Waksman — 48 — Actinomycetes 



fungi and bacteria, certain special methods have also found application. 

 Among these, it is sufficient to mention the following: 



1. Method of Henrici.—A drop of melted agar medium is placed on a slide, 

 allowed to cool somewhat, and inoculated with the actinomyces culture. The 

 agar is then spread in a thin film on the slide. The agar may also he allowed 

 to cool first before being inoculated with a sharp needle. The slide is then 

 incubated in a sterile moist chamber. After growth has taken place, the slides 

 are allowed to dry, are fixed in alcohol, and stained. The entire colony, with both 

 vegetative and aerial mycelium can thus be stained and examined in an undis- 

 turbed condition. 



2. Method of Drechsler.— "The culture is grown on a synthetic medium, and 

 the fully developed colony is cut from the agar as carefully as possible. A slide 

 smeared with albumin fixative is brought into firm contact with the surface 

 mycelium of the colony, then separated from it, precautions being taken to avoid 

 any sliding of the two surfaces on each other. If the growth is not too young, 

 the upper portions of the aerial mycelium will be left adhering to the slide without 

 much disarrangement. The adhered growth is then killed and fixed at once, and 

 the preparation is stained and mounted in balsam. Preparations in which the 

 spore chains have commenced to disintegrate are impaired by the large masses of 

 free spores. The most convenient fixative agent is 95 per cent alcohol. As a 

 stain, Haidenhain's iron-alum haemotoxylin is good for protoplasmic structures. 

 Delafield's haemotoxylin, allowed to act for 24 hours with the proper degree of 

 decolorization, yields deeply stained, clear preparations showing distinctly the 

 various mycelial structures of the organism. 



3. Other methods.— Various special methods have been utilized for preparing 

 actinomyces cultures for staining. It is sufficient to mention the use of a drop of 

 liquid synthetic medium placed on a cover slip, which is then inoculated with a 

 few actinomyces spores and incubated in a sterile moist chamber or in the form 

 of a hanging drop preparation. The liquid medium may also be allowed to flow 

 around an actinomyces colony, which has been removed from an agar plate 

 and placed on a cover slip; the peripheral growth may be stained. 



All actinomycetes are gram-positive, although certain thermophilic 

 forms, according to Lieske, may be gram-negative at temperatures above 

 50°C. 



Most actinomycetes are non-acid-fast. Some of the Nocardia species, 

 however, especially many of the pathogenic forms, are acid-fast (145, 

 432). 



The mycelium stains uniformly, except in older cultures. The 

 presence of metachromatic granules has been observed by Brussov (49) 

 and Drechsler (97); this was believed by some investigators to indi- 

 cate that the cultures possess the property of pleomoi-phism. The gran- 

 ules in the mycelium can be readily stained with methylene blue (1:1,- 

 000) and decolorized by sulfuric acid. Droplets of fat, often pig- 

 mented, can be seen frequendy in the mycelium (242). The forma- 

 tion of vacuoles has also been reported (97). Neukirch (321) dif- 

 ferentiated the ectoplasm of the actinomycetes from the endoplasm, on 

 the basis of staining with dilute methylene blue, the first being dark 

 blue and the second light blue. 



The presence of nuclei in actinomycetes has aroused considerable 



