This study aimed to investigate the bacteriological quality of river Rima, kwalkwalawa, Wamakko Local Government Area of Sokoto State, Nigeria. Standard plate count and Most Probable Number (MPN) methods were used in the study. The identification of the organisms was determined by their morphology, culture characteristics, and biochemical profile. A total of 27 river water samples were randomly collected for 3 months .Result ofbacterial count ranged from 3.x106 CFU/ml to 7.2x106 CFU/ml. The total coliform counts ranged from 120 to 250 MPN/100ml. Coliform bacteria identified were Escherichia coli, Enterobacter cloacae, Salmonella typhi, Serratia mercensces, Proteus mirabilis, and Vibrio cholera. Based on World Health Organization (WHO) standard for the coliform count, all the samples were contaminated with microbes exceeding regulatory limits (100 coliforms 100/mL).However, on the density and isolates, the water is unfit for human consumption. Hence it can be suggested that the water from River Rima is not potable based on bacteriological quality except when treated using suitable treatment techniques such as chlorination and boiling Background: Access to portable water remains a major global concern due to the increased rate of water pollution contributed by climate change and human activities. Rivers and streams have been exposed to toxic and pathogenic pollutants released from untreated organic and inorganic waste that causes water pollution and is detrimental to aquatic plants, animals, and human consumers who depends on such a valuable ecosystem. Control of water-borne diseases is of primary importance in developed and developing countries.
Water plays an essential role in the sustenance of life and it is a key pillar of health determinants since 80% of diseases in developing countries are due to a lack of good quality water [WHO, 2004]. Water exists as surface water, groundwater, and rainwater [8]. Surface water especially freshwater also classified based on its size viz creek, pond, lake, stream, river, rivulets, etc.Freshwater is mostly used for potablewater and other domestic purposes including washing, cooking, bathing,etc [21,23]. Other economic activities carried out on surface water include fishing [16], dredging, etc. Fishing activities and irrigation area are common in communities aligning to the community of Kwalkwalawa Village. Over time it was studied that the groundwater gets polluted significantly because of improved human activities [1] and the growth of human population and swift industrialization has led to compromising other uses. Water sources could be the increasing use of urban waters as sewers, contaminated by microbial and chemical pollutants that come with domestic wastewater, sewage, etc [20]. Increases in urbanization, industrialization and various anthropogenic activities have increased surface and groundwater pollution.
Not all bacteria present in water are harmful but it is the kind of specific microorganisms that are pathogenic causes water-borne diseases. Their presence in water indicates contamination of water supply with fecal matter. Throughout the world, about 2.3 billion people suffer from diseases linked to water-related problems which continue to kill millions of people yearly. The quality of drinking water is a powerful environmental determinant of health [WHO, 2010]. Coliforms are the major microbial indicators for monitoring water quality. These are rod-shaped, none spore, motile or non-motile gram-negative, aerobic and facultative or anaerobic bacteria that ferment lactose with gas formation within 48 hours at 370C. They are commonly found in the intestinal tract of men and animals and considered as indicator organisms used all over the world to establish the degree of fecal pollution in water. Fecal polluted materials always contain coliforms. Most Probable Number (MPN) test is a specific test to determine the
presence of coliform in a given sample. Coliforms ferment lactose with the production of gas, bacteria other than coliforms do not ferment lactose and they do not produce gas. Fecally contaminated drinking water can spread diseases like hepatitis, cholera, dysentery, typhoid, and diarrhea. Diarrhea is considered as the most important waterborne disease, which affects 40% of children under 5 years old. Data on the research topic in the study area is scarce. Microorganisms are ubiquitous. They are alsoknown to cause different diseases. Few others have a beneficial role in several industries. Microorganisms suchas bacteria are found in water and could cause disease conditions in humans if such water is consumed. As suchpoor quality water is one of the medium through whichpathogenic microbes including bacteria, viruses, etc can betransmitted. Several diseases have been attributed topoor water with notable symptoms being enteric fever anddiarrhea. Therefore, this present study is designed toassess the bacteriological quality of river Rima at Kwalkwalawa, Wamakko, Sokoto State, Nigeria.
