Wolff (1905) usually found anaerobes in 1 cc. portions of milk ANAEROBIC BACTERIA IN DAIRY PRODUCTS

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1 ANAEROBIC BACTERIA IN DAIRY PRODUCTS I. NUMBERS OF SPORES OF ANAEROBIC BACTERIA IN MILK AND CREAM R. V. HUSSONG AND B. W. HAMMER Department of Dairy Industry, Iowa State College, Ames, Iowa Received for publication, July 5, 1929 INTRODUCTION Organisms belonging to the genus Clostridium have frequently been found in dairy products and have been considered to be causally related to certain of the changes taking place in these materials. Their growth is undoubtedly influenced by the other organisms present. Presumably it is favored by the using up of oxygen and, on the other hand, it may be inhibited by some of the products, such as acids, that are formed by certain species of bacteria. The presence of anaerobes in a dairy product is no assurance that they will develop but it would appear that if large numbers were involved the chances for their growth would be better than if only small numbers were included. The investigation herein reported was undertaken with the idea of securing information as to the numbers of spores of anaerobes that could be found in milk and cream produced under Iowa conditions. The work was limited to the spores because of the difficulties in including the vegetative cells. Moreover, it would seem that, because of the sources from which they come, most of the anaerobes gaining entrance to milk or cream would be in the spore stage. HISTORICAL The investigations of Rodella (1904) indicated that anaerobes are present in milk to the number of from one to three per 0.1 cc. Wolff (1905) usually found anaerobes in 1 cc. portions of milk 89 JOURNAL OF BACTERIOLOGY, VOL. XIX, NO. 2

2 90 R. V. HUSSONG AND B~. W. HAMMER but only once in a 0.1 cc. sample. Barthel (1910) investigated milk for the presence of anaerobes and concluded that they occurred in small numbers in commercial milk. He reported that they often could not be found even in 15 to 20 cc. portions. A method of examining milk for spores of BaiUu8 enteritidissporogenes was described by Savage (1912); he concluded that it was of value in determining the purity of milk samples. Weinzirl and Veldee (1915) examined 5, 10 and 15 cc. quantities of milk for the spores of anaerobic bacteria. With market milk the following results were secured: 90 samples were examined using 5 cc. portions and 28 per cent were positive; 112 samples were examined using 10 cc. portions and 37.5 per cent were positive; 34 samples were examined using 15 cc. portions and 50 per cent were positive. It is evident from these data that the numbers of spores of anaerobic bacteria in the milk were small. The data secured on pasteurized milk are as follows: 110 samples were examined using 5 cc. portions and 18 per cent were positive; 110 samples were examined using 10 cc. portions and 57 per cent were positive. Ayers and Clemmer (1921) carried out investigations on the anaerobic spore test. From the results they secured it is evident that the numbers of spores were never very large. Rawlinson (1926) emined 35 mples of London ice cream during July and August and found Clostridium welchii present in numbers ranging from 1 to 100,000 per cubic centimeter. METHODS The procedure used to estimate the numbers of spores present in the samples examined was an adaptation of widely used methods and involved the inoculation of different volumes of the materials to be examined into tubes of sterile litmus skim milk, after which heat was used to kill the vegetative cells; the tubes were then incubated at 37 C. and observations made for the growth of anaerobic bacteria. Two different sized tubes were used in which to inoculate the samples. Those used for quantities ranging from 5 to 100 cc. measured 3.8 by 25 cm. and had a capacity of 200 cc., while those

3 ANAEROBIC BACTERIA IN DAIRY PRODUCTS for volumes of 1 cc. or less measured 1.5 by 12 cm. and had a capacity of 20 cc. Litmus skim milk was added to each of the larger tubes in such an amount that the volume after the addition of the sample would be about 100 cc., except in the case of tubes that were to receive 100 cc. of sample when 50 cc. of litmus skim milk were used. Each of the small tubes received 10 cc. of the litmus skim milk. The milk was sterilized in the tubes, cooled by holding in cold water, inoculated and then heated to 80 C. for fifteen minutes after which it was quickly cooled to 370C. and incubated at this temperature. No seals were used on the large tubes but the material in the small ones was flooded with a sterile mixture of one part paraffin and two parts vaseline. The volumes used for the examination of a sample, the number of tests with each volume and the amount of sterile litmus milk employed with each volume are shown in the following tabulation: VOLUMES OF A SAMPLE USED N'MB FTBS AMO'UNT OF STERILE LITM'US FOR EXAMINATION MI U8ED CC. CC Observations for evidence of the growth of anaerobes were made daily for five days. Such growth was first judged on the basis of gas production, coagulation and odor, but all positive findings were checked by the actual isolation of an anaerobe from one or more tubes of each set. From the tubes in which anaerobes developed conclusions were drawn as to the number of spores in the sample. RESULTS OBTAINED The data secured on the numbers of spores of anaerobic bacteria in pasteurized and raw milk from the market milk laboratory of the Iowa State College are given in table 1. None of the 15 91

