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An evaluation of the standard biochemical and elevated temperature tests for differentiating faecal and non-faecal coliforms
Water Research Pergamon Press 1968 Vol. 2 pp. 575-585 Printed in Great Britain
A N E V A L U A T I O N OF THE S T A N D A R D B I O C H E M I C A L A N D ELEVATED T E M P E R A T U R E TESTS F O R D I F F E R E N T I A T I N G F A E C A L A N D N O N - F A E C A L COLIFORMS R. P.
MISHRA,S. R. JOSHI and P. V. R. C. PANICKER
Central Public Health Engineering Research Institute, Nagpur 3, India (Received 25 March 1968) Abstract--In this study the relative efficacies of the biochemical IMViC and the elevated temperature (FC) tests have been compared for differentiating the faecal and non-faecal coliforms. Altogether 1628 coliform strains comprised of 427 from 143 samples of human faeces, 301 from 101 samples of cattle faeces, 631 from 113 samples of sewage and 269 from 120 samples of soil were classified as to their IMViC types and response to elevated temperature reactions. Parr's Escherichia group (+ + - - , + - - and - + - - ) was represented by 83 and 85 per cent of the coliform strains isolated from human and cattle faeces respectively. Of the coliform strains isolated from human and cattle faeces 82"0 and 76.4 per cent respectively were found positive for the FC test. Hence, neither the IMViC nor the FC tests by themselves can be considered adequate for differentiating coliforms of faecal and non-faecal origin. Based on the results obtained in this study the authors Have interpreted the sanitary significance of the coliforms present in various sources. INTRODUCTION THE SEPARATIONof the coliform organisms into faecal and non-faecal types is based on two generally accepted procedures viz. (a) PARR'S (1938a, b) tests based on the biochemical characteristics using indole, methyl red, Voges-Proskauer and citrate utilization reactions; also designated as IMViC tests and (b) modification of EIJKMAN'S (1904) elevated temperature reactions, better known as FC test, described by PERRY and HAJNA (1944) and VAUGHN et aL (1951). The former requires the isolation of a pure culture on a solid medium for performing the tests while the latter can be applied to the 24 hr presumptive positive tubes of lactose broth. Besides, the FC test can be completed within 48-72 hr whereas the IMViC tests may take 7-10 days. According to the definition in the STANDARDMETHODS(1965), "The coliform group includes all of the aerobic and facultatively anaerobic, gram negative, non-spore forming, rod shaped bacteria which ferment lactose with gas formation within 48 hr at 35°C ''. Bacteria conforming to the above definition are found abundantly in faeces but they are also frequently encountered in non-faecal habitats such as soil, vegetation and water. While Eschericha coli I is predominantly faecal in origin (ESCHERICH, 1885; ROGERS et al., 1914; PARR, 1938 and FOOTE, 1937), Aerobacter aerogenes has been found quite commonly in soils and on vegetation (CHEN et al., 1920; FRANK et aL, 1941 ; BARDSLEY,1934; ROGERS et aL, 1915; THOMASet al., 1952 and THOMAS et aL, 1955). Because of their frequency of occurrence, E. coliI is considered faecal and .4. aerogenes as non-faecal or soil and grain type. The fact that certain species of coliforms can survive in soils is well established. Hence, the mere presence of coliforms in a water source may not necessarily indicate fresh excretal pollution. The recent trend in sanitary water bacteriology, therefore, is to enumerate both the faecal and non-faecal coliform densities in water. 575
576
R.P. MISHRA,S. R. JOSHIand P. V. R. C. PANICKER
The Intermediate Aerogenes Cloacae (IAC) group of coliforms survive comparatively longer periods in soil and in other non-faecal environments (PARR, 1937; PLATT, 1935 and TAYLOR, 1942). JOHNSON (1917), CHEN and RETTGER (1920), KOSER (1926), RAGHAVACHARI (1926) and FRANK and SKINNER (1941) reported Aerobacter aerogenes to be predominant in soils with percentages varying from 45.0 to 93.5. They are reported to be more resistant to chlorination than E. coli (TONNEYe t al., 1928; HEATHMAN et al., 1936; BUTTERFIELD et al., 1943 and KAHLER, 1951). The sanitary significance of the different types of coliforms, therefore, should not be judged on the same footing. Detection of faecal coliforms in water, either in the presence or absence of IAC groups of organisms, indicate recent excretal pollution. The preseiice of IAC group of organisms in the absence of faecal coliforms, however, may be due to remote excretal pollution or addition of soil run off water after precipitation. In so far as the raw water sources are concerned for drinking water supplies the enumeration of faecal coliforms is of importance. In treated drinking waters, however, the presence of any type of coliforms faecal or non-faecal, is not considered desirable. In this study the biochemical characteristics (IMViC) of the coliform strains isolated from human and cattle faeces, sewage and soils were compared with the lactose fermentation reactions at elevated temperature to find out whether the elevated temperature test is a suitable substitute to the IMViC test for differentiating the faecal from non-faecal coliforms. EXPERIMENTAL Samples of human faeces were collected from volunteers who were staff members of the Ceiatral Public Health Engineering Research Institute, Nagpur. Samples were collected in sterile glass containers in the morning and brought to the laboratory within an hour after collection. A loopful of the sample was transferred to a 10 ml sterile buffered distilled water blank and shaken well to make it as homogenous as possible. Two Eosin Methylene Blue Agar (EMB) plates (Difco Labs.) were streaked with this suspension. One of the plates was incubated at 37°C and the other at 44.5°C for 24 hr. Individual and discrete colonies, one of each type, whether typical or atypical, were picked up, transferred to peptone water and incubated at 37°C for 24 hr. From this, a loopful of the culture was transferred to an EC broth tube, incubated at 44.5°C for 24 hr in a constant temperature water bath and observed for the production of gas. All the individual strains picked up were also subjected to the IMViC tests in parallel as adopted by GELDREICH et al. (1964). Cattle faeces included mainly faeces of buffaloes and cows from Ajni, a locality specializing in milk trade. Samples were collected before the actual starting of the work. The procedures for streaking, incubation and other tests were the same as for the human faeces. Raw domestic sewage samples were collected in sterile glass containers. After making the samples homogenous by shaking they were streaked on EMB agar plates in duplicate and incubated at 37°C and 44.5°C temperatures as was done for human and cattle faeces. Individual strains were subjected to FC and IMViC tests. All strains isolated from human faeces, cattle faeces and sewage were tested for their ability to ferment lactose in lactose broth tubes at 37°C within 48 hr.
