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Livestock plays a pivotal role in the economy of Pakistan being contributing approximately 51.8 % to the Agricultural value addition and 11.3% to National GDP with a Gross value addition of Rs. 1287 billions. The livestock pole of the country includes 33.0 million cattle, 29.9 million buffaloes, 27.4 million of sheep, 58.3 million goats and 1.0 million camels. (Eco. Survey of Pak, 09).
Besides Milk and Milk products, the livestock industry also contributes to fulfill the meat demand of the country with a total production of 2.51 million tons including beef, mutton and poultry meat contribution of 1.6, 0.59 and 0.65 million tons, respectively. Other by products of the meat industry includes 0.325 million tons of edible offal's, 46.82 million No.'s of guts, 13.43 million No.'s of casings and 0.202 million tons of head and trotters. (Eco. Survey of Pak, 09).
In past farmers were rearing livestock only for subsistence but now-a-days with increasing challenges of prices hike, this trend has been changed and now is a commercial business. There is no specific breed for beef production in the country. The farmers keep cattle and buffaloes merely for milk production and when its production become vanishes due to old age or disease or any other problem it is then presented to the meat industry for slaughtering. The meat industry also depends on males of cattle and buffaloes. Scientists have made efforts to develop beef bread. They were successful to develop Narimaster, Charolais and Simmental crosses with Sahiwal, Dajal or Thari but their production and reproduction traits were not up to the standards of well known exotic beef breeds (Bhatti and Khan, 1999).
In urban areas slaughtering is done in government and private slaughter houses whereas in most of the rural areas, it is in private small butcheries owned by butchers themselves. Furthermore, slaughtering is done in absolutely unhygienic conditions. The entire animal slaughtering operation is performed under same shed right from restraining of animal till its conversion into meat carcasses. These meat carcasses are then transported to retailer meat shops in open trucks or pickups where displayed in open air or stored without adopting hygienic measures and proper storage facility. Chances of microbial contamination in meat cuts are much more under these conditions (Rehman, 2010).
Microbes have the ability to multiply rapidly, show physiological differences and withstand with unfavorable environmental conditions thereby found everywhere in the universe and can contaminate the un-hygienically processed and stored food products. These contaminated items causes food born diseases in humans when consumed. In addition some microbes can produce potent chemicals and toxins in food items which may lead to food borne poisoning. One of such type of most important bacteria is Escherichia coli 0157:H7 which is most prevalent serotype in food born outbreaks. The other most important serotypes of Escherichia coli are O26, O111 which contribute in spread diseases through contaminated food (Garcia, 2002). Shiga toxin (Stx)-producing Escherichia coli (STEC) is an emergent pathogen associated with food born diseases, especially foodstuffs of animal origin (Roldan et al., 2007).
Although Escherichia coli is one of the main inhabitants of the intestinal tract of most mammalians species, including humans and birds but still Verocytotoxin-producing Escherichia coli (VTEC) O157 also known as Enterohaemorrhagic E. Coli (EHEC), may cause watery diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome in humans (Fairbrother and Nadeau, 2006). Blanco et al. 1996 revealed that EHEC causes haemorrhagic colitis by the consumption of uncooked minced meat. Meat and meat products, drinking water and vegetables contaminated with animal feces are probably major sources of E. coli O157 infection (Abong'o and Momba, 2008).
Cattle are main reservoir for VTEC O157 (Nielsen et al., 2002) and bacteria can survive on cattle farms for years (Hancock et al., 2001). Arthur et al. (2007) stated that cattle hides become contaminated with Escherichia coli O157:H7 via pathogen transmission in feedlot, during transport and in lairage environment and bacteria can be transferred into beef carcasses at processing site.
This study is designed to find the bacterial contamination level especially of Escherichia coli on animal body coat, carcass soon after slaughter, processing tools (cutting knife, meat cleaver & chopper axe), water used for washing & slaughterhouse environment.