Study Area
Wamakko is located on latitude 13.0367ON and 5.0945OE. The river source is near Funtua in the south of Katsina State, some 275 km in a straight line from Sokoto, it flows north-west through Gusau in Zamfara State. Further downstream the river enters Sokoto State where it passes by Sokoto town and is joined by River Rima. The river lies between the university’s main entrance and the kwalkwalawa village; it is about 2km from the Usmanu Danfodiyo University. The inhabitants are predominantly farmers or fishermen and of the Islamic faith.
Study Design/Sampling Technique
The study is design for two season considering the month of June for rainy season and April for dry season with experimental design for water sample collection and bacterial analysis.
Twenty seven samples were randomly collected from river Rima at the side bank, the middle of the river, and in the opposite bank of the river collected in a sterile 1000 ml container for 3 months (9 samples per months). All the samples were labeled for easy identification and were immediately transported to the Microbiology Laboratory, Usmanu Danfodiyo University Sokoto for bacteriological analysis.
Sample Preparation
Ten ml of each collected sample was aseptically poured into sterile bottles containing 90mls of distilled water. The mixture was shaken vigorously to mix followed by serial dilution. Serial dilutions for each sample was prepared before subsequent culturing according to the microbiological techniques of Harley [2002].Six dilution tubes were prepared each containing 9.0 ml of sterile saline. 1 ml of sample was aseptically remove with a sterile pipette and transferred intothe first dilution tube. The tube was mix thoroughly by holding the tube in one hand while vigorously tapping the bottom with the other hand. 1.0 ml of the sample from the first dilution tube was aseptically removed and dispensed into the second dilution tube then mix. The procedure was continued serially from tube to tube until the dilution was completed (Harley2002).
Microbial Examination of Water Samples
Examination of total and feacal coliform
The total and feacal coliform test (presumptive,confirmatory, and completed test) of the water was carriedout using three tubes most probable number previously described by Pepper & Gerba, Benson, [8,34]. The result based on gas production and color change was compared with the standard table.
Enumeration of Total Heterotrophic Bacteria
Nutrient Agar medium for Standard Plate Count and MacConkey Agar medium for Coliform count were employed for isolation and enumeration of bacteria. Both media were preparedand used according to the manufacturers’ instruction following the pour plate method previously described by Benson, 8, Pepper and Gerba, 34. 1.0 ml of serially diluted sample was aseptically plates in both media and incubated inverted at 37ºC for 24- 48 hours. The resultant colonies were counted and expressed as colony-forming units per meal of water sample. The different colonies were isolated into nutrient Agar.
Characterization of Bacterial Isolates
Indole test
The bacterial growths were inoculated into 5mls of peptone water incubated at 370C for 24hours. three to eight drops of Kovac's reagent were added and shaken gently. A positive reaction is indicated by the development of red or pink color in the reagent layer above the broth within one minute, yellow color indicates a negative reaction [6].
Citrate utilization
This was carried out by inoculating the test organism in test tubes containing Simon's citrate medium and incubated for 24hours or 72 hours. The formation of deep blue color after incubation indicates a positive test, while no color changes indicate a negative result.
Methyl red –Voges Proskauer test
Five [5] mlsof Mr-Vp broth was inoculated with the test organism and incubated at 350C for 48-72 hours. 1ml of the broth transferred into a small serological tube. 2-3 drops of methyl red were added. The red color on the addition of an indicator signifies a positive methyl red test. A yellow color signifies a negative test. For the Vp test, 5 drops of 40% potassium hydroxide (KOH) were added to the rest of the broth in the original tube, followed by 15 drops of 5% naphthol in ethanol. The cap of the tube was shaken, loosens, and placed in a sloping position. The developments of red color starting from the liquid-air interface within 1 hour indicate a Vp positive test. No color changes indicate Vp negative. [15].
Motility test
The test organism was inoculated into a nutrient ager medium by making a fine stab with a needle to a depth of 2cm short at the bottom of the tube. The tubes were incubated for 48hours at 370C. After the incubation, the tubes were examined. The line of inoculation would not be sharply defined and the rest of the medium would be somewhat cloudy if the organism is motile while if the organism is not motile growth would be restricted to the line of inoculation which becomes sharply define. The test of the medium remains clear [6].
