Karyotyping, Chromosome 
     Banding and  
     Chromosome Painting
    1.Karyotyping
                   Karyotyping
   Definition: “It is a process of arranging 
   each pair of homologous chromosomes  in 
   a  sequence,  the  longest  chromosomes 
   being  placed  at  the  beginning  and  the   
   shortest at the end”.
   Indications for chromosome analysis:
         oMultiple congenital 
           abnormalities
         oUnexplained mental 
           retardation
         oSexual ambiguity
         oInfertility
         oRecurrent miscarriage
         oStill birth
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         oMalignancy & chromosome 
            breakage syndrome
       Introduction to Karyotyping
  Greek: Karyon- kernel, seed or nucleus.
    The number of chromosomes in human cells is 46 
  with 22 autosomal  pairs and 2 sex chromosomes – 2 X 
  chromosomes  for  females  and  one  X  and    one  Y 
  chromosome for males.
    The chromosomes are visible only at the metaphase 
  stage of mitosis.
    Each  chromosome  has  a  characteristic  size  and 
  shape in the “normal” cell.
    During  most  of  the    cell  cycle,  interphase,  the   
  chromosomes are  somewhat less condensed and are 
  not  visible  as  individual  objects  under  the    light 
  microscope.
                                 Method:
           Samples which can be used:
                Skin
                Bone marrow  
                Peripheral 
                  blood
                Amniotic fluid 
                  (Chorionic villi 
                  sampling)
           Blood culture 
             media preparation:
           Blood culture media; 500 ml RPMI 1640 with 
         100ml fetal bovine serum,  6.5ml penicillin – 
         streptomycin and 7ml glutamine.
             Dispense 10ml aliquots into sterile tube and 
           add 2% (0.2ml) PHA to each tube. Store at 4°C 
           for as long as 2 weeks.
           Lymphocyte  cells  do  not  normally  undergo 
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         subsequent  cell  divisions.  In  the    presence  of  a 
         mitogen (PHA), lymphocytes are stimulated to enter into 
         mitosis  by DNA replication. After 48-72 hours, a mitotic 
         inhibitor (colcemid) is added  to the culture to stop 
         mitosis in the metaphase stage.
             Other culture media which can be used are TC 
           199 and HAM F10.