Livestock plays a polar function in the economic system of Pakistan being lending about 51.8 % to the Agricultural value add-on and 11.3 % to National GDP with a Gross value add-on of Rs. 1287 one million millions. The livestock pole of the state includes 33.0 million cowss, 29.9 million American bisons, 27.4 million of sheep, 58.3 million caprine animals and 1.0 million camels. ( Eco. Survey of Pak, 09 ) .
Besides Milk and Milk merchandises, the farm animal industry besides contributes to carry through the meat demand of the state with a entire production of 2.51 million dozenss including beef, mouton and domestic fowl meat part of 1.6, 0.59 and 0.65 million dozenss, severally. Other by merchandises of the meat industry includes 0.325 million dozenss of comestible offal ‘s, 46.82 million No. ‘s of backbones, 13.43 million No. ‘s of shells and 0.202 million dozenss of caput and trotters. ( Eco. Survey of Pak, 09 ) .
In past husbandmans were rise uping farm animal merely for subsistence but now-a-days with increasing challenges of monetary values hike, this tendency has been changed and now is a commercial concern. There is no specific strain for beef production in the state. The husbandmans keep cowss and American bisons simply for milk production and when its production become vanishes due to old age or disease or any other job it is so presented to the meat industry for butchering. The meat industry besides depends on males of cowss and American bisons. Scientists have made attempts to develop beef staff of life. They were successful to develop Narimaster, Charolais and Simmental crosses with Sahiwal, Dajal or Thari but their production and reproduction traits were non up to the criterions of good known alien beef strains ( Bhatti and Khan, 1999 ) .
In urban countries butchering is done in authorities and private slaughter houses whereas in most of the rural countries, it is in private little abattoirs owned by meatmans themselves. Furthermore, butchering is done in perfectly unhygienic conditions. The full animate being slaughtering operation is performed under same shed right from keeping of animate being boulder clay its transition into meat carcases. These meat carcases are so transported to retailer meat stores in unfastened trucks or pickups where displayed in unfastened air or stored without following hygienic steps and proper storage installation. Opportunities of microbic taint in meat cuts are much more under these conditions ( Rehman, 2010 ) .
Microbes have the ability to multiply quickly, show physiological differences and withstand with unfavourable environmental conditions thereby found everyplace in the existence and can pollute the un-hygienically processed and stored nutrient merchandises. These contaminated points causes nutrient born diseases in worlds when consumed. In add-on some bugs can bring forth powerful chemicals and toxins in nutrient points which may take to nutrient borne toxic condition. One of such type of most of import bacteriums is Escherichia coli 0157: H7 which is most prevailing serotype in nutrient born eruptions. The other most of import serotypes of Escherichia coli are O26, O111 which contribute in spread diseases through contaminated nutrient ( Garcia, 2002 ) . Shiga toxin ( Stx ) -producing Escherichia coli ( STEC ) is an emergent pathogen associated with nutrient born diseases, particularly groceries of animate being beginning ( Roldan et al. , 2007 ) .
Although Escherichia coli is one of the chief dwellers of the enteric piece of land of most mammalians species, including worlds and birds but still Verocytotoxin-producing Escherichia coli ( VTEC ) O157 besides known as Enterohaemorrhagic E. Coli ( EHEC ) , may do watery diarrhea, hemorrhagic inflammatory bowel disease and hemolytic azotemic syndrome in worlds ( Fairbrother and Nadeau, 2006 ) . Blanco et Al. 1996 revealed that EHEC causes hemorrhagic inflammatory bowel disease by the ingestion of uncooked minced meat. Meat and meat merchandises, imbibing H2O and veggies contaminated with carnal fecal matters are likely major beginnings of E. coli O157 infection ( Abong’o and Momba, 2008 ) .
Cattles are chief reservoir for VTEC O157 ( Nielsen et al. , 2002 ) and bacteriums can last on cowss farms for old ages ( Hancock et al. , 2001 ) . Arthur et Al. ( 2007 ) stated that cowss hides become contaminated with Escherichia coli O157: H7 via pathogen transmittal in feedlot, during conveyance and in lairage environment and bacteriums can be transferred into beef carcases at treating site.
