Journal of Invertebrate Pathology 79 (2002) 203–204
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Note
Usefulness of staining parasporal bodies when screening for
Bacillus thuringiensis
The natural toxins produced by Bacillus thuringiensis
(Bt) are extremely popular for the control of insect pests,
both as a direct application and in transgenic organisms
(Prieto-Samsonov et al., 1997). Screening for novel en-
vironmental isolates of Bt almost universally incorp-
orates the use of phase-contrast microscopy for the
detection of toxin-containing parasporal bodies (Bravo
et al., 1998). We report on the benefits of using stained
specimens and light microscopy instead of phase-con-
trast microscopy during an evaluation of approximately
10,000 bacterial colonies for the presence of parasporal
bodies.
A straight inoculating wire was used to transfer an
aliquot of a sporulated colony to a grid on a microscope
slide that had been gridded with a wax pencil to give 60
squares, then replica-plated onto a nutrient agar plate.
The gridded slide was then heat fixed and stained
(0.133% Coomassie Blue stain in 50% acetic acid), rinsed
with distilled water, dried, and observed with bright-
field microscopy using a 100 oil immersion objective.
The presence of parasporal bodies was immediate and
strikingly evident by the presence of numerous dark-
blue staining objects (Figs. 1B and D) requiring only
approximately 3–5 s to move to and evaluate each
specimen. When a candidate was observed, the candi-
date was easily recovered from the corresponding grid
on the replica plate.
Our experience has been that stained specimens of-
fered two significant advantages over phase-contrast
microscopy. The first, unlike phase-contrast micros-
copy, very small parasporal bodies were readily evident
with stained specimens (Figs. 1A and B). It has in fact
been suggested that the unusually high proportion of cry
1 proteins detected may result from their crystals being
more easily viewed with phase-contrast microscopy
(Bravo et al., 1998). Second, the presence of stained
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PII: S 0 0 2 2 - 2 0 1 1 ( 0 2 ) 0 0 0 1 8 - 6
parasporal bodies was striking and instantaneously
recognizable, much more so than with phase-contrast
microscopy (Fig. 1), thus readily adaptable to quick,
high throughput screening methodologies. In fact, we
found that it was generally not necessary to stop on a
grid and evaluate the candidate. Rather it was possible
to move from grid to grid in a steady movement until
large numbers of blue objects were seen. Phase-contrast
microscopy is unquestionably useful when performing
Bt screens and for some applications it may be the best
method to observe parasporal bodies. However, it
should not be placed in such high regard that other
methods are not considered. Clearly, our experience has
demonstrated that stained specimens offer the investi-
gator advantages unique to those of phase-contrast
microscopy and should be considered when undertaking
large-scale screens for novel isolates of B. thuringiensis.
Acknowledgment
The work was supported by a grant from the
University of the West Indies’ Campus Research and
Publications Fund Committee.
References
Bravo, A., Sarabia, S., Lopez, L., Ontiveros, H., Abarca, C., Ortiz,
A., Ortiz, M., Lina, L., Villalobos, F.J., Pena, G., Nunez-Valdez,
M., Soberon, M., Quintero, R., 1998. Characterization of cry genes
in a Mexican Bacillus thuringiensis strain collection. Applied and
Environmental Microbiology 64 (12), 4965–4972.
Prieto-Samsonov, D.L., Vazquez-Padron, R.I., Ayra-Pardo, C., Gonz-
alez-Cabrera, J., de la Riva, G.A., 1997. Bacillus thuringiensis: from
biodiversity to Biotechnology. Journal of Industrial Microbiology
and Biotechnology 19, 202–219.
204 Note / Journal of Invertebrate Pathology 79 (2002) 203–204

Fig. 1. Panels (A) and (B) correspond, respectively, to a phase-contrast microscopy and a stained, brightfield image of a B. thuringiensis candidate
that produced a very small, oval parasporal body. Similarly, panels (C) and (D) present phase-contrast microscopy and stained images of a
B. thuringiensis candidate that produced a bipryamidal parasporal body.

David Ammons*
School of Veterinary Medicine
The University of the West Indies
Mt. Hope, West Indies
Trinidad and Tobago
E-mail address: ammonstt@netscape.net
(D. Ammons)

Joanne Rampersad
Ayub Khan
Department of Life Sciences, School of Agriculture
The University of the West Indies
Trinidad and Tobago
Received 12 March 2002; accepted 12 April 2002

*
Corresponding author. Fax: +868-645-7428.