The preservation of gonococci in liquid nitrogen

Abstract

SYNOPSIS Gonococci suspended in 1 % proteose peptone containing 8 % glycerol can be snap frozen in liquid nitrogen without detectable loss on thawing. This recovery rate has allowed the use of frozen organisms as the starting inoculum for the bulk growth of gonococci and to preserve gonococci both in urethral pus and after primary subculture for use in studies on gonococcal virulence.The method described for freezing urethral pus on charcoal swabs should make it possible to transport infected specimens from areas lacking adequate laboratory facilities.The long-term storage of standard strains of Neisseria gonorrhoeae is conveniently achieved by freeze drying but the recovery rates are poor, usually less than 5 % (Brookes and Heddn, 1966 CULTUREUrethral pus was cultured on Difco G.C. medium base containing dextrose 4 mg/ml, glutamine 100 ,ug/ml, ferric nitrate 10 ,ug/ml, and co-carboxylase 0-2 ,ug/ml (White and Kellogg, 1965 (Kocka and Bretz, 1969) as protective agents. Duplicate 0-1 ml volumes containing about 107 gonococci were dropped directly into liquid nitrogen contained in disposable polythene beakers. The frozen pellets were stored in screw-capped plastic ampoules (Sterilin Ltd) in a liquid nitrogen refrigerator.Urethral pus was suspended in 8 % glycerol peptone and 0.1 ml volumes were frozen either immediately after incubation at 37°C for 15 minutes or after homogenization using a Teflon grinder (Pierce, Dubos, and Schaefer, 1953). A further 0 1 ml of exudate was incubated at 37°C for 15 min in 8% glycerol peptone containing 5 % saponin. Pellets were thawed rapidly by dropping into 0-9 ml of 1% proteose peptone maintained at 37°C in a water bath. In both sets of experiments the results of duplicate viable counts on each of two frozen samples were averaged and expressed as a percentage of the starting inoculum.In a further series of experiments urethral exudates were collected on charcoal-impregnated swabs which were broken off into ampoules containing 0 4 ml of 8% glycerol peptone. After remaining at room temperature for periods of one to three hours, the swabs were cultured and then frozen by immersing the ampoules in liquid nitrogen. 122 on 11 May 2018 by guest. Protected by copyright.

Antigenic Heterogeneity Associated with Pilus Aggregation and Autoagglutinability in Neisseria gonorrhoeae

“…Strains a, p, y, 6 and 6 were obtained from the urethrae of male patients with acute purulent.gonorrhoea, with the assistance of Dr J. C. Clay and her staff at Birmingham General Hospital. Samples of urethral exudate were suspended (Ward & Watt, 1971), and several portions were frozen in liquid nitrogen.…”

A new colonial type of N. gonorrhoeae.

“…Isolated strains, in addition to daily subculturing, were also preserved in ampoules submerged in liquid nitrogen according to the method of Ward and Watt (1971). Strains to be frozen were suspended in a solution of 8 per cent.…”

Morphological, Biological and Antigenic Properties of Neisseria gonorrhoeae Adapted to Growth in Guinea-Pig Subcutaneous Chambers

“…An 18 h culture on modified Amies & Garabedian medium (see below) was suspended in Trypticase Soy Broth (TSB; BBL) and mixed with an equal volume of 2 : , ; (w/v) proteose peptone no. 3 (Difco) containing 16 % (v/v) glycerol; 0.5 ml portions were frozen and stored in liquid nitrogen (Ward & Watt, 1971). In some experiments thawed samples were used directly: these organisms are designated BS.…”