## Preparation of Blood Smears

1.  Flick the blood sample tube a few times to mix the blood.

2.  Dispense 5 μl of blood near the label end of a microscope slide
    (Fisher Premium Microscope Slides-Superfrost, cat \# 12-544-7 is one
    slide type that is used).

3.  Angle a second glass slide about 45° and allow the glass tip to
    contact the drop of blood. Allow the blood to spread across the
    slide.

4.  Then smoothly drag the second slide away from the label end of the
    first slide, creating a blood smear. Note: The angle of the second
    slide and the rate at which you drag the slide impacts the quality
    of the blood smear. Anemia makes it more difficult to prepare a good
    blood smear.

5.  Immediately place the blood smear under a blowing fan to dry.

## Preparation of Bone Marrow Smears 

1.  Harvest marrow by using a 5 or 10 ml syringe fitted with a 26 ½ or
    25 gage needle and flush the bones with cold Hepes buffered sterile
    saline without calcium or magnesium into a 6 cm petri plate on ice.

2.  Dispense 10 μl of blood near the center of a microscope slide
    (Fisher Premium Microscope Slides-Superfrost, cat \# 12-544-7 is one
    slide type that is used).

3.  Using a P10 or P20 hand pipettor, collect a marrow cluster and an
    additional smaller clump in approximately 7-10 μl volume. Note:
    Avoid collecting bony debris, as this interferes with the
    preparation of the smear.

4.  Gently release the marrow into the blood on the slide.

5.  Carefully sandwich a second glass slide (label ends of slides in
    opposition) over the first and allow the blood to spread almost
    completely throughout the slides (about 30 seconds).

6.  Smoothly draw the slides apart and place the bone marrow smear under
    a blowing fan to dry.

Note: Watching a hematologist or laboratorian prepare aspirate smears of
human bone marrow may be helpful.

# Cytospin Slide Preparation

> **[Materials]{.underline}**
>
> Glass microscope slides. (One slide type that is used is: Fisherbrand
> Superfrost/Plus Microscope Slides, Fisher Scientific, Cat #:
> 12-550-15)
>
> A cytospin that is commonly used is from Shandon:
>
> Shandon Cytospin 3
>
> Shandon Filter Cards (5991022)
>
> Shandon CytoFunnels
>
> Buffer. (One buffer that is used is Hepes Buffered Sterile Saline,
> Ca/Mg free, Phenol Red Free, with 2%heat inactivated fetal bovine
> serum and 2.5% Gibco-BRL Cell Dissociation Buffer)

### Procedure

1.  Label glass slides using a pencil. Prepare an even number of slides
    as the Cytospin needs to be balanced. Keep cell samples on ice prior
    to loading into Cytospin.

2.  Insert each slide face-up into the Cytospin cartridge, slip a filter
    card over the slide, followed by a cytofunnel over the filter card.
    Clasp the cartridge together. View the backside of the cartridge to
    ensure that the opening from the filter card and funnel are
    lined-up. If not, unclasp the cartridge and align.

3.  Dispense 200 μl of cold buffer into each funnel..

4.  Then load 50,000 to 150,000 cells in a volume of 200 μl per slide
    into the cytofunnel

5.  Centrifuge at 400 rpm for 8 minutes on medium acceleration.

6.  After centrifugation, it is important that any remaining moisture be
    rapidly dissipated. A small fan can be useful for this.

#### [Cleaning CytoFunnels]{.underline}

1.  Submerge cytofunnels in a container of distilled water with about 5%
    bleach.

2.  Rinse cytofunnels with distilled water at least twice.

3.  Pat cytofunnels dry.

# 

# 

## 

##### 

## 

## Wright Giemsa Staining, One Approach

###### Materials

Fisher Protocol Wright Giemsa Stain (cat #: 264-984)

Fisher Protocol pH 6.8 Phosphate Buffer (cat #: 262-236)

Fisher Protocol Giemsa Stain Original Azure Blend Type (cat #: 027-273)

Methanol

5 Coplin Jars

Mounting medium. One mounting medium that is used is Cytoseal 60
(Fisher, cat #: 8310-4)

Glass Microscope Slides. One type that is used is Fisher Premium
Microscope Slides-Superfrost (cat \# 12-544-7)

### Procedure

Prepare the following Coplin Jars:

Jar 1: Fresh Methanol

Jar 2: 30 ml Wright Giems Stain

Jar 3: 5 ml Wright Giemsa Stain

0.4 ml Azure Blend

30 ml ddH20

Jar 4: 3ml Wright Giemsa Stain

30 ml pH 6.8 Phosphate Buffer

Note:

Upon mixing of solutions in Jars 3 and 4, an oxidate forms at the
surface of the solutions. This oxidate needs to be removed for a clean
staining. To do so, use an electronic pipettor fitted with a 25 ml
pipette, and sample up and down (generating bubbles helps to lift the
oxidate which will then attach to the walls of the pipette). Repeat this
procedure until any visible masses of oxidate disappear (usually twice
is sufficient). Then place 5 microscope slides and let sit for a minute.
Repeat this. The slides should now be free of oxidative debris when
lifted out of the Giemsa mix. Slides pulled out from Jar 4 should
exhibit a pink blush. The stains are now ready for use.

1.  Fix slides in methanol for 30 seconds. Then dab the slides onto an
    underpad or Kimwipe to remove excess methanol.

2.  Place the slides in Jar 2 for 3 minutes. Then dab the slides onto an
    underpad or Kimwipe to remove excess stain.

3.  Place the slides in Jar 3 for 10 minutes. Then dab the slides onto
    an underpad or Kimwipe to remove excess stain.

4.  Place the slides in Jar 4 for 2 minutes. Then dab the slides onto an
    underpad or Kimwipe to remove excess stain.

5.  Place the slides into a Coplin jar containing distilled water.
    Agitate the jar containing the slides and then pour out the wash.
    Repeat this wash. Remove the slides and then briefly rinse both
    surfaces with distilled water.

6.  Use a large Kimwipe to clean the undersurface of the slides, then
    place the slides under a blowing fan to air dry.

7.  Coverslip with Cytoseal 60

Note: Prepare new Giemsa staining solutions weekly.

#