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ThesisItem Open Access Assessment of quality of well water in Eloor, Kerala(Department of Veterinary Public Health,College of Veterinary and Animal Science, Mannuthy, 2010) Divya Rani, Thomas; KAU; Sunil, BA comparative assessment of physical, chemical and microbiological quality of well water from Eloor, an industrial area in Ernakulum District of Kerala, India and Ollukara, a non industrial area, in Thrissur district of Kerala, India, was carried out to correlate the impact of industrialization on quality of well water. A total of 200 well water samples consisting of 100 each from both areas were taken for the study. Of the 100 samples, 25 samples each were collected during four different seasons of the year viz., summer (February), pre-monsoon (March-May), monsoon (June-September) and post-monsoon (October-November), to assess change in the quality of well water with the seasons. Mean temperature of well water was higher in Eloor than in Ollukara. The lowest temperature was recorded during monsoon in Eloor and Ollukara, and the highest temperature was recorded during pre-monsoon in Eloor and summer in Ollukara. Acidic pH was observed in both areas, with significantly lower pH in Ollukara. Higher pH values were observed during monsoon and post monsoon and lower during summer and pre-monsoon seasons. Mean total hardness of water was higher in Eloor than in Ollukara and the difference was highly significant. Highest value of total hardness was observed during summer in Eloor and monsoon in Ollukara. Mean COD of water samples showed no significant difference between two areas. In Eloor, the highest and lowest COD were observed during summer and monsoon, respectively. While in Ollukara, highest and lowest COD were observed during post monsoon and pre monsoon, respectively. Mean nitrate concentration was similar in water samples collected from Eloor and Ollukara with lowest concentration observed during pre - monsoon and summer in Eloor and Ollukara respectively, whereas it was highest during post monsoon in both areas. Mean fluoride concentration in well water samples from Eloor was significantly higher than that of Ollukara and no significant seasonal difference was observed in fluoride concentration in well water from Eloor. However, significant seasonal variations were observed in fluoride concentration in Ollukara viz., lower during monsoon and post monsoon, and higher during summer and pre-monsoon. Mean iron concentration was higher in Eloor and had highly significant difference with mean concentration in Ollukara. Significant difference in mean iron concentrations between four seasons could not be observed in Eloor, whereas in Ollukara, four different seasons showed significant difference. Lower concentration was observed during summer and pre-monsoon and higher during monsoon and post monsoon. A significantly higher lead concentration was recorded in well water from Eloor than that from Ollukara. In Ollukara, there was significant seasonal difference in mean lead concentration with highest during monsoon and the lowest during pre-monsoon seasons respectively. Throughout the entire period of study no mercury could be detected in well water samples from both areas. There was no significant difference in mean zinc concentration between well water samples from Eloor and Ollukara. In Eloor, the concentration in summer season was significantly higher than during other seasons. In Ollukara mean zinc concentration in post monsoon was significantly higher than during other seasons. Mean cadmium concentration was similar in both areas and was found to be significantly higher during monsoon season at both Eloor and Ollukara. It was observed that mean Aerobic Plate Count was higher in Eloor than that of Ollukara and the difference was highly significant. There was no significant difference observed between four seasons in Eloor. In Ollukara, significant difference between seasons could be observed with highest count during summer. The mean coliform count of well water from Eloor was significantly higher than that of Ollukara. There was no seasonal variation in mean coliform count in Eloor. Whereas significant variation in mean colifrom count between seasons was observed in Ollukara with highest count during monsoon and lowest during summer. The mean E. coli count in well water from Eloor and Ollukara did not differ significantly. There was no difference in mean E. coli count between four seasons in well water from Eloor and Ollukara. The mean enterococcal count of well water samples from Eloor and Ollukara did not show significant difference. The mean count was significantly higher in Eloor during summer, but there was no seasonal difference in enterococcal count in Ollukara. From the survey conducted among 25 households having wells in Eloor and Ollukara, it could be concluded that 20 and 100 per cent of households, used well as source of drinking water in Eloor and Ollukara respectively. Eighty percent wells were pucca wells in Eloor, whereas only 60 per cent of wells were pucca in Ollukara. Disinfection of wells was practiced by 48 and 52 per cent respectively in Eloor and Ollukara. Only 32 and 36 per cent wells respectively had distance more than 15 metre from nearest polluting source. Among the major human health problems in Eloor, 88, 72, 60, 40, 12, 4 and 16 per cent of household reported respiratory problems, skin diseases, musculoskeletal diseases, headache, ophthalmic problem, neoplasm and congenital anomalies and mental retardation, respectively, which were suggestive of iron, lead and cadmium toxicity and poor quality of air. Whereas in Ollukara, health problems were comparatively less and only 12 per cent house hold reported respiratory problem. Among the major animal health problems in Eloor were digestive disorders, reproductive disorders, skin diseases and lameness indicating iron, lead, cadmium and fluoride toxicity. Retrospective analysis of cases recorded in Eloor veterinary hospital, from January 2005 to December 2009, also revealed symptoms of iron, lead and cadmium toxicity in animals. It was observed that Kuzhikandam creek in Eloor was heavily polluted and acted as a potent source of groundwater pollution. From the comparative study, it was clear that the groundwater contamination in Eloor was purely chemical of industrial origin, while in Ollukara it was attributed to the soil type and household pollution. Construction of sanitary wells, keeping adequate distance from polluting sources, with adequate platform, drainage and parapet is recommended. Steining of wells and covering the wells with nets should also be adopted. Disinfection of wells with sufficient quantity of suitable disinfectant at regular interval also helps to minimize pollution mainly of microbial origin.ThesisItem Open Access Application of polymerase chain reaction for rapid evaluation of hygienic status of milk(Department of Veterinary Public Health, College of Veterinary and Animal Sciences,Mannuthy, 2008) Deepa Mary, J J; KAU; Sunil, BRapid assessment of the bacterial load and detection of pathogens in milk is of public health significance. