Microbial analysis of milk: a. Dye reduction test (using methylene blue and resazurin) b. Total bacterial count. c. Brucella ring test and tests for mastitis. d. Somatic cell count
Microbial analysis of milk:
a. Dye reduction test (using methylene blue and resazurin)
b. Total bacterial count.
c. Brucella ring test and tests for mastitis.
d. Somatic cell count
a. Dye reduction test (using methylene blue and resazurin)
Methylene Blue Reduction Test (MBRT):
The methylene blue reduction test is based on the fact that the color imparted to milk by the addition of a dye like methylene blue will disappear more or less quickly. The removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism cause the color to disappear. The agents responsible for the oxygen consumption are the bacteria. Though, certain species of bacteria have considerably more influence than others, it is generally assumed that the greater the number of bacteria in milk, the quicker will the oxygen be consumed, and in turn the color will disappear. Thus, the time of reduction is taken as a measure of the number of microorganisms in milk. Although, it is likely that it is more truly a measure of the total metabolic reactions proceeding at the cell surface of the bacteria. The test is useful in assessing the bacteriological quality of milk by determination of the time taken for the reduction of methylene blue in milk indicated by its colour change.
Principle
Oxidation reduction potential of a substrate may be defined generally as the chemical process in which the substrate either loses or gains electrons. When an element or compound loses electrons the substrate is said to be oxidized, while a substrate that gains electrons becomes reduced.
Milk, as it exists in the udder has a sufficiently low redox potential to reduce the methylene blue immediately. The processes like milking, cooling, dumping etc. raise the oxidation reduction potential of milk to +0.3V, because of the incorporation of atmospheric oxygen. At this particular O-R potential, methylene blue is in oxidized state. When bacterial cells multiply in milk these, consume dissolved oxygen and as more and more oxygen is used and gets depleted, the dye starts acting as electron acceptor instead of oxygen. As the oxidation reduction potential decreases from + 0.06 to 0.01 V, methylene blue gets reduced. One atom of hydrogen is taken up by the double bonded nitrogen of the dye that converts it into colourless state. The greater is the number of microorganisms in milk, the greater is the metabolic activity and the faster is the reduction of methylene blue.
MBRT is a rapid, sensitive and low cost, yet a simple quantification method to evaluate viable count during a growth experiment. It is widely used in dairy industry to determine the microbial load in the milk. This test involves the addition of methylene blue into a milk sample and measuring the time required for decoloration. The disappearance of color in a short time indicates a high microbial load. The disappearance of color is due to the removal of oxygen from milk and formation of reducing substances during bacterial metabolism.
Methylene blue in a sample containing microorganisms gets reduced to dleukoT or colorless form of the dye at the cell surface via reductase enzymes present in the cell membrane. This colorless form of methylene blue is uncharged, lipophilic, and enters cells by diffusion across the plasma membrane, where it is re-oxidized and thus sequestered within the cells. If oxygen is available, reduced methylene blue can be oxidized by the mitochondrial electron transport system that will result in the reappearance of the blue color.
Although the exact mechanism of dye reduction is not clearly known, some reports available suggest that MB is reduced by transmembrane reductases. This mechanism is applied to evaluate the microbial load in a liquid medium. The shorter time required for the disappearance of the blue color is indicative of a higher microbial load. It is assumed that greater the number of microorganisms, more the oxygen demand and lesser the oxygen concentration in medium resulting in the faster disappearance of the color. This fact has been used as a broad indicative test of a microbial load representing microbial quality of milk.
Standard solution of methylene blue
One tablet of methylene blue thiocyante or chloride is dissolved in 200 ml of cold sterile glass distilled water by gentle heating to facilitate dissolving and then add another 600 ml distilled water.
Procedure
The samples of milk are mixed thoroughly. If the milk is in a bottle/ sachet, it shall be inverted at least 25 times to mix the fat uniformly with the milk. Take 10 ml of milk into a test tube and add 1 ml of standard methylene blue solution. Invert the test tube to mix the milk and methylene blue solution. Place the test tube in a thermostatically maintained water bath at 37degree C and note down the time of incubation. Observe the test tubes after 30 min for decolourization reduction of dye. If there is no decolourization the tubes are inverted once and transferred to the water bath for further incubation. After 30 min, continue to observe for the reduction of dye at an interval of every one-hour. The milk shall be regarded as decolorized, when the entire column of milk is completely decolorized or is decolorized up to 5 mm of the surface.
