Research articles

By Mr. Shanmugavel Lingathurai , Dr. P. Vellathurai
Corresponding Author Mr. Shanmugavel Lingathurai
Entomology Research Institute, ERI, Loyola college,Chennai, India - India 600034
Submitting Author Mr. Shanmugavel Lingathurai
Other Authors Dr. P. Vellathurai
Department of Biotechnology, The American College, Madurai- 2,Tamil Nadu, Assistant Professor, Department of Biotechnology, The American College, Madurai- 2,Tamil Nadu - India 625002


Indian cow milk, Microbial quality, Safety, TPC, E.coli, S.Aureus

Lingathurai S, Vellathurai P. Bacteriological Quality And Safety Of Raw Cow Milk In Madurai, South India. WebmedCentral MICROBIOLOGY 2010;1(10):WMC001029
doi: 10.9754/journal.wmc.2010.001029
Submitted on: 20 Oct 2010 10:11:07 AM GMT
Published on: 20 Oct 2010 03:19:29 PM GMT


The microbiological quality and safety of raw milk from 60 dairy farms in Madurai were determined. Milk samples were collected at 60 centers from four regions, namely northern, eastern, western and southern (NEWS) according to stratified random sampling design. Samples were analyzed for Total plate count (TPC), psychrotrophs, thermophiles, Staphylococcus aureus, coliform, Escherichia coli 0157: H7 and Salmonella. The mean counts per ml for TPC, psychrotrophs and thermophiles were 12.5x106, 5x103 and 6.85x103 respectively. From the 60 milk samples tested, coliform bacteria contaminated approximately 90% and 70% were E. coli positive, with mean counts ranged from 103to 104 cfu ml-1. S. aureus was isolated from more than 61.7% of the samples and the mean count per ml was 6.2x103. Meanwhile, E. coli 0157: H7  was also detected in 39 (65%) samples. However, Salmonella was only detected in 8 (13.3%) of the samples with the southern region having the highest frequency of isolation.


Raw or processed milk is a well-known good medium that supports the growth of several microbes with resultant spoilage of the product or infections / intoxications in consumers (1 & 2). Microbes may gain entry into raw milk directly from dairy cows experiencing sub clinical or clinical mastitis (3), from the farm environment particularly the water source (4) and utensils used for the storage of milk on farm or during transportation (5).

A number of bacteria including S. aureus, Escherichia coli and Salmonella have been recovered from raw milk (6) and some of these have been determined to be pathogenic and toxicogenic, and implicated in milk- borne gastroenteritis (6, 7,& 8). In recent year’s E. coli 0157: H7 strain has become very important milk-borne pathogen and cattle are considered its main reservoir (9 &10).

In India raw milk is traditionally consumed at the small farms where it is produced or fermented into different products. During scaling up, the hygienic aspects are not always sufficiently considered. The risk of contaminated and pathogen containing products could therefore be even greater than when the milk is processed at household level (11). The delayed time of milking process performance and low hygienic conditions were possible to grow the microorganisms. The contamination leads to pathogenic microbes grows well the milking media. No matter how fast the microorganisms multiply, the contamination would not be detected until the incubation time is over and the contamination sample is taken for analysis.

The importance of various etiological agents in milk borne disease has changed dramatically over time. However, more than 90% of all reported cases of dairy related illness continued to be of bacterial origin, with at least 21 milk borne or potentially milk borne diseases currently being recognized (12). Pathogens that have been involved in food borne outbreaks include Salmonella, Staphylococcus aureus and E. coli. The presence of these pathogenic bacteria in milk emerged as major public health concerns, especially for those individuals who still drink raw milk (13). Most recently E. coli 0157: H7 has become serious threat to the dairy industries ranging from mild diarrhoea to potentially fatal hemolytic uremic syndrome (HUS), hemorrhagic colitis and thrombotic thrombocytopenic purpura (14, 15 & 16). Keeping fresh milk at an elevated temperature together with unhygienic practices in the milking process may also result in microbiologically inferior quality. Apparently, these are common practices for small-scale Asian produce fresh milk and sell it to consumers (17).

The output of dairy and dairy products from India is increasing day by day in their international market. Considering its economic potential, extensive and intensive exploitation of cow milk can both contribute to the nutrient requirements of the Indian public and increase the income of farmers. In view of the growing public awareness about food safety and quality, knowledge of the microbial and chemical composition of milk is of great significance for further development of its hygienic processing into high quality consumer products. Until now, information on such aspects is scant and scattered. Thus this study was carried out to investigate the microbiological quality and safety of local cow milk.