Standard Biochemical and Elevated Temperature Tests
577
Samples of soil were collected from the premises of the Institute which comprise about 100 acres of grazing field. Before collecting the samples, 2.5 cm of the surface growth was scraped with a knife and then a few grammes of soil were scooped out and collected in sterile containers. The soil sample was pulverized in an all glass sterilized pestle and mortar. It was then sieved through a sieve retaining pebbles and stones above 2 mm dia. Ten grammes of soil was added to a 100ml sterile buffered dilution water blank in a round bottom flask and shaken with the help of a rotating shaker for 5 min. After this, 10 ml of the suspension was transferred to a 10 ml double strength lactose broth tube and incubated for 48 hr at 37°C. The presumptive positive tubes were subjected to confirmatory test in two Brilliant Green Lactose Bile broth (BGLB) tubes, one of which was incubated at 37°C for 48 hr, and the other at 44.5°C for 24 hr. EMB agar plates were streaked from the confirmatory BGLB positive tubes at 37°C and 44.5°C respectively, incubated for 24 hr at 37°C and observed for growth. Discrete, typical and atypical colonies apparently looking different, were inoculated into peptone water and then subjected to the FC and IMViC tests. Soil samples which showed positive reactions in confirmatory Brilliant Green Lactose Bile broth (BGLB) tubes at 37°C within 48 hr were considered as coliform positive. When the samples showed positive indole tests for corresponding tryptone broth tubes, they were considered positive for E. coli I. But, final confirmation of the presence of coliforms and E. coli I was done on individual strains subjecting them to lactose fermentation, IMViC and FC tests as described before. It was not considered necessary to subject the faeces (human and cattle) and sewage samples to the presumptive and confirmatory tests, as were done for soil samples because the presence of coliform was a certainty. Appearance of typical and atypical colonies on EMB agar plates and lactose fermentation at 37°C gave positive evidence of the presence of coliforms. I M V i C tests
The indole, methyl red, Voges Proskauer and citrate utilization tests were conducted as described in the 12th edition of Standard Methods. R E S U L T S AND O B S E R V A T I O N S A total of 1628 coliform strains were examined, 427 from 143 samples of human faeces, 301 from the 101 samples of cattle faeces, 631 from 113 samples of sewage and 269 from 120 samples of soils. The samples were tested for the presence or absence of coliforms, E. coli I and A. aerogenes. The reactions of the strains isolated from these sources to the elevated temperature test were also determined. Samples from which any of the strains isolated showed + + - - IMViC reactions and positive FC test were considered positive for E. coli I. Further, samples from which any of the strains showed - - + + IMViC reactions, whether F C positive or negative, were considered positive for A. aerogenes. The results obtained are presented in TABLE 1. All the samples (100 per cent) of human faeces, cattle faeces and sewage were positive for coliforms. But for soils, only 70.8 per cent of the samples were positive. The presence of E. coli I was confirmed in 95.1, 96.0 and 86-7 per cent of human
578
R . P . MISHRA, S. R. JOSHI and P. V. R. C. PANICKER
faeces, cattle faeces and sewage respectively, whereas for soils 54.1 per cent only were positive. For A. aerogenes 23.1, 10.9, 63-4 and 71.6 per cent of strains from human faeces, cattle faeces sewage and soils samples respectively showed positive reactions. While a large percentage of samples from human, and cattle faeces and sew~tge showed positive FC tests, being 97.2, 96.0 and 97.3 per cent respect!vely, only 73.0 per cent of soil samples were positive for this test. TABLE 1. DISTRIBUTION OF COLIFORMS IN FAECES, SEWAGE AND SOILS
Coliform organisms E. coli 1 A. aerogenes Elevated temp. Source Sam. pies Positive Negative Positive Negative Positive Negative Positive Negative (No.) (No.) (%) (No.) (%) (No.) (%) (No.)(~o) (No.)(%) (No.)(Y.) (No.) (%) (No.)(%) Human faeces 143
143 100
0
0
136 95"1
7
4-9
33 23.1
Cattle faeces 101
I01 100
0
0
97 96"0
4
4"0
ll
Sewage
ll3
113 100
0
0
98 86.7
Soils
120
85 70.8
35 29.1
46 54-1
l l 0 76.9
139 97"2
4
2.8
10'9
90 89"1
97 96'0
4
4-0
15 13"3
72 63.4
41 36.5
l l 0 97.3
3
2.7
39 45-9
61 71.8
24 28.2
63 73-0
22 27.0
TABLE 2. GROUP TYPING OF THE COLIFORM STRAINS
Parr's IMViC group
Human faeces (No.) (~)
Cattle faeces (No.) (~)
Sewage (No.) (~)
355
83.1
256
85"0
311
51
11.9
37
12"3
Intermediate group 21
4"9
8
2'6
100.0
301
100.0
Soils
(No.)
(%)
49"3
96
35.7
283
44.9
159
59.1
37
5"9
14
5.2
631
100.0
269
100.0
Escherichia group + -t- -- -
-- + -- Aerobacter group - - + +
-
-
-
+
Total
427
Based on the four biochemical tests, viz., indole methyl red, Voges-Proskauer and citrate utilization: PARR (1938a) interpreted that strains showing + + - - , + - - and + -- - - reactions may be included under Escherichia group indicative of faecal origin, strains showing - - + + , - - - + and - - + - reactions under Aerobactor group indicative of soil and vegetation origin. All the strains isolated were classified into three groups, the results of which are presented in TABLE 2. It is interesting to note that only 83.1 and 85.0 per cent of the strains isolated from human and cattle faeces respectively are included under Escherichia group. About -
Standard Biochemical and Elevated Temperature Tests
579
49 per cent of the strains isolated from sewage are included under this group. In soils 35.7 per cent of the strains isolated are included under this group. It is of interest to note that 11.9 and 12.3 per cent of the strains isolated from human and cattle faeces were positive for Aerobacter group in contrast to 44-8 and 59.1 per cent in sewage and soils (TAnLE 2). The intermediate group of organisms were encountered in less than 5 per cent of the strains examined from human and cattle faeces. Most of the strains isolated (95 per cent) from faeces, either cattle or human, are included under Escherichia and Aerobacter group. The organisms of Intermediate group were present in 5.8 and 5.2 per cent of the strains obtained from sewage and soil. TABLE3. IMViC T Y P I N G Test
AND ELEVATED TEMPERATURE REACTIONS OF COLIFORM STRAINS
Human faeces Cattle faeces (No.) (~) (No.) (Yo)
Sewage (No.) (Yo)
Soils (No.) (~) 93
+ + - ++ --
320
74.9
246
81.7
286
45.3
- + - Indole positive Methyl red positive Voges Proskauer positive Citrate utilizers Elevated temperature positive Number of cultures
355 329 367 64 60 350 427
83-1 79.4 85"9 15.0 14.1 82"0 100.0
256 256 259 30 40 230 301
85.0 85"0 86-0 I0"0 13.3 76.4 100.0
311 310 326 210 289 350 631
49.3 49-1 51 "7 33.3 45.8 57.1 100.0
34.6
96 35.7 101 37.5 104 38.7 114 42.4 158 58.7 150 55"8 269 100.0
In TABLE 3 are presented individual reactions of the strains isolated from different sources. Besides, IMViC reactions representing Parr's Escherichia group have also been tabulated. The individual tests along with the combined IMViC reactions were compared with the elevated temperature test for all strains. It will be seen that 74.9 and 81.7 per cent of strains from human and cattle faeces were positive for E. coli I. The combined IMViC reactions of Parr's Escherichia group for human and cattle faeces represent 83.1 and 85.0 per cent respectively. In sewage and soils, strains showing this type of combined reactions constitute 49.3 and 85.7 per cent respectively. A large percentage o f the strains (82.0 per cent) of human faeces were positive for F C test. This percentage, however, was little less in cattle faeces. Surprisingly, the percentage of coliform strains showing elevated temperature positive reactions isolated from sewage and soils were almost identical; 57.0 per cent in sewage and 55-8 per cent in soils. The different kinds of IMViC combinations encountered with coliform strains isolated from these four sources and their relation to FC test are presented in TABLE 4. Out of 427 strains obtained from human faeces, 320 (74-9 per cent) showed + + - - reactions and from this number 292 (91.2 per cent) gave FC test positive reactions. In cattle faeces out of 301 strains isolated 246 (81.7 per cent) showed + + - - reactions and from this 213 (86.8 per cent) strains responded positively to the FC test. In sewage 286 out of 631 strains tested gave + + - - reactions and from this 225 (78.7 per cent) strains were found FC positive. In soils, only 93 strains
427
t Out of each I M V i C type.
* Out o f total strains isolated.