Factors affecting the MBRT
These are factors that affect the MBRT and therefore, the steps of operation should be uniform.
1. Since, the oxygen content must be used up before the colour disappears; any manipulation that increases the oxygen content affects the test.
a.Cold milk holds more oxygen than warm milk
b. Pouring milk back and forth from one container to another increases the oxygen, and
c. During milking time much oxygen may be absorbed.
2. The rate of reduction of dye depends on the type of microorganism
a. Coli forms appear to be the most rapidly reducing microorganisms,
b.Closely followed by Lactococcus lactis spp. lactis, some of the faecal Streptococci, and certain micrococci.
c. Psychrotrophs reduce methylene blue very slowly.
3. Presence of a large number of leucocytes as in mastitic milk will affect the reduction time materially.
4. Light hastens reduction process and therefore, the tests should be carried out in relatively low light.
5. Concentration of dye should be uniform as an increased concentration lengthens the time of reduction.
6. Increasing the incubation temperature augments the activity of the bacteria and therefore shortens the reduction time.
7. Creaming of milk causes a number of micro-organisms to be removed from of milk and brought to the surface with the rising fat. This factor causes variations in the reduction time, since the bacteria are not evenly distributed.
8. The accuracy of test is increased, reduction time shortened and decolourization more uniform, if the samples are periodically inverted during incubation.
Resazurin Reduction Test (RRT)
One tablet of Resazurin is dissolved in 50 ml of cold sterile glass distilled water by gentle heating to facilitate the dissolving. This is the bench solution for direct use and should always be used as fresh.
Alternatively dissolve 0.05 g of resazurin powder in 100 ml of distilled water and boil the contents for 30 min. This will make a standard solution of 0.05%, which should always be kept in a cool and dark place, stored in an amber coloured bottle. The bench solution (0.005%) for regular use should be prepared freshly by diluting the standard solution with distilled water i.e. 1 ml of standard solution with 10 ml of distilled water.
Procedure
� Take 10 ml of milk into a test tube and add 1 ml of working solution of Resazurin solution.
� Put air tight closure to prevent oxygen entry
� Invert the test tubes to mix the milk and Resazurin solution.
� Place the test tubes in a thermostatically maintained water bath at 37 C and note down the time of incubation (10 min or 1 h).
� At the end of incubation match the colour of the milk with one of the colour standards of Resazurin disc.
Advantages of dye reduction test
� Used for estimating the suitability of milk for liquid consumption.
� These tests are cheaper and less time is required.
� In case of SPC, clumps of microbes are recorded as one colony, whereas the rate of decolorizing of dye is due to the combined metabolic effect of each bacterium in the clump.
� With the help of these tests the activity is measured rather than the number of bacteria.
� Unlike the artificial media used in SPC, in milk the natural environment for microbes is present.
� In case of RRT, the results can be measured in a shorter time.
� Some of the bacteria capable of reducing the dye may not develop colonies on the medium used in SPC.
Disadvantages
� Rate of reduction of dye varies considerably and is related to species and the rate at which different micro-organisms grow at a particular temperature.
� Inhibitory substances like penicillin and other antibiotics prevent the growth of bacteria and thus increase the reduction time.
� Not suitable for classifying milk with low bacterial counts of less than 105 /ml.
� Reduction capability may vary because of variation in proportion of bacteria carried into cream layer by the rising fat globule.
� These tests do not give indication for the type of micro-organisms present.
� Temperature of incubation used during these tests is not the optimum for majority of the micro-organisms present in milk.
� Not suitable for testing quality of pasteurized milk intended for processing because of the low number of micro-organisms.
� Require continuous attention until reduction takes place.
b. Total bacterial count.
Total bacterial count (TBC) is the count of the number of bacterial colony-forming units present in the milk sample, giving a quantitative evaluation of the total number of bacterial colony-forming units per milk milliliter
. TBC is an indicator of hygienic and cleaning conditions in milk production and handling in the farm, as well as of its adequate refrigeration.
