While experimenting with crystal violet bile media for use in

Transcription

1 GROWTH OF ANAEROBES IN CRYSTAL VIOLET BILE MEDIA CHARLES F. POE Division of Sanitary Chemistry, Department of Chemistry, University of Colorado, Boulder, Colorado Received for publication, July 22, 1929 While experimenting with crystal violet bile media for use in water analysis, it was thought that some information could be obtained by testing the growth of anaerobes in media containing crystal violet and bile. An ideal medium for the detection of pollution in water supplies would be, of course, a medium which would prevent the growth of all bacteria except members of the colon group. This, however, is far from possible. B. welchii and other anaerobes are responsible for many false presumptive tests in waters. This fact was first reported by Frost (196) in 196. Many investigators have observed this condition in recent years. The question arose as to whether crystal violet bile broth would be effective in preventing the growth of anaerobes. Apparently this subject has not been investigated in regard to water analysis as no reference could be found in the literature. Hall and Ellefson (1918) (1919) were the first to call attention to the fact that gentian violet, when added to lactose broth, would eliminate the false presumptive test due to anaerobes. They found that one part in 2, was effective in eliminating all gas-forming anaerobes. Wagner and Monfort (1921) confirmed the work of Hall and Ellefson. EXPERIMENTAL Lactose broth was prepared and varying amounts of crystal violet and bile were added. The strength of the lactose and peptone was 1 per cent each. The ph value of all media was 117 Downloaded from on September 23, 218 by guest JOURNAL OF BACTEROGY, VOL. x, No. 2

2 118 CHARLES F. POE between 7.2 and 7.3. The media, in 1 cc. lots, were placed in Durham fermentation tubes. Some of the media, without the dye, was also prepared so that the effect of the bile could be studied. Dye in different concentrations was also added to the plain lactose broth. Suspensions of the different anaerobic cultures to be tested were made in sterile distilled water. Five cubic centimeters of this dilution were added to one tube from each of' the different lots of media. The tubes were allowed to incubate at 37 C., and gas readings were made in twelve, eighteen, twenty-four, and forty-eight hours. In case there was no gas production in fortyeight hours, another reading was made in seventy-two hours. In the routine methods for water analysis 1 cc. of water are added to 2 cc. of medium. These proportions were used throughout this work. All results are expressed in terms of the amount of dye present in the original medium before the sample of inoculum was added. The cultures used were mostly obtained from Dr. Ivan C. Hall, University of Colorado Medical School, Denver, Colorado. Three cultures of B. welchii were obtained from F. E. Greer, Department of Health, Chicago. In all, nineteen cultures were used as follows: B. multifermentans, two cultures; B. tertius, two cultures; B. sphenoides, two cultures; B. welchii, Type I, two cultures, Type II, three cultures, Type III, four cultures, and Type IV, four cultures. The growth of the anaerobes in the different media will be discussed under three headings: bile media, crystal violet lactose media, and crystal violet bile media. BILE MEDIA In order to study the effect of bile on the growth of anaerobes each culture was tested with media ranging in bile content from 1 to 15 per cent. Table 1 represents the gas production for all cultures except those of B. welchii. From table 1 may be seen that the growth in all dilutions of bile media was very good in the case of B. multifermentans and B. tertius. With one culture of B. 8phenoide8 no gas production was Downloaded from on September 23, 218 by guest

3 GROWVTH OF ANAEROBES IN CRYSTAL VIOLET BILE MEDIA 119 apparent in any of the media, while the gas production with the other culture was very weak even in forty-eight hours. TABLE 1 Gas production in per cent. Anaerobes in bile media B. MULTIFERMIEMTAN B. TBRTIUS B. SPHENOIDES PIR A B A B A B CENT BILE o 8. a 4 A C AO A A A A A A A A AO A A A A A A A A eq co C4 eq cc C4 eq cc eq co ". eq co cc eq Oc 14 _ co eqeqeqqeqq q 4 cq, e q eq - eq c' TABLE 2 Gas production in per cent with B. welchii, Type IV A B _D PER BILE B J _ a a a o a a a a eqc cco4 cq cc cc e cc - cc eq c O O Downloaded from on September 23, 218 by guest

4 12 CHARLES F. POE The gas production with most of the strains of B. welkhii was very good. One of the cultures of Type I gave an average of 65 per cent gas in twelve hours. The other gave 3 per cent gas in twelve hours. Two of the strains of Type II gave an average of over 4 per cent gas in twelve hours, while the other two strains averaged about 3 per cent gas in eighteen hours. All four strains of Type III averaged over 3 per cent gas in all the different strengths of bile in twelve hours. The gas production in the strains of Type IV varied considerably. The gas production for these is given in table 2. Table 3 gives the average gas production for the other strains of B. welchii. TABLE 3 Average gas production in per cent. B. welchii, Types I, II, III PER CENT BILE 12 HoIUs 18 HOuRS 24 HOURS 48 HouRs From tables 1, 2, and 3 we may conclude in general that no amount of bile present in lactose broth up to 15 per cent has the power to inhibit the growth of anaerobes. CRYSTAL VIOLET LACTOSE MEDIA As stated in the introduction Hall and Ellefson (1918) (1919) successfully used gentian violet in lactose broth to inhibit anaerobes. Fourteen different samples of lactose broth, each containing a different amount of crystal violet, were made up. The dye content ranged from one part in 3, to one part in 1. Downloaded from on September 23, 218 by guest

