Preparation and Storage of High-Titer Lactic Streptococcus Bacteriophages1

Abstract

Various techniques were employed for preparation of high-titer bacteriophage lysates of Streptococcus lactis, S. cremoris, and S. diacetilactis strains. Infection of a 4-h host culture in litmus milk at 30 C yielded the highest titers (2 x 109 to 4 x 1011 plaque-forming units/ml) for most phages. Host infection in lactose-containing broth produced similar virus numbers only when 0.1 M tris(hydroxymethyl)aminomethane buffer stabilized the pH. The pH at the time of infection as well as the inoculum phage titer were critical in obtaining high titers. Optimum conditions for infection in broth were coupled with a polyethylene glycol concentration procedure to routinely produce milligram quantities of phage from 1 liter of lysate. Neutralization of whey lysates, as a means of storage, offered no survival advantage over unneutralized samples. Storage of phage lysates in a 15% glycerol whey solution at-22 C yielded a high rate of survival in most cases, even with repeated freezing and thawing, over a period of 24 months.

Improved Medium for Lactic Streptococci and Their Bacteriophages

“…Acid-producing activity of S. cremoris AM, and S. lactis ML. as revealed by pH attained after 6 h at 30 C and 15 h at 22 C in SM inoculated (1%) from M16, M17, or lactic broth (LB) culture lines maintained by daily subculture (1%) and incubation at 30 C for 24 a pH values taken after incubation at 30 C for 6 h. "Coagulation (at least pH 4.7) observed after incubation at 22 C for 15 h; -, no coagulation; 1+, weak but definite coagulation; 2+, firm coagulation.…”

Cheese starters

“…Preparation of high titre phage stocks is improved by incorporation of buffer into the medium, whether broth or milk (96,426). Concentration of lactic streptococcal phages has been accomplished by dialysis (5), centrifugation (5,401,412) and phase separation in polyethylene glycol (380,426), but no direct comparison of the relative recoveries of viable particles by these procedures has been made.…”

“…Phages have remained viable after extended storage at room temperature, 4 °C, or -1 8 to -2 2 °C (426,427), particularly when stored in buffered medium (426) or frozen in glycerol (426,428). A pH of 6-0 to 7-0 was optimal for phage viability or high titre stock preparation (96, 426,429).…”

Method for Growth and Purification of Bacteriophage 643 on Streptococcus lactis ML3

“…Many lytic bacteriophages specific for streptococci have been isolated from dairy factory whey samples as a consequence of problems arising through the infection of starter cultures during cheese manufacture. Various authors (5,8,11) have published studies of host range, morphology, and serology of streptococcal phage isolates, but little is known about the genetics, genome size, and genome structure of these viruses. This is due, in part, to difficulties encountered in preparing sufficient quantities of phage DNA.…”

“…Normal phage maturation and lysis in infected cultures is inhibited by low pH conditions which arise as a result of lactic acid production by the culture, and as a consequence, final phage titers are generally low. Nyiendo and co-workers (8) reported a general method for growing lactic streptococcal phages in a complex medium buffered with Tris and obtained titers of up to 1013 PFU/ml of lysate for some strains. The phage particles were precipitated and concentrated from crude lysates with polyethylene glycol.…”