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LABORATORY EXERCISES 



LESSON 22 



Isolation of a Leaf Wilt Fungus in Pure Culture. — Given a fungus causing leaf 

 wilt, to obtain a pure culture by excluding the non-pathogenic forms. 



I. Look for the fruiting stage of the suspected fungus, or fungi. Transfer some 

 of the spores with a sterile needle into a tube of 5 c.c. of sterile water. (If pycnidia 

 or perithecia are present, transfer a whole pycnidium or perithecium into sterile 

 water, and crush the fruit body to cause the escape of the spores). Then with a 

 sterile needle transfer some of the water with the spores into a tube of agar-agar 

 which is made liquid by putting in a vessel of hot water and then allowed to cool. 

 This tube is marked A. Then from tube A transfer a drop of agar with a sterile needle 

 to another similar test-tube with liquid agar designated as B (Fig. 221). Then 

 perform the same sort of transfer to a third tube C. Distilled water or nutrient 

 bouillon can be used for these dilutions instead of agar. 



Fig. 221. Fig. 222. 



Fig. 221. — Method of holding test-tubes in transfer of fungi from one test-tube 

 to another. {After Lohnis.) 



Fig. 222. — Cylindric form of wire basket for holding test-tubes during steriliza- 

 tion and other operations. {After Schneider, Pharmaceutical Bacteriology, p. 37.) 



^, J5 and C are thoroughly shaken and each is transferred to Petri dishes marked 

 A, B and C. If water is used to dilute, or bouillon, it must be mixed with the material 

 poured into the Petri dishes. These are observed for any growth that may take place 

 on the surface of the agar-agar. Transfers are made from the single colonies into 

 agar slants in test-tubes. 



If no spore forms are present, cut out pieces of the affected leaf and place in a 

 tube containing i per cent, mercuric chloride diluted in equal amounts in 50 per cent, 

 alcohol. Shake the tube so that the material is bathed in the disinfectant. Do 

 this for half a second to two minutes according to the thickness of the leaf. Pour 

 off the disinfectant and wash the material three times in sterile water, care being 

 taken to keep out foreign infection. Then with a sterile forceps, take each piece 

 of the material and crush it thoroughly at the mouth of a tube containing melted 



