What is the principle of capsule staining?
The principle of capsule staining is based on staining of background with an acidic stain and staining of bacterial cell with a basic stain. As a capsule is non-ionic, it will not stain by either of the two dyes.
Can You stain a capsule with an acidic stain?
As a result, acidic and basic stains will often fail to adhere on to them. For this reason, it becomes necessary to stain the background using an acidic stain while the cell is stained using a basic stain. Two of the most commonly used methods used in capsule staining include: India ink method.
How can I visualize the stain of a bacterial capsule?
Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Therefore, the best way to visualize them is to stain the background using an acidic stain (e.g., Nigrosine, congo red) and to stain the cell itself using a basic stain (e.g.,crystal violet, safranin, basic fuchsin and methylene blue).
Why is capsule stain considered a differential stain?
Why is capsule stain considered differential stain? Because it's going to differentiate bacteria that has capsule and that dose not have. Define capsule It's a slime layer that is outside the bacteria Do all bacteria produce capsule? No, not all bacteria produce capsule. Those bacteria that produce capsule can have what?
What is the acid dye used in the capsule stain?
India InkThe basic dye such as Crystal Violet, will stain the bacteria, while the acidic dye, such as India Ink, provides a colored contrasting background. The capsule appears as a clear-white halo area around the bacteria.
Is capsule stain acidic or basic?
The capsule stain employs an acidic stain and a basic stain to detect capsule production. Capsules are formed by organisms such as Klebsiella pneumoniae .
Is acidic dye a capsule stain?
Capsule stain is a type of differential stain which uses acidic and basic dyes to stain background & bacterial cells respectively so that presence of capsule is easily visualized. Capsule is synthesized in the cytoplasm and secreted to the outside of the cell where it surrounds the bacterium.
What is the purpose of the capsule stain quizlet?
What is the purpose of the basic stain (positively charged)? To be able to stain the cell membrane. It is going to pass the nucleoid layer, and pass the cell wall, and it's going to stick to negatively charged cell membrane.
How does acid fast stain work?
The cells in the sample hold onto the dye. The slide is then washed with an acid solution and a different stain is applied. Bacteria that hold onto the first dye are considered "acid-fast" because they resist the acid wash. These types of bacteria are associated with TB and other infections.
What staining method is used to study the capsule bacteria?
Capsules do not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is typically used for capsule staining. The dye stains the background but does not penetrate the capsules, which appear like halos around the borders of the cell.
Which staining method is used for capsule staining?
In the capsule staining method, copper sulfate is used as a decolorizing agent rather than water. The copper sulfate washes the purple primary stain out of the capsular material without removing the stain bound to the cell wall.
What are the two things that are stained in a capsule stain?
Some of the organisms that are likely to have a capsule in place include:Streptococcus pneumoniae,Haemophilus influenzae,Neisseria meningitidis,Klebsiella pneumoniae,Escherichia coli,Group B streptococci.
Why is crystal violet used in capsule staining?
Accordingly, capsule stains are not heat-fixed, and water is never used to rinse. The primary stain applied is crystal violet, which stains both the bacterial cell and the surrounding capsule. A 20% copper sulfate solution is then applied, which serves a dual function as both decolorizer and counterstain.
What is acid-fast staining quizlet?
Acid Fast Stain. *A differential stain which distinguishes bacteria based on cell wall properties. *Gram positive microorganisms that are resistant to gram stain, acid fast allows us to see this bacteria. mycolic acid. a waxy substance in cell walls of acid fast bacteria.
What is the purpose of skipping the heat fixation step in the capsule stain procedure quizlet?
Why Do You NOT Heat Fix A Capsule Stain Slide? Heat Fixing Causes The Cells To Shrink Leaving An Artificial White Halo Around Them That Could Be Mistaken For A Capsule. Gram Staining is A Differential Staining Method Used To Determine Between Gram Positive Or Gram Negative.
What does a capsule do?
It has several functions: promote bacterial adhesion to surfaces or interaction with other organisms; act as a permeability barrier, as a defense mechanism against phagocytosis and/or as a nutrient reserve. Among pathogens, capsule formation often correlates with pathogenicity.
Which staining method is used for capsule staining?
In the capsule staining method, copper sulfate is used as a decolorizing agent rather than water. The copper sulfate washes the purple primary stain out of the capsular material without removing the stain bound to the cell wall.
Why do capsules repel most stains?
Capsules are composed of mucoid polysaccharides or polypeptides, repel most stains because of it's charge. - This technique stains around the cells.
What color is a capsule at the end of the capsule stain?