The main objectives of the present study are;
To determine the concentration of Escherichia coli in meat at various stages by contaminated hair coat, water used, processing tools and unhygienic slaughterhouse environment.
To identify toxin producing pathogens like Escherichia coli that causes spoilage of meat.
To develop possible methods for butchers to have hygienic red meat by reducing microbial load.
Chapter # 2. REVIEW OF LITERATURE
Meat and meat products are extremely perishable, so special care must be taken in handling during all operations. It is generally recognized that the post mortem changes associated with the conversion of muscle into meat and the subsequent handling and storage are accompanied by some deterioration irrespective of precautions taken during processing and handling.
Gorman et al. (2002) elevated the incidence of Salmonella, Campylobacter, Escherichia coli and Staphylococcus aureus to become disseminated from infected foods, such as fresh chickens, to hand and food contact surfaces in the domestic kitchen, reiterating the need for consumer awareness and knowledge of effective hygiene procedures in the domestic kitchen.
Microorganism on animal coats:
Castillo et al., (1999) compared the efficacy of a phosphoric acid-activated acidified sodium chloride (PASC) spray and a citric acid-activated acidified sodium chlorite (CASC) spray applied at room temperature (22.4 to 24.7 degrees C) in combination with a water wash with that of a water wash only treatment for reduction of Escherichia coli O157:H7 and Salmonella Typhimurium inoculated onto various hot-boned individual beef carcass surface. Initial counts of 5.5 and 5.4 log CFU/cm2 were obtained after inoculation with E. coli O157:H7 and Salmonella Typhimurium, respectively. Pathogens were reduced by 3.8 to 3.9 log cycles by water wash followed by PASC spray and by 4.5 to 4.6 log cycles by water wash followed by CASC spray. Results of this study indicate that acidified sodium chlorite sprays are effective for decontaminating beef carcass surfaces.
Reid et al. (2001) conducted a study for the microbial contamination of beef, with microorganisms transferred onto the carcass from the hide, during the slaughter and dressing processes. The most contaminated area was the brisket with one in five animals testing positive for E. coli O157 (22.2% prevalence on average) and approximately one in 10 animals testing positive for Salmonella spp. (10.0% prevalence on average). The least contaminated area on the cattle hides was the rump area (3.3% prevalence for E. coli O157, 2.2% prevalence for Salmonella spp.). Brisket area is therefore most likely to lead to cross-contamination of beef during the de-hiding process.
Fegan et al. (2005) investigated Escherichia coli O157 contamination of cattle during slaughter at an abattoir. E. coli O157 was detected using automated immunomagnetic separation (AIMS) and cell counts were determined using a combination of most probable number (MPN) and AIMS. E. coli O157 was isolated from 44% hides. The prevalence in different groups ranged from less than 1 to 41%. The numbers of E. coli O157 differed among the animals groups. The group which contained the highest fecal (7.5 x 10(5) MPN/g) and hide (22 MPN/cm2) counts in any individual animal was the only group in which E. coli O157 was isolated from carcasses, suggesting a link between the numbers of E. coli O157 present and the risk of carcass contamination. Processing practices at this abattoir were adequate for minimizing contamination of carcasses, even when animals were heavily contaminated with E. coli O157.
