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

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

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

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.

5

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's Syndrome X None

Kleinfelter's Syndrome XXY 1

6

Methylene blue

Objectives

Students will be able to

1. recognize cell structures.

2. compare and contrast male and female cheek cells.

3. identify the Barr body in 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 microscope slide.

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

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 microscope slide 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 dH 20 in the center of

the very clean slide.

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 scrape chunks. 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

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. 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 both powers. 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

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

Giemsa stain

The study of the Barr body from 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.

13

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 body is

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 chromosome is enough for metabolic activity. It is absent in male somatic

cells, because there only 1 X chromosome is 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

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.

15

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.