Composition and method for staining cellular DNA, comprising thiazine derivative metabisulfite and methanol or ethanol

A composition and method for staining cellular DNA are disclosed. The composition of the present invention is an aqueous alcoholic solution that includes a cationic stain, a metabisulfite, and an alcohol preferably selected from methanol, ethanol, and mixtures thereof. In a preferred embodiment, the cationic stain is thionin.

FIELD OF THE INVENTION 
The present invention generally relates to a composition and method for 
staining cellular DNA, and more particularly, to a composition that 
includes a cationic stain and methods for its use. 
BACKGROUND OF THE INVENTION 
Image cytometry has been increasingly used in cytopathology and 
histopathology to detect aneuploid cell populations within preneoplastic 
or neoplastic lesions. DNA image cytometry has gained wide acceptance in 
pathology and cytopathology as a means to obtain diagnostic and prognostic 
information for human cancer. Such diagnoses require the accurate 
determination of cellular DNA content. Accordingly, various methods for 
quantitatively staining nuclear DNA have been developed. The 
reproducibility and reliability of several quantitative DNA stains has 
been recently reported. "A Comparative Study of Quantitative Stains for 
DNA in Image Cytometry," Mickel, U. V. and Becker, Jr., R. L., Analytical 
and Quantitative Cytology and Histology 1991, 13:253-260. 
Included among the various techniques for staining cellular DNA are Feulgen 
staining techniques. Feulgen, R. and Voit K., Z. Physiol. Chem. 1924, 
136:57-61. Feulgen discovered that hydrolysis of fixed tissues (i.e., DNA 
hydrolysis) exposed the deoxypentose present in cell nuclei in an aldehyde 
form. Subsequently, Feulgen found that the mild acid hydrolysis of 
cellular DNA followed by the addition of a Schiff reagent provided a 
reddish-purple color to DNA-containing structures. The Feulgen technique 
continues to be practiced as a method for quantitating cellular DNA and 
generally involves the steps of oxidizing nucleic acids to provide 
aldehydes, and reacting these aldehydes with a Schiff reagent to form a 
purple-red color indicative of the presence of DNA. Because the reaction 
is stoichiometric, the intensity of the color is directly related to the 
amount of DNA in the sample, provided that excess reagents are washed out. 
In 1954, a thionin-sulfite reagent was found to exclusively stain nuclei of 
hydrolyzed sections of mouse kidney and liver cell nuclei. Van Duijn, P., 
J. Histochem. Cytochem. 1956, 4:55-63. The aldehyde reagent contained 
thionin and sulfur dioxide in a medium of t-butanol and water. This 
thionin reagent was prepared by acidifying a solution of thionin in 
aqueous t-butanol (water:t-butanol, 1:1) with aqueous hydrochloric acid 
followed by the addition of sodium metabisulfite. Van Duijn concluded from 
the histochemical data that the thionin-sulfite/t-butanol reagent contains 
one or more components that react with aldehydes, although the exact 
chemical nature of the reaction between thionin and sulfur dioxide and the 
nucleic acids was unclear. The use of Feulgen staining methods has 
continued to present, and various compounds have been used as Feulgen 
stains. Thionin, generally regarded as a nuclear stain, is one of the more 
commonly used stains in the Feulgen procedure. 
Despite the many years that have passed since the original report of the 
thionin-Feulgen stain reagent, the composition of the reagent has remained 
unchanged (i.e., a solution of thionin and sodium metabisulfite in aqueous 
t-butanol adjusted to about pH 1.5 with aqueous hydrochloric acid). 
Although the Feulgen thionin staining method developed in the 1950s does 
facilitate quantitative DNA measurements, the use of t-butanol as a 
solvent for the traditional thionin staining reagent is not without its 
drawbacks. First, t-butanol is an irritating substance and presents a work 
hazard. Second, because t-butanol has a melting point of about 25.degree. 