1
Total
-
0
+
+
+ + +
-
320 0 35 42 6 3 4 4 1 I 8 2 0
-
--+ -+ + + + + +
+ + + -+ + +
-
-+ + + -+ + + --
0
0"2
74"9 0 8'2 9"8 1"4 0"7 0"9 0"9 0-2 0"2 1"9 0-5 0
350
0
0
81-9
0
0
292 91.2 0 0 23 65.7 22 52.4 1 16"7 2 66.7 3 75-0 4 100.0 1 100.0 0 0 0 0 2 100.0 0 0
F C test (No. ¢) ( ~ )
H u m a n faeces
Strains isolated (No.*) ( ~ )
+ -+ --+ + + + --
+ + -
I M V i C types
301
0
0
246 3 7 23 3 11 2 3 1 1 1 0 0 0
0
81"7 1"0 2"3 7"6 1"0 3"6 0"7 1"0 0-3 1"3 0-3 0 0
Strains isolated (No. *) ( % )
230
0
0
213 1 0 9 0 I 2 3 0 0 1 0 0
76.4
0
0
86"8 33"3 0 39"1 0 9"0 100.0 100-0 0 100"0 100.0 0 0
F C Test (No. t ) ( ~ )
Cattle faeces
631
5
3
286 3 22 167 23 93 2 3 2 8 8 0 6 0"8
0'5
45"3 0'5 3'5 26"5 3"6 14-7 0'3 0"5 0-3 1"3 1'3 0 0.9
Strains isolated (No. *) ( ~ )
360
2
3
57.0
40.0
100
225 78.6 1 33"3 15 68.1 68 40-1 10 43'4 21 22"5 2 100 2 66"6 1 50.0 2 25.0 3 37.5 0 0 5 83.3
F C Test (No. ¢) ( ~ )
Sewage
TABLE 4. T h E RELATION BETWEEN I M V i C AND ELEVATED TEMPERATURE TESTS
269
0
0
0
0
93 34-6 0 0 3 1"1 94 34"9 13 4"8 52 19"3 0 0 0 0 2 0"7 6 2"2 4 1"5 2 0'7 0 0
Strains isolated (No. *) ( ~ )
Soils
150
0
0
55.7
0
0
91 97"9 0 0 0 0 41 43"6 3 23-1 10 19-2 0 0 0 0 1 50"0 0 0 2 50"0 2 100 0 0
F C Test (No. t) ( ~ )
t~
Standard Biochemical and Elevated Temperature Tests
581
from 269 strains isolated were included under + + - - IMViC type and amongst these 91 strains (97.7 per cent) were FC positive. The Aerobactor aerogenes type of reactions viz; IMViC - - + + type were met with in 42 (9.8 per cent) strains from human faeces, 23 (7.6 per cent) strains from cattle faeces, 167 strains (26-4 per cent) from sewage and 94 (35 per cent) strains from soils. Among - - + + type strains isolated from various sources 22 strains (52.4 per cent) from human faeces, 9 (39.2 per cent) from cattle faeces, 68 (40.2 per cent) strains from sewage and 41 (43.6 per cent) strains from soils responded positively to the FC test. The FC positive test reactions were shown by 350 (81.9 per cent) strains from human faeces, 230 (76.4 per cent) strains from cattle faeces, 360 (57.0 per cent) strains from sewage and 150 (55.8 per cent) strains isolated from soils. D I S C U S S I O N AND C O N C L U S I O N S Various biochemical reactions such as indole, methyl red, Voges-Proskauer, or citrate utilization tests had been proposed to differentiate the coliform strains from faecal and non-faecal environments. Individually none of these tests were found to be specific for coliform derived from any one source--faecal or non-faecal. PARR (1938) suggested the combination of indole, methyl red, Voges-Proskauer and citrate utilization tests for differentiating coliform strains into faecal and non-faecal. The series of four reactions was designated by the mnemonic "IMViC" tests. EIJKMAN (1904) divided the coliforms into a faecal group that would produce gas from glucose at 46°C and a non-faecal group that would produce no change in the medium. He believed that all the coliform bacteria derived f r o m the gut of warmblooded animals would respond positively to this test. This test suggested by him was highly specific for the faecal group but had low sensitivity. P~RRV and HAJNA (1944) improved the Eijkman test by the addition of bile salts and a buffer system and by reducing the air incubation temperature to 45.5°C. GELOP~ICH et al. (1962) studied 8747 strains of coliforms isolated from faeces of warm-blooded animals, polluted and unpolluted soils with IMViC reactions and the elevated temperature test in EC medium at 44-5°C. They observed excellent correlation between the IMViC tests and the FC tests of coliform strains. In their study 93.3 per cent of the coliform strains isolated from faeces of warm-blooded animals came under Parr's Escherichia group whereas 96-4 per cent of the strains gave FC positive reactions. In the present study 95 per cent of the human faeces samples were positive for E. coli I and 5 per cent were negative. Similarly, 96 per cent of the cattle faeces were positive for E. coli I and 4 per cent were negative. Thus, 4-5 per cent of the faeces samples may contain coliforms but not E. coli I. If E. coli I is taken as representing faecal pollution, in such instances, the polluted water may pass out as unpolluted since most of the coliforms there will be of non-faecal type belonging to the IAC group. The situation is similar for the sewage samples in which 13.3 per cent of the samples were found negative for E. coli I. It was but natural to expect that all the samples (100 per cent) of faeces and sewage contained coliforms. In our study, 70.8 per cent of the soil samples contained coliforms and 54.1 per cent samples were positive for E. coli I. The presence of E. coli I in such large percentage can be attributed to the frequent excretal pollution of the D
W
582
R.P. MISHRA,S. R. Josm and P. V. R. C. PANICKER
area by cattle and also to some extent by human beings. Soil run off water from such an area may add to the coliform density of a water source particularly after heavy precipitation. Although a large percentage of human and cattle faeces samples showed complete absence of A. aerogenes, quite a sizeable percentage showed their presence. The presence of A. aerogenes in faeces, human and cattle, would suggest that these calmot be entirely considered exclusively soil or grain type. Whether they are transient in faeces and occur there by accident is immaterial as long as they survive and adapt themselves in the gut of the animals, and human beings. Nearly 72 per cent of soil samples were positive for this group. It would appear, therefore, that this group of organisms can adapt themselves easily to the conditions in soils as well in the alimentary tract. Over 96 per cent of human and cattle faeces and sewage samples contained coliform strains which gave positive reactions at elevated temperature (TABLE 1). It is doubtful, however, whether the coliform strains present in faeces and sewage can continue to maintain the FC test positive characteristic after they are excreted and are submerged for long periods in water. This aspect needs further investigation. On the basis of the number of coliform strains isolated, only 74.9 per cent of the strains from human faeces and 81.7 per cent of the strain from cattle faeces showed ++-IMViC reactions. Considering Parr's Escherichia group , ( + + - - , -~ , +--) as representing the faecal coliform strains, still only 83 per cent and 85 per cent of coliform strains isolated from human and cattle faeces respectively could be included under this category (TABLE 3). Similarly, 12 per cent of the coliform strains isolated from these two sources showed reactions of Parr's Aerobacter group. The Aerobacter group and Intermediate group combined together form 17 per cent and 15 per cent of the coliform strains isolated from human and cattle faeces. From the results, it is clear that the IMViC reactions by themselves are not perfect biochemical tests which conclusively distinguish between the coliform strains derived from faecal and non-faecal environments. In sewage samples, there is a sharp fall in Escherichia group and a consequent increase in Aerobacter group. This may happen in two ways. Possibly the Escherichia group of organisms die out at a faster rate compared to the Aerobacter group after they are discharged in the faeces. It is likely that outside the gut, the Aerobacter group of coliform organisms may actually be able to multiply in sewage to some extent. But the latter possibility appears to be remote. In sewage, the Escherichia, Aerobachter and Intermediate groups form 49.3, 44.9 and 5.9 per cent respectively. The .presence of Aerobacter group in a water source may be due to the pollution either by faeces, sewage or soils. It is of importance, therefore, to find out whether the coliform strains others than E. coli I, present in water, were due to the addition of soil run-off water. It will also be interesting to find out the percentage of coliforms derived from faeces which fail to give FC positive results and strains of coliform derived from unpolluted non-faecal sources which give FC positive reactions. It was evident from the present investigation that quite a large percentage of coliforms present in faeces and sewage failed to respond positively to the elevated temperature reactions. In our study 18 per cent of the coliform strains from human faeces, 24 per cent from cattle faeces, and 43 per cent from sewage failed to give FC positive reactions (TABLE4). These results are in contrast to the results obtained by GELO-
Standard Biochemical and Elevated Temperature Tests
583
REICH et al. (1962) wherein they f o u n d that 3.6 per cent o f the coliform strains f r o m the faeces o f w a r m - b l o o d e d animals gave F C negative reactions. These results are, however, in agreement with the results obtained by MoussA (1965) who observed that the total n u m b e r o f coliform strains f r o m faeces and sewage which p r o d u c e d gas after 48 hr at 44.5°C did not exceed 62.1 per cent. It is evident, therefore, that m a n y coliform strains actually derived f r o m faeces o f warm-blooded animals fail to respond positively to the F C test. Hence, the F C test procedure cannot absolutely distinguish the coliform strains f r o m faecal and non-faecal origin. A large percentage (55.7) o f the coliform strains isolated f r o m soils showed F C positive reactions. This is likely because the area f r o m which the soil samples were drawn was frequently polluted. GELDREICH et al. (1962), however, f o u n d 83 per cent o f the coliform strains f r o m polluted soils as F C positive. Individually, indole and methyl red tests give positive reactions for quite a large percentage of strains isolated f r o m h u m a n and cattle faeces. In our study o f coliform strains f r o m h u m a n faeces, 79.4 per cent were indole positive and 85.9 per cent M R positive. MoussA (1965) f o u n d 38.8 per cent o f coliform strains f r o m h u m a n faeces as indole positive and 81.7 per cent as M R positive. GELDREICH et al. (1962) f o u n d 94 per cent o f the coliform strains f r o m faeces of w a r m - b l o o d e d animals as indole positive and 96.9 per cent as M R positive. They, however, discovered that o f the coliform strains isolated f r o m unpolluted soils 19.4 and 75-6 per cent gave indole and methyl red positive reactions respectively. The specificity o f these two tests for coliform strains derived f r o m faeces is therefore, doubtful. In the present study it was f o u n d that 37.5 per cent of the coliform strains f r o m soils were indole positive and 38.6 per cent o f the strains as methyl red positive. These reactions are not unusual since the land was subjected to frequent pollution. Altogether 15 I M V i c types o f coliform strains were isolated f r o m these four sources (TABLE4). The m o s t c o m m o n I M V i C type f o u n d in h u m a n faeces, cattle faeces, and sewage was + + - - , being 74-9, 81.7, and 45.3 per cent respectively. The other c o m m o n combinations in h u m a n faeces were - - + + (9.8 per cent) and - + - (8.2 per cent), in cattle faeces - - + + (7.6 per cent) and - - - + (3-6 per cent), in sewage - - + + (26.5 per cent). - - - + (14.7 per cent) and - + - - (3.5 per cent). In soils + + - and - - + + types were encountered approximately in equal numbers, being 93 (34.5 per cent) and 94 (34.9 per cent) respectively. The other c o m b i n a t i o n frequently seen was - - - + (19.0 per cent). F r o m the different I M V i C types isolated f r o m all sources, + + - type give m a x i m u m correlation with the F C positive tests. O f the + + - - types isolated f r o m h u m a n s and cattle faeces; sewage and soils 91.2, 86.8, 78.7 and 98.0 per cent respectively gave F C positive reactions. More than 52 per cent of the - - + + type o f coliforms f r o m h u m a n faeces gave F C positive reactions. It, therefore, follows that the F C positive test reactions m a y not be confined to Parr's Escherichia g r o u p alone. Quite a large percentage (43.6 per cent) o f the - - + + types isolated f r o m soil gave positive F C tests. During the course of' these studies it was observed that m a n y of the noncoliform organisms could also p r o d u c e + + - - I M V i C reactions at 37°C as well as at 44°C. Such strains, however, failed to ferment lactose at 37°C and 44.5°C. Few coliform strains f r o m h u m a n faeces showed + + - - reactions but failed to ferment lactose at 44-5°C although they fermented lactose at 37°C.
