Tsuchiya, pp. 71-72

III.1 An improved aceto-carmine squash method, with special reference to the modified Rattenbury's method of making a preparation permanent.

T. Tsuchiya, Agronomy Department, Colorado State University, Fort Collins, Colorado 80521, U.S.A.

Rattenbury (1956) proposed a simple and rapid method to make a squashed temporary preparation into a permanent one by replacing 45% acetic acid with a mixture of 10 parts of 45% acetic acid and 1 part of glycerine. The Rattenbury's method was slightly modified by the present writer in 1958 as shown in the following procedure:

1. Pre-treatment with 8-oxyquinoline or ice water (only root or shoot tip mitosis)
2. Fixing with 3:1 alcohol-acetic acid mixture
3. Treatment with a mordant, 2% iron-alum
4. Staining with 0.5-1.0% aceto-carmine
5. Maceration and destaining the overstained cytoplasm by applying a drop of a mixture of 1 part 1% acetocarmine and 1 part 1N HC1.
6. Apply the above-mentioned mixture of 45% acetic acid and glycerine (10:1) (Rattenbury's fluid)
7. Apply a cover slip and gently heat the slide. Should not be boiled.
8. Squashing to complete preparation.

Some excellent points of this squash technique will be given below:

(1) Most of the prophase cells could be used for chromosome counts and karyotype analysis to some extent. Generally speaking mitotic metaphase plates tend to be destroyed by the pressure applied at the squashing. However, the prophase nuclei are kept intact by the nuclear membrane and not so easily destroyed but just flattened to spread the chromosomes widely. Chromosomes could be counted readily.

The prophase nucleus is therefore especially suitable for the exact determination of chromosome numbers.

Furthermore, metaphase nuclei are sometimes very difficult to obtain in some root tips, especially roots from growing plants, probably because of the inadequate time of collecting materials (cf. Sakai 1935) and/or unknown reasons. Prophase nuclei at various stages are, however, rather frequent even in those roots just mentioned above. Prophase nuclei are useful for the chromosome counts in such cases as mentioned above and also in some cases for the karyotype analysis thanks to their specific gradient of the stainability at prophase (cf. Takemoto, 1962). This method has been used exclusively by the present author for more than ten years in the study of chromosomes in Hordeum, and many other plant species with successful results.

(2) No need to seal the edge of cover slip


1. Pretreatment with 0.002 mol/l   8-oxyquinoline at 18° C for 4 hr. (cf. Tjio and Levan 1950) or ice water for 12 to 72 hr. (cf. Tsunewaki and Jenkins, 1960). Only for root or shoot tips.

2. Fixing the material with 1:3 acetic-alcohol for 5 minutes or more, sometimes several months to about 2 years in the refrigerator.

3. Transfer the materials to 2% iron alum mordant, especially for the study of nucleolus in mitotic and meiotic cells.

4. Staining the materials in 0.5-1% acetocarmine by keeping several days or more.

5. Squashing the stained materials by the usual method: Here use a mixture of 45% acetic acid and glycerine (10:1) for mounting (see below, d).

    a. Cut the growing points of the stained roots at 0.1 mm thick or squeeze out the meristematic tissues with the tip of a forceps after removing the root cap. Anthers are used without cutting but a large anther such as lily or rye is better cut into several pieces.

    b. Put a piece of cut or squeezed meristematic tissues or anthers on a slide glass.

    c. Apply a drop of mixture of concentrated acetocarmine (1 or 2%) and 1N HC1 (1:1 ratio) for 1-3 seconds (depending on the stains), and remove it soon with a piece of blotting paper.

    d. Apply a drop of a mixture of 10 parts of 45% acetic acid and 1 part of glycerine mentioned above (Rattenbury's fluid).

    e. Put a cover slip on.

    f. Heat the slide gently on a small alcohol flame or gas burner at about 80° C.    (Should not be boiled here!!)

    g. Tap the cover slip gently after fixing the corner of the cover slip with a finger to avoid the cover glass slipping.

    h. Heat again for a second. Should not be boiled here either!!

    i. Apply a blotting paper on the cover glass and press with the finger after fixing the corner of the cover glass with another finger. Thus the preparation is finished and the excess fluid is removed by the covered blotting paper.

No need to seal here the edge of cover glass with valap or paraffin-balsam.

    j. Observe the preparation.

    k. If cells are crowded and/or the cytoplasm is still overstained apply a small drop of the Rattenbury's fluid and gently heat again and repeat the procedures from f to i.

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