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogens. Therefore efficient extraction of DNA directly from milk is a major step. DNA could be efficiently extracted directly from milk by a prior sample preparation so as to remove the fat and milk proteins. The phenol chloroform method of DNA extraction was modified to reduce the time require for the procedure. The use of lysozyme helped the release of DNA from lysed gram positive Staphylococcus aureus. The extracted DNA was used as template in PCR. PCR was carried out with already published primers. PCR was modified with the use of PCR buffer containing PCR facilitators (BSA and Tween 20) to overcome PCR inhibition. The standardized procedure was used to assess the bacterial load and to detect Escherichia coli and S. aureus directly from milk. To assess the bacterial load dilutions of milk were made upto10-10. DNA was extracted from each dilution with which PCR was carried out with primers specific for Pseudomonas. Aerobic Plate Count was also done for the same samples and compared with PCR. It could be concluded that the approximate APC of the milk sample by PCR is next lower dilution to the dilution giving the PCR amplification. The total time taken for the analysis was approximately five hours. Extraction of DNA and PCR was done with primers for detection of E. coli from the same milk samples and compared with culture. Percentage of samples positive both by culture and PCR was 50 and negative by both methods were 30. Twenty percentage of the samples were positive by PCR and negative by culture. Extraction of DNA and PCR was done with primers for detection of S. aureus from the same milk samples and compared with culture. Percentage of samples positive both by culture and PCR was 60 and negative by both methods were 20. Twenty percentage of the samples were negative by PCR and positive by culture. Hence, protocol developed for detection of S. aureus needs further refinement to take care of false negative results by PCR, probably due to the low number of organisms present in milk.ThesisItem Open Access Assessment of bacterial quality and shelf life pasteurized milk(Department of Veterinery Public Health, College of Veterinary and Animal Science, Mannuthy, 2007) Asha, K; KAU; Nanu, EIn the present study raw and pasteurized milk samples were collected from two processing plants viz., DP1 and DP2 and pasteurized milk from retail shops. A total of 254 samples were analyzed for the bacterial quality by estimating various bacterial counts and also assessed the presence of certain bacteria of public health importance. The bacterial, physical and organoleptic qualities of pasteurized milk samples from two dairies stored under refrigeration (4 ± 1oC) were evaluated. Raw milk revealed an inferior bacterial quality with 50 per cent samples graded as fair (based on total viable count) and 85.7 per cent as poor quality (based on coliform count). The total viable count from both dairies was obtained at the level of 7 log10 cfu/ml but coliform count was high in the samples obtained from DP1 (3.34 ± 0.05 log10 cfu/ml). The psychrotrophic count and faecal streptococcal count in the samples belonging to both sources were at the level of 7 and 3 log10 cfu/ml, respectively. Bacteria of public health significance like Escherichia coli, Staphylococcus aureus and Pseudomonas was detected from a few samples. Pasteurization reduced the level of total viable count, coliform count, psychrotrophic count and faecal streptococcal count to a highly significant (P<0.01) level. Pasteurized milk under refrigeration (4 ± 1oC) showed an increase in total viable count and psychrotrophic count throughout the storage period with a difference of more than 3 log with that of fresh sample. However, coliform count, Escherichia coli count, and faecal streptococcal count of samples belonging to DP1 initially showed increasing tendency up to six days and thereafter the counts decreased. The increase in total viable count, coliform count, Escherichia coli count, psychrotrophic count and faecal streptococcal count between zero and 10th day from DP2 was 4.8, 1.95, 2.08, 4.78 and 2.32 log10 cfu/ml, respectively. The increase in the counts during storage may lead to the reduction in shelf life due to bacterial deterioration of milk. Isolates of Escherichia coli was obtained from DP1 on all days except eighth and 10th day. A total of six isolates were obtained from DP2. The isolates belonged to O116 (3), O22, O46, O65 (2), O95 and the rest were rough variety. Staphylococcus aureus was also isolated from two samples stored on sixth day and three from the samples stored on zero, second and fourth day, respectively (DP1). From DP2, three isolates were obtained from the samples stored on 10th day and one from fresh samples. A total of 22.62 and 20.24 per cent Pseudomonas were isolated from DP1 and DP2, respectively and the isolates were identified as Pseudomonas putida, Pseudomonas aeruginosa and Pseudomonas flourescens. Sensory and physico-chemical (COB test) analyses of refrigerated milk samples showed an overall reduction in the score of colour and appearance, flavour, odour and body as the storage period increased. The mean total scores from DP1 revealed that the samples were of excellent quality for up to second day of storage. The sensory quality of the samples stored on fourth day was good and then the quality of milk remained fair till eighth day and on 10th day the quality became poor. In DP2 samples had excellent quality for upto second day of storage. The sensory quality of the sample stored up to sixth day was good and thereafter the quality of milk remained as fair till the end of storage period. COB test of samples from DP1 showed positive test on all samples stored on 10th day. However, one sample stored on day six was COB test positive. The samples belonging to DP2 showed that three samples stored on 10th day and one sample stored on eighth day was COB positive. The bacterial profile of the retail milk samples of the brands A, B, C, D, E and F was assessed and the samples belonging to the brand D had highest mean total viable count (5.94 ± 0.09 log10 cfu/ml), psychrotrophic count (5.09 ± 0.16 log10 cfu/ml) and faecal streptococcal count (2.87 ± 0.24 log10 cfu/ml). Highest coliform count was seen in the samples of brand A (2.40 ± 0.14 log10 cfu/ml) and Escherichia coli count (3.44 ± 0.72 log10 cfu/ml) in samples of the brand C. Low counts especially total viable count (4.89 ± 0.79 log10 cfu/ml) and coliform count (1.19 ± 0.42 log10 cfu/ml) were seen in the samples of the brand F. Escherichia coli were detected from 20.8 per cent samples and the isolates consisted of the serotypes O46, O65, O95, O116, O166 and O171. Out of 15 isolates obtained six showed a positive congo red reaction indicating their property of invasiveness. Staphylococcus aureus was isolated from only six samples (6.94 per cent). All retail milk samples were also tested for the isolation and identification of Pseudomonas