Biochemical Characterisation of Microbial Isolates from Berger Area Abattoir at Lagos/ O gun State Boundary Nigeria

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1 2013; 1 (1): ISSN xxxxxxxxx EJBB 2013; 1 (1): AkiNik Publications Received Accepted: Biochemical Characterisation of Microbial Isolates from Berger Area Abattoir at Lagos/ O gun State Boundary Nigeria Adeyinka A *, Fagade O.E, Ogunjobi A.A. ABSTRACT Adeyinka A Fagade O.E Ogunjobi A.A Correspondence: Adeyinka, A joseph_adeyinka35@yahoo.co.uk Abattoir samples (water, sediment and soil) were collected and analysed. Thirty bacteria isolates were obtained from samples collected and analysed. Various biochemical tests were carried out on the isolates, the probable identity of each genera include: Staphylococcus was gram positive cocci in clusters, catalase positive, oxidase negative and non- motile. was gram positive in chains, catalase negative and motile. Bacillus, was gram positive rod with terminal spores, catalase positive and hydrolsed starch. Proteus was gram negative rod, methyl red positive, voges-proskauer negative and unable to utilized mannitol sugar to produce acid and gas. Citrobacter was methyl red positive, voges-proskauer negative, indole negative and citrate positive. Enterobacter was gram negative rod, methyl red negative, voges-proskauer positive and indole negative. Escherichia coli was gram negative rod, methyl red positive, voges-proskauer negative and indole positive. Samonella was gram negative rod, methyl red positive, Vogesproskauer negative, citrate positive and utilized mannitol sugar with acid and gas production. The probable identity of the isolates are Staphylococcus,, Bacillus, Proteus, Citrobacter,Enterobacter, Escherichia coli and Samonella with reference to Bergey s manual of determinative bacteriology. Keywords: Abattoir samples, Bacteria Isolates, Biochemical characterisation, Media Sterilisation, Incubation. 1. Introduction An abattoir has been defined as a premise approved and registered by the controlling authority for hygienic slaughtering and inspection of animals, processing and effective preservation and storage of meat products for human consumption [1]. Abattoir waste can be defined as waste or waste water from an abattoir which could consist of the pollutants such as animal faeces, blood, fat, animal trimmings, paunch content and urine [2]. The different sources of waste (solid and liquid) in red meat abattoirs could be categorized as animal pens; bleeding, carcass processing, offal and by-products processes and processing. Slaughtering of animals result in meat supply and useful by-products like leather and skin, livestock waste spills can introduce enteric pathogens and excess nutrients into surface waters and can also contaminate ground waters [3]. These leachates consist largely of solids, microbial organisms and in special situations chemicals and shallow wells like hand-dug wells are more dangerously polluted [4]. Abattoir operations produce a characteristic highly organic waste with relatively high levels of suspended solid, liquid and fat. The solid waste includes condemned meat, undigested ingesta, bones, horns, hairs and aborted foetuses. The liquid waste is usually composed of dissolved solids, blood, gut contents, urine and water. Animal food is always microbiologically contaminated by organisms living in it naturally or entering it from the surrounding, such as those resulting from processing operations [5]. This research analysis looks at biochemical characterisation of bacterial isolates from water, soil and sediment samples of Berger Area abattoir situated at Lagos/ Ogun state boundary ~ 20 ~

