How do you make malachite green for endospore staining? 1) Prepare a smear on a clear, dry glass slide. 2) Allow it to air dry and fix it with gentle heat. 3) Flood the slide with 1% w/v Malachite Green (S020). 4)Allow the stain to be in contact with the smear for 2-3 minutes and heat the preparation for 3-6 minutes and then allow to cool.
Full Answer
Principle of Endospore Staining
Bacterial endospores are metabolically inactive, highly resistant structures produced by some bacteria as a defensive strategy against unfavorable...
Reagents Used For Endospore Staining
Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution)0.5 gm of malachite green 100 ml of distilled waterDecolorizing agentTap water or Di...
Procedure of Endospore Staining
1. Take a clean grease free slide and make smear using sterile technique. 2. Air dry and heat fix the organism on a glass slide and cover with a sq...
Result of Endospore Staining
Endospores: Endospores are bright green. Vegetative Cells: Vegetative cells are brownish red to pink.Spores may be located in the middle of the cel...
Endospore Staining by Dorner’S Method
Carbolfuchsin stain0.3 gm of basic fuchsin10 ml of ethanol, 95% (vol/vol)5 ml of phenol, heat-melted crystals95 ml of distilled waterDissolve the b...
Examples of Endospore Staining
Positive Clostridium perfringens, C. botulinum, C. tetani, Bacillus anthracis, Bacillus cereus, Desulfotomaculum spp, Sporolactobacillus spp, Sporo...
Quality Control of Endospore Staining
Positive control: Clostridium perfringens (ATCC 13124) Negative control: Escherichia coli (ATCC 25922)
Schaeffer-Fulton stain technique
It is the most widely used technique for endospore staining. The technique was first described by Alice B. Schaeffer and MacDonald Fulton in the 1930s. The method utilizes malachite green as the primary stain and safranin as counterstain.
Other techniques of endospore staining
Although the principle of endospore staining is the same, variations exist in the choice of primary stain, counterstain, and whether or not decolorizer is used. Some of them are summarized below
What are the principles of endospore staining?
Image 2: Endospore staining procedure. Picture Source: microbiologyinfo.com
How is Endospore Staining done?
The staining method for endospores is done in two ways: Dorner’s Method staining technique (traditional method) and Schaeffer-Fulton staining technique (modified method).
What do you need?
Malachite green – it is used as the primary stain. The malachite green (0.5 grams) should be dissolved in water (100 ml).
Principle of Endospore Staining
Bacterial endospores are metabolically inactive, highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions. The bacteria can remain in this suspended state until conditions become favorable and they can germinate and return to their vegetative state.
Reagents used for Endospore Staining
Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution) 0.5 gm of malachite green 100 ml of distilled water
Procedure of Endospore Staining
Take a clean grease free slide and make smear using sterile technique.
Result of Endospore Staining
Endospores: Endospores are bright green. Vegetative Cells: Vegetative cells are brownish red to pink.
Examples of Endospore Staining
Positive Clostridium perfringens, C. botulinum, C. tetani, Bacillus anthracis, Bacillus cereus , Desulfotomaculum spp, Sporolactobacillus spp, Sporosarcina spp, etc.
Introduction
Endospore Staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample, which can be useful for classifying bacteria.Within bacteria, endospores are protective structures used to survive extreme conditions, but this protective nature makes them difficult to stain using normal techniques such as simple staining and Gram staining.
Principle Of Endospore Staining
The primary dye for endospore staining is malachite green. It takes a long time for the spores to stain due to their density, so time acts as the mordant when performing this differential stain; the slide with the bacterium should be soaked in malachite green for at least 30 minutes and then rinsed off with water which acts as the decolorizer.
Uses of the Endospore stain
For detection of Firmicute groups of bacteria i.e Clostridium spp and Bacillus spp
Introduction
A normal bacterial cell is known as a vegetative form of a bacterial cell. It can multiply freely when provided with conducive environmental conditions like the provision of nutrients, temperature, water, and oxygen.
Principle
Endospore staining is a differential stain that aims at detecting, identifying and differentiating an endospore from the vegetative cell (an underdeveloped endospore).
1. Schaeffer Fulton Stain
In basic Laboratories, the simplest endospore staining technique is the Schaeffer-Fulton technique because of its easy and it is rapid to identify the bacteria.
Disadvantages
Another endospore staining technique that is not commonly used is known as the Klein method of endospore staining.