MacConkey Broth Research Articles

A study of coliform organisms in the Melbourne water supply and in animal faeces, with observations on their longevity in faeces and in soil

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.

Recent Experiences in the Rapid Identification of Bacterium coli Type I

Summary: When using MacConkey broth medium incubated at 44° as a confirmatory test for Bacterium coli Type I, positive reactions were produced by anaerobic lactose-fermenting bacteria such as Clostridium welchii. The use of brilliant green bile broth instead of MacConkey broth was completely successful in suppressing the growth of these organisms at 44°, but did not influence the growth of Bact. coli Type I. Two other coliform organisms, namely, Irregular Type II and Irregular Type VI were found to ferment lactose at 44°. A rapid test for their differentiation from Bact. coli Type I has been based upon the ability of that organism to produce indole at 44°, whereas the two irregular types are indole negative. By subculture of presumptive positive tubes in brilliant green bile broth and peptone water together with incubation at 44°, an estimation of the Bact. coli content of a water sample may be obtained within 48 hr. of primary inoculation.

The effect of temperature of incubation on the results of tests for differentiating species of coliform bacteria

1. A study has been made of different types of coliform bacteria with particular reference to (a) their ability to ferment lactose in MacConkey broth at different temperatures, and (b) the effect of using different temperatures of incubation for the indol, methyl-red, V.P., and citrate tests.2. It was found that 97% of the cultures ofBact. coli(indol positive, methyl-red positive, V.P. negative, citrate negative) examined could ferment lactose with production of acid and gas between 40 and 44° C. The number was not appreciably reduced at 45° C. but was markedly reduced at 46° C. 28% of the cultures ofBact. coli(indol negative, methyl-red positive, V.P. negative, citrate negative) and 15% ofBact. aerogenes(indol negative, methyl-red negative, V.P. positive, citrate positive) were found to be positive at 44° C.3. The adoption of a temperature of incubation of 30°C. for the V.P. test as advocated by Levine (1941) and the use of O'Meara's test showed that many cultures previously regarded as unable to produce acetylmethylcarbinol were in fact able to do so. Employing a temperature of 30°C. for 5, or in some cases 7, days for the methyl-red test, it was found that with nearly all the cultures tested there was an inverse correlation between the results of the methyl-red test and those of the V.P. test. With these modifications in technique some cultures originally designated as Intermediate type I were found to have reactions corresponding with those ofBact. aerogenestype I. Similarly, many cultures originally classified as Intermediate type II should have been typed asBact. aerogenestype II.4. It was found that all cultures of Intermediate type I classified as such by the new technique were incapable of using the nitrogen of uric acid for growth, but that the majority produced hydrogen sulphide. Cultures ofBact. aerogenestype II, on the other hand, grew well in uric acid medium, but produced no hydrogen sulphide.The investigation described in this paper was carried out by the Freshwater Biological Association, as part of the programme of the Water Pollution Research Board of the Department of Scientific and Industrial Research. The paper is published by permission of the Department.

298. The occurrence of slow-reducing coliform organisms in milk

Milk samples may frequently contain coliform organisms which give very slow reduction of methylene blue at 37° C.This slow reduction seems to be due to two factors present in raw milk, both of which are destroyed by heating milk to 70° C. for 1 hr. These factors are (a) a specific bactericidal substance, and (b) a growth-inhibitory factor.All the cultures examined appeared from differential tests to be intermediate types of coliform organisms. All were citrate negative and failed to produce acid or gas when grown in MacConkey's broth at 44° C. This tends to confirm the findings of Taylor (4) that the use of certain tests for the differentiation of coliform bacteria is not justified.

Further studies of incubation at 44° C. as a test for ‘faecal coli’

1. The authors concluded in a previous paper that the most reliable single test for the detection ofBact. coliwas incubation in MacConkey's broth at 44° C, such a test forBact. colialone being necessary in shellfish because of occasional multiplication of other coliforms in purified mussels. The present investigation is an attempt to verify this finding on a broader basis.2. A description is given of the isolation of 1600 cultures of coliform organisms from polluted mussels, sewage, and faeces (human, cow, sheep and sea birds). Their classification according to ‘IMViC’ reactions and the production of gas in MacConkey's broth at temperatures of 37, 41, 44, 45 and 46° C. is given.3. Of the + + − − type, 1036 out of 1065 cultures (97·3%) were found to produce gas in MacConkey's broth at 44° C. A balance is noted between the + + − − type which does not produce gas at 44° C. (irregular I) and the two irregular types which do so (irregular II and irregular VI). Such a balance was reported by Bardsley (1938).4. From the cultures examined it is confirmed that incubation at 44° C. is the most suitable temperature for use with MacConkey's broth for permitting the maximum number ofBact. colito produce gas while inhibiting the maximum number of other coliforms.

Bacteriology of fresh water: II. The distribution and types of coliform bacteria in lakes and streams

