Module 4. Microbiological methods of milk testing


Lesson 15


15.1 Introduction

Microbiological quality control test of milk can be divided into two groups: for example ‘direct tests’ (Quantitative) and ‘indirect tests’ (Qualitative).

15.2 Quantitative Tests

The direct tests are helpful for assessment of the actual number of bacteria present in milk by microscopic examination, direct microscopic count (DMC) or by enumeration of the colonies formed by viable cells of bacteria, Standard plate count (SPC).

The microscopic examination of raw milk sample provides a rapid indication of the quality of liquid milk. It must, however, be noted that, because of the small sample volume which is being examined, any direct microscopic count is insensitive if the bacterial load is less than 500000 per ml.

By the microscopic method, clump counts can be determined. When determining the individual count, all bacterial cells within clumps or in isolation are counted. Dead bacteria can also be stained in this method. However, bacteria killed by heat usually disintegrate soon after, or lose their staining ability. In addition, there are stain solutions that permit the recognition of dead cells, e.g. acridine orange only stains viable cells, and then the microscopic count can be applied to heated milk, or reconstituted samples of powdered milk, to furnish the information concerning the history of product.

The microscopic method can also be used for counting the somatic cells. For milk from individual quarters of cows in normal lactation, a somatic cell count of 125 x 103 – 250 x 103 per ml, and for bulk milk a somatic cell count of 500 x 103 per ml, has been recommended by IDF, although many countries accept counts of 500 x 103 to 100 x 104 per ml.

15.3 Pour Plate Methods

The plate count or pour plate method is often used for estimating the number of viable number of micro-organisms in liquid, reconstituted or suspended dairy products. Due to a wide range of bacterial counts occurring in dairy products, bacteria can often only be counted after diluting the liquid. A number of 10-fold serial dilutions of the sample are prepared.

One ml of the dilution is mixed with the liquefied sterile agar medium in a sterile Petri dish. After solidification of the agar, the Petri dishes are incubated at a specific temperature and for a suitable period of time. The bacterial cells grow to recognizable colonies that can be counted. Plates with 30-300 colonies are selected for counting. The loop method (plate loop count) is less time-consuming than the traditional pipetting, is adversely influenced by the following factors:

·         Depth and angle of loop penetration

·         Spread of loop removal

·         Temperature of milk and

·         Wetting ability of the loop and bacterial morphology

The manual plate loop count was suitable for routine lab that analyzes 4000 samples per month. Various automated and semi-automated apparatuses have been developed in an attempt to fully standardize the plate loop method with respect to the angle at which the loop is removed from the sample, the depth of immersion, and the speed of movement of the loop. When these precautions are taken, the very small amount (0.1 µl) of the sample may not be representative of the product tested, except perhaps for the 0·1 µl, hook.

15.4 Colony Counters

These are designed to count colonies on agar plates.  Most of these colony counters consist of a television camera to detect the colonies on the illuminated Petri dish, and a small electronic computer for detection, counting and control. Up to 150 plates per hour may be counted using these counters. The counting of impurities, lumps, un-dissolved parts in the media or air bubbles can be avoided, as these only occur due to the carelessness of the personnel. More serious problem is of counting pinpoint colonies, which are sometimes formed by thermodurics. Colonies smaller than 0·20 mm are usually not counted, while the human eye can see and count colonies exceeding 0·10 mm. Another disadvantage is that, automatic counters cannot distinguish colors or colored colonies. Colonies on media that absorb light, e.g. blood agar, are difficult to count, while diffuse media, e.g. agar containing milk, may cause difficulties because the contrast between colonies and background is too limited. When Petri dishes known to be difficult to count by an automatic colony counter are eliminated, the counts obtained are as accurate as those obtained manually, although the counts are usually 10-15% lower, owing to the exclusion of peripheral colonies.