This survey is designed to happen the bacterial taint degree particularly of Escherichia coli on animate being organic structure coat, carcase shortly after slaughter, processing tools ( cutting knife, meat chopper & A ; chopper axe ) , H2O used for rinsing & A ; slaughterhouse environment.
The chief aims of the present survey are ;
To find the concentration of Escherichia coli in meat at assorted phases by contaminated hair coat, H2O used, processing tools and unhygienic abattoir environment.
To place toxin bring forthing pathogens like Escherichia coli that causes spoilage of meat.
To develop possible methods for meatmans to hold hygienic ruddy meat by cut downing microbic burden.
Chapter # 2. REVIEW OF LITERATURE
Meat and meat merchandises are highly perishable, so particular attention must be taken in managing during all operations. It is by and large recognized that the station mortem alterations associated with the transition of musculus into meat and the subsequent handling and storage are accompanied by some impairment irrespective of safeguards taken during processing and handling.
Gorman et Al. ( 2002 ) elevated the incidence of Salmonella, Campylobacter, Escherichia coli and Staphylococcus aureus to go disseminated from infected nutrients, such as fresh poulets, to manus and nutrient contact surfaces in the domestic kitchen, repeating the demand for consumer consciousness and cognition of effectual hygiene processs in the domestic kitchen.
Microorganism on carnal coats:
Castillo et al. , ( 1999 ) compared the efficaciousness of a phosphorous acid-activated acidified Na chloride ( PASC ) spray and a citric acid-activated acidified Na chlorite ( CASC ) spray applied at room temperature ( 22.4 to 24.7 grades C ) in combination with a H2O wash with that of a H2O wash merely intervention for decrease of Escherichia coli O157: H7 and Salmonella Typhimurium inoculated onto assorted hot-boned single beef carcase surface. Initial counts of 5.5 and 5.4 log CFU/cm2 were obtained after vaccination with E. coli O157: H7 and Salmonella Typhimurium, severally. Pathogens were reduced by 3.8 to 3.9 log rhythms by H2O wash followed by PASC spray and by 4.5 to 4.6 log rhythms by H2O wash followed by CASC spray. Consequences of this survey indicate that acidified Na chlorite sprays are effectual for decontaminating beef carcase surfaces.
Reid et Al. ( 2001 ) conducted a survey for the microbic taint of beef, with micro-organisms transferred onto the carcase from the fell, during the slaughter and dressing procedures. The most contaminated country was the brisket with one in five animate beings proving positive for E. coli O157 ( 22.2 % prevalence on norm ) and about one in 10 animate beings proving positive for Salmonella spp. ( 10.0 % prevalence on norm ) . The least contaminated country on the cowss hides was the rump country ( 3.3 % prevalence for E. coli O157, 2.2 % prevalence for Salmonella spp. ) . Brisket country is hence most likely to take to cross-contamination of beef during the de-hiding procedure.
Fegan et Al. ( 2005 ) investigated Escherichia coli O157 taint of cowss during slaughter at an butchery. E. coli O157 was detected utilizing automated immunomagnetic separation ( AIMS ) and cell counts were determined utilizing a combination of most likely figure ( MPN ) and AIMS. E. coli O157 was isolated from 44 % fells. The prevalence in different groups ranged from less than 1 to 41 % . The Numberss of E. coli O157 differed among the animate beings groups. The group which contained the highest faecal ( 7.5 x 10 ( 5 ) MPN/g ) and fell ( 22 MPN/cm2 ) counts in any single animate being was the lone group in which E. coli O157 was isolated from carcases, proposing a nexus between the Numberss of E. coli O157 nowadays and the hazard of carcase taint. Processing patterns at this butchery were equal for minimising taint of carcases, even when animate beings were to a great extent contaminated with E. coli O157.
