Microbiological Examination of Dairy Products

GS/M.Sc./FOOD/3608/08

B.K.K.K.Jinadasa

Introduction

Milk contributes a greater number of the essential nutrients for human nutrition than any other

single food, some in relatively large amounts. It is an outstanding source of calcium, which is

needed all through life for healthy bones and teeth. It also supplies many other vitamins

including riboflavin and, when fortified, vitamin-D which helps structural and tissue

development. The protein in milk is one of the best quality proteins that any food offers. And

milk is not only a beverage. It can be used in or on cooked food such as gravies, puddings and

cereals. It can be consumed in the form of cheese, butter, ice cream or milk drinks, during meals

or snacks.

Due this highly nutritious nature of the milk and milk foods, it serves as an excellent growth

medium for a wide range of microorganisms. The microbiological quality of milk and dairy

products is influenced by the initial flora of raw milk, the processing conditions, and post-heat

treatment contamination. The most significant source of contamination is dairy utensils and milk

contact surface including milk pall or milking machines. It can also be subject to contamination

during transport, storage and manufacturing processes. Milk is an excellent culture medium for

many kind of microorganism. Its high water activity, moderate pH and available nutrients are the

principal factors which contribute to microbial growth. Therefore, it is necessary to maintain

high standards of hygiene during its production and related manufacturing processes. The

predominant microorganisms living in milk under improper handling and storage conditions are

Gram positive. Spoilage occurs when microorganisms degrade the carbohydrates, proteins, fats

of milk and produce noxious, end products. It may be seen that Lactobacillus or Streptococcus

species ferment the lactose to lactic acid and acetic acids turning the mi1k sour.

The importance of microbiology to the dairy industry has been demonstrated by foodborne

illness associated with consumption of milk and dairy products that had been contaminated with

pathogenic organisms or toxins. Undesirable microorganisms constitute the primary hazard to

safety, quality, and wholesomeness of milk and dairy foods. Consequently, increased emphasis

has been placed on the microbiological analysis of milk and dairy products designed to evaluate

quality and to ensure safety and regulatory compliance.

The focus of dairy microbiology, there are three microbiological analysis methods that can be

carried out to standardize milk.

1. Total colony count

2. E.coli type count

3. Methylene blue dye reduction test

3.1 Dye reduction test for milk

Introduction

Dye reduction methods depend on the ability of microorganisms to alter the oxidation-reduction

potential of the medium. They are in consequence a measure of the activity of microorganisms in

the test system rather than of the numbers in the sample. Suitable indicator dyes include

methylene blue and resazurin. The length of time taken to reduce the dye depends upon the mass

and activity of bacteria present in the sample. The greater the number present shorter the time

required for reduction.

The methylene blue reduction test is based on the fact that the colour imparted to milk by the

addition of the dye. Methylene blue will disappear more quickly. The removal of oxygen from

milk and the formation of reducing substances during bacterial metabolism cause the colour to

disappear.

Methylene blue Leuco methylene blue

Materials

Glass tubes with rubber stoppers

Two milk samples

Methylene blue

Water bath

Procedure

10 ml from each milk sample were added to sterilized glass tubes and 1 ml of methylene blue

was added.

Sterile stoppers were fitted to those test tubes and gently mixed (do not shake).

Then the tubes were placed in the water bath maintained at 36ºC in an inverted position for

incubation.

Tubes were covered to keep out of light.

A blank tube was placed without methylene blue to compare the colour.

Samples were checked for discoloration after 30 minutes of incubation. Readings were taken at

hourly intervals.

After each reading, tubes were inverted before placing in the water bath.

Time taken for methylene blue discoloration was recorded.

Results

Milk Sample Time taken for Decolourization

Raw Milk<20 minutes

Sterile milkMore than 8 hours

Discussion

Fresh milk sample contained considerable amount of microorganisms whereas sterilized milk

sample didn’t contain such amount of microorganisms to reduce the colour.

According to the data table we can say that there were 4 × 106 - 2× 107 microorganisms present

in the fresh milk sample.

3.2. Enumeration of bacterial numbers in milk sample by the plate counting

Materials

Sample Milk, Sterile pipettes (1.0ml)

Sterile Petri dishes

Sterile molten nutrient agar

Procedure

A dilution series of the sample to be tested was made by transferring aseptically 1.0ml of the

homogenized sample to the tubes containing 9.0ml of sterilized Ringer’s solution. Tubes were

mixed well by rolling tubes between the palms of the hands to ensure even dispersal of cells in

the mixture. 1.0ml of 10-3, 10-4, 10-5 dilution series was transferred to separate three Petri

dishes and 15ml of molten agar was poured to each of them. Petri dishes were gently rotated on

the table top clockwise and anticlockwise to ensure to ensure uniform distribution of the cells in

the medium.

Petri dishes were kept to solidify 15 to 20miniutes and they were incubated at 37 0C for 48 hrs.

After the incubation period colonies were counted.

Results and Readings

Sterilized milk

No of colonies:Not found in plates

Raw milk

Dilution which had 30 - 300 colonies = 10-5

Number of colonies per plate = 107

Calculation

Dilution factor = 10-5

Number of colonies / 1ml of sample = 107 × 105

Average Number of colonies/1ml of sample = 1.07 x 107

Conclusion.

Given Sterilized milk sample is considerably lower number of Microorganisms or no viable

microorganism found due to sterilization process.

Estimated number of microorganisms in the 1ml of the given raw milk sample is 1.07 × 107

Discussion

The microbiological diagnostic procedures used for assessing the overall quality and safety of

the Dairy foods products and the standard plate count is one of most common technique use all

over the world. By this method only viable cells are counted and it allows isolation of discrete

colonies that can be subculture in to pure culture, which may be then easily studied and

identified. In conclusion, all methods have limitations. One of the major limitations to the plate

count method is the relatively narrow countable range (generally considered to be 25-250 CFU

bacteria on a standard petri dish). The currently prevailing confusion between the Limit of

Detection (1 CFU) and Limit of Quantification (25 CFU) for the plate count method creates a

larger degree of variability in microbiology data than is necessary.

In other way the major disadvantages of this method are; time consuming, more experience

labour concern, relatively expensive when analyze a large number of samples. Also it is having

overnight incubation and is not suitable for rapid assessment of bacterial count.

The most of milk products are bearing short shelf life and the long testing procedure is not

suitable for these products. If food must be held until testing is complete, distribution is delayed

and the storage cost can add considerably to the cost of processing.

It is necessary to use more glassware in this procedure. It needs for greater manipulation May

result in erroneous counts due to personal errors in dilution or plating. Some pathogens do not

grow under conditions provided. (Ex: Mycobacterium tuberculosis) and this method not suitable

to detect pathogenic bacteria count.

The actual number of bacteria obtained by the plate method does not represent because of

various biological factors. The air supply and the temperature are not suitable for all types of

bacteria. Therefore most of the time value obtained from the plate count is lower than actual

number of viable bacteria presence in the product.