hihi, im going to share about slide making of blood specimen for chromosome study.
when the blood culture is ready for harvesting...
1. centrifuge the tube of cell suspension at 1200rpm for 10minutes
2. remove the supernatant and resuspend the cell pellet with fresh fixative
[do not cause too much mechanical disruption to the cells]
3. centrifuge the tube at 1200rpm for 10minutes
4. place the tubes ready for slide making in a rack and prepare a clean recycle tube with pipette for each tube in a rack
5. remove the supernatant into the recycle tube, leaving a suitable amount of fixative to resuspend the cell pellet
6. take a slide from a beaker in the fridge and flick to remove excess water leaving an evenly thin coat of water on the surface of the slide.
7. hold the pipette approximately 1-2 inches above the slides
[tilt the slides at about 45 degree angle]
8. gently drop the cells suspension on the slide
9. dry the sides and edge of the slide and leave it at room temperature to dry
10. check the mitotic index and spread length under the microscope
11. bake the slides at 90 degree celsius for approximately 1 hour 30 minutes
Factors affecting slide making
a) Proportion of fixative
if the metaphases does not spread well, use a higher proportion of acetice acid to methanol. this will soften the cell "sac", thus chromosome spread is better. however, this must be handled carefully as increase amount of acetic acid can cause staining problem later on.
b) Cell density
if the cell density is low, make a denser cell suspension by resuspending the cells with a lesser volume of fixative and vice versa.
c) Temperature of slides
warm slides allow more spreading, thus use it when the spreading is tight. cold slides prevent over spreading of the chromosome. however, cold but dry slides is used to prevent further spreading of the chromosomes. it is normally use when cells are "severely broken".
d) Aging of slides
slides can be sged at 90 degree celsius for 1hour 30minutes or 60-65 degree celsius ovenight.
e) Angle of slides
the angle of the slides is adjusted according to the condition of the metaphase. for example, a steeper angle will create a greater flow direction, thus enhanced the spreading.
by,
yeo sok kian jocelyn
0703359j
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Hi jocelyn! :D
ReplyDeleteCan I know what is the fixative used in step 2?
And, I don't understand what you mean by aging of the slides...
Thanks!
Siew Ming
0702862D
TG01 Group 2
Hello,
ReplyDeleteMay i know the importance of the mitotic index and the spread lenght.What do we do after checking both of the under the microscope and so what if it is high or low?
Stanley
Tg01 Grp 2
hi, can i know when would you know that the blood culture is ready for harvesting? do you grow the blood culture on agar/broth, or the blood culture means blood obtained from the patient and you just put in incubator?
ReplyDeletefor step 6, why do you remove the slide from the fridge? is the slide immersed in water or some special chemical?
i think it's better if you can post some pictures up (cause i dont really understand) thanks.
LIM JIA HUI JOEY
tgo1 0703605F
Hi Jocelyn =)
ReplyDeleteCool post =) How's your experience making slides ? Is it easy ? Sounds difficult cuz there are many factors affecting slide making =)
Hmm i would like to ask about the drying of slides. How does the environment (temperature and humidity etc) affect the drying process and chromosome spreading ?
Other questions to clarify. How many drops of cell suspension do you drop ? Also how many slides would you make per culture ? =)
Thanks =) take care, hope you are enjoying yourself =)
Ng Tze Yang Justin
0703747F
to SIEW MING,
ReplyDeletethe fixative used is methanol and acetic acid in the ratio of 3:1 respectively.aging of slides takes place in the last step after you drop the cells. the purpose of aging slides is to fix biological material to the glass surface.
to Stanley,
there must be sufficient mitotic index (MI) so that the lab technologist can analyse and produce an accurate result. for instance, in my lab, there must be at least more than 4 to be acceptable. whereas for spread length,it better for chromosome to be spread out rather than cluster together. this is because, if there is overlapping between the chromosome, some abnormalities might be missed out. however, there spread length cannot be too far apart as this might result is mixing up the chromosome from different cells.
after checking microscope, if the MI is not acceptable (<4), there is nothing much that can be done except during harvesting, ensure that there is sufficient cells for harvesting. whereas for spread length, if the chromosome are clustered, use a wet slides to enhance the spread. the angle of the slide can also be altered to facilitate the spreading better.
to Jia Hui,
for blood specimen, it is usually harvest at the forth day. the blood specimen is not grown on any agar, it is in a falcon tube with media in the incubator.
the slides are immersed in water in the fridge. this is to moist the slide before use.
to Justin,
if the humidity is high, there are more water vapor in the air, thus the slides takes longer time to dry. however, if the temperature is high, this shorten the drying process and the chromosome might not spread very well due to fast drying process.
the number of cells drop is dependent on each medical technologist. it is approximately 1 to 2 drops.
2-3 slides are made per culture.
=)
Yeo sok kian jocelyn
0703359j