Resistance to Tetracycline in Bacteria isolated from the organs of fishes belonging to Sardinella Carangidae and Serranidae sp.

Name of the student: Veena.S Name of the guide: Mrs.Swapna Karthik Name of the institution: Rajalakshmi engg coll

INTRODUCTION
1. Antibiotics are used to treat bacterial infections. 2. Antibiotic Resistance: Incorporation of sub-therapeutic dosages of antibiotics into feeds for cultured organisms.

3. Antibiotic resistance is due to the presence of R-Plasmid.

Major objectives
1. Isolation and identification of the Bacteria from each species of fish. 2. To study the hemolytic activity 3. To determine the antibiotic resistance. 4. Detection of the plasmids and calculation of the molecular weight.

Work design
Collection of fish samples

Identification of Bacteria from the fish

Biochemical tests to identify the bacteria Antibiotic sensitivity test Hemolysis test Plasmid detection and molecular mass determination

Fish samples to be used:

Sardinella sp. Carangidae sp. Serranidae sp.

Antibiotic to be used: TETRACYCLINE

MATERIALS AND METHODS

Materials:
1. Samples: Fish samples collected from known
source.

2. Media:
GSP Agar Peptone Water LB Broth Mueller Hinton Agar Tryptic Soy Agar

3. Antibiotic Discs
4. Reagents for Plasmid isolation and Agarose Gel Electrophoresis

Methods:
1. Sample collection and isolation of bacteria:
* Fish samples collected from know source
*Gills and intestines were removed and homogenized. *Inoculated into peptone water for pre-enrichment (37C, 24 hrs)

*Streaked on GSP Agar plates and incubated. (37C, 24 hrs)

*Colonies isolated and biochemically tested to confirm the genus and species.

2. Antibiotic sensitivity test: Single disc diffusion method

*Bacteria cultivated in LB Broth.
*Cells harvested by centrifugation. * Suspended in saline and spread on Mueller Hinton Agar *Spotted with discs containing antibiotics.

3. Hemolysis tests:
Strains streaked on Tryptic Soy Agar plates containing 5% human defibrinalated RBC and incubated. (37C, 24 hrs)

4.Plasmid isolation:
*Plasmid DNA was isolated. *AGE was carried out. *Approx molecular mass was determined by comparison with plasmids of known molecular mass of E.Coli.

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