Arthur et al. (2007) conducted a study on transportation and lairage environment effects on prevalence, numbers and diversity of Escherichia coli O157:H7 on hides and carcasses of beef cattle at processing. On three separate occasions, samples were obtained from cattle at the feedlot and again after cattle were stunned and exsanguinated at the processing plant (286 total animals). The prevalence of E. coli O157:H7 on hides increased from 50.3 to 94.4% between the time cattle were loaded onto tractor-trailers at the feedlot and the time hides were removed in the processing plant. Before transport, nine animals had E. coli O157:H7 in high numbers (> 0.4 CFU/cm2) on their hides. When sampled at the slaughter facility, the number of animals with high hide numbers had increased to 70. Overall, only 29% of the E. coli O157:H7 isolates collected postharvest (221 of 764) matched pulsed-field gel electrophoresis types collected before transport. Results indicated that transport to and lairage at processing plants can lead to increases in the prevalence and degree of E. coli O157:H7 contamination on hides and the number of E. coli O157:H7
Dayna et al. (2008) examined hide and carcass hygiene of cull cattle at slaughter, in U.S by measuring the aerobic plate count (APC) and the prevalence and load of Salmonella and E. coli O157:H7. The geometric mean Log10 APC colony forming unit (CFU)/100 cm2 levels on hides, pre-evisceration and post-intervention carcasses ranged from 6.17 to 8.19, 4.24 to 6.47 and 1.46 to 1.96, respectively. Average prevalence of Salmonella on hides, pre-evisceration and post-intervention carcasses was 89.6% and 50.2% and 0.8%, respectively. Prevalence of E. coli O157:H7 was 46.9% and 16.7% on hides and pre-evisceration carcasses, respectively. Examination of the concomitant incidence of Salmonella and E. coli O157:H7 showed that on average, 33.3% of cattle hides and 4.1% of pre-evisceration carcass samples were contaminated with both pathogens. Pathogen prevalence on hides and carcasses was not significantly affected by season, however, significant differences were observed between plants with respect to incoming pathogen load and the ability to mitigate hide to carcass transfer.
Prevalence of microorganism in the abattoir:
Guyon et al. (2001) identified hazard points and critical points during beef slaughtering, which is a necessary first step toward developing a hazard analysis and critical control point system to control meat contamination by Escherichia coli O157:H7, samples (n = 192) from surfaces, work tops, worker's hands, and beef carcasses were collected from a slaughterhouse in Calvados, France. This study has shown that pre-evisceration and de-fatting post and associated worker's materials are critical points for carcasses contamination by E. coli O157:H7 during beef slaughtering.
Barkocy et al. (2003) studied the seasonal prevalence of E. coli and found that 60% of hides and 26% of carcasses samples have E. coli which was sampled before the pre-evisceration wash. Salmonella prevalence peaked in feces in the summer and was highest on hide and preevisceration carcasses in the summer and the fall. Non-O157 STEC prevalence also appeared to vary by season, but the efficiency in the recovery of isolates from stx-positive samples ranged from 37.5 to 83.8% and could have influenced these results.
Gun et al. (2003) studied the contamination of bovine carcasses and abattoir environment by Escherichia coli O157:H7 in Istanbul. 3.6% of E. coli O157 were isolated from the cattle carcasses 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 number of E. coli O157H:7 strains isolated from the environmental samples was two from the knife, two from the hands, one from the apron and one from the floor. No E. coli O157 was isolated from the abattoir water.
Woerner et al. (2006) determined the prevalence of Escherichia coli O157 in cattle and beef from the feedlot to the cooler. Fecal pats from the feedlot pen floor were collected within 3 days before slaughter. During cattle processing at the slaughter facility, additional samples were collected from the hide, from the colon, and from the carcasses before and after evisceration and after final decontamination. Of 15 lots (a group of cattle from the same pen from a feedlot) sampled, 47% had a positive hide sample and 47% had a positive carcass sample pre-evisceration; however, only 8% of lots had a positive carcass sample postevisceration or after final intervention. Of the total samples tested (n = 1,328) 14.7, 10.1, 1.4 and 0.3% of fecal pats from the feedlot hide, pre-evisceration, postevisceration and final intervention samples, respectively, were positive for E. coli O157. Pens with greater than 20% positive fecal pats from the feedlot floor had 25.5% hide and 14.3, 2.9 and 0.7% carcass samples positive at pre-evisceration, at postevisceration and after final intervention, respectively. However, the fecal pats from feedlot floor samples that contained less than 20% positive fecal samples showed lower pathogen prevalence, with 5.0% hide and 6.3, 0 and 0% carcass positive samples at pre-evisceration, postevisceration and post-final intervention, respectively.
