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Methods for the isolation of Leuconostoc mesenteroides and a study of its mineral requirements Triggs, Rosalie Elizabeth

Abstract

Methods for the isolation of Leuconostoc mesenteroides and the establishment of its mineral requirements have been studied. Nine natural materials were employed as probable sources for the isolation of species of Leuconostoc. Sauerkraut and ensilage yielded typical cultures. No cultures were obtained from cheese, milk, soured potato, cow manure and sliming cabbage, lettuce and cucumber. The microorganisms were isolated by diluting and then plating onto solid media. Yeast Tryptic Digest Agar, Tomato Juice Agar and Yeast Tryptic Digest Gelatin, each at pH 6.9, 6.2 and 5.6 were employed as the plating media. The Yeast Tryptic Digest Agar at a pH between 6.2 and 6.8 proved to be the most effective medium for the isolation of species of Leuconostoc. An enrichment technique was also carried out employing a variety of media. The tubes were held under both aerobic and anaerobic conditions. However, this method resulted in failure. Proteolytic bacteria predominated to such an extent that the relatively weaker Leuconostoc, if present at all, were unable to survive even under the favourable anaerobic conditions employed. The cultures isolated from sauerkraut and ensilage were found to be similar in all respects to known species of Leuconostoc mesenteroides. Due to the difficulties involved in the isolation of the genus Leuconostoc a selective method for the isolation of lactic acid bacteria employing hydrogen peroxide was evolved. Preliminary conditions for isolation were worked out using S. lactis and E. coli. It was found that the minimum amount of E. coli cells (.0058%) and the maximum amount of S. lactis cells (19.2%) remained viable when treated with hydrogen peroxide at a concentration of 0.05% at 15°C for sixty minutes. A concentration of 0.2% hydrogen peroxide, while destroying all E. coli after sixty minutes treatment was also very deleterious to S. lactis. It was found that with an increase of temperature over 15°C, the rate of destruction of hydrogen peroxide and the rate of growth of the bacteria served to complicate the findings obtained. Fresh raw milk, employed as the medium of suspension, was found to be necessary for obtaining the optimum effect with hydrogen peroxide. Reconstituted milk and nutrient broth appear to lack the unknown protective factor present in fresh raw milk. Using the conditions worked out for S. lactis and E.coli, eight additional organisms were tested for the effect of hydrogen peroxide inhibition. It was demonstrated that these conditions permit the isolation of other catalase negative organisms and at the same time drastically reduce the number of catalase positive bacteria present. The two Micrococci cultures studied are an exception to the general observation that catalase positive organisms are sensitive to peroxide. The efficiency of this method when mixed cultures were employed was tested. It was found that all E. coli cells were destroyed while 5,700,000 S. lactis cells remained viable. To determine the practical application of this technique, a sample of fresh cow manure was treated with hydrogen peroxide. By using this agent the percentage of catalase negative bacteria arising on a plate increased from 3% to 40-45%. The mineral requirements of a defined strain of Leuconostoc mesenteroides have been determined. By depleting the basal medium (McLeod and Snell, 1947) with the strain of L. mesenteroides understudy the essential nature of manganese but not magnesium or iron could be shown. However, by depleting the basal medium with A. niger for twelve days the need for magnesium was demonstrated. Manganese must be present before the magnesium has a significant influence. A combination of A. niger and L. mesenteroides depletion had no significant advantage over the depletion by the mould alone. No stimulatory effect could be observed by the addition of calcium, zinc or cobalt at a concentration of 1.0 ppm., copper at a concentration of 0.05 ppm., and molybdenum at a concentration of 0.02 ppm. Molybdenum and zinc showed a slight inhibitory effect. Basal medium and depleted basal medium were extracted with 8-hydroxyquinoline and chloroform. Part of both media underwent further treatment; hydrolysis for thirty minutes with HCI (final concentration of 0.1N). In all cases the effect of added iron at a concentration of 0.1 mg. FeSO₄/10 ml. medium was very pronounced if manganese was also present. The addition of 0.01 FeSO₄ was as influential as 0.001 mg. or 0.01 mg. FeSO₄)10 ml. medium. The additional procedure of hydrolysis apparently removes some factor other than iron, manganese or magnesium which is necessary for the optimal growth of L. mesenteroides. It was shown that this factor was not tryptophane, zinc or copper. The effect of iron on two other heterofermentative and two homofermentative bacteria was determined. One heterofermentative organism (species of Leuconostoc) showed stimulation with the addition of iron and manganese; however, the other one belonging to this type (E. coli)and the two homofermentative bacteria (S. lactis and S. faecalis) gave very little response even after the addition of manganese, magnesium and iron. Respiration studies of L. mesenteroides were carried out. No difference in acid production or carbon dioxide evolution per cell could be observed with varying amounts of manganese added to the growth medium.

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