It is, therefore, of extreme importance to control and follow the procedures of dairy systems.
Bulk tank total bacterial count (TBC) is the first and principal tool used by technicians and farmers to evaluate the efficiency of production processes, cleaning and sanitation practices, and to predict the keeping quality and shelf life of milk and dairy products.
It is therefore a very useful variable for monitoring and improving flock milk quality in dairy cattle.
However, little information is available on TBC in dairy sheep and there are no known studies that empirically investigate the effect of variation factors on TBC under field conditions in this species.
Indeed, TBC are affected by a number of sources of variation and an attempt should be made to identify them and assess some of their implications in hygiene practices or milk payment schemes
Standard Plate Count (SPC)
The most basic milk bacteria test is the Standard Plate Count (SPC). Other names for this test are the raw count or the total bacteria count (TBC). There are several approved ways of conducting the Standard Plate Count, but the time and temperature conditions used for this test are standardized.
The reason for running an SPC is to determine how many bacteria are present in the farm bulk tank. However, test results show bacteria in the sample when it was received at the lab. If the sample is contaminated or mishandled, bacterial numbers will increase between the bulk tank and the lab. The sample will then fail to accurately reflect the tank count.
A sample for SPC is placed on growth media and incubated at 90 degrees F (32.2° C) for 48 hours. Under these conditions, all bacteria rapidly grow in the presence of adequate food to form visible colonies. After 48 hours, visible bacteria colonies are counted. Based on the sample dilution, a final number is calculated and expressed as the number of colony-forming units per milliliter or CFU’s/ml.
Bacteria counts in raw milk should be compared to appropriate benchmark numbers. The legal maximum based on PMO (Pasteurized Milk Ordinance) (USA) guidelines is 100,000 cfu/ml. A realistic and achievable goal is less than 10,000 cfu/ml. Many producers consistently attain 5,000 cfu/ml.
Bacterial species that grow rapidly under warm conditions (90°F, 32.2°C) produce high SPC counts. This includes all of the typical mastitis pathogens including Strep agalactiae, Staph aureus, Strep non-ag species and most other mastitis causing bacteria. Species that are shed from infected quarters cause elevated bacterial counts in raw milk. Strep bacteria tend to be released in very large numbers and can create elevated bulk tank SPC counts. Staph aureus is not normally released in large quantities into raw milk so it is not likely to elevate the SPC. Coliforms normally are not shed in large numbers into raw milk, plus cows with a serious case of coliform mastitis are typically diverted from the tank.
Cold conditions significantly reduce the growth rate of most mastitis-causing bacteria. Therefore, keeping milk very cold at all times is the best way of minimizing growth. Any milk cooling problems will also increase the SPC.
Common environmental bacteria may also cause elevated bulk tank bacteria counts.
These bacteria can enter the milking system via dirt, contaminated water or manure. Fall-offs, liner slips or careless rinsing of the milking cluster can carry contamination into the system.
Dirty milking systems provide a place for any of these bacteria to lodge, grow and develop into large numbers. The bacterial buildup may be transferred to the tank as fresh milk passes over it and becomes inoculated. Dirty pipelines, unwashed zones in the milk handling system, long milking times (8-10 hrs), no sanitizing, etc., are all factors that can cause bacterial buildup in the system. When these occur, the tank SPC can rise.
c. Brucella ring test and tests for mastitis.
