DS 23.30 



DYE STAINS OF SPECIAL APPLICATION 



499 



23.3 Other Parasites and 

 Commensals 



The techniques here given are not only 

 those which are specificall}- intended for 

 the differentiation of parasite from host in 

 sections, but inchide also those techniques 

 which have been developed for the stain- 

 ing of specific parasites, and which cannot 

 reasonably be employed for the staining of 

 any free-living organism. The four classes 

 which have been set up are obviously the 

 only four into which parasites can be di- 

 vided. The techniques which are given in 

 each of the four classes are, without excep- 

 tion, so specific that their individual ap- 

 plication is given under each, 



23.30 TYPICAL EXAMPLES 



Demonstration of myceUa of Peni- 



cillium in orange rind using the 



thionin-hght green-orange G-eryth- 



rosin stain of Margolena 1932 



The method here described is one of the 

 easiest and most certain methods of dem- 

 onstrating the penetration of the myceUa 

 of parasitic fungi through the tissues of 

 their plant hosts, though the example se- 

 lected for demonstration is quite possibly 

 an example of a saprophytic, rather than a 

 parasitic, fungus. This material, however, 

 has the advantage that it may be procured 

 without the slightest difficulty, if it is re- 

 quired for class-demonstration purposes, 

 and also that it permits a clear definition 

 of the invading myceha. 



To secure a growth of Penicilhum on 

 orange rind it is only necessary to take an 

 orange and to remove the wax, with which 

 the marketer has protected and polished 

 it, by rubbing any very fine form of grit 

 gently onto the surface with the fingers. 

 One of the most readily available forms of 

 grit are the scouring powders sold for the 

 cleaning of domestic utensils. If an orange 

 be scrubbed with some of this scouring 

 powder, it will not appreciably damage 

 the surface, but will roughen it sufficienth' 

 to permit the spores of Penicilhum, which 

 are always in the air, to fight directly on 

 the damaged surface. The orange should 

 then be placed under a bell jar on top of 



moist blotting paper and kept at about 

 85°F. After a few days, a vigorous growth 

 of Penicilhum will be seen on the surface, 

 and a piece of about j'i-inch side should 

 be cut from the rind and dro])ped into any 

 of the alcohol-acetic-formaklehj^do mix- 

 tures (Chapter IS, F 0000.1010) which 

 may be available. It should remain in this 

 fixative for about three days and then be 

 placed in absolute alcohol in which it 

 should remain, with frequent changes, 

 until such time as the oil has ceased to 

 leave it. The piece should then be em- 

 bedded in paraffin (Chapter 12) and cut 

 into sections of from ten to twelve microns 

 in thickness. These sections are mounted 

 on a slide, deparaffinized, and brought 

 down to water. It is also possible, though 

 not so convenient, to use this same tech- 

 nique on unmounted sections cut on a 

 hand microtome. 



The staining solutions required are: 

 first, a 0.1% solution of thionin in 5% 

 phenol; second, a 0.5% solution of hght 

 green in 95% alcohol; third, a mixture in 

 the proportion of 1:2 of a saturated al- 

 cohohc solution of orange G with a satu- 

 rated solution of erythrosin in clove oil. 



The sections are taken from water and 

 placed in the thionin solution for one hour. 

 They are then rinsed in water to remove 

 the excess thionin, or, if many sections are 

 being dealt with, accumulated in water 

 while the next stage is passed through. 

 The sections are then dehydrated before 

 being dipped up and down in the light 

 green until the color changes from blue to 

 green. They may then be returned to 

 water until no more color comes away. 

 All the sections are then taken together 

 and passed to absolute alcohol, in which 

 they remain until they are dehydrated. 

 Several changes of absolute alcohol may 

 be required, but graded alcohols cannot 

 be used because they would extract the 

 stain. The alcohol-clove-oil stain mixture 

 is then dropped, from a drop bottle, on 

 each individual slide and allowed to act 

 for from one to two minutes. The excess 

 stain is then washed off the shde with 

 fresh clove oil, the clove oil removed with 

 xylene, and the section mounted in 

 balsam. 