Arthur et Al. ( 2007 ) conducted a survey on transit and lairage environment effects on prevalence, Numberss and diverseness of Escherichia coli O157: H7 on fells and carcases of beef cowss at processing. On three separate occasions, samples were obtained from cowss at the feedlot and once more after cowss were stunned and exsanguinated at the processing works ( 286 sum animate beings ) . The prevalence of E. coli O157: H7 on fells increased from 50.3 to 94.4 % between the clip cowss were loaded onto tractor-trailers at the feedlot and the clip fells were removed in the processing works. Before conveyance, nine animate beings had E. coli O157: H7 in high Numberss ( & gt ; 0.4 CFU/cm2 ) on their fells. When sampled at the slaughter installation, the figure of animate beings with high fell Numberss had increased to 70. Overall, merely 29 % of the E. coli O157: H7 isolates collected postharvest ( 221 of 764 ) matched pulsed-field gel cataphoresis types collected before conveyance. Consequences indicated that conveyance to and lairage at treating workss can take to additions in the prevalence and grade of E. coli O157: H7 taint on fells and the figure of E. coli O157: H7
Dayna et Al. ( 2008 ) examined fell and carcase hygiene of cull cowss at slaughter, in U.S by mensurating the aerophilic home base count ( APC ) and the prevalence and burden of Salmonella and E. coli O157: H7. The geometric mean Log10 APC settlement organizing unit ( CFU ) /100 cm2 degrees on fells, pre-evisceration and post-intervention carcases ranged from 6.17 to 8.19, 4.24 to 6.47 and 1.46 to 1.96, severally. Average prevalence of Salmonella on fells, pre-evisceration and post-intervention carcases was 89.6 % and 50.2 % and 0.8 % , severally. Prevalence of E. coli O157: H7 was 46.9 % and 16.7 % on fells and pre-evisceration carcases, severally. Examination of the attendant incidence of Salmonella and E. coli O157: H7 showed that on norm, 33.3 % of cowss fells and 4.1 % of pre-evisceration carcase samples were contaminated with both pathogens. Pathogen prevalence on fells and carcases was non significantly affected by season, nevertheless, important differences were observed between workss with regard to incoming pathogen burden and the ability to extenuate fell to carcass transportation.
Prevalence of micro-organism in the butchery:
Guyon et Al. ( 2001 ) identified jeopardy points and critical points during beef slaughtering, which is a necessary first measure toward developing a jeopardy analysis and critical control point system to command meat taint by Escherichia coli O157: H7, samples ( n = 192 ) from surfaces, work tops, worker ‘s custodies, and beef carcases were collected from a abattoir in Calvados, France. This survey has shown that pre-evisceration and de-fatting station and associated worker ‘s stuffs are critical points for carcases taint by E. coli O157: H7 during beef slaughtering.
Barkocy et Al. ( 2003 ) studied the seasonal prevalence of E. coli and found that 60 % of fells and 26 % of carcases samples have E. coli which was sampled before the pre-evisceration wash. Salmonella prevalence peaked in fecal matters in the summer and was highest on fell and preevisceration carcases in the summer and the autumn. Non-O157 STEC prevalence besides appeared to change by season, but the efficiency in the recovery of isolates from stx-positive samples ranged from 37.5 to 83.8 % and could hold influenced these consequences.
Gun et Al. ( 2003 ) studied the taint of bovine carcases and abattoir environment by Escherichia coli O157: H7 in Istanbul. 3.6 % of E. coli O157 were isolated from the cattle carcases and eight ( 2.4 % ) of them gave positive reaction with anti-H: 7. Six strains of E. coli O157 were isolated from the environmental samples and all strains were positive for H7. The figure of E. coli O157H:7 strains isolated from the environmental samples was two from the knife, two from the custodies, one from the apron and one from the floor. No E. coli O157 was isolated from the abattoir H2O.