Brucellosis is primarily a disease of dairy cows causing economic losses to the livelihoods of many farmers around the world. In recent years, the cases of bovine brucellosis have been increased in India, possibly due to increased trade and rapid movement of livestock. Despite of various preventive and control measures being followed in India, there is still a high potential for the transmission and spread of B. abortus due to its widespread prevalence (Renukaradhya et al., 2002). Further, the risk of acquiring infection from unpasteurized milk is a major cause as raw milk is traditionally consumed in India where the hygienic aspects are not always sufficiently considered A number of serological tests are widely used for the diagnosis of brucellosis because infected cattle may or may not produce all antibody types in detectable levels. The MRT, first described in Germany by Fleischhauer (1937), is used as a routine periodic test for brucellosis free herds and for identifying infected herds. The MRT is an agglutination test conducted on fresh milk collected from dairy cattle, but it does not work on pasteurized or homogenized milk (Fleischhauer, 1937). The MRT, which detects IgM and IgA antibodies bound to fat globules, may have wide acceptability as it is cost effective, easy to perform and can cover a large population in a short time
Milk Ring Test (MRT) for identifying infected cows: The milk ring test is the most practical method for locating infected dairy animals and for surveillance of brucellosis-free herds. The test was performed by adding 30 μl (0.03 ml) of B. abortus Bang Ring Antigen (hematoxylin-stained antigen manufactured by the State Biological Laboratory, Institute of Veterinary Preventive Medicine, Ranipet, India). The height of the milk column in the tube was kept up to 25 mm. The milk (antigen) mixtures were incubated at 37°C for 1 h, together with positive and negative control samples. Agglutinated Brucella cells were picked up by fat globules as they rose, forming a dark cream layer on the top of the sample. A strongly positive reaction was indicated by formation of a dark blue ring above a white milk column. The test was considered negative if the color of the underlying milk exceeded that of the cream layer and when the cream layer was normal. Samples were read as negative, 1+, 2+, 3+ and 4+ depending on the intensity of color in the cream layer.
d. Somatic cell count
The Somatic Cell Count (SCC) is a main indicator of milk quality. The majority of somatic cells are leukocytes (white blood cells) - which become present in increasing numbers in milk usually as an immune response to a mastitis-causing pathogen - and a small number of epithelial cell which are milk-producing cells shed from inside of the udder when an infection occurs.
The SCC is quantified as the number of cells per ml of milk. In general terms:
- An individual cow SCC of 100,000 or less indicates an 'uninfected' cow, where there are no significant production losses due to subclinical mastitis.
- A threshold SCC of 200,000 would determine whether a cow is infected with mastitis. Cows with a result of greater than 200,000 are highly likely to be infected on at least one quarter.
- Cows infected with significant pathogens have an SCC of 300,000 or greater.
The SCC in the milk increases after calving when colostrum is produced before the cow settles into lactation, and tends to rise towards the end of lactation, most likely due to the concentrating effect of lower amounts of milk being produced. SCCs vary, however, due to many factors, including seasonal and management effects.
Dairy farmers are financially rewarded for low herd SCCs and penalised for high ones, because cell counts reflect the quality of the milk produced and how mastitis can affect its constituent parts, having implications for its keeping abilities, its taste and how well it can be made into other dairy products such as yoghurt or cheese. Milk contracts often define several SCC 'thresholds' and any respective bonus for attaining them. Milk with an SCC of more than 400,000 is deemed unfit for human consumption by the European Union.
Essentially, a lower SCC indicates better animal health, as somatic cells originate only from inside the animal's udder. SCC monitoring is important because as the number of somatic cells increases, milk yield is likely to fall, primarily due to the damage to milk-producing tissue in the udder caused by mastitis pathogens and the toxins they produce, particularly when epithelial cells are lost.
A particularly low SCC is sometimes regarded as a sign of poor immune response, but in general terms this need not be necessarily true; it may be the case that there issimply a low level of current infection. Immune response is best measured by how quickly the immune system reacts to the disease challenge, not how many white blood cells are present before infection occurs.
Cell counts tend to reflect a response to contagious mastitis pathogens: the Bactoscan count, on the other hand, indicates the level of bacterial contamination from external sources, such as insufficient cleaning of the milking equipment or poor udder and teat preparation prior to milking, and can indicate a high level of environmentalpathogens.
Referene link: http://ecoursesonline.iasri.res.in/mod/resource/view.php?id=5014
https://www.tandfonline.com/doi/full/10.4081/ijas.2013.e75#:~:text=Total%20bacterial%20count%20(TBC)%20is,Agricultura%2C%20Pecu%C3%A1ria%20e%20Abastecimento%2C%202003
https://www.sciencedirect.com/science/article/pii/S0022030206721178
http://www.milkproduction.com/Library/Scientific-articles/Milk--milking/Bulk-tank-bacteria-concerns/
file:///C:/Users/STUDENT%20211/Downloads/19045-Article%20Text-68368-1-10-20140602%20(1).pdf
https://ahdb.org.uk/somatic-cell-count-milk-quality-indicator
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