A total 60 raw cow milk samples were collected from 60 dairy farmers who send their milk-to-milk centers (MC) in Madurai. Farmers involved in the study were chosen according to stratified experimental design, where by Madurai was divided in to four regions. Samples were collected in the early morning.

Approximately 100-200ml milk was aseptically sampled from containers (Pails, buckets or Churns) of bulk milk from each individual farmer into a sterile bottle. It was collected immediately after milking using hand or machine in to bulk milk containers at ambient temperature (28-30oC). Samples were delivered to the laboratory in a cool box at less than 4oC within 1-2 h of collection and tested immediately upon arrival.

 Microbiological analysis: Initially, 25ml of sample were dispensed into a sterile bag containing 225ml of sterile water and homogenized with stomachar (Bagmixer 400. Interscience). Subsequent serial decimal dilutions of milk were prepared in saline water. Enumeration of total plate count, psychrotrophs, thermophiles, coliform, E. coli and S. aureus were carried out as described by standard methods of the American Public Health Association (18). To enumerate the numbers of coliforms bacteria and E. coli in milk, a three tube most probable number (MPN) technique was employed. Positive tube from MPN was streaked onto Eosine Methylene Blue (EMB) agar and then incubated overnight at 35oC. Typical isolates were confirmed based on their iMViC pattern. Baird Parker Agar (Hi Media, India) was used for quantitative detection of S. aureus. Representative colonies with typical black appearance and surrounded by clear zone were picked and subjected to catalase and coagulase tests (Staphylex, Oxoid).

Detection of Salmonella was carried out according to the International Standard Organization protocol (19), and typical Salmonella colonies were confirmed using API 20E test kit (This kit used as identification for Enterobacteriaceae and other non-fastidious Gram-negative rods, which uses 21 standardized and miniaturized biochemical tests and a database). Milk samples (25ml) were inoculated into 225ml modified tryptic soy broth with Novobiocin (Hi Media, India) and incubated overnight at 35oC. Approximately 0.1ml of the broth then was streaked on to the surface of sorbitol MacConkey Agar (Hi Media, India) colorless colonies from SMAC Agar were streaked onto a modified EMB agar before confirmed with E. coli 0157: H7 latex test (Hi Media, India).

Statistical analysis

Bacterial load and mean counts of coliform, E. coli and S. aureus were statistically analyzed by one way Analysis of Variance. Significant differences between treatments were determined using Tukey’s multiple range test at P =0.05 with the help of SPSS 11.5 software.

Results & Discussion

Fresh cow milk collected from different farms were heavily contaminated by bacteria with a mean total plate count (TPC) of 12.5x106 cfu ml-1 (Table. 1). The highest mean value of TPC was found in milk from the eastern region with 13.9 x106 cfu ml-1, while the lowest mean value of 11.7x 106 cfu ml-1 was detected in milk obtained from the western region. Results from the analysis of variance (ANOVA) suggested that there was a significant difference (p≤0.05) in bacterial loads between the two regions. The presence of bacteria in milk samples may not be due to infection of the udder itself, but arise from the teat duct (20). The bacteria can be carried into milk duct of the cow during milking by suction of the milking machine and then flushed out during subsequent milking without causing clinical symptoms of infection. A TPC less than 106 cfu ml-1 is used as a basic standard by milk centers in the price incentive program.

The milking process, especially the equipment associated with it introduces the greatest proportion of microorganism in cow milk (21). According to Aumaitre, 1999 (22) the health of the dairy herd, milking and pre storage conditions are also basic determinants of milk quality. Bacteria may enter milk through the udder and most of the organisms in raw milk are contaminants from the external surface of udder, milking utensils and handlers (23). Various types of equipment and utensils, such as milking machines, pails, cans and milk churns are used in handling milk on the farm. In order to reduce contamination of milk, utensils used for milking should be rinsed, cleaned using detergent and disinfected immediately after use (11 & 24). The results for psychrotrophs and thermophile contamination in raw milk are shown in table 1. Counts for psychrotrophs and thermophiles ranged between103 and 104 cfu ml-1 with an average count of 5.0x103 and 6.85x103 cfu ml-1, respectively. Samples taken from the eastern region had a significantly higher (p<0.05) psychrotrophic count as well as thermophilic load, as compared with other regions. Nevertheless, the high TPC was not significantly correlated with the number of psychrotrophs(r = 0.42) and thermophiles (r = 0.48).

