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An Investigation of the Bacterial Flora Causing Spoilage of Fishes at Board Fish Market Peshawar Pakistan.

Byline: Arif Jan Zaigham Hasan Hussain Shah Rooh Ullah Iftikhar Ahmad and Muhammad Younas

Abstract

Bacterial flora from the epidermis of different species of carp fishes were isolated and identified as potential causes of spoilage of fish at Board fish market Peshawar Pakistan from November to December 2013. Nutrient agar medium was used for mixed culture of bacteria. Other selective media such as MacConkey agar Blood agar medium EMB medium Pseudomonas medium and Mannitole salt agar medium were used for culture and identification of specific bacteria. Five bacterial groups that are pathogenic to humans (Staphylococcus aureus Staphylococcus epidermidis Pseudomonas spp Klebsiella spp. and Escherichia coli) were reported to reside on the fish epidermis. Staphylococcus aureus was isolated from Labeo rohita Tor putitora Hypophthalmichthys molitrix Wallago attu Ctenopharyngodon idella and Cyprinus carpio. Staphylococcus epidermidis was also found on the epidermis of Labeo rohita and Wallago attu.

Bacillus species like E. coli Pseudomonas and Klebsiella spp. were isolated from the epidermis of Maha Sher (Tor putitora) Silver carp (Hypohthalmichthys molitrix) Rohu (Labeo rohita) Malli (Wallago attu) Gulfam (Cyprinus carpio) and Grass carp (Ctenopharyngodon idella). Harvesting handling transportation and storage of local market fish should be performed hygienically to reduce the risk of spoilage.

Key Words: Carp fishes spoilage bacteria Nutrient agar medium EMB (Eosin Methylene Blue) medium.

INTRODUCTION

Microbial degradation of fish often results in spoilage which is the quality deterioration due to changes in sensory properties rendering the fish unusable for human consumption. Along with chemical changes and physical damage microbial growth and metabolism lead to the formation of aldehydes ketones alcohols sulfides organic acids and amines which make the fish off-flavor (Gram and Dalgaard 2002). Spoilage is the deterioration of food which make its taste and smell bad (e.g. when it is sour rotten or mouldy) and/or makes it a carrier of disease organisms (Brigitte et al. 2004). Spoilage of fish not only results in a loss of protein for human consumption but may also lead to great economic loss due to food borne illness (Abbas 2014). Microbial contamination was reported to be dependent on water fishing conditions and unsuitable processing distribution and storage applications following the capture of fishes.

Expansion of culture fishery enterprises has also increased the consumption of fish. Concurrent with the increase of aquaculture there has been an increase in bacterial diseases of fish. Nearly 70 bacterial species have been reported pathogenic to humans (Yagoub 2009). However bacteria recovered from the skin and gills of fish may be transient rather than resident on those surfaces (Cahill 1990). The micro flora of fish is influenced by water in which the fish are grown as well as the water that is sprawed over fishes to keep them fresh as in some fish markets (Liston 1980).

The pollution of fresh water resources in densely populated areas by various anthropogenic activities is a matter of grave concern (Mehboob et al 2014). Each year a considerable amount of fish is lost due to spoilage. According to Eyo (1997) during post harvesting of fishes approximately 1 out of 14 Kg of fish are spoiled and discarded due to improper handling. This is a significant economic loss and there is a great need to identify the species of bacteria responsible and to develop effective methods of fish handling that will minimize the loss of this important food product due to spoilage.

MATERIALS AND METHODS

Six species of fishes namely Rohu (Labeo rohita) Mahasheer (Tor putitora) Silver carp (Hypophthalmichthys molitrix) Malli (Wallago attu) Grass carp (Ctenopharyngodon idella) and Gulfam (Cyprinus carpio) were sampled from Board Fish Market which is located at University Road in the west of Peshawar City of Pakistan. The study area is located approximately 3 km from the laboratory of PCSIR (Pakistan Council of Scientific and Industrial Research). The temperature of market fluctuated between 14C to 28C during the study period. Samples i.e. pieces of muscles with skin on were brought in ice packed polythene bags to the PCSIR laboratory within 15 minutes of its collection. Fishes were taken and brought to the laboratory for culturing and identification of bacterial flora on their epidermis.

