Biochemical and Physiological Characteristics of...
Transcript of Biochemical and Physiological Characteristics of...
146
University of Africa Journal of Sciences
Biochemical and Physiological Characteristics of Escherichia coli isolated from Different Sources
Sherfi S., A.1, Dirar, H., A.2, Ibrahim F. Ahmed3
1 Department of Basic Sciences, Faculty of Medical Laboratories Sciences, International University of Africa, Khartoum, Sudan. 2 Department of Botany andAgricultural Biotechnology, Faculty of Agriculture, University of Khartoum, Khartoum, Sudan. 3 Dean of the Faculty of Medical Laboratories Sciences, International University of Africa, Khartoum, Sudan.
ABSTRACT
In this study ninety four isolates of Escherichia coli were obtained
from different sources of water, sewage, and human and animal feces
by using multiple tube fermentation technique or by direct streaking
on MacConkey agar. All isolates were first identified biochemically
using a Japanese API like system.
The physiological characteristics of the E. coli isolates were examined
by studying their growth in different culture media, i.e., E. coli
medium (EC), brilliant green bile broth (BGB), MacConkey broth,
and lauryl sulphate tryptose broth, at different temperatures, viz., 30,
37 and 44.5°C. The results of these tests showed that EC broth
medium yielded the highest growth density of E. coli at all
temperatures. Brilliant green bile broth gave the second best growth
density, followed by MacConkey broth, then lauryl sulphate tryptose
broth.
147
University of Africa Journal of Sciences
The incubation Temperature 37°C gave the highest growth density in
all four growth media, the lowest growth density being obtained at
44.5°C.
The results of this study indicated that E. coli can be used as a fecal
indicator for contaminated water in Sudan but the choice of media
used as well as the survival of the organism in pure water should be
taken into consideration.
Key words: E. coli, Fecal indicator
1. INTRODUCTION Escherichia coli is inhabitant of the intestinal tract of humans and
animals.
For this reason scientists argued that if E. coli was found in any
habitat other than the intestine, that habitat must have been somehow
contaminated with human or animal feces. This led to the suggestion
that E. coli should be used as an indicator of the contamination of
foods, particularly of water, with human excreta, hence, possible with
pathogens (Clark, J. A. and Pagel, J. E. 1977). The test has been
developed in temperate countries. The later application of the test in
the Third World meant its application in a new environment such as
tropical countries whose inhabitants eat different kinds of foods not
necessarily made of wheat or potatoes as in cold countries. It is now
well known, even in general microbiology (Tortora et al. 1992;
Madigan et al. 1997) that the gut flora in humans can change in
response to an altered environment and can vary qualitatively, and
quantitatively depending on the diet.
148
University of Africa Journal of Sciences
A great amount of research data has questioned the suitability of E.
coli as an indicator of fecal contamination of tropical water. Some
authors have argued that Vibrio cholerae is a natural inhabitant of
tropical waters and the absence of E. coli in this water does not show
that the cholera organism is not present (Perez-Rosas and Hazen,
1989). Others showed that pathogens like Salmonella survived for a
longer time in tropical waters and that E. coli might not be suitable as
indicator of recent contamination (Jimenez et al.1989). It has even
been suggested that E. coli is a normal inhabitant of tropical waters
and therefore is unsuitable as a fecal indicator (WHO 1995). These are
just some examples of research carried out in tropical countries.
The Sudan has recently published, through Sudan Standards and
Metrology Organization (SSMO), its first preliminary microbiological
standards for foods. Naturally, E. coli is adopted in these standards as
the major indicator of fecal contamination as generally recommended
by the World Health Organization (WHO, 1964) and other
international institutions.
But Sudan is a tropical country with temperatures soaring up to near
50°C or more in the summer. The major diet of the general rural
population is sorghum and pearl millet. It is likely that the micro- flora
of the intestines of humans and animals and the survival of E. coli in
natural waters is different in this country from Europe and North
America.
The workers of Sudan preliminary microbiological standards of foods
were very much aware of this and wrote: “SSMO should sponsor and
149
University of Africa Journal of Sciences
encourage research on the microbiology of foods as related to food
standards and safety”.