584
R . P . MISHRA, S. R. JOSHI and P. V. R. C. PANICKER
C o l i f o r m o r g a n i s m s a r e p r e s e n t in faeces a n d soils. Escherichia g r o u p o f o r g a n i s m s usually p r e d o m i n a t e in h u m a n a n d cattle faeces a n d the Aerobacter g r o u p in soils. H u m a n a n d cattle faeces also c o n t a i n coliforms o f the Aerobacter a n d I n t e r m e d i a t e g r o u p s in a p p r e c i a b l e numbers. Similarly, soils often c o n t a i n coliforms representing P a r r ' s Escherichia g r o u p . T h e r e is no m e t h o d at present to differentiate the Aerobacter g r o u p o f o r g a n i s m s d e r i v e d f r o m faeces a n d soils. F o r this reason, differentiation o f the c o l i f o r m strains into faecal a n d non-faecal origin on the basis o f the I M V i C tests is n o t fully justified a l t h o u g h these tests d o give a g o o d i n d i c a t i o n o f their origin. These tests have n o t been used in a r o u t i n e scale because they are c o m p l i c a t e d a n d time consuming. M o r e o v e r , these tests can be p e r f o r m e d only on p u r e cultures isolated f r o m the samples. T h e elevated t e m p e r a t u r e test m a y give a g o o d i n d i c a t i o n o f the origin o f the c o l i f o r m strains b u t this test t o o was n o t f o u n d very specific for c o l i f o r m strains f r o m faeces. I n o u r s t u d y m a n y o f the c o l i f o r m isolated f r o m soils gave F C positive reactions. GELDREICH et al. (1962) t o o n o t e d t h a t m a n y coliforms isolated from unp o l l u t e d soils gave F C positive reactions. T h e y f o u n d t h a t 96.4 p e r cent o f the c o l i f o r m strains f r o m faeces o f w a r m - b l o o d e d animals gave F C positive reactions whereas 93.3 p e r cent only gave P a r r ' s Escherichia g r o u p I M V i C c o m b i n a t i o n s . In o u r study, we c o u l d find t h a t o u t o f the c o l i f o r m strains isolated f r o m h u m a n a n d cattle faeces 82.0 p e r cent a n d 76.0 p e r cent o f the strains gave F C positive reactions (TABLE 3). But P a r r ' s Escherichia I M V i C c o m b i n a t i o n s were observed in 83.1 a n d 85.4 p e r cent o f strains f r o m h u m a n a n d cattle faeces. Based on the results o b t a i n e d in this study it is n o t possible to consider the F C test to be specific for the coliform strains derived f r o m the faeces a n d sewage a n d thus there is still m u c h scope to develop a m e t h o d for differentiating faecal a n d nonfaecal coliforms. Acknowledgement--The authors are grateful to the Director, Central Public Health Engineering
Research Institute, Nagpur (India) for having given all the facilities for the work and permission to publish the data. REFERENCES BARDSLEYD. A. (1934) The distribution and sanitary significance of B. cull, B. Lactis aerogenes and the Intermediate types of coliform bacilli in water, soil, faeces and ice cream. J. Hyg., Camb. 34, 38. BUTTERFIELDC. T. (1943) Influence of pH and temperature on the survival of coliforms and enteric pathogens when exposed to free chlorine. Publ. Hlth Rep. 58, 1837. CHEN C. C. and RETTGERL. F. (1920) A correlation study of the colon aerogens groups of bacteria with special reference to the organisms occurring in the soil. J. Bact. 5, 253-298. EIJKMAN C. (1904) Die G~irungsprobe bei 46 ° als Hilfsmittel bei der Trinkwasseruntersuchung. Centr. Bakteriol. Parasitenk, Abt. L Orig. 34, 742-748. ESCHERICHT. (1885) Die Darmbakterien des Neugeborenen und Sauglings. Fortschr. Med. 3, 515-547. FOOTE H. B. (1937) The possible effects of wild animals on the bacterial pollution of water. J. Am. Wat. Wks Ass. 29, 72-74. FRANK N. and SKINNERC. E. (1941) Coli-aerogenes bacteria in soil. J. Bact. 42, 143. GELDRICHE. E., BORDNERR. H., HUFF C. B., CLARKH. F. and KABLERP. W. (1962) Type distribution of coliform bacteria in the faeces of warm-blooded animals. J. Wat. Pollut. Control Fed. 34, 295-301. GELDREICHE. E., HUFF C. B., BORDNER R. H., KABLERP. W. and CLARK H. F. (1962) The faecal coli-aerogenes flora of soils from various geographical areas. J. appl. Bact. 25, 87-93.
Standard Biochemical and Elevated Temperature Tests
585
GELDREICH E. E., KENNER B. A. and KABLER P. W. (1964) Occurrence of coliforms, faecal coliforms and Streptococci in vegetation and insects. App. Microbiol. 12, 63-69. HEATHMAN L. S., PIERCE G. O. and KABLER P. W. (1936) Resistance of various strains of E. typhi and coli-aerogenes to chlorine and chloramine. Publ. Hlth Rep. 51, 1367. JOHNSON B. R. and LEVINE M. (1917) Characteristics of coli-like microorganisms from the soil. J. Bact. 2, 379401. KABLER P. W. (1951) Relative resistance of coliform organisms and enteric pathogens in the disinfection of water with chlorine. J. Am. Wat. Wks Ass. 43, 553-568. KOSER S. A. (1926) The coli-aerogenes group in soil. J. Am. V/at. Wks Ass. 15, 641-646. MoussA R. S. (1965) Type distribution of coliforms isolated from faecal and non-faecal habitats. Ind. J. med. Res. 53, 629-637. PARR L. W. (1937) Viability of coli-aerogenes organisms in cultures and in various environments. J. infect. Dis. 60, 291-301. PARR L. W. (1938a) Coliform intermediates in h u m a n faeces. J. Bact. 36, 1-15. PARR L. W. (1938b) The occurrences and succession of coliform organisms in human faeces. Am. J. Hyg. 27, 67-87. PERRY C. A. and HAJNA A. A. (1944) Further evaluation of EC Medium for the Isolation of Coliform Bacteria and Escherichia coli. Am. J. pubL Hlth, 34, 735-748. PLATT A. E. (1935)The viability of Bact. coli and Bact. aerogenes in water: A method for the rapid enumeration of these organisms. J. Hyg., Camb. 35, 437. RAGHAVACHARI T. N. S. (1926) The application of koser's citrate utilization test to sanitary water analysis in India. Indian J. med. Res. 14, 47. ROGERS L. A., CLARK W. M. and EVANS A. C. (1914) The characteristics of bacteria of the colon type found in bovine faeces. J. infect. Dis. 15, 100. ROGERS L. A., CLARK W. M. and EVANS A. C. (1915) Characteristics of bacteria of the colon type occuring on grains. J. infect. Dis. 17, 137. STANDARD METHODSFOR THE EXAMINATIONOF WATERAND WASTEWATER. (1965) 12th edition. Amer. Pub. H#h Ass. New York. TAYLOR C. B. (1942) The ecology and significance of the different types of coliform bacteria found in water. J. Hyg., Camb. 42, 23-36. THOMAS S. B. and HOBSON P. M. (1955) Coli-aerogenes bacteria isolated from ears and panicles of cereal crops. J. appl. Bact. 18, 1. THOMAS S. B. and McQUILLIN J. (1952) Coli-aerogenes bacteria isolated from grass. Proc. Soc. appl. Bact. 15, 41. TONNEY F. O., GREER F. E. and DAUFORTH T. F. A. (1928) The minimal chlorine death point of bacteria. Am. J. Pub. Hlth 18, 1259-1263. VAUGHN R. H., LEVINE M. and SMITH H. A. (1951) A buffered boric acid lactose medium for enrichment and presumptive identification of E. coil Fd Res. 16, 10-19.
