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Isolation and Identification of Vibrio Nereis and Vibrio Harveyi in Farm Raised Penaeus Monodon Marine Shrimp

Sadhan Kumar Mondal
Department of Microbiology, Jessore University of Science and Technology, Bangladesh.

Md. Bakhtiar Lijon
Modern Food testing Laboratory, Chittagong City Corporation, Chittagong, Bangladesh.

Md. Rubayet Reza
Department of Microbiology and hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Tasneema Ishika
Department of Microbiology, Jessore University of Science and Technology, Bangladesh.

Abstract/ Executive Summary:

The present research work was conducted for the isolation and identification of Vibrio nereis and Vibrio harveyi in farm raised Penaeus monodon shrimp on three commercial ghers. Shrimp (n=6) were collected from three ghers located at Satkhira district of Bangladesh. Intestinal (n= 6) samples were collected and the intestine of shrimp was taken into a test tube containing 10 ml of sterile distilled water and mixed well by vortex mixer machine. The resulting solution was then used to prepare serial dilution. 1ml of this suspension was transferred to 9 ml of sterile distilled water for tenfold (1:10) dilution and further diluted up to 104 dilutions. For enumeration of bacteria 1ml of diluted samples were inoculated on petri plate aseptically before pouring the nutrient agar on the plates and incubated at 10°C, 27°C, 37°C and 45°C for 24-48 hours. After incubation total bacteria was counted and well-spaced colony was marked for isolation. Isolated colony was then streaked on nutrient agar for pure culture. For isolation of Vibrio spp. pure bacterial culture was then streaked on TCBS agar plate. Identification of bacteria was performed by cultural, staining and biochemical properties. One Vibrio harveyi and one Vibrio nereis isolates were identified in Penaeus monodon shrimp. The results of this study indicate that Penaeus monodon shrimp harbor Vibrio harveyi and Vibrio nereis which might cause vibriosis in shrimp and public health problem if enter into human food chain.

Keywords: Enumeration, Identification, Penaeus monodon, Vibrio nereis, Vibrio harveyi

Location: Satkhira district of Bangladesh.

Start Date: 00-01-2014

End Date: 00-06-2014

Program Area: Variety and Species

Commodity: Shrimp

Objectives:

(i) Isolation of Vibrio harveyi and Vibrio nereis from farm raised Penaeus monodon shrimp.

(ii) Identification of Vibrio harveyi and Vibrio nereis from farm raised Penaeus monodon shrimp collected from Satkhira district of Bangladesh.

Methodology:

A total of 6 shrimps were collected from three ghers which were located at Satkhira (n=2, Satkhira Sadar and Assasuni Upazilla) district of Bangladesh in the period of January to June, 2014. The samples were placed into the sterile polyethene bags and transported to the Department of Microbiology at the Jessore Science and Technology University, Jessore using an ice box aseptically for bacteriological analysis. The intestine of shrimp was taken into a test tube containing 10 ml of sterile distilled water and mixed well by vortex mixer machine. The resulting solution was then used to prepare serial dilution. One ml of this suspension was transferred to 9 ml of sterile distilled water for tenfold (1:10) dilution and further diluted up to 10⁴ dilutions. For the enumeration and isolation of bacteria, serial dilution was carried out. For this purpose 1 ml of each diluted samples were inoculated on sterilized petri dish by using a sterile pipette and 15 - 20 ml of melted Nutrient Agar Media was poured on the petri dishes. Media plates were incubated at 10°C, 27°C, 37°C and 45°C for 24 to 48 hours for enumeration of bacterial colony. After 1 to 2 days of incubation, the plates having well - spaced colonies of different bacteria were selected for counting. The selected plates were placed on a colony counter and the colonies were counted precisely by naked eye. The counts of colonies were considered as per ml and were calculated by multiplying the average number of colonies per plate by the reciprocal of the dilution factor. The calculated results were expressed as colony forming units (cfu) per ml of sample.

After enumeration of the plates, highly well - spaced colonies were selected for isolation. The selected colonies were marked and their morphological (colony) characteristics were studied and recorded. Selected bacterial colonies were transferred into slope of the slants prepared with the corresponding plating media for further studies. The culture tubes (slant tubes) were kept in polythene bags. The bags were tied up and preserved as stock culture in a refrigerator at 2 to 8°C. These isolates were transferred to fresh medium periodically. When all plate shown only one type of colony distinctly, it was considered as pure. Uniform vegetative and reproductive structures were also indicative of purification of the isolates. For bacteria, isolates were purified by repeated streaking on to nutrient agar plate. The pure culture of the isolates was coded according to the number of colonies and the serial of the sample used. The code numbers were maintained and followed till identification of the isolates after through characterization.

Morphological characters of the selected isolates can be observed by culture and microscopic methods. By the culture method, colony characteristics on agar plate, agar slants, and growth in liquid or in deep media were observed. But microscopic methods such as: Gram’s staining and acid fast - staining generally carried out for demonstration of size, shape, arrangement and color of isolates. Finally, 6 strains of bacteria were selected by comparing their growth, color and size of the colony in culture media and on the basis of their morphology and nature of arrangement through microscopic study. Identification of bacteria was performed on the basis of cultural characteristics and colony morphology on the Nutrient agar, agar slant and TCBS agar. Gram’s staining, acid fast staining, motility test, sugar fermentation test and biochemical tests such as: oxidase test, catalase test, gelatin hydrolysis test, citrate utilization test, indole test, Voges - Proskaur (VP) test, methyl red reaction and production of hydrogen sulphide were performed to identify bacteria.

Source of Information: Int. J. Biosci. 8(4), 55-61, April 2016. ISSN: 2220-6655

Article Link (Online Journal): DOI: http://dx.doi.org/10.12692/ijb/8.4.55-61

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

This study was performed to isolate and identify Vibrio nereis and Vibrio harveyi from shrimp intestine that are collected from gher of coastal water of Satkhira district of Bangladesh. The present research work indicated that intestine of Penaeus monodon shrimp harbor Vibrio harveyi and Vibrio nereis which might cause Vibriosis in shr imp and public health problem if enter into human food chain.

Knowledge Management: Journal