and the organism was isolated from 16 (22.22 per cent) samples. The isolates were identified as Pseudomonas putida (7), Pseudomonas aeruginosa (6) and Pseudomonas flourescens (3). Polymerase Chain reaction was employed to identify and confirm the Escherichia coli isolates obtained from the milk samples and a 366 bp product was obtained.ThesisItem Open Access Bacterial quality of milk at the point of production with special emphasis o the quality assuarance programme(Department of Veterinery Public Health, College of Veterinary and Animal Science, Mannuthy, 2006) Lekha, Chacko K; KAU; Nanu, EIn the present study a total of 108 milk samples were collected from farmers of three societies (S1, S2 and S3) and analysed for microbial quality, by estimating various microbial counts and also by assessing the presence of certain bacteria of public health importance. Antibiotic susceptibility of Escherichia coli and Staphylococcus aureus was also evaluated. The samples were graded based on total viable count and also by methylene blue reduction test. The critical control points of bacterial contamination of milk at various stages of production were also assessed during the investigation. Analysis of variance test of the data revealed highly significant difference (P0.01) between the mean counts of milk samples collected from three societies. Milk samples from S2 had the highest mean total viable count (6.57 ± 0.13 log10 cfu/ml), coliform count (3.89 ± 0.08 log10 cfu/ml), Escherichia coli count (1.11 ± 0.19 log10 cfu/ml), faecal streptococcal count (3.86 ± 0.08 log10 cfu/ml) and psychrotrophic count (5.57 ± 0.12 log10 cfu/ml). But the highest mean yeast and mould count (4.08 ± 0.01 log10 cfu/ml) was observed in samples collected from S1. Milk samples from S2 revealed maximum contamination. Analysis of data revealed that highly significant (P<0.01) and positive correlation between various bacterial counts. Highly significant (P<0.01) and positive association was observed between total viable count and psychrotrophic count and also between the former count and faecal streptococcal count. Microbial analysis of milk samples collected from six farmers of S1 revealed that samples from F4 had highest mean total viable count (7.44 ± 0.01 log10 cfu/ml) and the lowest in samples of F3 (4.82 ± 0.03 log10 cfu/ml). Similarly, the other bacterial counts, viz., coliform count, Escherichia coli count, faecal streptococcal count, psychrotrophic count was also seen in the highest level in the samples of F4. Whereas the highest mean yeast and mould count was observed in the samples of F1. Microbial analysis of the samples collected from farmers of S2 revealed that the highest mean total viable count (7.42 ± 0.08 log10 cfu/ml) and coliform count (4.44 ± 0.08 log10 cfu/ml) was seen in the samples of F5. Whereas these counts were lowest in the samples of F2. But the Escherichia coli count was highest in the samples collected from F3. However, the organism was not detected in the samples of F2 of S2. The highest mean faecal streptococcal count and psychrotrophic count was also seen in samples of F5, indicating that the hygienic practices followed by the farmer is very poor. Microbial analysis of the milk samples collected from six farmers of S3 revealed that the samples from F4 had the highest mean total viable count, coliform count, Escherichia coli count, faecal streptococcal count and psychrotrophic count indicating the poor hygienic practices followed by the farmer. The highest yeast and mould count was observed in the samples of F6. The presence of these organisms revealed the unsanitary condition of production of milk. The public health impact on the consumer was assessed by isolation and identification of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Aeromonas and Salmonella. The presence of coliforms and faecal streptococci indicate unhygienic handling of milk and possible faecal contamination. Escherichia coli was isolated from 41.67 per cent of the samples examined. A total of 45 isolates were obtained. Only 22 (48.89 per cent) out of 45 isolate were serotyped. The isolates fell into 10 serotypes viz., O116, O24, O84, O145, O172, O125, O79, O87, O103 and O157. Out of these serotypes except 024, 079 and 087 showed a positive congo red binding test which indicate the property of pathogenisity of isolates. Staphylococcus aureus was isolated from 33 (30.56 per cent) samples. The highest number of isolates was obtained from S2 (14) followed by S1 (12) and S3 (7). Listeria monocytogenes was isolated from 3 (2.70 per cent) samples. All isolates were obtained from F5 of S1. Bacillus cereus was isolated from 3.70 per cent of the total samples. Of the four isolates obtained, one was from S3 and three from S2. The per cent of isolation of Aeromonas was 18.51. Two species of Aeromonas were obtained viz., Aeromonas hydrophila and Aeromonas caviae. None of the samples tested revealed the presence of salmonella and Yersinia enterocolitica. Antibiogram of Escherichia coli isolates obtained from the samples belonging to various sources revealed that all isolates were sensitive to chloramphenicol, co-trimoxazole and gentamicin. None of the isolates showed resistance to all the antibiotics used. Antibiogram of Staphylococcus aureus isolates revealed that all the isolates were sensitive to chloramphenicol. Highest per cent (36.36) of organisms showed resistance against erythromycin followed by oxytetracycline (21.22). None of the isolates revealed resistance to all the antibiotics used. Milk samples collected from three societies were graded based on total viable count. Out of the 108 samples analysed 16.67, 29.63, 20.37 and 33.33 per cent were graded as very good, good, fair and poor quality based on BIS (1977). However 16.67 per cent from S1 and 33.33 from S3 were graded as very good. The per cent of samples graded as good, fair and poor from S1 was 19.44, 30.56 and 33.33, respectively. However, the corresponding per cent from S2 was 33.33, 16.67 and 50.00. The per cent of good, fair and poor samples from S3 was 36.11, 13.89 and 16.67, respectively. Milk samples collected from three societies were graded based on methylene blue reduction test and the result showed that 22.22, 34.26, 25.00 and 18.52 per cent of samples were graded as very good, good, fair and poor, respectively. The various critical control points of bacterial contamination of milk was evaluated by collecting samples of air, water, rinsinging of utensils, hand wash of the milker and udder washes of the animals and estimating their bacterial load. The highest bacterial counts were observed in the hand wash samples of the milker, followed by udder wash of the animal and these two were considered as the point of major source of contamination of milk. The highest bacterial counts in water, utensil rinsings, hand wash of milker and udder washes of animal were observed in samples collected from S2. Thus it may be inferred that the high bacterial load of milk samples from S2 might be due to the contamination of milk from these sources. Therefore, utmost care should