This research has been carried out to see if E. coli is a suitable
indicator of fecal contamination of food. The strategy to do this is to
isolate, characterize and study the physiology of E. coli from different
water sources and human and animal feces in Khartoum State where
practically all Sudanese communities are represented.
2. MATERIALS AND METHODS
2:1 Collection of samples: -
Samples from different sources such as human and animal feces and
different types of water (drinking water from zeer, stagnant rain
water, irrigation water, and sewage) were collected and used to
obtain 94 suspected E. coli isolates. The samples were collected in
clean and sterile screw- capped bottles or test tubes from different
sites of Khartoum State. The bottles and test tubes were placed in ice.
Samples were examined on the same day and those which were not
processed within one hour after collection were stored at zeroºC for a
maximum of two days.
2:2 Isolation of E. coli: -
2:2:1 Isolation from feces: -
E. coli isolates from human and animal feces were obtained by direct
streaking of the sample on MacConkey, s agar (Mast Diagnostic U.K.)
as they needed no enrichment because of their large number. The
150
University of Africa Journal of Sciences
plates were incubated at 37ºC aerobically for 24hrs. All plates were
inspected for growth and colonial morphology. The isolates were
subjected to confirmation tests on Eosin Methylene Blue (EMB)
medium.
2:2:2 Isolation from water and sewage: -
Standard multiple tube fermentation technique described by Geldreich
(1975) was used for water examination, which is divided into three
stages:-
presumptive test on Lauryl sulphate tryptose broth (LST), confirmed
test on EC medium and completed test on Eosin Methylene Blue
(EMB) agar. Typical E. coli colonies picked from EMB agar including
isolates from feces were transferred to nutrient agar plates to remove
their colors. The Gram-stain was performed on the new growth; E.
coli appeared as Gram-negative, short rods occurring singly or in
pairs.
2:3 Biochemical identification of E. coli: -
All 94 cultures appearing as Gram- negative, short rods or
coccobacilli, were subjected to various biochemical tests (special
Japanese scheme similar to the API system) (Colle et al., 1996). This
biochemical scheme includes triple- sugar- iron (TSI) test, motility
test, indole test, urease test, lysine decomposition test, Voges-
proskauer (VP) test, and DNAse production test. All media and
reagents used in these tests were prepared in the laboratory (Health
Laboratory, Khatoum State). Based on the outcome of the tests, each
151
University of Africa Journal of Sciences
isolate was given a code number consisting of four digits, e. g., 1370.
The digits were determined as follows: code number of lactose
represented the first digit, the summation of the (TSI) codes
represented the second digit, the summation of lysine, indole and
motility codes represented the third digit and the summation of Urea,
(V-P) and DNAse codes represented the fourth digit (Colle et al.,
1996). Isolates scoring the same code number represent the same
strain.
2:5 Physiological tests: -
2:5:1 Effect of the culture media and the incubation temperature
on the growth of E. coli:-
The media selected were those commonly used in the examination of
water for E. coli. One loopful of 18- 24 hrs culture (standard loop; a
loop of known dimension -1/400 ml-) of each identified ninety four
isolates of E. coli was used to inoculate 10 ml nutrient broth and
incubated at 37º C for 24 hours. One loopful of this culture was
inoculated into 10 ml of different types of media, viz., lauryl sulphate
tryptose broth (LST), EC medium, MacConkey,s broth and brilliant
green bile broth. The media were incubated at different temperatures,
i.e., 30º C, 37º C, and 44.5º C for 24 hours. The degree of growth was
measured by turbidometric method at the visible range of the spectrum
at average wavelength 557 nm, using Spectrophotometer Model 6300
(Jenway Ltd., U.K.) to determine the optical density of the culture,
using sterile medium as control.
