3.4. In vitro sporulation of Paenibacillus larvae
Paenibacillus larvae and Paenibacillus popilliae, “catalase-minus” paenibacilli as defined by the loop test (i.e. scraping growth from a slant or plate with an inoculating loop, placing into 3 % H2O2, and examining for bubble formation), have the characteristic of sporulating efficiently in their insect hosts, but usually exhibit very poor sporulation when general in vitro growth conditions are used. For P. larvae, suppression of in vitro sporulation has been postulated to result from oxygen toxicity (Dingman and Stahly, 1984). Growth of strain NRRL B-3650 under limiting O2 improves sporulation in liquid culture. Also, nutrient availability at time of sporulation has been shown to influence spore production (Dingman and Stahly, 1983).
Procedures promoting effective sporulation of P. larvae on solid, and in liquid, growth media have been developed (Dingman, 1983). By limiting colony number, many strains have been observed to sporulate well on MYPGP agar plates (see section 3.1. for recipe). However, Mueller-Hinton broth – one of its ingredients – was inhibitory to sporulation of strain NRRL B-3650 in liquid culture, rendering use of this growth medium in liquid form ineffective for sporulation. Development of a liquid growth medium (TMYGP; 1.5 % Difco yeast extract; 0.4 % glucose; 0.1 % sodium pyruvate; 0.03M Tris-maleate, pH 7.0; in distilled water) and conditions that aided sporulation of P. larvae NRRL B-3650 has been reported (Dingman and Stahly, 1983). Unfortunately, other strains of this bacterium sporulated poorly, if at all, in this medium. However, Genersch et al. (2005) reported using the liquid part of Columbia sheep-blood agar slants for production of endospores from different P. larvae strains. Following are protocols using solid and liquid media growth conditions for in vitro sporulation of P. larvae.