Effect of Aeration and Lack of CO 2 on Growth of Bacteria-Free Cultures of Protozoa. ∗

It has been known for some time that CO₂ is necessary for the growth of bacteria, yeast, and molds on solid media 1 , 2 and recent investigations have shown that it is also necessary for bacteria when synthetic liquid media are used. 3 , 4 In liquid media containing hydrolyzed protein growth is not completely inhibited, but it may be delayed considerably by the absence of CO₂. 4 , 5 It has also been suggested that lack of sufficient CO₂ might be a cause of the lag period in growth of cultures of free-living protozoa and a cause of the failure of various investigators to obtain bacteria-free cultures of intestinal species. 6 For these reasons information concerning the effect of lack of CO₂ on protozoa seems highly desirable.

In the present experiments bacteria-free cultures of Glaucoma piriformis † and Chilomonas paramecium ‡ were grown in a heavily buffered medium of 0.5% hydrolyzed casein and 0.25% KH₂PO₄ at pH 6.0 under the following conditions: (a) unaerated, in flasks with cotton stoppers, (b) aerated, in Drechsel gas washing bottles with ordinary air which had been washed through 2 bottles of water and an indicator solution (brom thymol blue), (c) aerated, in Drechsel bottles with CO₂,-free air prepared by passing air through 2 Milligan spiral gas washing bottles of strong KOH and then through 2 bottles of water and an indicator solution. These conditions are similar to those used by Walker 5 for bacteria. The rate of bubbling was a but 5 liters per hour per flask. The cultures were immersed in a stirred water bath, and the temperature was thereby maintained equal in all cultures and approximately that of the room (2O°-25°C.).