be given to monitor the microbial quality of milk at various stages of production, and also to identify various sources of bacterial contamination of milk. Then only we can improve the quality and shelf life of milk produced.ThesisItem Open Access Bacterial quality of raw milk at the co-operative society level with special reference to quality assuarance programme(Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, 2006) Jaibi, K; KAU; Nanu, EA total of 144 raw milk samples, consisting of individual and pooled milk samples collected from three societies, viz. S1, S2 and S3 were tested to assess the microbial quality by determining Total Viable Count (TVC), Coliform Count (CC), Escherichia coli Count (ECC), Faecal Streptococcal Count (FSC), Psychrotrophic Count (PC), and Yeast and Mould Count (YMC). The presence of certain bacterial pathogens like Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Aeromonas and Salmonella was also assessed. Statistical analysis of the data revealed highly significant difference (P<0.01) in microbial counts of individual samples of the three sources. The overall mean total viable count, coliform count, faecal streptococcal count, psychrotrophic count, and yeast and mould count was 6.12 ± 0.07, 3.27 ± 0.04, 3.06 ± 0.05, 3.81 ± 0.06, 3.48 ± 0.07 log10 cfu/ml, respectively. Escherichia coli was not detected in 61.11 per cent of individual samples. The overall Escherichia coli count of the samples was 1.23 ± 0.15 log10 cfu/ml. Samples of S2 had the highest mean count. Highly significant (P<0.01) and positive correlation was observed between all the bacterial counts. Association between total viable count and psychrotrophic count, total viable count and coliform count and coliform count and psychrotrophic count was the highest. Anlaysis of variance test of the data revealed highly significant (P<0.01) difference between the bacterial count of the 36 pooled milk samples obtained from the three sources. The overall mean total viable count, coliform count, faecal streptococcal count and psychrotrophic count were 6.52 ± 0.08, 3.47 ± 0.07, 1.52 ± 0.27, 3.37 ± 0.07 and 4.26 ± 0.07 log10 cfu/ml, respectively. Significant (P<0.05) difference was observed in the mean yeast and mould count of pooled milk samples and the overall mean count was 3.85 ± 0.09 log10 cfu/ml. The highest mean microbial count was observed in the samples of S2. Highly significant (P<0.01) and positive correlation was observed between total viable count and other bacterial counts. The correlation between psychrotrophic count and other counts was also very high. Among the individual samples of S1, highest mean total viable count was seen in the samples of F4 (6.75 ± 0.07 log10 cfu/ml) and the lowest mean count was in the samples of F2 (4.68 ± 0.05 log10 cfu/ml). The highest coliform count and psychrotrophic count were observed in the samples of F4 and the lowest count was seen in the samples of F2. Samples of F4 had the highest Escherichia coli count (2.68 ± 0.54 log10 cfu/ml). and the lowest count was observed in the samples of F6 (1.04 ± 0.66 log10 cfu/ml). None of the samples of F2 and F3 revealed the presence of the organism. Faecal streptococcal count was the highest in the samples of F5 and the lowest mean count was in the samples of F2. The mean yeast and mould count was the highest in samples of F4 (4.26 ± 0.06 log10 cfu/ml) and the lowest in the samples of F2 (2.25 ± 0.07 log10 cfu/ml). The association between psychrotrophic count and the other bacterial counts was highly significant (P<0.01). Highly significant (P<0.01) association was also observed between total viable count and coliform count. The total viable count of individual samples of S2 showed highly significant (P<0.01) difference. Samples of F2 had the highest mean count (6.72 ± 0.06 log10 cfu/ml). The lowest mean count was observed in the samples of F6 (6.09 ± 0.08 log10 cfu/ml). All bacterial count, except Escherichia coli count was highest in the samples of F2. The highest Escherichia coli count was in the samples of F3. All the microbial counts, except faecal streptococcal count was lowest in the samples of F6. Faecal streptococcal count was lowest in the samples of F3. Total viable count of samples of farmers belonging to S3 showed highly significant (P<0.01) difference. The samples of F1 had the lowest count (4.74 ± 0.04 log10 cfu/ml) and the highest count was observed in the samples of F6 (6.72 ± 0.05 log10 cfu/ml). All microbial counts were highest in the samples of F6. Escherichia coli was isolated from 38.89 per cent individual samples and 47.22 per cent of pooled milk samples. Only 23 isolates from individual samples and eight isolates from pooled milk samples were serotyped. The serotypes obtained were O5, O24, O25, O68, O84, O87, O103, O116, O157 and O172. Out of the isolates, 14 and five from individual samples and seven and two from pooled samples were untypable and rough, respectively. Congo red binding property was shown by 17 serotyped and 10 untypable isolates obtained from individual milk samples and five serotyped and four untypable strains obtained from pooled milk samples. Staphylococcus aureus was isolated from 28.70 per cent of individual and 27.78 per cent of pooled milk samples. Listeria monocytogens was isolated from two individual milk samples from S3. Bacillus cereus was isolated from two milk samples obtained from S2. Aeromonas was isolated from 12.04 per cent of individual samples and 25.00 per cent of pooled milk samples. From the individual samples, seven isolates of Aeromonas sobria and six isolates of Aeromonas caviae were obtained. From the pooled milk samples, three isolates of Aeromonas hydrophilia and six isolates of Aeromonas caviae were obtained. Antibiogram of the Escherichia coli isolates revealed that the isolates were highly sensitive to cephotaxime, chloramphenicol, ciprofloxacin and gentamicin whereas 11.86 per cent isolates were resistant to streptomycin. Staphylococcus aurues isolates were sensitive to cephotaxime, chloramphenicol, ciprofloxacin and gentamicin. But 19.51 per cent and 9.76 per cent isolates were resistant to erythromycin and penicillin, respectively. Grading of individual milk samples based on total viable count as per the standards prescribed by Indian Standards (1977), revealed that 42.59 per cent samples were graded as fair. Only 16.67 per cent samples were graded as very good whereas 20.37 per cent each were graded as good and poor. None of the pooled milk samples was graded as very good, while 16.67, 58.33 and 25.00 per cent samples were graded as good, fair and poor, respectively. Based on methylene blue reduction test, 21.30, 30.56, 39.81 and 8.33 per cent individual samples were graded as very good, good, fair and poor, respectively while, 13.89, 30.56, 47.22 and 8.33 per cent pooled milk samples were graded as very good, good, fair and poor, respectively. The various critical control points of microbial contamination of milk was evaluated by collecting samples of air, water, hand wash of the handler and utensil wash and subjecting to estimation of the