152
University of Africa Journal of Sciences
3. RESULTS AND DISCUSSION
3:1 Isolation of E. coli:-
E. coli isolated from human and animal feces gave smooth pink
colonies on the solid isolation medium MacConkey’s agar, indicating
lactose fermentation. Lactose is included in this medium as a
fermentable carbohydrate together with a pH indicator, usually neutral
red. Strong acid producers, like Escherichia, Klebsiella, and
Enterobacter produced red colonies on this solid medium (Adams and
Moss, 2000), whereas those isolated from water sources and sewage
produced gas in the liquid enrichment EC medium at 44.5˚C. Gas
production among identically prepared subcultures varies in amounts
from sample to other in the multiple tube tests. Meadows et al. (1990)
referred this variability to the erroneous interpretation of the results of
coliform multiple tube tests or the growth of E. coli without gas. This
has been interpreted as the chance cultivation of anaerogenic or
environmentally damaged strains. Meadows et al. (1990) reported that
good gas production was obtained in buffered media.
Their confirmed test by streaking on EMB agar produced the dark-
centered colonies with metallic green sheen. EMB agar is a popular
selective and differential medium. The aniline dyes, eosin and
methylene blue, are the selective agents but also serve as an indicator
for lactose fermentation by forming a precipitate at low pH. Strong
lactose fermenters produce green black colonies with a metallic sheen
(Adams and Moss, 2000).
153
University of Africa Journal of Sciences
3:3 Identification of E. coli:-
3:3:1 Biochemical tests:-
Results obtained from the biochemical tests differentiate five groups
of E. coli which differ in (1) gas production from glucose fermentation
(2) lysine decarboxylation (3) indole production, and (4) motility test.
As shown in Tables 1 to 5, the code numbers (see Material and
Methods) obtained for our isolates were: 1370 (61.7%; 58/ 94), 1360
(28.7%; 27/ 94), 1330 (5.3%; 5/ 94), 1170 (3.2%; 3/ 94) and 1350
(1.1%; 1/ 94). Thus the biochemical tests grouped our E. coli isolates
into five groups. .
(i) Triple sugar – iron (TSI):
All isolates of E. coli showed a yellow butt and a yellow slope to
indicate the fermentation of lactose, sucrose and possibly glucose. The
bubbles in the medium indicate gas production from glucose
fermentation. About 96.8% of E. coli isolates produced gas from
glucose fermentation and gave code number of (2) and about 3.2%
isolates did not produce gas from sugar fermentation, and were given
code number of (0). It means that most isolates of E. coli produced gas
from glucose fermentation and a few of them did not. All isolates
obtained from water sources and sewage showed a positive gas
production from TSI whereas those obtained from animal and human
feces showed 89.47% and 97.44% positive gas production,
respectively. We thus notice that gas production from glucose
fermentation in E. coli was affected by the source of isolate. This may
be related to the nutrients or oxygen available in the source. Farmer et
154
University of Africa Journal of Sciences
al. (1985) reported that 95% of E. coli isolates produced gas from
glucose fermentation, 90- 100% of normal E. coli and 0- 10% of
inactive E. coli produced gas from fermentation.
All isolates showed no blackening along the stab line or throughout
the medium indicating no production of H2S and were given a code
number of (0).
(ii) Lysine test:
About 64.9% of E. coli isolates showed violet color throughout the
medium as positive result of lysine decarboxylation to the
corresponding amine with the liberation of carbon dioxide, and were
given code number of (1). About 35.1% of E. coli isolates kept the
yellow color of media as it was as a negative result of lysine
decarboxylation and were given (0) as a code number. Human feces
isolates gave 38.5% negative lysine decarboxylation, animal feces
isolates gave 36.8%, water source isolate gave 32.0% and isolates
obtained from sewage gave 27.3%.
Farmer et al., (1985) reported that 90% of E. coli isolates
decarboxylated lysine, 90- 100% of normal E. coli and 25.1- 74% of
inactive E. coli gave positive lysine decarboxylation.
(iii) Urease test
The positive result of urease test is indicated by a change in the color
of the indicator to red pink. All isolates of E. coli did not change the
color of the medium to red pink; all E. coli strains did not produce
urease (negative urease) and were given a code number of (0).
