18
NUCLEIC ACIDS OF EIMERIA TENELLA
[Vol. XXV, Part I,
at ±4°C. for 2 hours) as recommended by Kurnick [1950 b]. They were washed
wherever necessary (not the alcohol fixed ones which were given only a couple of
changes in 95 per cent alcohol) dehydrated, cleared in toluol and embedded for
45 minutes only in tissue mat (Fishcer & Co.). Temperature of the paraffin bath
was kept at 52-53°C. to avoid depolymerisation of DNA. Sections were cut 4-6 µ
thick.
For the demonstration of DNA, Feulgen technique [Cowdry, 1928], and to test
the veracity of this reaction method of Oster and Mulinos [1944] was adopted. The
latter procedure confirmed the ' truth' of Feulgen reaction. In Feulgen's technique
by missing acid hydrolysis, it was shown that the performed aldehydes were absent
in the parasite and the host cells as well. Preparations made from the variously
fixed tissues gave comparable results except in those that were fixed in formalin
after the method of Kurnick. Here, DNA in nuclei of both host cells and parasites
appeared as a solid homogeneous mass [ thus confirming the observations of Kurnick
(loc. cit.) on the nuclei of chick red blood corpuscles and mouse liver cells ]. Al-
cohol fixed tissues were stained with Unna-Pappenheim's stain [ after Brachet,
1953 ] to distinguish between the state of polymerisation of DNA, and also for the
location of RNA. Tissues fixed in alcohol were treated with ribonuclease [ Kunitz,
1950] and subsequently stained with Unna-Pappenheim's stain. Comparison of
preparations stained in this manner with similarly stained counterparts but not
submitted to enzyme digestion provided confirmation of the results obtained by
staining with pyronin. After enzyme digestion both the parasite and host cells
lost all affinity for pyronin, except where it has been indicated otherwise in the
text of this article, proving the reliability of pyronin to stain nucleic acids of
low polymerisation. As the specificity of Feulgen's technique has now been
universally accepted [ Oster and Mulinos, 1946 ], confirmation of observations thus
made after digestion with desoxyribosenuclease was found unnecessary.
NOTE.—As different methods give different reactions, a description in respect of colour sequences of
various structures of the parasite (and also the host cell) would create confusion, a procedure of giving
the net significance of each staining reaction regarding the state of polymerisation of nucleic acid has
been adopted here. Concepts which have led to these deductions need a brief mention here. Feul-
gen's reaction indicates the presence of DNA only in tissues without reference to the state of poly-
merisation. Latter is a function of the two dyes: (1) methyl-green, and (2) pyronin. The former
preferentially stains nucleic acid of high polymerisation (i.e. a high polymer of DNA), and the latter
dye has affinity for nucleic acid of low polymerisation (i.e. RNA ; and also a low polymer or depoly-
merised DNA Kurnick, 1950). The reader is specially referred to works of Pollister and Leuchten-
berger [1949], Kurnick [1949-50; 1950 a, 1952], Kurnick and Foster [1950], and Devereux, et al.
[1951] in support of these remarks.
OBSERVATIONS
A low polymer of DNA (Feulgen positive methyl-green negative but pyronin
positive after ribonuclease digestion) occurred in nuclei of both the schizogonous
cycles (Plate II, Fig. 1, 2). However, its pattern of distribution differed in schizonts
and merozoites. In the former nuclei were solid mass of DNA, and in the latter
the acid was distributed sparsely in the peripheral chromatin and roundabout the
karyosome which remained ' unstained', i.e. negative for DNA. Sporozoites en-
countered both in the lumen and epithelial cells always revealed the presence of a
low polymer of DNA in the peripheral chromatin and roundabout the karyosome.
Karyosome of the first generation schizont gave moderately positive reaction for