STAIN TECHNOLOGY VOLUME 28 SEPTEMBER 1953 NUMBER 5

GOMORI'S HEMATOXYLIN AS A CHROMOSOME STAIN YNCVE htELANDER and KARL GEORC WINGSTRAND, institutes for

Genetics and Zoology resp., University of Lund, Sweden Received for publication Jan. 15, 1953

ABsTRAcT.-The chromic hematoxylin of Gomori (1941) can be used as an excellent chromosome stain after hydrolysis of the tissue in warm 1-N hydrochloric acid. The hydrolysis must be accurately timed for different material as in the case of the Feulgen reaction. The staining of sections can be performed at room temperature and requires about 15 minutes. For pieces of tissue and whole prepara- tions, it is recommended to stain at 60°C. for 40 minutes. Sections stained at room temperature can be differentiated in 1% hydro- chloric acid alcohol for one minute and can be counterstained with phloxine according to Gomori's formula. Whole preparations or sections stained at 60°C. must be differentiated in 45% acetic acid for half an hour or more. Tissue pieces may, after staining, be squashed and examined in the acetic acid, but the preparations can also be made permanent. The blue-black stain is very selective and has the advantage of giving high contrast, and it is nonfading, and insoluble in water and other common reagents. It proved definitely superior to other chromosome stains for difficult material such as planarians, rabbit blastocysts, and cleavage stages of sea urchins. Though both the procedure and the result of this method show some similarity to the Feulgen reaction nothing can be said with certainty about its chemical basis.

The observations leading to this staining method were the follow- ing. It was found that the leuco basic fuchsin (Schiff's reagent) and the chromic hematoxylin of Gomori (1941) stain essentially the same structures in sections treated in the same way. Thus, after treatment of sections of the pituitary and hypothalamus of birds with potas- sium permanganate and bisulfite (Gomori, 1941) both stain selec- tively the neurosecretory substance (cf. Hargmann, 1949). Untreated sections showed little staining in both cases. After treatment with warm hydrochloric acid, both solutions stain the nuclei in a selec- tive way. Some difference was noted, however, after oxidation of the

STAIN TECHSOI.OW, VOL. 28, So. 5, SFPTFXBFR 1953

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21 8 STAIN TECHNOLOGY

sections with periodic acid (PAS-schedule, Pearse, 1950). In such sections, the Schiff solution stains she mucoproteins distinctly whereas the Gomori hematoxylin gives an unsatisfactory result. Also in some other cases the two staining solutions give somewhat different result, particularly if the fixation of the specimens has been less perfect. This indicates some difference in the chemistry of the staining with Schiffs reagent and the Gomori hematoxylin. Perhaps the affinity of the tissues to Gomori’s hematoxylin depends on the presence of carboxyl or other acid groups, whereas Schiffs reagent is known to react with aldehyde groups only. This would explain the different result after treatment with periodic acid, since this reagent is known to produce aldehyde groups only and does not oxidize the latter further.

The clear and selective nuclear staining of the chromatin with Gomori hematoxylin after hydrolysis in hydrochloric acid made us try the stain for different material. It was found that though chro- mosome staining can be obtained at the first attempt, the time of hydrolysis must be adjusted to the material for an optimal result. The time of hydrolysis varies from one organism to another and is also dependent on the method of fixation as in the case of Feulgen reaction. We give some schedules below, which we found satisfac- tory for a number of test objects. If a new material is to be stained, it is recommended to try one or more of these schedules and then to adjust the time of hydrolysis to the material in question.

A. PREPARATION OF THE GOMORI HEMATOXYLIN (AFTER GOMORI, 1941) Mix equal parts of 1% aqueous solution of hematoxylin and 3%

aqueous solution of chrom alum. Add to each 100 ml. of the mixture 2 ml. 5% aqueous solution of potassium bichromate and 2 ml. 0.5 N sulfuric acid. The solution may be used after one day and works well for fourteen days if it is not kept too warm. A good solution is covered by a film with metallic luster on its surface after one day’s standing, and must be filtered immediately before use. Different hematoxylins (Merck, Ciba) have been used without noticeable dif- ferences in the result.

B. SQUASH PREPARATIONS Different material will require minor modifications in the technic

for an optimal result. Some examples are given here.

A. ROOT TIPS OF ALLIUM CEPA (FIG. 4) 1. Treat root tips at 20°C. for 25 minutes with the following mix-

ture: 4.5 parts (by volume) glacial acetic acid, 4.5 parts 1 N hydro- chloric acid, and 1 part 25% formalin (commercial formalin diluted with water 1:3).

2. Wash tips rapidly in distilled water. 3. Stain in Gomori’s hematoxylin at 60°C. for 40 minutes.

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GOXIOR1 \ H E \ l i TO\YLIS IS I CHRO\lOFO\lE ST4IN 219

PLATE I. Chromosome preparations stained with Gomori’s hematoxylin.

FIG. 1. - Mouse testicle, first meiotic di\ision, beginning ol anaphase. .Ihe sex chromosomes have already migrated toward the poles. Squash preparation according to formula R:b. X670. -

FIG. 2. - Euplaiiarin l u g u h i s ; squash preparation of a sonlatic cell. Treatment ac- cording to B:c. X670.

