Lab 7 Flashcards
Why are Anionic Dyes used and what are some examples
▪︎These are negatively charged chromophores
▪︎They are repelled by the negatively charged cell wall, so it stains the background instead of the cell.
▪︎Thet are called Negative Stains.
▪︎Eosin, Congo Red, and Nigrosin are all examples of negative stains.
Bacterial capsule
A thick polysaccharide or polypeptide layer around the cell wall of a bacterial cell called glycocalyx. This layer is made inside the cell and is secreted to the cell surface. Once the substance is organized and firmly attached, it is called a capsule.
What is the significance of Congo red in capsule staining? How is the capsule visualized under the microscope
Congo red is an acidic dye that cannot penetrate the outer layers of the capsule. This will cause the cell to appear transparent on a (blue) colored background. When the Maneval’s stain is added, the bacterial cell will stain red. The capsule appears as a colorless layer between the red bacterium and blue background.
What role do capsules play in the life of bacteria
It adds to the virulence of bacteria. It prevents pathogenic bacteria from phagocytosis. It also protects bacteria from desiccation by maintaining moisture, protects from protozoa, and serve as nutrients in times of need. The capsules also help bacteria adhere to surfaces, create biofilms, colonize surfaces, and cause disease.
What are 3 bacterial species that make capsules
- Bacillus anthracis
- Streptococcus pneumonia
- Serratia marcescens
What are Negative stains
Stains that stain the background, are acidic, and are repelled by bacterial cells
Capsule staining procedure
- add 1 drop of Congo red to slide
- Aseptically remove a loopful of cx from broth/plate and add to Congo dye. Mix only if from solid cx.
- Use clean slide to spread drop of stain across slide, producing a broad, even smear.
- Allow to air dry without heating
- Flood slide with acid alcohol for 20 seconds
- Dip in water
- Flood with Manevals stain for 2 minutes
- Let stain air dry at angle or gently blot dry
- View under oil immersion
Why is heat fixing not used for capsule staining
It dehydrates the cells and cause them to shrink, resulting in false capsule-like areas around the cell. It can also destroy protein capsules
What is the purpose of using Maneval’s stain
The acid lowers the pH in the sample to a 3 or lower, causing the Congo Red to change from red to blue/purple. The Maneval’s stain penetrates through the capsule and stains the cell bright red/pink.
What is in Maneval’s stain
Acetic acid and acid fuchsin
What does positive capsule stain mean
Bacterial cell with a capsule
What does negative capsule stain mean
No capsule
Why do capsulated bacteria that lose their capsules become non pathogenic
Because they are susceptible to the hosts immune response such as phagocytosis and they can no longer adhere to surfaces easily which will prevent them from colonizing and causing disease.
What would happen if a basic dye was used for capsule staining
Basic dye has a positive charge and is attracted to structures with a negative charge such as the bacterial cell. The cell would stain against a white background and it would be difficult to visualize the capsule which would also be white or clear.
Why is it important to use a basic dye and acidic dye to demonstrate capsules
By using both dyes, one is able to differentiate between the cell, capsule, and background. The basic dye stain the cell red, the acidic dye stains the background blue, the capsule repels all dyes and stays clear. This way the capsule is seen around the bacterial cell.
What would be the outcome if you used an acidic dye but not a basic dye for capsule staining
This would result in a transparent bacterial cell on a dark background. You would not be able to see the capsule around the bacterial cell.
When is capsule formation induced in a lab grown media
When bac6are grown in media rich in sugars and low nitrogen and in older cultures.
When would Congo remain red
As long as pH remains at 5 or above, it will stain red