15.5 Electronic Colony Counters

Another approach to count colonies is electronic micro-colony counting using particle counters (coulter principle) the micro-colonies grow in a solid nutrient medium containing gelatin that is melted before counting. This method can also be applied to bacteria grown on selective media.

15.6 Surface Count

Surface colonies grow faster and can be counted after 24 h. Where it is desired to produce surface colonies, the spread method or the drop method can be applied. In the spread method, 0·1 ml of the 10-fold dilutions are transferred to, and spread over, the dry surface of a solid agar medium. After incubation, plates on which the overall growth has been retarded due to overcrowding of the colonies must be discarded and the rest are counted.

15.7 Membrane Filtration

When the bacterial count of a sample is low, and the sample or its dilution can be efficiently filtered, the membrane filtration method is most suitable. After the sample is passed through the membrane, the latter is placed on a solid agar medium, or on a filter pad that has been saturated with liquid medium. After incubation, the bacteria grow into colonies on the membrane. The test is only effective, if the number of colonies per membrane is in the range of 10-200 (optimum: 50). When the medium does not contain an indicator, it is advisable to stain the membrane, e.g. by gently flooding the surface with a 0·01 % aqueous solution of malachite green-oxalate, to confirm the count.

15.8 Most Probable Number (MPN)

MPN test makes use of a statistical technique to determine low counts of bacteria in dairy products. Three sets of three or five tubes, each containing sterile medium, are prepared and inoculated from each of three consecutive, 10-fold dilutions. Tubes showing bacterial growth after incubation are positive. From the number of positive tubes in each set of three or five tubes, the MPN of bacteria are counted per unit of sample, as per MPN table. When more than three dilutions are made, only the results from three consecutive dilutions are significant. The highest dilution which gives positive results in all of the tubes, and the next two succeeding higher dilutions, should be chosen. When the weight or volume of sample in the first dilution is 10 or 100 times less than the weight or volume listed in MPN tables, then the count tabled will be multiplied by 10 and 100, respectively.

15.9 Counting Single Bacteria

Apparatuses have been developed that are able to, rapidly, count the number of viable single bacteria in milk. The bacteria, stained with a fluorescent dye (acridine orange), are detected by an image analyzer­ microscope or alternatively, a small quantity of the diluted 'milk' is applied to a rotating disc that spins in front of a narrow slit in the detector. The fluorescent light emission of the bacteria is transformed into pulses of electricity that in order to avoid interference due to other particles, like fat globules, protein micelles and somatic cells, these materials may be initially separated by centrifugation; the bacteria in the 'milk' are then clearly visible. The separation, dilution, chemical treatment and staining are performed automatically. The same principle is also applied for the counting of somatic cells in milk.

15.10 Qualitative Methods

Dye reduction tests are indirect methods of assessing the microbiological quality of milk. These are based on the metabolic activity of the microorganisms. A correlation is made between the time required for the reduction of dye and probable number of bacteria in milk.

The principle of these tests is to add dyes, like methylene blue, resazurin or trimethyltetrazolium chloride, to milk or liquid dairy products, and to measure the color change after incubation. The color change is based on the dehydrogenase activity of the bacteria present in sample.

Dehydrogenases are mainly flavin enzymes that transfer hydrogen ion from a substrate to biological acceptor or redox dye which then undergo a color change. When bacterial cells multiply in milk these consume dissolved oxygen that acts as an electron acceptor. As more and more oxygen is used, it gets depleted, and the dye starts acting as electron acceptor instead of oxygen. Consequent to the lowering of oxygen, the redox potential shows a decline and this decrease in redox potential changes the color of the dye. Such dyes, methylene blue and resazurin are known as leucoform dyes. The rate of reduction of dyes that depends on the enzyme activity has been used as an index of number of microbes present in milk. The time needed to change or to decolorize the dye is an index of the bacterial load of the product tested.