FIG. 3. - Mitotic metaphase in a bull testicle. Pi-epared accoi-ding to formula B:h. X1300.

FIG. 4. - Root tip of All iutn cepa; squashed and stained according to B:a. X670.

FIG. 5. - Salivary gland chromosomes ol Dr-osophila rrielnuogaslrt; treated accorcl-

FIG. 6. -Sheep testicle, cross section of tubule; stained according to D:a. X150.

ing to B:d. x670.

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220 STAIN TECHNOLOGY

4. Place in 45% acetic acid, 30 minutes or more. 5. Disintegrate root tips on a slide in a drop of 45% acetic acid by

6. Put on a cover glass, and press i t so that the cells are flattened. The preparations may be successfully studied in the acetic acid, or

they may be made permanent in the following way: 7. Place the slide upside down on an even surface of frozen carbon

dioxide and, after freezing, lift it and remove the cover glass with a razor blade. Work quickly so that the preparation remains solid all the time.

8. Drop the slide with the frozen preparation immediately into 95-9670 alcohol. Pass to absolute alcohol and mount in euparal or other alcohol-miscible medium. Avoid toluene and xylene.

Since the stain is resistant to acetone, permanent preparations may be made at once after step 4 by squashing under a celluloid cover slip in agreement with Melander (1948).

grinding it with the convex side of a watch glass.

B. TESTICLES OF MAMMALS (FIGS. 1 AND 3) 1. Fix very small pieces of tubules in glacial acetic acid, i part, and

absolute alcohol, 3 parts, I hour at room temperature or store at -20°C. for as long as desired.

2. Transfer to 1 N hydrochloric acid, 20 minutes at room tempera- ture in order to allow the HC1 to penetrate the tissues. Very small pieces do not require this treatment.

3. Hydrolyze in 1 N hydrochloric acid at 60°C. for 8-13 minutes. After long preservation in the fixing solution the optimal time for hydrolysis will decrease.

4. Dry pieces on filter paper and stain for 40 minutes in Gomori's hematoxylin at 60°C.

5. Place in 45% acetic acid, 2 hours. 6. Squash and mount in some of the ways mentioned above. For rabbit blastocysts it proved necessary to treat with cold hydro-

chloric acid (step 2) and to wash for 30 minutes in distilled water after the hydrolysis (step 3).

c. CHROMOSOMES OF PLANARIANS (e.g. Euplanaria polychroa) (FIGS. 1 AND 2)

1. The animals are cut into pieces in a fresh solution consisting of: glacial acetic acid: absolute alcohol (1:3), 9 parts, and 25% forma- lin, l part. The pieces are left in the fixative for one hour at room temperature or indefinitely at -20°C.

Steps 2-6 as for mammalian testicles, but the optimal time for hy- drolysis is 12 minutes. If the formalin is excluded from the fixative the squashing is easier but the intensity of the staining decreases a little. The optimal time of hydrolysis will then be 8 minutes.

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GOMORI’S HEMATOXYLIN AS A CHROMOSOME STAIN 221

The above schedule has also been successfully applied to pollen meiosis in plants and to testicles of crustaceans.

D. SALIVARY GLAND CHROMOSOMES OF DROSOPHILA (FIG. 5) 1. Dissect the larvae in alcohol - acetic acid (3: 1). 2. Within 2 minutes transfer salivary glands to 1 N hydrochloric

3. Hydrolyze in 1 N HCI 6 minutes at 60°C. Dip in distilled water. 4. Place in Gomori’s hematoxylin, 20 minutes at 60°C. 5. Transfer to 4570 acetic acid for two hours. 6. Make squash preparations in the common way. I t is necessary to shorten the time in the fixative, lest the chromc-

somes become fragile and difficult to spread. The chromomeres in the chromosomes become particularly distinct after this treatment.

acid (room temperature).

C. WHOLE PREPARATIONS This was tried only for cleavage stages of the sea urchin Psam-

mechinus miliaris.1 Since the eggs and embryos are difficult to mount on slides it is recommended to pipette a sample of specimens into a centrifuge tube holding about 2 ml. and to centrifuge and pour off every time the solutions are to be changed. The speed of the centri- fuge must not be too high, for then the embryos will be crushed.

1. Fix in Carnoy (absolute alcohol : chloroform : glacial acetic acid, 6 : 3 : 1 by volume), 1-3 hours. Preserve in 70% alcohol.

2. Change to 50% and 30% alcohol, and to 2 portions of distilled water.

3. Change to 3 portions of cold 1 N hydrochloric acid. The speci- mens are left for 30 minutes in the last change to let the acid pene- trate.

4. Dip the tube in water, heated to 60”C., for 5-7 minutes. 5. Change to distilled water, two portions. 6. Stain in Gomori’s hematoxylin for 40 minutes, with the tube

dipping in water of 60°C. 7. Place in 45% acetic acid. The acetic acid must be changed 3 4

times with at least 30-minute intervals. The differentiation is com- plete after 3-4 hours and then the material may be transferred to a slide with a pipette and can be examined in the acetic acid.

