- Step 2: Smear Preparation (Review) ...
- Cover the smear with carbolfuchsin dye. ...
- Dry heat for 2 minutes.
- Cool and rinse with water. ...
- Wash the top and bottom of slide with water and clean the slide bottom well.
- Counterstain with Methylene Blue for 30 seconds to 1 minute.
What is the counterstain in the Ziehl-Neelsen acid-fast stain procedure?
22 What is the counterstain in the Ziehl-Neelsen acid- fast stain procedure? a Carbolfuscin Crystal violet c. Methylene blue . Malachite green Safranin 2. What is the usual decolorizer in the Ziehl-Neelsen acid-fast stain procedure? Acetone 1. Water clodine . Acid-alcohol Ethyl alcohol 2s.
What is Ziehl Neelsen staining technique?
Neelsen in 1883 used Ziehl’s carbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques was developed. The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups.
What is acid-fast staining?
The use of acid-alcohol in the technique earned it the name Acid-Fast Stain and the application of heat in the technique gives it the name the hot method of Acid-Fast staining which is a synonymous name for the Ziehl-Neelsen Staining technique.
What is the difference between Neelsen and Rindfleisch's method of staining?
Rindfleisch heated the slide for few minutes instead of putting into a hot solution and reduced the staining time (1882). Neelsen combined Basic fuchsin and carbolic acid together and used these as a single solution (1883).
What is stained by the counterstain in the acid-fast stain procedure?
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 will be red, while nonacid fast bacteria will stain blue/green with the counterstain with the Kinyoun stain.
What is acid-fast staining or Ziehl-neelsen method?
This method became known as the Ziehl-Neelsen method in the early to mid 1890s. In this method heat is used to help drive the primary stain into the waxy cell walls of these difficult-to-stain cells. The use of heat in this method has been the reason that this technique is called the “hot staining” method.
What is Ziehl-Neelsen stain procedure?
Ziehl-Neelsen Staining ProcedurePrepare the sputum smear. ... Place slides on the dryer with smeared surface upwards, and air dry for about 30 minutes.Heat fix dried smear. ... Application of a primary stain. ... Heat the smear until vapor just begins to rise (i.e. about 60°C). ... Wash off the stain with clean water.More items...•
What is the mordant used in the Ziehl-Neelsen acid-fast stain procedure?
In the Ziehl–Neelsen stain, heat acts as a 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.
Why is carbol fuchsin used in acid-fast staining?
Carbol fuchsin is used as the primary stain dye to detect acid-fast bacteria because it is more soluble in the cells' wall lipids than in the acid alcohol.
Which stain is used in acid-fast staining?
carbolfuchsinThe primary stain used in acid-fast staining, carbolfuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall.
What is the counterstain in the Ziehl-Neelsen technique for AFB smear quizlet?
In the Ziehl-Neelsen method, heat is applied to the smear as a penetrating agent and methylene blue is used as the counterstain.
What is Ziehl-Neelsen stain made of?
The reagents used for Ziehl–Neelsen staining are carbol fuchsin, acid alcohol, and methylene blue. Acid-fast bacilli are bright red after staining.
Which is the primary stain used in the Ziehl-Neelsen technique quizlet?
The primary stain in the Ziel-Neelsen method is carbol fuchsin, and basic fuchsin in the Kinyoun method.
What is the counterstain in the Ziehl-Neelsen acid-fast stain procedure quizlet?
What is the function of the counterstain in the acid-fast stinging procedure? The counter stain (methylene blue) will turn the non acid fast organisms blue.
What is the use of safranin solution?
Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endospore staining. It can also be used for the detection of cartilage, mucin and mast cell granules.
Why is Safranin used in Gram staining?
The safranin is also used as a counter-stain in Gram's staining. In Gram's staining, the safranin directly stains the bacteria that has been decolorized. With safranin staining, gram-negative bacteria can be easily distinguished from gram-positive bacteria.
What is the Ziehl Neelsen stain used for?
The Ziehl-Neelsen stain uses basic fuchsin and phenol compounds to stain the cell wall of Mycobacterium species.