C., it is often a solid at room temperature and requires heating and 
melting so that it may be dispensed as a liquid in the preparation of the 
reagent. Unlike most other low molecular weight alcohols, t-butanol is 
difficult to dispose of properly. Because of its hazardous nature, there 
are also shipping restrictions associated with t-butanol. In contrast to 
other simple alcohols, t-butanol is extremely expensive (e.g., $45 per 
liter compared to methanol or ethanol at about $2 per liter). Perhaps most 
importantly, the useful shelf life of thionin/t-butanol staining solutions 
is about two days. Such a short shelf life precludes the storage and 
therefore commercial utility of such a reagent, and requires one who 
wishes to use the thionin reagent in a Feulgen staining method to prepare 
the reagent immediately prior to use. 
Accordingly, there exists a need for a thionin-based reagent that offers 
the advantages of cellular DNA quantitation afforded by the traditional 
thionin/t-butanol staining reagent without the accompanying disadvantages 
associated with t-butanol, a key component of the reagent. More 
specifically, there exists a need for a thionin staining reagent that has 
a long and stable shelf life. The present invention seeks to fulfill these 
needs and provides further related advantages. 
SUMMARY OF THE INVENTION 
In one aspect, the present invention provides a composition that is useful 
for staining cellular DNA. Generally, the composition is an aqueous 
alcoholic solution that includes a cationic stain having a formula: 
##STR1## 
where R.sub.1 and R.sub.2 are independently selected from hydrogen and 
methyl, and X is a counterion; a metabisulfite; and an alcohol selected 
from either methanol ethanol, and mixtures of methanol and ethanol. In a 
preferred embodiment, the cationic stain is thionin acetate (i.e., R.sub.1 
and R.sub.2 are hydrogen and X is acetate). 
In another aspect of the present invention, a method for staining cellular 
DNA is provided. In the method, the composition noted above is applied to 
a cell to provide a cell having a stained nucleus. The present invention 
also provides a method for quantitating cellular DNA that includes the 
steps of staining the cell with the composition noted above to provide a 
cell having a stained nucleus, and then measuring the optical density of 
the stain nucleus to determine the presence or amount of DNA present in 
the nucleus. 
In still another aspect, the present invention provides a kit for staining 
cellular DNA that includes the composition noted above. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The present invention generally relates to a composition and method for 
staining cellular DNA. More specifically, the present invention provides a 
composition that is useful as a reagent in a Feulgen staining method and 
useful in both staining and determining the presence or amount of cellular 
DNA. The composition of the present invention is an aqueous alcoholic 
solution that includes a cationic dye, a metabisulfite, and an alcohol 
preferably selected from methanol, ethanol, and mixtures of ethanol and 
methanol. 
The cationic stain useful in the composition of the present invention is a 
phenothiazine derivative, and more specifically, a 
3,7-diamino-5-phenothiazine derivative. The cationic stain is represented 
by the following formula: 
##STR2## 
where R.sub.1 and R.sub.2 are either hydrogen, methyl, or combinations of 
hydrogen and methyl, and X is a counterion. While the cationic stain 
useful in the composition of the present invention is represented by the 
formula above, it will be appreciated that the cationic stain may also be 
represented by other equivalent formulas including: 
##STR3## 
Suitable cationic stains include Azure A (R.sub.1 and R.sub.2 are methyl) 
and Azure C (R.sub.1 is methyl and R.sub.2 is hydrogen). In a preferred 
embodiment, the cationic stain is thionin (R.sub.1 and R.sub.2 are 
hydrogen). Suitable counterions X include anions that do not adversely 
affect the performance of the cationic stain in the staining procedure and 
include acetate, chloride, and bromide. In a preferred embodiment, the 
counterion is acetate. In a particularly preferred embodiment, the 
cationic stain is thionin acetate (R.sub.1 and R.sub.2 are hydrogen and X 
is acetate). 
