64 



Marine Microbiology 



Stationary Pure Cultures 



In a 500 ml Erlenme) er flask, 250 ml of the following sterile 

 agar medium was allowed to solidify: seawater, 2 per cent agar 

 (Bacto, Difco), 0.01% NH^Cl, 0.01% KH.PO., 0.01% Na.COs, 

 0.02% H2S, pH 8.0. The H^S was added as a saturated solution 

 in sterile seawater; Na^COs was autoclaved separately as a 1 per 

 cent solution; both ingredients were added to the agar just be- 

 fore it had gelled. After the agar had solidified, 250 ml sterile 

 seawater was added; the flask was closed with a sterile rubber 

 stopper containing an outlet with a cotton plug and a device for 

 localized aeration, consisting of a cotton-plugged Pasteur pipette 

 with a glass tube sealed around its drawn-out end. The glass tube 

 was open underneath and had a hole above the water level 

 (Fig. 1). When a slow current of air was passed through the 

 pipette, the resulting aeration was restricted to the liquid in the 

 tube, the dissolved oxygen slowly diffusing into the bulk of the 

 medium. The device, by preventing turbulence, provided for the 

 establishment of a concentration gradient of both HjS and O2, 

 the fomier being highest at the agar surface, the latter at the aer- 



Fig. 1. Schematic drawing of culture vessel for stationary cultivation of 

 Thiovtilum. A = sulfide-containing agar layer; B = mineral seawater 

 medium; C = protective tube for localised aeration; D = Pasteur pipette; 

 E = cotton plug; F = position of TJiioviilum web in initial stage of cul- 

 tivation. 



