Development of techniques for the recovery and enumeration of two mosquito pathogens

Media for the selective recovery of the mosquito pathogens Bacillus sphaericus 1593 and B. thuringiensis serovar. israelensis were developed. Streptomycin at 100 µg/ml and 500 µg/ml in NY agar (nutrient agar with 0.05% yeast extract) successfully selected for B. sphaericus, and allowed counts equivalent to those obtained on the nutrient medium NY without antibiotics. The medium containing 100 µg/ml of streptomycin (NYST) was used to recover B. sphaericus 1593 from a mixed microbial population in pond water. Sodium chloride, penicillin G, and pH adjustment of the medium were found to be unsatisfactory selective agents. Two selective media for the recovery of B. thuringiensis serovar. israelensis gave counts equivalent to those obtained on the nonselective NY medium. One medium contained 100 µg/ml of polymyxin with 1.0 µg/ml chloramphenicol (NYPC), and the other contained 500 µg/ml of polymyxin alone. The use of the higher level of polymyxin with chloramphenicol reduced the number of viable B. thuringiensis serovar. israelensis. NYPC was used to recover B. thuringiensis serovar. israelensis from a mixed microbial population in pond water. The selective media reduced the number of pond water microorganisms on plates by 90 to 99%. A heat treatment of 50ºC for 10 minutes also reduced pond water microbiota by approximately one log. The use of heat treatment plus either NYST or NYPC reduced the pond water microbiota further. The heat treatment had little effect on sediment microbiota. A selective-differential medium for B. thuringiensis serovar. israelensis was developed for use when heat treatment of samples would be undesirable. This medium, PEMBAC, permitted the observation of peptone deamination and hydrolysis of lecithin, which are characteristic of B. thuringiensis serovar. israelensis. The medium contained 50 µg/ml of polymyxin and 1.0 µg/ml chloramphenicol as selective agents. The parasporal crystals of B. thuringiensis serovar. israelensis are the site of the mosquito larval toxin. Because the crystals are not viable, another method for their enumeration was examined. Antisera to whole crystals and to solubilized crystal antigens were prepared in rabbits for use in the indirect fluorescent antibody technique. Because of the small size and irregular shape of the parasporal crystals of B. thuringiensis serovar. israelensis, the crystals were difficult to distinguish from other small fluorescing particles. The antisera prepared precipitated several antigens in solubilized crystals, but did not adsorb to the majority of the antigens in whole crystals.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Microbiology

Department dc:contributor.department

Microbiology

Grantor dc:publisher

Virginia Polytechnic Institute and State University

Year dc:date.issued

1982

Author dc:creator

• Jones, Margaret Ellen

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10919/80125

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/80125