Dye reduction tests however, are of little value as an index of the bacterial count of refrigerated milk, because this relationship is poorly correlated. The reason is that most of the bacteria of refrigerated milk are in a dormant state. Furthermore, a relatively large proportion of bacteria present are psychrotrophs. These micro-organisms, compared with lactic acid bacteria, have a low dehydrogenase activity, a characteristic that contributes to the low correlation between bacterial count and methylene blue reduction time or resazurin disc reading. In order to achieve the same reliability of, methylene blue test for non-refrigerated milk, approximately twice as many samples of refrigerated milk 'have to be tested. Pre-incubation (13-18°C for 16-24h) has been shown to be unsuccessful in improving the relationship between bacterial count before incubation, and the results of a metabolic activity test after pre-incubation. Similar results were obtained with the nitrate reductase test, which is only suitable as a method of detecting samples with a content of coliforms, psychrotrophs and other contaminating bacteria in excess of acceptable standards.

Because of these problems, the suitability of other metabolic activity tests is also investigated. The break-down process that takes place in milk can be classified as glycolysis, proteolysis and lipolysis and often the heat-resistant enzymes, that can survive processing, are produced by bacteria. A measurement of catabolic activity of the bacteria present would thus give a better index of the total bacterial load than either dye reduction tests or the colony count.

The key substance of most microbial metabolic processes is pyruvate. Thus, pyruvate content of milk products is a function of types, numbers and activity of the micro-organisms that are present. The natural pyruvate content of raw milk is approximately 0·5 mg per kg, that would correspond to a plate count of 50000-100000.

Adenosine triphosphate is present in all living cells and serves as the universal energy carrier during catabolic reactions. Milk contains free ATP originating from somatic and bacterial cells. The ATP content of bacterial cells is fairly constant and has been used as an index of the bacterial load of milk. The ATP determination is used as an indirect assay of the bacterial quality of milk sample. The ATP test does, however, have certain limitations that prevent it from being recommended as a rapid bacteriological test. These limitations include lack of sensitivity regarding bacterial loads of less than 100000 ml-1, over estimation caused by residual somatic ATP, and difficulties regarding standardization because of residual somatic ATPase.

During active growth, microorganisms alter certain non-ionic sub­stances (e.g. lactose and glucose) to ionic compounds, like pyruvic or lactic acid. An accumulation of these ionic compounds leads to increased resistance to the flow of an alternating electrical current (impedance). The electrical impedance in a culture medium remains fairly constant until a threshold of 106-107 cells per ml is reached, when major changes in impedance start to occur. The time taken to reach the threshold value is indicative of the initial bacterial load in raw milk. A limitation of this method is that while a very good correlation exists between the impedance detection time and the standard plate count, if the predominating micro-organisms are mesophilic, a very poor relationship exists, if psychrotrophs are dominant.

Another interesting metabolic activity test is the oxygen test carried out by means of an O2 electrode. The magnitude of decrease in oxygen increases in relation to the initial bacterial count. However, due to the different procedures of handling milk and liquid dairy products, such as different time lapses until testing, and/or the time and speed of stirring, a direct measure of O2 content does not give a clear indication of bacterial count. The samples have to be saturated with air (e.g. by shaking) followed by a specified period of incubation to obtain a close relationship between O2 and bacterial counts. The multiple regression relationship between O2 and bacterial counts was consistent enough (r = 0·70) to recommend the 6 h O2 test as an index of total mesophilic and psychrotrophic count.

In conclusion, it may be stated that metabolic activity tests give an indication of the relative metabolic rates of the various microorganisms, while the bacterial count gives information about the effectiveness of production hygiene. Metabolic reactions caused by bacteria can be measured when the bacterial count exceeds 50000-100000 per ml, but they are difficult to measure in high quality products.

Pre-incubation of samples will increase the activity of the bacteria present, but this increase is, however, not consistently related to the initial bacterial count. When the bacteriological quality of product has reached a high level, the counting of bacteria or the determination of specific groups of bacteria (psychrotrophs, coliforms, thermoduric bacteria, etc.) is recommended in preference to metabolic activity tests.