155
University of Africa Journal of Sciences
(iv) Indole production:
About 93.6% of E. coli isolates gave a red color in the upper layer of
the medium, indicating a positive result for indole production and
were given a code number of (2), while 6.4% of isolates gave negative
results of indole production, and were given the code number (1).
Isolates obtained from sewage source showed high percentage of
positive indole production up to 100.0%, whereas human and animal
feces isolates gave almost equal percentages, 94.87% and 94.74%,
respectively, and isolates obtained from water gave 88.0% indole
positive. The results obtained from this test showed that indole
production in E. coli varied, as reported by Jay (1992). The IMViC
reaction + + - - designates E. coli type 1 and E. coli type 2 are - + - -.
Water samples contain more of E. coli type 2 than feces and sewage
samples.
Farmer et al (1985) reported that 98% of E. coli were indole positive,
90- 100% of normal E. coli and 75- 89% of inactive E. coli were
indole positive.
(v) Voges Proskauer test:
All isolates showed a negative reaction towards V P test. No pink
color was formed during the test which means that the isolates did not
form acetoin. A code number of (0) was given.
(vi) Motility:
About 94.7% of E. coli isolates were motile showing a turbidity
throughout the medium and were given a code number of (4), whereas
156
University of Africa Journal of Sciences
5.3% were non-motile, producing growth only along the line of
inoculation, and were given a code number of (2). Isolates of E. coli
obtained from water sources gave 92.0% motile. Human and animal
feces isolates gave 94.87 and 94.74%, respectively, and all isolates of
E. coli obtained from sewage gave 100.0% motile, similar to that of
indole positive results. We notice that all indole – positive isolates
were also motile except SW8 which had a code number of 1350
identified as E. coli according to code sheet Table 1 Appendix. That
means indole production is correlated with the motility of E. coli. This
disagreed with Krieg (1984) who reported that 90- 100% of E. coli
strains were indole positive, whereas 76- 89% of E. coli strains were
motile.
Colle et al., (1996) reported that at least 80% of E. coli strains were
motile, though sometimes only weakly, on primary isolation.
Contrasting with the typical, biochemically active types, some strains
are lactose non-fermenting, non-motile, anaerogenic and biochemcally
inactive, like Shigella. Farmer et al., (1985) reported that 95% of E.
(vii) DNAse test:
DNAse- producing colonies growing on DNA medium were
surrounded by clear areas, indicating DNA hydrolysis, while
unhydrolyzed DNA is precipitated by added HCl acid. All isolates of
E. coli did not produce DNAse and their colonies were not surrounded
157
University of Africa Journal of Sciences
by clear areas indicating absence of hydrolyzed DNA, and were given
a code number of (0).
Table 1: The confirmatory biochemical tests of E. coli isolates
obtained from sewage.
(numbers are codes given according to the Japanese system. See text
for explanation) Tsi = Triple sugar- iron Lysine = Lysine test Suc = Sucrose fermentation V. P =Voges proskauer test Gas = Gas production from glucose H2S = production of H2S Urea = Urease test DNA = DNAse test
158
University of Africa Journal of Sciences
Table 2: The confirmatory biochemical tests of E. coli isolates obtained from human feces (first batch)
159
University of Africa Journal of Sciences
Table 3: The confirmatory biochemical tests of E. coli isolates obtained from human feces (second batch)
160
University of Africa Journal of Sciences
Table 4: The confirmatory biochemical tests of E. coli isolates obtained from different sources of water
161
University of Africa Journal of Sciences
Table 5: The confirmatory biochemical tests of E. coli isolates obtained from animal feces
3:4 Physiological tests:- 3:4:1 The effect of culture media and incubation temperature on
the growth of E. coli:-
The extent of growth for each of the 94 isolates in different media at
different temperatures is given as optical density. These values are
presented as average values in Figs 1- 7.
As shown in Figs 1-3 EC medium yielded the highest growth density
of all isolates of E. coli at the three temperature degrees of 30, 37 and
44.5°C. Isolates of E. coli obtained from human feces and sewage
showed higher growth densities than isolates obtained from water and
162
University of Africa Journal of Sciences
animal feces. Brilliant green bile broth (BGB) medium gave high
growth density at different temperature degrees for all isolates of E.
coli obtained from the different sources. On this medium isolates of E.
coli obtained from sewage and water sources showed higher growth
densities than those isolated from human and animal feces.
