Hello Godfred!
I confirm Nallasivams hints. Furthermore you can optimize spreading in dropping procedure. Give cells time to soak completely with fixative. Moreover, a muggy atmosphere (by moist chamber at 37°C) in which the fixed cells should drop will help. Fixative is hydrophilic. In a moist atmosphere the fixed cell will soak with atmosphere water like a balloon. Thus, the chromosomes are separated more effectively in metaphases.
Good luck, Jörg.
You can also use slides stored in the water, in refrigerator, or even (shortly) in deep-freezer. Then let water quickly "go down" and use wet slides to make your preparations.
Good luck!
Dear Anna Gutkowska
I would like to ask whether we should use slides stored in the water or Methanol, in refrigerator, or even (shortly) in deep-freezer??
Thank you all. Please can you provide some references/ links with crucial points.
Claussen U; Michel S; Mühlig P; Westermann M; Grummt U-W; Kromeyer-Hausschield K; Liehr T (2002) Demystifying chromosome preparation and the implications for the concept of chromosome condensation during mitosis. Cytogenet Genome Res 98:136–146.
Role of water in chromosome
spreading and swelling induced
by acetic acid treatment:
a FTIR spectroscopy study
[European Journal of Histochemistry 2014; 58:2330]
For me in plant slide preparation, the good roots that are growing actively with lots of mitotic cells are very important. After pretreatment (collect metaphase chr if you need), that wil be easy to get slide with lots of metaphase chr. You know, we cannot overcome totally the inconsistency in slide preparation, what for me to get more accuracy results in the classical cytogenetics is to check more samples/chr.
Kai
It is right that slide making is an art, but science behind this is very well elaborated in the Bible for cytogeneticists, 'AGT manual', the blue book {ISBN-10: 0397516517
ISBN-13: 978-0397516513}. By knowing how to provide optimum drying time by sensing the temperature/humidity of the day and using steam Vs. hot plate to arrive at right combination is the trick. Not everyone can afford the slide making chamber, but it is possible to get 55-60% RH and 25 degree C temperature in the area of slide making. In addition to that, one can aim to have enough cell suspension, make maximum number of slides with cells on a small area, and get maximum selection of good metaphase spreads.
Vaporise slides before dropping cells on slide. The vapours can be mouth vapours also or place dry slides on wet tissue paper and then drop cell suspension to optimise the humidity. Temperature and humidity plays a major role in proper spread of metaphase spread.
For optimum chromosome spread in conventional cytogenetics is the fixative. After the hypotonic solution, and the spin, remove the supernatant, mix thoroughly and, drop by drop, 10ml fresh fixative made up of 1 part acetic acid to 3 parts methanol or ethanol. Repeat twice more the fixative, if the chromosome isn’t spread, it’s important increase with acetic acid. Use the fix 2 part acetic acid 2 part methanol/ethanol, or 1 part acetic acid 3 part methanol/ethanol. For optimum chromosome spread in conventional cytogenetics is important to use fixative with more acetic acid, se the chromosomes are closest you have to use more methanol/ethanol.
This is a recent article about cryopreservation. I believe it is interesting and might solve some of your questions.
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