Yoghurt is a cultural diary product produced by lactic fermentation of milk (Hui, 1992). Yoghurt is produced by the controlled fermentation of milk by two species of bacteria; Lactobacillus bulgaricus and Strptococcus thermophilus.
These two species of bacteriae have now been established as the yoghurt starters.
Any sort of milk may be used to make yoghurt, but modern production is dominated by cow milk. It is the fermentation of the milk sugar (Lactose) into lactic acid that gives yoghurt its gel-like texture and characteristics tang (Davis, 1974).
Milk is often regarded as being nature’s most complete food. It earns this reputation by providing many of the nutrients which are essential for the growth of the human body. It is an excellent source of protein, vitamins, minerals particularly calcium and also some anti-bacterial substances such as lysozyme, ladoferin (transferin) and lactoperoxidases. In addition to the major constituent, milk contains a large amount of lactose sugar, phosphate, peptone and nitrogenbase enzyme (Clurance et al., 1963). Yoghurt has practically the same food value as the basic milk product (Ruud and Bert, 2004).
Fermented milk products such as yoghurt contain bacteria from the lactobacilli group. These bacteria occur naturally in the digestive tract and have a cleansing and healing effect. Therefore, the introduction of fermented products like yoghurt into the diet can help prevent certain pathogenic bacteria, which may cause illness. Many people suffer from a condition known as “LACTOSE INTOLERANCE”. This means that they are unable to digest the milk sugar (lactose). Such people can however, tolerate milk if it is fermented to produce foods such as yoghurt. During fermentation, lactic acid producing bacteria breakdown lactose and in doing so eliminate the cause of irritation (Ruud and Bert, 2004).
In countries where food borne illness are investigated and documented, the relative importance of pathogens like Staphylococcu aureus and Escherichia coli in yoghurt borne infection is well known (Obende, 1999). Diary foods are frequently contaminated with staphylococci and cases of staphylococcal contamination of milk and milk products like yoghurt have been recorded (Umoh et al., 1985). More than 50% of the strains of S. aureus are able to produce enterotoxin associated with food poisoning. The most important source of contamination by S. aureus is the human. (Payre and Wood, 1974).
The coli forms bacteria are also reported to be contaminants of yoghurt. The study of Obende, (1999) shows that 60% of yoghurt samples analyzed in Enugu Urban are contaminated with coli forms. The source of contamination of coli forms bacteria is through water used or through the workers or utensil used in processing (Gillies and Bodds, 1973).
Interestingly, most of the yoghurts are produced in little less than the cottage industries at homestead level under different brand names and are found in stalls, or more commonly hawked along the streets, motor parks and in the market. In view of this, the objective of this study was to investigate the level of microbial contamination of yoghurts sold in Enugu Urban.
Chemical Composition Of Milk
Milk is composed of milk fat, lactose (milk sugar), proteins, vitamins, minerals and water. The non-fat portion of milk is termed skimmed milk even when not separated from the whole milk. Normal milk also contains the immunoglobulins, which protect the young from infectious diseases. The composition of milk is influenced by several factors, which include: type of the animal, its quality/health, time between milking, animal feeds, age and season of the year (Jennes and Patton, 1999).
Table 2.1: AVERAGE COMPOSITION (%) OF MILK OF
History of Yoghurt
Yoghurt is traditionally believed to be an invention of the Bulgar people of Central Asia, although there is evidence of cultured milk products as far back as 2000 BC. The earliest yoghurt was probably spontaneously fermented, perhaps by wild bacteria residing inside goatskin bags used for transportation.
Yoghurt remained primarily a food of Central and Eastern Europe until the 1900s when a Russian biologist named Llyich Mechnikov theorized in 1990that heavy consumption of yoghurt was responsible for the unusually long lifespan of the Bulgar people. Believing LACTOBACILLUS to be essential for good health, Mechnikov worked to popularize yoghurt as a foodstuff throughout Europe. It fell to a Spanish entrepreneur named Isaac Carasso to industrialize the production of yoghurt. In 1919, he started a commercial yoghurt plant in Barcelona.
Traditional Method of Yoghurt Production
Vander berg (1988), indicated that the manufacture and composition of cultures used for the fermentation of milk vary from one area to the other. From skimmed milk C1-5-2.0% fat, the milk is heated to 70oC in a covered pot and held for two minutes to destroy pathogens and spoilage organisms to provide a clearer medium in which the desired microorganisms can be established. The heat denatures the proteins, which increases the viscosity of the product.