The cationic stain is present in the composition at a concentration from 
about 1.0.times.10.sup.-3 M to about 2.5.times.10.sup.-2 M, and preferably 
from about 4.0.times.10.sup.-3 M to about 8.0.times.10.sup.-3 M. 
In addition to the cationic stain, the composition of the present invention 
also preferably includes a metabisulfite. While embodiments of the present 
invention that include bisulfite are useful as staining solutions, it has 
been found that metabisulfite containing solutions provide the best 
staining results. Suitable metabisulfites do not adversely affect the 
staining performance of the composition of the present invention. Suitable 
metabisulfites include metal metabisulfites such as lithium, sodium, and 
potassium metabisulfites. Preferably, the composition includes either 
sodium or potassium metabisulfite. Preferably, the metabisulfite is 
present in the composition at a concentration from about 
1.0.times.10.sup.-3 M to about 2.5.times.10.sup.-2 M, and preferably from 
about 4.0.times.10.sup.-3 M to about 8.0.times.10.sup.-3 M. In contrast to 
Feulgen staining solutions known in the art that have a molar ratio of 
metabisulfite:thionin of 4-10:1, in the composition of the present 
invention, a molar metabisulfite:thionin ratio of 1:1 is preferred. 
The composition of the present invention includes a cationic stain and a 
metabisulfite in aqueous alcohol. The aqueous alcohol solution serves to 
solubilize the stain and metabisulfite as well as any active DNA staining 
components formed in the solution. Therefore, the alcohol is preferably 
miscible with water and, for ease of reagent preparation, a liquid at room 
temperature. Alcohols useful in the composition include C1 to C3 alcohols 
(i.e., methanol, ethanol, n-propanol and isopropanol), and mixtures of 
these alcohols. Preferred alcohols include methanol, ethanol, and mixtures 
of methanol and ethanol. Preferably, the aqueous alcohol solution contains 
from about 20 to about 60 percent alcohol, and more preferably, about 40 
percent alcohol. The composition of the present invention is preferably an 
aqueous acidic solution having a pH of from about 1.0 to about 2.0, and 
more preferably, from about 1.3 to about 1.5. While the pH of the 
composition can be adjusted with any one of a number of acids, the pH of 
the solution is preferably adjusted with aqueous hydrochloric acid. 
The preparation of a representative composition of the present invention, a 
thionin/methanol staining solution, is described in Example 1. 
Generally, the reaction of the above-noted cationic stain with 
metabisulfite results in the formation of an active ingredient that binds 
to hydrolyzed DNA. Thus, the composition of the present invention is 
useful for staining cellular DNA. Accordingly, in another aspect, the 
present invention provides a method for staining cellular DNA. In the 
method, the composition described above is applied to cells to provide 
cells having stained nuclei. Basic procedures, such as cell fixation, 
dehydration, clearing, staining, and mounting, as well as specific 
staining methods, including Feulgen techniques, nucleic acid staining, and 
the staining of cellular elements, are well known to those of skill in the 
art. Basic cytological and histochemical techniques are described in 
Histological and Histochemical Methods: Theory and Practice, 2nd Ed., J. 
A. Kierenan, ed., Pergamon Press, New York, 1990, and Animal Tissue 
Techniques 4th Ed., Gretchen L. Humason, W.H. Freeman & Company, San 
Francisco, 1979, both expressly incorporated herein by reference. 
As noted above, the method of the present invention includes applying the 
composition described above to cells. The composition and method of the 
present invention are useful in staining virtually any cell having a 
nucleus including, for example, epithelial, muscle, nerve, and connective 
tissue cells. Any one of a number of cell preparations may be used in the 
method, for example, preparations of cells of interest include 
conventional smears, histological preparations, and monolayer 
preparations. Generally, cells are deposited on a microscope slide for 
staining. After deposit on the slide, the cells are fixed by immersing the 
slide in a fixative solution and then rinsed. Alternatively, fixed cells 
may also be deposited on the slide for staining. After rinsing, the 
cellular DNA is then hydrolyzed by immersing the slides in an acidic 
solution. Following another rinse, the cells are stained by immersing the 
slides in a staining solution. After additional rinses, the cells are then 
dehydrated by immersion in ethanol solutions, followed by clearing in 
xylene. At this point, the staining results may be visualized by any one 
of a number of techniques including manual and automated techniques 
including, for example, microscopic and image cytometric techniques. A 
detailed method for a representative staining procedure that includes the 
method of the present invention is described in Example 1. Although the 
staining procedure may be a manual procedure, the method of the present 
invention may also be incorporated into an automated staining procedure. 
