Culicidae 389 



THE CULTURE OF MOSQUITO LARVAE FREE FROM 

 LIVING MICRO-ORGANISMS 



William Tracer. Rockefeller Institute for Medical Research 



A METHOD has recently been described (Trager, 1935) whereby 

 bacteria- free larvae and adults of the yellow fever mosquito, Aedes 

 aegypti, may be readily obtained. The essentials of this method will be 

 briefly summarized here. 



STERILIZATION OF THE EGGS 



This is accomplished by a slight modification of the method of Mac- 

 Gregor (MacGregor, 1929). "Boats," made out of coverslips by heating 

 their edges in a flame to make them curl down and sterilized by dry heat, 

 are placed in small sterile petri dishes containing a 5% solution of Cas- 

 tile soap. From 5 to 20 eggs (as desired) are put in each boat and left in 

 the soap solution 5 to 7 minutes. With sterile forceps each boat is then 

 lifted out (the eggs coming with it), drained of excess liquid and placed 

 in a sterile petri dish holding 80% alcohol. The eggs are left 15-17 

 minutes in the alcohol and are then transferred, as before, to a sterile 

 petri dish holding sterile water. Finally, the boat with its contained eggs 

 is lifted with sterile forceps and dropped into a tube of culture medium. 

 This method is nearly 100% successful as long as the eggs used have 

 been laid recently (within 1 to 3 days) on filter paper partly immersed in 

 distilled water. 



THE CULTURE MEDIUM 



Liver extract. To every 100 cc. of distilled water, 0.5 gm. of Eli Lilly 

 and Company liver extract #343 is added. The somewhat turbid mix- 

 ture, when filtered through paper, gives a clear amber-colored filtrate. 

 The pH of this solution is adjusted to 7.0 by the addition of N/i NaOH 

 (about 0.25 to 0.3 cc. per 100 cc. of solution). The medium is then 

 sterilized either by passage through a Berkefeld "N" filter or by auto- 

 claving. 



Yeast. A strain of Fleischmann's bakers' yeast may be used. Two- to 

 4-day growths from the surface of Blake bottles of dextrose agar are 

 suspended in sterile tap water, centrifuged down, re-suspended in sterile 

 distilled water, again centrifuged, and finally suspended in enough sterile 

 distilled water so that 10 to 11 cc. of suspension contain the yeast from 

 one Blake bottle. The yeast suspension is then killed by heating at 

 8o'°-8s° C. for 30 minutes. Ordinarily, 1 cc. of such a yeast extract is 

 added to each large test tube (22 x 180 mm.) containing 12 to 14 cc. 

 of the liver extract. 



An even simpler but equally effective medium may be made by using 