MacConkey broth gave a low growth density of all isolates of E. coli
at different temperature degrees compared with EC and BGB media,
on which isolates of E. coli obtained from human feces showed
highest density of growth than isolates obtained from sewage, water
sources or animal feces.
LST medium yielded the lowest growth density of E. coli at all
temperature degrees tested. E. coli isolates obtained from water
sources gave higher growth density than E. coli isolates obtained from
human feces, animal feces or sewage.
With respect to the effect of growth medium on the growth density of
E. coli, our results showed that EC medium yielded the highest growth
of E. coli. This may be due to its content of buffers (K2HPO4 and
KH2PO4) and the bile salts No.3 and tryptose instead of peptone.
Hajna and Perry (1943) reported that the EC medium was a highly
buffered lactose broth modified by the addition of 0.15% of Difco
Bacto bile salts No. 3 and the substitution of Bactotryptose for Bacto
peptone. The virtue of the bile salts mixture lies in its enhancement of
the growth of coliform bacteria, and its ability to inhibit more or less
completely the growth of fecal streptococci and spore formers. The
EC medium can be used equally well for the isolation of coliform
bacteria at 37º C or for the isolation of E. coli at 45.5º C. It can be used
163
University of Africa Journal of Sciences
either as a primary medium for the growth of E. coli or as a
satisfactory secondary medium for the confirmation of E. coli.
The specificity of the EC medium is 100 percent. The superiority of
EC medium for fecal coliforms was also reported by Jay (1986).
Colle et al., (1996) reported that entero- hemorrhagic E. coli (EHEC)
strains would not grow at 44.5°C in the standard EC medium
formulation, but would grow when the bile salts content in the
medium was reduced from 0.15% to 0.112%.
Brilliant green bile broth gave high growth of E. coli but less than did
EC medium. This lower growth may be due to the substitution of
lactose with glucose, also bile salts No.3 with dried bovine bile in
BGB medium. Black and Klinger (1936) found that brilliant green bile
broth did not perform uniformly with all strains of E. coli.
MacConkey broth gave a growth rate less than that of BGB medium,
in which peptone was used instead of tryptose and there are no buffers
in its content. This result agreed with WHO (1995) who reported that
MacConkey agar was not strongly selective and would support the
growth of a number of non- Enterobacteriaceae including Gram –
positives such as enterococci and staphylococci. Also Grabow and Du
Preez (1979) found that the lowest counts of E. coli were usually
recorded on MacConkey’s agar.
Lauryl sulphate tryptose broth yielded the lowest growth rate of E.
coli. This may be due to the absence of bile salt in its ingredients.
Hajna and Perry (1943) found that the specificity of lauryl sulphate
tryptose broth was 98.7% compared with 100% specificity of EC
medium.
164
University of Africa Journal of Sciences
The growth of E. coli at different temperature degree was influenced
by the source of E. coli isolate and the type of the growth medium. As
shown in Fig. 1, all isolates of E. coli gave highest growth density at
30°C in EC. Isolates obtained from human feces and sewage gave
highest growth density followed by the isolates obtained from the
water source, whereas the animal feces isolates gave the lowest
growth density of E. coli.
Fig. 2 shows that at 37°C human feces and sewage isolates
enumerated in EC medium yielded the highest growth density,
whereas human feces and sewage isolates cultured on LST medium
yielded the lowest growth density.
Fig. 3 shows that at 44.5°C, human isolates and sewage isolates in EC
medium gave the highest growth density, whereas in LST medium
human feces and sewage isolates gave the lowest growth density.
As shown in Figs. 4 to 7 the effect of the different temperatures on the
growth of all isolates of E. coli shows that the temperature degree
37°C gave the best growth density for the isolates of E. coli obtained
from animal feces and sewage enumerated on all growth media used
in this experiment. Incubation at 30°C gave the best or the same
growth density for isolates of E. coli obtained from water or human
feces, whereas 44.5°C showed a weak growth for all isolates on
different growth media.