The capsule appears as a clear halo between the pink-stained bacterium and the bluish-grey stained background. The background stain is the acidic stain Congo red (which changes colour to bluish-grey due to the pH), and the pink bacterial stain is due to acid fuchsin.
What is the Hiss method?
The Hiss method is also a type of positive staining method that stains the capsule and the bacterial cell with a brighter background. As a result, a capsule appears as a light violet colour between a dark violet coloured bacterial cell and colourless background.
What is the color of the capsule in Anthony's method?
Anthony’s method is a type of positive staining method, which stains the capsule along with the bacterial cell (not the background). As a result, a capsule appears as a faint blue halo between the violet bacterial cell and purple background.
What is the primary stain of CuSO4?
This method makes the use of two reagents, namely crystal violet as a primary stain, 20% of CuSO4 solution as a decolouring agent and counterstain. Crystal violet stains the bacterial cell and background. The CuSO 4 solution stains the non-ionic capsule. After staining: A bacterial cell appears violet.
What is India ink?
India ink method is a type of negative staining method, which stains both the bacterial cell and its background (but not a capsule). As a result, a capsule appears as a bright halo between the violet bacterial cell and a darker background.
What is the principle of capsule staining?
The principle of capsule staining is based on staining of background with an acidic stain and staining of bacterial cell with a basic stain. A capsule being non-ionic will not stain by either of the two dyes. Thus, capsule staining creates contrast by staining a bacterial cell along with its background and leaving a capsule as a colourless halo.
What is capsule staining?
A capsule staining is a special staining method, which uses differential capsule stains that either highlight the capsule or stains the bacterial cell along with the background.
What is the difference between positive and negative staining?
Positive staining method either stain a capsule only or stain both the capsule and bacterial cell. The negative staining method stains both the bacterial cell and its background. The capsule staining is employed to know the presence or absence of the capsule, after which you could classify the type of bacteria, i.e.
What is the primary stain for capsules?
In this type of capsule staining procedure, the primary stain is crystal violet, and all parts of the cell take up the purple crystal violet stain. There is no mordant in the capsule staining procedure. A 20% copper sulfate solution serves a dual role as both the decolorizing agent and counterstain. It decolorizes the capsule by washing out the crystal violet, but will not decolorize the cell. As the copper sulfate decolorizes the capsule, it also counterstains the capsule. Thus, the capsule appears as a faint blue halo around a purple cell.
How to remove capsules from a cell?
Tilt the slide and rinse with 20% copper sulfate solution. DO NOT RINSE WITH WATER! Water will remove the capsule from the cell.
How long does it take for a crystal violet slide to dry?
Allow the film to air dry (will take 5-7 minutes). DO NOT heat or blot dry! Heat will melt the capsule! Saturate the slide with crystal violet for 1 minute and rinse slightly & very gently with water. Be cautious w ater may remove the capsule from the cell. Let the slide air dry for a few minutes.
How to spread crystal violet?
This is done by contacting the drop of crystal violet with the clean microscope slide and using the capillary action of the dye/ slide to spread the crystal violet across the smear.
How to spread India ink on a microscope?
This is done by contacting the drop of India ink with the clean microscope slide and using the capillary action of the dye/ slide to spread the India ink across the smear.
What is the best way to visualize a cell?
Therefore, the best way to visualize them is to stain the background using an acidic stain (e.g., Nigrosine, congo red) and to stain the cell itself using a basic stain (e.g.,crystal violet, safranin, basic fuchsin, and methylene blue). Image 1: Capsule Staining (source-microbugz)
Why do capsules stain?
Capsules are associated with virulence in several microorganisms, including Streptococcus pneumoniae and Neisseria meningitides, because capsules resist phagocytosis thus evading the host immune system. In capsule staining procedure “we do not heat fix and rinse the smear with water” as heat and water may dislodge capsules from bacteria.
What is a Capsule?
Some bacteria like Bacillus anthracis have a well organized layer of material lying outside the cell wall referred to as a capsule (or K antigen ). What really differentiates a capsule from any other material that may form on the surface of a cell wall of other bacteria is the fact that capsules are well organized layers that cannot be easily washed off.
Why is capsule stain important?
For this reason, capsule stain is of great benefit in that it helps identify the presence of capsules and thus allowing healthcare professionals to recommend the right treatment. Cell Staining in Microscopy.
Why do bacteria need capsules?
With a capsule in place, the bacteria is protected from such body defense cells as macrophages. Therefore, it becomes easier for the bacteria to thrive and cause diseases. For this reason, capsule stain is of great benefit in that it helps identify the presence of capsules and thus allowing healthcare professionals to recommend the right treatment.