Woerner et Al. ( 2006 ) determined the prevalence of Escherichia coli O157 in cowss and beef from the feedlot to the ice chest. Faecal raps from the feedlot pen floor were collected within 3 yearss before slaughter. During cattle processing at the slaughter installation, extra samples were collected from the fell, from the colon, and from the carcases before and after evisceration and after concluding decontamination. Of 15 tonss ( a group of cowss from the same pen from a feedlot ) sampled, 47 % had a positive fell sample and 47 % had a positive carcase sample pre-evisceration ; nevertheless, merely 8 % of tonss had a positive carcase sample postevisceration or after concluding intercession. Of the entire samples tested ( n = 1,328 ) 14.7, 10.1, 1.4 and 0.3 % of faecal raps from the feedlot fell, pre-evisceration, postevisceration and concluding intercession samples, severally, were positive for E. coli O157. Pens with greater than 20 % positive faecal raps from the feedlot floor had 25.5 % fell and 14.3, 2.9 and 0.7 % carcase samples positive at pre-evisceration, at postevisceration and after concluding intercession, severally. However, the faecal raps from feedlot floor samples that contained less than 20 % positive faecal samples showed lower pathogen prevalence, with 5.0 % fell and 6.3, 0 and 0 % carcase positive samples at pre-evisceration, postevisceration and post-final intercession, severally.
Bosilevac et Al. ( 2009 ) studied the prevalence and numbering of Escherichia coli O157: H7 and Salmonella in U.S. butcheries that process fewer than 1000 caput of cowss per twenty-four hours. Across all workss, hide prevalence of E. coli O157: H7 and Salmonella was 71 and 91 % , severally. Twelve per centum of fells had E. coli O157: H7 at countable degrees ( & gt ; or =40 CFU/100 cm2 ) , while 36 % of fells had Salmonella at countable degrees. Across all workss, the prevalence of E. coli O157: H7 on pre-evisceration carcases was 33 % , with 2 % at an countable degree ( & gt ; or = 0.8 CFU/ 100 cm2 ) . Across all workss, Salmonella prevalence on pre-evisceration carcases was 58 % , with 8 % at an countable degree. Significant plant-to-plant fluctuations in degrees and prevalence of pathogens on carcases were detected. Reduced degrees of pathogens on carcases were noted among little processors that had incorporated a hide-directed intercession.
Carcass microbic taint
Chapman et Al. ( 1993 ) investigated cowss as a possible beginning of verocytotoxin-producing Escherichia coli O157 infections in adult male. During probe of the butchery, bovid rectal swabs and samples of meat and surface swabs from beef carcases were examined for E. coli O157, isolates of which were tested for toxigenicity, plasmid content and phage type. E. coli O157 was isolated from 84 ( 4 % ) of 2103 bovine rectal swabs ; of these 84, 78 ( 93 % ) were VT+ , the most common phage types being 2 and 8, the types implicated in the bunch of human instances. Positive cowss were from diverse beginnings within England. E. coli O157 was isolated from 30 % carcases of rectal swab-positive cowss and from 8 % carcases of rectal swab-negative cowss. The survey has shown that cowss may be a reservoir of VT+ E. coli O157 and that taint of carcases during slaughter and processing may be how beef and beef merchandises become contaminated and thereby convey the being to adult male.
In European states, nutrient safety policy and ordinance are really rigorous therefore one may non see the microbic taint in meat in these states as reported by the Richards et Al. ( 1998 ) . He studied the presence of verocytotoxic Escherichia coli O157 in bovine fecal matters submitted for diagnostic intents in England and Wales and on beef carcasss in butcheries in the United Kingdom. Contamination with verocytotoxin-producing E. coli ( VTEC ) O157 was confirmed in 0.47 % of the 4067 ( 95 % assurance limits 0.22-1.00 % ) of cervix musculus samples. A important inclination for carcasss present in the same butchery on the same twenty-four hours to hold similar consequences was found, therefore proposing cross taint. VTEC O157 was found in 0.83 % of 6495 bovine fecal matters samples routinely submitted for diagnostic intents to Veterinary Investigation Centres in England and Wales. Of the samples from cowss less than 6 months old, 3.7 % of 68 samples from animate beings without GI disease were positive for E. coli O157, in contrast to 0.75 % of 2321 samples from instances of GI disease. No association with season or herd type ( beef or dairy ) was found.
Madden et Al. ( 2001 ) conducted a survey to find the incidence of Escherichia coli O157: H7 in beef carcase. Analysiss were based on excised samples of cervix meat taken less than 48 h post-kill. Overall, 780 carcases were sampled and all were negative for E. coli O157: H7. A sub-set of samples was analyzed for the presence of Listeria monocytogenes ( n=200 ) , Salmonella ( n=200 ) and Campylobacter spp. ( n=100 ) . L. monocytogenes was non detected but Listeria innocua was found on five carcases and Listeria seeligeri on one. Three carcases carried salmonellas ; Salmonella Mbandaka was found on two and Salmonella Thompson on one.
