Barr Body Staining

1. 1
BARR BODY STAINING
CONTENTS
Introduction
Mechanism
Barr body stain
Methylene blue
 Objectives
 Materials
 Procedure
 Interpretation
Scrapping of Cheek cells
Giemsa stain
 Materials
 Procedure
 Observations
 Precautions
Thionin staining
 Procedure

2. 2
BARR BODY STAINING
Introduction
A Barr body (named after discoverer Murray Barr) is the inactive X chromosome
in a female somatic cell, rendered inactive in a process called lyonization, in those
species in which sex is determined by the presence of the Y (including humans) or
W chromosome rather than the diploidy of the X or Z. The Lyon hypothesis states
that in cells with multiple X chromosomes, all but one are inactivated during
mammalian embryogenesis. This happens early in embryonic development at
random in mammals, except in marsupials and in some extra-embryonic tissues of
some placental mammals, in which the father's X chromosome is always
deactivated.
In men and women with more than one X chromosome, the number of Barr bodies
visible at interphase is always one less than the total number of X chromosomes.
For example, men with a 47,XXY karyotype have a single Barr body, whereas
women with a 47,XXX karyotype have two Barr bodies. Barr bodies can be seen
on the nucleus of neutrophils.
Barr Bodies: heterochromatinized X-chromosomes

3. 3
Mechanism
A normal human female has only one Barr body per somatic cell, while a normal
human male has none.
Mammalian X-chromosome inactivation is initiated from the X inactivation centre
or Xic, usually found near the centromere. The center contains twelve genes, seven
of which code for proteins, five for untranslated RNAs, of which only two are
known to play an active role in the X inactivation process, Xist and Tsix. The
centre also appears to be important in chromosome counting: ensuring that random
inactivation only takes place when two or more X-chromosomes are present. The
provision of an extra artificial Xic in early embryogenesis can induce inactivation
of the single X found in male cells.
The roles of Xist and Tsix appear to be antagonistic. The loss of Tsix expression on
the future inactive X chromosome results in an increase in levels of Xist around the
Xic. Meanwhile, on the future active X Tsix levels are maintained; thus the levels
of Xist remain low. This shift allows Xist to begin coating the future inactive
chromosome, spreading out from the Xic. In non-random inactivation this choice
appears to be fixed and current evidence suggests that the maternally inherited
gene may be imprinted.
It is thought that this constitutes the mechanism of choice, and allows downstream
processes to establish the compact state of the Barr body. These changes include
histone modifications, such as histone H3 methylation and histone H2A
ubiquitination, as well as direct modification of the DNA itself, via the methylation
of CpG sites. These changes help inactivate gene expression on the inactive X-
chromosome and to bring about its compaction to form the Barr body.

4. 4
Nucleus of a female amniotic fluid cell. Top: Both X-chromosome territories are detected by
FISH. Shown is a single optical section made with a confocal microscope. Bottom: Same nucleus
stained with DAPI and recorded with a CCD camera. The Barr body is indicated by the arrow, it
identifies the inactive X (Xi).
Left: DAPI stained female human fibroblast with Barr body (arrow). Right: histone macroH2A1
staining. Arrow points to sex chromatin in DAPI-stained cell nucleus, and to the corresponding
sex chromatin site in the histone macroH2A1-staining.

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Barr body stain
In 1949 Barr and Bertram reported that nerve cells from cats showed a sexual
difference (dimorphism). Cell nuclei from female cells consistently showed a
small darkly staining body near the nuclear envelope, while cell nuclei from males
showed no such structure. It is now known that this difference is present in most
cell nuclei from most of the mammalian groups, including humans. The darkly
staining structure found only in the nuclei of cells from females is known as the
Barr body.
Further research has shown that the Barr body is actually an inactivated X
chromosome. For proper functioning, most cell types must have one and only one
active X chromosome at any given time. Since each female cell has two X
chromosomes, one of them must be inactivated for normal cell functioning. Male
cells have only one X, so they don't have a spare to inactivate and display as a Barr
body. Infrequently, some females end up with three X chromosomes (XXX or
trisomy X). The cell nuclei of these individuals show two Barr bodies.
The fact that the number of X chromosomes is directly related to the number of
Barr bodies in the nucleus is commonly used to confirm the sex of women athletes.
Condition Sex Chromosomes Number of Barr
Bodies
Normal Female XX 1
Normal Male XY None
Trisomy X XXX 2
Turner'sSyndrome X None
Kleinfelter's Syndrome XXY 1

6. 6
Methylene blue
Objectives
Students will be able to
1. recognize cell structures.
2. compare and contrastmale and female cheek cells.
3. identify the Barr bodyin a female cheek cell.
Materials
Light microscope Beaker Paper towel
Glass slide Dropper pipette Lens paper
Cover slip Water
Flat toothpick Methylene blue (0.3%)
Procedure
1. Obtain a clean glass microscopeslide.
2. Place a drop of water in the center of the slide.
3. Gently scrape the inside of your cheek with the flat edge of a
toothpick.
4. Spread cells in the drop of water.
5. Place cover slip on top of specimen. Use correct technique to prevent
air bubbles.
6. Observe, diagram, label, and record magnification.
7. Apply one drop of methylene blue along one side of the cover slip.

7. 7
8. Along the opposite edge of the cover slip, gently touch with a piece of
paper towel. This will draw the methylene blue under the cover slip
to stain the cells.
9. Observe, diagram, label, and record magnification.
10. Switch slide with someone of the opposite sex.
11. Observe, diagram, label, and record magnification.
Preparation of slide and index fingernail:
1. Clean a microscopeslide well with soap &
water, dry with a non-linty paper towel.
2. Cleanse very thoroughly under the nail of
your index finger.
3. Place a small drop of dH20 in the center of
the very clean slide.