A N E V A L U A T I O N OF THE S T A N D A R D B I O C H E M I C A L A N D ELEVATED T E M P E R A T U R E TESTS F O R D I F F E R E N T I A T I N G F A E C A L A N D N O N - F A E C A L COLIFORMS R. P.
MISHRA,S. R. JOSHI and P. V. R. C. PANICKER
Central Public Health Engineering Research Institute, Nagpur 3, India (Received 25 March 1968) Abstract--In this study the relative efficacies of the biochemical IMViC and the elevated temperature (FC) tests have been compared for differentiating the faecal and non-faecal coliforms. Altogether 1628 coliform strains comprised of 427 from 143 samples of human faeces, 301 from 101 samples of cattle faeces, 631 from 113 samples of sewage and 269 from 120 samples of soil were classified as to their IMViC types and response to elevated temperature reactions. Parr's Escherichia group (+ + - - , + - - and - + - - ) was represented by 83 and 85 per cent of the coliform strains isolated from human and cattle faeces respectively. Of the coliform strains isolated from human and cattle faeces 82"0 and 76.4 per cent respectively were found positive for the FC test. Hence, neither the IMViC nor the FC tests by themselves can be considered adequate for differentiating coliforms of faecal and non-faecal origin. Based on the results obtained in this study the authors Have interpreted the sanitary significance of the coliforms present in various sources. INTRODUCTION THE SEPARATIONof the coliform organisms into faecal and non-faecal types is based on two generally accepted procedures viz. (a) PARR'S (1938a, b) tests based on the biochemical characteristics using indole, methyl red, Voges-Proskauer and citrate utilization reactions; also designated as IMViC tests and (b) modification of EIJKMAN'S (1904) elevated temperature reactions, better known as FC test, described by PERRY and HAJNA (1944) and VAUGHN et aL (1951). The former requires the isolation of a pure culture on a solid medium for performing the tests while the latter can be applied to the 24 hr presumptive positive tubes of lactose broth. Besides, the FC test can be completed within 48-72 hr whereas the IMViC tests may take 7-10 days. According to the definition in the STANDARDMETHODS(1965), "The coliform group includes all of the aerobic and facultatively anaerobic, gram negative, non-spore forming, rod shaped bacteria which ferment lactose with gas formation within 48 hr at 35°C ''. Bacteria conforming to the above definition are found abundantly in faeces but they are also frequently encountered in non-faecal habitats such as soil, vegetation and water. While Eschericha coli I is predominantly faecal in origin (ESCHERICH, 1885; ROGERS et al., 1914; PARR, 1938 and FOOTE, 1937), Aerobacter aerogenes has been found quite commonly in soils and on vegetation (CHEN et al., 1920; FRANK et aL, 1941 ; BARDSLEY,1934; ROGERS et aL, 1915; THOMASet al., 1952 and THOMAS et aL, 1955). Because of their frequency of occurrence, E. coliI is considered faecal and .4. aerogenes as non-faecal or soil and grain type. The fact that certain species of coliforms can survive in soils is well established. Hence, the mere presence of coliforms in a water source may not necessarily indicate fresh excretal pollution. The recent trend in sanitary water bacteriology, therefore, is to enumerate both the faecal and non-faecal coliform densities in water. 575
576
R.P. MISHRA,S. R. JOSHIand P. V. R. C. PANICKER
The Intermediate Aerogenes Cloacae (IAC) group of coliforms survive comparatively longer periods in soil and in other non-faecal environments (PARR, 1937; PLATT, 1935 and TAYLOR, 1942). JOHNSON (1917), CHEN and RETTGER (1920), KOSER (1926), RAGHAVACHARI (1926) and FRANK and SKINNER (1941) reported Aerobacter aerogenes to be predominant in soils with percentages varying from 45.0 to 93.5. They are reported to be more resistant to chlorination than E. coli (TONNEYe t al., 1928; HEATHMAN et al., 1936; BUTTERFIELD et al., 1943 and KAHLER, 1951). The sanitary significance of the different types of coliforms, therefore, should not be judged on the same footing. Detection of faecal coliforms in water, either in the presence or absence of IAC groups of organisms, indicate recent excretal pollution. The preseiice of IAC group of organisms in the absence of faecal coliforms, however, may be due to remote excretal pollution or addition of soil run off water after precipitation. In so far as the raw water sources are concerned for drinking water supplies the enumeration of faecal coliforms is of importance. In treated drinking waters, however, the presence of any type of coliforms faecal or non-faecal, is not considered desirable. In this study the biochemical characteristics (IMViC) of the coliform strains isolated from human and cattle faeces, sewage and soils were compared with the lactose fermentation reactions at elevated temperature to find out whether the elevated temperature test is a suitable substitute to the IMViC test for differentiating the faecal from non-faecal coliforms. EXPERIMENTAL Samples of human faeces were collected from volunteers who were staff members of the Ceiatral Public Health Engineering Research Institute, Nagpur. Samples were collected in sterile glass containers in the morning and brought to the laboratory within an hour after collection. A loopful of the sample was transferred to a 10 ml sterile buffered distilled water blank and shaken well to make it as homogenous as possible. Two Eosin Methylene Blue Agar (EMB) plates (Difco Labs.) were streaked with this suspension. One of the plates was incubated at 37°C and the other at 44.5°C for 24 hr. Individual and discrete colonies, one of each type, whether typical or atypical, were picked up, transferred to peptone water and incubated at 37°C for 24 hr. From this, a loopful of the culture was transferred to an EC broth tube, incubated at 44.5°C for 24 hr in a constant temperature water bath and observed for the production of gas. All the individual strains picked up were also subjected to the IMViC tests in parallel as adopted by GELDREICH et al. (1964). Cattle faeces included mainly faeces of buffaloes and cows from Ajni, a locality specializing in milk trade. Samples were collected before the actual starting of the work. The procedures for streaking, incubation and other tests were the same as for the human faeces. Raw domestic sewage samples were collected in sterile glass containers. After making the samples homogenous by shaking they were streaked on EMB agar plates in duplicate and incubated at 37°C and 44.5°C temperatures as was done for human and cattle faeces. Individual strains were subjected to FC and IMViC tests. All strains isolated from human faeces, cattle faeces and sewage were tested for their ability to ferment lactose in lactose broth tubes at 37°C within 48 hr.
Standard Biochemical and Elevated Temperature Tests
577
Samples of soil were collected from the premises of the Institute which comprise about 100 acres of grazing field. Before collecting the samples, 2.5 cm of the surface growth was scraped with a knife and then a few grammes of soil were scooped out and collected in sterile containers. The soil sample was pulverized in an all glass sterilized pestle and mortar. It was then sieved through a sieve retaining pebbles and stones above 2 mm dia. Ten grammes of soil was added to a 100ml sterile buffered dilution water blank in a round bottom flask and shaken with the help of a rotating shaker for 5 min. After this, 10 ml of the suspension was transferred to a 10 ml double strength lactose broth tube and incubated for 48 hr at 37°C. The presumptive positive tubes were subjected to confirmatory test in two Brilliant Green Lactose Bile broth (BGLB) tubes, one of which was incubated at 37°C for 48 hr, and the other at 44.5°C for 24 hr. EMB agar plates were streaked from the confirmatory BGLB positive tubes at 37°C and 44.5°C respectively, incubated for 24 hr at 37°C and observed for growth. Discrete, typical and atypical colonies apparently looking different, were inoculated into peptone water and then subjected to the FC and IMViC tests. Soil samples which showed positive reactions in confirmatory Brilliant Green Lactose Bile broth (BGLB) tubes at 37°C within 48 hr were considered as coliform positive. When the samples showed positive indole tests for corresponding tryptone broth tubes, they were considered positive for E. coli I. But, final confirmation of the presence of coliforms and E. coli I was done on individual strains subjecting them to lactose fermentation, IMViC and FC tests as described before. It was not considered necessary to subject the faeces (human and cattle) and sewage samples to the presumptive and confirmatory tests, as were done for soil samples because the presence of coliform was a certainty. Appearance of typical and atypical colonies on EMB agar plates and lactose fermentation at 37°C gave positive evidence of the presence of coliforms. I M V i C tests
The indole, methyl red, Voges Proskauer and citrate utilization tests were conducted as described in the 12th edition of Standard Methods. R E S U L T S AND O B S E R V A T I O N S A total of 1628 coliform strains were examined, 427 from 143 samples of human faeces, 301 from the 101 samples of cattle faeces, 631 from 113 samples of sewage and 269 from 120 samples of soils. The samples were tested for the presence or absence of coliforms, E. coli I and A. aerogenes. The reactions of the strains isolated from these sources to the elevated temperature test were also determined. Samples from which any of the strains isolated showed + + - - IMViC reactions and positive FC test were considered positive for E. coli I. Further, samples from which any of the strains showed - - + + IMViC reactions, whether F C positive or negative, were considered positive for A. aerogenes. The results obtained are presented in TABLE 1. All the samples (100 per cent) of human faeces, cattle faeces and sewage were positive for coliforms. But for soils, only 70.8 per cent of the samples were positive. The presence of E. coli I was confirmed in 95.1, 96.0 and 86-7 per cent of human
578
R . P . MISHRA, S. R. JOSHI and P. V. R. C. PANICKER
faeces, cattle faeces and sewage respectively, whereas for soils 54.1 per cent only were positive. For A. aerogenes 23.1, 10.9, 63-4 and 71.6 per cent of strains from human faeces, cattle faeces sewage and soils samples respectively showed positive reactions. While a large percentage of samples from human, and cattle faeces and sew~tge showed positive FC tests, being 97.2, 96.0 and 97.3 per cent respect!vely, only 73.0 per cent of soil samples were positive for this test. TABLE 1. DISTRIBUTION OF COLIFORMS IN FAECES, SEWAGE AND SOILS
Coliform organisms E. coli 1 A. aerogenes Elevated temp. Source Sam. pies Positive Negative Positive Negative Positive Negative Positive Negative (No.) (No.) (%) (No.) (%) (No.) (%) (No.)(~o) (No.)(%) (No.)(Y.) (No.) (%) (No.)(%) Human faeces 143
143 100
0
0
136 95"1
7
4-9
33 23.1
Cattle faeces 101
I01 100
0
0
97 96"0
4
4"0
ll
Sewage
ll3
113 100
0
0
98 86.7
Soils
120
85 70.8
35 29.1
46 54-1
l l 0 76.9
139 97"2
4
2.8
10'9
90 89"1
97 96'0
4
4-0
15 13"3
72 63.4
41 36.5
l l 0 97.3
3
2.7
39 45-9
61 71.8
24 28.2
63 73-0
22 27.0
TABLE 2. GROUP TYPING OF THE COLIFORM STRAINS
Parr's IMViC group
Human faeces (No.) (~)
Cattle faeces (No.) (~)
Sewage (No.) (~)
355
83.1
256
85"0
311
51
11.9
37
12"3
Intermediate group 21
4"9
8
2'6
100.0
301
100.0
Soils
(No.)
