25 points total possible

Table of Contents: 

     Background Information 
     Directions 
     Test Questions 
     Teacher's Notes 
     Teacher's Answer Key 
 
 

Background Information 

Occasionally it is useful to determine the sex of a person by doing a sex chromatin test. This is done in cases such as the birth of a baby with "ambiguous genitalia" in which the sex of the baby is not immediately apparent.  By taking a few cells from the lining of the baby's mouth (buccal mucosa) it is possible in about an hour to determine the "genetic" sex of the child by looking for X inactivation as evidenced through the presence of "Barr Bodies".

If the child is genetically female then a Barr body should be evident in approximately 10-20 percent of the cells.  It is not present in all cells since the viewing of the Barr Body is dependent on the position of the cell on the slide to which it is fixed. A male will show 0-2 percent evidence of Barr Bodies. The two percent represents a small proportion of false positives. Occasionally it is possible to detect abnormal karyotypes such as Turners
syndrome and Klinefelter syndrome (see attached descriptions) using a buccal smear. In Turners syndrome a phenotypically female exhibits no Barr Bodies because only one X chromosome is present. Her karyotype is 45,X. In Klinefelter syndrome a phenotypic male exhibits one Barr Body since the karyotype is 47,XXY and one of the X's must inactivate. In rare cases such as 48,XXXX, multiple Barr Bodies may be observed in the same cell. Only one X can be active in somatic cells so all of the others will become inactive heterochromatic
Barr bodies.

Chromosomal Syndromes

     Klinefelter Syndrome

          Features: 
               Tall stature 
               Disturbed body proportions 
               Deficient maturation of male secondary sexual characteristics 
               More or less pronounced gynecomastia 
               Later disturbances of sexual function - infertility 
               Average intelligence may be slightly reduced.

          Cytogenetics: 
               Each male has two X chromosomes and one Y chromosome (47,XXY)

          How?: 
               Nondisjunction (possibly maternal)

          Incidence: 
               1/1000 Caucasian population - 1 in about 50 infertile men

          Detection: 
               Usually found due to infertility

          Prognosis: 
               Life span presumably normal

     Turner Syndrome

          Features: 
               Shortness of Stature 
               Atypical faces 
               Webbed Neck 
               Shield Chest 
               Cubitus valgus 
               Sexual infantilism 
               Primary amenorrhea 
               Sterility 
               (Mental retardation is slight but increased over general population)

          Cytogenetics: 
               Monosomy for the short arm of the X chromosome (one X chromosome is present) 45,X

          How?: 
               Monosomy X may be caused by nondisjunction in gametogenesis in the mother or the father, or may be the result of postfertilization errors

          Incidence: 
               1/10,000 live births but 95 percent of cases are miscarried.

          Detection: 
               In newborn - by webbed neck or lymphedema 
               In childhood - by short stature and cardiovascular malformation 
               In adolescence - by short stature, primary amenorrhea

          Prognosis: 
               Presumably a normal life span - may need therapy for psychological

Directions 

The presence of the sex chromosomes of man can be observed in interphase nuclei by means of certain staining techniques. One of the two X chromosomes in female cells is facultatively heterochromatic and is condensed during interphase forming the "X chromatin body" or "Barr body". A Barr body is about 1 micrometer in diameter and is located at the periphery of the nuclear membrane. There is one Barr body per diploid set of chromosomes, but the number of Barr bodies is one less than the number of X chromosomes. Barr bodies can be determined most easily in buccal mucosa, hair roots and fibroblast cells. The normal positive range for sex chromatin bodies is 20-60 percent. A female patient shall be considered normal if the range is +15 percentage points from normal controls. Negative must be less than 4% - any number between 4-15 may be suggestive of mosaicism (meaning some normal cells and some abnormal cells). The reason Barr bodies are not seen in all cells even though they should be present is that you are observing a three dimensional cell in a two dimensional way -
the Barr body may simply be out of your line of view.

This laboratory will take three days - with a little help from the teacher in between. We will be determining whether Barr bodies are present in buccal mucosa cells. These are the cells that make up the lining of your mouth.

Purpose: To identify X-chromatin and to determine its frequency in the interphase nuclei of the buccal mucosa.  To explore the differences in X-chromatin between the sexes and in genetic disorders involving the sex chromosomes.

Materials:

     Cresyl violet acetate (substitute for cresyl echt violet) working solution 
     Ethanol (95%) 
     Ethanol (50%) 
     Fixative 
     Xylol 
     Microscope Slides 
     Plastic Spoons or Wooden Spatulas 
     Microscope (Oil immersion is best but a good light-microscope with 400x magnification will suffice if necessary)

Procedure:

     Day 1 

        1.Rinse your mouth vigorously with tap water. 
        2.Using medium pressure scrape the inside of your cheek with the plastic spatula or spoon. Discard the first sample by wiping the tool on a clean towel. This will cut down on the bacterial content of the sample you are testing. 
        3.With the same tool, sample the area again. 
        4.Spread the material across the surface of a clean glass slide. 
        5.Prepare a second slide from the same cheek in the same manner. 
        6.Place the slides for at least one hour in a jar or beaker containing 3:1 fixative (methyl alcohol:acetic acid). 
        7.Remove the slides and air dry. (Your teacher will do this part for you so be certain you have labeled your slides before you leave).

     Day 2 

        1.Place both slides with your fixed buccal mucosa smears into a jar containing 1% creyl violet acetate stain solution for 5 minutes. 
        2.Rinse slides by dipping quickly into three separate jars (called changes) of 95% ethanol. 
        3.Rinse the slides by dipping into two changes of xylol. Then place in fresh xylol for two minutes. 
        4.Permit the slides to air-dry overnight

     Day 3 

        1.Examine the slides by transmitted light (oil immersion is best if it is available). 
        2.Count 200 nuclei (100 per slide). Count those that are not broken, folded, or excessively granular.
          Avoid areas heavily contaminated by debris or bacteria. 
        3.A nucleus is regarded as "positive" if it contains an X chromatin body that is clearly defined, dark staining, and touching the nuclear membrane. 
        4.If the X chromatin score in 200 nuclei is 0-2%, it is "negative". If the score is between 11% and 19% it is "positive".

Score Sheet 

Questions 

   1.Which chromosome does the Barr body represent?

   2.How many X chromosomes would a normal male have?

   3.How many Barr bodies would a normal male have?

   4.How many X chromosomes would a normal female have?

   5.How many Barr bodies would a normal female have?

   6.List at least two diseases that are inherited as sex linked and are carried on the X chromosome.

   7.Will the inheritance of these diseases be affected in any way by X-inactivation? How?

   8.Would a buccal smear study help determine the likelihood of a person carrying these abnormal genes?
     How?

   9.When would a doctor want to have a buccal smear done?

  10.Why do only some cells show the presence of the Barr body?

  11.Why is one X inactivated in females? Why not have two active ones?

  12.What is a fixative and why did you need to use it?

    13.- 20. Fill in the following chart.