Citrus Systemic Pathogen Research

CTV Inclusion Virus Detection

 

Stains & Reagents     < Previous -- Next >

  • Calcomine Orange 2RS
  • Luxol Brilliant Green BL
  • Azure A
  • 2-methoxyethanol (Ethylene glycol monomethyl ether or Methyl cellusolve)
  • 0.2 M dibasic sodium phosphate
  • 2-methoxyethyl acetate
  • 95% ethanol
  • Euparal mounting media

Stains

  • 0.1 g Azure A in 100 ml 2-methoxyethanol.  Stir to dissolve and filter before use.
  • 1 g Calcomine Orange in 100 ml 2-methoxyethanol.  Stir to dissolve and filter before use.
  • 1 g Luxol Brilliant Green in 100 ml 2-methoxyethanol.  Stir to dissolve and filter before use.

Azure A staining procedure

  • Mix the following in a small staining dish:
  • 2 parts 0.2 M dibasic sodium phosphate
  • 18 parts Azure A

Float tissue sections on or in dye mixture for 10 to 15 minutes.  After that time, remove stain and de-stain by adding and quickly removing (5-10 seconds per change) 95% ethanol for a total of about 30 seconds or until tissue is properly de-stained.  Replace ethanol with 2-methoxyethyl acetate which stops the de-staining process.  Carefully remove tissue sections and place on a drop of Euparal on a glass slide.  Place a coverslip over the tissue sections.

Calcomine Orange/Luxol Brilliant Green (0-G) staining procedure

Mix the following in a small staining dish:

  • 1 part water
  • 1 part Calcomine Orange
  • 8 parts Luxol Brilliant Green

Float tissue sections on or in dye mixture for 10 to 15 minutes.  After that time, remove stain and de-stain by adding and quickly removing (5-10 seconds per change) 95% ethanol for a total of about 30 seconds or until tissue is properly de-stained.  Replace ethanol with 2-methoxyethyl acetate which stops the de-staining process.  Carefully remove tissue sections and place on a drop of Euparal on a glass slide.  Place a coverslip over the tissue sections.

Staining reactions of host cell constituents (from: Christie & Edwardson, 1994)

Staining reactions of host cell constituents

References

Brlansky, R. H. and R. F. Lee.  1990.  Numbers of inclusion bodies produced by mild and severe strains of citrus tristeza virus in seven citrus hosts.  Plant Dis.  74:297-299.

Christie, R. G. and J. R. Edwardson.  1994.  Light and electron microscopy of plant virus inclusions.  Fla. Agric. Exp. Stn. Monogr. Ser. 9.  164 pp.

Lee, R. F., R. H. Brlansky, S. M. Garnsey and Y. K. Yokomi.  1987.  Traits of citrus tristeza virus important for mild strain cross protection of citrus: The Florida approach.  Phytophylactica.  19:215-218