Topic 2: Microscopy & Staining Flashcards
The quality of an image under the light microscope depends on:
- Magnification
- Contrast
- Resolution
Three lenses in a compound microscope
- Condenser
- Objective
- Ocular
Magnification
The product of the individual magnifications of the ocular and objective lenses
Condenser lense function
Focuses the light on the specimen
What minimum magnification is needed to view cells
100 x
Resolving power (d)
The ability of a lens system to allow you to see two points as being distinct
Do higher or lower values of ‘d’ mean greater resolution
Lower
Resolution formula
-d = wavelength/ (2 NA) N = refractive index of space between specimen & lens A = numerical aperture
Resolving power can be maximized by:
- Using a higher magnification
- Use a shorter wavelength of light
Oil immersion
The refractive index of oil is much closer to that of glass
- Capture more refracted light
Brightfield
Dark objects are visible against a bright background
- Contrast from light being refracted and absorbed
- Stain kills specimen
Darkfield
Light objects are visible against a dark
- Only light that is reflected/refracted by the specimen will enter the objective lens
- Poor detail
Fluorescence Microscopy
Adhere fluorescent dyes to the specimen
- Fluorescent substances absorb u.v. light, and emit visible light
Confocal Microscopy
Short wavelength light progressively focused at different depths of dyed specimen
- Produces 3-dimensional image
Electron Microscopy
Similar to brightfield except that an electron beam is used rather than visible light
- Wavelength is smaller (magnification is better)
- Magnification of up to 100,000x
- TEM & SEM
TEM (Transmission Electron Microscopy)
Electron beam passes through specimen then an electromagnetic lens, to a screen/film
- Embedded in special resin
SEM (Scanning Electron Microscopy)
Electron beams knocks other electrons off the surface of a specimen
- Specimen usually coated with an electron dense medium
- Detectors capture the secondary electrons to produce the image
Simple Stains
- Use a basic dye
- Adds contrast based on colour
- Chromophore has a positive charge
- Require heat to be fixed
- Heat causes cells to be distorted
What overall charge do bacteria have?
Negative
What charge do chromophores have?
Positive
Examples of basic dyes
Crystal violet & safranin
Negative (acidic) Stains
- Use an acidic dye
- Chromophore has a negative charge
- Dye falls to sides of the cells & they appear colourless against a dark background
- See true size and shape of cells
Differential Stains
Use simple stains in different ways to visualize specific structure and features of the specimen
- Use mordants & counterstains
Mordants
Chemicals to modify and enhance the action of the simple stain
Counterstains
Simple stains that stain structures that weren’t stained the first time
- Makes contrast between these structure clearer
Gram Stains
Target differing drug resistance characteristics
Gram Positive
- Thicker cell wall
- Tend to be killed by Penicillin
Gram Negative
- Have an external layer of lipopolysaccharides
- More resistant to antibiotics
Counterstain of crystal violey
Safranin
What color is gram positive bacteria?
Purple
What colour is gram negative bacteria?
Pink/Red
Acid-fast Stain
- Used to identify Mycobacteria and Nocardia
- Have waxy material in cell wall
- Uses carbol fuschin & counterstains with methylene blue
What color do Mycibacteria and Nocardia appear in acid-fast stains?
Red
Endospore stain
- Bacillus & Clostridium
- Treat with malachite green
- Counterstain with safranin
- Endospores appear green
Flagella Stain
- A positive stain from flagella indicates that the specimen cells are motile
- If flagella present they will appear under brightfield microscopy
