Glass preparation prior to dropping the cell suspension
Commercially pre-cleaned slides tend to have a very good quality. Two brands of pre-cleaned slides, Gold Seal (Becton, Dickinson Co, Portsmouth, NH) and Superfrost (Erie Scientific, Portsmouth, NH) were commonly used in our laboratory and both performed well. They did not require any extra cleaning steps or washes prior to use. Cell suspensions were dropped on slides taken directly out of their original boxes and results were always consistent.
Batches of such already pre-cleaned slides were sometimes passed through successive washes in acetone, HCl/ethanol and triple distilled water and their quality compared. In our hands, there were no visible differences in chromosome spreading, GTG banding and FISH quality between slides taken out of the box and slides pre-cleaned in the laboratory.
The only time when slides did not work well, was when there had been dust or cardboard/paper residua on the glass surface, more commonly on the two outermost slides in the package.
Other glass surfaces
Various non-precleaned brands of slides and coverslips were tested in time. In general, good metaphase spreading was achieved on all types of glass used, although spreading was somewhat easier when these glass surfaces were precleaned in the lab. This was achived by soaking the slide/coverslip in acetone, HCl, acetic acid and water prior to use.
Chromosome spreading depends on the drying speed of the cell suspension after it is placed on the slide, and it is influenced by the amount of water on the slide and by the drying temperature.
Several tests were performed in which cell suspension was dropped onto slides kept at -20º C, 0º C, room temperature and 50 C (warm plate) respectively, and drying was done at room temperature. Results indicated that, in general, with a good quality cell suspension, and in "standard" conditions (see above), good quality chromosome spreads can be obtained on dry slides, at room temperature. By lowering the initial temperature of the slides, more atmospheric water will condense on them and will slow down fixative evaporation. A similar but not quite identical situation is that in which slides are kept at room temperature (no water condensation on them) but the atmospheric humidity is high (thus slowing down fixative evaporation). When slides are preheated above room temperature, water will not condense on them and drying will be much faster.
Based on these observations, it can be concluded that, when drying is done at room temperature, cold slides are more useful when atmospheric humidity is low, whereas room temperature or slightly warm slides work better when atmospheric humidity is high.
To reduce the influence of the variable atmospheric humidity on the drying process, slides can be soaked in (cold) water prior to pipetting the cell suspension. Careful selection of the drying temperature (in this case a warm/hot plate) allows good spreading of the chromosomes. However, too much water on the slides may prevent chromosome spreading when drying is performed at room temperature. Although simple and widely used, this procedure for slide preparation still shows enough variability in results, and does not use critical information resulting from an intimate observation of the drying process.
Fixative on slide
Especially in very humid atmospheric conditions, soaking the slide in fixative prior to adding the cell suspension may improve chromosome spreading as it "dilutes" out the water condensing on the slide. In such situations is advisable to use room temperature slides and room temperature or 37º C fixative to speed up drying.
Acetic acid on slide
Increased acetic acid concentration is known to improve chromosome spreading, as it slows down the drying process (acetic acid evaporates slower than methanol) and increases the fragility of the cell membranes. Thus, 1:1 methanol : acetic acid or even a few drops of glacial acetic acid can be spread on the slide right before the cell suspension is added. With this procedure, metaphases may look "cleaner", with less debris around the chromosomes. This technique also improves chromosome spreading in high humidity conditions, probably because of the increased membrane fragility.
Dropping technique. Is height important?
The spreading process was observed live at the microscope on slides where the cell suspension was dropped from about 1m distance and on slides where the cell suspension was placed directly on the glass (fig. 3). Based on these experiments, it became obvious that the height from which the cell suspension is dropped has no influence on chromosome spreading !. The only potential benefit is that the cells may be more uniformly distributed across the surface of the slide. However, uniform cell distribution can be easily achieved by placing a few drops of cell suspension (20-30 µl) on the slide with a micropipette and then immediately spreading the suspension by tilting the tip parallel to the slide surface and gently moving it across the surface. This procedure saves a tremendous amount of cell suspension, which otherwise may get wasted either by "missing" the slide or by flooding the slide with cells.
Post-dropping technique. Drying procedures.
Drying at room temperature vs. heat plate.
Drying the cell suspension on the slides can be performed at room temperature (RT) when the atmospheric humidity is not high. On rainy days or when atmospheric humidity reaches values between 90-100%, drying at room temperature is too slow, and chromosomes don’t spread well. In such cases, a humidity controlled environment is very useful. Otherwise, the drying speed needs to be increased by placing the slides on a worm/hot plate.
Water or water vapors
In dry atmospheric conditions, the slide-drying process is fast and needs to be slowed down by humidifying the slides AFTER dropping the cell suspension. A common procedure is to blow on the slides or expose them to a source of water vapors. One important observation made was that exposing the slides to very hot vapors (75-80º C) is the most beneficial, as it allows the deposition of similar amounts of water on slides independent of the atmospheric humidity. It is also possible that the heat by itself has a positive action on spreading.
Especially in high humidity conditions, adding a few drops of fixative or acetic acid to the slide just before the cell suspension starts drying, helps chromosome spreading and cleaning the metaphases. The reason for this is, probably, the sudden increase in the overall ratio of acetic acid to water, which makes cellular membranes more fragile.