Prophage Induction in Natural Populations of Heterotrophic Bacteria
For unconcentrated seawater samples , add 25 mL each to a control or treatment , 50 - mL sterile , conical centrifuge tubes .
Take an additional sample ( 25 mL ) and fix with 1 % 0.02 - µm filtered formalin .
For the treatment samples , add 1 µg / mL mitomycin C ( or 0.5 µg / mL in oligotrophic environments ) .
The samples are incubated for 16–24 h at room temperature and fixed with either 2 % glutaraldehyde ( for TEM ) , 1 % formalin ( epifluorescence microscopy ) , or 1 % formalin / 0.5 % glutaraldehyde ( flow cytometry [ FCM ] ) .
Samples for enumeration by epifluorescence microscopy should be counted within 24 h of collection or stored as frozen slides stained with SYBR Gold .
Count both bacteria and viruses in control and treated samples .
Calculate the % lysogenic bacteria as follows : % lysogens = [ ( VDCT - VDCC ) / Bz ] / BDC T = o' .
The average burst size can be derived by TEM observation of bacterial bursts ( i . e . , when viruses become visible in the cell at the end of the latent period ; Ackermann / Heldal , this volume ) .