The psychrotrophs count was considered lower than the count for milk produced in temperate countries, which could reach as high as 106 cfu ml-1 (25). Generally, psychrotrophic organisms were represented by both Gram-negative and Gram-positive bacteria such as, Pseudomonas, Flavobacterium, Bacillus, Clostridium and Mycobacterium (26 & 27). Champagne et al., 1994 (28) indicated that the quality of dairy products may be affected by heat resistant enzymes or metabolites secreted by psychrotrophs in raw milk during the cold storage.

Table 2 displays mean counts of coliform, E. coli and S. aureus of locally produced raw milk. Nearly 90% of the samples collected were contaminated by Coliform bacteria (Table 3), with a mean number of colonies 88.3 per cent. The existence of coliform bacteria may not necessarily indicate a direct fecal contamination of milk, but more precisely as an indicator of poor hygienic and sanitary practices during milking and further handling. E. coli was isolated from 42 (70%) of the milk samples tested, with none of the regions supplying milk free from the organism (Table 3). Samples with the highest prevalence (80%) of E. coli originated from the Western zone, while the lowest prevalence (60%) was detected in milk from northern region. Although global importance of E. coli as a causative agent for diarrhoeal illness has decreased markedly over the past 50 years following the implementation of improved sanitary practices, it is still the major cause of illness in under- developed nations (13). Detecteion of E. coli in milk often reflects fecal contamination although environmental coliforms have also been detected in milk (29).

Nearly 61% of the milk samples analyzed were positive S. aureus with a frequency of detection ranging from 53% in Eastern region to 67% in western and northern regions which showed a significantly higher S. aureus count than other regions (Table 3). These, may most probably due to some of the samples from the regions were highly contaminated with S. aureus and also due to the differences in milking technique. However, the rate of isolation of the organism was very much lower than (˃40%) reported from other tropical countries (30). Leonard and Markey, 2008 (34) stated that S. aureus is widely recognized as a major causative agent of clinical and subclinical mastitis in dairy cattle. Overall 39 of 60 (65%) milk samples tested were positive for E. coli 0157: H7; in raw milk samples collected from the northern region was the highest 73.3% followed by samples from eastern and western regions with prevalence of 66.7% respectively (Table 4). The prevalence of E. coli 0157: H7 in local milk seems to be higher than (˂76%) the published data reported by (31 & 32). The difference in the frequency may be partially due to the fact that in the present study, selective enrichment medium was used before streaking onto Sorbitol MacConkey agar.

Although the consumption of undercooked group beef is still the traditional mode for E. coli 0157: H7 infection, illness resulting from ingestion of contaminated raw milk is increasing. The environmental niches for E. coli 0157: H7 have not yet been clearly established. However, dairy cattle appear to be a major reservoir for this pathogen, even though with a very low prevalence (14 & 33). E. coli 0157: H7 is apparently confined to the intestinal tract of dairy cattle and perhaps other animals as well. Given the higher possibility for contamination of milk at dairy farms, consumption of such raw milk should be avoided. Flushing animal houses with water to remove manure are fairly common practice in most dairy farms. Although it is effective and quickly removes manure, this practice may distribute fecal flora throughout the farm environment, thus exposing large number of animals to the organism. All aspects of hygienic handling, strict maintenance of refrigeration at lower than 4oC and effective control measures are all primary concerns for quality assurance in the dairy industry (27).

The incidence of Salmonella spp in local raw milk was still low, as only 8 of 60 milk samples were found positive for this organism (Table 4). Samples from southern region of the district seem to have a higher rate of isolation (3%), while the lowest (1%) was milk samples from eastern region. All salmonellae are of public health concern having the ability to produce infection ranging from a mild self-limiting form of gastroenteritis to septicemia and life threatening typhoid fever (2). Thus, although their occurrence in local milk is low, they still pose a health risk to consumer if milk is consumed without any heat treatment. This problem is particularly evident in developed countries like England and Wales, where the most frequently reported out-breaks were salmonellosis associated with the consumption of raw milk and products (6).

Since the microbiological limits of raw milk are not established in this country: it is very likely that milk should often be tested, if found positive for pathogens then withheld from human consumption. The production of high-quality milk and safe milk should be of great importance to the economy of the farmer and the sustainability of the dairy industry in this country.


Therefore, poor milk quality has often been considered as one of the major reasons for losses and results in deduced income for the stallholder dairies in Madurai.


The authors are grateful to Department of Biotechnology, The American College, Madurai for providing lab facilities and encouragement during the study period.