Pasteurized milk is immediately immersed in a basin of cold water to bring the milk temperature to 40oC – 47oC. This is to subject the thermoduric organisms in the milk to thermal shock resulting in autolysis and to provide convenient environment for the inoculation with starter cultures.
The cooled milk is inoculated with mixed cultures of Streptococcus thermophilus and Lactobacillus bulgaricus of 1 – 2% to produce desired fermentation and to flavour the product. This can be achieved by inoculating with a freshly prepared yoghurt purchased from a reliable store.
Yoghurt is usually classified into two types; The set and stirred yoghurt. Set yoghurt is the yoghurt packed immediately after inoculation with starter cultures and incubated in the container whereas the “stirred yoghurt” is packed after inoculation and incubation also, the coagulum is broken and cooled before packaging (Alfa – Lawal, 1984).
Processing Of Yoghurt
The heating process kill contaminating organisms in yoghurt, lowers redox potential of the mixture and produce factors and conditions favouvrable to the development of the inoculated bacteria. Heating also causes the denaturation of the whey proteins and alterations to casein which gives the final product improved and more uniform consistency.
Many variations can be made to the basic procedure first, it is common practice to add 3 – 5% skimmed milk to the milk prior to inoculation. This raises the nutritive value of the yoyhurt and gives a product of better body and consistency. Homogenization of the ingredients prior to pasteurization has also been found to improve product consistency and physical stability by producing a firm, uniform curd. Stabilizers such as gelatin (0.1 – 03%), alginate and agar may also be added to improve the flavours and fruits may be added prior to or after fermentation has been completed.
The flavour and quality of yoghurt is very much related to the fernentation carried out by the inoculated species of bacteria. Streptococcus thermophilus initiate fermentation of lactose to lactic acid, reduces and causes some milk protein breakdown through the production of proteolytic enzymes. This also creates favourable conditions for the development of lactobacillus bulgaricus which begins to develop when the pH has dropped to a value of about 4.5. The final product usually contains some 10 cells per milliliters of the product. The characteristic flavour of yoghurt is due to the lactic acid and trace amount of elhanol, dimethylpropanol, ethanoic acid and other volatiles produced by bacterial fermentation. Lactobacillus bulgaricus is mostly responsible for the production of acetyldehyde.
The word quality has to do with degree of excellence. The quality of yoghurt is very related to the fermentation carried out by the inoculated bacterial species of streptococcus thermophilus. Artherton (1977) indicated that good quality yoghurt should have some features like being firm and cohesive enough, gelation, consistency, smooth in appearance and in the mouth. Finally, it should not have any unatural sweetness or vinegar taste.
Low- temperature –longtime (LTLT) pasteurization of yoghurt requires a minimum heat of 630c for 30 minutes while high –temperature –short time (HTST) pasteurization of yoghurt requires 720C for 15 seconds held as minimum heat treatment. (Ihekeronye and Ngoddy, (1985). Heat pasteurization of yoghurt is necessary to prevent the transmission of disease and to prevent microbial and enzymatic spoilage i.e. the enzymes that affects physical properties of yoghurt (taste and colour). The principal problem with pasteurization of yoghurt is the associated destabilization of the product by heat through the destruction of the casein (whey protein). Pasteurized yoghurt have a shelf –life of months and does not need refrigeration. If the yoghurt is not heated to kill the bacteria after fermentation it is sold as containing “live active culture” cor just as “live” in some countries, which some believe to be nutritionally superior (Ruud and Bert, 2004).
The presentation of yoghurt also encourage or initiates intensive production practices and at the same time reduces losses due to spoilage sine quality food of man which are in great demand are also highly perishable (Desrosier, 1995).
Yoghurt is acidic due to lactic acid produced which is effective at inhibiting the growth of bacteria (Pederson 1979) Varman and setherland (1994) reported that yoghurt can be preserved by adding chemical preservative like ascorbic acid, benzoic acid, which are permitted
Refrigeration of Yoghurt
Products like cream, yoghurt and butter milk need to be preserved by low temperature storage of about 40C and definitely not up to freezing point. yoghurt is a perishable drink, it hardly last for 48 hours if not kept in cool place (refrigerator or freezer) due to its nutritive quality which aids the growth of microorganisms (Ihekonony and Ngoddy, 1985)
The major components in yoghurt are the carbonyl compounds, acetaldehyde, acetone and diacetyl. The most important among all is acetaldehyde. Hui, (1992) reported that good flavoured yoghurt results when proper level of acetaldehyde (20 – 40ppm) is present.
