ISSN: 2278-778X Research Article www.ijbio.

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A NEW MEDIUM FOR SCREENING OF GRAM NEGATIVE BACTERIA IN PORTABLE WATER

Mohamed Younes Abdelftah Hassan1, Omar Abdelateef Omar Saad2, Magda Hassan Mahran3, Ahmed Ismail Madkour4, Suliman S Abdelrawaf5 and Idress Hamad Attitalla*5,6 1 Minia Company for Drinking Water and Sanitation, Minia, Egypt 2 Department of Agricultural Microbiology, Faculty of Agriculture, Minia University, Egypt. 3 Assalam International Hospital, Egypt 4 Ophthalmology Research Institute, Egypt 5 Department of Microbiology, Faculty of Science, Omar Al-Mukhatr University, Box 919, Al-Bayda, Libya 6 Agriculture Research Centre (ARC), Libya *Corresponding Author: Dr. Idress Hamad Attitalla, Professor, Department of Microbiology, Faculty of Science, Omar Al-Mukhatr University, Box 919, Al-Bayda, Libya Received for publication: October 22, 2012; Accepted: January 03, 2013. Abstract: A series of laboratory experiments were performed to screen the presence of gram negative bacteria (coliform group) in drinking water to determine its suitability for human consumption and in clinical, pathological, and ophthalmology purpose. A new medium (Makka medium) with methylene blue as an indicator which inhibit Gram positive bacteria was developed, evaluated and used in the present study. Composition of this medium doesnt contain lactose found as in m-Endo agar medium. Generally lactose is used by the bacteria during fermentation process but in Makka medium fermentation occurred in the absence of lactose which was recorded as the change in the color of the medium due to pH drift and Dipotassium phosphate dibasic serves as buffer and enhance organisms growth (coliform group). In comparison with the commercially available synthetic media, Makka medium is unique in composition, economical, safe and easy to use for detection of contamination of drinking water with coliforms. Keywords: Coliform, Gram Negative Bacteria, Makka Medium, M-Endo Agar, Portable Water

INTRODUCTION
Coliforms such as Escherichia coli, E. aurescens, E. freundii, E. intermedia; Aerobacter, Aerogenes and A. cloacae are aerobes and facultative anaerobic which are classified as Gram-negative, non-spore-forming, rod shaped bacteria which ferment lactose and produce gas within 48 hours at 35C. This group has been used as indicator organisms in most of the water testing laboratories. An indicator organism, by itself is considered to cause no diseases in man or animals, but its presence usually indicates the presence of pathogenic or disease-causing organisms. Approved traditional methods for coliform detection include the multiple-tube fermentation (MTF) technique and the membrane filter (MF) technique using different specific media and incubation conditions. However these methods have their own limitations, such as duration of incubation, antagonistic organism interference, lack of specificity and poor detection of slow growing or viable but non-culturable (VBNC) microorganisms. Nowadays, the simple and inexpensive membrane filter technique is the most widely used method for routine enumeration of coliforms in drinking water. The membrane filter technique (MF) is used for the counting of coliforms. The filtration process is designed in such a way as to retain coliform bacteria present in the sample onto a membrane filter. The entire filter with the retained bacteria is placed on a specially

prepared medium in a Petri dish. The Petri dish and its content are then incubated for 24 hours at 35C. At the end of the incubation period, the filter is examined with a 10-15X stereoscopic optical device. All blue colored colonies on the filter will then be counted and each blue colored colony is assumed to be the result of one faecal coliform originally in the sample. From the colony counting procedure and the sample volume filtered, it is possible to calculate the number of faecal coliforms/ 100 mL present in the sample to be tested (Mc fetrsetal, 1986; Apha, 1998). Eosin Methylene Blue (EMB, also known as "Levine's formulation") is used for testing clinical materials, food, and dairy products; it is a blend of two stains, eosin and methylene blue in the ratio of 6:1. A common application of this stain is in the preparation of EMB agar, a differential microbiological medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., E. coli") and those that do not (e.g., Salmonella, Shigella). Organisms that ferment lactose display nucleated colonies" colonies with dark center (Differential Media (Levine's Formulation)) and E. coli will give distinctive metallic green sheen (due to the metachromatic properties of the dyes.

