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

Chilcott, C. N.; Wigley, P. J.

doi:10.1111/j.1472-765x.1988.tb01254.x

Cited by 10 publications

(6 citation statements)

References 2 publications

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“…Preliminary screening based on colony morphology was carried out for Bt‐like colonies (Wigley et al, 2005) after 4 days and the presence of the Bt marker was confirmed using stained bacterial smears prepared as described by Chilcott & Wigley (1988). A speck of each colony was removed from the plate, smeared on to a glass slide, air‐dried, and baked at 100 °C for 1 h. The slide was dipped in 1.5% (wt/vol) naphthalene black (BDH Laboratories, Poole, UK) in 37% acetic acid for 2 min, rinsed in water, and stained in 2% (vol/vol) Gurr’s Improved R66 Giemsa stain solution for 2 min.…”

Section: Methodsmentioning

confidence: 99%

Dispersal of potato tuber moth estimated using field application of Bt for mark‐capture techniques

Cameron¹,

Wigley²,

Elliott³

et al. 2009

Entomologia Exp Applicata

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A new mark‐capture technique involving field applications of Bacillus thuringiensis Berliner (Bt) to study the dispersal of potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), was investigated as a tool to improve information on the potential impact of insect pest dispersal on crop infestation and insecticide resistance. The acquisition and persistence of Bt on moths were characterized and potential contamination of moths from naturally occurring Bts was examined. This mark‐capture technique was developed to mark larger numbers of moths than had been previously achieved with laboratory marking using fluorescent dyes in mark‐release‐recapture experiments. Applications of commercial preparations of Bt to 0.3 and 1.0 ha potato fields were estimated to have marked ca. 50 000 moths in each experiment. Pheromone trap catches of potato tuber moths in the Bt‐sprayed fields and in potato fields at distances of ca. 80, 200, 350, and 750 m were assayed for the Bt marker using selective microbiological media and identification of characteristic Bt crystal inclusions. Marking rates of moths were 78–100% in the sprayed fields and, compared with our previous mark‐release‐recapture studies, marking at ca. 200 m was increased by 15–18‐fold to >3.0 moths per trap. This capture rate allowed the calculation of a dispersal curve that improved the reliability of estimates of movement at farm‐scale distances. These estimates indicated that 10% of the population dispersed to 240 m in 3 days, and suggested that moths can potentially disperse throughout a typical potato‐growing area in one growing season. This level of dispersal has implications for the spread and management of potato tuber moth populations, especially if insecticide resistance is present.

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Section: Methodsmentioning

confidence: 99%

Dispersal of potato tuber moth estimated using field application of Bt for mark‐capture techniques

Cameron¹,

Wigley²,

Elliott³

et al. 2009

Entomologia Exp Applicata

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“…The colonies resembling Bt cream-colored and have the appearance of a fried egg on a plate were selected and gram stained, sub-cultured as ribbon streak four colonies per plate on T 3 agar medium tryptone-3 g, tryptose-2 g, yeast extract-1.5 g, sodium phosphate-0.05 M, manganese chloride-0.005 g, agar-15 g, distilled water-1000 mL, pH-6.8 . After 72 h of incubation crystal staining was done Chilcott and Wigley, 1988 . The isolates showing the presence of crystalline inclusions were selected as Bt and streaked on T 3 agar medium for single colony purification. Broth culture was made from the isolated single colonies of crystal positive Bt isolates.…”

Section: Consistency Of Tree Was Checked By a Bootstrap Valuementioning

confidence: 99%

Diversity and Toxicity of <i>Bacillus thuringiensis</i> from Shifting Cultivation (Jhum) Habitat

2016

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Bacillus thuringiensis (Bt) strains were isolated from jhum-agriculture, jhum-forest, aquatic and fallow soil samples from Mizoram by acetate selection method. Isolates were characterized for biochemical typing, cry gene and protein profiling, growth curve study and toxicity against Culex tritaeniorhynchus. Bt frequency was high in jhum-agriculture land (69.56%) whereas low in jhum-forest soils (31.57%). Bt was found to be abundant in jhum shifting cultivation soil with an index ranging between 0.010 and 0.015. Majority of the isolates from jhum soils produced oval and spherical crystals and showed eleven types of crystal proteins groups. PCR analysis revealed predominance of dipteran-active cry genes (cry4 and cry9). The variations in crystal morphology, cry genes and Cry protein (s) from the isolates of Bt revealed molecular diversity. Higher mortality, lower lethal dose, and lesser time to kill were observed in Bt isolates from jhum soils than aquatic and fallow habitats. Based on the toxicity test, SC1 and HP7 isolates containing cry 4 and cry 9 genes showed higher activity. Growth curve analysis showed significant variations among Bt isolates to reach the sporulating stage. Higher growth index and lower mean generation time were observed in SC1 and HP7 Bt isolates. Bt strains express different endotoxin genes and crystal proteins and their harvesting time also varied from strain to strain. Significant variation was found in Bt isolates from jhum habitats in relation to the cry gene composition, protein profiling and toxicity. Results from this study suggest that novel Bt entomopathogens may complement for regulating mosquito vectors.

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“…Preliminary screening based on colony morphology was carried out for Bt-like colonies after 4 days and confirmatory evidence of presence of the Bt marker was sought using stained bacterial smears prepared as described by Chilcott & Wigley (1988). A speck of the colony was removed from the plate using a sterile toothpick, smeared onto a water droplet placed on a glass slide, allowed to air-dry and baked at 100°C for 1 h. The slide, while still hot, was dipped in 1.5% naphthalene black (BDH Laboratories) in 37% acetic acid for 2 min, rinsed in water, and stained in 2% Giemsa stain for 2 min.…”

Section: Laboratorymentioning

confidence: 99%

Bacillus thuringiensisas a marker for insect dispersal studies

Wigley¹,

Madhusudhan²,

Cameron³

et al. 2005

New Zealand Journal of Crop and Horticultural Science

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Laboratory studies confirmed that commercial formulations of Bacillus thuringiensis (Bt) could be used to mark potato tuber moths, Phthorimaea operculella. Moths were successfully marked by direct application or through indirect acquisition by contact with marked surfaces such as a leaf or a Petri dish. Marked moths were identified by the distinctive crystalline morphology of different subspecies of Bt, allowing the separation of individual moths marked with either Btk (kurstaki) or Bti (israelensis). A simple microbiological method for processing the trapped moths before definitive, microscopic identification of the subspecies is described. Field marking was verified by capturing light brown apple moths, Epiphyas postvittana, from areas that had been subjected to large-scale spraying of Btk for control of an exotic pest species. Btk was also detected in moths from isolated traps at c. 1 km from the treated area, but not in moths from traps at greater distances. These results indicate that Bt preparations could be used to mark naturally occurring populations as well as laboratory-reared individuals to study their dispersal.

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

Abstract: 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.

Cited by 10 publications

References 2 publications

Dispersal of potato tuber moth estimated using field application of Bt for mark‐capture techniques

Dispersal of potato tuber moth estimated using field application of Bt for mark‐capture techniques

Diversity and Toxicity of <i>Bacillus thuringiensis</i> from Shifting Cultivation (Jhum) Habitat

Bacillus thuringiensisas a marker for insect dispersal studies

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