ENUMERATION OF SULPHITE-REDUCING CLOSTRIDIA FROM DRINKING WATER AND RIVERS IN BASRAH CITY USING MODIFIED DRCM MEDIUM
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1 J.Basrah Researches (Sciences) Vol.. No SEP. (2007) ENUMERATION OF SULPHITE-REDUCING CLOSTRIDIA FROM DRINKING WATER AND RIVERS IN BASRAH CITY USING MODIFIED DRCM MEDIUM Asaad M. Ridha Al-Taee Marine Science Centre Basrah University ISSN ( Received 4//2006, Accepted /7/2007 ) ABSTRACT The enumeration of sulphite-reducing clostridia from water upon modified DRCM was evaluated against DRCM and RCM using 80 of treated drinking waters and river waters with or without heat treatment of the. The recoveries of presumptive sulphite-reducing clostridia were significantly higher on modified DRCM (8.7% on drinking water and 8.2% on rivers), while 48.7% and 28.% respectively on DRCM, and.2% and 2.4% on RCM. Heat treatment of to 70 C for minutes prior to filtration reduced both clostridia and background on modified DRCM but did not affect counts on DRCM. Four species were identified as Clostridium perfringens, C. baratii, C. ramosum, and C. difficile. Key Words: RCM, DRCM,, Clostridium INTRODUCTION The sulphite-reducing clostridia are normal inhabitants of the intestinal microbiota of humans and other mammals. These microorganisms form endospores, which allow the bacteria to survive in almost any habitat, either terrestrial or aquatic, waiting for favorable conditions for growth []. The enumeration of sulphite-reducing clostridia is used in assessing drinking water quality in Europe and UK. [2-8]. Some researchers proposed to use several types of media, which include TSC, MCA and m-cp for recovery of C. perfringens and other sulphite-reducing clostridia from water and shellfish [9-2]. In this study the reinforced clostridia agar (RCM) and differential reinforced clostridia agar (DRCM) failed in isolating the species of clostridia, so a new medium was introduced and compared with RCM and DRCM with and without sample pretreatment at 70 C for the enumeration of sulphite-reducing clostridia from rivers and drinking water. MATERIALS AND METHODS Samples Field A total of 80 were collected from drinking water (60 ), Ashar river ( ) and Garma river ( ). The were collected in sterile 20 ml. Nalgene polycarbonate conical flasks to which a solution of 2.% (W/V) sodium thiosulphate was added as a reducing agent for any residual chlorine (). Enumeration Media Reinforced clostridium medium and DRCM were prepared according to the manufactures instructions and the new medium, which is called modified DRCM () (Table ).
2 Asaad M. Ridha Al-Taee Table : Comparison between RCM, DRCM, and media. Components RCM(Oxoid) DRCM[4] Meet extract 9 Yeast extract 2. Peptone 7. D-Glucose Starch Sodium acetate 4 Sodium chloride "Lab lemco" powder L- cystein-hcl Sodium sulphite 0.0 Ammonium ferric 0.04 (III)citrate Resazurine Na 0.02 Agar Polymyxin B 0.02 The media were autoclaved at. bar for min. A polymyxin was added to the after sterilized by filtration. Analytical Procedures All were filtered in duplicate through Millipore WCN type membrane filters size 0.4µ (Whatman Corp. Japan). One hundred and fifty milliliters were filtered for each medium, with and without sample heat treatment at 70 C for min. The plates were incubated anaerobically in gas jar using either Oxoid anaerobic gas generating kits (Code No. BR 8) or Al-Razi anaerobic gas generating kits (B. No. 47) for 48 h. at 7 C. All black colonies were counted as presumptive sulphite-reducing clostridia. They were subjected to Gram staining and test for motility, nitrate reduction, gelatin liquification and fermentation of lactose, mannose, galactose, mannitol, sorbitol, raffinose, salicin and arabinose. Further more they were identified using API 20 A strips (BioMerrieux). RESULTS From Table (2) the isolation of presumptive sulphite-reducing clostridia on was higher than RCM and DRCM for all types of water analyzed. The counts of presumptive sulphite-reducing clostridia on were higher using unheated (Table ). Heating of the to 70C for min. reduced the counts of sulphite-reducing clostridia and the background counts. Both procedures produced higher counts than DRCM and RCM. 69
3 ENUMERATION OF SULPHITE-REDUCING CLOSTRIDIA Twenty isolates from and from each DRCM and RCM were examined by Gram staining and subjected to biochemical tests and API 20 A strips, only four species were identified as Clostridium perfringens (8 from, 2 from RCM and 2 from DRCM), C. ramosum ( from, from RCM and 2 from DRCM), C. baratii (2 from m- DRCM, from RCM and from DRCM) and finally C. difficile (4 from and from DRCM). The remaining one isolate from, four from RCM and four from DRCM could not be identified to species level. DISCUSSION In this study two media have been used for isolating clostridia from drinking water and rivers. RCM is based on a basic nutrient medium developed in the 90s []. The use of this medium resulted in non-selective growth of other bacteria (Table 2). Sodium sulphite and ferric citrate are added to RCM to become differential RCM which has been recommended for the detection of sulphite-reducing clostridia in water [4].These two substrates were used as indicator of sulphite reduction by Clostridium which produces black colonies. However, also this medium failed to decrease the background bacteria in comparison with the new medium m- DRCM which was shown high selectivity for sulphitereducing clostridia and this may be related to that, the increasing of