Anaerobic analysis

The production of fermented milk products such as cheese, yogurt, and fermented milks have developed over the centuries as a means of preserving the nutritive value of milk and providing a safe, attractive food (Roberts and Skinner, 1983). It is important to note that the products of fermentation apart from contributing to flavor also prevent the growth of pathogens and other bacteria whose metabolism would cause undesirable flavor or textural changes.

Yogurt is made from milk, skimmed milk or fortified milk usually from cows but sometimes from other animals such as goats or sheep (Adams and Moss, 2000). Yogurt fermentation is brought about by the inoculation of the milk with Lactobacillus bulgaricus and Streptococcus thermophilus in a 1:1 ratio. The Streptococcus grows faster than the Lactobacillus and is primarily responsible for acid production. The Lactobacillus on the other hand is responsible for the development of flavor and aroma. This associated growth of the two organisms result in lactic acid production at a greater rate than either when growing alone. Sometimes Lactobacillus acidophilus may also be included in the inoculum.

In this laboratory exercise, yogurt will be made and analysis of the product for bacterial content using plate count method will be done. Materials and Methods Four (4) milk samples were prepared: (a) skim milk (500ml), (b) skim milk (500ml) + 25g of powdered skim milk, (c) low fat milk (500ml), and (d) low fat milk (500ml) + 25g powdered low fat milk. The different milk samples were heated to 45oC in a water bath, stirring constantly. The milk samples were then kept at 450C using a thermometer and starter culture added. This starter culture was prepared by mixing 2.55g of the starter culture with 10ml of the skim milk for the skim milk yogurt and 2.55g of starter culture with 10ml of the low fat milk for low fat yogurt. The inoculated milk samples were then kept at 45oC for 4 hours. It was then chilled to 10oC so that the ph did not go lower.

Man Rogosa Sharpe (MRS) Agar and Plate Count Agar (PCA) were prepared as outlined by the manufacturer and autoclaved at 121oC for 15 minutes. Serial dilutions of 10-5, 10-6 and 10-7 were prepared from yogurt samples for spread plate on the MRS agar. There was a dilution each for an aerobic and an anaerobic analysis. Serial dilutions of 10-6, 10-7 and 10-8 were prepared from the yogurt samples for pour plate on the PCA. Control (blank) samples of agar and water were prepared for each agar.

From the laboratory exercise done, a relatively good quality yogurt was obtained. It was observed that the yogurt samples to which powdered milk was added were harder than the others. This was due to the fact that the added powdered milk increased the total soluble solids and thus produced a harder yogurt. The exercise was successful with regards to identifying the total bacterial count in the yogurt. Using the MRS agar, counts were obtained for those media anaerobically stored and none for those stored in an aerobic environment. This may be due to the fact that the microorganisms under investigation grow best under anaerobic conditions.


The lab exercise was successful in producing yogurt. Using the different growth media, observations were made of the two major organisms responsible for fermenting the milk and thus making yogurt – Lactobacillus bulgaricus and Streptococcus thermophilus. Title: The Most Probable Number (MPN) Technique Aim: To Estimate Bacteria in Potable and Contaminated Water Introduction The Most Probable Number (MPN) is one method of enumerating low numbers of viable organisms. The method is usually based on inoculating replicate tubes of an appropriate liquid medium 9usually 3, 4, or 5) wit three different sample sizes or dilutions of the material to be studied (Adams and Moss, 2000). The medium used should be designed to make it possible to decide whether growth or no growth occurred and the number of positives (gas production) at each dilution is determined after incubating the tubes.

The MPN is not a precise measure, however, a specific value can be computed for a single dilution or for multiple dilutions. The MPN is obtained by referring to an MPN index table. This laboratory exercise seeks to use the MPN technique to estimate bacteria in potable and contaminated water. Materials and Methods Lauryl Tryptose Broth (LTB), EC Media (for fecal coliform) and Brilliant Green Blue (BGB) Broth (for total coliform) were prepared based on manufacturers guidelines.

Twenty-five (25) test tubes each containing 10ml of the Lauryl Tryptose Broth were prepared. Ten (10) ml of potable water was dropped in each of 10 of the test tubes containing the (LTB). Also, 10 ml of the contaminated water was dropped into each of another 10 of the test tubes containing LTB. The other 5 test tubes remained blank and were kept as the control. Here there was one tube with lauryl tryptose broth and E. coli and another with Lauryl tryptose broth and 10 mls of sterile water. The tubes were then incubated at 37oC for 48 hours (Presumptive Test).

After incubation, 1drop of solution was taken from tubes that were deemed positive for microorganisms and placed in 10 ml of BGB and EC Medium respectively. There were 10 replications for BGB and the EC Medium (Confirmative Test). The BGB medium was incubated at 44.5oC and the EC at 37oC for 48 hours. The principal components of milk are water, fat, protein and lactose. Because of its high water activity, moderate pH (6.4 – 6.6), and ample supply of nutrients, milk is an excellent medium for microbial growth (Adams and Moss, 2000). Thus, milk demands high standards of hygiene in its production and processing.

Milk normally contains bacteria that originate from the udder of the cow and from handling before and after pasteurization. Raw milk has undergone no treatment, other than the desired cooling. Pasteurized milk on the other hand, is very often homogenized and heat-treated. There could be treatment at 60 – 65oC for 30 minutes, or at 72oC for 15 seconds (high temperature, short time). Psychrotrophs are bacteria that are able to grow at or near 0oC and are found in milk. Psychrotrophs are usually killed by pasteurization. Thus psychrotrophic counts are used on pasteurized milk to predict shelf life. Thermoduric bacteria on the other hand, are able to survive the pasteurization temperature. There presence in raw milk is indicative of the presence of soil contamination or poorly sanitized milking equipment among other possibilities. When present after pasteurization they signify improperly cleaned equipment.

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