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This medium is important in medical laboratories for distinguishing pathogenic microbes in a short period of time (Bachoon et al., 2008) (Cunnif, 1995; Murtay et al, 1995). This medium is primarily used for detection and confirmation of coliforms. In the membrane filter technique, them-Endo Agar medium is usually used for detection of coliforms. In addition to the high cost of this medium it is reported that this medium may cause cancer if used without precautions (Enne de Boer (2004)). Furthermore, some components in this medium irritate eyes, skin, and respiratory system. Hence, developing an economical safe alternative medium for such purpose is a challenge. The present study aims at finding a new economical and safer method for detection of coliform group in portable water.

1and4). But in m-Endo agar medium, coliforms form pink to red colonies and some with metallic sheen were observed (Figure 5). The first medium is selective in nature due to the presence of methylene blue, which inhibits the growth of Gram +ve bacteria but allowing the growth of Gram ve ones. It is different in that lactose fermenting bacteria give colored colonies due to the formation of complex in this method. Differential media or indicator media distinguish one microorganism type from another growing on the same media Washington (1996). This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients as indicators (such as neutral red, phenol red, eosin y, or methylene blue) which are added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.

MATERIALS AND METHODS

High levels of environmental contamination, often associated with improper waste and excreta management, are widespread among informal settlements within urban areas in developing countries. We determined the level of fecal contamination in domestic water sources collected from parts of Egypt and Libya, evaluated the potential contribution of these water sources to coliforms using an economical and indigenous media (Makka media) developed in our lab. Membrane filtration technique was used for enumeration of total and fecal (Escherichia coli) coliform bacteria in water samples collected from different municipal sources, which is used for drinking and clinical purposes. A comparative study was conducted to check if the Makka media developed is on par with the existing commercially media (m-Endo Agar) for the detection of Gram negatives especially coliforms. i. Sample Collection: The samples chosen for the study were randomly selected from different locations across Libya and Egypt during a 2-year period and checked for routine total coliform analysis. All samples were collected by accepted procedures Clinical and Laboratory Standards Institute guidelines (CLSI)) and shipped to the laboratory at ambient temperatures. Samples from municipal water supplies were tested by the membrane filter total coliform technique. Isolation of the Microbes Biochemical characterization Growth on Makkah medium and m-Endo Agar Colony characteristics

DISCUSSION
In the 1stexperiment we used Endo agar medium to confirm the presence of coliform group in the sample. The coliform group appears with metallic sheen. This result agrees with Enne de Boer (2004) who reported that, E. coli also has a characteristic color (red with green metallic sheen) on Endo agar. In the 2ndexperiment we used methylene blue and applied on this medium to see the effect on colony numbers and characteristics. The coliform group appear with dark green to pale green due to consumption of methylene blue. In the 3rdexperiment we compare the two media through streaking on filter disc. Data shown in Figs.2 and 3 indicated that, the growth of the coliform group was identical on both media in the 4th experiment we compare both the media through spread culture technique. These results (figures 6 and 7) showed that, the efficiency of new medium (Makka medium) enhance coliform group growth as Endos agar medium. Finally we cultivated typical microorganisms and compared biochemical tests to determine the ability of Makka medium towards its selection of gram negative bacteria. Generally there is no need to sterilize media for coliforms. Pasteurization of these media results in sufficient decontamination, as surviving spore-forming bacteria will not be able to develop in media with bile salts and dyes. Moreover sterilization may inactivate some of the inhibiting substances, making the media less selective Enne de Boer (2004). These results showed that Makka medium is recommended to be used instead of M-Endo agar medium for detection of coliforms and gram negative

ii. iii. iv. v.

RESULTS
On Makka medium the coliforms appear blue green color and some have dark green color (Figure

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bacteria. To conclude we demonstrated that Makka medium is equally good in comparison with Eosin methylene blue agar, Levine used for the isolation and differentiation of Gram-negative enteric bacilli Madigan and Martnko (2005), however makka medium showed the following benefits over EMB agar.
An easy method Its components' are locally available which are safe and economical. It is also guarantees occupational health and safety. It is no toxic hence can be considered a best alternative for EMB agar.

