Mumtaz Begurn ( Biological Production Division National Institute of Health, Islamabad. )
Six hundred samples, 300 each of mutton and beef were collected under aseptic conditions from meat shops of Rawalpindi/Islamabad. They were examined bacteriologically under aerobic and anaerobic conditions at different temperatures for varying periods of time. Different species of bacteria isolated were Escherichia coli (520), Staphylococcus aureus (437), Staphylococcus epiderimis (200), Streptococcus faecalls (105), Corynebacterium pyogenes (50), Bacillus subtiis (84), Salmonella typhi (50), Proteus mirabiis (140), Pseudomonas aerugenosa and Aerobacter aerogenes (80).(JPMA 34 : 214, 1984).
The study was carried out to find out the type of bacterial contaminants and their mode of action and prolification during storage and transportation upto the disposal to the consumers under the prevailing conditions in twin city Rawalpindi/Islamabad.
Material and Methods
Three hundred each of fresh beef and mutton samples were collected from randomly selected animal carcasses at various meat shops of Rawalpindi/Islamabad areas.
Two samples, each weighing about 20 gms were taken from each carcass comprising of (i) inner portion of the muscles and (ii) external surface of the carcass. Each sample was taken in a clean sterilized wide mouth glass stoppered bottle using sterilized forceps and scissors. The samples were handled carefully to avoid contamination and transported to the laboratory for bacterial and cultural examination on selected media.
a) Selenite - F - enrichment medium (Oxoid)
b) Thioglycollate Broth ( “ )
c) Cooked meat medium ( “ )
d) MacConkey’s agar (Difco)
e) Brillian gree n agar ( “ )
f) Salmonella Shigella agar (Oxoid)
g) Staphylococcus medium No. 110 ( “ )
h) Eosin methylene blue agar ( “ )
i) Triple sugar iron agar ( “ )
j) Nutrient agar ( “ )
k) Blood agar (10% defibrinated sheep blood
plus Nutrient agar)
The following enrichment, selective, differential and normal culture media were used in this study:
Diagnostic Antigens and Anti Sera for Identification of Different Species of Organisms were
1. Rabbit serum -- used for coagulase test.
2. Diagnostic salmonella antisera (Difco)
Samples of meat (Beef and Mutton) were emulsified in sterile solvents like phosphate buffer or physiological saline alongwith sterile sand in pestle and mortar in perfect aseptic conditions. The homogeiiate was allowed to stand for 10-15 minutes in order to get clear supernatant. One ml of supernatant was dispensed aseptically in three tubes, each containing liquid medium like, cooked meat medium, thioglycollate and selenite-Fenrichment medium. Shaked well and incubated, cooked meat medium tubes were incubated under anaerobic condition at 37°C for 24 hours, while Thioglycollate and Selenite-F-enrichment medium tubes were incubated under aerobic condition at 37°C for 24 hours.
The growth in liquid medium was examined microscopically by gram staining and streaked on solid media like nutrient agar, blood agar and MacConkey’s agar. The inoculated plates of nutrient agar and blood agar were incubated anaerobically at 35.37°C for 24.-48 hours, while three plates of each of solid media i.e. Nutrient agar, Blood agar and MacConkey’s agar were incubated under aerobic conditions at 35-37°C, at room temperature (24-26°C) and at refrigeration temperature (4-10°C) for varying periods as shown in table 1. The morphology of representative colonies was studied from the plates and organisms were identified by biochemical reactions and slide agglutination tests using specific antisera. Coagulase positive Staphylococcus aureus were isolated on blood agar and identified by Pigment Production, mannitol fermentation and coagulase test.1 Isolation of anaerobes was done using the method of Dowell et al2.
Salmonellae were isolated according to the procedure described by Mildred et a13.
From the bacteriological study of 600 meat samples, a total of 1841 isolates were obtained, of which 981 (53.28%) were gram-positive Cocci and Bacilli, where as 860(46.72%) were gram-negative rods of bacterial isolates, 1736 (94.30%) were aerobés while 105 Isolates (5.70%) were strictly anaerobes. The species identified were, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus faecalis, Clostridium Perfringens, Bacillus subtiis, Corynebacterium pyogenes, Salmonella typhi, Proteus mirabiis, pseudomonas aerugenosa, and Aerobacter aerogenes (Table I).