Because the staining method of the present invention is a quantitative 
method for staining cellular DNA, the combination of the steps of staining 
cells with the staining solution of the present invention and measuring 
the optical density of the resulting stained nuclei permits the 
determination of the presence or amount of DNA present in a cell's 
nucleus. Thus, in another aspect, the present invention provides a method 
for quantitating cellular DNA that includes the steps of staining the cell 
with the composition described above, and then measuring the optical 
density of the stained nuclei to determine either the presence or the 
amount of DNA present in the nucleus. The optical densities of stained 
nuclei may be measured by manual and automated techniques including, for 
example, optical scanners, microscopes, and other image cytometers. 
Suitable instruments for measuring the optical densities of stained cells 
are known in the art and include those described in "The Design and 
Development of an Optical Scanner for Cell Biology," Jaggi, B. and Palcic, 
B., IEEE Proc. Eng. Med. Biol. 1985; 2:980-985; "Cell Recognition 
Algorithms for the Cell Analyzer," Jaggi, B. and Palcic B., IEEE Proc. 
Eng. Med. Biol. 1987; 4:1454-1456; and "Design of a Solid State 
Microscope," Jaggi, B., Deen, M. J., and Palcic B., Opt. Engineer 1989; 
28(6):7675-682, each expressly incorporated herein by reference. 
The methods of the present invention are useful in techniques in which 
stained cells are digitally imaged and the resulting digital intensity 
images manipulated by computer to perform a variety of cell feature 
measurements. Cellular features, such as the size, shape, and DNA content 
of the nucleus, and other features that describe the spatial distribution 
of chromatin within the nucleus, can be calculated. Features that describe 
the chromatin distribution are collectively referred to as texture 
features, and texture features can be selected for their ability to 
differentiate between the various descriptive classes of chromatin 
patterns and to further provide quantitative information regarding the 
extent of changes in chromatin patterns associated with certain diseases 
including, for example, malignancy. Thus, the composition and methods of 
the present invention are useful in nuclear texture measurements, and 
consequently the diagnosis and prognosis of certain diseases. Nuclear 
texture measurements in image cytometry and their utility in the diagnosis 
and prognosis of human cancer are described in A. Doudkine, C. MacAulay, 
N. Poulin, and B. Palcic, "Nuclear Texture Measurements in Image 
Cytometry," Pathologica 1995; 87:286-299, expressly incorporated herein by 
reference. 
The integrated optical density (IOD), a nuclear feature measurement, for 
cells stained by representative staining solutions of the present 
invention and the traditional thionin/t-butanol stain are presented in 
Example 3. 
As noted above, one of the most significant drawbacks to the traditional 
thionin/t-butanol Feulgen staining technique is the short shelf life of 
the t-butanol-based staining solution. In contrast, the methanol- and 
ethanol-based compositions of the present invention have considerable and 
significantly longer shelf life. A comparison of the staining performance 
of representative compositions of the present invention (i.e., 
thionin/methanol and thionin/ethanol staining solutions) and the 
traditional Feulgen staining reagent (i.e., thionin/t-butanol staining 
solution) is described in Example 2. The results summarized in Tables 1 
and 2 clearly demonstrate that (1) the composition of the present 
invention provides cellular DNA staining comparable to the standard 
thionin/t-butanol staining solution, and (2) the ability of the 
composition of the present invention to effectively stain cellular DNA 
remains essentially constant over a period of about five weeks. In 
contrast, the conventional thionin/t-butanol staining solution loses about 
40 percent of its staining capacity in only four days. The enhanced 
stability of the composition of the present invention compared to the 
conventional staining solution offers significant practical and commercial 
advantages. For example, while the conventional staining solution must be 
freshly prepared immediately prior to staining by the practitioner or 
clinician, the composition of the present invention, by virtue of their 
stability and long shelf life, may be prepared, stored, and used 
repeatedly over a relatively long period of time. 