(%)
49"3
96
35.7
283
44.9
159
59.1
37
5"9
14
5.2
631
100.0
269
100.0
Escherichia group + -t- -- -
-- + -- Aerobacter group - - + +
-
-
-
+
Total
427
Based on the four biochemical tests, viz., indole methyl red, Voges-Proskauer and citrate utilization: PARR (1938a) interpreted that strains showing + + - - , + - - and + -- - - reactions may be included under Escherichia group indicative of faecal origin, strains showing - - + + , - - - + and - - + - reactions under Aerobactor group indicative of soil and vegetation origin. All the strains isolated were classified into three groups, the results of which are presented in TABLE 2. It is interesting to note that only 83.1 and 85.0 per cent of the strains isolated from human and cattle faeces respectively are included under Escherichia group. About -
Standard Biochemical and Elevated Temperature Tests
579
49 per cent of the strains isolated from sewage are included under this group. In soils 35.7 per cent of the strains isolated are included under this group. It is of interest to note that 11.9 and 12.3 per cent of the strains isolated from human and cattle faeces were positive for Aerobacter group in contrast to 44-8 and 59.1 per cent in sewage and soils (TAnLE 2). The intermediate group of organisms were encountered in less than 5 per cent of the strains examined from human and cattle faeces. Most of the strains isolated (95 per cent) from faeces, either cattle or human, are included under Escherichia and Aerobacter group. The organisms of Intermediate group were present in 5.8 and 5.2 per cent of the strains obtained from sewage and soil. TABLE3. IMViC T Y P I N G Test
AND ELEVATED TEMPERATURE REACTIONS OF COLIFORM STRAINS
Human faeces Cattle faeces (No.) (~) (No.) (Yo)
Sewage (No.) (Yo)
Soils (No.) (~) 93
+ + - ++ --
320
74.9
246
81.7
286
45.3
- + - Indole positive Methyl red positive Voges Proskauer positive Citrate utilizers Elevated temperature positive Number of cultures
355 329 367 64 60 350 427
83-1 79.4 85"9 15.0 14.1 82"0 100.0
256 256 259 30 40 230 301
85.0 85"0 86-0 I0"0 13.3 76.4 100.0
311 310 326 210 289 350 631
49.3 49-1 51 "7 33.3 45.8 57.1 100.0
34.6
96 35.7 101 37.5 104 38.7 114 42.4 158 58.7 150 55"8 269 100.0
In TABLE 3 are presented individual reactions of the strains isolated from different sources. Besides, IMViC reactions representing Parr's Escherichia group have also been tabulated. The individual tests along with the combined IMViC reactions were compared with the elevated temperature test for all strains. It will be seen that 74.9 and 81.7 per cent of strains from human and cattle faeces were positive for E. coli I. The combined IMViC reactions of Parr's Escherichia group for human and cattle faeces represent 83.1 and 85.0 per cent respectively. In sewage and soils, strains showing this type of combined reactions constitute 49.3 and 85.7 per cent respectively. A large percentage o f the strains (82.0 per cent) of human faeces were positive for F C test. This percentage, however, was little less in cattle faeces. Surprisingly, the percentage of coliform strains showing elevated temperature positive reactions isolated from sewage and soils were almost identical; 57.0 per cent in sewage and 55-8 per cent in soils. The different kinds of IMViC combinations encountered with coliform strains isolated from these four sources and their relation to FC test are presented in TABLE 4. Out of 427 strains obtained from human faeces, 320 (74-9 per cent) showed + + - - reactions and from this number 292 (91.2 per cent) gave FC test positive reactions. In cattle faeces out of 301 strains isolated 246 (81.7 per cent) showed + + - - reactions and from this 213 (86.8 per cent) strains responded positively to the FC test. In sewage 286 out of 631 strains tested gave + + - - reactions and from this 225 (78.7 per cent) strains were found FC positive. In soils, only 93 strains
427
t Out of each I M V i C type.
* Out o f total strains isolated.
