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A comparative study of coliform organisms found in chlorinated and in non-chlorinated swimming bath water
- Doris A. Bardsley
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- Journal:
- Journal of Hygiene / Volume 38 / Issue 6 / November 1938
- Published online by Cambridge University Press:
- 15 May 2009, pp. 721-731
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The investigation deals with the bacteriological examination of 386 samples of water from Manchester swimming baths where the method of purification could be studied.
There were 339 samples from chlorinated pools of which 160 gave acid and gas in MacConkey broth at 37° C. and 2130 strains were isolated. The remaining forty-seven samples were from untreated open-air pools. Thirty of these samples were positive and 270 strains were isolated. The strains were classified according to Wilson's method into the coli, intermediate, aerogenes and irregular groups, and only 1·78% of the total were found to be irregular. Both series of samples included more than 72% which contained coli, and among the chlorinated samples no less than 70% yielded aerogenes and nearly 49% intermediate type, while among the non-chlorinated baths there were 43% with aerogenes and 40% with intermediate type. There was a similar difference in the proportions of coli, aerogenes and intermediate type in chlorinated and in non-chlorinated baths when the results were considered on the basis of strains (Table II).
Since these differences in proportion were considered significant, and since the chief variation in conditions between outdoor and indoor baths consists of chlorination, a series of experiments was devised to test the effect of chlorine on pure cultures of coli, aerogenes, and intermediate type. There was very little change after adding 0·1 part of chlorine per million, but the addition of 0·3 and 0·5 part was followed by very rapid reduction in numbers with diminution in free chlorine. This reduction was on the whole least with aerogenes. The numbers continued to fall for about 48 hr. until all trace of chlorine had disappeared and then regrowth occurred. This regrowth was most vigorous and most rapid with aerogenes (see figures) but was very slow in all cases.
A comparison of two methods of assessing the number of different types of coliform organisms in water
- Doris A. Bardsley
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- Journal:
- Journal of Hygiene / Volume 38 / Issue 3 / May 1938
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- 15 May 2009, pp. 309-324
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1. A bacteriological examination was made of 1108 samples of water submitted to the laboratory for routine analysis. Of these, 550 contained coliform bacilli and were examined by two different methods. The first method, referred to as method I and officially recommended by the Ministry of Health, involves enrichment in MacConkey broth at 37°C., followed by plating and subsequent identification of selected colonies. The second method, described by G. S. Wilson and his colleagues in their report on milk and referred to as method IV, consists in subculturing every positive 37°C. MacConkey tube into MacConkey broth at 44°C. for Bact. coli I and into Koser's citrate at 37°C. for organisms of the iritermediate-aerogrenes-cloacae group (I.A.C.). In both methods the absolute numbers of coliform bacilli and of the different types are calculated from probability tables.
2. From every fermented tube of MacConkey broth at 37°C. that was plated coliform organisms were isolated. In not a single instance was a false positive, due to non-coliform bacilli, encountered. This merely confirms the general experience in this country of the high degree of specificity of MacConkey broth for members of the conform group. Altogether 2840 strains were isolated by method I and classified according to the scheme given in Table I (p. 310).
3. In the 550 samples Bact. coli I was demonstrated 417 times by method I and 446 times by method IV. The corresponding numbers for I.A.C. were 213 and 449. Method IV therefore proved rather more delicate for the detection of Bact. coli I, and very much more delicate for the detection of I.A.C.
4. Evidence is brought to show that the frequent failure of method I to detect I.A.C. is due partly to a dilution effect and partly to the overgrowth of I.A.C. by Bact. coli I. The chances of demonstrating I.A.C. by method I are remarkably small unless these organisms are present alone, or in numbers greater than or approximately equal to those of coli I.
5. The specificity of the 44°C. MacConkey test for Bact. coli I, and for the infrequent Irregular II which closely resembles it, is very high. For instance, among 2840 strains tested, only fifteen produced gas in MacConkey at 44°C. which could not be classified as Bact. coli I or Irregular II. Only six out of a total of 1086 I.A.C. strains reacted positively to this test. Finally not a single strain, other than Bact. coli I, isolated from 100 samples of human faeces was found capable of producing gas in MacConkey broth at 44°C.
6. The specificity of the citrate test for I.A.C. is not so high as that of the 44° C. MacConkey test for Bact. coli I. In eight out of eighty-one samples of water a positive citrate test was given by organisms other than I.A.C. Examination showed that these consisted of Bact. coli I, non-lactose-fermenters, or of irregular types. Since neither Bact. coli I nor the non-lactose-fermenters grew in citrate in pure culture, and since they were found in citrate tubes that had been subcultured from fermented MacConkey broths inoculated with relatively large quantities of polluted water, it seems possible that their growth was due to the carrying over of small amounts of organic matter into the citrate medium.
