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


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


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


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


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


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


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


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


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


(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


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


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


Table 2: The confirmatory biochemical tests of E. coli isolates obtained from human feces (first batch)

159


Table 3: The confirmatory biochemical tests of E. coli isolates obtained from human feces (second batch)

160


Table 4: The confirmatory biochemical tests of E. coli isolates obtained from different sources of water

161


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


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


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


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


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


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


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


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


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


Fig. 7 The effect of incubation temperature on the growth of E. coli isolated form the animal feces

171


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


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


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


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.

biochemical and physiological characteristics of...

Documents