bacterial load. Hand wash was found to be a major source of contamination. The highest microbial count in the samples of air and hand wash was observed in S2. The higher mean microbial count in the milk samples of S2 might be attributed to the contamination from these sources. The microbial count in water and utensil wash was the highest in the samples of S1.ThesisItem Open Access Microbial quality and safety of raw milk with reference to sources of contamination(Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, 2007) Gini, George; KAU; Nanu, EIn the present investigation a total of 180 raw milk samples, consisted of 108 individual milk samples obtained from farmers belonging to three co- operative societies (S1, S2 and S3) and 72 pooled milk samples from the three societies were collected and evaluated the microbial quality. The samples were also tested to detect the presence of Escherichia coli, Staphylococcus aureus and Yersinia. The isolated Escherichia coli cultures were confirmed using Polymerase Chain Reaction (PCR) technique. The pooled milk samples obtained from the societies were also tested to detect the adulterants and preservatives added in the milk. During the investigation the factors contributing the bacterial contamination of milk from various sources were also evaluated to identify the critical control points. Statistical analysis of the data revealed highly significant difference (P<0.01) in microbial counts of individual samples of the three sources. The overall mean total viable count, coliform count, Escherichia coli count, faecal streptococcal count and yeast and mould count was 6.01 ± 0.07, 4.44 ± 0.07, 0.86 ± 0.11, 3.14 ± 0.10 and 2.09 ± 0.12 log10 cfu/ml, respectively. Samples of S2 had the highest mean count on the basis of total viable count, coliform count, Escherichia coli count and faecal streptococcal count. Milk samples from S2 revealed maximum contamination. Escherichia coli was not detected in 63.89 per cent of individual samples. Analysis of the data revealed significant (P<0.05) and positive correlation between total viable count and faecal streptococcal count and also between total viable count and coliform count. A similar correlation was observed between coliform count with faecal streptococcal count. Microbial analysis of milk samples collected from six farmers of S1 revealed that samples from F1 had highest mean total viable count (6.29 ± 0.15 log10 cfu/ml) and the lowest count was observed in samples of F5 (5.58 ± 0.37 log10 cfu/ml). Highest mean coliform count (4.77 ± 0.19 log10 cfu/ml) and yeast and mould count (2.74 ± 0.25 log10 cfu/ml) were seen in the samples of F3, whereas lowest coliform count (3.52 ± 0.77 log10 cfu/ml) and yeast and mould count (1.75 ±0.56 log10 cfu/ml) was observed in the samples of F2 and F5, respectively. Samples obtained from F6 did not revealed the presence of Escherichia coli, but the count was highest in the samples of F5 (1.50 ±0.49 log10 cfu/ml). The highest (3.97 ± 0.09 log10 cfu/ml) and lowest (1.80 ± 0.59 log10 cfu/ml) faecal streptococcal count was observed in the samples of F6 and F5, respectively. Critical difference test of the data revealed that none of the bacterial association was significant in the samples of S1. Microbial analysis of individual milk samples collected from the farmers of S2 revealed that samples from F2 had highest mean total viable count (7.08 ± 0.20 log10 cfu/ml) and coliform count (5.33 ± 0.15 log10 cfu/ml) and the samples from F5 showed lowest values for the above two counts. Similarly, the bacterial counts, viz., faecal streptococcal count (4.10 ± 0.18 log10 cfu/ml) and yeast and mould count (2.99 ± 0.24 log10 cfu/ml) were highest in the samples of F3. Lowest values for faecal streptococcal count (3.12 ± 0.31 log10 cfu/ml) and yeast and mould count (0.96 ± 0.61 log10 cfu/ml) were in the samples of F4 and F6, respectively. Samples obtained from F2 did not revealed the presence of Escherichia coli, but the count was highest in the samples of F6 (2.23 ± 0.49 log10 cfu/ml). Analysis of the data of the samples obtained from S2 revealed that significant (P<0.05) and positive correlation between total viable count and faecal streptococcal count and also between total viable count and coliform count. A similar correlation was observed between coliform count with faecal streptococcal count and yeast and mould count. Analysis of variance test of the data of the samples belonging to the farmers of S3 revealed highly significant (P<0.01) difference between the mean total viable count and coliform count. The samples of F2 had lowest total viable count (4.85 ± 0.18 log10 cfu/ml), but the highest count was in the samples of F6 (6.66 ± 0.38 log10 cfu/ml). The samples belonging to F4 had the highest mean coliform count (4.96 ± 0.17 log10 cfu/ml) while the lowest count was observed in samples of F1 (3.74 ± 0.13 log10 cfu/ml). The highest mean Escherichia coli count (0.80 ± 0.51 log10 cfu/ml) was seen in the samples belonging to F1 and F5. The samples belonging to F2, F4 and F6 had the mean count of 0.33 ± 0.33 log10 cfu/ml. The highest mean faecal streptococcal count (3.66 ± 0.14log10 cfu/ml) was seen in the samples of F4. The samples of F3 had the lowest mean count (2.13 ± 0.68 log10 cfu/ml). The samples belonging to F3 had the highest mean yeast and mould count (2.77 ± 0.11 log10 cfu/ml) and the lowest mean count was observed (1.41 ± 0.64 log10 cfu/ml) in the samples from F1. A significant (P<0.05) and positive correlation was observed only between the total viable count and coliform count of the samples of S3. Analysis of variance test of the data revealed highly significant (P<0.01) difference in the bacterial count of the 72 pooled milk samples obtained from the three sources. The overall mean total viable count, coliform count, Escherichia coli count, faecal streptococcal count and yeast and mould count was 6.19 ± 0.09, 4.65 ± 0.09, 1.27 ± 0.16, 3.50 ± 0.06 and 2.37± 0.11 log10 cfu/ml, respectively. Escherichia coli was not detected in 50.00 per cent of pooled samples. Samples of S2 had the highest mean count based on total viable count, coliform Count and Faecal Streptococcal Count. Escherichia coli count and yeast and mould count showed highest values in the samples of S3. Significant (P<0.05) and positive correlation was observed only between total viable count and coliform Count of the pooled milk samples. Escherichia coli was isolated from 36.11 per cent individual samples and 50.00 per cent of pooled milk samples. Fifteen isolates from individual samples and eighteen isolates from pooled milk samples were serotyped. The serotypes obtained were O12, O29, O60, O68, O75, O79, O96, O107, O116, O131, O160 and O172. Twelve isolates each from individual samples were untypable and rough. Among the pooled samples three isolates were untypable and fifteen isolates were rough. Congo red binding property was shown by nine and sixteen serotyped isolates obtained from individual and pooled milk samples, respectively. Staphylococcus aureus was isolated from 40.74 per cent of individual and 34.72 