hi everyone, i am here to blog again...anyway i realised that many people tend to spell my name wrongly...my name is spelt as Rachael not Racheal:) haha...anyway...in the previous post i talked about blood culturing...in this post i would like to continue from the previous post to talk about blood harvesting...

In the harvesting process, the purpose of the mitotic arrestment is to prevent spindle fibre formation so that the cell division can be blocked at metaphase stage. After which, there will be hypotonic treatment which is to increase the cell volume so that the chromosomes can be spread out and lastly, it will be the addition of fixative to remove water from the cells so as to preserve them.

Method:

1.The hypotonic solution was warmed in 37ºC water bath before the start of harvest process.
2.50µl of colcemid was added to each tube and one tube was exposed to colcemid for 20minutes while the other was exposed to colcemid for 2hours.
3.The tubes were centrifuged at 1200rpm for 10minutes and the supernatant was aspirated.
4.The pellet was loosen gently and 14ml of warmed hypotonic solution was added into each tube and the tubes were inverted 2-3 times to ensure that the suspension is well mixed.
5.The tubes were left at room temperature for 5minutes.
6.After 5minutes, the tubes were transferred into a covered centrifuge bucket and spun at 1500rpm for 6minutes.
7.The supernatant was aspirated and discarded.
8.The pellet was loosen gently and approximately 12ml of warmed hypotonic solution was added into each tube and the tubes were inverted 2-3 times to ensure that the suspension is well mixed.
9.Step 5 was repeated.
10.The required amount of fresh fix was prepared based on the number of cases to be harvested. (40ml per case at room temperature)
11.After 5minutes, 1pipetteful of fixative was added and the tubes were inverted 2-3 times to mix well.
12.Steps 6 and 7 were repeated.
13.The cell pellet was resuspended by flicking the tubes until no visible clumps can be seen.
14.Another 10-12ml of fixative was added and the tubes were inverted 2-3 times to mix well.
15.The tubes were stand at room temperature for at least 15minutes.
16.The tubes were centrifuge at 1000rmp for 10minutes and the supernatant was aspirated and discarded.
17.The pellet was resuspended in 8-10ml of fixative and step 16 was repeated. This step would be repeated if there clumpy or jelly like pellets are still present.
18.The pellet was resuspended in 5-7ml of fixative and was stored in refrigerator at 4ºC until ready to make slide.

when the slides are made, the chromosomes are ready to be analysed under the fluorescence microscope.thats about it:)

Rachael
0606168C
Tg01