1. Microbiology: Bacterial Structures and their Functions (3) Flashcards
What is resolving power?
Refers to the ability of the lenses to discriminate between two adjacent objects.
What is light microscopy?
Refers to the use of any kind of microscope that uses visible light to observe microorganism.
Most used today are Compound Microscopes, containing two sets of lenses: the objective lens and the ocular lens.
What is the absolute limit of the resolving power?
Roughly 1/2 the wavelength of light used.
What is the range of the wavelength of visible light?
Ranges from 400nm to 800nm, and thus the smallest object that can be observed in a light microscope must be at least 200nm (0.2 um)
Why can light microscopes not be used to provide information on the internal structures of bacterial cells?
Because of the size and length of the bacteria.
most bacteria range in size from 0.2 micrometers to 2 micrometers and 2 micrometers to 8 micrometers in length.
What are the different types of light microscopy?
Brightfield microscopy phase-contrast microscopy darkfield microscopy ultraviolet microscopy fluorescence microscopy
What is electron microscopy?
an electron microscope uses a beam of electrons rather than visible or ultraviolet light to illuminate the object.
The shorter wavelengths of electrons at the voltages commonly used does what to the resolving power?
increases the resolving power to about 0.001 micrometers (1nm).
What are the types of electron microscopes?
the transmission electron microscope (TEM)
the scanning electron microscope (SEM)
Most microorganisms usually appear what when viewed through a light microscope?
Colorless
Most stains are composed of what?
are salts composed of a positive and a negative ion, one of which is colored (referred to as the chromophore)
What are basic dyes?
colored cation (+ charge) colorless anion (- charge) includes crystal violet, safranin, malachite green, and methylene blue
What is acidic dyes?
colorless cation (+ charge) colored anion (- charge) includes Cango red, eosin, and nigrosin
At pH 7, bacteria are charged how?
negatively charged
therefore, the colored positive ion in a basic dye is attracted to the negatively charged bacterial cell.
What is known as negative staining?
the colored negative ions in an acidic dye are repelled by the negatively charged bacterial cell, so the stain colors the background while the bacterial cell remains colorless.
How do you prepare bacteria for staining?
a small amount of culture is dispersed in a drop of water on a glass slide.
The smear is allowed to dry at room temperature and the bacteria are fixed to the glass by gentle heating in a flame, smear side up.
Fixing also kills the bacteria.
Facts about gram stains
was developed in 1884 by the Danish bacteriologist Hans Christian Gram.
the basis of the gram reaction is the differences in the structure of the cell walls of gram-positive and gram-negative bacteria.
What is the primary stain?
Crystal violet (violet to purple)
What is the counterstain?
Safranin (red to pink)
What is the Decolorizing agent?
Alcohol or acetone-alcohol
What is the Mordant?
Iodine.
a mordant increases the affinity of a stain for a biological specimen; it can also be used to coat a structure (eg. flagellum) to make it thicker and easier to see after it is stained with a dye.
List the steps of a gram stain
1) application of crystal violet (purple dye)
2) application of iodine (mordant)
3) alcohol wash (decolorization)
4) application of safranin (counterstain)
Why can the crystal violet-iodine complex inside the cells of gram-positive bacteria not be washed out by alcohol?
because of the thick peptidoglycan layer and thus the gram-positive cells retain the dye and stain purple.
How do gram-negative bacteria remain colorless when crystal violet-iodine complex is used?
the decolorizing alcohol disrupts the lipopolysaccharide layer and the crystal violet-iodine complex is washed out through the peptidoglycan layer. This leaves the gram-negative bacteria colorless until counterstained with safranin, after which they appear pink.