Urease test
The various test organisms were inoculated on urea agar sloped and incubated at 370C for 48 hours. The development of bright pink or red color indicates a positive reaction.
Triple–sugar iron agar test
This was done by using a sterile needle to obtain the test organism, the surface of the slant was streaked and but was stabbed to 2 times. The cap was closed loosely and incubated at 350C for 24hours. [15]. Other tests conducted were Vp, H2S, spore, glucose, sucrose and lactose.
Identification of The Microbial Isolates
The different pure isolates found in the water samples were subjected to microscopic observation, gram staining procedure, and standard biochemical tests following the scheme of Cheesbrough, [10,12] and Benson, 2002. Thereafter, the resultant appearances were compared with those of known taxa using the scheme of Cheesbrough, [10-12] and Bergey’s Manual of Determinative Bacteriology by Holt. Based on gram reaction, the gram-positive organisms were streaked in a Mannitol Salt Agar plate and incubated inverted at 37°C for 24 hours. The presence of yellowish pigments in Mannitol Salt Agar indicates Staphylococcus aureus [Kigigha & Baraseibai, 21,23]. Tubes with color change and gas production were shaken and streaked in Levine’s eosin Methylene Blue (EMB) Agar and incubated at 37ºC for 24 hours. The presence of small nucleated colonies with greenish metallic sheen indicates E. coli [8,34]. The colonies were streaked in blood agar, the presence of swarming growth and haemolytic properties on medium after incubation indicates Proteus species and Streptococcus species respectively [21,23]. The presence of black and pink colonies in Salmonella-Shigella agar suggests the Salmonella and Shigella species respectively [28]. A Triple Sugar Iron Agar was prepared into slants and the colonies were aseptically transferred into the slants. A positive tube was confirmed by the presence of cracks and blackening of the medium [16-18].
Statistical analysis
Data obtained from the study were analyzed using IBM SPSS version 23 to determine frequency counts and percentages. Also subjected to analysis of variance [ANOVA] and Duncan Multiple Range Test to separate the means where there was significant difference.
The result of Biochemical characteristics and identification of bacterial isolates from the river rima was coliform bacteria identified as E. coli (11%). Enterobacter cloacae (3.7%), Salmonella typhi (7.4%), Serratia emergences (7.4%), Proteus mirabilis (3.7%) and Vibrio cholera (7.7%).This study corroborates with Ogbondeminu and Mudassiru who reported similar bacterial isolates as shown in table 3. However, various bacteria species tentatively isolated have been reported from potable water sources (surface water i.e.fresh water, groundwater i.e. borehole and rainwater) in Nigeria. In a recent review study, Izah and Ineyougha, [18] reported similar bacterial isolates from the groundwater.
These bacteria are microbes of public health importance. Some of the isolates have been linkedto several disease conditions such as diarrhea (E. coli,Enterobacter, Salmonella species, etc). Other bacteria are also associated with infectious diseases including gastroenteritis, typhoid fever, dysentery, cholera, andurinary tract infections, etc. The presence of these bacteriaisolates is an indication that such water sources are notpotable.
Table 1: Result of bacterial count of river rima
Sample | Count in cfu/ml Total | Mean |
AP | 4 x 106 | 2.0 x106 |
JU | 14 x 106 | 7.2 x 106 |
MA | 4.2 x 106 | 3.0 x 106 |
Table 2: MPN Determination from multiple test tubes
| S/No. | 3 of 10mls | 3 of 1ml | 3of 0.1ml | MPN/100ml Index per 100 ml |
| AP | 3 | 1 | 2 | 120 |
| JU | 3 | 2 | 1 | 250 |
| MA | 3 | 2 | 2 | 210 |
According to the parameters given by APHA, the presence of coliforms in given water samples indicates that the water is polluted and not of potable quality. The presenceof coliform in river water may cause various diseases like cholera and typhoid that are fatal. Water from the river site should be boiled and filtered or treated before use. The public should be enlightened on the health hazard associated with spreading manure as fertilizer on-field during the dry period close to water bodies. Animals (livestock, duck, swan) should not be allowed to graze near water sources such as rivers, streams, etc. The provision of proper wastewater disposal should be designed so that the down water supply will not be contaminated. Further research on the river should be carried out to study the seasonal changes in the bacteriological qualities of the river.
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