The following steps describe what is observed at the phase microscope during the dropping/drying process:
Step 1 (Fig 3a and 3aa): while the cell suspension spreads on the slide, cells float wildly in all directions but ultimately touch the glass surface and immediately become immobile. No chromosome spreading takes place. At the microscope, for as long as there is plenty of fixative, the cells look like small gray spheres.
Step 2 (Fig 3b and 3bb): the fixative starts drying. At the point where the surface of the liquid touches the surface of the spherical cells, each cell on the slide starts reflecting the light and acquires a bright halo. Chromosomes are still not spread at all. In macroscopic terms, this is the moment where the surface of the slide, when examined with the naked eye, becomes "grainy".
Step 3 (Fig 3c and 3cc): very rapidly, the cells with metaphase chromosomes start flattening and spread their content (chromosome spreading), BEFORE the nucleated cells do so. At this point, the chromosome-containing cells lose their halo of light, and the chromosomes become dark and visible. The nucleated cells continue to have some halo of light, indicating that the nuclear membrane/content is much more resistant to the flattening force of the drying fixative. Most of the chromosome spreading is achieved at this step.
Step 4 (Fig 3d and 3dd): as the fixative continues to dry, the nucleated cells continue to flatten slowly, whereas the metaphase-containing cells which are already spread increase just a little more their spreading (by less than 7%, if one measures the change in the diameter of various metaphase spreads). Fixative evaporates first from the glass areas lacking biological material and, at this time, every cell on the slide continues to be surrounded by a puddle of fixative. As all these puddles continue to dry, for a brief moment there is another burst of more intense light halo around every nucleated cell.
Step 5 (Fig 3e and 3ee): The puddles of fixative, surrounding the cells, are vanishing almost synchronously from the slide. There is no more chromosome spreading visible at this time, but it is likely that minor changes may still take place.
The following conclusions can be derived from this experiment:
1Dropping cells from any height will not influence in any way chromosome spreading. It only helps distribute the cells more evenly on the slide. Chromosome spreading takes place much later, when the surface of the slide becomes "grainy" and the drying process is already well under way.
2 Over 90% of chromosome spreading takes place at the time when the fixative surface, as it evaporates, comes in contact with the spherical cell surfaces (Step "c" above). This is the CRITICAL moment to intervene in order to increase or decrease the degree of spreading. Water vapors, heat or variable acetic acid concentrations can be used.
3 After step "c", very little chromosome spreading takes place. During steps "d" and "e" described, the true benefit one can achieve is to clean more of the cytoplasmic residua still surrounding the chromosomes.
Based on observations made during the drying of the cell suspension (Fig. 3a-3ee), we developed a slide preparation procedure which eliminates entirely the influence of atmospheric humidity on chromosome spreading, and allows customizing the level of spreading as desired by the cytogeneticist. Spreading can vary from "condensed" to "broken" metaphases. Nuclei become very flat and acquire a larger diameter, making them suitable for interphase FISH. Slides prepared by this procedure worked well in both banding and FISH/CGH analyses.
The only required setting is a waterbath at 75-80? C (Fig 1), equipped with a metal plate partially covering the opening of the bath. If the water level is 1-2 cm below the plate, one end of this plate becomes hot in just a few minutes, whereas the other end is roughly at room temperature. 25-30 µl cell suspension are placed in the desired places on the slide using an automatic pipette, almost by touching the tip to the glass. As son as the drops start spreading, the tip can be tilted until it is parallel to the glass surface and can be used to gently spread the suspension uniformly on the slide. This allows a very precise control of the areas of the slide where the cells will be placed and eliminates the losses of biologic material due to over-flooding the slide.
The cell suspension/fixative is allowed to spread until it covers the entire surface, then excess liquid (if any) at the edges of the slides is quickly drained on a paper towel. The slide is kept under observation until, as the fixative evaporates, the surface of the slides becomes GRAINY (corresponding to steps 2 and 3 in fig. 3). At this moment, the cells are visible and resemble fine specs of sand spread on the glass surface. Immediately thereafter, the thin layer of fixative starts drying out, a process always beginning at the slide edges. It is at this precise moment that intervention is necessary in order to achieve the desired spreading of the chromosomes. As soon as dry glass areas are being noticed, the slide needs to be immediately placed face-down, close to the hot water surface for 1-3 seconds, then placed on the hottest area of the metal plate covering the waterbath and allowed to dry. To achieve uniform heat transfer, a drop or two of water or another liquid can be placed between the metal and the slide.
This procedure controls the speed of fixative evaporation twice, first by placing roughly the same amount of water on the slide regardless of environmental humidity and second, by using roughly the same drying temperature. The slides and the cell suspension are at room temperature, thus atmospheric water condensation is not a factor. The main unknown variable remains the quality of the cell suspension, which depends greatly on the harvesting technique. In case of fragile cell pellets, with metaphases "breaking" too easily, spreading is controlled by using the temperature gradient across the metal plate. Lowering the drying temperature should restrict spreading. In the case of cells which do not spread well, chromosome spreading is improved by a controlled extension of the drying period. In this case, the slide is passed as usual through the hot water steam, but instead of drying on the hot plate, it is kept again under observation until it starts drying at the edges. Then, the slide is passed again through the hot steam and observed again. This process of passing through the hot steam followed by partially drying can be repeated 3-4 times. After the last pass, the slide is immediately placed on the hottest area of the metal plate for a fast drying.
This procedure has been tested on a variety of cell types: human lymphocytes and lymphoblastoid cell lines, fibroblasts, solid tumors (germ cell tumors and germ cell tumor lines), leukemias and mouse fibroblasts. A good understanding of the theoretical considerations above and proper usage of the experimental data allowed very good slide preparations using all cell types regardless of climate conditions (from dry to very humid).