Bosilevac et al. (2009) studied the prevalence and enumeration of Escherichia coli O157:H7 and Salmonella in U.S. abattoirs that process fewer than 1000 head of cattle per day. Across all plants, hide prevalence of E. coli O157:H7 and Salmonella was 71 and 91%, respectively. Twelve percent of hides had E. coli O157:H7 at enumerable levels (> or =40 CFU/100 cm2), while 36% of hides had Salmonella at enumerable levels. Across all plants, the prevalence of E. coli O157:H7 on pre-evisceration carcasses was 33%, with 2% at an enumerable level (> or = 0.8 CFU/ 100 cm2). Across all plants, Salmonella prevalence on pre-evisceration carcasses was 58%, with 8% at an enumerable level. Significant plant-to-plant variations in levels and prevalence of pathogens on carcasses were detected. Reduced levels of pathogens on carcasses were noted among small processors that had incorporated a hide-directed intervention.
Carcass microbial contamination
Chapman et al. (1993) investigated cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infections in man. During investigation of the abattoir, bovine rectal swabs and samples of meat and surface swabs from beef carcasses 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 cluster of human cases. Positive cattle were from diverse sources within England. E. coli O157 was isolated from 30% carcasses of rectal swab-positive cattle and from 8% carcasses of rectal swab-negative cattle. The study has shown that cattle may be a reservoir of VT+ E. coli O157 and that contamination of carcasses during slaughter and processing may be how beef and beef products become contaminated and thereby transmit the organism to man.
In European countries, food safety policy and regulation are very strict therefore one may not see the microbial contamination in meat in these countries as reported by the Richards et al. (1998). He studied the presence of verocytotoxic Escherichia coli O157 in bovine faeces submitted for diagnostic purposes in England and Wales and on beef carcases in abattoirs in the United Kingdom. Contamination with verocytotoxin-producing E. coli (VTEC) O157 was confirmed in 0.47% of the 4067 (95% confidence limits 0.22-1.00%) of neck muscle samples. A significant tendency for carcases present in the same abattoir on the same day to have similar results was found, thus suggesting cross contamination. VTEC O157 was found in 0.83% of 6495 bovine faeces samples routinely submitted for diagnostic purposes to Veterinary Investigation Centres in England and Wales. Of the samples from cattle less than 6 months old, 3.7% of 68 samples from animals without gastrointestinal disease were positive for E. coli O157, in contrast to 0.75% of 2321 samples from cases of gastrointestinal disease. No association with season or herd type (beef or dairy) was found.
Madden et al. (2001) conducted a study to determine the incidence of Escherichia coli O157:H7 in beef carcass. Analyses were based on excised samples of neck meat taken less than 48 h post-kill. Overall, 780 carcasses 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 not detected but Listeria innocua was found on five carcasses and Listeria seeligeri on one. Three carcasses carried salmonellas; Salmonella Mbandaka was found on two and Salmonella Thompson on one.
Chapman et al. (2001) investigated Escherichia coli O157 in cattle and sheep at slaughter, on beef and lamb carcasses and in raw beef and lamb products in South Yorkshire, UK. Samples of rectal faeces were collected immediately after slaughter from 400 cattle and 600 sheep, and 400-430 samples of raw meat products were purchased from butchers' shops. Meat samples were also obtained from 1500 beef and 1500 lamb carcasses. All samples were examined for E. coli O157 by enrichment culture, immunomagnetic separation and culture of magnetic particles onto cefixime tellurite sorbitol MacConkey agar. Raw meat products were also examined for numbers of generic E. coli by a standard membrane culture method. E. coli O157 was isolated from 620 (12.9%) of 4800 cattle, 100 (7.4%) of 7200 sheep, 21 (1.4%) of 1500 beef carcasses, 10 (0.7%) of 1500 lamb carcasses and from 22 (0.44%) of 4983 raw meat products. E. coli O157 was isolated more frequently from lamb products (0.8%) than from beef products (0.4%). Numbers of generic E. coli in meat products reached seasonal peaks in July and August with counts of > 10(4)/g occurring more frequently in lamb products (50.8 and 42.4%, respectively) than in beef products (19.3 and 23.8%, respectively). The majority of E. coli O157 strains, from animals, carcasses and meat samples, were isolated during the summer. Most were verocytotoxigenic as determined by Vero cell assay and DNA hybridisation, eaeA gene positive and contained a 92 kb plasmid. The isolates were compared with 66 isolates from human cases 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 only from bovine faecal samples. However, 61 (92%) of the 66 isolates from humans belonged to 13 subtypes which were also found in the animal population.