2 2. Materials and Methods Standard methods were used throughout the course of this analysis 3. Description of the Study Area The study area is located in Isheri- Olofin after Berger bus stop in Lagos/ Ogun state boundary along Lagos Ibadan express way. The slaughter area consist of slab, which is about 10m 2 surface area with two major drains that lead directly into Ogun river. Liquid waste like blood, urine, waste water and gut content flow directly untreated into the river. Also solid wastes like dead carcass, trimmed meat, animal dung, undigested ingesta and aborted foetus also find their way into Ogun River. About cattles are slaughtered on daily basis. 4. Sampling Area and Design Sampling was carried out on 3 rd November 2008 and 29 th November 2008 respectively. Samples collected include water, sediment and soil. The river course was divided into three sections namely, upstream, midstream and downstream. Nine sampling points were chosen along Ogun river course for water and sediment samples (3 points from each section of the river). The distance between two consecutive sampling points was fixed for 20meters; hence the study area covered about 200 meters. Also nine sampling points were chosen for soil samples collection on the basis of river course division (Three points per river s section). 5. Samples Collection Water samples were collected by carefully opening sterilized McCartney bottle and lowered into the river from the canoe at the point of collection and covered immediately. Sub-surface water collection was made at three different points at about 20meters interval per each section of the river. Water samples were appropriately labeled. Upstream water were labeled UHO 1, 2 & 3, while midstream water labeled MHO 1,2 &3 and downstream water labeled DHO 1,2,3 with the aid of marker. Sediment samples were collected with vanveen grab. The grab was lowered into the bottom of the river from canoe at three different locations per each section of the river. Three samples collected were mixed together to form a composite sample per section of the river to make three samples in all. Sediment samples were labeled USE for upstream sediment, MSE for midstream sediment and DSE for downstream sediment. Soil samples were collected with the aid of surgical rubber gloves at three different locations per section of the river. Three samples collected were mixed together to form a composite sample per river section to make three samples in all. Soil samples were labeled USO for upstream soil, MSO for midstream soil and DSO for downstream soil. 6. Handling and Transportation of Samples Samples collected were immediately stored and presevered in coolers containing ice crystals and conveyed down to the Laboratory from the sampling site the same day. On reaching the Laboratory, samples were kept in the refrigerator at < 4 0 C prior laboratory analysis. 7. Microbiological Analysis 7.1 Sterilization of culture media All culture media used were prepared according to the manufacturer s instructions and then sterilized at 121 o C for 15 minutes. Sugars solutions were autoclaved at 115 o C for 10 minutes. The media used were Nutrient Agar, MacConkey agar, Eosine methylene agar, and Salmonella Shigella agar. The total heterotrophic bacteria count of each sample was determined using Nutrient agar. MacConkey agar was used for Coliform detection. Eosine methylene agar was used for Escherichia coli and Samonella Shigella agar was used for detection of Samonella. 7.2 Sterilization of glassware All glassware used: Petri- dishes, beakers and conical flasks were sterilized in an hot air oven at c for 1hour. 7.3 Isolation of Bacteria from water sample Sterile pipette was used to transfer 1ml of each water sample, into 9ml of sterile distilled water, shaken properly to obtain 10-1 dilution. Further serial dilutions were similarly carried out to obtain dilution of up to 10-4 for higher dilutions. 1ml was taken from dilution (10-2 and 10-4 ) and plated in duplicate using Nutrient Agar, MacConkey agar, Eosine methylene agar, and Salmonella Shigella agar using pour plates techniques. The plates were swirled and allowed to solidify. All inoculated plates were incubated at 37 0 C for 24 hours. 7.4 Isolation of Bacteria from sediment and soil sample One pooled sample per river s section was analyzed. 1g each of soil and sediment sample was weighed respectively into 9ml of sterile distilled water, shaken properly to obtain 10-1 dilution. Further serial dilutions were similarly carried out to obtain dilution of up to 10-5 for higher dilutions. 