Preparation of specimen
Although living microorganisms can be directly examined with light microscopy, they often must be fixed and stained to increase visibility, accentuate specific morphological features and preserve them for future study
Fixation:
• Is a process by which the internal and external structures of cells and microorganisms are preserved and fixed in position
Two different types of fixation
• Heat-fix bacterial smears by gently flame heating an air-dried film of bacteria. This preserves overall morphology but not structures within cells
• Chemical fixatives such as ethanol, acetic acid, mercuric chloride, formaldehyde and glutaraldehyde penetrate the cells and react with cellular components, usually proteins and lipids to render them inactive, insoluble and immobile
Dyes:
Dyes have two features in common:
• They have chromophore groups (groups with conjugate double bonds that give the dye its color)
• They can bind with cells by ionic, covalent, or hydrophobic bonding. Ex. +vely charged dye binds to –vely charged structures of the cell
Ionizable dyes are divided into two classes based on their charged group
Basic dyes – methylene blue, basic fuchsin,crystal violet, safranin, malachite green – have +vely charged groups. These bind to –vely charged molecules like nucleic acids and many proteins. Surfaces of bacterial cells are –vely charged, so most often used in bacteriology
Acid dyes – eosin, rose Bengal and acid fuchsin – posses –vely charged groups such as carboxyls (-COOH) and phenolic hydroxyls (-OH). These bind to +vely charged cell structures.
Staining Methods:
Preparation of bacterial smears:
All the methods start by preparing 'fixed' smears on the microscope slides. An aqueous suspension is first made, in the case of a non-liquid specimen, by taking a small amount of the sample and suspending it in a single drop of distilled water on a 25 mm x 75 mm slide. Do not make the smear too thick. If you have a liquid sample, a single drop is used directly from the culture container. The suspension made by either method is air dried, then "fixed" by passing rapidly through the Bunsen flame two or three times. Cover the smears with 95% methyl alcohol for 1 min, and then allow the smear to cool before staining.
Staining of specimens
Staining technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. Stains may be used to define and examine bulk tissues, cell populations, or organelles within individual cells.
Stains
A stain is a substance that adheres to a cell, giving the cell color. The presence of color gives the cells significant contrast. Different stains have different affinities for different organisms, or different parts of organisms. They thus may be used to differentiate different types of organisms or to view specific parts of organism.
Simple stains
Is a stain using only a single dye that does not differentiate between different types of organisms. There is a single staining step and everything that stains is stained the same color.
Simple staining:
- • A simple stain is an aqueous or alcohol solution of a single basic dye
• Simple stains usually highlight the entire microorganism so that cellular shapes and structures are visible.
• Stain is applied to the fixed smear for a certain length of time and then washed off, and the slide is dried and examined.
• Basic dyes like crystal violet, methylene blue and carbolfuchsin are frequently used to determine the size, shape and arrangement of bacteria
A differential stain uses more than one dye and stains different kinds of organisms with different colors. The differential stains are Gram stain and Ziehl - Neelsen acid fast stains.