Antibiotics

The use of antibiotics is not recommended when isolating strains that will be used for physiological or ecological studies as mutant clones may be produced that do not reflect populations in the wild. However for species used in aquaculture, population genetics, molecular biology, toxicology or bioproduct screening it may be necessary to work with axenic strains and sometimes this may only be achieved through using antibiotics.

The choice of antibiotics is made based on their efficacy against Gram (+) and/or Gram (-) bacteria and for their mode of action, either as inhibitors of cell wall synthesis or of cell growth via inhibition of protein synthesis. For the cell wall inhibitors to be truly effective the bacteria need to be actively growing therefore it is advisable to add a small amount of organic matter to the treatment media. For this reason adding additional antibiotics that slow cell growth may be counter productive hence a cascade treatment where several antibiotics are administered sequentially is more effective than a cocktail treatment where a single mixed dose is given. Cell wall inhibitors generally belong to the penicillin family, such as Penicillin G that acts primarily against Gram (+) bacteria or the cephalosporin family of beta-lactams that disrupt the integrity of the bacteria cell wall, making it more porous and include broad spectrum antibiotics such as Imipenem. Aminoglycosides such as gentamycin, kanamycin, neomycin and streptomycin attach to the bacterial cell’s ribosomes and impair protein production. Using a B-lactam first to increase cell wall porosity may then allow a faster passage of aminoglycoside to then disrupt protein synthesis.

Antifungal agents include cycloheximide (= acti-dione), Nystatin or Amphotericin B

Germanium dioxide (GeO₂) was used as a media supplement by Lewin (1966) as a means to remove diatoms at a final concentration of 10 mg/L. It acts as a cell division inhibitor rather than a toxin. Suggested concentrations vary widely (https://listserv.heanet.ie/cgi-bin/wa?A2=ind9706&L=ALGAE-L&P=R1150) and Markham and Hagmeier (1982) found concentrations as low as 0.045—0.179 mg/L were sufficient to kill diatoms contaminating their kelp cultures. At ANACC we use final concentrations of 0.4—1 mg/L. As GeO₂ is difficult to dissolve, lower initial stock concentrations improve dissolution so we make up a stock solution of 2 mg GeO₂ in 100 mL of MilliQ water and filter-sterilise. We add the stock solution at a concentration of 1—2.5 mL per 50 mL enrichment medium. If removing diatoms is the goal then all sources of silica should be removed from the culture environment therefore the stock solution is stored in a polycarbonate bottle and the enrichment or isolation cultures performed in plastic culture ware.

References

Lewin (Phycologia 6: 1-12,1966)

Markham, J.W. & Hagmeier, E. (1982) “Observatons on the effects of germanium dixoide on the growth of macro-algae and diatoms.” Phycologia 21(2):125-130