and Laboratory Methods. '2591 



The Agar-agar Method for Embedding Plant Tissues. 



Some time ago 1 noticed an account of a method for tlie rapid fixing and em- 

 bedding of fresh animal tissues in a five per cent, solution of agar-agar. The 

 method was worked out by Drs. Bolton and Harris of the St. Louis University, 

 and first appeared in Am. Med. 5: S3S-839, and later in Science, 17: 1007, 

 1903. It occurred to me to try this method on plant tissue. I wrote to Dr. 

 Bolton for particulars of the method, and he informed me that he had not tried 

 it on plant tissue, and sent me a small amount of the prepared agar-agar. 



According to Bolton and Harris, " the method consists essentially in placing 

 the fresh tissues in a hot 2 per cent, solution of agar-agar to which 10 per cent, 

 of formalin has been added. The temperature of this fluid should be kept at 

 about 70° C. After remaining in the solution from one to several hours, the 

 tissues are removed and attached to blocks with a 5 per cent, solution of agar- 

 agar containing 10 per cent, of formalin. The heat and the formalin harden 

 and fix the tissues at the same time the agar-agar impregnates it. After fixing 

 the tissues to blocks these are placed in 95 per cent, alcohol and allowed to 

 remain from two to four hours, and the tissues are then ready to be cut into sec- 

 tions, which can be stained, cleared, and mounted on slides in the usual way 

 employed for celloidin sections." 



The following are some of the plant tissues on which the agar-agar method 

 was tried : Young ovules of water lily showing the embryos ; cross sections of 

 geranium stems ; cross and longitudinal sections of begonia stems ; cross sec- 

 tions of young ovularies of canna and fig; cross section of the leaf of Fiscus 

 elastica; cross section of the sori of fern leaf: longitudinal section of petiole of 

 Boston ivy; cross sections of stamens of canna; longitudinal section of moss 

 plant (Bryum roseum) ; cross section of leaf of Asimina triloba showing Phyllosticta 

 asiminae ; cross section of raspberry leaf showing rust. 



At first the 5 per cent, solution of agar-agar was used for the fixing and 

 embedding of the tissues. Later the 2 per cent, solution was used for the fixing 

 and found to be more satisfactory. 



The 2 per cent, solution of agar-agar can be made as follows: Take 10 

 grams of agar-agar to 500 c. c. of distilled water and boil for two hours. Then 

 pour the hot solution into a high cylinder and allow it to cool slowly until the 

 cloud has fallen. After the solution has cooled, cut off the clear upper portion 

 and put it in a glass jar. Place the jar in a basin of water and heat it until the 

 agar-agar is melted. Then add formalin in the proportion of 1 part of formalin 

 to 9 parts by volume of the melted agar-agar. 



The 5 per cent, solution is made in the same way as the 2 per cent., only 25 

 grams of agar-agar to 500 c. c. of distilled water are taken. Formalin should be 

 added in the same manner and proportions as in the 2 per cent, solution. The 

 5 per cent, solution when melted is quite fluid, but when cold it is about the con- 

 sistency of new cheese. It becomes much firmer on the blocks after exposure 

 to the action of strong alcohol. Large quantities of the agar-agar solution can 

 be prepared and preserved in air tight vessels to prevent evaporation. 