5 GROWTH OF ANAEROBES IN CRYSTAL VIOLET BILE MEDI 121 Each of the anaerobes was tested in these media. The organisms showing growth in forty-eight hours are given in table 4. It will be noted from table 4 that the only organisms producing gas in forty-eight hours were the B. welchii cultures belonging to Type II. One culture of B. sphenoides produced a small amount of gas in seventy-two hours in the three greatest dilutions of dye. Two cultures of other types of B. welchii produced a small amount of gas in seventy-two hours. One of these produced gas in a dye content as high as 1 to 75,. In the other case the dye content was 1 to 15,. It seems TABLE 4 Gas production in per cent. Anaerobes in crystal violet lactose broth, B. welchii, Type II Day CONTENT A B3 C hours hours hours hours hours hours hours hours hours hours hours hours 1:3, :2, :15, :1, :75, :5,, :35, :3, :25, :2, 5 25 rather strange that B. welchii, Type II, was the only type which showed gas production in the crystal violet lactose broth. This fact might afford a means of differentiating this type of B. welchii from the other types. Further work should be conducted along this line by some one interested. CRYSTAL VIOLET BILE MEDIA The gas production with anaerobes in crystal violet bile media varied considerably. With the two cultures of B. multtifermentans a dye content of 1: 1 was required to inhibit growth in most of the concentrations of bile. With the cultures of B. tertius a Downloaded from on September 23, 218 by guest

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7 GROWTH OF ANAEROBES IN CRYSTAL VIOIET BILE MEDIA 123 dye dilution of 1:1 was required to inhibit growth in most of the higher concentrations of bile. Even with this dye concentration, gas was produced in eighteen hours with the media having a bile concentration of 9 per cent and more. With the cultures of B. sphenoides the growth was very scant. There was no gas production with one of the cultures. In case of the other culture no gas appeared until after seventy-two hours and this was confined to bile percentages of 3 to 7 per cent inclusive, dye content 1: 1,. In analyzing the results from the study of the cultures of B. welchii we find considerable variance. One of the cultures of Type I required a dye content of 1: 35 to'prevent growth. All the cultures of Type II gave excellent results in twelve to eighteen hours in a dye concentration of 1: 1. Three strains of Type III required a dye concentration of 1: 35 to prevent growth. In the other strain, excellent gas production was produced in forty-eight hours in a dye concentration of 1:1. The members of Type IV varied the most. Two of the strains required a dye content of 1: 35. Another produced no gas in forty-eight hours in a dye concentration of 1: 1, and the fourth strain was inhibited by a dye content of 1:1, in the percentages of bile below 9 per cent. In table 5 are summed up the results with the bile dye media. In this table are shown the reciprocals of the dye concentration required to prevent gas formation in forty-eight hours. A number of the cultures were not inhibited by a dye concentration of 1:1, the greatest concentration tried. In such cases these are indicated by > 1, meaning that a dye concentration greater than 1: 1 was required to inhibit growth. Table 5 seems to indicate that a very high concentration of dye is required to inhibit the growth of anaerobes. The concentration of dye would be far too great to use in media for water analysis. CONCLUSIONS 1. In general, a bile content up to 15 per cent is not effective in eliminating the growth of anaerobes. Downloaded from on September 23, 218 by guest

8 124 CHARLES F. POE 2. With most strains of B. welchii the gas production was much better in the higher percentages of bile. 3. A crystal violet dye content of over 1:75, in lactose broth will eliminate most anaerobes. 4. B. welchii, Type II seems to be very resistant to crystal violet in lactose broth. A dye content of more than 1: 2, is required to inhibit growth in this type of B. welchii. 5. The addition of bile to crystal violet lactose broth destroys the inhibitive power of the dye as far as the growth of anaerobes is concerned. REFER4ENCES FROST 196 Confirmatory test for B. coli in routine water examination. Amer. Jour. Pub. Health, 6, 585. HALL AND ELLIEPSON 1918 The elimination of spurious presumptive tests for B. coli in water by the use of gentian violet. Jour. Bact., 3, 329. HALL AND ELLEF5ON 1919 Further studies on gentian violet as a means of eliminating spurious presumptive tests. Jour. Amer. Water Works Assoc., 6, 67. WAGNER AND MONFORT 1921 Lactose broth for isolating Bacterium coli from water. Amer. Jour. Pub. Health, 11, 23. Downloaded from on September 23, 218 by guest