1. The distribution of coliform bacteria in lakes and streams has been studied. Weekly samples have been collected from different depths from the north and south basins of Windermere, from Thirlmere, and Bsthwaite Water, dnd from streams flowing into these lakes. Nearly 300 cultures have been isolated from positive tubes of MacConkey broth and their relationship to the coliform group has been studied and considered.2. In relatively unpolluted streams the counts of coliform bacteria and the plate counts on sodium casemate agar tended to fluctuate in the same direction as river level, but in polluted streams the increased flow of water accompanying a higher river level reduced the numbers of bacteria per unit volume. Counts of coliform bacteria were, in general, much higher in summer than in winter, despite the lower rainfall in summer.3. During the winter period of circulation of the water, samples of water from different depths in the lakes gave approximately the same count for coliform bacteria. When stratification of the water became established numbers of coliform bacteria in the hypolimnion (the lower stratum) dropped to very low figures and remained fairly constant; much higher counts were found in the epilimnion (the upper stratum).Large fluctuations in numbers of coliform bacteria which occurred from week to week in the lakes were related to fluctuations in plate counts at only one of the seven sampling points, and were not related to previous rainfall. The count of coliform bacteria was greatest in summer and autumn, a phenomenon that was by no means entirely a result of the bacteria which were washed in being concentrated in the epilimnion. It is suggested that temperature may have affected viability and proliferation.4. Of the total number of coliform organisms producing acid and gas from lactose at 37° C, approximately 70% wereBact. colitypes I and II, and the remainder were members of the I.A.C. group. In the relatively pure waters of Thirlmere and its inflowsBact. coli, type I, made up 86 and 98% respectively of the total coliform cultures isolated, whereas in the relatively impure waters of Windermere and Esthwaite Water the percentages were 39 and 37 respectively. The differences were due to the greater proportions ofBact. aerogenes, type I, and of intermediate and irregular types in the impure waters. The significance of greater proportions of the I.A.C. group in polluted rather than in unpolluted water is discussed. The actual counts of coliform bacteria were very much lower in Thirlmere than in the other lakes examined.5. Of the total cultures isolated from positive tubes of MacConkey broth 27% did not produce acid and gas in MacConkey broth at 37° C. Approximately one-half of these cultures, however, fermented lactose at 30° C, and gave varied results with the standard differential tests. The majority of these cultures could, by gradually increasing the incubation temperatures of successive inoculations, be “trained” to ferment lactose at 37° C but not at 40° C. They were more frequently isolated from the polluted lakes and inflows than from unpolluted waters. The remaining organisms isolated proved to be non-lactose-fermenting species belonging to the generaProteus, Salmonella, EberthellaandAlkaligenes(Bergey).

Wilson Method for the Bacteriological Examination of Water

AMONG the numerous methods proposed for determining the coliform count of water the two most widely used are those of the American Public Health Association and the British Ministry of Health. In the American Standard Method the water is inoculated into lactose broth; tubes showing gas are plated on Endo or eosine-methylene-blue agar; and one or more typical colonies are picked off into lactose and studied microscopically to see whether the contained organisms have the morphological and staining properties of coliform bacilli. By this means it is possible to determine the number of coliform bacilli present in the water, but not to form any idea as to whether these organisms belong to the fecal coli, the intermediate-aerogenes-cloacae, or irregular type.* In the British Ministry of Health (1934) method, lactose broth is replaced by MacConkey's broth. Since

A Comparison of MacConkey's Broth and Standard Lactose Broth as Media for Detection of Coliform Organisms in Water

A Comparison of MacConkey's Broth and Standard Lactose Broth as Media for Detection of Coliform Organisms in Water M. H. McCrady CopyRight https://doi.org/10.2105/AJPH.29.11.1250 Published Online: August 29, 2011

Incubation at 44°C. as a test for faecalcoli

1. The use ofEsch. colialone as an index of faecal pollution for shellfish, and the correlation between the 44° C. MacConkey test and citrate tests are discussed.2. The mercury-toluene thermo-regulator used in these experiments, which gives a maximum variation of ± 0.1° C., is discussed briefly and illustrated.3. Experiments are described in which 522 colonies from polluted shellfish were isolated, inoculated into MacConkey's broth and incubated at temperatures of 37° C. and at successive 1° intervals from 41 to 46° C., in accurately controlled water-baths. An almost perfect negative correlation was found to exist between 44° C. incubation and the citrate test.4. It appeared that temperatures above 44° C. are detrimental to the growth ofEsch. coli.5. Certain cultures of citrate-negative lactose fermenters at 37° C., which were inhibited at 44° C., were found on further investigation to be mostly intermediate types.

A comparative study of coliform organisms found in chlorinated and in non-chlorinated swimming bath water

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 thecoli, intermediate,aerogenesand irregular groups, and only 1·78% of the total were found to be irregular. Both series of samples included more than 72% which containedcoli, and among the chlorinated samples no less than 70% yieldedaerogenesand nearly 49% intermediate type, while among the non-chlorinated baths there were 43% withaerogenesand 40% with intermediate type. There was a similar difference in the proportions ofcoli, aerogenesand 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 ofcoli, 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 withaerogenes. 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 withaerogenes(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

A comparison of two methods of assessing the number of different types of coliform organisms in water

Further Studies on the Bacterial Flora of the “Kingston Cheese”

Summary A brief resume is given of the system adopted in the making of the “Kingston cheese” and the specific characteristics of the cheese are noted. Bacteriological analyses have been made of twenty cheeses manufactured on ten different days, ten cheeses when one day old and ten cheeses of the same day's “make” when mature at ten days after making. The results of the analyses made of these twenty cheeses are recorded in table 1. A list of the media employed is recorded and a description of the method of examination of the cheese is given. Several varieties of media were used with the object of determining which were best adapted for the determination of the bacterial analyses of the “Kingston cheese,” glucose-agar and glucose-gelatin proving to be most satisfactory. One hundred seventeen organisms have been isolated from plates made of the cheese and 5 from plates made of the starter used in the making of the cheese. As far as possible the organisms were those which appeared to occur with the greatest degree of frequency. Though 18 of the 122 organisms isolated were of the genus Escherichia this does not necessarily represent the percentage of organisms of that Genus present in the cheese. The strains were taken from MacConkey's broth tubes and MacConkey's agar plates in order to determine the species of gas formers occurring with the greatest frequency. The morphology, cultural features and physiological reactions of organisms isolated from “Kingston cheese” are given in table 2. The 122 organisms isolated are placed in five main groups, and are classified according to Bergey's Determinative Bacteriology (13).