What is the name of the staining technique used by Ziehl and Neelsen?
What is Ziehl-Neelsen staining? The Ziehl-Neelsen staining technique is a differential staining technique that was initially developed by Ziehl and modified later by Neelsen, hence the name Ziehl-Neelsen stain. Neelsen used carbol-fuschin from Ziehl’s experiment, with heat and added a decolorizing agent using acid-alcohol ...
How to get pink smear out of a smear?
Add 20% sulphuric acid and leave it for 1-2 minutes. Repeat this step until the smear appears pink in color. Wash off the acid with water. Flood the smear with methylene blue dye and leave it for 2-3 minutes and wash with water. Air dry and examine the stain under the oil immersion lens.
What is the name of the method of staining Ziehl-Neelsen?
The use of acid-alcohol in the technique earned it the name Acid-Fast Stain and the application of heat in the technique gives it the name the hot method of Acid-Fast staining which is a synonymous name for the Ziehl-Neelsen Staining technique.
What was the name of the chemical used in the Ziehl-Neelsen stain?
Neelsen used carbol-fuschin from Ziehl’s experiment, with heat and added a decolorizing agent using acid-alcohol and a counterstain using methylene blue dye, thus developing the Ziehl-Neelsen Technique of staining. The use of acid-alcohol in the technique earned it the name Acid-Fast Stain and the application of heat in the technique gives it ...
What is the cell wall of mycobacterium made of?
Mycobacterium cell wall contains high lipid content made up of mycolic acid on its cell wall making it waxy, hydrophobic, and impermeable. These are ß-hydroxycarboxylic acids made up of 90 carbon atoms that define the acid-fastness of the bacteria.
What color do acid fast bacteria take up?
The acid-fast bacilli take up the red color of the primary dye, carbol-fuschin. While non-acid-fast bacteria easily decolorize on the addition of the acid-alcohol and take up the counterstain dye of methylene blue and appear blue.
How long does carbol fuchsin stain stay on slide?
Allow the heated stain to remain on the slide for 5 minutes. Heating the stain: Great care must be taken when heating the carbol fuchsin especially if staining is carried out over a tray or other container in which highly fiammable chemicals have collected from previous staining.
How to kill M. tuberculosis?
M. tuberculosis is killed by bleach and during the staining process. Heat-fixation of untreated sputum will not kill M. tuberculosis whereas alcohol-fixation is bactericidal. Cover the smear with carbol fuchsin stain. Heat the stain until vapour just begins to rise (i.e. about 60 u0002C). Do not overheat.
What happens when you smear a bacterial cell?
When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the Mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal wall and enters into cytoplasm. Then after all cell appears red. Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol) ...
What is Ziehl Neelsen staining?
So this method is also called Ziehl-Neelsen staining techniques. Neelsen in 1883 used Ziehl’s carbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques was developed. The main aim of this staining is to differentiate bacteria into acid fast group ...
Why are non-acid fast cells colorless?
The non-acid fast organism lack the lipoidal material in their cell wall due to which they are easily decolorized, leaving the cells colorless. Then the smear is stained with counterstain, methylene blue. Only decolorized cells absorb the counter stain and take its color and appears blue while acid-fast cells retain the red color.
How to acid fast stain a sputum?
Procedure of Acid-Fast Stain. Prepare bacterial smear on clean and grease free slide, using sterile technique. Allow smear to air dry and then heat fix. Alcohol-fixation: This is recommended when the smear has not been prepared from sodium hypochlorite (bleach) treated sputum and will not be stained immediately.
How to clean a malachite smear?
Caution: Acid alcohol is fiammable, therefore use it with care well away from an open fiame. Wash well with clean water. Cover the smear with malachite green stain for 1–2 minutes, using the longer time when the smear is thin. Wash off the stain with clean water.
What color is the smear after decolorization?
The ability of the bacteria to resist decolorization with acid confers acid -fastness to the bacterium. Following decolorization, the smear is counterstained with malachite green or methylene blue which stains the background material, providing a contrast colour against which the red AFB can be seen.