1
Total
-
0
+
+
+ + +
-
320 0 35 42 6 3 4 4 1 I 8 2 0
-
--+ -+ + + + + +
+ + + -+ + +
-
-+ + + -+ + + --
0
0"2
74"9 0 8'2 9"8 1"4 0"7 0"9 0"9 0-2 0"2 1"9 0-5 0
350
0
0
81-9
0
0
292 91.2 0 0 23 65.7 22 52.4 1 16"7 2 66.7 3 75-0 4 100.0 1 100.0 0 0 0 0 2 100.0 0 0
F C test (No. ¢) ( ~ )
H u m a n faeces
Strains isolated (No.*) ( ~ )
+ -+ --+ + + + --
+ + -
I M V i C types
301
0
0
246 3 7 23 3 11 2 3 1 1 1 0 0 0
0
81"7 1"0 2"3 7"6 1"0 3"6 0"7 1"0 0-3 1"3 0-3 0 0
Strains isolated (No. *) ( % )
230
0
0
213 1 0 9 0 I 2 3 0 0 1 0 0
76.4
0
0
86"8 33"3 0 39"1 0 9"0 100.0 100-0 0 100"0 100.0 0 0
F C Test (No. t ) ( ~ )
Cattle faeces
631
5
3
286 3 22 167 23 93 2 3 2 8 8 0 6 0"8
0'5
45"3 0'5 3'5 26"5 3"6 14-7 0'3 0"5 0-3 1"3 1'3 0 0.9
Strains isolated (No. *) ( ~ )
360
2
3
57.0
40.0
100
225 78.6 1 33"3 15 68.1 68 40-1 10 43'4 21 22"5 2 100 2 66"6 1 50.0 2 25.0 3 37.5 0 0 5 83.3
F C Test (No. ¢) ( ~ )
Sewage
TABLE 4. T h E RELATION BETWEEN I M V i C AND ELEVATED TEMPERATURE TESTS
269
0
0
0
0
93 34-6 0 0 3 1"1 94 34"9 13 4"8 52 19"3 0 0 0 0 2 0"7 6 2"2 4 1"5 2 0'7 0 0
Strains isolated (No. *) ( ~ )
Soils
150
0
0
55.7
0
0
91 97"9 0 0 0 0 41 43"6 3 23-1 10 19-2 0 0 0 0 1 50"0 0 0 2 50"0 2 100 0 0
F C Test (No. t) ( ~ )
t~
Standard Biochemical and Elevated Temperature Tests
581
from 269 strains isolated were included under + + - - IMViC type and amongst these 91 strains (97.7 per cent) were FC positive. The Aerobactor aerogenes type of reactions viz; IMViC - - + + type were met with in 42 (9.8 per cent) strains from human faeces, 23 (7.6 per cent) strains from cattle faeces, 167 strains (26-4 per cent) from sewage and 94 (35 per cent) strains from soils. Among - - + + type strains isolated from various sources 22 strains (52.4 per cent) from human faeces, 9 (39.2 per cent) from cattle faeces, 68 (40.2 per cent) strains from sewage and 41 (43.6 per cent) strains from soils responded positively to the FC test. The FC positive test reactions were shown by 350 (81.9 per cent) strains from human faeces, 230 (76.4 per cent) strains from cattle faeces, 360 (57.0 per cent) strains from sewage and 150 (55.8 per cent) strains isolated from soils. D I S C U S S I O N AND C O N C L U S I O N S Various biochemical reactions such as indole, methyl red, Voges-Proskauer, or citrate utilization tests had been proposed to differentiate the coliform strains from faecal and non-faecal environments. Individually none of these tests were found to be specific for coliform derived from any one source--faecal or non-faecal. PARR (1938) suggested the combination of indole, methyl red, Voges-Proskauer and citrate utilization tests for differentiating coliform strains into faecal and non-faecal. The series of four reactions was designated by the mnemonic "IMViC" tests. EIJKMAN (1904) divided the coliforms into a faecal group that would produce gas from glucose at 46°C and a non-faecal group that would produce no change in the medium. He believed that all the coliform bacteria derived f r o m the gut of warmblooded animals would respond positively to this test. This test suggested by him was highly specific for the faecal group but had low sensitivity. P~RRV and HAJNA (1944) improved the Eijkman test by the addition of bile salts and a buffer system and by reducing the air incubation temperature to 45.5°C. GELOP~ICH et al. (1962) studied 8747 strains of coliforms isolated from faeces of warm-blooded animals, polluted and unpolluted soils with IMViC reactions and the elevated temperature test in EC medium at 44-5°C. They observed excellent correlation between the IMViC tests and the FC tests of coliform strains. In their study 93.3 per cent of the coliform strains isolated from faeces of warm-blooded animals came under Parr's Escherichia group whereas 96-4 per cent of the strains gave FC positive reactions. In the present study 95 per cent of the human faeces samples were positive for E. coli I and 5 per cent were negative. Similarly, 96 per cent of the cattle faeces were positive for E. coli I and 4 per cent were negative. Thus, 4-5 per cent of the faeces samples may contain coliforms but not E. coli I. If E. coli I is taken as representing faecal pollution, in such instances, the polluted water may pass out as unpolluted since most of the coliforms there will be of non-faecal type belonging to the IAC group. The situation is similar for the sewage samples in which 13.3 per cent of the samples were found negative for E. coli I. It was but natural to expect that all the samples (100 per cent) of faeces and sewage contained coliforms. In our study, 70.8 per cent of the soil samples contained coliforms and 54.1 per cent samples were positive for E. coli I. The presence of E. coli I in such large percentage can be attributed to the frequent excretal pollution of the D
W
582
R.P. MISHRA,S. R. Josm and P. V. R. C. PANICKER
area by cattle and also to some extent by human beings. Soil run off water from such an area may add to the coliform density of a water source particularly after heavy precipitation. Although a large percentage of human and cattle faeces samples showed complete absence of A. aerogenes, quite a sizeable percentage showed their presence. The presence of A. aerogenes in faeces, human and cattle, would suggest that these calmot be entirely considered exclusively soil or grain type. Whether they are transient in faeces and occur there by accident is immaterial as long as they survive and adapt themselves in the gut of the animals, and human beings. Nearly 72 per cent of soil samples were positive for this group. It would appear, therefore, that this group of organisms can adapt themselves easily to the conditions in soils as well in the alimentary tract. Over 96 per cent of human and cattle faeces and sewage samples contained coliform strains which gave positive reactions at elevated temperature (TABLE 1). It is doubtful, however, whether the coliform strains present in faeces and sewage can continue to maintain the FC test positive characteristic after they are excreted and are submerged for long periods in water. This aspect needs further investigation. On the basis of the number of coliform strains isolated, only 74.9 per cent of the strains from human faeces and 81.7 per cent of the strain from cattle faeces showed ++-IMViC reactions. Considering Parr's Escherichia group , ( + + - - , -~ , +--) as representing the faecal coliform strains, still only 83 per cent and 85 per cent of coliform strains isolated from human and cattle faeces respectively could be included under this category (TABLE 3). Similarly, 12 per cent of the coliform strains isolated from these two sources showed reactions of Parr's Aerobacter group. The Aerobacter group and Intermediate group combined together form 17 per cent and 15 per cent of the coliform strains isolated from human and cattle faeces. From the results, it is clear that the IMViC reactions by themselves are not perfect biochemical tests which conclusively distinguish between the coliform strains derived from faecal and non-faecal environments. In sewage samples, there is a sharp fall in Escherichia group and a consequent increase in Aerobacter group. This may happen in two ways. Possibly the Escherichia group of organisms die out at a faster rate compared to the Aerobacter group after they are discharged in the faeces. It is likely that outside the gut, the Aerobacter group of coliform organisms may actually be able to multiply in sewage to some extent. But the latter possibility appears to be remote. In sewage, the Escherichia, Aerobachter and Intermediate groups form 49.3, 44.9 and 5.9 per cent respectively. The .presence of Aerobacter group in a water source may be due to the pollution either by faeces, sewage or soils. It is of importance, therefore, to find out whether the coliform strains others than E. coli I, present in water, were due to the addition of soil run-off water. It will also be interesting to find out the percentage of coliforms derived from faeces which fail to give FC positive results and strains of coliform derived from unpolluted non-faecal sources which give FC positive reactions. It was evident from the present investigation that quite a large percentage of coliforms present in faeces and sewage failed to respond positively to the elevated temperature reactions. In our study 18 per cent of the coliform strains from human faeces, 24 per cent from cattle faeces, and 43 per cent from sewage failed to give FC positive reactions (TABLE4). These results are in contrast to the results obtained by GELO-
Standard Biochemical and Elevated Temperature Tests
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REICH et al. (1962) wherein they f o u n d that 3.6 per cent o f the coliform strains f r o m the faeces o f w a r m - b l o o d e d animals gave F C negative reactions. These results are, however, in agreement with the results obtained by MoussA (1965) who observed that the total n u m b e r o f coliform strains f r o m faeces and sewage which p r o d u c e d gas after 48 hr at 44.5°C did not exceed 62.1 per cent. It is evident, therefore, that m a n y coliform strains actually derived f r o m faeces o f warm-blooded animals fail to respond positively to the F C test. Hence, the F C test procedure cannot absolutely distinguish the coliform strains f r o m faecal and non-faecal origin. A large percentage (55.7) o f the coliform strains isolated f r o m soils showed F C positive reactions. This is likely because the area f r o m which the soil samples were drawn was frequently polluted. GELDREICH et al. (1962), however, f o u n d 83 per cent o f the coliform strains f r o m polluted soils as F C positive. Individually, indole and methyl red tests give positive reactions for quite a large percentage of strains isolated f r o m h u m a n and cattle faeces. In our study o f coliform strains f r o m h u m a n faeces, 79.4 per cent were indole positive and 85.9 per cent M R positive. MoussA (1965) f o u n d 38.8 per cent o f coliform strains f r o m h u m a n faeces as indole positive and 81.7 per cent as M R positive. GELDREICH et al. (1962) f o u n d 94 per cent o f the coliform strains f r o m faeces of w a r m - b l o o d e d animals as indole positive and 96.9 per cent as M R positive. They, however, discovered that o f the coliform strains isolated f r o m unpolluted soils 19.4 and 75-6 per cent gave indole and methyl red positive reactions respectively. The specificity o f these two tests for coliform strains derived f r o m faeces is therefore, doubtful. In the present study it was f o u n d that 37.5 per cent of the coliform strains f r o m soils were indole positive and 38.6 per cent o f the strains as methyl red positive. These reactions are not unusual since the land was subjected to frequent pollution. Altogether 15 I M V i c types o f coliform strains were isolated f r o m these four sources (TABLE4). The m o s t c o m m o n I M V i C type f o u n d in h u m a n faeces, cattle faeces, and sewage was + + - - , being 74-9, 81.7, and 45.3 per cent respectively. The other c o m m o n combinations in h u m a n faeces were - - + + (9.8 per cent) and - + - (8.2 per cent), in cattle faeces - - + + (7.6 per cent) and - - - + (3-6 per cent), in sewage - - + + (26.5 per cent). - - - + (14.7 per cent) and - + - - (3.5 per cent). In soils + + - and - - + + types were encountered approximately in equal numbers, being 93 (34.5 per cent) and 94 (34.9 per cent) respectively. The other c o m b i n a t i o n frequently seen was - - - + (19.0 per cent). F r o m the different I M V i C types isolated f r o m all sources, + + - type give m a x i m u m correlation with the F C positive tests. O f the + + - - types isolated f r o m h u m a n s and cattle faeces; sewage and soils 91.2, 86.8, 78.7 and 98.0 per cent respectively gave F C positive reactions. More than 52 per cent of the - - + + type o f coliforms f r o m h u m a n faeces gave F C positive reactions. It, therefore, follows that the F C positive test reactions m a y not be confined to Parr's Escherichia g r o u p alone. Quite a large percentage (43.6 per cent) o f the - - + + types isolated f r o m soil gave positive F C tests. During the course of' these studies it was observed that m a n y of the noncoliform organisms could also p r o d u c e + + - - I M V i C reactions at 37°C as well as at 44°C. Such strains, however, failed to ferment lactose at 37°C and 44.5°C. Few coliform strains f r o m h u m a n faeces showed + + - - reactions but failed to ferment lactose at 44-5°C although they fermented lactose at 37°C.
