Graduate Thesis Or Dissertation


Host range and chemical composition of lactic streptococcal bacteriophages Public Deposited

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  • Comparisons were made of the abilities of Streptococcus lactis, Streptococcus cremoris and Streptococcus diacetilactis bacteriophages to endure various laboratory isolation and storage treatments. Neutralization of Cottage cheese whey containing phages offered no survival advantage over unneutralized samples, refrigeration of neutral and acid whey samples increased the survival of only the S. diacetilactis phage. About 50 percent of the phages could be recovered from milk coagulated by addition of lactic acid to provide casein-free, clear, phage-containing whey. Sterilization of phage lysates by membrane filtration allowed collection of 92 to 99 percent of the phages; Seitz filtration allowed collection of a maximum of 24 percent. Whey samples containing phages could be concentrated by removing water using polyethylene glycol. Lyophilization decreased phage titers at least 50 percent. Cross reaction patterns of phages for strains of lactic streptococci revealed that similar strains were present in cultures supplied by several companies. For example, phages which were lytic for strains isolated from one brand of commercial mixed strain starter cultures would lyse strains isolated from as many as three different brands of starter cultures. The lytic patterns of 60 bacteriophage races toward 100 single lactic streptococcal strains allowed the establishment of eight phage groups, A through H. The phages within a particular group were generally species specific. However several exceptions were noted. Group A phages attacked primarily strains of S. diacetilactis. Groups B through H phages mainly attacked strains of S. lactis and S. cremoris. Groups G and H consisted of one phage that attacked only the homologous host. Lytic reactions determined by multiple printing of phages on lawns seeded with possible hosts were employed for selecting strains to be used in mixed-strain starter cultures. A computer was used in selecting strains for blending into mixed-strain cultures with resulting maximum protection against phage. The strains selected by the computer were arranged in a sequence or rotation system suitable for use in dairy plant fermentations. Protection from phage infection as determined by a resistance index remained high for six successive cultures, but the addition of the seventh culture to the rotation system increased the likelihood of phage attack significantly. Neutralization of representatives of each host range phage group by standardized rabbit antisera prepared against four different phages revealed similarity among several groups. However the neutralization of phages was not constant within each group. Electron photomicrographs of a phage for S. diacetilactis revealed a tadpole-shaped particle. The head width and length were each about 60 mμ , the tail width was about 10mμ and the tail length was about 170 mμ . There also were suggestions for a polyhedral head structure in enlarged (84,000X) pictures of the viruses. Chemical compositional studies of the phage studied by electron microscopy revealed that phosphorus represented 4.45 percent of the phage dry weight. Nitrogen was found to constitute 12.8 percent of the phage protein. A ratio of phage phosphorus to phage protein nitrogen of 2.7 was determined. Bacteriophages prepared in broth medium were recovered by differential centrifugation and purified in cesium chloride density gradients. An amino acid analysis revealed the following mole percentages for the amino acids quantitated: lysine, 8.1; histidine, 1.7; arginine, 3.8; aspartic acid and asparagine, 10.0; threonine, 7.5; serine, 6.8; glutamic acid and glutamine, 12.1; proline, 3.4; glycine, 9.0; alanine, 9.5; valine, 8.7; methionine, 1.1; isoleucine, 7.9; tyrosine, 2.2 and phenylalanine, 2.8. The nucleic acid of the phage was determined to be double-stranded DNA with a thermal melting point of 84.3°C, indicating an average base composition of 35.7 percent guanine plus cytosine. Calculated values for percentage DNA and protein composition of the phage were 44 and 56 percent respectively. The density of the phage was calculated to be 1.47g/cc.
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