How does carbol fuchsin stain?
Staining by carbol fuchsin is further enhanced by steam heating the preparation to melt the wax and allow the stain to move into the cell. Acid is used to decolorize nonacid-fast cells; acid-fast cells resist this decolorization. The ability of the bacteria to resist decolorization with acid confers acid -fastness to the bacterium.
How to clean a slide with methylene blue?
Wash well with clean water. Cover the smear with methylene blue or mala chite green 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 (do not blot dry).
What oil did Ziehl use to stain bacillus?
He stained bacillus with basic fuchsin in the presence of aniline oil as mordant and used dilute mineral oil for decolorization. Ziehl used carbolic acid as the mordant instead of aniline oil (1882). Rindfleisch heated the slide for few minutes instead of putting into a hot solution and reduced the staining time (1882).
Why is Gram stain so difficult to use?
Organisms such as Mycobacteria are extremely difficult to stain by ordinary methods like Gram Stain because of the high lipid content of the cell wall. The phenolic compound carbol fuchsin is used as the primary stain because it is lipid soluble and penetrates the waxy cell wall.
How much ethanol to dissolve fuchsin?
Primary stain : 0.3% Carbol Fuchsin – Dissolve 50g phenol in 100ml ethanol (90%) or methanol (95%). Dissolve 3g basic fuchsin in the mixture and add distilled water to bring the volume to 1 L.
How long did Robert Koch soak tubercle bacilli in blue?
Robert Koch stained tubercle bacilli by immersing in a hot alkaline solution of methylene blue for 24 hours (1882)
What is the color of the counterstain?
Counterstain (Methylene Blue or malachite green): This is used as the final reagent to stain previously decolorized cells. As only non–acid-fast cells undergo decolorization, they may now absorb the counterstain and take on its blue color or green, while acid-fast cells retain the red of the primary stain.
What is the decolorizing agent in AFB?
Decolorizing Agent (Acid-Alcohol; 3% HCl + 95% Ethanol): Prior to decolorization, the smear is cooled, which allows the mycolic acid to solidify such that AFB will be resistant to decolorization. Primary stain is more soluble in the cellular waxes than in the decolorizing agent. So, in this event, the primary stain is retained and the mycobacteria will stay red while non-acid fast bacteria become decolorized (colourless).
What is carbol fuchsin?
Carbol fuchsin, a dark red stain in 5% phenol that is soluble in the lipoidal materials that constitute most of the mycobacterial cell wall, does penetrate these bacteria and is retained. Penetration is further enhanced by the application of heat, which drives the carbol fuchsin through the lipoidal wall and into the cytoplasm. ...
What is the primary stain for mycobacteria?
1.Primary Stain (Carbol Fuchsin): Unlike cells that are easily stained by ordinary aqueous stains, most species of mycobacteria are not stainable with common dyes such as methylene blue and crystal violet. Carbol fuchsin, a dark red stain in 5% phenol that is soluble in the lipoidal materials that constitute most of the mycobacterial cell wall, does penetrate these bacteria and is retained. Penetration is further enhanced by the application of heat, which drives the carbol fuchsin through the lipoidal wall and into the cytoplasm. This application of heat is used in the Ziehl-Neelsen method. The Kinyoun method, a modification of the Ziehl-Neelsen method, circumvents the use of heat by addition of a wetting agent (Tergitol) to this stain, which reduces surface tension between the cell wall of the mycobacteria and the stain. After primary staining, all cells will appear red.
What is acid fast bacilli?
After staining, an acid alcohol is applied as decolorizing agent which removes the stain from the background cells, tissue fibres, and any organisms in the smear except mycobacteria which retain (hold fast to) the dye and are therefore referred to as acid fast bacilli, or simply AFB.
What is the stain method used to stain bacteria?
A special staining method is developed to stain such organism called as Acid-fast sta ining method. It is also known as Ziehl-Neelsen method. In this method, bacteria is stained with carbol fuchsin combined with phenol. The stain binds to the mycolic acid in the mycobacterial cell wall.
