BIOL 2421 Microbiology Lecture Notes Dr. Weis
Microscopy/Staining
Microorganism Size
Microorganisms range in size from the smallest [virus at 0.01μm or 10 ήm] to the largest [protozoa at 100µm].
In order to view most of these microorganisms, an instrument called the microscope is used. Specific microscopy techniques involve light microscopy, atomic-force microscopy, scanning electron microscopy (SEM), and transmission microscopy (TEM).
Light microscopy uses electromagnetic radiation in the visible or ultraviolet wavelength range to obtain a magnified image of an object. The resolution of the image is limited by the minimum forces of the radiation due to diffraction. The unaided human eye can see objects as small as 0.1mm in diameter. Most cells can not be seen without the aid of the microscope, since their diameters range between 0.01 and 0.1 mm. The two most commonly used light microscopes are the binocular dissecting microscope for use in viewing entire objects in three dimensions. Use of this scope can help to magnify microorganism growth on an agar plate. The other light microscope is the compound light microscope, which is used in the lab extensively to view cells, fungi, and bacteria.
Viruses are too small to view using this instrument, so other techniques are used.
The simplest light microscope consists of an objective lens and an eyepiece.
The objective lens forms a real image which is then magnified by the eyepiece to create a virtual image.
Illumination comes from below at the base and is a light source for the microscope. The light passes through the translucent sections of the sample to the objective lens.
High magnification requires very bright illumination of the sample and the condenser lens is usually placed between the light source and the stage to help focus light onto the sample. This is done through the iris diaphragm and its control lever.
The objective lens is identified by its magnification properties: scanning (4X), low (10X), high dry (40X), and oil (100X), while the ocular lens magnification is usually 10X.
Total magnification of an object is the result of the objective lens magnification x the ocular lens magnification.
The mechanical stage allows light to pass through a center hole and is also used as a means to support and anchor the slide that is viewed. Stage clips aid in this process.
Other microscopy principles:
a) Resolving power: ability to distinguish between two separate objects
b) Field of view: diameter area visible for a particular objective lens
c) Depth of Field: vertical depth of an object that still remains in focus
d) Parfocal: ability of a microscope to remain in focus while switching between objectives
e) Working Distance: clearance between slide and bottom of objective lens
f) Oil immersion microscopy: use of oil to capture refracted light and concentrate
it through the specimen
g) Refractive Index: the bending power of light as it travels from one medium to another
Staining is used to increase the contrast and resolution of a specimen.
Definition: Coloring the microorganism with a dye that emphasis certain structures.
Preparation for Staining:
1) Create smear: film of specimen spread over slide and allowed to air dry
2) Fixing: adhere organism to slide by heat. Also kills microorganism
3) Stain: dyes applied and washed off.
Types of Dyes:
Stains are salts composed of positive and negative ions.
One of these ions is colored and called the chromophore.
Basic dyes consist of the positive ion that stains negatively charged structures
Acid dyes consist of the negative ion that stains positively charged structures
Examples
Basic Dyes:
Crystal violet (violet)
Methylene blue (blue)
Malachite Green (green)
Safranin (red)
Acidic Dyes:
Nigrosin (black)
Congo Red (red)
Eosin (red)
Carbol (Acid) Fuschin (red)
Since
bacteria are negative charged, basic dyes are used since the positive
ion in the dye is attracted to the bacteria. Acid
dyes are not normally used since the dye’s negative ion is repelled by
the bacteria’s negatively charged cytoplasm. Acid dyes are used in a negative
staining technique, in which the bacterial cells remain clear against a
colored background. Since the bacteria are not heat fixed, the cells normal
size and shape are maintained.
Sudan Black stain does not form ionic bonds but is lipid soluble and is used to stain the phospholipid membrane.
Types of Staining:
Simple Staining: single basic dye used to highlight a microorganism shape and arrangement.
Differential Staining: distinguish between bacterial types
Special Stains: use color to isolate and identify specific parts of a bacterium
Mordant: a chemical additive used to intensify the stain. Can be used in all types of staining.
Gram Stain
Differential Stain
Classifies bacteria into two groups: Gram (+) and Gram (-)
Four different reagents are used
|
Solutions and Use |
G (+) reaction |
G(-) reaction |
|
Crystal Violet (CV) Primary stain |
Cells stain violet |
Cells stain violet |
|
Gram’s Iodine (I) Mordant |
CV-I complex formed within cells, cells remain violet |
CV-I complex formed within cells, cells remain violet |
|
Alcohol Decolorizer |
Cell walls dehydrate, pores shrink, permeability decreases, CV-I complex retained, cells remain violet |
Lipid extracted from cell walls; porosity increases, CV-I removed from cells; Cells are colorless |
|
Safranin counterstain |
Cells not affected Remain violet |
Cells take up the stain, Become pink to red |
The composition of the cell wall can vary among species of bacteria, but it is important in the staining characteristics that help identify and classify each organism.
Those bacteria with a thick cell wall have a peptidoglycan structure made up of carbohydrate polymers crossed linked by proteins. Such bacteria retain a purple color with the crystal violet dye.
Other bacteria have a double cell wall consisting of a thin inner wall made up of peptidoglycan and an outer wall made up of carbohydrates, proteins, and lipids. These bacteria do not retain the crystal violet dye, but take up the red color from the safranin stain.
The composition of the cell wall also determines their susceptibility to chemotherapeutic agents.
Acid Fast Technique
Differential Stain
Waxy material in cell wall makes it difficult to identify bacteria with Gram stain
Used to identify Mycobacteria (TB and Leprosy) and Nocardia species.
Carbol Fuscin (red) dye penetrates waxy lipid material in cell wall
Alcohol decolorizer removes stain from non-acid fast bacteria
Special Stains
Capsule Stain
Capsules are an additional layer of polysaccharides and proteins secreted by some bacteria on the cell surface as an additional barrier for protection against dehydration, nutrient loss, and phagocytosis.
Identify presence of capsule can be useful in determining virulence
Negative stain and /or Simple Stain can be used in combination
Negative Stain such as Nigrosin or Congo Red with
Simple Stain such as Crystal Violet
Also can use other Staining Techniques
Crystal Violet
Copper Sulfate as decolorizer and counterstain
Endospore Stain
Regular dyes do not penetrate the dormant capsule structure of organisms such as Bacillus and Clostridum species
Malachite Green is used as the primary stain and heat is used to drive in the dye
Water as decolorizer
Safranin counterstain
Endospores appear green within red/pink vegetative cells
Flagella Stain
Carbol Fuschin with a mordant
Identify structure used for locomotion, number and arrangement are
useful in identification.
Since staining flagella can be complicated, other methods can be used
to determine motility:
a) Hanging Drop Technique (cover slip with special slide)
b) Motility Test agar