8. 8
Harvest the cells, prepare the smear:
4. Gently scrap the inside ofyour cheek to pick
up some of the shed stratified squamous cells.
Do not scrapechunks. A toothpick may be
used if you have no fingernails. Gentle
scraping is the watchword, there should be no
discomfort.
5. Express the material from under your nail
by pressing with your thumb, and press the
material into the drop of water on the slide,
mix and spread the material around to the size
of a dime:

9. 9
Fix the smear:
6. Pass the slide through the flame several times
to fix the smear. Do not heat the slide above a
temp which is comfortable.
You are merely "gluing" the smear to the slide.
Stain the smear:
7. Place a drop of 0.3% methylene blue on
the specimen. Let sit for 1 minute.
8. Rinse off the excess stain with tap
water. (Do not splash on your white shirt!)
9. Blot dry with an non-linty paper towel
or bibulous paper. Do not rub.

10. 10
10. Flame again briefly to dry slide.
Examine under the microscope:
11. Examine first with the 4x objective,
scanning the entire field to find a well-
distributed region. Avoidregions where
cells may be piled up to thickly . Then view
with the 10x and 40x objectives, illustrating
the view at bothpowers. Note 1) the
nucleus, 2) nucleolus, 3) cell boundary and
4) the variety of bacteria colonizing the
surface of the cells.
Interpretation
1. Barr Body: Crumpled dark chromosome seen in cell
2. Suggests normal female karyotype 46XX

11. 11
Scrapping of Cheek cells
A buccal smear is a test where cells are taken from the tongue. Cells are collected
by scraping the tongue with a spatula. The cells are then placed on a slide and the
sample is taken to the laboratory for evaluation. The cells are evaluated for the
presence of Barr bodies (a mass seen in a normal female sex chromosome). The
buccal smear test can confirm whether the patient is a male or female.
The cells which line the inside of your cheeks are classified as a stratified
squamous epitheliumtissue and are the surface of a mucous membrane. These flat,
scale-like buccal cells (pronounced "buckle") are shed constantly as the tissue is
renewed. By gently scraping the inside of your cheek, these cells can be harvested,
and when smeared and stained, may be used to illustrate a number of important
biological phenomena including cell and tissue structure, oral bacterial flora and
morphology, etc. This tissue is non-keratinized and therefore the surface cells are
living and still possess their nuclei, in contrast with shed epidermal cells.

12. 12
Giemsa stain
The study of the Barr bodyfrom the (female) smear of Buccal epithelial cells.
Materials
 Buccal epithelial cells
 Giemsa stain
 Carnoy’s fixative
 Slides
 Cover slip
 Microscope, etc.
Procedure
1. Gently rub the inside of the cheek with a flat rounded piece of wood and
transfer the scraping over a clean glass slide.
2. Then, made a thin film of cells on the slide and keep them for air-drying.
3. Air-dried smear was kept in Carnoy’s fixative for 30–35 minutes.
4. Then, the Giemsa stain was poured and allowed to stand for 20–25 minutes.
5. After staining, the slide was washed with distilled water to remove the
excess stain.
6. Finally, the slide was kept for air-drying and then observed under the
microscope.

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Observations
It is found that very lightly stained cells are scattered here and there in the smear.
In the cells, violet-Barr bodies are observed inside a pink nucleus. A Barr bodyis
nothing but an inactivated (heterochromatinized) X chromosome. It was first
observed by Murray Barr in 1949. It is found only in female cells, because in those
1 X chromosomeis enough for metabolic activity. It is absent in male somatic
cells, becausethere only 1 X chromosomeis present, which is in an active state.
Giemsa stained Barr bodies
Precautions
The smear or film should be uniform and thin over the glass slide so that the cells
will not overlap each ot Giemsa stain

14. 14
Thionin staining
In this section, you will make a cheek cell smear as above, but use a sensitive DNA
stain (Thionin) that will make Barr bodies visible.
Procedure:
The Thionin staining protocol is a good deal more delicate and involved than the
methylene blue stain you used previously. Follow these directions carefully.
1. Wash your slide in detergent and rinse well in deionized water. Rinse the slide
in a stream of 70% ethyl alcohol from a squeeze bottle. Allow the slide to air dry
on a clean paper towel.
2. To avoid contamination with bacteria, which stain nicely and might look like a
Barr body, rinse your mouth very well with tap water at least three times.
3. Immediately after rinsing, Gently scrape the inside of your cheek with the
toothpick and then smear it across the slide.
4. Allow the slide to air dry for 1 minute.
5. Treat slides very gently from now on because excess agitation or a direct stream
of water will wash away cells.
6. Gently wet the cells with an indirect stream of deionized water.
7. While holding the slide over the sink, cover the cells with several drops of 6N
HCl and gently rinse off with an indirect stream of deionized water after 10
seconds. This strength of HCl will burn eyes and skin. Use protective eyeware.
This step breaks down RNA which also stains with thionin and can resemble or
obscure Barr bodies.

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8. Gently wash the cells with an indirect stream of deionized water for about 1
minute.
9. Cover the cells with several drops of thionin stain and place slide in covered
petri dish to prevent evaporation. Caution! Stains fingers and clothing.
10. Stain for 15 minutes.
11. Gently wash the cells with an indirect stream of deionized water for about 1
minute.
12. Remove excess water and place cover slip over cells.
13. Examine cells under oil immersion. Barr bodies should be stained dark blue or
black and appear near the nuclear envelope.
14. Switch slides with someone of the opposite sex. Make sure you observe Barr
bodies in female cells. If you're a male and you find a Barr body in your nuclei,
don't panic. About 2% of male cheek cells show dense chromatin that resembles a
Barr body.
15. Sketch a representative of each sex.