Preparation of sample

The fish samples were brought to the laboratory and kept in a petri dish. The sample was directly brought to the laboratory and streaking was done through the use of a sterilized stick swab (Bie and Berntsen Copenhagen) on the prepared media under aseptic conditions. The stick swab had been soaked in peptone water and was then used to collect the bacterial sample by rubbing the stick against the fish tissue (Kumari and Ichhpujani 2000). The stick was then streaked on the prepared media.

Then a small piece (2-3cm) of fish epidermis was removed from various regions of the body and was kept in another sterilized petri plate under the aseptic environment of a laminar air flow hood (Technical Scientific Supply Lahore Standard: US Federal Standard 209 E) in an effort to avoid post- collection bacterial contamination.

Culturing bacteria from the sample

Nutrient agar medium was used to culture the suspect spoilage bacteria. Other selective media such as MacConkey agar medium Pseudomonas agar medium Minitol salt agar EMB medium Blood agar and Simmon citrate agar medium were also used for identification of specific bacteria isolated individually on the epidermis of the fish.

Incubation

The medium plates were then sealed with parafilm and kept in an incubator (Banstead lab Model number 4951) at 35C for about 24 (2) h for the growth of bacteria (Kumari and Ichhpujani 2000). This resulted in the initial mixed bacterial culture.

Pure/sub culture for identification of bacteria

Media used for identification of bacterial pathogens were: Mannitol salt agar medium Pseudomonas agar medium Blood agar medium EMB and MacConkey agar.

Confirmation of bacterial species by different tests

Biochemical tests including catalase coagulase oxidase indole haemolysis and citrate utilization tests were performed for bacterial species identification as recommended in Prescott (2002). In addition morphological characteristics such as Gram staining was performed to properly confirm identification of microbial isolates.

RESULTS

In present study 65 samples of fishes were collected from the study site (Board Fish Market) for the identification or prevalence of bacteria on the epidermis of fishes Table I reveals the morphology (colour form and elevation) of the pathogen and the type of media used to culture it and finally identified. Table II shows the details of the media used for the culture

Table I.- Morphology of bacterial colonies on different media.

###Colony morphology

S.No.###Media###Bacterial species

###Colour###Form###Elevation

1###Mannitole###Pale yellow milky white###Circular and rod shaped###Convex###Staphy. aureus/epidermidis

2###EMB###Metallic green###Circular###Convex###Escherichia coli

3###MacConkey###Creamy white###Circular###Convex###Klebsiella spp

4###Pseudomoneas###Creamy white###Circular and fringed###Convex###Pseudomonas spp

5###Nutrient agar###Creamy/Pale yellow###Circular and rod###Convex###For all above bacteria

Table II. - Different media used for identification of different bacterial species

###Media used

###S.No.###Bacterial species###Mannitol###Pseudomonas###MacConkey###EMB###Fish names

###agar###agar###agar

###1###S. aureus###+###--###--###--###Wallago attu

###Hypophthalmichthys molitrix Labeo

###rohita Ctenopharyngodon idella Tor

###putitora and Cyprinus carpio.

2###S. epidermidis###+###--###--###--###Wallago attu and

###Labeo rohita.

###3###Pseudomonas spp###--###+###--###--###Wallago attu

###Hypophthalmichthys molitrix

###Labeo rohita and Tor putitora.

###4###Klebsiellas sp###--###--###+###--###Wallago attu

###Hypophthalmichthys molitrix

###Labeo rohita Tor putitora and

###Cyprinus carpio.