How to prepare a smear?
Procedure for Smear preparation 1 Take a clean glass slide. 2 Using aseptic technique, prepare a smear of given sample organisms 3 Heat-fix the dried smear 4 Alcohol-fixation: This is recommended when the smear has not been prepared from sodium hypochlorite (bleach) treated sputum and will not be stained immediately. M. tuberculosisis killed by bleach and during the staining process. Heat-fixation of untreated sputum will not kill M. tuberculosis whereas alcohol-fixation is bactericidal.
How long does it take for acid alcohol to decolorize a slide?
11.decolorize with acid alcohol for about 30 sec. or until the acid alcohol dripping off the slide is a light red color.
What does heating the slide do?
heating the slide "melts" or increases the fluidity of acid-fast cells and permits the penetration of the primary stain.
Does the Kinyoun method use steam?
the Ziehl-Neelsen method uses steam, Kinyoun method does not.
What Is Ziehl-Neelsen Staining?
Objectives of The Ziehl-Neelsen Staining
- To differentiate between acid-fast bacilli and non-acid-fast bacilli.
- To stain Mycobacteriumspecies.
Principle of The Ziehl-Neelsen Staining
- The Ziehl-Neelsen stain uses basic fuchsin and phenol compounds to stain the cell wall of Mycobacterium species.
- Mycobacterium does not bind readily to simple stains and therefore the use of heat along with carbol-fuschin and phenol allows penetration through the bacterial cell wall for visualization.
- Mycobacterium cell wall contains high lipid content made up of mycolic acid on its cell wall m…
- The Ziehl-Neelsen stain uses basic fuchsin and phenol compounds to stain the cell wall of Mycobacterium species.
- Mycobacterium does not bind readily to simple stains and therefore the use of heat along with carbol-fuschin and phenol allows penetration through the bacterial cell wall for visualization.
- Mycobacterium cell wall contains high lipid content made up of mycolic acid on its cell wall making it waxy, hydrophobic, and impermeable. These are ß-hydroxycarboxylic acids made up of 90 carbon a...
- Use of Carbol-fuschin which is basic strongly binds to the negative components of the bacteria which include the mycolic acid and the lipid cell wall. addition of acid alcohol along with the applic...
Reagents Used in The Ziehl-Neelsen Stain
- Carbol-Fuschin (Primary dye)
- 20% sulphuric acid or acid-alcohol (Decolorizer)
- Methylene Blue dye (counterstain) or malachite green
Procedure For Ziehl-Neelsen Staining
- Image Source: Elizabeth Gray. 1. On a clean sterile microscopic slide, make the smear of the sample culture and heat fix the smear over blue heat. 2. Over the smear, pour and flood the smear with carbol fuschin and heat gently until it produces fumes. 3. Allow it to stand for 5 minutes and wash it off with gently flowing tap water. 4. Add 20% sulphuric acid and leave it for 1-2 minut…
Results and Interpretation
- Figure: Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain. Image Source: CDC/Dr. George P. Kubica. 1. Acid-fast bacteria retain the primary dye, carbol-fuschin, and stain pink. 2. Non-acid fat bacteria take up the methylene blue dye and appear blue.
Applications of Ziehl-Neelsen Staining
- Used for examination and identification of Mycobacteriumspecies.
- Used to differentiate between acid-fast and non-acid fast bacilli
- Used for the identification of some fungal species such as Cryptosporidium.
Limitations of Ziehl-Neelsen Staining
- It can only be used to identify acid-fast bacilli.
- The physical morphology of the organism is distorted.
References and Sources
- 2% – https://labhelpline.com/2019/05/23/ziehl-neelsen-staining-technique-for-acid-fast-bacilli/
- 1% – https://www.uwyo.edu/virtual_edge/units/acidfast_stain.html
- 1% – https://www.ncbi.nlm.nih.gov/pubmed/17533853/
- 1% – https://www.differencebetween.com/difference-between-acid-fast-and-vs-non-acid-fast-bacteria/