4 92 R. V. HUSSONG AND B. W. HAER TABLE 1 Numbers of spores of anaerobic bacteria in milk NUMBERS OP SPORES SUBEo NUMBR OW Pasteurized milk: 1 spore in 10 cc spore in 5 cc spores in 5 cc spores in 5 cc spores in 1 cc... 2 Raw milk: 4 spores in 5 cc... 4 TABLE 2 Numbers of spores of anaerobic bacteria in cream NUMBBW or amnzs NUBERS OF SPOREDS ~~~NUMBERS SAMPLESP 0F Pasteurized cream: 1 spore in 25 cc. 2 1 spore in 10 cc.1 I spore in 5 cc.1 3 spores in 5cc. 1 Raw cream: 1 spore in 50 cc. 2 1 spore in 25 cc. 2 1 spore in 10 cc. 3 1 spore in 5 cc. 1 2 spores in 5cc. 1 3 spores in 5cc. 4 4 spores in 5cc. 3 2 spores in 1 cc spores in 1cc. 3 6 spores in 1 cc.1 10 spores in 1 cc.1 TABLE 3 Numbers of spores of anaerobic bacteria in sour skim milk NlJlBwaS OJ SKRM ~~~NMEROP NUMBERS OV SIPORES SAnmPLESi 2 spores in 1 cc.1 10 spores in 1 cc.1 samples, 11 pasteurized and 4 raw, contained the spores in large numbers; the maximum number was 4 spores in 1 cc., while the minimum was 1 spore in 10 cc.

5 ANAEROBIC BACTERIA IN DAIRY PRODUCTS Table 2 gives the results obtained on 27 samples of sweet cream, 5 pasteurized and 22 raw, that came from either the market milk or butter laboratory of the Iowa State College. As with the milk, the numbers of the spores present in the cream were comparatively small. The maximum was 10 spores in 1 cc. and the minimum was 1 spore in 50 cc. Two samples of condensed sour skim milk from an Iowa condensery were also examined for the numbers of spores of anaerobes contained. This product is regularly put up in barrels. No attempt is made to sterilize it, the high acidity being relied on to inhibit bacterial action. From the results obtained, which are given in table 3, it is evident that in this material also the spores were present in comparatively small numbers. The tubes in which anaerobes developed ordinarily showed extensive changes due to these forms. Gas production and characteristic odors were conspicuous and often definitely preceded coagulation. The types of changes in the tubes varied considerably and were undoubtedly influenced by such factors as whether one or more species were present and the combinations of organisms in the mixed cultures. In some instances aerobic spore formers developed and played a part in the changes that took place. Experience made it possible to recognize such organisms by organoleptic tests, although microscopic examinations and agar shake cultures were very helpful in drawing definite conclusions. DISCUSSION OF RESULTS Spores of anaerobes were present in each of the 44 samples studied, 15 of milk, 27 of cream and 2 of condensed sour skim milk, but the numbers were always comparatively small. The maximum was 10 spores in 1 cc. and the minimum was 1 spore in 50 cc. No definite difference between the pasteurized and the raw products was evident; the 11 samples of pasteurized milk showed both higher and lower numbers of spores than the 4 samples of raw milk, while the 22 samples of raw cream showed both higher and lower numbers than the 5 samples of pasteurized cream. This indicates that, as would be expected from the 93

6 94 R. V. HUBSONG AND B. W. HAMMER general resistance of bacterial spores, pasteurization has no effect on the spores of anaerobes. The rapid development of anaerobes in milk in which conditions have been made favorable for these organisms suggests that the restraining action of various factors is important in preventing their growth under practical conditions. The objectionable fermentations produced by some of the anaerobes are evidence that the rapid development of acid commonly occurring in milk and cream on holding is to be preferred to some of the changes that can take place in these products. REFERENCES AYERS, S. HENRY, AND CLEMMER, PAUL W The sporogenes test as an index of the contamination of milk. U. S. D. A., B. A. I., Bul BARTHEL, CHR Obligat anaerobe Bakterien in Milch und Molkereiproduktin. Cent. f. Bakt. 2 abt., 26, RAWLINsoN, E. G A note on the bacterial content of ice cream. Lancet, 211, RODELLA, A tyber die in der normalen Milch vorkommenden Anaerobien und ihre Beziehungen zum KLsereifungsprozesse. Cent. f. Bakt., 2 abt., 13, SAVAGE, W. G Milk and the Public Health. Macmillan and Co., London, p WEINZIRL, JOHN, AND VELDEE, MILTON V A bacteriological method for determining manurial pollution of milk. Amer. Jour. Pub. Health, 5, WOLFF, ARTHEUR 1908 Zur Kenntnis der Verinderungen in der Bakterienflora der frischen Milch wahrend des sogenannten Inkubationsstadiums. Cent. f. Bakt., 2 abt., 20,