1ml was taken from dilution (10-3 and 10-5 ) each for sediment and soil and plated in duplicate using Nutrient Agar, MacConkey agar, Eosine methylene agar, and Salmonella Shigella agar using pour plates techniques. The plates were swirled and allowed to solidify. All inoculated plates were incubated at 37 0 C for 24 hours. 7.5 Bacterial Count Bacteria colonies were counted on all the media used after 24 & 48hours of incubation respectively and results recorded. 7.6 Morphological studies of Isolates The bacteria colonies were examined and grouped on the basis of the type of growth pattern, shape, elevation, size, pigmentation, edges, colour, colonial surface and optical characteristics were recorded after 24 hours of incubation. 7.7 Purification of Isolates Randomly selected bacteria colonies were picked and sub - cultured by streaking on fresh solidified Nutrient agar medium until pure cultures were obtained. 7.8 Storage of Pure Isolates for Further Test The pure cultures were prepared on Nutrient agar slant and stored in refrigerator and sub - culturing techniques was done for pure isolates before carrying out further tests on them. 7.9 Biochemical Characterisation of Bacterial Isolates Pure cultures of bacteria isolates were subjected to various biochemical tests to determine the probable identity of the bacteria isolates. The result of each test was recorded and the probable identity of the isolates was deduced [6] Biochemical tests are: ~ 21 ~

3 8. Gram s staining Gram staining was carried out as described [7] using Crystal Violet, Gram s iodine, Alcohol and Safranin. A thin smear of hours culture of each isolate was made on a clean grease free slide and heat fixed by passing it gently over a flame. Each smear was stained with crystal violet solution for one to two minutes and poured off. It was flooded with Gram s iodine solution and allowed to react for 1 minute. The iodine solution was poured off and the slide washed with 95% ethanol until no more violet runs from the slide and the slide was washed with water and counterstained with safranin for 1 minute and was washed with water, allowed to air dry and observed under the microscope using the oil immersion objective lens. Gram positive cells appeared purple while gram negative cells are red or pink. This technique also showed the different shapes of bacteria and their arrangements. 8.1 Motility Test Semi solid Nutrient agar medium (consisting 1% of agar) was prepared and dispensed into tubes and sterilized by autoclaving at 121 o C for 15 minutes and allow to set on slope. Each of the tubes was inoculated using a sterile inoculating needle, by making a simple stab down the centre of the tubes to about half the depth of the medium. The tubes were incubated for 48hours and examined for diffuse growth from the line of streak, which is index for motility. 8.2 Catalase Test A thick emulsion of each isolate from 18hours old culture was made on a clean grease-free glass slide. A drop of 3% hydrogen peroxide was added to each plate [8]. Positive result was indicated by the production of gas bubbles, while the absence of bubble indicated negative reaction. 2H 2O 2 2H 2O + O Oxidase Test Few drops of oxidase reagent (1% solution of tetramethyl-pphenylene diamine dihydrochloride) were added to the smear of each 24 hours cultured isolate on Whatman No. 1 filter paper. The phenylene-diamine dye is oxidized to give a deep purple colour within 10 seconds indicating positive result. A delayed reaction was regarded as negative [8]. 8.4 Indole Test The isolates was inoculated into peptone water medium and incubated at 37 o C for 48 hours, 5-6 drops of Kovac s reagent were then added to the culture. If a rose pink red colour develops on the surface of the medium, this indicates a positive reaction. No change in colour indicates a negative reaction [9]. 8.5 Starch Hydrolysis Starch hydrolysis test was used to determine the ability of organisms to hydrolyse starch. Starch agar was prepared by dissolving 1g of soluble starch in 100ml of distilled water and then added to 2.8g of Nutrient Agar, the mixture was homogenized in water bath and sterilized at 110 o C for 15 minutes. This was dispensed into sterile plates and allowed to solidify, each isolate was streaked across the plate once and incubated for 3 days at 37 o C [7]. Uninoculated starch agar plate served as control. The plates were flooded with Gram s iodine after 72 hours of growth. A positive result was indicated by the retainment of the iodine colour as a clear zone while unhydrolyzed starch formed a blue-black colour. 