8. If permanent preparations are desired, leave the material in fresh 45% acetic acid till the next day, then transfer to SO%, SO%, SO%, 95-96% and to two portions of absolute alcohol. Change to alcohol-benzene (1: 1) and through two portions of benzene. Then pipette some specimens into a drop of balsam on a slide and put on a cover glass. The staining becomes darker when the preparations are made permanent. It is therefore important that the differentiation be

‘Material kindly supplied by Dr. I. Agrell, Zoological Institute, Lund.

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STAIN TECHNOLOGY 222

complete. The acetic acid must be almost uncolored before dehydra- tion is started.

It niay be noted also that the chromosome plates of the first cleav- age stages become distinct after this staining, and that chronlosome counts can be made.

D. SECTIONS A. TESTICLES OF MAMMALS (Bos, Mus, OvisJ (FIG. 6)

1. Fix small pieces of testicles in alcohol-acetic-acid (3:l) for 1

2. Embed in paraffin, cut 6-8 p sections, mount with albumen and

3. Hydrolyze for 8-13 minutes in 1 N hydrochloric acid at 60°C. 4. Wash in distilled water, 1 minute. 5. Stain in Gomori’s hematoxylin, 40 minutes at 60°C. 6. Differentiate in 45% acetic acid, 2 hours. 7. Wash under the tap, 5 minutes. Run through alcohols to

toluene and mount in balsam. This method gives a very sharp contrast and is useful for studies

of chromosomes. Other structures are completely unstained. For in- vestigations of cytoplasmic structures, nucleoli, spindles, etc., the following method is recommended, though the contrast is not so sharp.

hour at room temperature (longer in cold).

run through toluene and graded alcohols to water.

B. TESTICLES, DUODENUM AND OTHER PARTS OF THE PIGEON, COLUMBA LIVIA

1. Fix in Bouin-Allen fluid for 1 hour (Romeis, 1948, 8310). 2. Wash in 90% alcohol, dehydrate, embed in paraffin, cut 8 p sec-

tions, mount with albumen and run through toluene and alcohols to water.

3. Wash sections in two portions of distilled water, 2 minutes each. 4. Hydrolyze in hydrochloric acid for 20-25 minutes at 60°C. 5. Rinse in distilled water. 6. Place in Gomori’s hematoxylin, 15 minutes at room tempera-

7. Transfer to hydrochloric acid alcohol, 1 minute (95-96y0 alco-

8. Wash in running tap water, 5 minutes. 9. Counterstain in phloxine (Gurr), 0.5% aqueous solution, 5

10. Rinse in distilled water. 11. Mordant in 5% phosphotungstic acid in aqueous solution, 1

12. Wash in running tap water, 5 minutes. 13. Differentiate the phloxine in 95-96% alcohol, run through

absolute alcohol to toluene and mount in balsam.

ture.

hol, 100 ml. and concentrated hydochloric acid, 1 ml.).

minutes.

minute.

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GOMQRI’S HEMATOXYLIN AS A CHROMOSOME STAIN 223

After this treatment the nuclei are blue-black whereas nucleoli, spindles, and cytoplasmic structures in general are stained in differ- ent shades of red.

DISCUSSION In all preparations stained according to the above formulae the

chromosomes and the chromatin of the resting nuclei are stained a deep blue-black color. Cytoplasmic structures are in most cases com- pletely unstained, but the Bouin-Allen fixative causes a bluish tint to remain in the cytoplasm. The latter fixative preserves the cyto- plasm very well, however, and is to be preferred for studies of nucleoli, cytoplasmic structures and spindles. These structures may be counterstained with phloxine according to the last formula.

For some organisms which are difficult to stain (planarians, rab- bit blastocysts, cleavage stages of sea urchins) the Gomori hematoxy- lin gives definitely better results than all other chromosome stains we have used. It gives a better or equally good contrast also in ordi- nary material. 4 great advantage of this method is that the Gomori hematoxylin gives a constant result independently of the kind of hematoxylin used for the preparation of the staining solution. Fur- ther the staining is permanent (in contrast to orcein, Feulgen, crystal violet, etc.) so the slides may be preserved for years without bleach- ing. Figures 1 - 6 give some idea of the selectivity of the method.

REFERENCES

BARCMANN, W. 1949. Ueber die neurosecretorische Verkniipfung von Hypothal-

GOMORI, G. 1941. Observations with differentia1 stains on human islets of

MELANDER, Y. 1948. The use of dissolvable cover slips when making permanent

PEARSE, A. G. E. 1950. Differential stain for the human and animal anterior

ROMEIS, B. 1948. Mikroskopische Technik. 15. AuP. Miinchen, 1948.

amus und Neurohypophyse.

Langerhans. Amer. J. Path., 17, 395406.

squash preparations of chromosomes.

hypophysis. Stain Techn., 25, 95-102.

Ztschr. Zellforsch., 34, 61&-34.

Hereditas, Lund, 34, 512-3.

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