Results obtained in this experiment show that E. coli isolates gave best
growth densities at 37°C, a temperature of incubation recommended
by WHO (1995). E. coli is a typical mesophile growing from 7°C-
10°C up to 50°C with an optimum around 37°C. According to Bergey’s
165
University of Africa Journal of Sciences
Manual (Krieg, 1984) and (Colle et al., 1996), the optimal temperature
of E. coli is 36°C-37°C, though growth occurs over a fairly wide
temperature range (18- 44°C).
The growth rate of E. coli at 30°C was less than that at 37°C, whereas
44.5°C gave the lowest growth rate. This may be due to E. coli being a
mesophilic bacterium or due to the tropical climate of Sudan in which
temperature degrees are higher than that in Western countries. Similar
results were reported by Leroi et al. (1994) although 32°C and 42°C
are symmetric around the temperature 37°C at which E. coli had been
propagated, relative to 37°C, 42°C reduces both maximal growth rate
and yield, whereas 32°C reduces only the former. The temperature
42°C is approximately 0.5°C off the critical thermal limit for extinction
of the bacterial population under standard culture conditions, whereas
32°C is more than 10°C away from both the upper and lower thermal
limits. Whereas 42°C induces expression of stress proteins that
mediate a protective response, 32°C does not. Raina et al. (1995)
reported that in E. coli the classical heat shock response appears as a
transient acceleration in the synthesis of 20 proteins.
APHA (1937) found that greater numbers of E. coli were detected
with lactose broth at 37°C over the Eijkman medium at 46°C.
166
University of Africa Journal of Sciences
Fig. 1 The effect of culture medium on the growth of E. coli isolated from different sources at 30°C
0
0.5
1
1.5
2
SA SH SS SW
Optical density
Dif ferent sources of E. coli
LST EC Mac BGB
SA: samples obtained from animals feces SH: samples obtained from human feces Ss: samples obtained from sewage Sw: samples obtained from water
167
University of Africa Journal of Sciences
Fig. 2 The effect of culture medium on the growth of E. coli isolated from different sources at 37°C
0
0.5
1
1.5
2
SA SH SS SW
Optical density
Different sources of E. coli
LST EC Mac BGB
168
University of Africa Journal of Sciences
Fig 3 The effect of culture medium on the growth of E. coli isolated from different sources at 44.5°C
0
0.5
1
1.5
2
SA SH SS SW
Optical density
Dif ferent sources of E. coliLST EC Mac BGB
Fig. 4 The effect of incubation temperature on the growth of E. coli isolated from water sources
1.61.611.621.631.641.651.661.671.681.691.7
1.711.721.73
30C 37C 44.5C
Optical density
Temperature
Fig. 5 The effect of incubation temperature on the growth of E. coli isolated from sewage
169
University of Africa Journal of Sciences
1.6
1.61
1.62
1.63
1.64
1.65
1.66
1.67
1.68
1.69
1.7
1.71
30C 37C 44.5C
Optical density
Temperature
Fig. 6 The effect of incubation temperature on the growth of E. coli
isolated from human feces
1.61.611.621.631.641.651.661.671.681.69
1.71.71
30C 37C 44.5C
optical density
Temperature
170
University of Africa Journal of Sciences
Fig. 7 The effect of incubation temperature on the growth of E. coli isolated form the animal feces
171
University of Africa Journal of Sciences
CONCLUSION
The results obtained from this research show that E. coli can be used
as fecal indicator of water contamination in Sudan. But some
reservations should be taken into consideration:
- EC medium gave best growth density of E. coli than Mac, LST
and BGB.
- The biochemical tests differentiated E. coli isolates into five
groups.
- Some isolates of E. coli does not produce indole also some were
not motile and others do not produce gas from glucose
fermentation.
- Indole production in E. coli is linked to motility.
- Growth of E. coli at 44.5˚C is very weak.
172
University of Africa Journal of Sciences
REFERENCES
Adams, M.R. and Moss, M.O. (2000). Food Microbiology, 2nd
Edition, Royal Society of Chemistry, Cambridge.
