Staining Methods And Aseptic Techniques

 INTRODUCTION

The microscopic examination of stained preparation.

1. Gives idea of the morphology of bacteria ( cocci, bacilli, spirilla, etc.).
2. Helps to determine the staining affinity of bacteria  ( Gram positive, Gram negative, acid fast, etc.).
3. Demonstrates the purity of the culture.
4. Demonstrates the bacteria present in the sample material to be cultured.
5. Helps to diagnose organisms such as gonococci, acid fast bacilli, etc. By direct microscopic ex- amination.

Staining requirements

• Stains: They can be basic, acidic or neutrals salts. Most of the stains that are used to stain bacteria are of the basic type. The nucleic acid of bacterial cells attracts the positive ions of basic stains. Ex- Basic fusion, methylene blue, crystal violet, etc. Acidic stain are mainly used in microbiology to provide background staining. Example - Eosin.  Neutral stains are used to stain nucleic acid and cytoplasm. Example - Giemsa stain. The majority of stains used in microbiology are of the  synthetic types and are manufactured from aniline. Hematoxylin is, however a neutral dye.
• Mordants: The chemical which is required to bring about the staining reaction is called a mordant. Basic mordants react with acidic stains and acidic mordant react with basic stains. Examples - phenol in corbol - fuchsin ( Ziehl - Neelsen technique) and iodine which is added after the application of crystal violet in the Gram's staining method.
• Decolorization( differentiation):  During the regrassive staining produce, all the constituents of the smear are stained.  This is then followed by washing out and selective removal of excess stain. This procedure is referred to as decolorization, or differentiation. For basic stains an acidic decolorizer is used and for acidic stain an alkaline decolorizer is used. In progressive staining technique, several stains are used in sequence for specified times, without the use of decolorizer.