SPIROCHETE STAINING

INTRODUCTION
Spirochete are gram negative,flexible,spiral shaped organisms who have distinct morphoplogy and mechanism of locomotion. Length of spirochete varies from 6500m and width ranges from 0.1-3 m. Instead of having flagella projection from the cell, the flagella of spirochetes form central axial fibrils(endoflagella) that runs through the periplasm. Periplasmic flagella (axial fibrils , axial filaments) are wound around the helical cells between the protoplast and the outer sheath. There may be two to more than 100 flagella per cell. One end of each flagellum is anchored to a point at the pole of the cell while the other end is unanchored. The outer sheath contains lipid, protein and carbohydrate and varies in structure between different genera. Since spirochetes are very delicate and extremely thin, special staining methods are employed for stsining of spirochete.

FONTANAS STAINING METHOD Principle

Due to delicate nature of spirochetes, heat fixation is avoided as it destroys the shape of organisms. Chemical like formalin and glacial acetic acid are used for fixation. Moreover,the use of mordant containing tannic acid increases the affinity of stain towards the cell. Finally, when the stain containing ammoniacal silver nitrate is heated up, silver oxide is formed which precipitates on the organism thereby the apparent diameter is adequately increased and organisms are visible.

Requirements
1.Fontanas fixative, Fontanas mordant, Fontanas stain and 95% ethanol

Procedure
1. Sterilize a pin by dipping in alcohol and flaming it Bunsens burner. 2. Allow the pin to cool down, and scrap out tartar from interspaces between teeth . 3. Emulsify tartar in a drop of distilled water placed in center of the slide. 4. Spread and prepare a smear over a small area. Allow it to air dry ; dont heat fix the smear. 5. Immerse the slide in coplin jar containing Fontanas fixative for 30 seconds. Repeat the process for three at interval of 1 mintue each. 6. Add Fontanas mordant on the smear, and heat till rises for 30 seconds. 7. Wash well in distilled water and allow the slide to dry. 8. Add Fontanas stain, and heat till rises for 30 seconds. 9. Wash well in distilled water, drain , blot dry and exmine.

RESULT
Spirochete are stained brownish-black in color against brown background.

BACKERS STAINING METHOD

Principle
Mechanism of fixation and mordant is similar to that of Fontanas method.Here , organisms are stained with Beckers stain which is made up of basic fuchsin dissolved in alcohol and as the alcohol evaporates, colloidal precipitate formed are deposited on spirochetes. This leads to increase in size of organisms and makes them visible.

Requirements
1.Fontanas fixative, Fontanas mordant and Beckers stain and 95% Ethanol

Procedure
1.Prepare smear in the manner similar to that of Fontanas technique. 2.Immerse the slide in coplin jar containing Fontanas fixative for 30 seconds. Repeat the process for three at an interval of 1 minute each. 3. Pour Fontanas mordant on the smear and allow it to act for 3-5 min. 4.Rinse the slide in running water for 30 seconds. 5.Treat the smear with Beckers stain for 3-5 min. 6.Rinse in tap water , drain, blot and exmine. Result Spirochetes stained pink in color. OTHER METODS 1.Rues method: In this method the apparent thikness of the organism is increased by application of basic dyes in presence of mordant like sodium bicarbonate, which settles onto the cell surface. 2.Levaditis method: This method is used to detect spirochetes in tissue. It is modification of Beckers method in which silver nitrate-pyridine is used instead of basic stain.

EXAMPLE OF SPIROCHETES
Treponema denticola :found in oral cavities of animal Treponema macrodenticum :commensal present in gums of teeh. Treponema oralis :found in oral cavities of animals. Treponema palladium :casues syphilis. Spirillum minus : causes rat bite fever.