Quality Control of Yoghurt
This is the application of this knowledge to the manufacturing, marketing and distribution of a product with the optimum level of quality. It assures product conformity, uniformity, wholeness, reliability and quality assurance (Ihekonnye and Ngoddy (1999).
In quality control of yoghurt, two basic parameters have to be put into consideration they are microbiological analysis and biochemical analysis.
Processed yoghurt is a potential habitat for microorganisms such as yeast, mold and bacteria species. But fro the purpose of this project work only bacteria are considered.
Pathogenic bacteria are not supposed to be present in any successfully manufactured yoghurt because of heat treatment and low acidity of yoghurt. Their presence in yoghurt is sign of re-contamination. The occurrence of enterococci, coliforms, salmonella species, clostridium species and bacillus species is a sign of re-contamination 9speck, 2000).
Pathogenic organisms in milk products we yoghurt have been a matter of public health concern since early days of diary industry. Many diseases such as Tuberculosis, Brucellosis, Diphtheria, Scarlet fever and Gastroenteritis are transmitted via milk product.
Milk products have frequently been implicated in transmission of human pathogen (Bryan, 1990)’, Contamination of dairy products by coliforms is attributed to three reasons. Firstly, the wide distribution of coliform in nature makes them useful indicators of the extent of re-contamination after pasteurization and also the sanitary quality of the pasteurized product. Secondly, coliforms themselves cause rapid spoilage of food held under conditions conducive to the microbial growth. Thirdly, some strains are bacterial enteric pathogen which may be disseminated by diary products (Rutzun and Marth, 1980).
Coliform bacteria include all the aerobic and facultative anaerobic gram negative non sporulating bacilli that produces acid and gas from fermentation of lactose.
Davis, (1974) reported that escheriachia coli should not be present in a good and well prepared yoghurt as high acidity of the fermented product should restrict their survival. Slow acidity development can allow sufficient build up to give a “taint” or “off-flavour” to this retail product which is a clear indication of gross contamination.
Umol et al., (1990) reported that dairy foods are frequently contaminated with staphylococcus and cases of staphylococcal contamination in milk and milk product have been recorded. The presence of staphylococcus aureus in milk and manufactured dairy products, have made them involved in outbreak of staphylococcal food poisoning which is an intoxication caused by enterotoxin produced by strains of S. aureus in food.
Staphylococcus aureus is a non-motile, gram positive cocci, non-sporing, aerobic and facultative anaerobic organism. It produces entertoxin which is thermostable and cases of food poisoning are due to the enterotoxin produced when growing in food such as milk and milk product prior to ingestion.
Clostridium perfringens was isolated from milk and milk products by various investigators (Mechran, 1975). The feasibility of using Clostridium perfringens as an index of fecal contamination of milk was studied and it was shown that there was definite correlation between the hygienic condition of milk production and content of C. perfringens spores in the milk.
Food poisoning occur due to ingestion of food containing large populations of viable vegetative cells of C. perfringens and the subsequent production of an enterotoxin in the intestine.
Kim and Goepfer, (1971) reported that Bacillus cereus can sometimes be found in large number in dairy products. The role of B. cereus in outbreak of food borne illness in becoming well documented B. cereus is recognized as causing diarrhoeal and emetic type of food borne outbreak and has a high incidence in dairy products. Multiplication of B. Cereus in dairy products is not only of concern as public health hazard but also a cause of economic losses through spoilage of contaminated products.
The aetiology of yoghurt spoilage is fairly well established (FAO, 1979). Improper pasteurization and post pasteurization contamination of yoghurt by microorganisms like yeasts, molds and bacteria lead to yoghurt spoilage.
Biochemically, lactose (milk sugar is converted to lactic acid as a result of enzyme activity produced by the microorganisms. Lactose (B-gulactosidase) converts lactose to glucose and galactose. The bacteria utilize only a small part of sugar for its energy requirement but converts most of it to lactic acid through glycolysis.
The bacteria are microaerophillic because if carry out essential metabolic biological process with oxygen by means of complex series of intermolecular oxidation and reduction. The charges they accomplish does not result in decomposition of milk to their basic components such as carbon dioxide, water, simple nitrates and sulphate (Pederson 1979).
