week 15
Subject : Cytogenetics
Hi friend, now already week 15, 5 more weeks of SIP. Hope you all doing well at your workplace.
In the previous blog I shared with you the harvest method and the next step which I am going to shared with you in this blog is the Slide Making method.
Slide making
The ultimate goal of Slide making
to obtain chromosome that have the following characteristics on the phase contrast microscope:
-Mostly medium gray to dark gray chromosome. Light gray chromosomes yield cells with poor contrast between bands and very black images may have increase cytoplasmic background.
-Little chromosome scattering.
-Minimal number of chromosome overlaps, for accurate counting and bnads analysis.
-Very thin cytoplasmic background so that trypsin banding will be optimal.
-Proper concentration of cell fixative suspension. Too dilute a suspension yields slides that are time consuming to scan for metaphase, and too concentrated a suspension may interfere with the banding.
-Chromatids that are together and not split apart.
Materials
-Clinical Lab Reagent Water (CLRW)
-Sterile 10 ml round-bottomed tube
-1ml and 10 ml serological pipet
-Glass Pasteur pipet
-Disposable plastic sterile and non-sterile transfer pipet (3ml)
-Microscope glass slide, frosted end
Methods
1. Remove from the fridge a beaker of cold, wet pre-cleaned slides kept in CLRW.
2. Take a slide and flick off excess water and wiped with toweling paper before dropping the cell suspension.
3. From a height of 10-20 cm from the slide surface, drop about 3-6 drops of the cell suspension from a glass pipet along the upper edge of the slide, while holding the slide horizontally to the bench at 45°angle.
4. Wipe the back of the slide with a towel and firmly bang the slide on the bench top several times (this may help in chromosome spreading).
5. Air dry before placing it on a 56℃ warming tray.
6. Check the spread under phase contrast microscopy, adjust cell density or height from which the suspension is dropped accordingly.
7. Baked slide at 90℃ oven for 2 hours.
Now the slides are ready for staining, which I will share with you all in my next blog entry.
In order to achieve the goal of the slide making, there are some variable that we have to take note of:
Wet VS dry slide
In our laboratory, wet slides are preferred, by dropping the fixed cells on wet slides, it facilitates spreading due to the immediate reaction of the water meniscus. The energy of dehydration from fixation is returned as a change in the free energy of mixing between fixative and water, which spreads the cells. Wet slides may further facilitate spreading, control by the use of different temperature of water coating to speed up or slow down drying time. Cold wet slides will slow drying, increase spreading. While warm slides will accelerate drying time.
Angle of the slides
Slides made with long edge down tend to be more uniform, especially if the angle is kept 20-30°, cells are placed in the upper one third of the slides and allowed to move downward. Greater tilt angles may speed up the drying process at the upper end of the slide compared to the lower end which gives uneven, inconsistent slides. Uniform drying can be achieved by tilting slides at one angle for part of the drying time and another for the final drying.
Hope my blog entry is clear for everyone, any question feel free to ask. =)
CHEN TING JIE
TG02
0608495H
6 Comments:
Hi Tingjie,
I am also attached to cytogenetics lab now. There are some areas that my lab does differently from yours. So just thought of clarifying some questions.
Will 3-6 drops of cell suspension cause the cells to be very packed? Or your suspension is very diluted? Cos in my lab we only drop one drop.
Then what is the purpose of placing the slide on the 56 deg celcius warming tray? Is it to increase the spread of metaphases?
Thanks.
Ka Hang
TG02
hi ting jie
what is the reason behind storing the slides in the fridge before use? does the cold temperature enhance adhesion of the cells to the slide surface?
and is the baking of the slide a step in fixation of the cells to the slide, or is it done for other reasons?
thanks
rusydiana
tg02
to rusydiana,
1.what is the reason behind storing the slides in the fridge before use? does the cold temperature enhance adhesion of the cells to the slide surface?
answer:
this question actually already anser in the blog...
is under "wer VS dry slide", at the end of the paragraph.
Cold wet slides will slow drying, increase spreading. While warm slides will accelerate drying time.
2.and is the baking of the slide a step in fixation of the cells to the slide, or is it done for other reasons?
answer:
it is one of step to fix the cells to the slides.
the principle behind baking/aging the slide:
in the past the slides have to be sit or 3 or 4 days before it is suitable for banding. after experimenting, it was discovered by many laboratorians that heating the mosome preparation ont eh slides in an oven or on a hot plate for 12-24 hours at 40-60 degree celcius 20 minutes at 90-95 degree celcius can prevent choromosome resisting banding procedures ad decrease turnaround time, as the G-banded can be done after aging of slides. aging of slides involves driving off water. the aging process is very important, as it gives better contrast and crispnss of chromosome.
reference:
the agt cytogenetics laboratory manual
hope i answered yor question
tingjie
tg02
0608495h
to ka hang:
answer for question one:
yap, our cell suspension is very diluted, so that it allow us to drop more drops, but most of the time we drop 3 drops, seldom up to 6 drops.
answer to question 2 please refer to my comment to rusydiana question 2.
thank you
hope i answer your question
tingjie
tg02
0608495h
Hi TIng Jie,
What is usually the height in which the cell suspension is dropped onto the slide? What are the components of CLRW or is it just DI water?
Thanks!
From: Benjamin Ma
Class: tg01
0606181F
to benjamine,
1.What is usually the height in which the cell suspension is dropped onto the slide?
answ:
the height we usually drop the cell suspension is about 3 cm.
2.What are the components of CLRW or is it just DI water?
answ:
it is atucally just DI water, we call it CLRW is just the scientific name the laboratory name the DI water, it is to act as standardization.
hope i answer your question=)
tingjie
tg02
0608495h
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