1. Murinda SE, Nguyen LT, Man HM Almedia RA.  Detection of sorbitol negative and sorbitol-positive shiga toxin-producing Escherichia coli, Listeria monocytogenes, Campylobacter jejuni and Salmonella species in dairy farm environments. Foodborne Pathogens and Disease 2004; 1: 97-104.
2.Oliver SP. Jayarao BM. Almedia RA. Food borne pathogens in milk and the dairy environment food safety and public heatlth implications. Foodborne Pathogens and Disease 2005; 2: 1115-1129.
3. Rodojcic-Prodaova D, Necev T. Most common agents of subclinical mastitis in cows on private and communal farms in the republic of Macedonia vet glasnik 1991; 45: 745-747.
4. Eberhart RJ. Coliform mastitis. Journal of American Veterinary Association 1977; 170: 1160-1163.
5. Freedman, B. (1977) Milk quality. In sanitarials handbook: theory and administrative practice for environmental health fourth ed. New Oreleans, USA, Peerless publishing. pp564-589
6. De Buyser, M.L. Dufour, B. Marie, M. and Lafarage, V. (2001) Implications of milk and milk products in food borne diseases in France and in different industrialized countries. International Journal of Food Microbiology 67:1-17.
7. Bergdoll, M.S. (1979). Staphylococcal intoxications. In Food-borne Infections and Intoxications, 2nd Ed. (H. Reimann and F.L. Bryan, eds.) pp. 443–490, Academic Press, New York, NY.
8. Maguire, H., Cowden, J., Jacob, M., Rowe, B., Roberts, D., Bruce, J., Mitchell, E. (1992) An outbreak of Salmonella dublin infection in England and Wales associated with a soft unpasturized cows milk cheese, Epidemiology Infection 109: 389-396.
9. Betts, G.D. (2000) Controlling E. coli 0157: H7. Nutrition and Food Science 30: 183-186.
10.  Karmali, M.A. (1989) Infection by verocytotoxigenic producing Escherichia coli. Clinical Microbiology 2: 15-38.
11.  FAO and WHO (1997) General requirements (food hygiene). Codex Alimentarius, Vol.1B (suppl). Food and Agriculture Organization, Rome.
12.  Bean, N.H. Goulding, J.S. Lao, C. and Angulo, F.J. (1996) Surveillance of food borne disease outbreaks- United States, 1988-1992, Morbidity Weekly Report. 45: 55-5.
13.  Riser, E.T. (1998) Public health concerns in: Marth, E. H Steele, J. L.(Eds.), Applied Dairy Microbiology, Marcel Dekker, Inc, New York, pp263-403.
14.  Wells, J.G. Shipman, L.D. Gren, K.D. Sowers, E.G. Green, J.H. Cameron, D.N. Downers, P.P. Martin, M.L. Griffin, P.M. Ostroff, S.M. Potter, M.E. Tauxe, R.V. and Wachsmuth, I.K. (1991) Isolation of Escherichia coli serotypes 0157: H7 and other shiga like toxin producing E. coli from dairy cattle. Journal of Clinical Microbiology 29: 985-988.
15.  Bleem, A. (1994) E. coli 0157: H7 in raw milk a Review. In: Colins, C.O.F (Eds.), Animals Health Insight. USDA, APHIS, VS Center for Epidemiology and Animal Health. pp 12-58.
16.  Coia, J.E. Johnston, Y. Steers, N.J and Hanson, M.F. (2001) A survey of the prevalence of Escherichia coli 0157: H7 in raw cow’s milk and raw milk cheeses in southeast Scotland. International Journal of Food Microbiology, 66:63-69.
17.  Chye, F.Y. Aminah, A. and Khan, A.M. (1994) Microbiological quality of milk produced by three types of milking methods. In proceeding of the fifth ASEAN food conferences, Kuala Lumpur, Malaysia, pp26-29.
18.  Vanderzant, C. and Splittstoesser, D.F. (1992). Compendium of methods for the microbiological examination of Foods 3rd edition. American Public Health Association, Washington, DC, pp 32-45.
19.  ISO (1990) Microbiology- General guidance on the method for the detection of Salmonella . International Standard Organizations 150, ISO Geneeva, 6579.
20.  Ledford, R.A. (1998) Raw milk and fluid milk product. In : Marth, E. H., Steele, J. L (Eds.), Applied Dairy Microbiology, Marcel Dekker, New York, pp 55-64.
21.  Olson, J.C. and Mocquot, G. (1980). Milk and milk product. In : International Commission on microbiological specification for foods (Eds.). Microbial Ecology of Foods: Food Commodities, vol.2. Academic press, New York, pp 470-490.
22.  Aumaître, A. (1999). Quality and Safety of Animal Products. Livestock Production Science, 59: 113-124.
23.  Ayres, J.C. Mundt, J.O. and Sandinc, W.E. (1980) Microbiology of Foods. W. H Freeman, San Francisco. pp 42-56
24.  Dodd, F.H. and Phipps, R.H. (1994) Dairy management and health. In : Smith, A. J, (Eds.) milk production in developing countries. Centre for Tropical Veterinary Medicine, University of Edinburgh, Scotland, UK pp 258-271.
25.  Reinheimer, M.R. Demkow, M.R. and Calabrese, L.A. (1990) Characteristic of psychrotrophic microflora of bulk collected raw milk from the Santa Fe Area (Argentina). Australian Journal of Dairy Technology 45: (2) 41-46.
26.  Cousin, M.A (1982) Presence and activity of psychrotrophic microorganisms in milk and dairy products: a review. Journal of Food Protection, 45: 172-207.
27.  Sorhaug, T. and Stepaniak, L. (1997) Psychrotrophs and their enzymes in milk and dairy products : Quality Aspects. Trends in Food Science Technology 8: 35-40.
28.  Champagne, C.P, Laing, R.R. Mafu, D. and Griffiths, M.W. (1994) Psychrotrophs in dairy products their effect and their control. Critical Review in Food Science and Nutrition. 34:1-30.
29.  Shehu, L.M. and Adesiyun, A.A. (1990) Characteristic of strains of Escherichia coli isolated from locally fermented milk (‘nono;) in Zaria, Nigeria, Journal Food Protection 53: 574-577.
30.  Umoh, V.T. Adesiyun, A.A. and Gomwalk, N.E. (1990) Antibiogram of staphylococcal strain isolated from milk and milk products. Journal of veterinary Medical B 37:701-706.
31.  Adesiyun, A.A., Webb, L. and Rahman, S. (1995) Microbiological quality of raw cow’s milk at collection centers in Trinidad. Journal of Food protection, 58 (2): 139-146.
32.  Padhye, N.V. and Doyle, M.P. (1991) Rapid procedure for detecting enterohemorrhagic Escherichia coli 0157: H7 in food, Applied Environmental Microbiology 57: 2693-2696.
33.  Garber, L. Wells, S. Schroeder-tucker, L. and Ferris. K. (1999) Factors associated with fecal shedding of verotoxin-producing Escherichia coli 0157 on dairy farms. Journal of Food Protection 62 (4), 307–312.
34.  Leonard FC, Markey BK. (2008). Meticillin-resistant Staphylococcus aureus in animals: a review. Vet J.175:27–36.

