Abstract

Addition of KH2P04 salts to Fraser’s Medium produced higher titers of Salmonella potsdam bacteriophage P3/2, but it failed to maintain stable titers under normal cold storage conditions. The addition of ethylenediaminetetraacetate (EDTA) to the medium also produced high titers and helped to maintain phage viability indefinitely in storage. The presence of KH2P04 with the EDTA did not increase the titers nor better the storage properties and was discontinued in favor of EDTA alone. This provided for the accumulation of the necessary amounts of P3/2 for DNA studies.

The phage P3/2 adapted well to purification by the differential centrifugation technique. From 50-60 percent of the phage in a suspension can be concentrated by this method. In density gradient centrifugation, phage P3/2 maintained the most optimum viability in potassium tartrate. In potassium tartrate, P3/2 was calculated to have a density of 1.26 and a density of 1.42 in cesium chloride. The virulent mutant, P3/2vir, had a density of 1.476 in cesium chloride.

Phage P3/2vir was employed in experiments on DNA characterization, DNA estimations by Dische method were reproducible over a period of time for a single phage preparation, but the titer increased upon initial storage in the cold. This made a DNA per pfu determination impractical. Calculations of protein per pfu, employing the Lowry technique, were likewise inconclusive due to their titer increase and the presence of extraneous protein material in the purest density gradient preparations.

The average extinction to phosphorus value for two preparation of P3/2vir DNA was found to be 6065 which is comparable to the 6130 value for the highly polymerized calf thymus DNA.

Formalin denaturation and heat denaturation test indicated that the P3/2vir DNA was double-stranded.

The Tm, the thermal denaturation temperature of P3/2vir DNA, was determined to be 91.8°C. From the Tm value, the G-C content of the DNA was calculated to be 53.0 percent.

By means of deoxyribonuclease and snake venom phosphodiesterase enzyme degradation and mononucleotide separation by ion-exchange chromatrography, the following molar ratios for the P3/2vir DNA were determined: G-C 54.7-55.1 percent; A-T 44.9-45.3 percent.

LLU Discipline

Microbiology

Department

Microbiology

School

Graduate School

First Advisor

Robert L. Nutter

Second Advisor

Leonard R. Bullas

Third Advisor

Richard E. Beltz

Degree Name

Master of Science (MS)

Degree Level

M.S.

Year Degree Awarded

1968

Date (Title Page)

8-1968

Language

English

Library of Congress/MESH Subject Headings

Salmonella; Bacteriophages

Type

Thesis

Page Count

iii; 72

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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Included in

Microbiology Commons

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