The utility and success of the composition and method of the present 
invention in quantitatively staining intracellular DNA is noteworthy in 
view of the well established sensitivity of quantitative DNA methods to 
various chemical treatments. Quantitative DNA measurements are known to be 
highly sensitive to, for example, cell fixation methods. Fixation methods, 
and more specifically the reagents and solvents used in a particular 
method, have been shown to dramatically affect intracellular DNA 
quantitation. The variation in the amount of cellular DNA available for 
staining has been suggested as one reason for the variation observed in 
certain methods. Within a cell, DNA is associated with protein. Cell 
fixation serves, in part, to denature the associated protein and to expose 
the DNA thus making the DNA available for staining. Fixation methods 
typically employ solvents, including alcohols, to effect protein 
denaturation. Generally, the extent to which the associated protein is 
denatured, and the extent to which DNA is made available for measurement, 
is highly dependent on the reagents and methods employed. For instance, in 
formalin fixation methods, the concentration of alcohol in the fixation 
reagent has been found to have a critical effect on protein denaturation 
and, consequently, the quantitative measurement of intracellular DNA. In 
general, the effect of a specific solvent or reagent on a quantitative DNA 
method is largely unpredictable and may typically be determined 
empirically through experimentation. 
Despite the well-known sensitivity of quantitative DNA methods to various 
reagents and solvents, the composition and method of the present invention 
have been found to be effective in quantitating intracellular DNA. As 
demonstrated by the staining results presented in Examples 2 and 3, the 
present composition is as effective in staining intracellular DNA as the 
standard t-butanol-based composition. Furthermore, the present composition 
provides a staining reagent that has a stable shelf life considerably 
greater than the standard staining composition. 
Accordingly, in another aspect, the present invention provides a kit for 
staining cellular DNA. The kit of the present invention includes the 
composition described above. Generally, composition components are 
contained in one or more vessels in the kit. For example, in one 
embodiment, the kit includes a staining reagent that incorporates all of 
the components of the composition of the present invention in a single 
solution (i.e., an aqueous alcoholic solution of a cationic dye and a 
metabisulfite adjusted to about pH 1.5). Alternatively, in another 
embodiment, the kit includes a staining reagent in dry (i.e., powdered) 
form. In such an embodiment, the kit can contain solid cationic stain in 
one vessel and solid metabisulfite in a second vessel. For this 
embodiment, the liquid components of the composition are added to the dry 
reagents which are then combined to provide the staining solution of the 
invention. In yet another embodiment, the kit can include a combination of 
solid cationic stain and metabisulfite in a single vessel. 
In addition to the staining reagent, the kit also includes a rinse reagent 
(i.e., an aqueous acidic solution of metabisulfite). As noted above for 
the staining reagent, the rinse reagent may be provided as a solid or in 
solution. The kit can optionally include unstained, cultured cells on 
microscope slides (control slides) to, for example, monitor quality 
control of the staining procedure. The kit can also include an instruction 
booklet that provides directions for the staining procedure and, for 
applicable embodiments, directions for preparing the staining solution 
from the kit's reagents. 
The following examples are provided for the purposes of illustration, and 
not limitation.

EXAMPLES 
Example 1 
Representative Procedure for Cellular DNA Staining 
In this example, a representative procedure for staining cellular DNA with 
alcohol solutions of thionin is described. The reagents used in the DNA 
staining procedure, including methanol and t-butanol solutions of thionin, 
and fixative and rinse solutions, were prepared as described below. 
