Differential Staining
* Differential staining is a staining process which uses more than one chemical stain.
* Using multiple stains can better differentiate between different microorganisms or structures/ cellular components of a single organisms.
* Two stains are used. One is called primary stain while other one is counterstain.
* Examples : Gram's Staining , Acid - fast staining
Gram's Staining
* The Gram staining is fundamental to the phenotypic characterization of bacteria.
* It is a method of staining used to distinguish and classify bacterial species into two large groups : gram - positive bacteria and gram - negative bacteria.
* The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.
* This gram stain technique continues to be a standard procedure in medical microbiology.
Principle :
"The Gram staining procedure differentiates organisms of the domain Bacteria according or cell wall structure. Gram- positive cells have a thick peptidoglycan layer and stain blue to purple. Gram - negative cells have a thin peptidoglycan layer and stain red to pink".
Steps :
Mechanism :
Gram - positive cell wall :
* Gram - Positive bacteria have a thick mesh - like cell wall which is made up of peptidoglycan (50 - 90% of cell wall), which stains purple.
* Peptidoglycan is mainly a polysaccharide composed of two subunits called N- acetyl glucosamine and N- acetyl muramic acid.
* As adjacent layers of peptidoglycan are formed, they are cross linked by short chains of peptids by means of a transpeptidase enzyme, resulting in the shape and rigidity of cell wall.
* The thick peptidoglycan layer of Gram - positive organisms allows these organisms to retain the crystal violet - iodine complex and stains the cells as purple.
Gram - Negative Cell wall :
* Gram - negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall).
* Therefore, they lose the crystal violet - iodine complex during decolorization with the alcohol, rinse, but retain the counter stain Safranin.
* As a result, gram negative cells appear reddish or pink.
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| Gram positive VS Gram negetive cell wall |
Procedure :
* Take a clean, grease free slide.
* Prepare the smear of suspension on the clean slide with a loopful of sample.
* Air dry and heat fix.
* Crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.
* Flood the gram's iodine for 1 minute and wish with water.
* Then, wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.
* Add Safranin for about 1 minute and wash with water.
* Air dry, Blot dry and observe under microscope.
Stain Reaction :
The four basic steps of the Gram Stain are :
1) Application of the primary stain crystal violet (CV) to a heat fixed smear of bacterial culture.
* CV dissociates in aqueous solutions into CV+ and Cl- ions.
* These two ions then penetrate through the cell wall and cell membrane of both Gram - positive and Gram - negative cells.
* The CV+ ions later interacts with negatively charged bacterial components and stains the bacterial cells purple.
2) Addition of Gram's Iodine
* This step is known as "fixing the dye".
* Iodine (I - or - I3 -) acts as a mordant and as a trapping agent.
* A mordant is a substance that increases the affinity of the cell wall for stain by binding to the primary stain, thus forming an insoluble complex which gets trapped in cell wall.
* In the Gram stain reaction, the crystal violet and iodine form an insoluble (CV - I) which serves to turn the smear a dark purple color.
* At this stage, all cells will turn purple.
3) Decolorizations with 95% ethyl alcohol
* This step is known as "Solvent treatment".
* Alcohol/ acetone dissolves the lipid outer membrane of Gram negative bacteria, thus leaving the peptidoglycan layer exposed and increases the porosity of the cell wall.
* The CV - I complex is then washed away from the thin peptidoglycan layer, leaving Gram negative bacteria colorless.
* On the other hand, alcohol has a dehydrating effect on the cell walls of gram positive bacteria which causes the pores of the cell wall to shrink.
* The CV - I complex gets tightly bound into the multi-layered Gram positive cell wall thus staining the cells purple.
4) Counterstain with safranin
* The decolorized Gram negative cells can be rendered visible with a suitable counterstain, which is usually positively charged safranin.
* Safranin stains them red.
* Red colour which adheres to the Gram positive bacteria is masked by the purple of the crystal violet.
* Basic fuschin is sometimes used instead of Safranin in rare situations.
Observation :
Summary
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| Gram's staining Summary |
Acid-Fast staining
* Acid fast stains are used to differentiate acid fast organisms such Mycobacteria.
* Acid fast bacteria have a high content of mycolic acids in their cell walls.
* Acid- fast bacteria are also known as acid-fast bacilli of simply AFB.
