Different techniques of handling microorganisms

Staining methods

In bright field microscopy, the microorganism background appears light and microorganism appear dark as they absorb some of the light.microorganisms are transparent and cannot observe easily on the transparent background.Therefore microorganism first treated with some colour dye which gives them characteristic colour,such coloured microorganism is easily seen in the transparent background.The process of treating microorganism with coloured dye is called staining.And the major purpose of staining is to increase the contrast between microorganism and background.

Types of staining

The most dye used for staining are synthetic which are derived from aniline and nitrobenzene.Each dye consists three components.

Benzene ring+chromophore+Auxochrome

Each dye contains the colourless benzene ring,chromophore, and auxochromes.The chromophore is functional that import colour to the dye and auxochromes is the functional group that gives the iconic property to the dye.The coloured portion of dye is called chromogen.Based on nature of chromogen dyes types are

Types of dye

Acidic dye(anionic dye):Coloured part is negatively charged.It is used to stain basic (Positively charged)components such as histone protein.It is used in negative(background)staining.Since most bacterial cells are negatively charged,an acidic dye cannot stain bacterial cell due to repulsion between similar charges eg eosin,Indian ink,nigrosin

Basic dye(cationic dye):coloured part of this dye is positively charged,an acidic dye is used to stain negatively charged(acidic)component such as the bacterial cell.Eg methylene blue,crystal violet,safranin,malachite green and carbon fuchsin.

Natural dye:in neutral dye both anion and cation are coloured.These are the salt of acidic and basic dye eg include Giemsa stain.

Mechanism of staining

The process of staining involves ion exchange ion between the stain and active site of the cell.For eg bacterial cell contain a large number of negatively charged proteins due to COO group.These negative group from salt with the positive ion such as Na.So,bacterial cell can be represented by

(Bacterial cells)(Na⁺)

These negatively charged bacteria are stained by cationic dye whose coloured positive ion replaces the ion.For eg methylene blue (which is cationic dye)can be represented as

MB⁺ Cl^-

When bacteria is stained by methylene blue,staining reaction os given by

(Bacterial cell)(Na)+(MB)(CL) →(Bacterial cell)(MB)+NaCl

Therefore,due to ion exchange reaction,an acidic dye can stain only basic components and basic dye can stain only acidic components.if material or cell component is nonionic,it cannot be stained.Difficulty in capsule staining is due to its nonionic property.

Types of staining

There are three types of staining technique.They are given as below

• Simple staining:

The main purpose of simple staining is to elucidate the morphology and arrangement of the bacterial cell.In this staining,the bacterial smear is stained with a single reagent.The commonly used basic stains are methylene blue,crystal violet, and carbon fuchsin.Bacteria are stained with the single solution of stain.bacteria retain the colour of stain and seem deeply stained.in simple staining,the first smear is fixed on the slide,then fixed smear is flooded with the single solution of stain and then excess stain is washed off with water and slide blotted dry and smear is observed under the microscope under oil immersion.Simple staining makes all microorganism appear similar.So cannot be differentiating bacteria into the different group.

• Negative staining

The main purpose of negative staining is to study the morphological shape ,size and arrangement of bacterial cells that is difficulties to stain.At first,few drops of nigrosin is placed in some of the slides when loopful inoculum is added .Then thin smear is made with the help of another slide and air dried.the slide is placed under a microscope to examination.Here slide is stained but not the microorganism,In negative staining, the acidic dye is used as it cannot stain negatively charged bacteria due to repulsion.So bacteria appear transparent on the deeply stained background.

• Differential staining

It is one which can differentiate bacteria into different groups or which can different cell organelles.In differential staining, the specimen is subjected to series of stains in which different organism and different parts of cell stain differently.So that they can be distinguished from each other.Some Eg of differential staining are

1. Gram staining
2. Spore staining
3. Capsule staining
4. Acid fast staining

Advantages of negative staining over simple staining

1. Heat fixation is done in simple staining which is not done in negative staining.So due to heat fixation,shrinkage of the cell occurs and morphology of cell is distorted .In negative staining, the morphology of the organism is not distorted to a greater extent.
2. Negative staining can visualise the structures which are difficult to stain.For Eg capsule cannot be stained by simple staining because it is nonionic.Such structures can be visualised by negative staining.Some staining methods

• Spore staining

A bacterial endospore is very difficult to stain because spore contains hard Spore coats and exosporium which prevents entry of dye into the Spore.Spore is stained only by the extreme condition.First, Spore is stained with an extreme condition.For eg by applying heat.Once stained it is destined only with difficulty.So such a condition is chosen that will detain cell but not spore.One spore staining method is Schaeffer-Fulton method.In this technique, cell Is first heat fixed than smear is flooded with malachite green and heated till staining.Heating increases permeability and spore walls and dye enters into the Spore.At this stage,safranin enters into the cell and cell appears red.

• Capsule staining:

The capsule is nonionic and very delicate structure.Due to nonionic property, a capsule cannot be stained.So, negative staining technique Is applied to visualised the capsule.In this method,background stained by acidic dye and bacterial cell is stained by basic dye and capsule appears colourless between dark background and coloured bacterial cell

Here a drop of the acidic dye such as nigrosin is placed at one end of solid.Then one loop full of broth culture of the organism is placed on this drop and rubs by another slide.now smear is air dried and lastly flooded by methylene blue and crystal violet which are basic dye and bacterial cell.Therefore capsule appears transparent between the dark background and blue cell.Washing is not done in capsule staining.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.