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Subject: Veterinary Public Health × End date: 2008 × Start date: 2008 × Type: Thesis × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen 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, B
    Rapid 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.
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    ThesisItemOpen Access
    Assessment of microbial quality, adulterants and preservatives in pasteurized milk
    (Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, 2008) Bhagyalakshmi, P S; KAU; Nanu, E
    In the present study 200 pasteurized milk samples were collected from the retail shops of Thrissur and Palakkad districts. From Thrissur district, 20 samples each were collected from five brands viz., A, B, C, D and E, and an equal number of samples were obtained from the brands F, G, H, I and J of Palakkad district. All the samples were analyzed for the microbial quality by estimating various bacterial counts and yeast and mould count and also assessed the presence of certain pathogenic and spoilage bacteria. The milk samples were also tested to detect the presence of adulterants viz., starch and cane sugar and preservatives like carbonates and formaldehyde. The study revealed that the samples belonging to Thrissur district had an overall mean total viable count, coliform count, Escherichia coli count, psychrotrophic count, faecal streptococcal count and yeast and mould count of 5.08 ± 0.05, 2.89 ± 0.09, 0.53 ± 0.11, 5.30 ± 0.01, 3.40 ± 0.14 and 1.89 ± 0.08 log10 cfu/ml, respectively. The corresponding count in the samples of Palakkad district was 5.24 ± 0.04, 3.01 ± 0.20, 0.78 ± 0.12, 4.99 ± 0.05, 3.20 ± 0.07 and 2.03 ± 0.09 log10 cfu/ml. According to the total viable count limit prescribed by BIS (1992) 50 per cent samples from brand E were graded as satisfactory and the samples from all other brands were graded as unsatisfactory. The highest mean total viable count was seen in the samples of brand A (5.39 ± 0.04 log10 cfu/ml). Of the samples collected from Thrissur district, the lowest count (4.51 ± 0.08 log10 cfu/ml) was recorded from the samples of brand E. The samples collected from I brand of Palakkad district had the highest mean total viable count (5.35 ± 0.05 log10 cfu/ml) and the lowest count (5.19 ± 0.09 log10 cfu/ml) was observed in the samples of brand G. Of the 100 samples collected from Thrissur district, the samples belonging to brand A had the highest mean coliform count (3.40 ± 0.17 log10 cfu/ml). An equal number of samples collected from Palakkad district revealed that the highest mean count (4.39 ± 0.02 log10 cfu/ml) was observed in the samples belonging to brand I. According to the bacterial count limit prescribed by BIS (1992) 70 per cent of the samples from brand H were graded as satisfactory and the samples belonging to all other brands were graded as unsatisfactory. The overall mean coliform count of the samples belonging to various brands from Thrissur and Palakkad districts were at the level of two and three log10 cfu/ml, respectively. The samples belonging to brand E of Thrissur and brand H of Palakkad had the lowest mean count. The samples collected from brand A of Thrissur district had the highest mean Escherichia coli count (0.92 ± 0.31 log10 cfu/ml) and the lowest count (0.10 ± 0.10 log10 cfu/ml) was observed in the samples belonging to the brand E. Among the samples collected from Palakkad district, the highest mean count (1.54 ± 0.11 log10 cfu/ml) was observed in the samples of the brand I and the lowest count (0.43 ± 0.23 log10 cfu/ml) was seen in the samples belonging to the brand H. The highest mean psychrotrophic count (5.39 ± 0.02 log10 cfu/ml) was seen in the samples belonging to brand C of Thrissur district and the lowest count (5.29 ± 0.05 log10 cfu/ml) was observed in the samples of the brand B. Among the samples from Palakkad district, the highest mean count (5.43 ± 0.01 log10 cfu/ml) was seen in the samples of the brand I and the lowest count (4.63 ± 0.10 log10 cfu/ml) was observed in the samples of the brand H. Of the samples collected from Thrissur district, the lowest mean faecal streptococcal count (2.95 ± 0.17 log10 cfu/ml) was seen in samples belonging to brand C and the highest count (3.78 ± 0.16 log10 cfu/ml) was observed in the samples of the brand B. Among the samples belonging to various brands of Palakkad district, the lowest mean count (2.88 ± 0.12 log10 cfu/ml) was seen in samples of the brand G and the highest count (3.51 ± 0.21 log10 cfu/ml) was observed in the samples of the brand I. Among the samples collected from the five brands of Thrissur district, the highest mean yeast and mould count (2.46 ± 0.17 log 10 cfu/ml) was seen in the samples of the brand A and the lowest count (1.35 ± 0.08 log 10 cfu/ml) was observed in the samples of the brand E. Of the samples belonging to the five brands of Palakkad, the highest mean count was seen in the samples of the brand J (2.40 ± 0.24 log10 cfu/ml) and the lowest count was observed in the samples of the brand F (1.66 ± 0.09 log10 cfu/ml). A highly significant (P<0.01) difference was noticed among the mean total viable count, coliform count, faecal streptoccal count and yeast and mould count of various brands of pasteurized milk from Thrissur district. Similarly a highly significant (P<0.01) difference was noticed among the mean coliform count, Escherichia coli count and psychrotrophic count of the samples belong to the five brands of pasteurized milk from Palakkad district. Escherichia coli was isolated from 10 per cent of the samples belonging to Thrissur and the isolates consisted of serotype of O4 (1), rough strains (2) and untypable strains (7). The organism was isolated from 11 per cent of the samples collected from Palakkad. One of the isolates was serotyped as O4 and three isolates were serotyped as O60. One isolate fell in the class rough and six isolates were untypable. Two isolates each from Thrissur and Palakkad districts revealed congo red binding charecteristics. Staphylococcus aureus could not isolate from the samples obtained from Thrissur and Palakkad districts. Pseudomonas organism was isolated from four and six per cent of the samples from Thrissur and Palakkad. The isolates were identified as Pseudomonas aeruginosa, Pseudomonas flourescens, Pseudomonas cepacia and Pseudomonas putida. Bacillus cereus was isolated from three samples obtained from Thrissur district and two samples belonging to Palakkad district. None of the samples from Thrissur and Palakkad districts revealed the presence of the adulterants like starch and cane sugar and preservative like bicarbonates. But formaldehyde was detected from 19 per cent of the samples from Thrissur and 47 per cent of the samples from Palakkad.