* Stained bacteria that are not decolorized by acid alcohol are called acid fast bacteria.
* They are a group of bacteria sharing the characteristic of acid fastness.
* Acid fastness is a physical property that gives a bacterium the ability to resist decolorization by acids during staining procedures.
* This means that once the bacterium is stained, it cannot be decolorized by acids routinely used in the process.
* Bacteria displaying acid fastness include:
1) Genus Mycobacteria - M. leprae, M. tuberculosis, M. smegmatis etc.
2) Genus Nocardia - N. brasiliensis, N. cyriacigeorgica, N. farcinica etc.
* There are two types of acid fast staining : hot method (e.g. Ziehl-Neelsen method) and cold method (e.g. Kinyoun method)
Principle : (some for both hot cold acid fast staining method )
" The cell wall of acid fast bacteria contains a lipid called Mycolic acid which makes them resistant to simple aqueous stains. Thus, a lipid- soluble stain (primary stain) is required to stain them which is retained even after decolorization step. whereas primary stain is lost from the non acid fast bacteria upon decolorization and cells get stained with counterstain".
Mechanism : (same for both hot and cold acid fast staining method)
* The cell wall of Mycobacterium sps typically contain waxy substance (Mycolic acid) that makes it impermeable to staining by aqueous staining solutions.
* These bacteria can not be stained by simple or even by Gram staining.
* They can however be stained by lipid-soluble stain and one stained can not be readity decolourized by weak mineral acid.
* Hence, these bacteria are called acid fast bacilli and the staining method is called acid fast staining.
Reagents: (same for both hot and cold acid fast staining method)
* Primary stain : 0.3% Carbol-fusion,(Dissolve 50g phenol in 100ml ethanol (95%) or methanol (95%). Dissolve 3g Basic fushsin in the mixture and add distilled water to bring the volume to 1L)
*Decolorization Solution : Add 30ml hydrochloric acid to 1L of 95% denatured alcohol. Cool and mix well before use. Alternate decoloring reagent (without alcohol): slowly add 250ml sulfuric acid (at least 95%) to 750ml distilled water. Cool and mix well before using.
Counterstain: 0.3% methylene blue (Dissolve 3.0g methylene blue in 1L distilled water).
Ziehl - Neelsen method
* Acid fast staining technique was developed in 1882 by Paul Ehrlich.
* It is named for two German dactors who modified the stain : the bacteriologist Franz Ziehl and the pathologist Friedrich Neelsen in 1890.
* Z.N. staining or Ziehl - Neelsen staining is a bacterilogical stain used to identify acid-fast organisms mainly mycobacteria.
* Ziehl - Neelsen is a not method of acid fast staining.
* The primary stain used in acid-fast staining is carbolfuchsin.
* It is lipid - soluble and contains phenol, which helps the stain penetrate the cell wall.
* This is further assisted by the addition of heat.
* The smear is then rinsed with a very strong decolorizer, which removes the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms.
* The decolorized non-acid-fast cells then take up the counterstain.
Procedure:
* Prepare and fix the specimen smear prior to staining.
* Heat-fix the dried smear.
* Flood the smear with carbol fuchsin stain.
* Heat the stain to evaporate; replenish stain as needed. Also, prevent stain from boiling. Do not overheat. Allow the heated stain to remain on the slide for 5 minutes.
*Wash off the stain with clean distilled water. Heated slides must be cooled prior to washing.
* Decolorize the smear with acid alcohol for 5 minutes or until the smear is sufficiently decolorized.
* Wash well with clean water.
* Flood the smear with methylene blue stain for 1-2 minutes.
* Wash off the stain with clean water.
* Wipe the back of the slide clean and place it in a draining rack for the smear to air dry.
* Examine the smear microscopically, using the 100X oil.
Observation:
*Acid fast : Bright red colored
*Non-acid fast: Blue color
* The kinyoun method or kinyoun stain cold method) developed by joseph J. Kinyoun, is a peocedure used to stain acid-fast species of the bacteria.
* It is a variation of a method developed by Robert Koch in 1882.
* It does not require heating.
* In the Ziehl-Neelsen stain, heat acts as physical mordant while phenol (carbol of carbol fuschin) acts as the chemical mordant.
* Since the kinyoun stain is a cold method (no heat applied), the concentration of carbol fuschin used is increased.