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Koneman EW, SD Allen, VR Dowell, Jr., WM Janda, HM Sommers and WC Winn, Jr. (eds) 1988. Color Atlas and Textbook of Diagnostic Microbiology. (4th ed). American Society for Microbiology, Washington. Martin WJ and JA Washington II, 1985, Enterobacteriaceae, Chap. 16, IN: Lennette, E.H., et.al. (eds) Manual of Clinical Microbiology, (4th ed) American Society for Microbiology, Washington. Edwards PR and WH Ewing, 1972, Identification of Enterobacteriaceae. (3rd ed) Burgess Publishing, Philadelphia. Kaplan RL, 1985, Campylobacter, Chap. 19. IN: Lennette, E.H., et.al. (eds) Manual of Clinical Microbiology, (4th ed) American Society for Microbiology, Washington. Kaplan RL and Barrett JE, 1981, Monograph: Campylobacter. Anon, 1987, Policy and Procedure Manual for Inpatient and Outpatient Care in Laboratory Department. Naval Medical Center, Portsmouth, VA. Shatter JG and M Goldin, 1974, Medical Microbiology. Chapt. 18, IN: Davidson, I., and J.B. Henry. (eds) Todd-Sanford Clinical Diagnosis by Laboratory Method (15th ed) W.B. Saunders, Philadelphia. Sonnenwirth AC, 1970, Collection of Culture Specimens and Guide for Bacterial Identification. Chap. 63. IN: Frankel, S., et.al (ed) Gradwohls Clinical Laboratory Methods and Diagnosis, (17th ed) C.V. Mosby, St. Louis, MO.

13.

14.

15.

REFERENCES
1. Apha AW, 1998, Standard methods for the examination of water and wastewater, 20th Edition, 21st Edition (2005).American public Health Association, Washington, DC, USA. Cunnif P, 1995, Official Methods of Analysis AOAC International, 16th ed. AOAC International, Gaithersburg, MD. 18. 3. Enne de Boer, 2004, Entero bacteriaceae, coliforms and E. coli. Classical and Modern Methods for Detection/Enumeration. Inspectorate for Health Protection, Zutphen, The Netherlands Available online 17 November P. 610-617. 19. 4. Madigan M and Martinko J, (editors). 2005. Brock Biology of Microorganisms (11th ed.ed.). Prentice Hall. ISBN 0131443291. MacFaddin JF, Biochemical tests for identification of medical bacteria. 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2000. Mcfetrs GA, Kippin IS and Lechevaller MW, 1986, Injured coliforms in drinking water. APPL. Environ. microbiol.51:1. Murtay PR, Baron EJ, Pfaller MA, Tenover FC and Yolken RH, 1995, Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C. 22. 8. Washington JA, 1996, Principles of Diagonsis. Baron s Medical microbiology (Baron S el al., eds) (4th ed. Ed.) Univ. of Texas Medical Branch. ISBN O- 9631172-1-1. 23. 9. Isenberg HD and JA, Washington II, A Balows and AC Sonnenwirth, 1985, Collection, Handling and Processing of Specimens. Chap. 6. In: Lennette, EH, et.al. (eds) Manual of Clinical Microbiology, (4th ed) American Society for Microbiology, Washington. Isenburg HD and BG Painter, 1985, Indigenous and Pathogenic Microorganisms of Humans. Chap. 4. IN: Lennette, EH, et.al. (eds) Manual of Clinical Microbiology, (4th ed) American Society for Microbiology, Washington. Rosenberg T, 1961, Microorganisms Indigenous to Man. McGraw-Hill, New York. 16. 17.

2.

5.

20. Head CV, DA Whitty and S Ratma, 1982, Comparative Study of Selective Media for the Recovery of Yersinia enterocolitica. J. Clin. Micro, 16 (4) 615-621. 21. Gunn BA, JF Keiser and RD Alazan, 1987, Culture Media Tests and Reagents in Bacteriology, pp. 849. IN: Howard, B.J., et.al. (eds) Clinical and Pathogenic Microbiology. C.V. Mosby, St.Louis, MO. National Committee for Clinical Laboratory Standards (2000) Performance Standards for Antimicrobial Disk Susceptibility Tests. 4th Edn. Approved Standard M2-A7. NCCLS. Villanova Pa. Pollock HM, et al. (1986) J. Clin. Microbiol. 24. 1-6.

6.

7.

24. WHO, 1961, Standardization of Methods for Conducting Microbic Sensitivity Tests. Geneva. Tech. Rep. Ser. No.210. 25. WHO, 1977, Expert Committee on Biological Standardization. Geneva. Tech. Rep. Ser. No.610.

10.

26. Barry AL and Effinger LJ, 1974, Amer. J. Clin. Path. 62. 113-117. 27. Reller LB, Schoenknecht FD, Kenny MA and Sherris JC, 1974, J. Infect. Dis. 130. 454-463.

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Source of support: Nil Conflict of interest: None Declared

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