8.6 Methyl Red Test The methyl red test was done to detect the production of acid during the fermentation of glucose and maintenance of acidic condition that is shown by change in colour of the Methyl red. Glucose phosphate broth this consisted of peptone 0.5g, glucose 0.5g; dipotasium hydrogen phosphate, 0.5g all dissolved in 100ml of distilled water was prepared, 5ml of the medium was dispensed into screw capped tubes and sterilized at 115 o C for 10 minutes. The glucose phosphate broth was inoculated with each bacteria isolate and incubated at 37 o C for 24 hours. After which 2-3 drops of methyl red reagent was added, red colour indicated positive result (i.e. acid production) while yellow colouration indicated negative reaction [10]. Table 4.1: Biochemical Characterisation of Bacteria Isolates Obtained In Water, Sediment and Soil Samples from Abattoir Isolate Gram reaction Shape Catalase Motility Starch Hydrolysis Heamolysis Methyl red V.P. Indole Oxidase Citrate Arabinose Xylose Glucose Maltose Mannitol Galactose Fructose Sucrose Lactose Inulin Probable Indentity UHO1 + Cocci AG AG AG AG AG G A G - A UHO2 + Cocci AG AG AG AG AG - A G - A Staphyloccus UHO6 + Cocci AG AG AG AG AG AG A AG - - USE1 - Rod AG - AG AG - A AG AG - - Proteus USE2 + Rod A G A - G AG AG - A Bacillus USE3 + Cocci A A A AG AG AG G - USE4 + Rod A - - A A AG A + G - Bacillus USE7 - Rod AG AG AG AG AG AG A AG G A Enterobacter USO1 + Cocci AG AG AG AG AG - A - G - USO2 + Rod AG A AG AG AG G A - G - Bacillus MHO2 + Cocci AG AG AG AG AG AG AG AG - - ~ 22 ~

4 MHO3 + Rod AG AG AG AG AG G A AG - - Bacillus MHO6 + Cocci AG AG AG AG AG AG AG - - A MHO7 + Cocci AG A AG AG AG A A AG - - MHO8 + Cocci AG AG AG AG AG - AG AG - - MSE1 - Rod AG AG AG AG A - A AG - - Proteus MSE3 + Cocci AG AG AG AG AG AG A AG - A Staphyloccus MSE4 + Rod AG AG AG AG AG AG AG AG G A Bacillus MSO2 - Rod AG AG AG AG AG - AG AG AG - Echerichia coli MSO4 + Rod AG AG AG AG A G G Bacillus MSO5 + Rod AG AG AG AG A AG AG AG - AG Bacillus DHO5 - Rod AG AG AG AG AG AG AG AG - - Citrobacter DHO6 - Rod AG A AG AG AG G A A - - Citrobacter DSE1 + Cocci A AG A AG AG G AG - A DSE3 + Cocci AG AG AG A AG AG AG A - A Staphylococcus DSE4 + Rod AG A AG AG AG AG AG AG - A Bacillus DSO1 + Rod AG AG AG AG AG A - Bacillus DSO2 + Cocci AG AG A AG AG AG AG DSO4 - Rod AG AG AG AG AG A AG AG - A Salmonella DSO6 - Rod AG AG AG A AG A AG A - A Salmonella KEYS: + = Positive, W+ = Weakly positive, - = Negative, A = Acid production, AG= Acid and Gas. UHO = Upstream water, USE = Upstream sediment, USO = Upstream soil, MHO = Middle stream water, MSE = Middle stream sediment, MSO = Middle stream soil, DHO = Down stream water, DSE = Down stream sediment, DSO = Down stream soil. 8.7 Voges - Proskauer Test This is a test for the production of acetyl-methyl-carbinol (CH 3- CO-CHOCH 3) from glucose. To sterile glucose phosphate broth in screw-capped tubes, bacteria isolates were stabbed-inoculated and incubated for 72 hours at 37 o C, an uninoculated medium served as control test [11]. At the end of the incubation period, 0.5 ml of 60% α-napthol solution and 0.5ml conc. KOH were added to 1ml of the culture. These were shaken in the tube. A positive reaction was indicated by the development of red colouration within 5 minutes. 8.8 Citrate Utilization Test The medium used was Koser s citrate medium prepared according to specification, the medium was prepared and dispensed in 10ml amount in each test tube. These were sterilized. Each test tube was inoculated with a loopful of bacterial isolates and incubated at 48 hours. Uninoculated citrate medium served as control. Change in colour of the medium from green to blue indicated positive results. 9. Sugar Fermentation This test was carried out to investigate the ability of the isolates to ferment various sugars. The medium used consisted of 1% peptone water and appropriate sugar with suitable indicator (Phenol Red). All were dissolved in 100ml s of distilled water. 