per cent of pooled milk samples. Yersinia was isolated from 22.22 per cent of individual samples and 29.17 per cent of pooled milk samples. From the individual samples, five isolates of Yersinia enterocolitica was obtained. Yersinia frederiksenii, Yersinia intermedia, Yersinia aldovae and Yersinia kristensenii was isolated from ten, five, two and 1 of the individual milk samples. Yersinia pseudotuberculosis was obtained from one of the individual sample. From the pooled milk samples, three isolates each of Yersinia kristensenii and Yersinia aldovae was obtained. Yersinia intermedia and Yersinia frederiksenii was isolated from eight and six samples, respectively. Yersinia enterocolitica was obtained from one of the sample. Grading of individual milk samples based on total viable count as per the standards prescribed by Indian Standards (1977) revealed that 34.26 per cent samples were graded as good. The per cent of samples graded as fair was 32.41. Only 15.74 per cent samples was graded as very good, whereas 17.59 per cent was graded as poor. The highest (40.28) per cent of pooled milk samples was graded under the category fair, while 9.72, 31.94 and 18.06 per cent samples were graded as very good, good and poor, respectively. The various critical control points of microbial contamination of milk was evaluated by collecting samples of air, water, hand wash of the milker or milk handler and utensil wash and subjecting to estimation of the bacterial load. Hand wash was found to be a major source of contamination. The highest microbial count in the samples of water, utensil wash and hand wash was observed in the samples obtained from S2. The higher mean microbial count in the milk samples of S2 might be attributed to the contamination from these sources. Adulterants (starch and cane sugar) and preservatives (carbonates, formaldehyde and boric acid) were not detected in any of the 72 pooled milk samples examined. The Escherichia coli isolates obtained from raw milk were confirmed by Polymerase Chain Reaction (PCR) and the analysis of the electrophoresed gel under UV transilluminator revealed the presence of a 366 bp band in 93.33 per cent isolates.ThesisItem Open Access Microbial quality assurance of curd during production and storage(Department of Veterinary Public Health, College of Veterinary and Animal Science, Mannuthy, 2005) Praseeda, R; KAU; Nanu, EIn the present investigation, a total of 180 freshly prepared curd samples belonging to 10 batches were collected on the day of production. Two samples from each batch were selected at random and examined on zero day and the remaining samples were stored under refrigeration and duplicate samples examined on day 3, 5, 7, 9, 12, 15, 18 and 21 of storage. A total of 80 curd samples belonging to four brands viz. A, B, C and D were also collected from retail outlets in and around Thrissur Corporation. All samples of curd were tested to evaluate the microbial quality by estimating the total viable count (TVC), coliform count (CC), faecal streptococcal count (FSC), psychrotrophic count (PC) and yeast and mould count (YMC). Isolation and identification of Escherichia coli, Staphylococcus aureus, Salmonellae, Pseudomonas aeruginosa and Bacillus species were also carried out. The organoleptic qualities of curd such as colour and appearance, flavour, body and texture and product acidity and physico-chemical parameters such as pH and titratable acidity were also assessed. Microbial quality assurance of curd during its production and critical control points of microbial contamination in the production line were also evaluated during the study. Paired‘t’ test of the data revealed that mean TVC of the samples increased at a highly significant (p<0.01) level from zero day to fifth day of storage, after which it decreased gradually. The mean CC of curd samples showed a gradual decreasing trend till the 12th day of storage. Coliforms were not detected in 90 per cent of the fresh curd samples. The mean FSC of curd samples also decreased significantly through out the storage period. On 18th and 21st day, 70 per cent of the samples did not reveal the presence of faecal streptococci. The mean PC decreased subsequently during storage till 12th day, after which it slightly increased. The mean YMC of fresh curd was 3.91 ± 0.11 log10cfu/g and it increased gradually from third day of storage compared to the count of the fresh sample. The count increased by 1.5log10cfu/g on the 12th day of storage and the increase was highly significant (p<0.01). A highly significant (p<0.01) and positive association was observed between the mean TVC and FSC on 21st day of storage. A significant (p<0.05) and negative association was observed between the mean TVC and PC on day nine of storage. A similar association was observed between mean CC and PC of samples on 12th day of storage and mean YMC and PC of samples on 15th day of storage. E. coli was not detected from any of the fresh and refrigerated samples of curd. Staphylococcus aureus was isolated from 35 per cent of fresh curd samples. On day 3, 5 and 7 of storage, 25, 20 and 10 per cent of the samples revealed the presence of the organism and all isolates were coagulase and TNase positive. None of the curd samples revealed the presence of Salmonellae and Pseudomonas aeruginosa. Bacillus species were present in 70 per cent of the fresh samples and 20 per cent each of the samples tested on day 15 and 18. Bacillus cereus was not detected in any of the curd samples and among the isolates, 55.4 and 25.67 per cent were B. subtilis and B. coagulans. The mean titratable acidity showed an increase till ninth day of storage and then decreased throughout the storage period. The pH of fresh curd samples decreased on storage and after 15th day, it increased gradually. Analysis revealed that flavour scores decreased during storage. Body and texture score remained almost the same throughout the storage period except a slight decrease on the third day. Colour and appearance score decreased significantly during the storage period and product acidity score also got reduced during storage. Of the 80 samples collected from the four brands, 42.5, 37.5 and 20 per cent had mean TVC at the level of 109, 108 and 107 cfu/g, respectively. Highly significant (p<0.01) difference was observed in mean CC of the samples of the different brands. Coliforms were not detected in the samples of brand B and 80 and 70 per cent samples of the brands C and D, respectively. A highly significant (p<0.01) difference was observed between the mean FSC count of samples from the four brands. Of the samples, 40 per cent did not reveal the presence of the organism and the count in 50 per cent samples from brand A was at the level of 103cfu/g. E. coli was not detected in the samples of brands B and D. The organism was isolated from 5 and 15 per cent of samples of brand A and C and the four isolates belonged to the serotype O157, O5, O148 and rough type. None of the samples from the four brands revealed the presence of Salmonellae. Pseudomonas aeruginosa was present in 2.5 per cent