Stain Reagent Preparations: 
A. Representative Staining Solution of Present Invention 
Thionin/methanol Staining Solution 
1. Add 0.5 g thionin (Aldrich Chemical Co., Milwaukee, Wis.) and 0.5 g 
sodium metabisulfite to a 500 ml glass bottle with a stirring bar. 
2. Add 200 ml methanol. Mix well. 
3. Add 250 ml distilled water. 
4. Add 50 ml 1N hydrochloric acid and cap the bottle. 
5. Stir stain solution for one hour. Protect solution from light by 
wrapping the bottle with aluminum foil. Do not refrigerate. 
6. Filter stain solution through filter paper (No. 1 grade) in a fume hood 
immediately prior to use. 
B. Conventional Feulgen Staining Solution 
Thionin/t-butanol Staining Solution 
1. Add 0.5 g thionin to 435 ml distilled water in a 2000 ml Erlenmeyer 
flask. 
2. Heat solution to boiling for about 5 minutes and then allow to cool to 
about room temperature. 
3. Add 435 ml t-butanol. (If necessary, melt the t-butanol in a waterbath. 
The melting point of t-butanol is 25-26.degree. C. and therefore is a 
solid at temperatures below about 25.degree. C.). 
4. Add 130 ml 1N aqueous hydrochloric acid. 
5. Add 8.7 g sodium metabisulfite. 
6. Add stirring bar and seal container with Parafilm M. 
7. Stir stain solution for at least 1 hour. Protect from light and do not 
refrigerate. 
8. Filter stain solution through filter paper (No. 1 grade) in a fume hood 
just prior to use. 
Other Reagent Preparations: 
Bohm-Sprenger Fixative 
1. Combine 320 ml methanol and 60 ml aqueous formaldehyde (37%) in a 500 ml 
glass bottle. 
2. Add 20 ml glacial acetic acid. 
3. Mix well and seal with Parafilm M. 
Rinse Solution 
1. Dissolve 7.5 g sodium metabisulfite in 1425 ml distilled water in a 2000 
ml Erlenmeyer flask. 
2. Add 75 ml 1N aqueous hydrochloric acid. 
3. Add stirring bar and stir until dissolved. Seal flask with Parafilm M. 
1% Acid Alcohol 
1. Mix 280 ml of absolute ethanol and 120 ml distilled water. 
2. Add 4 ml concentrated hydrochloric acid. 
3. Mix well. 
The reagents prepared as described above were then used to stain cellular 
DNA by the following method. Preparations of cells of interest (e.g., 
cells from uterine cervix samples), including conventional smears and 
monolayer preparations, may be used in the method. In the method, cells 
are generally deposited on a microscope slide for staining. 
Staining Procedure: 
1. Deposit cells on a microscope slide. 
2. Fix cells by immersing slide in Bohm-Sprenger fixative: 30-60 minutes. 
3. Rinse slide in distilled water: 1 minute, agitate. 
4. Hydrolyze cellular DNA by immersing slide in 5N hydrochloric acid: 60 
minutes at room temperature. 
5. Rinse slides in distilled water: 15 dips, agitate. 
6. Stain cells by applying freshly filtered thionin stain solution: 75 
minutes. 
7. Wash slides in distilled water: 6 changes, 20 dips each. 
8. Rinse slides in freshly prepared rinse solution: 3 changes: 30 seconds 
for the first two rinses, 5 minutes for the last rinse. 
9. Rinse slides in distilled water: 3 changes, 20 dips each. 
10. For mucoidal samples only: 
Optionally rinse slides in 1% acid alcohol: 2 minutes. 
11. Rinse slides in distilled water: 3 changes, 20 dips each. 
12. Dehydrate cells by sequentially immersing the slides in 50%, 75% 
aqueous ethanol and two changes of 100% ethanol: 1 minute each. 