10ml of solution was dispensed into test tubes. Sterile Durham Tubes were inserted into each of the medium in a test tube and sterilized at 115 o C for 72 hours. Acid production was shown by a change in a colour of the medium from red to yellow. While gas production was indicated by the displacement of the solution in the Durham tube by air (carbondioxide) [12]. The various sugars used were Glucose, Fructose, Maltose, Lactose, Sucrose, Mannitol, Xylose, Inulin, Galactose and Arabinose. 9.1 Hemolytic test This test was carried out as screening for bacterial isolates to determine isolate s heamolytic ability. Blood agar was prepared by evenly mixing 5ml of fresh uncoagulated blood with 100ml of sterile Nutrient agar. Bacteria isolates were streaked on solidified blood agar and incubated at 37 o C for 24 hours. Complete clear ~ 23 ~

5 zone around line of streak indicates complete heamolysis known as Beta heamolysis. Incomplete clear zone indicate partial heamolysis known as alpha heamolysis. No clear zone around line of streak is termed as negative result. 10. Discussion of Results Coker et al. [13] identified seven pathogenic of bacteria in abattoir effluent in Southwestern These among others included Staphylococcus sp., sp., in harsh environmental conditions; hence they affect animal and human health. 12. Sharpe ME, Fryer TF, Smith DC. Identification of the Lactic Acid Bacteria In GIBBS, B.M., a Skinner, P.A. Identification Methods for Microbiologists Part A 1966; Coker AO, Olugasa BO and Adeyemi AO. Abattoir wastewater quality in South Western Proceedings of the 27th WEDC Conference, Lusaka, Zambia 2001; The table below shows the result of biochemical characterisation of the bacterial isolates. Thirty bacterial isolates were obtained from samples collected and analyzed (water, sediment and soil). In biochemical analyses carried out on the isolates, the probable identity of each genera include: Staphylococcus was gram positive cocci in clusters, catalase positive, oxidase negative and non- motile. was gram positive in chains, catalase negative and motile. Bacillus, was gram positive rod with terminal spores, catalase positive and hydrolsed starch. Proteus was gram negative rod, methyl red positive, vogesproskauer negative and unable to utilized mannitol sugar to produce acid and gas. Citrobacter was methyl red positive, vogesproskauer negative, indole negative and citrate positive. Enterobacter was gram negative rod, methyl red negative, voges-proskauer positive and indole negative. Escherichia coli was gram negative rod, methyl red positive, voges-proskauer negative and indole positive. Samonella was gram negative rod, methyl red positive, Voges-proskauer negative, citrate positive and utilized mannitol sugar with acid and gas production. The probable identity of the isolates are Staphylococcus,, Bacillus, Proteus, Citrobacter,Enterobacter, Escherichia coli and Samonella with reference to Bergey s manual of determinative bacteriology [6]. 11. Reference: 1. Alonge DO. Textbook of meat hygiene in the tropics Farmcoe press, Ibadan 1991; Adelegan JA. Environmental policy and slaughterhouse waste in Proceedings of the 28th WEDC Conference, Calcutta, India, 2002; Meadows R. Livestock Legacy Environmental Health Perspectives 1995; 103(12): Ifeadi CN. Contamination and control of water supply from borehole system in Nigeria, Proc. of The Third Nat. Conf. On Water Pollution, Port Harcourt, Nigeria 1982; Lewicki P. Higiena Produckcji. Czesc I. Przem. Spoz 1993; 47(10): Buchanan RE & Gibbons NE. Bergey s manual of Determinative Bacteriology, 8th ed. Williams & wilkins Co. Baltimore, Md Olutiola PO, Famurewa O and Sontag HG. An Introduction to general Microbiology. A Practical Approach Seeley HW and Van Denmark PJ. Microbes in Action. A Laboratory manual of Microbiology 2nd ed. W.A. freeman and co. San Francisco, CA ltd. W.H. Freeman Publishing Kovac N, Norris JR and Ribbons DW. Academic Methods of Microbiology Press, New York, Harrigan WF and Mc Cance ME. Laboratory Methods in Microbiology Academic Press. University of Michigan Barrit M. The intensification of the Voges- Proskauer reaction by the addition of naphthol. J. Path. Bact 1936; 42: ~ 24 ~