APHA (1937) American Public Health Association Part III. Am. J
Public Health; 27(3 Suppl): 114– 163.
Black, L.A. and Klinger, M.E. (1936). A Comparison of media for the
detection of Escherichia-Aerobacter. J Bacteriol.; 31(2): 171– 179.
Clark, J.A. and Pagel, J.E. (1977). Pollution indicator bacteria
associated with municipal raw and drinking water supplies, Canadian
Journal of Microbiology, 23: 456- 470.
Colle, J.G., Duguid, J.P., Fraser, A.G. and Marmion, B.P. (1996).
Practical Medical Microbiology, Second edition, Makie &
McCartney’s Churchill Living stone, London.
Farmer, J.J.; Davis, B.R.; Hickman-Brenner, F.W.; McWhorter, A.;
Huntley-Carter, G.P.; Asbury, M.A.; Riddle, C.; Wathen-Grady, H.G.;
Elias, C. and Fanning, G.R. (1985). Biochemical identification of new
species and biogroups of Enterobacteriaceae isolated from clinical
specimens. J Clin Microbiol. 21(1): 46–76.
Geldreich, E.E. (1975). Handbook for Evaluating Water
Bacteriological Laboratories, 2nd ed., Municipal Environmental
Research
Grabow, W.O.K., and Du Preez, M. (1979). Comparison of m-Endo
LES, macConkey, and teepol media for membrane filtration counting
173
University of Africa Journal of Sciences
of total coliform bacteria in water. Appl. and Environ. Microbiol, 38
(3): 351-358.
Hajna, A.A. and Perry, C.A. (1943). Comparative study of
presumptive and confirmative media for bacteria of the coliform
group and for fecal streptococci, American Journal of Public Health,
33: 550- 556.
Jay, J.M. (1986). Modern Food Microbiology, 3rd ed., Van Nostrand
Reinhold, New York.
Jay, J.M. (1992). Modern Food Microbiology, 4th ed., Van Nostrand
Reinhold, New York.
Jimenez, L.; Muniz, I.; Toranzos, G.A. and Hazen, T.C. (1989).
Survival and activity of Salmonella typhimurium and Escherichia coli
in tropical freshwater. J Appl Bacteriol.; 67(1):61- 69.
Krieg, N.R. (1984). Bergey’s Manual of Systematic Bacteriology,
Vol. 1, Williams & Wilkins, U.S.A.
Leroi, A.M.; Bennett, A.F. and Lenski, R.E (1994). Temperature
acclimation and competitive fitness: an experimental test of the
beneficial acclimation assumption. Proc Nat. Acad. Sci. U S A., 91(5):
1917–1921.
Madigan, M.T.; Martinko, J.M. and Parker, J. (1997). Brock Biology
of Microorganisms. 8th Edition. Prentice Hall, London.
Meadows, P.S.; Anderson, J.G.; Patel, K. and Mullins, B.W. (1990).
Variability in gas production by Escherichia coli in enrichment media
174
University of Africa Journal of Sciences
and its relationship to pH. Appl. and Environ. Microbiol, 40 (2), 309-
312.
Perez-Rosas, N. and Hazen, T.C. (1989). In situ survival of Vibrio
cholerae and Escherichia coli in a tropical rain forest watershed. Appl
Environ Microbiology 55(2):495- 499.
Raina, S.; Missiakas, D. and Georgopoulos, C. (1995). The rpoE gene
encoding the sigma E (sigma 24) heat shock sigma factor of
Escherichia coli. EMBO J. 14(5): 1043–1055.
Tortora, G.J.; Funke, B.R. and Case, C. L. (1992). Microbiology, an
introduction. 4th Edition. The Benjamin Cummings Publishing
Company, New York.
WHO (1964). Operation and control of water treatment processes, No.
49. Geneva.
WHO, (1995). Guidelines for Drinking- Water Quality, Second
edition, 1, Recommendations, Regional Center for Environmental
Health Activities (CEHA), Amman, Jordan.