Chapman et Al. ( 2001 ) investigated Escherichia coli O157 in cowss and sheep at slaughter, on beef and lamb carcases and in natural beef and lamb merchandises in South Yorkshire, UK. Samples of rectal fecal matters were collected instantly after slaughter from 400 cowss and 600 sheep, and 400-430 samples of natural meat merchandises were purchased from meatmans ‘ stores. Meat samples were besides obtained from 1500 beef and 1500 lamb carcases. All samples were examined for E. coli O157 by enrichment civilization, immunomagnetic separation and civilization of magnetic atoms onto cefixime tellurite sorbitol MacConkey agar. Raw meat merchandises were besides examined for Numberss of generic E. coli by a standard membrane civilization method. E. coli O157 was isolated from 620 ( 12.9 % ) of 4800 cowss, 100 ( 7.4 % ) of 7200 sheep, 21 ( 1.4 % ) of 1500 beef carcases, 10 ( 0.7 % ) of 1500 lamb carcases and from 22 ( 0.44 % ) of 4983 natural meat merchandises. E. coli O157 was isolated more often from lamb merchandises ( 0.8 % ) than from beef merchandises ( 0.4 % ) . Numbers of generic E. coli in meat merchandises reached seasonal extremums in July and August with counts of & gt ; 10 ( 4 ) /g happening more often in lamb merchandises ( 50.8 and 42.4 % , severally ) than in beef merchandises ( 19.3 and 23.8 % , severally ) . The bulk of E. coli O157 strains, from animate beings, carcases and meat samples, were isolated during the summer. Most were verocytotoxigenic as determined by Vero cell check and DNA hybridization, eaeA cistron positive and contained a 92 kilobit plasmid. The isolates were compared with 66 isolates from human instances over the same period. A combination of phage type, toxin genotype and plasmid analysis allowed subdivision of all the E. coli O157 isolates into 96 subtypes. Of these subtypes, 53 ( 55 % ) were isolated merely from bovid fecal samples. However, 61 ( 92 % ) of the 66 isolates from worlds belonged to 13 subtypes which were besides found in the carnal population.
Carney et Al. ( 2006 ) investigated the prevalence and degree of Escherichia coli O157 on samples of beef fixingss ( n=1351 ) , beef carcases ( n=132 ) and bovid caput meat ( n=132 ) in a beef slaughter works in Ireland. The study besides included an appraisal of the prevalence of virulency cistrons in the E. coli O157 isolates obtained. Samples were examined for the presence of E. coli O157 by direct plating on SMAC-CT and by enrichment/immunomagnetic separation ( IMS ) with plating of cured immunobeads onto SMAC-CT agar. Presumptive E. coli O157 isolates were confirmed by PCR aiming a scope of cistrons i.e. vt1, vt2, eaeA, hlyA, fliC ( h7 ) and parts of the rfb ( O-antigen encoding ) part of E. coli O157. Enterobacteriaceae on caput meat samples were estimated by direct plating onto Violet Red Bile Glucose agar. E. coli O157 was recovered from 2.4 % ( 32/1351 ) of beef fixingss samples, at concentrations runing from & lt ; 0.70-1.61 log10 cfu g ( -1 ) . Of the 32 positive isolates, 31 contained the eaeA and Hyla cistrons while 30/32 contained the fliC ( h7 ) cistron and 31/32 contained vt1 or vt2, or both vt cistrons. E. coli O157 was recovered from 3.0 % ( 4/132 ) of carcase samples, at concentrations runing from & lt ; 0.70-1.41 log10 cfu g ( -1 ) . All of the carcase isolates contained the eaeA, Hyla and fliC ( h7 ) cistrons. E. coli O157 was recovered from 3.0 % ( 3/100 ) of caput meat samples, at concentrations of 0.7-1.0 log10 cfu g ( -1 ) . All of the caput meat isolates contained the eaeA, Hyla, fliC ( h7 ) and vt2 cistrons. No caput meat isolates contained the vt1 cistron. Head meat samples ( n=100 ) contained Enterobacteriaceae, at concentrations runing from 0.70-3.0 log10 cfu g ( -1 ) . Overall, the qualitative and quantitative informations obtained for E. coli O157 on beef paring samples in this survey could be employed as portion of a quantitative hazard appraisal theoretical account.
