Bacterial Staining Flashcards
What is the product of magnification of the ocular lens of the magnification of an objective lens?
Magnification of a light microscope
When can oil immersion be using a light microscope?
Only use for samples that are fixed ordead
How do you calculate the resolution of a light microscope?
Resolution = 0.5 (wavelength) / numerical aperture
Which type of staining is the most used for staining procedure in microbiology?
Gram stain
What is the advantage of differential stain?
Does not stay in all kinds of cells the same colour
What colour does a Gram-positive bacteria show on the Gram stain?
Purple colour
What colour does a Gram-negative bacteria show on the Gram stain?
Pink colour
How do you prepare cells for Gram staining?
- thin-film of cells from culture applied to slide
- Air dried
- flame fixes cells glass
- first dye with crystal violet for one minute
- rinse with water
- flood with iodine for three minutes
- rinse with water
- flood with ethanol for 20 seconds
- Purple colour will show if Gram-positive
- For Gram-negative need to add safranin for 1-2 minutes
- Rinse with water before viewing
Why do we use crystal violet?
Positively charged and binds with high affinity to negatively charged molecules
What does crystal violet an iodine form?
Complex
Why is it important to use crystal violet and iodine in a Gram stain?
Difficult to remove dye from the cells if complex formed
What does ethanol extract from the Gram-negative bacteria?
Crystal violet
Why does ethanol not extract the crystal violet from a Gram-positive bacteria?
Gram-positive have a thick peptidoglycan cell wall
Ethanal causes dehydration causing pores to close and prevent crystal violet from leaving
What solutions can be used for simple staying?
Crystal violet/safranin/methylene blue
How do use a simple stain?
- Use stain
- Rinse of with water
Why do we use simple stain?
Size, shape and arrangement of cells
How does crystal violet stain?
Stains most bacteria uniformly
How does safranin stain?
Stains most bacteria pink or red
How does methylene blue stain?
Stains bacteria light blue
What are the different types of differential stains?
- Gram stain
- Acid-fast stain
- Endspore stain
What is the most used staining technique?
Differential stains
What makes a differential stain different to simple staining?
Uses more than one type of dye
Why do you use differential staining?
To distinguish between different structures
What is the most frequently used differential stain?
Gram stain
What is the procedure of an acid-fast stain?
- Stain with carbol fuchsin with heat
- Cooled and decolourised with hydrochloric acid
- counterstain with methylene blue
- Rinse with water
Why do we use acid-fast stain?
Identify a subset of bacterium genes - Mycobacterium has a waxy material in cell wall
Why do we use endospore stains?
To identify endospores inside of plasmids of bacteria
Why do we have to use endospores staining techniques to identify endospores?
-Highly resistant to dissection, heat and radiation
What is the procedure of an endospore stain?
- Malachite green stain
- Heat
- Rinse with water
- Counterstain with safranin
- Rinse with water
- Appears green if endospores present
What are the different types of special stains?
- Negative stains
- Flagellar stains
Why do we use special staining procedures?
Interact with special features such as the capsules or flagellum
What do negative stains do?
Negative stains stain the background leaving cells colourless as acid dye are repelled by negative charges on the surfaces of the cell
What is the negative stain procedure?
- Basic counterstain
- Rinse with water
- Eosin stain
- Rinse with water