Yoghurt is an acidic product, for this reason, if it is made under proper sanitary conditions, it can be kept without the need for preservatives. The percentage of acid (PH) present in milk is a rough indication of its age and the manner in which it has been handled/processed. Varman as Sulherland (1994) reported that PH of a standard yoghurt to be 4.0 – 4.5.
MATERIALS AND METHODS
The media used in this work include nutrient agar, Macconkey agar, Eosine methyleneblue agar (EMB), peptone water. The composition of the media, their method of production and the list of other equipment and reagents used in this work are presented in the appendix.
Six different brands of bottle packaged yoghurt were brought from hawkers and beverage stores in Enugu Urban. The two samples of each brand were used and the brands were designated A, B, C, D, E, F, giving a total of 12 yoghut samples.
The samples were brought to the laboratory and analysed within 6 hours of collection.
ANALYSIS OF SAMPLE
Each sample was serially diluted using sterile distilled water as diluents (Prescolt et al 2002) and Iml of 10-3 sample was plated in duplicate using the pour plate method on MacConkey agar and nutrient agar media the plates were incubated at 370C for the 24hrs. After incubation, pink colonies that developed in MacConkey plates were counted and used to estimate the coliform count (cfu/ml) of the yoghurt samples. The colonies that develop on the nutrient agar plates were counted and used to determine the total bacterial count of the yoghurt samples (cfu/ml) the representative colonies on the plates were sub-cultured on fresh nutrient agar to obtain pure cultures of the isolates. The pure cultures were then transformed into nutrient agar slants for biochemical identification.
IDENTIFICATION OF ISOLATES
Identification of the isolates were based on cultural, morphological and biochemical characteristics following standard methods.
The method used was that described by carpenter (1977) and Thomas (973). Smears of the isolates were prepared and heat fixed on clean grease free slides. The smears were stained for one minute with crystal violet. This was washed out with a gentle running tap water. the slides were flooded with dilute Gram’s iodine solution. This was washed off with water and the smears were decolorized with 95% alchohol till the blue colour no more dripped out (about 30 seconds)
The smears were then counter stained with saffranin solution for about 10 seconds finally, the slides were washed with tap water, air-dried and observed under oil immersion objective.
This test was used to determine which of the isolates were motile organisms from non-motile ones. For this lest, the hanging drop technique was employed and the technique was carried out as described by Kirk et al (1975).
A little Vaseline jelly was rubbed around the cavity of a hanging drop slide. A drop of peptone water containing the pure culture was placed on a cover slip. The hanging drop slide was then placed over the drop of peptone water in such a way that the center of the depression lies over the drop. The slide was quickly inverted and viewed under the microscope, using oil immersion objective.
This test was used to demonstrate the ability of the isolates to produce the enzyme urease which splits urea forming ammonia. The test is usuall used to differentiate organisms like proteus from other non urease positive organisms, (baker and Breach 1979). The method used was that described by speck (1976)
A loopfull of the isolates were used to inoculate a tube of urea agar. The tubes were incubated at 370C. A change in colour from yellow to red confirmed the presence of urease.
This test was used to demonstrate which of the isolates could produce the enzyme catalase that releases oxygen from hydrogen pereoxide. This test is usually used as an aid to differentiate staphylococci form streptococci and to differentiate other catalase positive organism from catalase negative (Barker 1976). The method employed here was that described by speck (1976).
A loopful of the pure colony was transferred onto a plane, clean glass slide. The sample was then mixed with a drop of 3% v/v hydrogen pereoxide. The reaction was observed immediately Gas production indicated by the production of gas bubbles confirmed the presence of catalse.
Methyl Red Test
This test was used to dectect which of the isolates could produce and maintain sufficiently a stable acid product from glucose fermentation. The test is usually used as an aid in the identification and differentiation of the enterobacteriaceae (Baker 1976). This test was carried out as described by Kirk et al (1975).
Tubes of buffered glucose –peptone broth were lightly inoculated with the isolates. The tubes were incubated at 370C for not less than 48 hours. About 5 drops of the methyl Red reagent was added into 5ml of the culture. The production of a bright red colour immediately on the addition of the reagent showed a positive test.
Methyl red test indicator consists of 0.1g methyl Red.
300ml of 95% ethyl alcohol.