Chahed et Al. ( 2006 ) studied the prevalence of enterohaemorrhagic Escherichia coli from serotype O157 and other attaching and obliterating Escherichia coli on bovine carcases in Algeria. Two-hundred and 30 carcases were swabbed and analysed by classical microbiological methods for entire E. coli counts and for the presence of infective E. coli. The E. coli counts were high, with a 75th percentile of 444.75 CFUs centimeter ( -2 ) . For infective E. coli, more than 7 % of the tried carcases were positive for E. coli O157. Eighteen E. coli O157 strains were isolated and typed by manifold PCR. The chief stray pathotype ( 78 % ) was eae+ stx2+ ehxA+ . In add-on to E. coli O157, other attaching and obliterating E. coli ( AEEC ) were besides detected from carcases by settlement hybridisation after pre-enrichment and plating on sorbitol MacConkey agar utilizing eae, stx1 and stx2 investigations. Thirty carcases ( 13 % ) on the 230 analyzed harbored at least one settlement positive for one of the tried investigations. These positive carcases were different from those positive for E. coli O157. Sixty-six settlements ( 2.9 % ) positive by settlement hybridisation were isolated. The bulk ( 60.6 % ) of the positive strains harboured an enteropathogenic E. coli-like pathotype ( eae+ stx- ) . Merely three enterohaemorrhagic E. coli ( EHEC ) -like ( eae+ stx1+ ) settlements were isolated from the same carcase. These strains did non belong to classical EHEC serotypes. The planetary hygiene of the abattoir was low, as indicated by the high degree of E. coli count. The prevalence of both E. coli O157 and other AEEC was besides high, stand foring a existent jeopardy for consumers.
Chapter. # 3.
Material and Methods:
This survey informations will be a baseline and in future microbic burden in meat at the slaughter may be monitored. This survey will be conducted in Peshawar metropolis. In this randomised sampling for microbic passenger car in abattoir ( private/government, located in Peshawar ) will be taken.
Beginnings of samples:
Cattles are the coinage of pick because it serves as reservoir for E-coli ( Nielsen et al. , 2002 ) among animate beings largely slaughtered at butcheries. All samples will be collected from predominating strains of cowss brought for butchering to the said butcheries. Cattle will be divided in two groups for sample aggregation i.e. washed animate beings ( Control Group ) and non-washed animate beings before slaughter. For control group, acidified sodium chlorite sprays ( phosphorous acid-activated acidified Na chloride ( PASC ) or citric acid-activated acidified Na chlorite ( CASC ) ) will be applied at room temperature ( 22.4 to 24.7 grades C ) in combination with H2O wash ( Castillo et al. , 1999 ) .
Random samples of ruddy meat will be collected from carcases at slaughter. Besides trying will be done from the processing tools ( used in butchering operation at the abattoir like cutting knife, meat chopper & A ; chopper axe ) , H2O used for carcase lavation and lairage environment. A sum of 80 samples will be collected. The item is as under ;
Table # 1. Animal Body Coat and Meat ( Carcass ) Sampling at the Slaughter House
Non- Washed Animals
15 Body Coat Samples
( 5 Animals )
10 Meat Samples
( 5 Animals )
Thigh ( Whole ) Meat
15 Body Coat Samples
10 Meat Samples
( 5 Animals )
Thigh ( Whole ) Meat
Table # 2. Samples from Processing Tools, Water & A ; Lairage Environment
Type of Sample
No. of Samples
Samples Collection & A ; Transportation system:
For trying from the animate being ‘s organic structure coat, a one-pass swab technique will be carried out to try a mensural country ( 12 inch ) on hindquarters, wing and brisket of each animate being before slaughter ( Reid et al. , 2001 ) . Same technique will be used for trying from liarage land.
