Lamellate Structures in the Nucleolus of the Cellular 
Slime mold Acrasis rosea 1 
Hans R. Hohl and Susan T. Hamamoto 2 
During a study on the morphogenesis of 
Acrasis rosea , a cellular slime mold, we have 
encountered lamellar structures appearing as 
part of the nucleolus of spores and stalk cells 
in the fruiting body. This report describes in 
detail these structures, their occurrence, and 
their possible nature. 
materials and methods 
Acrasis rosea originally obtained from Dr. 
A. Kahn, Syracuse University, was grown on 
Difco cornmeal-dextrose agar supplemented 
with 0.1% yeast extract (Weitzman, 1962) 
with Rhodotorula mucilaginosa as food orga- 
nism. Myxamoebae and fruiting bodies were 
most successfully prepared for electron micros- 
copy by one of the following two methods: 
(1) Fixation for 1 hour in osmium vapor, 
followed by 1 hour in 2.5% phosphate-buffered 
glutaraldehyde at pH 7.3, and post-fixation for 
1 hour in 1% phosphate-buffered osmium 
tetroxide at pH 7.3. (2) Fixation for l/ 2 hour 
in Karnovsky’s (1965) cacodylate-buffered 
glutaraldehyde-paraformaldehyde at pH 7.0, 
diluted to % (personal communication from 
Dr. S. Ito, Harvard University), followed by 
fixation in 1% Palade’s osmium tetroxide at 
pH 7.3 for l/ 2 hour. For both methods dehydra- 
tion was carried out in ethyl alcohol followed by 
propylene oxide, and the specimen was then 
embedded in epon. In some cases the tissue was 
incubated in 0.5% uranyl acetate in 70% ethyl 
alcohol for l/ 2 hour during dehydration. Sec- 
tions were cut on an LKB ultramicrotome, post- 
stained in lead citrate and viewed in a Hitachi 
HU-11 A electron microscope. 
1 This research was supported by a research grant 
(GM 11758-03) from the National Institutes of 
Health of the U. S. Public Health Service. 
2 Pacific Biomedical Research Center and Depart- 
ment of Microbiology, University of Hawaii, Hono- 
lulu, Hawaii 96822. Manuscript received May 23, 
1967. 
For demonstration of RNA, glutaraldehyde- 
paraformaldehyde fixed (% hour, 4°C, pH 6.0) 
samples were treated with 0.7 mg/ml RNAase 
(heated for 10 minutes at 90°C prior to use). 
After incubation, the cells were washed with 
5% trichloracetic acid for 30 minutes at 0°C, 
postfixed in osmium tetroxide, and embedded as 
described above. 
For histochemical purposes at the light- 
microscope level the cells were fixed for 10 
minutes in acetone at 4°C. The cells were 
stained for (1) RNA using pyronin Y accord- 
ing to Kurnick (1955), for (2) DNA and 
RNA with acridine orange followed by fluores- 
cence microscopy, and for (3) protein with 
mercury bromphenol blue following the proce- 
dure outlined by Pearse (I960). Controls were 
treated with DNAase or RNAase (Loh and 
Soergel, 1965) before staining. 
RESULTS 
The nucleolus of the myxamoebae of Acrasis 
rosea appears to be built from three morpho- 
logically distinct components. The most striking, 
and the one of particular interest for our dis- 
cussion, consists of a series of roundish masses 
of granular material (Figs. 1 and 2). These 
masses are either dispersed as separate units 
within the nucleus (Fig. 2) or condensed intc 
large aggregates (Fig. 3). Each mass is made 
up of a large number of electron dense granules 
which stain heavily with lead citrate and are 
comparable in size to cytoplasmic ribosomes. 
The granules are more densely packed at the 
periphery of each clump, the central part of 
which then appears as a light core revealing a 
somewhat fibrous matrix. 
The second component has not been observed 
frequently. It appears as an intensely stained, 
homogeneous body containing electron trans- 
parent cavities and is always situated within the 
first component (Figs. 1 and 5). The third 
component of the nucleolus consists of a large 
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