DIFFERENTIAL STAINING OF BLOOD FILMS 87 
require a strong stain with a mordant and once the cell is stained it is 
decolorized with great difficulty. Different reasons such as a thick 
membrane about the cell or peculiar structure of the cell contents, 
have been given to explain the acid fast staining procedure. Klebs 
(1896) has shown that the acid fast properties are lost if the tubercle 
bacillus is extracted with ether. Others (Bienstock, 1886) have stated 
that by increasing the fat content of the cell it may be made to 
take on acid fast properties. Miller (1916) grew tubercle bacilli in 
sperm oil and attributed the variations in the staining properties with 
carbol fuchsin to the production of free oleic acid in the interior of 
the rod. This acid was believed to be formed by the spores or round 
granules in the cell. Bulloch (1904) when studying the acid fast 
properties of bacteria isolated a wax and other substances in the nature 
of fats from acid fast bacteria. The other constituents of the cells 
were not acid fast. Baumgarten (1911) thought that unsaturated 
fatty acids were present in the acid fast bacteria which united with the 
dye. Benians (1912) claimed that the cell wall was the important 
factor in retaining the dye. He states “for the present the evidence 
points to the existence of a waxy layer enclosing a protoplasmic and 
fatty cell substance and conferring on the organism the property of 
resisting the penetration of acid and alcohol.” Aronson (1910) claims 
that the peculiar staining properties of the tubercle bacillus are due 
to the fat content and not to any peculiar kind of protoplasm. This 
waxy substance may be extracted by trichlorethylen. 
Ziehl-Nielsen Method of Acid Fast Staining. Flood the smear 
with the stain and heat for ten minutes. Decolorize in 25 per cent 
mineral acid and wash. Counterstain if desired with eosin or Bismarck 
brown. The staining solution is prepared as follows: 
Basic fuchsin... 0.0... cece eee ees 1 part 
Absolute alcohol... ............0.02 2-000 .. 10 parts 
Carbolic acid (1 to 20)...... 0.0.02... 00 eee 100 parts 
Wrighi’s Method for the Differential Staining of Blood Films and 
Malarial Parasites. The method of preparing the stain and applying 
it is outlined by Wright (1902) as follows: 
I. Preparation of the Staining Fluid. Make a 0.5 per cent solution 
of sodium bicarbonate in an Erlenmeyer flask and add to it 1.0 c.c. of 
methylene blue. Grubler’s BX or Koch’s or Erlich’s rectified methy- 
lene blue may be used. The sodium bicarbonate should be entirely 
dissolved before the methylene blue is added. The mixture should be 