Samples of ruddy meat ( 5 gm each ) will be collected in glass tubings incorporating 45ml of PBS ( Phosphate Field Buffer Solution ) / Normal Saline. On processing tools, taging 12 inch country on each tool will so be drained with normal saline/ PBS into the unfertile glass tubings. Samples from liarage air will be collected by exposing unfertile media home base in unfastened for 20 proceedingss.
All samples will be transported to Microbiology research lab, Department of Animal Health, NWFP Agricultural University, Peshawar harmonizing to the standard methods prescribed by Church and Wood, ( 1990 ) .
Meat Sample Processing:
For each meat sample collected from the abattoir, taking 5gm meat sample with 45ml of PBS in a high velocity winker jar will be blind it at 10,000 to 12,000 revolutions per minute to do 1:10 ( 10-1 ) dilution ( W/V ) . Using separate unfertile pipette fix the denary dilutions ( 10-2, 10-3aˆ¦ 10-10 ) by blending 1ml from the old dilution and 9ml of PBS in separate tubings. Shake all dilutions 25 times in 30cm arch for 7 seconds.
Isolation and Identification of E-coli:
Designation and verification of E.coli bacteriums will be performed following the standard protocols as per Bergey ‘s Manual of Determinative Bacteriology ( 9th edition ) . Basic parametric quantities for designation of bacteriums will be based on morphological features, microscopic characteristics and Biochemical profiles. Pure civilization will be maintained on Eosine Methylene Blue ( EMB ) agar. Indole Production Test, Methyl Red Test, Voges Prausker ‘s Test and Citrate Utilization Test will be applied for verification of E. coli isolates.
Determination of Pathogenicity:
Congo ruddy medium will be used to find pathogenicity of E. coli isolates. Growth of brick ruddy colour settlements will be declarative of infective E. coli while non-pathogenic will bring forth grey white settlements after 96 hours incubation ( Ahmad et al. , 2009 ) .
Data will be collected utilizing MS Excel Sheet.
MS Excel & A ; SPSS for summery statistics.
Using CRD ( Wholly Randomized Design ) , informations will be analyzed through SPSS ( version 18 ) .
Control group will be compared with Non-control group for E. coli prevalence.
DMR ( Duncan ‘s Multiple Range ) trial will be used for pair-wise comparing.
A multiple additive regressing will be fitted to set up the relationship b/w E. coli prevalence with clip, hygienic conditions, processing tools & A ; liarage environment.
Chapter. # 4. LITERATURE CITED
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Isolation and Identification of E-coli:
Protocol for E. coli Culturing and Counting:
Pipette 1ml of each dilution into separate extra suitably marked petri-dishes.
Add 20ml of molten Eosin Methylene Blue ( EMB ) agar holding a temperature of 45-46 A°C to each home base within 15 proceedingss of original dilution. ( EMB agar is selective for E-coli growing ) .
Immediately blend the sample dilution and agar medium exhaustively and uniformly.
Allow agar to solidify, invert petri-plate and incubate for 48 hours at 37A°C.
After incubation, select the dishes holding 25-250 settlements for settlements numbering and isolation of pure civilization. The E-coli settlements show a metallic sheen colour.
The entire feasible count/ml of the sample for E-coli will be calculated as ;
Entire Viable Count = Colonies X Dilution Factor.
When two home bases are in appropriate settlements range, take the norm of both and put in the above expression.
Gram ‘s staining:
Fix a vilification with the civilized beings on glass slide and arrested development by rapidly go throughing it 2 to 3 times over fire.
Pour Crystal Violet dye ( basic discoloration ) on the vilification for one minute and gently wash with H2O.
Apply Gram ‘s Iodine solution ( black ) on the vilification for one minute and gently wash with H2O.
Bleach with 95 % Ethyle intoxicant for 15 seconds and gently wash with H2O.
Counter discoloration with Safranine for 0.5 -1 minute and gently wash with H2O.
Blot dry with boozy paper or air dry and examine under microscope utilizing 100x oil submergence lens.
Consequence: Pink/Red colour and Rod shaped beings will give indicant of E-coli ( Gram negative ) .