###5###E.coli###--###--###+###+###Wallago attu

###Hypophthalmichthys molitrix

###Labeo rohita

###Ctenopharyngodon idella Tor putitora

###and Cyprinus carpio.

and identification of all the types of bacterial colonies from the epidermis of all the different types of fishes. The + sign in each column under the specific medium shows that the particular type of bacterial colony has cultured on that medium. The sign means the opposite. Table III shows the bacterial flora extracted from each fish species individually. Each fish species were found to carry more than one species of bacteria that may be capable of causing spoilage.

Gram's staining

Grams staining of the samples further indicated that Gram positive bacteria were Staphylococcus aureus and Staphylococcus epidermidis. Gram negative species were E. coli Pseudomonas spp. and Klebsiella sp. S. aureus and E. coli were found to be with high percentages followed by Klibsiella sp Pseudomonas sp. and S. epidermis (Table IV).

Table III.- List of various bacterial species from each procured specimen of fishes.

S.No.###Specimen###Bacterial species

1###Wallago attu###S.epidermidis E. coli and

###Pseudomonas spp.

2###Tor putitora###S.aureus E. coli and

###Klebsiella spp.

3###Hypophthalmichthys###S.aureus E. coli and

###molitrix###Klebsiella spp.

4###Labeo rohita###S.aureus E. coli and

###Pseudomonas spp.

5###Labeo rohita###S.epidermidis and E. coli

6###C. idella###S. aureus and E. coli

7###Tor putitora###S.aureus E.coli and Klebsiella

###spp

8###Hypophthalmichthys###S.aureus E.coli and

###molitrix###Pseudomonas spp.

9###Labeo rohita###S. aureus E. coli

###Pseudomonas and Klebsiella

10###Labeo rohita###S. aureus E. coli and

###Klebsiella spp.

11###Labeo rohita###S. aureus E. coli and

###Klebsiella spp.

12###Hypophthalmichthys###S. aureus E. coli and

###molitrix###Pseudomonas spp.

13###Tor putitora###S. aureus and Pseudomonas

###spp.

14###Cyprinus carpio###S. aureus E.coli and

###Klebsiella spp.

15###Wallago attu###S. aureus and Klebsiella spp.

Table IV.- The frequencies of occurrence of various bacterial pathogens

###Number of###Percentage

S.No.###Bacterial isolates###bacterial###of

###colonies###occurrence

1###S. aureus###13###30.95

2###S. epidermidis###2###04.76

3###Pseudomonas spp.###6###14.29

4###E. coli###13###30.95

5###Klebsiella spp.###8###19.05

DISCUSSION

The analysis of present work indicated that the bacteria that may play a role in fish spoilage were: S. aureus S. epidermidis Pseudomonas spp E. coli and Klebsiella spp. Our findings are consistent with the findings of Sinha et al. 1991 which also indicated the presence of Salmonella Staphylococcus Aeromonas Pseudomonas E. coli Micrococcus Streptococcus Proteus Klebsiella and molds in the marketed Labeo rohita. This study indicated that at Board Bazaar Peshawar Pakistan the conditions for the prolong time of storage and selling of fishes are not ideal and may lead to spoilage.

It is recommended that fish should not be kept beyond six hours at ambient temperature until it is iced if its shelf life is to be maintained to meet the market's quality demands. This recommendation is in agreement with the findings of Amos (2007). It is therefore recommended that good hygienic conditions and use of clean water during fish processing should be strictly adhered to. After harvest fresh fish should be properly stored at low temperatures to inhibit survival and growth of bacteria.

ACKNOWLEDGEMENTS

The authors are grateful for the assistance of the technical staff at Pakistan Council of Scientific and Industrial Research Peshawar.

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Article Details
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Author:Zaigham, Arif Jan; Hussain, Hasan; Rooh, Shah; Ahmad, Ullah Iftikhar; Younas, Muhammad
Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:9PAKI
Date:Oct 31, 2014
Words:2066
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