Spectinomycin

Preliminary experiments with single and mixed antibiotics (chloramphenicol, neomycin, ampicillin, streptomycin, rifampin [each at 50 mg/liter] and 60 mg/liter spectinomycin) were performed to establish the final protocol for the generation of sterile polyps. The remaining cultivable bacteria were analyzed based on CFU per milliliter on MB and R2A agar plates after 5 days of incubation. Consequently, sterile polyps were generated by treatment of 3-day-starved animals with an antibiotic mixture containing 50 mg/liter each of chloramphenicol, neomycin, ampicillin, streptomycin, and rifampin, and 60 mg/liter spectinomycin (all from Carl Roth, Karlsruhe, Germany) in sterile ASW. After 5 days, absence of cultivable bacteria was ensured by plating polyp homogenate on MB and R2A agar plates. In addition, the genomic DNA of the polyps was used as the template for full-length 16S rRNA gene amplification using GoTaq polymerase with universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The absence of amplicons in combination with the absence of colonies confirmed that the polyp, early strobila, and late strobila stages were sterile.