Technical note: an improved method for differential staining of Bacillus thuringiensis crystals

A new staining method is described using naphthalene black 12B and Gurr's improved R66 Giemsa for staining all known crystal types produced by Bacillus thuringiensis.     

The use of coconut wastes for the production ofBacillus thuringiensis var.israelensis

Summary Good yields of crystal/spores and larvicidal activity were obtained whenBacillus thuringiensis var.israelensis was grown in coconut water and endosperm extracts. Coconut water contained 2.2 mg protein/ml, while the endosperm extracts contained 1–4 mg protein/ml extract. The carbohydrate content of the media ranged from 6–38.4 mg/ml.

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Resumen Utilización de desechos de coco para la producción de Bacillus thuringiensisvar. israelensis Agua de coco y extractos de endosperma se han utilizado para el cultivo deBacillus thuringiensis var.israelensis obteniendo un buen rendimiento en cristales/esporas y una buena actividad larvicida. El agua de coco contenía 2.2 mg de proteína/ml y los extractos de endosperma 1–4 mg/protei'na/ml de extracto. El contenido en hidratos de carbono de los medios utilizados varió entre 6–38.4 mg/ml.

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Résumé Utilisation des déchets de noix de coco pour la production de Bacillus thurigiensisvar. israelensisDe bons rendements en cristaux, spores et activité larvicide ont été obtenus en cultivantBacillus thurigiensis var.israelensis dans l'eau et les extraits endospermiques de noix de coco. L'eau des noix contient 2,2 mg de protéines/ml et les extraits d'endosperme 1–4 mg. La teneur des milieux en carbohydrates était comprise entre 6–38,4 mg/ml.

     

Activities of Bacillus thuringiensis Insecticidal Crystal Proteins Cyt1Aa and Cyt2Aa against Three Species of Sheep Blowfly

The toxicity of Bacillus thuringiensis Cyt1Aa protein to sheep blowfly larvae depends on its solubilization and proteolytic activation. Cyt1Aa crystals were not toxic. Full-length and trypsin-digested Cyt1Aa proteins were toxic to larvae of three species of sheep blowfly. Neither full-length nor trypsin-digested Cyt2A soluble crystal proteins were toxic.     

Efficacy of native and recombinant Cry1B protein against experimentally induced and naturally acquired ovine myiasis (fly strike) in sheep

Several hundred strains of Bacillus thuringiensis (Bt), isolated in New Zealand from samples of soil and sheep fleece, were tested for toxicity to larvae of the blowfly Lucilia cuprina Wiedemann. Characterization of the Bt strains revealed that three of the more active strains produced Cry1Ba (an insecticidal protein present in Bt mother cell crystal inclusion) that was toxic to blowflies. These strains were evaluated for the ability to prevent experimentally induced fly strike in a bioassay by using first instars. Results with undiluted spore/crystal preparations were variable, but they generally prevented fly strike on sheep maintained on pasture for 3-6 wk. Spore viability was satisfactory throughout the trials and environmental factors (e.g., precipitation and UV radiation) seemed to have minimal effect on persistence. The loss of fly strike protection in these experiments correlated with the movement of spore/crystal toxicity away from the skin as a result of wool growth. Solubilized protein preparations were not as potent as spore/crystal preparations and fly strike protection lasted only from 1 to 3 wk. Vegetative forms of the Cry1Ba-producing strains of Bt did not establish on the fleece of sheep, did not produce significant sporulation, and no protection against fly strike was achieved. Escherichia coli expressing recombinant Cry1Ba protein was toxic to larvae in vitro but did not effectively protect sheep from fly strike because blowfly larvae were able to establish readily 8 d posttreatment. In a single field experiment involving 80 sheep per group, a spore/crystal preparation from a Bt strain expressing Cry1Ba provided less protection from naturally acquired fly strike than afforded by a commercially available dip.     

Safety assessment of the oral cavity probiotic Streptococcus salivarius K12

Streptococcus salivarius is a prominent member of the oral microbiota and has excellent potential for use as a probiotic targeting the oral cavity. In this report we document safety data relating to S. salivarius K12, including assessment of its antibiogram, metabolic profiles, and virulence determinants, and we examine the microbial composition of saliva following the dosing of subjects with K12.     

Safety Assessment of the Oral Cavity Probiotic Streptococcus salivarius K12

Streptococcus salivarius is a prominent member of the oral microbiota and has excellent potential for use as a probiotic targeting the oral cavity. In this report we document safety data relating to S. salivarius K12, including assessment of its antibiogram, metabolic profiles, and virulence determinants, and we examine the microbial composition of saliva following the dosing of subjects with K12.     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.     

Regeneration of Salvia sclarea via organogenesis

Summary The present work provides a system for regeneration of clary sage, (Salvia sclarea L.) via organogenesis using plant tissue culture techniques in a multistage culturing medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (9.05–181.00 μM). A higher frequency of organogenic tissue initiation was obtained from immature zygotic embryo cotyledons (IZEC) 2–3 wk after pollination on the medium supplemented with 9.05 μM 2,4-D. The organogenic tissues were then proliferated on media containing both indole-3-acetic acid (IAA) and 6-benzylaminopurine (BA). Organogenic lines were established via selection, isolation and continuous subculture of organogenic tissues on a medium containing 22.19 μM BA and 2.85 μM IAA. Shoots were regenerated from both the proliferated tissues and IZEC, and propagated in the presence of IAA or α naphthaleneacetic acid (NAA), BA and gibberellic acid (GA₃). Although roots were induced from regenerated shoots on the media containing a low concentration of IAA, IBA (0.98 μM) in combination with desiccation of regenerated shoots with a stem ∼10 mm in length promoted more and stronger root formation. After the root system was well established (20 mm in length), the regenerated plants were transferred to soil in plastic pots for further growth and production of R1 seeds in the greenhouse.