Staining (microscopy) Flashcards
What is staining?
With coloured/ fluorescent chemicals that bind to cell organelles and makes them visible allows to identify different types of cells and different organelles (providing contrast)
What are stains in microscopy?
Are used to increase the contrast of a sample by selectively binding to certain structures within the sample and changing their optical properties
What is a contrast in biology?
Refers to the degree of visibility or differentiation between different structures or cells within a sample
What would happen without staining?
Many structures within a sample may appear transparent or have similar refractive indices, making it difficult to distinguish between them
How do stains help address this issue?
By making certain structures or cells more visible and thus increasing the contrast
What are simple stains?
- they involve the use of a single dye to colour all of the cells or structures in a sample
- methylene blue can be used to stain DNA
- The stain is positively charged so attracted to the negatively charge DNA. All DNA will stain the same, from bacteria to humans
What can you see in simple stains?
1) the shape
2) some basic structures
3) relative size
What are differential stains?
Uses multiple dyes to distinguish different types of cells or structures based on their physical or chemical properties. The second dye is called a COUNTERSTAIN. Usually it’s a contrasting colour to the principal stain.
What are differential stains used for?
- contrast two cell types
- indicate cell parts (eg gram stain and endospore stain)
What are most stains in microbiology like and what does this involve?
- these involve the use of more than one dye (so that certain differences between cell type and structure can be distinguished)
What is the most important differential stain?
The Gram Stain
Why is staining used when preparing specimens to be examined under a microscope?
- so the cells and their contents are visible
- to increase contrast
- so organelles can be seen eg nucleus
What are the two different gram stain groups?
Gram positive (violet) and Gram negative (red)
What is the gram stain technique for?
It is used to separate bacteria into two groups, Gram positive bacteria and Gram negative bacteria
How is Gram - positive tested?
- Crystal violet is first applied to a bacterial specimen on a slide
- then iodine, which fixes the slide
- the slide is then washed with alcohol
-the gram positive bacteria retains the crystal violet stain and will appear blue/purple under the microscope
How are gram negative tested?
They have thinner cell walls, and therefore they lose the stain.
They are then stained with safranin dye, which is called a counterstain
These bacteria will appear red
How are gram positive bacteria when it comes to penicillin?
They are susceptible to the antibiotic penicillin, which inhibits the formation of cell walls
How is gram negative bacteria with penicillin?
They have much thinner cell walls that are not susceptible to penicillin
What is the Acid-Fast technique used for?
To differentiate species of Mycobacterium from other bacteria
Explain the Acid-Fast technique
A lipid solvent is used to carry carbolfuchsin dye into the cells being studied
The cells are then washed with the dilute acid-alcohol and retain the carbolfuchsin stain, which is bright red
Other bacteria lose the stain and are exposed to a methylene blue stain, which is blue
Explain the 4 stages involved in the production of these slides.
- FIXING : chemicals like formaldehyde are used to preserve specimens in as near-natural state as possible
- SECTIONING : specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. this can then be sliced thinly with a knife called a microtome
- STAINING : specimen are often treated with multiple stains to show different structures
- MOUNTING : the specimens are then secured to a microscope slide and a cover slip placed on top
Explain the Risk Management
- many stains are toxic / irritants.
- risk assessment must be carried out before any practical work is started (to identify specific procedures that can cause harm)
What is CLEADSS?
Provide student safety sheets that identify specific risks, advise on the measures to be taken to reduce these risks and the action to be taken in any emergencies
SUGGEST WHY GRAM-NEGATIVE INFECTIONS ARE MORE DIFFICULT TO TREAT THAN GRAM-POSITIVE INFECTIONS
- gram negative have a thinner wall
- penicillin disrupts cell wall
- less cell wall formation (in gram negative)
- membrane (around gram negative) prevents entry of penicillin
DESCRIBE PRECAUTIONS WHEN USING CRYSTAL VIOLET & POTASSIUM IODIDE (CLASSED AS IRRITANTS)
- avoid skin / eye contact
- wear gloves / goggles
In order to investigate onion cells, a stained temporary mount was made. Suggest what a good stain to use.
Iodine or methylene blue
Why did the onion cells need to be stained?
To observe the cells, nuclei and cell walls absorb the stain more strongly, so that they can be distinguished
What is the advantage of making a temporary mount of cells?
Temporary mounts are quick to prepare; the cells can be observed whilst alive
A scientist wishes to study yeast cells using a light microscope. How should the slide be prepared?
To create a temporary mount, yeast cells should be placed on a slide with a droplet of iodine solution should be added to stain the organelles in the cell. A cover slip should be placed over the cell. A squash slide can be prepared by gently pressing down on the cover slip.
Stains are used for a number of different purposes when preparing slides. Give 3 examples of how they can be used.
Increase contrast, so different parts of a cell can be distinguished.
to observe the location of a certain chemical in a cell
Differentiate between organisms that can be hard to tell apart
