Dunkin Donuts

Evaluating the advantages and uses of

Bacillus thurengiensis israelensis
Bhakta, Jatin, Brian Parkins, Keitia Ramos, and Vincent Vo*
California State University Fullerton, Department of Biological Science
Background
Bacillus thuringiensis
o Gram (+), rod shaped, spore forming, found naturally
in soil
o Endotoxin- insecticide
o Past study with SDS-PAGE
o cry2, cry9 genes
o 1976 first sign of insecticidal properties in Israel

Background
Study involved Summits Mosquito dunk and a sample of pond
water
What is a Mosquito Dunk?
o Spread on water surface, fall to the bottom
Drought resistance
o Biofilm
Impact on ecology
o -endotoxins may allow for Bt to outcompete other species
o Decrease in biodiversity
www.summitchemical.com
Hypothesis
If B. thuringiensis is drought resistant, then we predict the
formation of a thick biofilm to prevent desiccation

If B. thuringiensis is capable of producing potent endotoxins,
then we predict the formation of a zone of inhibition when
bacteria are exposed to this endotoxin

Materials and
Methods
Endotoxin extraction
750l of week-old Bt, 750l DI water
Centrifuged for 45 seconds @ 13K RPM
Loaded 1.5 mL of supernatant into SDS-PAGE

Modified Kirby Bauer
Normally test for antibiotic sensitivity to antibiotics

0.3l of E.coli spread plated onto MH media

Filter disk dipped into working culture solution

Filter disk placed on prepared MH plate and
incubated for 5 days

Materials and
Methods
Results - Establishing
Mosquito Dunks - Bacillus thuringiensis israelensis
Gram positive bacilli
Endospore positive
TSI - Yellow slant, red butt (ferments sucrose and lactose)
SIM - Sulfur and Indole negative, Motility positive
Pond Water
Mostly Gram positive bacilli (Bt)
Some Gram negative cocci
SIM - All negative
Results
Dot found on mosquito dunk culture
o Gram staining showed Gram positive Bacillus (expected) and Gram-
variable vibrio (unknown).
o Resulting cultures displayed Gram positive Bacillus, Gram positive
Staphylococcus (sp. unknown, assumed contamination), and Gram-
variable vibrio (unknown).

SDS-PAGE for -endotoxin yielded no results - unable to isolate.
Results
Biofilm test
o B. thurengiensis
o B. megaterium (+ control)
o E. coli (+ control)

Standardized using largest
absorbance value at 595nm
Results - The Dot
Gram-variable
vibrio resolved on
both MSA and
EMB, with stronger
growth on EMB.

Vibrio cell wall
structure is special.
Results
15mm zone of inhibition
found around disc E. coli
plated on Mueller-Hinton
agar.

Corresponds with
intermediate
resistance/susceptibility.
Conclusions
Bt produces biofilm to be drought
resistant

E. coli- intermediate susceptibility to -
endotoxin

Future Work
Compare bacteria populations in ponds
before and after putting in mosquito dunks

New hypothesis
o Bacillus thuringiensis decreases
biodiversity
Literature Cited
Bacillus mycoides, Bacillus pseudomycoides and Bacillus thuringiensis to 24 antimicrobials using Sensititre automated microbroth dilution and Etest
agar gradient diffusion methods J. Antimicrob. Chemother. June 2007 60 (3): 555-567

Ejiofor, AO, Johnson, T, Physiological and molecular detection of crystalliferous Bacillus thuringiensis strains from habitats in the South Central United
States J. Industrial Micro. & Biotech. (2002) 28, 284-290

Espinasse, S., M. Gohar, J. Chaufaux, C. Buisson, S. Perchat, and V. Sanchis. Correspondence of high levels of-exotoxin I and cry1B in Bacillus
thuringiensis. Appl. Environ. Microbiol. September 2002 68:41824186.

Hernandez-Rodriguez, C S., I.R. Escudero, A. C. Asensio, J. Ferre, P. Caballero. 2013. Encapsulation of the Bacillus thuringiensis secretable toxins
Vip3Aa and Cry1la in Pseudomonas fluorescens. Biological Control. Vol. 66 Issue 3. Pages 159-165

Shu, C., S. Huiqin, Z. Jie, H. Kanglai, D. Huang, F. Song. 2013. Characterization of cry9Da4, cry9Eb2, and cry9Ee1 genes from Bacillus thuringiensis strain
T03B001. Applied Microbiology and Biotechnology.
Sutherland, IW, Biofilm exopolysaccharides: a strong and sticky framework Microbiology January 2001 147:3-9

Luna, VA, King, DS, Gulledge, J, Cannons, AC, Amuso, PT, Cattani, Susceptibility of Bacillus anthracis, Bacillus cereus,

BROWNBRIDGE, M, Margalit, J. "New Bacillus thuringiensis strains isolated in Israel are highly toxic to mosquito larvae." Journal of Invertebrate Pathology
48.2 (1986):216-222.