these substrates (sodium sulphite and ferric citrate) which are incorporated with polymyxin B provident a high degree of selectivity and specificity for these bacteria. This medium also contains starch to promote spore germination and resazurine as a redox indicator. Some researchers [9, 2] have demonstrated that, the incubation temperature of 4 C appears to be selective for sulphite-reducing clostridia of fecal origin. But in this study the incubation temperature of 7 C was found more selective and specific for these bacteria. In this study the were heated at 70 C for min. to kill vegetative cells and enhance spore germination. From Table () heat treatment affects the counts of sulphitereducing clostridia on but does not affect the counts on DRCM this may be related to that the components of the have some inhibitors which may not enhance the spore germination and this agreed with Warnes and Keevil (2004) who found that the conventional methods for the detection of sulphite-reducing clostridia have incorporated heat killing of vegetative cells of clostridia and contaminating bacteria to identify the presence or quantify the clostridial spores present. This is followed by the use of rich medium to promote spore germination. We conclude from our study that the incubation of without heat treatment on m- DRCM at 7 C for 48 h. may be the best medium for the enumeration of sulphite-reducing clostridia from drinking water and rivers. Table 2: The percentage of presumptive sulphite-reducing clostridia on RCM, DRCM and m- DRCM. Count per 0 ml upon RCM DRCM Sample source No. of sample Ashar river Garma river Drinking water 70
4 Asaad M. Ridha Al-Taee Mean (80) Range Standard deviation Table : Impact of heat treatment of on recovery of sulphite-reducing clostridia on RCM, DRCM and. Count per 0 ml upon RCM DRCM Not heated Heated Not heated Heated Not heated Heated REFERENCES -S. Robles; J. M. Rodrigues; I. Granados; and M. C. Guerrero. Sulphitereducing clostridia in the sediment of a high mountain lake (Laguna Grands,Gredos, Spain) as indicators of fecal pollution. Internat. Microbiol. : (2000). 2- DOE (Department Of the Environment). The bacteriological examination of drinking water supplies. Reports on public health and medical subjects No. 7. HMSO. London. (98). - J. R. Postgate. The Sulphate-Reducing Bacteria. 2 ed. Cambridge University Press, Cambridge. (984). 4- K. M. Emde; D. W. Smith and R. Facey. Initial investigation of Microbially Influenced Corrosion (MIC) in a low temperature water distribution system. Water Res. 26: (992). - D. P. Sartory; A. M. Pritchard and P. Holmes. Enumeration of sulphite-reducing clostridia from potable water supplies. Wat. Sci. Tech. 27: (99). 6- J. M. Odom and Jr. R. Singleton. The Sulphate-Reducing Bacteria: Contemporary Perspective. Springer Verlag New York, USA. (99). 7- P. W. Adcock and C. P. Saint. Rapid confirmation of Clostridium perfringens by using chromogenic and fluorogenic substrates. Appl. Environ. Microbiol. 67: (200). 8- Environment Agency. The microbiology of drinking water Part 6 Methods for the isolation and enumeration of sulphite-reducing clostridia and Clostridium perfringens by membrane filtration. Methods for the Examination of Water and Associated Materials. UK. (2002). 9- J. S. Burger; E. M. Nupen and W. O. K. Grabow. Evaluation of four growth media for membrane filtration counting of Clostridium perfringens in water. Water SA. : (984). - D. P. Sartory. Membrane filtration enumeration of faecal clostridia and Clostridium perfringens in water. Water Res. 20: (986). - T.J. Easterbrook. and P. A. West. Comparison of most probable number and pour plate procedure for isolation and enumeration of sulphite-reducing Clostridium spores and Group D fecal streptococci from oysters. J. Appl. Bacteriol. 62: (987). 2- D. P. Sartory. Evaluation of two media for the membrane filtration enumeration of Clostridium perfringens from water. Lett. Appl. Microbiol. 27: (998). - APHA. Standard Methods for the Examination of Water and Wastewater. 7 th ed. American Public Health Association. Washington D. C. (989). 4- ISO Detection and enumeration of the spores of sulphite-reducing anaerobes (clostridia) Part 2. Method by membrane filtration. (986). - Sara L. Warnes and C. W. Keevil. Work package, Task 4: Desk studies on feasibility of horizontal standard rapid methods for the detection of Clostridium perfringens and enterococci in sludges, soil, soil improvers, growing media and biowastes. P: -0. University of Southampton. (2004). 7
5 ENUMERATION OF SULPHITE-REDUCING CLOSTRIDIA الكشف عن بكتريا الكلوستريديا المختزلة للكبريتيت باستخدام وسط من مياه الشرب ومياه الانهار أسعد محمد رضا الطاي ي الخلاصة استخدم الوسط المحور كوسط جديد وقورن مع الاوساط التقليدية DRCM و RCM للكشف عن بكتريا الكلوستريديا المختزلة للكبريتيت من مياه الشرب ومياه نهري العشار والكرمة في مدينة البصرة. أجريت الد ارسة على 80 عينة من المياه قبل معاملتها بالح اررة بدرجة 70 م وبعدها ولمدة دقاي ق أظهر العد الافت ارضي لبكتريا الكلوستريديا المختزلة للكبريتيت على الوسط % 8.7 لمياه الشرب و % 8.2 لمياه الانهر في حين % 48.7 و % 28. على الوسط DRCM و %.2 و 2.4 % على الوسط RCM على التوالي. انخفض عدد بكتريا الكلوستريديا المختزلة للكبريتيت على الوسط المحور عند معاملة العينات ح ارريا" في حين لم يتا ثر على الوسط. DRCM أظهر التشخيص النهاي ي باستخدام الاختبا ارت البيوكيمياي ية وأ شرطة الاختبا ارت السريعة API 20 A أربعة أنواع من الكلوستريديا وهي Clostridium perfringens و.C baratii و. Cو ramosum.c. difficile 72
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