ANDC DU/Biology Protocols/vital staining

To study mitochondria in striated muscle cells using vital stain Janus green B

Introduction

Mitochondria are energy-converting organelles that make ATP; thus, they are the "powerhouse" of the cell. A mitochondrion is divided into two compartments, the mitochondrial matrix and inter-membrane space, by two layers of membranes, the outer and inner membrane. Because a mitochondrion has its own DNA, it can be considered a semiautonomous organelle. Cytologists observed the presence of small granules in different types of cells. In 1890 Robert Altman noted that these subcellular granules were similar in size to bacteria; accordingly, he named them "bioblasts". He speculated that these structures were the basic units of cellular activity and that, by virtue of their colonial association in the cytoplasm of the host cell; the cell acquired the properties of life. This concept is still accepted today in a modified form, namely the endosymbiont theory of the origin of mitochondria. The term mitochondrion was coined by C. Bender in 1898; in Greek "mitos" is "thread" and "chondros" is "granule". Mitochondria in living cells can be stained using dyes.

In 1900 L. Michaelis selectively stained mitochondria with the dye Janus Green B. Since this is a redox dye, which must be oxidized to become colored, Michaelis proposed that mitochondria were cellular oxidizing agents. Another dye for mitochondria staining is the vital fluorescent dye rhodamine 123 (with vital dyes cells are stained in the living state) Staining of mitochondria in living cells has demonstrated the plasticity of mitochondria: the organelles change shape, move, coalesce and divide.

Vital stains are stains that do not kill cells; for example, Neutral red becomes concentrated in the vacuoles of plant cells and Janus green B is oxidized in the mitochondrion to form a blue stain. Such staining will kill the organisms eventually, but makes detailed analysis of cells easier. These dyes can be added as 0.02% aqueous solutions.

Some useful vital stains and what they colour are as follows: Neutral red - food vacuoles, Janus green B - mitochondria, Sudan black B and Sudan IV - lipids. Although the organisms will be killed rapidly, acidified methyl green can be used to stain nuclei.

Principle

The specificity of this stain for mitochondria is due to the activity of cytochrome oxidase which maintains the dye in its oxidised , blue green stqate while it is reduced in the reminder of the cell to a colourless leuco-compound. The chemical basis of the reaction has been studied by Lazarow NS and Cooperstein (1953). Janus geen B is chemically a diethyl safraanin-azo-dimethyl aniline with absorption maxima at 585, 390 and 285 mu at pH 7.4. reduction by sulphydryls or lack of oxygen can lead to reddish stain due to safranin after splitting of the molecule at the azo bridge.

Structure of Janus Green

Requirements

Biological: Live Insect
Chemicals:

• Insect Saline (100ml)
• NaCl 9.00 g/l
• KCl 0.20 g/l
• Cacl2.2H20 0.27 g/l
• Dextrose 4.00 g/l
• Janus green B 0.02% in insect saline

Procedure

• Collect a live insect from the garden/field.
• Dissect out the flight muscle from the thoracic region of the insect.
• Place the muscle on glass slide on a drop of saline.
• Tease the tissue with the help of needle.
• Incubate the tissue in 0.02% stain for 30 minutes in presence of oxygen.
• Place the coverslip and allow some air bubbles to get entrapped inside the coverslip.
• Observe the slide under microscope.

Observation and Result

Numerous spherical, round shaped blue green bodies were observed.

Precautions

• To avoid early death of tissue, keep the tissue in saline immediately after dissection.

• Stain the tissue immediately.

• Ensure the presence of air bubbles beneath the cover slip.

Work in progress, expect frequent changes. Help and feedback is welcome. See discussion page.