of the samples from the four brands. Staphylococcus aureus was detected in two of the curd samples, one (5%) each from brand A and C. Bacillus species was isolated from 62.5 per cent of the samples and among the isolates, 14.14 per cent was B. cereus. Analysis of variance test revealed a highly significant (p<0.01) difference in the mean titratable acidity and pH of the samples from the various brands. Organoleptic score analysed by Kruskal-Wallis test revealed that the product acidity score of the market curds varied significantly (p<0.01). Air samples collected from the various areas of curd production revealed highest bacterial count at the area of thermal treatment and lowest at the area of cream separation. The mean YMC was higher at the pasteurisation room. Hand washings of the personnel had the highest mean TVC and CC. E. coli was detected only in hand washings of personnel. Samples collected from the cream separator had the highest mean TVC and least in packaging material. Faecal streptococci were detected in all the samples tested and highest count was observed in the case of storage can. Samples from the double jacketed vat had the highest CC. Coliforms were not detected in the washings of the packaging material. Heat treated skim milk was free of coliforms and the mean FSC was significantly (p<0.05) lower than that of starter inoculated milk after one and six h of incubation. Yeast and moulds were not detected in heat treated skim milk, but present in starter inoculated one and six h milk samples. The mean pH of fresh curd samples was significantly (p<0.01) lower than that of heat treated skim milk. The starter culture was free of coliforms and faecal streptococci. The present study reflects the importance of quality assurance during every step of production and storage of curd to avoid early spoilage and to safeguard consumer health. Presence of pathogenic organisms in curd is of great public health significance as it is consumed as such paving way to food poisoning.ThesisItem Open Access Microbial quality assurance of milk in its production, processing and storage(Department of Veterinary Public Health, College of Veterinary and Animal Science, Mannuthy, 2005) Prejit; KAU; Nanu, EIn the present study 296 milk samples were collected from dairy farm, processing plant and retail shops and analysed for the microbial quality by estimating various microbial counts and assessing the presence of certain bacteria of public health importance. The microbial, physico-chemical and organoleptic qualities of pasteurized milk samples stored under refrigeration were evaluated. The critical points of bacterial contamination of milk at various stages of production, pasteurization, packaging and storage were also assessed during the investigation. Out of 60 samples of raw milk from individual animal, pooled milk and chilled milk analysed, 46.7 and 36.6 per cent samples were graded as very good and good based on BIS (IS 1977). However only 25 per cent samples were only considered satisfactory based on coliform count with none of the pooled milk samples meeting the standards. Pooled milk samples revealed maximum contamination with highly significant (p0.01) difference between mean TVC and CC and significant (p0.05) difference between mean PC and FSC of milk from individual animal. However mean E.coli count was more in individual animal milk sample when compared with pooled milk. The pooled milk sample was kept under chilled condition for 17 hours. The chilled milk had higher mean TVC, ECC and PC however the increase was not statistically significant. Pasteurization of milk was effective in reducing the microbial population of raw milk and there was highly significant (p0.01) reduction in TVC, CC, PC, FSC and YMC. From the sample of milk collected immediately after heating, after pasteurization and after packaging the mean TVC was high in packaged milk sample (4.76±0.15 log10 cfu/ml). Coliform was absent in cent percent of heated milk. However, 60 per cent of packaged milk had the organism with the mean count of 0.98±0.36 log10 cfu/ml. Only 40 per cent of packaged milk was graded satisfactory based on coliform standards prescribed by BIS (Indian Standards, 1992). E.coli could not be detected from milk collected after heating section and immediately after pasteurization. Maximum microbial contamination was seen in packaged milk. This indicates that contamination of milk occur in the storage tank or packaging section. Hence proper cleaning and sanitation of storage tank and packaging machine will reduce the microbial contamination to a considerable extent. The microbial analysis of the retail milk sample (56) of the brands A, B, C and D revealed that the samples of the brand B had highest mean TVC (5.91 ± 0.01 log10 cfu/ml) and FSC (1.52 ± 0.22 log10 cfu/ml). The highest mean coliform (3.08 ± 0.29 log10 cfu/ml), E. coli, (2.35 ± 0.38 log10 cfu/ml), psychrotrophic counts (5.68 ± 0.25 log10 cfu/ml) were seen in the samples of brand C. Brand A had highest mean YMC (1.21 ± 0.22 log10 cfu/ml). Only 23.2 and 26.8 per cent retail samples met the TVC and CC standards prescribed by BIS (IS-1992). However, 57.14 per cent of the samples of the brand A met the standard. On comparison of freshly packed milk obtained from dairy plant with the retail brands revealed that freshly packed milk sample had highly significant (p0.01) difference and lesser TVC, CC and PC in comparison with the brands B, C and D. Thus the microbial quality of retail milk available in the locality varied among different brands hence strict hygienic measures should be adopted to minimize microbial contamination. Pasteurized milk stored under refrigeration (4±1°C) showed an increase in TVC, CC, ECC, PC, FSC and YMC throughout the storage period. However, the initial growth rate of microorganism was slow due to sub lethal injury of microorganism during pasteurization and storage of milk under refrigerated condition. The increase in TVC, CC, ECC, PC, FSC and YMC between zero day and 12th day was 3.96, 1.76, 0.16, 3.76 and 2.37 log10 cfu/ml, respectively. The increase in the count of organism during storage may present the problem of shelf life deterioration of milk. The public health impact on the consumers was assessed by isolation and identification of E.coli, S. aureus and L. monocytogenes, the organisms of public health importance. Escherichia coli contamination of milk occurs through unhygienic handling of milk. The organism was detected in four samples (20 per cent) of the samples of raw pooled milk and was of serotypes 05 (2 samples), 0141 and 0172. Ten per cent of freshly packaged and refrigerated milk samples revealed the presence of the organism and all isolates were of serotypes 0148. A total of 38 E.coli isolates were obtained from retail milk samples. S. aureus was isolated from 60, 5, 12.5 per cent of the samples of raw, freshly pasteurized and retail milk, respectively. The organism was isolated from 5 per cent of the samples stored on sixth day and from 10 per cent samples stored on the eight day. L. monocytogenes was not isolated