Source(s) of Funding

The American college, Department of Zoology provided for funding

Competing Interests



This article has been downloaded from WebmedCentral. With our unique author driven post publication peer review, contents posted on this web portal do not undergo any prepublication peer or editorial review. It is completely the responsibility of the authors to ensure not only scientific and ethical standards of the manuscript but also its grammatical accuracy. Authors must ensure that they obtain all the necessary permissions before submitting any information that requires obtaining a consent or approval from a third party. Authors should also ensure not to submit any information which they do not have the copyright of or of which they have transferred the copyrights to a third party.
Contents on WebmedCentral are purely for biomedical researchers and scientists. They are not meant to cater to the needs of an individual patient. The web portal or any content(s) therein is neither designed to support, nor replace, the relationship that exists between a patient/site visitor and his/her physician. Your use of the WebmedCentral site and its contents is entirely at your own risk. We do not take any responsibility for any harm that you may suffer or inflict on a third person by following the contents of this website.

0 reviews posted so far

0 comments posted so far

Please use this functionality to flag objectionable, inappropriate, inaccurate, and offensive content to WebmedCentral Team and the authors.


Author Comments
0 comments posted so far


What is article Popularity?

Article popularity is calculated by considering the scores: age of the article
Popularity = (P - 1) / (T + 2)^1.5
P : points is the sum of individual scores, which includes article Views, Downloads, Reviews, Comments and their weightage

Scores   Weightage
Views Points X 1
Download Points X 2
Comment Points X 5
Review Points X 10
Points= sum(Views Points + Download Points + Comment Points + Review Points)
T : time since submission in hours.
P is subtracted by 1 to negate submitter's vote.
Age factor is (time since submission in hours plus two) to the power of 1.5.factor.

How Article Quality Works?

For each article Authors/Readers, Reviewers and WMC Editors can review/rate the articles. These ratings are used to determine Feedback Scores.

In most cases, article receive ratings in the range of 0 to 10. We calculate average of all the ratings and consider it as article quality.

Quality=Average(Authors/Readers Ratings + Reviewers Ratings + WMC Editor Ratings)