13. Clear slides by immersing in xylene: 5 minutes. 
14. Mount coverslips on slides. 
15. Identify slides with barcode labels if desired. 
Example 2 
Cellular DNA Staining Results: Performance Over Time 
This example compares the cellular DNA staining performance of 
thionin/methanol and thionin/t-butanol staining solutions over time. In 
the comparative study, thionin solutions were prepared in either methanol 
or t-butanol, as described in Example 1 above, and cellular preparations 
were stained and the optical densities of the stained nuclei measured. To 
determine the effect of time on the performance of each thionin staining 
solution, cellular stainings with each thionin solution were performed at 
regular time intervals (see Table 1 below) with the same stock staining 
solution. In effect, the comparative experiment determined the useful 
shelf life of each of the specific thionin staining reagents studied. 
Cellular preparations (HL-60 cells from American Type Culture Selection) 
were stained by the procedure described in Example 1 above. The integrated 
optical density (IOD) for each sample was determined by quantitative image 
cytometry using an optical scanner and by the methods described in 
"CytoSavant and Its Use in Automated Screening of Cervical Smears," 
Garner, D. M., Harrison, A., MacAulay, C., and Palcic, B., in Compendium 
on the Computerized Cytology and Histology Laboratory, Wied, G. L., 
Bartels, P. H., Rosenthal, D. L., and Schenck, U., eds., Tutorials of 
Cytology, Chicago, 1994; "The Design and Development of an Optical Scanner 
for Cell Biology," Jaggi, B. and Palcic, B., IEEE Proc. Eng. Med. Biol. 
1985; 2:980-985; "Cell Recognition Algorithms for the Cell Analyzer," 
Jaggi, B. and Palcic B., IEEE Proc. Eng. Med. Biol. 1987; 4:1454-1456; and 
"Design of a Solid State Microscope," Jaggi, B., Deen, M. J., and Palcic 
B., Opt. Engineer 1989; 28(6):7675-682, all expressly incorporated herein 
by reference. For each data point, three slides were stained followed by 
the measurement of their optical density. The performance (i.e., the 
capacity to stain cellular DNA and provide optically dense nuclei) of the 
two thionin staining solutions is summarized in Table 1 below. 
______________________________________ 
Thionin Solution 
Day t-Butanol Methanol Ethanol 
______________________________________ 
0 114.7 .+-. 4.2 
120.0 .+-. 2.0 
120.9 .+-. 1.6 
1 117.3 .+-. 3.5 
-- -- 
2 123.0 .+-. 5.7 
-- -- 
3 85.3 .+-. 3.1 
-- -- 
4 68.3 .+-. 4.7 
-- -- 
7 -- 119.7 .+-. 3.1 
116.2 .+-. 5.6 
14 -- 119.3 .+-. 2.1 
118.9 .+-. 1.2 
21 -- 117.7 .+-. 2.5 
113.3 .+-. 3.3 
28 -- 119.7 .+-. 2.1 
106.5 .+-. 1.8 
35 -- 112.0 .+-. 2.0 
114.4 .+-. 6.0 
______________________________________ 
As shown in Table 1, the performance of the t-butanol-based thionin 
staining solution decreases dramatically and rapidly over time. After only 
four days, the effectiveness of this solution, as measured by the optical 
density of cells stained, has decreased to about 60% of its original 
value. In contrast, the performance of the thionin/methanol and 
thionin/ethanol staining solutions remains essentially unchanged over a 
period of four weeks. These results demonstrate that methanolic and 
ethanolic solutions of thionin are significantly more stable than 
solutions of thionin in t-butanol. The results also demonstrate the 
effectiveness and comparability of methanol and ethanol as solvents for 
thionin staining, and their superiority to t-butanol as a thionin stain 
solvent. 
While the preferred embodiment of the invention has been illustrated and 
described, it will be appreciated that various changes can be made therein 
without departing from the spirit and scope of the invention.