Voges- Proskeur Test (V.P. Test)
This test was used to detect which of the isolates were able to produce a neutral end point acetyl methyl carbinol (acetoin) from glucose fermentation or its reductive product butylenes glycerol. The test is usually used to differentiate between Gram negative organisms especially members of the enterobacteriaceae, (baker 1975). Tubes of buffered glucose-peptone broths were lightly inoculated with a young culture of the isolates. The tubes were incubated at 37oC for not less than 48 hours. Burrits reagent was used for the test. 0.6% W/V of solution A and 0.2ml of solution B were added into 1ml of the culture in turns. The mixture were shaked well after each addition.
Positive reaction was indicated by a pink colour that appears immediately or within 5 minutes at the topmost part of the tube.
Solution A contains
5g of – naphlhol
100ml of absolute ethyl alcohol
Solution B contains
100ml Distilled water
40g potassium hydroxide
The alkali oxidized the acetylmethyl carbinol (acetoin) to daiacetyl which gives the pink colour.
This test was used to determine which of the isolates has the ability to split indole from tryptophan present in a buffered peptone water. the test is usually used as an aid in the differentiation of Gram negative, bacilli especially those of the enterobacteriacea (Baker 1976). The test was carried out as described by Kirk et al (1975).
Tubes of peptone water were inoculated with young culture of the isolates. The tubes were incubated at 370C for 48 hours. About 4 drops of Kovac reagent were added into 1ml of each of the culture tubes.
Positive test was indicated by a red colour that occurs immediately at upper part of the test tube.
Kovac’s reagent consists of the following
150ml of Amyl alcohol
10g of D-Dimethyl aminobenzaladhyde
50ml of concentrated Hydrochloric acid
Citrate Utilization Test
This test was used to identify which of the isolates can utilize citrate as the sole source of carbon for metabolism. The test is usually used as an aid in the differentiation of organisms in the enterobacteiraceae and most other genera. (Baker 1976). The medium used for this test was the simon’s citrate agar.
Slant tubes of simon’s citrate agar were inoculated with young culture of the isolates the inoculation was done by stabbing the medium on the tubes using sterile straight inoculating were containing the culture.
The tubes were then incubated at 370C for about 24 hours.
A change in colour from green to blue after about 24 hours of incubation indicated
Each of the isolates was tested for its ability to ferment a given sugar with the production of acid and gas or acid only. Since most bacteria especially Gram negative bacteria utilized different sugar as source of carbon and energy with the production of either acid and gas or acid only the test is used as an aid in their differentiation.
The growth medium used was peptone water and the method used was that described by Kirk et al (1975) peptone water was prepared in a conical flask and the indicators bromocresol purple was added. The mixture was dispensed into test tubes containing Durham tubes. The tubes with their content were sterilized by autoclaving at 1210C for 15 minutes. 1% solution of the sugar was prepared and sterilized separately at 1150C for 10 minutes. This was then ascetically dispensed in 5ml aliquot volume into the tubes containing the peptone water and indicator.
The tubes were inoculate with young culture of the isolates and incubated at 37%. Acid and gas production or acid only were observed after about 24 hours of incubation. Acid production was indicated by the change of the medium from light green to yellow colour while gas production was indicated by the presence of gas in the Durham’s tubes.
The control tubes were not incubated.
Slide and tube method was used (carpenter 1977). In slide test, a loopfull of the isolate was mixed with human plasana and allowed to stand for some minutes. Particles indicating aggregation was used as indication of coagulate reaction.
In tube method, plasma was added into a culture of the isolate in peptone water in a bijou bottles. The bottles were incubated at 370C for 24 hours a clumping/agglutination of the plasma was used to indicate presence of coagulase.
The malachite green staining method was used. The staining was carried out as described by (carpenter (1977). Smears of the pure isolates were made on grease –free glass slide and heat fixed. The slides were flooded with 5% w/V malachite green solution. The slide were flamed in such a way that the stain steamed but did not bil. The slides were then allowed to stand for 5minutes. The stains was then washed out in running tap water the smears were counter stained with saffron in for 30 seconds. This was washed off and the slides were blotted, dried and examined under the oil immersion objective. The spores stained green while vegetative ceils stained red.
The heterotrophic bacterial and coliform counts (cfulml) of the yoghurt samiles are shown is table 1. The mean bacterial counts of the six brands of yoghurt is 2.35 x 03 while the means coliform count is 0.14×03.