Bio-chemical Trials ( IMViC ) :
Indole Production Trial:
Inoculate the tryptophane stock with the trial being ( 1 bead ) from a 24 hours encephalon hearth extract ( BHI ) broth civilization.
Incubate at 35-37 A°C for 24-48 hours.
To 5 milliliter of incubated tryptone stock incorporating the trial being, add few beads ( 0.2-0.5 milliliter ) of Kovac ‘s reagent. Blend it and maintain the tubing in the base undisturbed for a few proceedingss.
Consequence: Appearance of a pink of distinguishable ruddy colour thin bed at the top of the stock is a positive indole production trial.
Voges Proskaur Trial:
Inoculate the 1 bead of test cultural beings from 24 hours BHI broth civilization to the MR-VP medium.
Incubate for 48-50 hours at 35-37 A°C.
Add 0.5 milliliter Voges Proskaur Reagent.
Consequence: Positive if pink colour and negative if colorless.
Methyl Red Trial:
Incubate MR-VP medium tubings for extra 48 hours at 35-37 A°C after executing Voges Proskaur Test.
Add 5 beads of methyl ruddy solution to each tubing and mix gently.
Consequence: Yellow colour is negative trial while ruddy colour is a positive trial.
Citrate Utilization Test:
Lightly inoculate the aslant tubing of Kauser Citrate Broth/ Simmon ‘s Citrate Broth with civilization being.
Incubate for 4-7 yearss at 35-37 A°C.
Consequence: Changing colour from green to blue or development of distinguishable turbidness in blue ( Cu like ) colour of citrate stock is a positive trial.
IMViC Pattern for E-Coli: ( + + – – ) i.e.
Indole Test = Positive, Methyl red Test = Positive, Voges Proskaur Test = Negative and Citrate Utilization Test = Negative.
Material and Equipments to be used in the said research are list below:
Sterile Stomacher Bags
Sterile Cotton Swabs
Disposable Baseball gloves
Microscopic holding 100x Oil Emersion Lens.
Screw Capped Bottles
Chemicals / Media to be used in the said research:
Normal Saline/ Phosphate Field Buffer Solution ( PBS ) .
Media ; Eosin Methylene Blue Agar ( EMB ) for culturing of E-coli.
Crystal Violet Dye ( for primary staining ) .
Iodine Solution ( repairing agent ) .
Safranine Dye ( counter staining ) .
Alcohol ( bleaching agent ) .
Kovac ‘s Reagent
Voges Proskaur Reagent
Methyl Red Solution
Kauser Citrate Broth/ Simmon ‘s Citrate Broth
Brain Heart Infusion ( BHI ) Broth
Congo Red Media
Media and Reagents Preparation:
Phosphate Field Buffer Solution:
Mix 34gm of KH2PO4 with 500 milliliters distilled H2O. Adjust PH to 7.2 with 1 normal NAOH solution. Bring volume to 1 litre with distilled H2O. Sterilize at 121A°C for 15 proceedingss and shop in a icebox.
Kovac ‘s Reagent:
Dissolve 5 gram p-Dimethyl-aminobenzaldehyde in 75 milliliter of isoamyl intoxicant. Then easy add 25 milliliter of ( conc. ) HCl. Shop at 4A°C.
Dissolve 20gm Tryptose, 5gm Lactose, 2.75gm KH2PO4, 2.75gm K2HPO4, 5gm NaCl and 1gm Sodium Laryal Sulphate in 800 milliliters Distilled Water with soft heat, so filter, cool to 20 A°C and dilute to 1 litre. Auto-Clave it and adjust concluding Ph to 6.8 A± 0.2.
Dissolve 7gm Buffered Peptone, 5gm Glucose and 5gm K2HPO4 in 800 milliliters Distilled Water with soft heat, so filter, cool to 20 A°C and dilute to 1 litre. Auto-Clave it and adjust concluding Ph to 6.8 A± 0.2.
Voges Proskaur Reagent:
Add 0.6 milliliter of alpha-nepthol solution to 0.2 milliliter of 40 % KOH solution, shingle, and so add few crystals of creatine.