Carney et al. (2006) investigated the prevalence and level of Escherichia coli O157 on samples of beef trimmings (n=1351), beef carcasses (n=132) and bovine head meat (n=132) in a beef slaughter plant in Ireland. The survey also included an assessment of the prevalence of virulence genes 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 recovered immunobeads onto SMAC-CT agar. Presumptive E. coli O157 isolates were confirmed by PCR targeting a range of genes i.e. vt1, vt2, eaeA, hlyA, fliC(h7) and portions of the rfb (O-antigen encoding) region of E. coli O157. Enterobacteriaceae on head 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 trimmings samples, at concentrations ranging from<0.70-1.61 log10 cfu g(-1). Of the 32 positive isolates, 31 contained the eaeA and hylA genes while 30/32 contained the fliC(h7) gene and 31/32 contained vt1 or vt2, or both vt genes. E. coli O157 was recovered from 3.0% (4/132) of carcass samples, at concentrations ranging from <0.70-1.41 log10 cfu g(-1). All of the carcass isolates contained the eaeA, hylA and fliC(h7) genes. E. coli O157 was recovered from 3.0% (3/100) of head meat samples, at concentrations of 0.7-1.0 log10 cfu g(-1). All of the head meat isolates contained the eaeA, hylA, fliC(h7) and vt2 genes. No head meat isolates contained the vt1 gene. Head meat samples (n=100) contained Enterobacteriaceae, at concentrations ranging from 0.70-3.0 log10 cfu g(-1). Overall, the qualitative and quantitative data obtained for E. coli O157 on beef trimming samples in this study could be employed as part of a quantitative risk assessment model.
Chahed et al. (2006) studied the prevalence of enterohaemorrhagic Escherichia coli from serotype O157 and other attaching and effacing Escherichia coli on bovine carcasses in Algeria. Two-hundred and thirty carcasses were swabbed and analysed by classical microbiological methods for total E. coli counts and for the presence of pathogenic E. coli. The E. coli counts were high, with a 75th percentile of 444.75 CFUs cm(-2). For pathogenic E. coli, more than 7% of the tested carcasses were positive for E. coli O157. Eighteen E. coli O157 strains were isolated and typed by multiplex PCR. The main isolated pathotype (78%) was eae+ stx2+ ehxA+. In addition to E. coli O157, other attaching and effacing E. coli (AEEC) were also detected from carcasses by colony hybridization after pre-enrichment and plating on sorbitol MacConkey agar using eae, stx1 and stx2 probes. Thirty carcasses (13%) on the 230 analyzed harbored at least one colony positive for one of the tested probes. These positive carcasses were different from those positive for E. coli O157. Sixty-six colonies (2.9%) positive by colony hybridization were isolated. The majority (60.6%) of the positive strains harboured an enteropathogenic E. coli-like pathotype (eae+ stx-). Only three enterohaemorrhagic E. coli (EHEC)-like (eae+ stx1+) colonies were isolated from the same carcass. These strains did not belong to classical EHEC serotypes. The global hygiene of the slaughterhouse was low, as indicated by the high level of E. coli count. The prevalence of both E. coli O157 and other AEEC was also high, representing a real hazard for consumers.
Chapter. # 3.