The physiochemical properties of bacteria isolated from the yoghurt samples are shown in table 2 while bacteria isolated from different brands of the yoghurt examined are presented in table 3. bacteria isolated from the yoghurt samples include. Klebsiella sp. Streptococcus species escherichia coli, bacillus sp. Staphylococcus aureus, psendomonas sp. Enterobacter sp, lactobacillus bulgarisius and Protons sp. Escherichia coli were isolated from 3 of the six brands of yoghurt examined.
Table 1: Total heterotrophic and coliform count of yoghurt sample
|Source of sample||Total heterotrophic count. (x103cfu/ml)||Total coliform count (x103cfu/ml)|
Table 3: Bacteria isolated from different yoghurt brands
|BACTERIA ISOLATED||YOGHURT BRANDS|
The result obtained from the microbiological analysis showed that product were contaminated by microorganisms of public health concern the high heterotrophic bacterial and coliform counts may be a consequence of low level of hygiere maintained during the processing by some of the producers of the product.
The detection of escherichia coli, enterobacter sp, klebsiella sp, and Proteus sp is an indication of possible faecal contamination of the product by potentially pathogenic organisms even though they were isolated from less than three of the six brands examined.
It is not surprising that lactosacillus and streptococcus were isolated from virtually all the sample examined because they are the organisms used as starter cultures for the production of yoghurt.
Similar microorganisms were isolated from yoghurt a similar product by Uzeh et al (2006).
The isolation of staphylococcus aureus is also of public health importance because of its ability to cause a wide range of infections especially food –borne intoxication some of these organisms were also isolated by Olasupo et al (2002) from kanunzaki, a cereal based, non-alcoholic beverage Engel et al (1980), Olpala and Jideani(2006) noted that some of the sources of microorganisms that contaminate yoghurt are problem of plant sanitation, the filling operation or the container and also the workers.
The conclusion drawn from this research work is that yoghurt if produced under a good hygienic condition, the microbial load could be reduced to be within acceptable limits of standard organization.
It is evident from this study that most of the samples of yoghurt analysed met the requirements compared to standard. However, yoghurt samples containing relatively low counts of Staphylococcus aureus and Escherichia coli may constitute a health hazard to consumers if mishandled.
Yoghurt should be prepared in a good hygienic condition so as to reduce the microbial load. Also care should be taken during processing and storage of the yoghurt samples to prevent contamination with unwanted materials and microorganisms such as staphylococcus species, (e.g. staphylococcus aureus) and coliforms (e.g. Escherichia coli).
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The following media were used.
- Nutrient Agar (NA)
This medium was used for the enumeration of bacteria cells and also to maintain pure cultures. Nutrient agar is a general medium. It was therefore used here on the assumption that as many organism as are on the samples will grow.
The medium is composed of the following
Lab- lemco powder 1g
Yeast Extract 200g
Sodium chloride 5.0g
Agar No. 3 15g
The powdered form was used and it was prepared as directed by the manufacturer.
Twenty three grams of the powdered nutrient agar (Oxiod) was suspended in 1000ml of freshly prepared distilled water and made to dissolve by heating. This was autoclaved at 1210C for 15 minutes. The sterilized medium was allowed to cool down to about 450C and was the poured into sterile petri dishes in about 20ml aliquots. The medium was allowed to solidify on these plates and were thereafter used.
- MacConkey Agar (M C A)
This medium was used primarily to differentiate lactose fementers from nonlactose fermenters and also to suppress the swarming activity of proteus and other spreading organisms.
Bile salt’s 5g
Neutral red 0.075g
Agar No.3 12g
Distilled water 1000ml
The powdered form was used and it was prepared as directed by the manufacturer. 51.5g of the powdered MacConkey medium (Oxoid) was suspended in 1000ml of freshly prepared distilled water and dissolved completely by heating. The medium was then sterilized by autoclaving at 1210C before being poured into sterile petridishes in 20ml aliquote. The medium was allowed to solidfy in these plates and were thereafter used.
iii. Peptone Water (PW)
This medium was used to enrich and develop the inoculums that were used to inoculate the agar plates. It was also used to maintain the cultures for some biochemical tests.
Sodium chloride 5g
Distilled water 1000ml
The powdered medium was used and it was prepared as directed by the manufacturer. Fifteen grams of the powdered medium (Oxoid) was dissolved in 1000ml of distilled water. the medium was sterilized by autoclaving at 1210C for 15 minutes.
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