from milk samples. Sensory analysis of refrigerated milk samples showed an overall reduction in the score of colour and appearance, flavour, odour and body as the storage period increased. Development of off odour, salty or stale flavour and presence of clotted particles indicated the sensory spoilage and the maximum shelf life obtained was eight days. There was reduction in mean pH value throughout the storage period. All the samples showed positive to clot on boiling test on the day 12 of storage but the sample showed sensory unacceptability earlier. The various critical points of bacterial contamination of milk was evaluated by collecting samples of air, water, rinse samples from utensils, equipments, hand washing of milker/personnel in the processing line, strainer and packaging material and were subjected to estimation of various bacterial counts. The mean total viable count and yeast and mold count of air samples were found to increase after the milking process or processing. Among the water samples, coliform and E. coli was detected more in the samples obtained from dairy farm. High microbial count was recorded for milk pail, milkers hand washings and package machine wash indicating an important sources of contamination. Strict hygienic practices followed by health education will minimize the microbial contamination to a considerable extent.ThesisItem Open Access Evaluation of bacteriological quality of beef carcasses in meat processing plant(Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, 2003) Sethulekshmi, C; KAU; Nanu, EDuring the present study, 40 beef carcasses were randomly selected from a meat processing plant located at Kochi in Kerala. The plant procures beef carcasses from two slaughtering units, viz., Source A and B, located in Tamil Nadu. From each carcass surface 500-cm2 area was swabbed which consisted of 100 cm2 each from neck, brisket, loin, flank and outer round. The samples from each carcass were examined for the bacterial quality by estimating the total viable count (TVC), coliforms count (CC), Escherichia coli count (ECC) and faecal streptococcal count (FSC). All samples were also subjected to the isolation and identification of Escherichia coli, Staphylococcus aureus, Salmonella and Listeria monocytogenes. The samples of air, water, equipment and hand wash of personnel were also collected and estimated the various bacterial loads of these samples. Analysis of variance test of the data did not reveal significant difference between the mean total viable count of the samples from two sources. The samples had an overall mean total viable count of 7.40 ± 0.17 10glO cfu/cm/. The mean count of samples from source B was slightly higher than that from source A. The count of the samples belonging to source A ranged from 106 to 109 cfu/cm2 while the count of the samples from source B varied between 106 and 108 cfu/cm/. Of the 40 carcass samples examined, 45 per cent had count at the level of 106 cfu/crrr', The counts in 30 per cent and 22.5 per cent were at the level of 108 cfulcm2 and 107 cfu/cnr', resp~ctively. Analysis of variance' test of the data revealed a significant (P<0.05) difference between the mean coliforms count of the samples from the two sources. The samples from source A had a higher mean count. The overall mean coliforms count of the samples was 3.41 ± 0.13 10glO cfu/cm". The count of the samples from both the sources varied from 102 to 104 cfu/cm/. The count in 21 (52.5 per cent) carcass samples was at the level of 103 cfu/crn", In 17.5 per cent carcasses the count was at the level of 104 cfu/crrr' and in 30 per cent carcass samples the count was at the level of 102 cfu/crrr'. Analysis of variance test of the Escherichia coli count revealed a significant (P<0.05) difference between the mean count of samples from the two sources. The samples belonging to source A had a higher mean count. The overall mean Escherichia coli count of the samples was 1.83 ± 0.22 10glO cfu/crrr'. The Escherichia coli count of the samples from both the sources varied from 101 to 103 cfu/cm2. The count in 16 (40 per cent) of carcass samples was at the level of 102 cfu/cm", In 17.5 per cent samples each had count at the levels of 101 and 103 cfu/cm", Analysis of variance test of the faecal streptococcal count revealed significant (P<0.05) difference between the mean counts of samples from the two sources. Source A had higher mean count. The overall mean count of samples was 3.27 ± 0.10 10glO cfu/cm". The count on the carcasses from the source A ranged between 102 to 104 cfu/crrr' whereas the count on the carcasses belonging to source B varied between 102 to 103 cfu/crrr'. Out of 40 carcasses, 18 (45 per cent) had count at the level of 103 cfu/crrr'. The counts on 40 and 15 per cent of the carcasses were at the levels of 102 to 104 cfu/cm/, respectively. A significant (P<0.05) positive correlation was observed between the mean CC and FSC of the carcasses belonging to source A. The association between the mean TVC and FSC in source B was significant (P<0.05). Escherichia coli was isolated from 10 carcasses belonging to source A and 5 carcasses from source B. Out of the 15 isolates, 14 were serotyped and were grouped under nine serotypes and one isolate was untypeable. The serotype 0157 was isolated from two of the carcass samples belonging to source A. Four isolates from this source belonged to serotype 036. The serotypes 036, 0156, 0157 and 0172 were isolated only from the samples obtained from the source A. The serotype 08 was isolated from both the sources. However, the serotypes 013,065,069 and 075 were isolated only from samples of source B. Staphylococcus aureus was isolated from two (5 per cent) carcasses belonging to source A and one (2.5 per cent) of the carcasses belonging to the source B. Of-the 40 carcass samples tested, salmonellae were isolated from one sample each from the source A and B. Listeria monocytogenes could not be isolated from any of the samples belonging to both the sources. The mean total viable counts on meat-cutting board and meat-cutting table were 4.94 ± 0.87 and 4.28 ± 0.87 10glO cfu/cm/, respectively. The mean coliforms count on the former was 1.24 ± 0.94 10glO cfu/crrr' and the latter was at the level of 1.26 ± 0.83 10glO cfu/crrr'. The mean faecal streptococcal count was at the level of 1.38 and 1.29 10glO cfulcm2 on meat cutting board and meat cutting table, respectively. Ice samples had a mean total viable count of 3.20 ± 0.11 10glO cfu/ml. The coli forms count in ice, pond, and tap water were 2.30 ± 0.08, 1.39 ± 0.77 and 0.50 ± 0.21 10glO cfu/ml, respectively. The mean total viable count, coliforms count, Escherichia coli count and faecal streptococcal count observed per ml of the hand wash of the personnel engaged in various operations were 4.96 ± 0.82, 1.26 ± 0.78, 0.80 ± 0.24 and 1.48 ± 0.77 IOglO cfu/ml, respectively. The mean bacterial load in the air samples of slaughter hall and chilling room obtained in the present study was 100.7 ± 8.17 and 8.75 ± 1.19 cfu/min, respectively.
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