Material and Methods:
This study data will be a baseline and in future microbial load in meat at the slaughter may be monitored. This study will be conducted in Peshawar city. In this randomized sampling for microbial carriage in slaughterhouse (private/government, located in Peshawar) will be taken.
Sources of samples:
Cattle are the specie of choice because it serves as reservoir for E-coli (Nielsen et al., 2002) among animals mostly slaughtered at abattoirs. All samples will be collected from prevailing breeds of cattle brought for slaughtering to the said abattoirs. Cattle will be divided in two groups for sample collection i.e. washed animals (Control Group) and non-washed animals before slaughter. For control group, acidified sodium chlorite sprays (phosphoric acid-activated acidified sodium chloride (PASC) or citric acid-activated acidified sodium chlorite (CASC)) will be applied at room temperature (22.4 to 24.7 degrees C) in combination with water wash (Castillo et al., 1999).
Random samples of red meat will be collected from carcasses at slaughter. Also sampling will be done from the processing tools (used in slaughtering operation at the slaughterhouse like cutting knife, meat cleaver & chopper axe), water used for carcass washing and lairage environment. A total of 80 samples will be collected. The detail is as under;
Table# 1. Animal Body Coat and Meat (Carcass) Sampling at the Slaughter House
Non- Washed Animals
15 Body Coat Samples
10 Meat Samples
Thigh (Whole) Meat
15 Body Coat Samples
10 Meat Samples
Thigh (Whole) Meat
Table# 2. Samples from Processing Tools, Water & Lairage Environment
Type of Sample
No. of Samples
Samples Collection & Transportation:
For sampling from the animal's body coat, a one-pass swab technique will be carried out to sample a measured area (12 inch) on rump, flank and brisket of each animal before slaughter (Reid et al., 2001). Same technique will be used for sampling from liarage ground.
Samples of red meat (5 gm each) will be collected in glass tubes containing 45ml of PBS (Phosphate Field Buffer Solution)/ Normal Saline. On processing tools, marking 12 inch area on each tool will then be drained with normal saline/ PBS into the sterile glass tubes. Samples from liarage air will be collected by exposing sterile media plate in open for 20 minutes.
All samples will be transported to Microbiology laboratory, Department of Animal Health, NWFP Agricultural University, Peshawar according to the standard methods prescribed by Church and Wood, (1990).
Meat Sample Processing:
For each meat sample collected from the slaughterhouse, taking 5gm meat sample with 45ml of PBS in a high speed blinder jar will be blind it at 10,000 to 12,000 rpm to make 1:10 (10-1) dilution (W/V). Using separate sterile pipette prepare the decimal dilutions (10-2, 10-3â€¦ 10-10) by mixing 1ml from the previous dilution and 9ml of PBS in separate tubes. Shake all dilutions 25 times in 30cm arch for 7 seconds.
Isolation and Identification of E-coli:
Identification and confirmation of E.coli bacteria will be performed following the standard protocols as per Bergey's Manual of Determinative Bacteriology (9th edition). Basic parameters for identification of bacteria will be based on morphological characteristics, microscopic features and Biochemical profiles. Pure culture 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 confirmation of E. coli isolates.
Determination of Pathogenicity:
Congo red medium will be used to determine pathogenicity of E. coli isolates. Growth of brick red color colonies will be indicative of pathogenic E. coli while non-pathogenic will produce grayish white colonies after 96 hours incubation (Ahmad et al., 2009).
Data will be collected using MS Excel Sheet.
MS Excel & SPSS for summery statistics.
Using CRD (Completely Randomized Design), data 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) test will be used for pair-wise comparison.
A multiple linear regressing will be fitted to establish the relationship b/w E. coli prevalence with time, hygienic conditions, processing tools & 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 duplicate appropriately marked petri-dishes.
Add 20ml of molten Eosin Methylene Blue (EMB) agar having a temperature of 45-46 Â°C to each plate within 15 minutes of original dilution. (EMB agar is selective for E-coli growth).