Why is India ink used in smears?
Because of its acidic nature, India ink (or Congo red, nigrosin) stains the background dark. On the other hand, crystal violet is used for number of reasons including: To act as a fixative. Increase penetration power. Stain the cells (being a basic dye) Decrease pH of smear.
Why are capsules important for bacteria?
As such, they protect the bacteria from phagocytic actions of such cells as neutrophils allowing the bacteria to thrive. This is achieved due to the fact that the capsule is very smooth and has a negative charge that prevents attachments/adherence.
What is capsule stain?
Capsule stain is a type of differential stain which involves the use of two stains; primary stain and the counterstain. Here, it's worth noting that for most part, capsules are non-ionic.
Why is my smear dark?
When viewed under the microscope, the background will be dark as a result of India ink, bacteria cells will be purple having taken the crystal violet dye while the capsule will be clear against a dark background given that it takes no stain.
What is the best stain for a slide?
Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on the slide.#N#Congo Red is easier to see, but it does not work well with some strains. India Ink generally works, but it has tiny particles that display Brownian motion that must be differentiated from your bacteria.Nigrosin may need to be kept very thin or diluted.
Why is a capsule stain misnomer?
Capsules stain very poorly with reagents used in simple staining and a capsule stain can be, depending on the method, a misnomer because the capsule may or may not be stained. Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with india ink ...
Capsule Staining Definition
Capsule Staining involves identification of capsular material. The primary function of capsule stain is to differentiate capsular material from bacterial cells. A capsule is a gelatinous outer layer that surrounds and adheres to the cell wall and is secreted by bacterial cells.
Capsule Staining Principle
Capsules stain very poorly with simple staining reagents, and a capsule stain can be a misnomer depending on the method because the capsule may or may not be stained.
Capsule Staining Procedure
Apply a little drop of a negative stain to the slide (India Ink, Congo Red, Nigrosin, or Eosin). Using a sterile technique, apply a loop of bacterial culture to the slide and smear it in the dye.
Capsule Staining Result
Capsule Staining appearance: Clear zone between refractile cell outline against the dark background of Nigrosin stain.
Where to add counter stain?
Add counter stain right on top of the negative stain.
Why do virulence factors have a virulence factor?
They have a virulence factors because those capsule can prevent phagocytosis.
Is a syringe extracellular or secreted?
It is secreted outside of cell wall therefore, it is extracellular.
Why do virulence factors have a virulence factor?
They have a virulence factors because those capsule can prevent phagocytosis.
Where to add nigrosin?
Add nigrosin to the far right hand side
Where to add counter stain?
Add counter stain right on top of the negative stain.
Can a monopolist make a profit?
A monopolist can earn a positive economic profit, even in the long run.
Is a syringe extracellular or secreted?
It is secreted outside of cell wall therefore, it is extracellular.
Principle of Capsule Stain
Materials and Reagents Required
- Test bacteria: 36-48 hour culture of capsulated bacteria e.g. Klebsiella pneumoniae growing on a slant ofEMB agar or culture of other capsulated bacteria and non-capsulated bacteria [Note: Growing...
- Stain solutions: Depending on the type of method used (crystal violet, India ink, Nigrosin, copper sulfate, Basic carbol fuschin solution, methylene blue solution, etc).
- Test bacteria: 36-48 hour culture of capsulated bacteria e.g. Klebsiella pneumoniae growing on a slant ofEMB agar or culture of other capsulated bacteria and non-capsulated bacteria [Note: Growing...
- Stain solutions: Depending on the type of method used (crystal violet, India ink, Nigrosin, copper sulfate, Basic carbol fuschin solution, methylene blue solution, etc).
- Microscopic slides
- Inoculating loop
Capsule Stain Procedure
- A. India Ink Method
1. Place a single drop of India inkon a clean microscope slide, adjacent to the frosted edge. 2. Using a flamed loop and sterile technique, remove some Klebsiella pneumoniaefrom culture tube or plate and mix it into the drop of India ink. Be sure there are no large clumps of organism, but t… - B. Anthony’s stain method
1. Place a single drop of crystal violeton a clean microscope slide, adjacent to the frosted edge. 2. Using a flamed loop and sterile technique, add three loopful of test bacterium (any capsulated bacteria such as Klebsiella pneumoniae, Streptococcus pneumoniae) from broth culture. If you a…
Points to Remember
- Clean your microscope with lens cleaner, removing all oil from lenses.
- Dispose of staining waste and slides in designated waste containers.
- Be cautious while handling the slide, since the organisms have not been killed.