In the present study only 35 bacterial strains were isolated from the Inner portion of the muscles of meat samples, which were identified as Staphylococcus aureus, Salmonella typhi,
Pseudomonas aerugenosa and Corynebacterium pyogenes.
Isolates identified from the surface meat samples of carcasses were, Escherichia Coli, Staphylococcus aureus, Staphylococcus epidermidis Proteus mirabiis, Streptococcus faecalis, Clostridium perfringens, Bacillus subtiia, Pseudomonas aerugenosa, Aerobacter aerogenes, Salmonella typhi and Corynebacterium pyogenes (Table II).
Bacteriological study of 600 beef and mutton samples showed bacterial contamination with species of the genera, Escherichia, Staphylococcus, Streptococcus, Clostridium, Bacillus, Salmonella, Proteus, Pseudomonas, Corynebacterium and Aerobacter. The presence of these Isolates in the higher percentage in meat samples collected from the retail market of Rawalpindi/Islamabad areas is an indication of unsatisfactory handling of meat and inadequate hygiene. Most of bacterial contamination of meat occurs during post slaughter handling in the slaughter house and during transportation.4-6
Escherichia Coli was the main contaminant. Its incidence rate was 90% and 83.33% in samples of beef and mutton respectively. Escherichia coli was isolated only from the surface meat samples of carcasses and not from the inner portion of the muscles, which means that the source of entry of these organisms may be through polluted water and unhygienic handling of the slaughtered animals. Similar high incidence of Escherichia coli in raw meats has also been reported in other series.7,8 Organisms next in order of frequency were, Staphylococcus aureus (67% and 78.66%) and Staphylococcus epidermidis (26.66% and 40%) in case of beef and mutton samples respectively. These organisms are mostly present in raw meats due to surface pollution by polluted water and unsatisfactory hygienic conditions in the abattoirs.9 Prevalence of Streptococcus faecalis in meat is an indication of faecal pollution.10,11 Streptococcus faecalis can cause illness among humans through ingestion, which means raw meat handlers are at. risk of infection by Streptococcus faecalis too.10,11 Of the anerobes only Clostridium Perfringens could be identified in 13 and 21% of beef and mutton samples respectively. Simlar figures were reported in another study12 . Clostridium perfringens are very widely distributed, inhabiting soil and intestinal tract of animals. Its occurrence indicate that meat could have been contaminated with these two sources.
The incidence of all other isolates, the Salmonella typhi, Corynebacterium pyogenes, Bacillus subtiis, Proteus mirabilis , Pseudomonas aerugenosa, and Aerobacter aerogenes, ranged between 5 to 18%. Salmonellae are important from public health point of view, being pathogenic microorganisms. They may be transferred to the carcass surface during slaughtering, handling and processing of meat.3,131,4
Species of the genera Proteus are constantly present in rotten meat, and sewage as well as human and animals excreta. Their incidence rate in meat is fairly high15. Pseudçmonas is also a common pyschrophile causing spoilage of meat15,17 This organism may warrant attention especially when meat is refrigerated.
Most of the microorganisms in the present study (1806 of 1841) were isolated from the surface meat samples of carcasses, while internal portion of the muscles yielded only 35 bacterial strains. Isolation of such a high number of bacterial strains from surface meat samples have also been made by other workers.16,17 Since only 35 isolates were identified from the inner portion of the muscle of meat samples, it is obvious that inner muscle portion are relatively clean.10,18 The frequency of bacterial contami nation of meat in present study is quite high. Species of the genera, Escherichia, Proteus Aerobacter and Pseudomonas are important in respect to their capabilities lot spoilage of meat and meat products at temperature varying between 4 to 37°C, whereas species of the genera, Salmonella, Clostridium, Corynebacterium, Staphylococcus and Bacillus subtiles are pathogenic and capable of producing disease through toxin production or direct infection through meat handling and processing. Thus consumption of highly polluted meat even after cooking appears to be unsafe due to heat stable toxins and spore bearing organisms.
The help and guidance of Dr Abdul Ban, Dept of Biological Sciences, Quaid-e-Azam University Islamabad in carrying out this work, Mr Zafar Mi, National Institute of Health, Islamabad in preparation of manuscript is gratefully acknowledged.
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