Development of a Defined Minimal Medium for the Growth of Neisseria gonorrhoeae

Abstract

This investigation describes the development of a solid and a liquid medium (Gonococcal Genetic Medium; GGM) which support the rapid growth of 41 gonococcal clinical isolates and laboratory strains with a minimum number of nutritional components. The complete medium contains minimal salts, eight amino acids, two nitrogen bases, vitamins, coenzymes, key metabolic intermediates, and some miscellaneous components. Results indicate that GGM can be modified and simplified even further than we described. In liquid GGM, several gonococcal strains grew logarithmically after a 2-to 3-h lag period with generation times ranging from 72 to 115 min, reaching optical densities of 175 to 320 Klett units in the presence of seven amino acids and in the absence of a CO2 atmosphere. The development of a solid and a liquid defined minimal medium such as GGM should greatly broaden the avenues of experimentation for biochemical genetic studies with N. gonorrhoeae, especially gonococcal genetic transformation. N. gonorrhoeae can be classified into eight major and minor phenotypic groups, depending on its growth responses on GGM to just five amino acids: cysteine and cystine, arginine, proline, isoleucine, and serine. Such results demonstrate the feasibility of using GGM as a simple, sensitive, rapid probe for investigating the epidemiological patterns of gonorrhea.

Growth of Neisseria Gonorrhoeae in Continuous Culture

“…The composition of the liquid medium was basically that described by La Scolea and Young [5] but it was modified in the light of the results obtained. The final medium contained (gll) NaC1, 5.25; MgC12, 0.2; NH4Cl, 0.3; Na,S04, 0.75; K2HP04, 10.5; KH2P04, 4.5; CaCl, 6H20, 0.005; Fe(NO,), 4 HzO, 0.005; sodium citrate, 1.13;L-glutamic acid, 0.19; L-arginine, 0.13; L-proline, 0.25; L-serine, 0.25; L-methionine, 0.19; L-aspartic acid, 0.25; L-isoleucine, 0.25; uracil, 0.005; hypoxanthine, 0.005; NAD, 0.01 ;thiamine hydrochloride, 0.02; cocarboxylase, 0.02; calcium pantothenate, 0.02; biotin, 0.004; pyruvic acid, 1.58; glycerol, 1.6; D-glucose, 7.5 ;sodium thioglycollate 0.025; cysteine, 0.3; cystine, 0.025; N-2-hydroxyethylpiperazine-N'-2ethanesulphonic acid (HEPES), 2.38.…”

A fully defined, clear and protein-free liquid medium permitting dense growth ofNeisseria gonorrhoeaefrom very low inocula

“…Chemically defined liquid media for N. gonorrhoeae are usually minimal media and none has been demonstrated to support the growth of low (e.g. o 10 3 CFU mL À1 ) inocula (Welton et al, 1944;Kenny et al, 1967;Hunter & McVeigh, 1970;Bacigalupi & Lawson, 1973;Catlin, 1973;La Scolea & Young, 1974;Elmros et al, 1976;Manchee et al, 1980;Morse & Bartenstein, 1980;Wong et al, 1980). Currently, therefore, there are no available liquid media, defined or undefined, that permit study of the growth of N. gonorrhoeae from low inocula (Reinhold et al, 1988).…”

The serological classification of Neisseria gonorrhoeae. I. Isolation of the outer membrane complex responsible for serotypic specificity.

“…These observations were confirmed by Danielsson (21) who also noted that N. sicca, Neisseria tiara, and Neisseria flavescens reacted with antigonococcal globulin. Recent investigations, most notably those of Carifo and Catlin (22) and LaScolea and Young (23), have dwelt upon the feasibility ofauxotyping, while others (24,25), though unsuccessful, have probed the possibility of bacteriocins as potential candidates for gonococcal serotyping. The isolation of pili as a discrete protein antigen of the virulent colonial types, T, and T2, by Buchanan et al (26) prompted the analysis of pill as having serotypic specificity.…”