7. Observations on the association of Bact. coli II, Irregular I and Irregular II with other coliform types render it probable that Bact. coli II is not mainly of faecal origin, that Irregular I is almost certainly of faecal origin, and that Irregular II may be of faecal origin, but that further evidence will be required to establish this definitively.
8. Owing to the fact that Irregular I does not produce gas in MacConkey broth at 44°C., the faecal coli count by method IV is underestimated, and owing to the fact that Irregular II does produce gas at 44°C., the faecal coli count is overestimated. In this series of examinations the faecal coli count was falsely estimated owing to the presence of Irregular I in 0·4% and owing to the presence of Irregular II in 0·5 % of samples. The combined error of 0·9 % is insignificant in relation to the experimental error of the technique as a whole.
9. By the use of method IV it is possible to demonstrate that (a) I.A.C. is nearly as frequently present in polluted water as Bact. coli I, and when the pollution is only slight may exceed it; (b) I.A.C. tends to be the dominant organism in polluted water that has been subjected to chlorination; and (c) I.A.C. is often present in human faeces (61% of 100 samples), and may sometimes constitute the dominant coliform type. In view of these facts it would seem unwise to neglect organisms of the I.A.C. type in the interpretation of water analyses in this country. Their presence is likely to be of most significance in water of a fairly high degree of purity in which, owing to chlorination or other causes, Bact. coli I can no longer be detected.
10. It is therefore concluded that it would be advisable in future to adopt a method of analysis, such as method IV, which affords a more delicate index of the presence of Bact. coli I, and particularly of I.A.C., in water than the method officially recommended at the present time. The fact that method IV is both simpler, quicker, and cheaper than Method I provides an additional recommendation for this change.
The Distribution and Sanitary Significance of B. coli, B. lactis aerogenes and Intermediate Types of Coliform Bacilli in Water, Soil, Faeces, and Ice-Cream
- Doris A. Bardsley
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- Journal:
- Journal of Hygiene / Volume 34 / Issue 1 / February 1934
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- 15 May 2009, pp. 38-68
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This section of the work deals with the routine bacteriological examination of 2144 samples of water, of which 1102 contained lactose-fermenting bacilli, 4333 strains being isolated. Twelve strains which liquefied gelatine and 24 which gave anomalous MR and VP reactions were excluded from the coliform group; the remaining 4297 cultures were classified on the basis of the MR, VP, indol, citrate and uric acid tests.
The bacterial content of ice-cream in relation to manufacture, storage and standards of purity
- Doris A. Bardsley
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- Journal:
- Journal of Hygiene / Volume 38 / Issue 5 / September 1938
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- 15 May 2009, pp. 527-546
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Ice-Cream has occasionally been proved to be the carrier of disease, and therefore a study of the ingredients used, the processes of manufacture and the bacterial content of the final product is an important branch of modern hygiene.
A study of coliform organisms in the Melbourne water supply and in animal faeces, with observations on their longevity in faeces and in soil
- Doris A. Bardsley
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- Journal:
- Journal of Hygiene / Volume 46 / Issue 3 / September 1948
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- 15 May 2009, pp. 269-279
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The bacteriological examinations of 1616 samples of polluted water from the Melbourne supply revealed that the production of acid and gas in MacConkey or in lactose broth was not always evidence of the presence of a coliform bacillus. There was, however, a marked difference in the number of confirmed samples in the two media. Among 1136 samples incubated in MacConkey broth coliform organisms were isolated by plating in 98·4%, but among 480 samples enriched in lactose broth 10·8% failed to confirm. MacConkey's broth is therefore recommended as the better medium for the presumptive coliform test in Melbourne water.
The technique used in the examination of 1345 samples of water and 73 samples of animal faeces was Wilson's method IV extended by plating from at least one tube giving a positive presumptive reaction at 37° C. to include method I also. In both series of tests coli I appeared to be by far the more numerous type by method I, but when method IV was used the intermediate, aerogenes, cloacae group (abbreviated to I.A.C.) was detected in almost as many samples in water and in 63% of the faeces where the numbers were often very low compared with coli I. Unless I.A.C. was present alone or in large numbers in relation to coli I it was frequently missed by the plating method. Even coli I was more often found by method IV which proved, therefore, to be a more delicate test than method I for both types of organism.
The 44° C. test for coli I proved to be highly specific in Victoria, since few organisms were able to produce acid and gas in MacConkey broth at 44° C. except coli I and organisms which had affinity with coli I rather than I.A.C. This contrasts with results reported from Madras (Raghavachari, 1939) and Singapore (Boizot, 1941) where the test appears to be less specific, but is in agreement with British (Bardsley, 1938; Batty-Smith, 1942) and Argentine (Ferramola, 1940) experience where the 44° C. test has also proved to be of high differential value.