584
R . P . MISHRA, S. R. JOSHI and P. V. R. C. PANICKER
C o l i f o r m o r g a n i s m s a r e p r e s e n t in faeces a n d soils. Escherichia g r o u p o f o r g a n i s m s usually p r e d o m i n a t e in h u m a n a n d cattle faeces a n d the Aerobacter g r o u p in soils. H u m a n a n d cattle faeces also c o n t a i n coliforms o f the Aerobacter a n d I n t e r m e d i a t e g r o u p s in a p p r e c i a b l e numbers. Similarly, soils often c o n t a i n coliforms representing P a r r ' s Escherichia g r o u p . T h e r e is no m e t h o d at present to differentiate the Aerobacter g r o u p o f o r g a n i s m s d e r i v e d f r o m faeces a n d soils. F o r this reason, differentiation o f the c o l i f o r m strains into faecal a n d non-faecal origin on the basis o f the I M V i C tests is n o t fully justified a l t h o u g h these tests d o give a g o o d i n d i c a t i o n o f their origin. These tests have n o t been used in a r o u t i n e scale because they are c o m p l i c a t e d a n d time consuming. M o r e o v e r , these tests can be p e r f o r m e d only on p u r e cultures isolated f r o m the samples. T h e elevated t e m p e r a t u r e test m a y give a g o o d i n d i c a t i o n o f the origin o f the c o l i f o r m strains b u t this test t o o was n o t f o u n d very specific for c o l i f o r m strains f r o m faeces. I n o u r s t u d y m a n y o f the c o l i f o r m isolated f r o m soils gave F C positive reactions. GELDREICH et al. (1962) t o o n o t e d t h a t m a n y coliforms isolated from unp o l l u t e d soils gave F C positive reactions. T h e y f o u n d t h a t 96.4 p e r cent o f the c o l i f o r m strains f r o m faeces o f w a r m - b l o o d e d animals gave F C positive reactions whereas 93.3 p e r cent only gave P a r r ' s Escherichia g r o u p I M V i C c o m b i n a t i o n s . In o u r study, we c o u l d find t h a t o u t o f the c o l i f o r m strains isolated f r o m h u m a n a n d cattle faeces 82.0 p e r cent a n d 76.0 p e r cent o f the strains gave F C positive reactions (TABLE 3). But P a r r ' s Escherichia I M V i C c o m b i n a t i o n s were observed in 83.1 a n d 85.4 p e r cent o f strains f r o m h u m a n a n d cattle faeces. Based on the results o b t a i n e d in this study it is n o t possible to consider the F C test to be specific for the coliform strains derived f r o m the faeces a n d sewage a n d thus there is still m u c h scope to develop a m e t h o d for differentiating faecal a n d nonfaecal coliforms. Acknowledgement--The authors are grateful to the Director, Central Public Health Engineering
Research Institute, Nagpur (India) for having given all the facilities for the work and permission to publish the data. REFERENCES BARDSLEYD. A. (1934) The distribution and sanitary significance of B. cull, B. Lactis aerogenes and the Intermediate types of coliform bacilli in water, soil, faeces and ice cream. J. Hyg., Camb. 34, 38. BUTTERFIELDC. T. (1943) Influence of pH and temperature on the survival of coliforms and enteric pathogens when exposed to free chlorine. Publ. Hlth Rep. 58, 1837. CHEN C. C. and RETTGERL. F. (1920) A correlation study of the colon aerogens groups of bacteria with special reference to the organisms occurring in the soil. J. Bact. 5, 253-298. EIJKMAN C. (1904) Die G~irungsprobe bei 46 ° als Hilfsmittel bei der Trinkwasseruntersuchung. Centr. Bakteriol. Parasitenk, Abt. L Orig. 34, 742-748. ESCHERICHT. (1885) Die Darmbakterien des Neugeborenen und Sauglings. Fortschr. Med. 3, 515-547. FOOTE H. B. (1937) The possible effects of wild animals on the bacterial pollution of water. J. Am. Wat. Wks Ass. 29, 72-74. FRANK N. and SKINNERC. E. (1941) Coli-aerogenes bacteria in soil. J. Bact. 42, 143. GELDRICHE. E., BORDNERR. H., HUFF C. B., CLARKH. F. and KABLERP. W. (1962) Type distribution of coliform bacteria in the faeces of warm-blooded animals. J. Wat. Pollut. Control Fed. 34, 295-301. GELDREICHE. E., HUFF C. B., BORDNER R. H., KABLERP. W. and CLARK H. F. (1962) The faecal coli-aerogenes flora of soils from various geographical areas. J. appl. Bact. 25, 87-93.
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GELDREICH E. E., KENNER B. A. and KABLER P. W. (1964) Occurrence of coliforms, faecal coliforms and Streptococci in vegetation and insects. App. Microbiol. 12, 63-69. HEATHMAN L. S., PIERCE G. O. and KABLER P. W. (1936) Resistance of various strains of E. typhi and coli-aerogenes to chlorine and chloramine. Publ. Hlth Rep. 51, 1367. JOHNSON B. R. and LEVINE M. (1917) Characteristics of coli-like microorganisms from the soil. J. Bact. 2, 379401. KABLER P. W. (1951) Relative resistance of coliform organisms and enteric pathogens in the disinfection of water with chlorine. J. Am. Wat. Wks Ass. 43, 553-568. KOSER S. A. (1926) The coli-aerogenes group in soil. J. Am. V/at. Wks Ass. 15, 641-646. MoussA R. S. (1965) Type distribution of coliforms isolated from faecal and non-faecal habitats. Ind. J. med. Res. 53, 629-637. PARR L. W. (1937) Viability of coli-aerogenes organisms in cultures and in various environments. J. infect. Dis. 60, 291-301. PARR L. W. (1938a) Coliform intermediates in h u m a n faeces. J. Bact. 36, 1-15. PARR L. W. (1938b) The occurrences and succession of coliform organisms in human faeces. Am. J. Hyg. 27, 67-87. PERRY C. A. and HAJNA A. A. (1944) Further evaluation of EC Medium for the Isolation of Coliform Bacteria and Escherichia coli. Am. J. pubL Hlth, 34, 735-748. PLATT A. E. (1935)The viability of Bact. coli and Bact. aerogenes in water: A method for the rapid enumeration of these organisms. J. Hyg., Camb. 35, 437. RAGHAVACHARI T. N. S. (1926) The application of koser's citrate utilization test to sanitary water analysis in India. Indian J. med. Res. 14, 47. ROGERS L. A., CLARK W. M. and EVANS A. C. (1914) The characteristics of bacteria of the colon type found in bovine faeces. J. infect. Dis. 15, 100. ROGERS L. A., CLARK W. M. and EVANS A. C. (1915) Characteristics of bacteria of the colon type occuring on grains. J. infect. Dis. 17, 137. STANDARD METHODSFOR THE EXAMINATIONOF WATERAND WASTEWATER. (1965) 12th edition. Amer. Pub. H#h Ass. New York. TAYLOR C. B. (1942) The ecology and significance of the different types of coliform bacteria found in water. J. Hyg., Camb. 42, 23-36. THOMAS S. B. and HOBSON P. M. (1955) Coli-aerogenes bacteria isolated from ears and panicles of cereal crops. J. appl. Bact. 18, 1. THOMAS S. B. and McQUILLIN J. (1952) Coli-aerogenes bacteria isolated from grass. Proc. Soc. appl. Bact. 15, 41. TONNEY F. O., GREER F. E. and DAUFORTH T. F. A. (1928) The minimal chlorine death point of bacteria. Am. J. Pub. Hlth 18, 1259-1263. VAUGHN R. H., LEVINE M. and SMITH H. A. (1951) A buffered boric acid lactose medium for enrichment and presumptive identification of E. coil Fd Res. 16, 10-19.