Уважаемый Vladpatholog!
Вот один из достаточно профессиональных ответов на Ваш вопрос. Его я нвшел в FAQ-list J. Kiernan:
(Да не обидятся, те, кто не может прочесть по-английски - просто нет времени на перевод). ** Giemsa staining of blood smears: several hints
Question.
My methanol-fixed blood smears are not staining reliably
with Giemsa. Some advice is needed, please.
Answer
Fixation of well dried (at RT) PB smears can vary from 1-10
minutes; automated systems tend to use about 1-2 minutes and use
the methanol only once. For manual staining, most labs would fix
for about ten minutes. Precautions must be taken against
absorption of water from humid air. The methanol is usually
replaced twice daily, but more frequently at those times of
the year when humidity is high.
The first sign of unacceptable water content in the fixing
methanol will be the appearance of clear refractive spaces on
the biconvave surfaces of erythrocytes: perhaps only a few cells
per high-power field, but this will increase further as the
water content increases, and eventually the films will lose all
diagnostic value. Replacement of the methanol when you see more
than say 1-2/HPF might not be a bad idea. This artifact may also
be seen in some automated systems where the stain pack is not
turned over very quickly. Rather than replacing the stain pack,
economy of reagent can be maintained by manually fixing the
slides before thay go on the machine. This is particularly so
for the older Hematek grey models.
Caution. Longer fixation times are required for bone marrow
smears: 15-20 minutes, and always use fresh methanol for these.
Most persons using Giemsa prefer to stain the smear first with
May Grunwald or Jenner stain, either using it neat or diluting
1:2 with buffer. This pre-step improves the granule definition
and clarity, and also changes the traditional reddish purple of
nuclei with plain Giemsa to a blue purple as seen with Wright's
stain.
The selection of Sorensen's buffer will vary form 6.4-7.2, with
the lower pH being most popular with Wright's rather than
Giemsa. The aim is to select a pH that produces a colour balance
that readily allows the user to differentiate between
normochromic and polychromic red cells and to distinguish toxic
granulation when present, this is usually pH 6.8. If looking for
malarial parasites, then a pH of 7.2 is preferable because it
allows better contrast to detect chromatin dots, trophozioites
etc.
Прододжение следует.
Добавлено (2010-08-22, 8:15 PM)
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Вот продолжение
Dilution of the Giemsa solution is best done immediately before
use and will vary from 1:8 to 1:12 depending upon your protocol.
As a general rule of thumb the higher dilutions require longer
staining times of about 20 minutes, and the less dilute stains
need between 6 and 12 minutes, depending upon tthe quality of
the Giemsa. It was frequently claimed that the longer times gave
better definition, but I must admit that I've seen short timed
smears that are every bit as good.
For many years good quality Giemsa would be stable after
dilution for 6 to 8 hours. For the last 2 or 3 yrs, however, the
best you can hope for is 3 to 4 hours. After dilution the
solution starts to deteriorate, with the appearance of floccules
and a subsequent loss of staining ability or strength. As the
time progresses you may need to compensate by increasing the
staining time, but after 3 hours you will need to replace it.
Recipes for Giemsa vary, whether it be that of Hayhoe or of
Dacie & Lewis, and measurements may be by weight or volume.
Stock solutions that have a 50% by volume content of glycerol
(Analar or USP) are the most stable. Under no circumstances ever
heat your glycerol to more than 45C, even though most texts say
56C. Above these temperatures there is a risk of oxidation, even
in the stock solution, I use 45C as a cut-off point to give me a
safety margin. Dye content will also vary from 0.45 to 0.8%.
Lillie's comments should considered here. After standing for up
to 5 days, filtration to remove undissolved material is
essential.
Differentiation, by giving the slides two rinses in buffer of
two minutes each, is fairly standard, but you can overdo it. A
single rinse of three quick dips may in fact suffice. It will
depend upon your Giemsa solution and tastes. If overstaining is
a problem then consider adding methanol to your buffer rinse,
starting at 5% and adjusting according to results, followed by a
water rinse to remove solvent.
Mike Rentsch, "Histomail," Downunder
Ссылка: IHCWorld