Growth in citrate in method IV was sometimes due to coli I, non-lactose fermenters or Pseudomonas pyocyanea, particularly when lactose broth was the enrichment medium.
Bact. coli II and irregular I were found in heavily and in lightly polluted waters, and were associated rather more often with coli I than with I.A.C. They were seldom found in animal faeces. Irregular II rarely occurred among the organisms cultured, but the evidence suggests that it may be of faecal origin.
The coliform counts on decaying leaves, algae and other vegetable debris found in the sumps and on the screens were very high, and the figures showed a heavy increase after incubation.
Periodic sampling of cow dung left to dry in the open meadow showed that reduction in coliform count was very gradual and largely dependent on the moisture content, and that coli I was still overwhelmingly dominant 3–7 weeks after the samples were voided. Laboratory experiments, in which cow faeces were stored in moist and in dry chambers, gave similar results.
Soil plots low in organic matter were watered with cultures of coli I, intermediate I or aerogenes I and sampled at intervals. All three types tended to die out gradually and there was no question of intermediate I or aerogenes I growing and multiplying under the conditions of the experiment, neither were any profound changes apparent in the biochemical reactions of the three types as a result of prolonged sojourn in soil. Soil which had been heavily contaminated with cow manure 3 years previously still contained large numbers of viable coli I.
B. coli as an Index of Faecal Pollution of Water Supplies
- Doris A. Bardsley
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- Journal:
- Journal of Hygiene / Volume 25 / Issue 1 / February 1926
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- 15 May 2009, pp. 11-25
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The present investigation deals with the routine bacteriological examination of 525 samples of water, 265 of which contained lactose fermenting bacilli; 1441 strains were isolated and various differential tests were used in the classification of these organisms.
All the strains were tested in dextrose phosphate culture for the methyl red and the Voges and Proskauer reactions, and although most of them gave normal results (M.R. + V.P. 0 or M.R. 0 V.P. +), there were 25 strains, 1·73 per cent., which gave either double positive or double negative reactions. Similar cases have already been discussed in an earlier portion of this paper, and they leave no doubt that there are types which may vary from the normal as far as the methyl red and the Voges and Proskauer reactions are concerned. At the same time these abnormal types are not sufficiently numerous to make any real practical difficulties in the use of these tests for routine purposes.
The Koser test, considered by many bacteriologists to be of great differential value, was found to give imperfect correlation with the other reactions.
1395 strains were isolated which could be classified in the B. coli group as a result of the production of acid and gas in lactose, the clotting of milk, and the non-liquefaction of gelatine, but when the methyl red and the Voges and Proskauer results were considered it was found that nearly 11 per cent. of these strains were of the B. lactis aerogenes type, and were therefore of no value as indicators of faecal pollution.
The indol reactions were somewhat variable. Among 1244 strains of the B. coli type (M.R. + V.P. 0), more than 10 per cent. failed to produce indol; while among 151 strains of the B. lactis aerogenes type (M.R. 0 V.P. +), nearly 51 per cent. were indol producers. Preliminary extraction with ether was carried out with 448 of these strains before Böhme's reagent was added. With this technique it was found that among 400 methyl red positive types there were 87 strains (22 per cent.) which produced no indol, and among 48 methyl red negative strains there were 12 (25 per cent.) which gave the reaction. Chen and Rettger (1920) were unable to establish any definite correlation between the indol test and the other reactions.
When the sample results were studied, it was found that although 262 waters contained organisms which were included in the B. coli group on the basis of the lactose, milk and gelatine tests, nearly 6 per cent. of these samples contained only organisms which gave a methyl red negative, Voges and Proskauer positive reaction, and were probably associated with pollution from soil washings rather than contamination from faecal sources. This means that the application of the methyl red and Voges and Proskauer reaction does make a considerable practical difference in the interpretation of results in the bacteriological examination of water.
More detailed examination as regards the fermentation of the various carbohydrates is useful for purposes of classification, but is of no significance, in the light of our present knowledge, in assessing the probability of excretal pollution.
It is suggested that, in considering the results of a bacteriological examination of water, organisms should be regarded as significant which are of typical morphology and staining reaction, produce acid and gas in lactose, fail to liquefy gelatine, clot milk, and give a positive methyl red and a negative Voges and Proskauer reaction. The production of indol in a medium containing peptone may be added as an extra test if desired, but the available evidence does not suggest that failure to produce indol is a sufficient basis for the exclusion of an otherwise typical bacillus from the B. coli group.
My thanks are due to Dr G. D. Dawson, in conjunction with whom the present investigation was started; and also to Prof. W. W. C. Topley whose interest throughout the course of the research was made manifest by his helpful advice and criticism.