3: Bacterial Demonstration Flashcards
why do we stain
direct visualization of the organism
process in which bacteria are stained to give color to them (because they are colorless and transparent).
bacterial staining
types of dyes
- Cationic/basic - combines with acidic cellular elements
- e.g., crystal violet, methylene blue, malachite green, safranin - Anionic/acidic - combines with alkaline cell elements
- e.g., acid fuchsin, eosin, nigrosine
!!! Bacteria have an affinity to BASIC dyes due to the acidic nature of their protoplasm
2 components of stains
positive ions
negative ions
section of a molecule in a given material that absorb particular wavelengths of visible light causing us to see color
chromophore
3 types of stains
simple stains
differential stains
special stains
a type of stain that use only one and stains all cells with the same color
give 3 examples
simple stain - e.g., methylene blue, crystal violet, safranin, carbol fuchsin
a type of stain that distinguish two kinds of microorganisms
give 2 examples
differential stains - e.g., gram stain, acid-fast stain
a type of stain that highlight specific cell structures
give 2 examples
special stains - e.g., Hansen’s, Anthony’s, Schaeffer-Fulton
– inactivate enzymes that might disrupt cell morphology and toughens cell structures so they will
not change during staining and observation
- microorganisms are killed and attached firmly to the slide during this method
fixation
2 fundamental types of fixation
heat fixation - gently flame heating an air-dried film of bacteria and adequately preserve overall morphology but not structures within the cells
chemical fixation - to protect fine cellular substructure and the morphology of larger and more delicate microorganisms
- differential stain that allows the determination of to protect fine cellular substructure and the morphology of larger and more delicate microorganisms
- devised by Hans Christian Gram (1884)
gram staining
name the reagents in gram staining
- primary stain
- mordant
- decolorizer
- counterstain
primary stain - crystal violet
mordant - Lugol’s iodine
decolorizer - acetone alcohol
counterstain - safranin
!!! study procedure !!!
3 mechanisms involved in gram staining
thickness of the cell wall
pore size
permeability of the intact cell
4 limitations of gram staining
- high number of microorganisms required
- liquid samples with low numbers of microorganisms (e.g., cerebrospinal fluid)
- requires centrifugation to concentrate pathogens
- pellet then examined after staining
a differential stain introduced by Paul Ehrlich (1882) and modified by Ziehl and Neelsen (1883)
used for microorganisms that are not staining
by simple or Gram staining method
acid-fast staining
name the reagents in acid-fast staining
- primary stain
- mordant
- decolorizer
- counterstain
primary stain - carbol fuchsin
mordant - heat
decolorizer - acid alcohol
counterstain - methylene blue
!!! study the procedure !!!
the substance behind why some bacteria retain the color of the primary stain (carbol fuchsin) in acid-fast staining
mycolic acid - a waxy substance that does not allow the decolorizer to enter into the cell wall due to its
waxy nature
- a special stain for bacterial capsules wherein colorless bacteria are observed against a dark background
- distortions of cell size and shape are minimized because heat fixing is not necessary and the cells do not pick up the stain
negative stain
- a special stain method for endospores wherein the endospore are stained with malachite green; heat is used to provide stain penetration
- rest of the cell is then decolorized, and counter stained a light red safranin
Schaffer-Fulton Method
!!! study method !!!
a special stain method where thin cells and structures are thickened with silver on the surface so they become visible under ordinary microscope
e.g., demonstration of spirochetes and bacterial flagella and the sue of FONTANA’S SILVER STAIN
impregnation
any technique for producing visible images of structures or details too small to be seen by the human eye using a microscope or other magnification too
microscopy
3 main branches of microscopy
- optical/light - use of any kind of microscope that uses visible light to observe specimens
- electron - uses electromagnetic lenses, electrons, and a fluorescent screen
- image captured on photographic film to create an ELECTRON PHOTOMICROGRAPH
- use 0.005 nm wavelength, 100, 000 SHORTER THAN VISIBLE LIGHT - scanning probe microscopy - photographic film to create an electron photomicrograph
- higher resolution than electron and sample does not need special preparation
3 things a microscope should provide
magnification
resolution
clarity
