Usefulness of staining parasporal bodies when screening for Bacillus thuringiensis

Ammons, David; Rampersad, Joanne; Khan, Ahmad Nawaz

doi:10.1016/s0022-2011(02)00018-6

Cited by 53 publications

(28 citation statements)

References 2 publications

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“…Evaluation could be done in a continuous motion without stopping to view each specimen. The stain also offered improved resolution over Phase Contrast, allowing the visualization of very small parasporal bodies [7]. A few of the crystals did not take up the stain well, however the contrast between spores and crystals was sufficient to easily differentiate the two, even when crystal morphology mimicked that of spores (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…Where more than one isolate from the same sample was observed possessing the same crystal morphology, they were considered clonal and not all such isolates were kept. The slide was heat fixed and stained with Coomassie Brilliant Blue [7] and viewed under Brightfield Microscopy using a 100X immersion oil objective. Cell morphology was noted for all candidates of B. thuringiensis , which was defined as a large spore-forming rod whose ellipsoidal spore did not swell the mother cell and which produced a parasporal body upon sporulation [20,21].…”

Section: Methodsmentioning

confidence: 99%

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A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results

Rampersad

Ammons

2005

BMC Microbiol

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Background: Bacillus thuringiensis is a bacterium known for producing protein crystals with insecticidal properties. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. There is great interest in isolating strains with improved or novel toxin characteristics, however isolating B. thuringiensis from the environment is time consuming and yields relatively few isolates of interest. New approaches to B. thuringiensis isolation have been, and continue to be sought. In this report, candidate B. thuringiensis isolates were recovered from environmental samples using a combination of a novel stain, high throughput and reduced selection. Isolates were further characterized by SDS-PAGE, light microscopy, PCR, probe hybridization, and with selected isolates, DNA sequencing, bioassay or Electron Microscopy.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results

Rampersad

Ammons

2005

BMC Microbiol

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“…The SEM observation was conducted on a HITACHI S-800 (Hitachi, Tokyo, Japan) following instructions for the device. For light microscopy, spore-crystal mixtures were spread on a glass slide, heat-fixed, stained with Coomassie blue (0.133% Coomassie blue in 50% acetic acid) [37] and observed under 100× magnification using immersion oil.…”

Section: Methodsmentioning

confidence: 99%

Identification of Distinct Bacillus thuringiensis 4A4 Nematicidal Factors Using the Model Nematodes Pristionchus pacificus and Caenorhabditis elegans

Iatsenko

Nikolov

Sommer

2014

Toxins

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Bacillus thuringiensis has been extensively used for the biological control of insect pests. Nematicidal B. thuringiensis strains have also been identified; however, virulence factors of such strains are poorly investigated. Here, we describe virulence factors of the nematicidal B. thuringiensis 4A4 strain, using the model nematodes Pristionchus pacificus and Caenorhabditis elegans. We show that B. thuringiensis 4A4 kills both nematodes via intestinal damage. Whole genome sequencing of B. thuringiensis 4A4 identified Cry21Ha, Cry1Ba, Vip1/Vip2 and β-exotoxin as potential nematicidal factors. Only Cry21Ha showed toxicity to C. elegans, while neither Cry nor Vip toxins were active against P. pacificus, when expressed in E. coli. Purified crystals also failed to intoxicate P. pacificus, while autoclaved spore-crystal mixture of B. thuringiensis 4A4 retained toxicity, suggesting that primary β-exotoxin is responsible for P. pacificus killing. In support of this, we found that a β-exotoxin-deficient variant of B. thuringiensis 4A4, generated by plasmid curing lost virulence to the nematodes. Thus, using two model nematodes we revealed virulence factors of the nematicidal strain B. thuringiensis 4A4 and showed the multifactorial nature of its virulence.

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“…Briefly, a straight inoculating wire was used to transfer an aliquot of a sporulated culture onto a microscopic slide. Then the slide was heat fixed and stained (0.133% CBB stain in 50% acetic acid), rinsed in distilled water, dried and observed under light microscope using 100X oil immersion objective (Rampersad and Ammons, 2002). The presence of parasporal inclusions were clearly observed as dark blue stained bodies.…”

Section: Phenotypic Characterization Through Sporulation Gram Stainimentioning

confidence: 99%

Soil enrichment and dominant occurrence of Bacillus thuringiensis subsp. israelensis in a garden sprayed with Bacillus thuringiensis based biopesticide

Chai¹,

Rathinam²,

Krishnaveni³

et al. 2014

MJM

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Aims: Bacillus thuringiensis (Bt) based biopesticides have been successfully used to control agricultural pests and vectors of human diseases. B. thuringiensis subsp. israelensis (Bti) based formulations have been used as anti-dipteran agents to control the mosquito larvae. The study was conducted to understand the occurrence and relative dominance of Bti in a botanical garden, a non-agricultural ecosystem previously sprayed with Bt based formulation. Methodology and results:The preliminary phenotypic investigations revealed that all the isolates were Bt. The Coomassie Brilliant Blue staining of the isolates revealed the presence of spherical crystals, a striking characteristic of Bt. The SDS-PAGE analysis provided basic information on the similarity of all the isolates based on the protein profile. The similarity between native isolates and Bti was further confirmed by RAPD-PCR. Conclusion, significance and impact study: Through phenotypic and molecular methods, this study has conclusively proved the occurrence and the dominance of Bt isolates similar to the Bti used for spraying. This study provided new insight into the possible mechanism exerting continued pressure on the target insects, natural cry gene transfer because of the dominant presence.

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Usefulness of staining parasporal bodies when screening for Bacillus thuringiensis

Cited by 53 publications

References 2 publications

A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results

A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results

Identification of Distinct Bacillus thuringiensis 4A4 Nematicidal Factors Using the Model Nematodes Pristionchus pacificus and Caenorhabditis elegans

Soil enrichment and dominant occurrence of Bacillus thuringiensis subsp. israelensis in a garden sprayed with Bacillus thuringiensis based biopesticide

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