCTION OF ALFALFA BACTERIA E. B. FRED AND N. E. LOOMIS Department of Bacteriology, University of Wisconsit The value of the hydrogen electrode in determining the reaction of culture media has been shown by Michaelis (1914), Clark (1915, 1917), and others. Their results indicate that bacterial processes are influenced to a marked degree by the hydrogen-ion concentration of the medium. It appears that certain species of organisms are very sensitive to slight changes, while others will develop in a medium having considerable variation in reaction. The question arises whether the reaction of a medium within certain limits exerts an important influence on the growth of legume bacteria. In this laboratory an attempt has been made to study the effect of reaction on the growth of B. radicicola from alfalfa. METHODS Mannitol solution was used as a culture medium. Mannitol grams Magnesium sulfate gram Monobasic potassium phosphate.0.2 gram Sodium chloride.0.2 gram Calcium sulphate.0.1 gram Distilled water cc. The phosphate was dissolved separately in a little water and the solution made neutral to phenolphthalein with N/i sodium hydroxid. After sterilizing, the culture medium was again adjusted to the neutral point of phenolphthalein. Hydrogen-ion concentrations were measured electrometrically. A modification of the apparatus used and described in. detail by Loomis and Acree (1911) was used. All readings were made at 25 C. and are expressed in terms of the symbol PH, 629

2 630 E. B. FRED AND N. E. LOOMIS (Sckrensen (1909)) representing the log I/C{. Corrections were made for hydrogen pressure and for variations of the calomel electrodes, as suggested by Loomis and Acree (1916). REACTION TO PHENOLPHTHAL!9IN AND HYDROGEN-ION CONCENTRATION Twenty-four 100 cc. portions of mannitol solution in 750 cc. Erlenmeyer flasks were inoculated with alfalfa bacteria. The cultures were then arranged in pairs and the reaction changed by the addition of sterile N/10 sulphuric acid or sodium hydroxid. Column 2 of table 1 shows the variations in reaction. Immediately after inoculation the hydrogen-ion concentration was determined, and again after two weeks. (See chart 1.) In connection with the measurements of reaction, plate counts of the total number of bacteria were made after two and four weeks. The averages of triplicate plates, aaid the hydrogen-ion concentration, as well as the titrated acidity or alkalinity are given in table 1. TABLE 1 Reaction and growth of legume bacteria NORMAL ACID OR A,IXALI IN 100 CC. P BACTERIA IN 1 CC. OF CULTURE MEDIUM OF MEDIUM FLASKBeing NUM- Beginning After BER two After two weeks After four weeks Cubic centimeter Theory Found weeks water In culture Total rela- Total Rieamedium ti've tive H2SO No growth No growth H2S No growth No growth H2S ,580, ,000, H2SO ,520, ,500, Neutral ,900, ,200, NaOH ,280, ,200, NaOH ,280, ,100, NaOH NaOH ,220, ,800, NaOH NaOH ,540, ,900, NaOH ,100, ,600,000 21

3 HYDROGEN-ION CONCENTRATION AND ALFALFA BACTERIA 631 The results show clearly the difference between the concentration of hydrogen ions and the total concentration of acid or alkali, From the hydrogen electrode measurements, it is evident that change in the hydrogen-ion concentration is much greater with a given increase in acidity than with a corresponding increase in akalinity, especially after bacterial growth has continued for two weeks. Consequently large amounts of alkali have little effect on the hydrogen-ion concentration of the mannitol medium, while even small amounts of acid cause a decided change in true acidity. The difference in the PH for a given amount of acid and alkali in water solutions and in the mannitol medium is very noticeable from the figures of table 1. Here the vertical column 3 gives the theoretical PH in water solutions and columns 4 and 5 the PH found in the culture medium when inoculated. The buffer action may be due to the mannitol or to products of the bacterial growth. The latter seems more probable, because it is so much more noticeable after two weeks',growth. HYDROGEN-ION CONCENTRATION AND GROWTH OF BACTERIA The most important point shown by the data of table 1 is the relation between hydrogen-ion concentration and development of alfalfa bacteria. Plate counts after two and four weeks indicate that B. radicicola is much more sensitive to sulphuric acid in mannitol solution than to gram equivalent amounts of sodium hydroxid. Somewhat similar results have been reported by Prucha, (1915), who found that normal hydrochloric acid was much more injurious to the multiplication of B. radicicola of alfalfa than equivalent amounts of normal sodium hydroxid. It was assumed in previous investigations that B. radicicola from alfalfa was especially resistant to alkali and likewise especially sensitive to acid. In view of the curve of hydrogen-ion concentration, it seems that the apparent resistance of the legume bacteria to alkali is due to the slight concentration of hydroxyl-ions in the mannitol solution. From the data of table 1 it will be seen that alfalfa bacteria in mannitol solution

4 632 E. B. FRED AND N. E. LOOMIS II - H1dro en Ion Conceniratiorol Cu.ture medium Inoculdted alter 2 ureeks InOCUldted dt beginning C.C. of 0.1 Ilormal 5odium Hqdroxid $ulphur'i CHXRT 1 G.C. of 0.1 Ilormdl acid

5 HYDROGEN-ION CONCENTRATION AND ALFALFA BACTERIA 633 gave the highest, count in a neutral solution, although small amounts of alkali had very little effect on the number of bacteria. On the other hand, acid in gram equivalent amounts seriously retarded or prohibited growth. The relation of growth to hydrogen-ion concentration remained almost the same after two and four weeks. EFFECT OF BACILLUS RADICICOLA ON HYDROGEN-ION CONCENTRATION Chart 1 gives the curves of the hydrogen-ion concentration of mannitol solution before and after the growth of legume bacteria. The hydrogen-ion concentration is plotted in terms of PH on the ordinate and the cubie centimeters of acid or alkali on the abscissa. It should be remembered that as PH increases the concentration of hydrogen-ions decreases and vtce versa. One of the most striking facts noted by these curves is the difference in the hydrogen-ion concentration of the culture media before and after growth of bacteria. It will be seen that this change is most marked in the case of high PH of the medium. Apparently the alfalfa bacteria bring about changes in the reaction of the medium which are favorable for their reproduction. The growth of different species of legume bacteria in culture media of varying hydrogen-ion concentration will be discussed in a future paper. The authors are indebted to Dr. S. F. Acree for many helpful suggestions. REFERENCES CLARK, W. M Jour. Inf. Dis., 17, 109. CLARK AND LUBS 1917 Jour. Bact., 2, 1. LOOMIS AND ACREE 1911 Am. Chem. Jour., 46, 585. LOOMIS, N. E. AND ACREE, S. F Jour. Am. Chem. Soc., 38, MICHAELIS 1914 Die Wasserstoffionenkonzentration. PRUTCHA, M. J N. Y. Agr. Exp. Sta. Cornell Mem. 5, S0RENSEN 1909 Compt. rend. Lab. Carlsberg, 8, 1.