MATERIALS AND METHODS Bacterial strains. B. thuringiensis H serotype reference strains were obtained from the collection of Dulmage (7) or from the Entomopathogenic Bacteria collection of the Institut Pasteur, Paris, France. Growth. For the production of crystal proteins, B. thuringiensis strains were grown as shake flask cultures in DSMG medium [0.4% (wt/vol) nutrient broth (Difco), 0.5% (wt/vol) glucose, 25 mM K2HPO4, 25 mM KH2PO4, 0.5 mM Ca(NO3)2, 0.5 mM MgSO4, 10 ,uM MnCl2, 10 ,uM FeSO4] at room temperature (21 to 24°C) for 4 to 5 days until sporulation and cell lysis had occurred. For the preparation of flagella, B. thuringiensis strains were grown as static liquid cultures in nutrient broth (Difco) plus 0.1% (wt/vol) glucose. Colony hybridization. A modified colony hybridization procedure of Grunstein and Hogness (12) was used. Environmental samples, consisting of soil, crop dust, and organic material, were suspended in spore buffer (0.06% [wt/vol] sodium phosphate [pH 7.0], 0.05% [wt/vol] potassium phosphate [pH 6.6], 0.005% [wt/vol] sodium dodecyl sulfate [SDS], 1 ,u1 of 1-octanol per ml; final pH of buffer approximately 7) to a concentration of 1 g of material per 10 ml of buffer, heated for 30 min at 60°C, diluted in spore buffer, and spread on NSM (19) agar medium. After overnight growth at 25°C, colonies were blotted from the agar medium to nitrocellulose filters and treated with 0.5 M NaOH-1.5 M NaCl for 30 min and then with 1 M ammonium acetate-0.02 M NaOH for 30 min. The filters were probed at 65°C in a solution containing 3x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), lOx Denhardt's solution (lx Denhardt's solution is 0.02% [wt/vol] bovine serum albumin, 0.02% [vol/vol] Ficoll, 0.02% [wt/vol] polyvinylpyrrolidone), 200 ,ug of heparin per ml, and 0.1% SDS with the 2.0-kb HindIII-XbaI cryIlIA fragment of strain EG2158 (6) or with 3337