Immediately mix the sample dilution and agar medium thoroughly and uniformly.
Allow agar to solidify, invert petri-plate and incubate for 48 hours at 37Â°C.
After incubation, select the dishes having 25-250 colonies for colonies counting and isolation of pure culture. The E-coli colonies show a metallic sheen color.
The total viable count/ml of the sample for E-coli will be calculated as;
Total Viable Count = Colonies X Dilution Factor.
When two plates are in appropriate colonies range, take the average of both and put in the above formula.
Prepare a smear with the cultured organisms on glass slide and fixation by quickly passing it 2 to 3 times over flame.
Pour Crystal Violet dye (basic stain) on the smear for one minute and gently wash with water.
Apply Gram's Iodine solution (mordant) on the smear for one minute and gently wash with water.
Decolorize with 95% Ethyle alcohol for 15 seconds and gently wash with water.
Counter stain with Safranine for 0.5 -1 minute and gently wash with water.
Blot dry with bibulous paper or air dry and examine under microscope using 100x oil immersion lens.
Result: Pink/Red color and Rod shaped organisms will give indication of E-coli (Gram negative).
Bio-chemical Tests (IMViC):
Indole Production Test:
Inoculate the tryptophane broth with the test organism (1 drop) from a 24 hours brain hearth infusion (BHI) broth culture.
Incubate at 35-37 Â°C for 24-48 hours.
To 5 ml of incubated tryptone broth containing the test organisms, add few drops (0.2-0.5 ml) of Kovac's reagent. Mix it and keep the tube in the stand undisturbed for a few minutes.
Result: Appearance of a pink of distinct red color thin layer at the top of the broth is a positive indole production test.
Voges Proskaur Test:
Inoculate the 1 drop of test cultural organisms from 24 hours BHI broth culture to the MR-VP medium.
Incubate for 48-50 hours at 35-37 Â°C.
Add 0.5 ml Voges Proskaur Reagent.
Result: Positive if pink color and negative if colorless.
Methyl Red Test:
Incubate MR-VP medium tubes for additional 48 hours at 35-37 Â°C after performing Voges Proskaur Test.
Add 5 drops of methyl red solution to each tube and mix gently.
Result: Yellow color is negative test while red color is a positive test.
Citrate Utilization Test:
Lightly inoculate the slanted tubes of Kauser Citrate Broth/ Simmon's Citrate Broth with culture organism.
Incubate for 4-7 days at 35-37 Â°C.
Result: Changing color from green to blue or development of distinct turbidity in blue (copper like) color of citrate broth is a positive test.
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
Microscopic having 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 (fixing agent).
Safranine Dye (counter staining).
Alcohol (decolorizing agent).
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 ml distilled water. Adjust PH to 7.2 with 1 normal NAOH solution. Bring volume to 1 liter with distilled water. Sterilize at 121Â°C for 15 minutes and store in a refrigerator.
Dissolve 5 gm p-Dimethyl-aminobenzaldehyde in 75 ml of isoamyl alcohol. Then slowly add 25 ml of (conc.) HCl. Store at 4Â°C.
Dissolve 20gm Tryptose, 5gm Lactose, 2.75gm KH2PO4, 2.75gm K2HPO4, 5gm NaCl and 1gm Sodium Laryal Sulphate in 800 ml Distilled Water with gentle heat, then filter, cool to 20 Â°C and dilute to 1 liter. Auto-Clave it and adjust final PH to 6.8 Â± 0.2.
Dissolve 7gm Buffered Peptone, 5gm Glucose and 5gm K2HPO4 in 800 ml Distilled Water with gentle heat, then filter, cool to 20 Â°C and dilute to 1 liter. Auto-Clave it and adjust final PH to 6.8 Â± 0.2.
Voges Proskaur Reagent:
Add 0.6 ml of alpha-nepthol solution to 0.2 ml of 40% KOH solution, shake, and then add few crystals of creatine.