Chapter 3 - Staining and Microscopy Flashcards
Why are slides heat fixed?
To kill the microorganisms and adhere them to slide while preserving the microbe in its natural state
Why do we stain slides?
To make organisms more visible and identifiable
What are stains?
Salt - either basic (+) or acidic (-)
What kind of stain is used most of the time and why?
Basic, positive ions are attracted to the negative bacteria cells, the bond is stronger
Which 4 basic stains will we use?
Methylene blue, malachite green, safranin, crystal violet
What are 3 acidic stains?
Eosin (Red), India ink (purple), nigrosin (brown)
Which kind of stain requires no heat fix?
Acidic stain
What part of the slide does negative stain color?
The background
What is the purpose of negative staining?
To study overall morphology and size of the organism, to observe the presence/absence of capsules
Where in a cell is the capsule found?
Outside the cell wall
What is an aqueous or alcohol solution of a single basic dye color specimen?
Simple stain
What is the purpose of a simple stain?
To help see the microorganism, see shape and structure, does not help I.D.
What is a differential stain?
A stain that utilizes more than one dye of contrasting colors
Which stain helps distinguish different kinds of bacteria and identify bacteria?
Differential
Who developed the Gram stain?
Hans Christian Gram
What is G+?
A cell with a cell wall with 40+ layers of peptidoglycan
A cell whose cell wall is one thin layer of peptidoglycan is what kind of cell?
G-
What happens if too much decolorizer is used on a Gram stain?
A G+ can read as a G-
When might a G- look like a G+?
If too little decolorizer is used
What kind of stain is a Gram stain?
Differential
Which kind of differential stain binds strongly to bacteria with waxy material in their cells?
Acid Fast
What does an acid fast stain test for?
The presence of a lipid coat outside the cell wall
What color does a lipid coat show up as on an acid fast stain?
Red
If a bacteria is not acid fast, what color will it appear?
Blue
What is the purpose of a special stain?
To color and isolate specific parts of a microorganisms such as endospores or flagella
What are the three kinds of special stain?
Negative, Endospore, and Flagella
Which special stain demonstrates the presence of capsules to determine the virulence of a cell?
Negative stain
How does a negative stain work?
It uses a color background and an acidic dye
Under adverse conditions, what can happen to some cells?
They can transform into endospores
What is an endospore?
Dormant cell, more resilient, can revert back to cell in ideal conditions
What is the purpose of an endospore stain?
To stain the endospore wall and detect it
What is the purpose of a flagella stain?
to make the flagella visible because the number and location can help diagnose
What kind of substance is iodine?
A mordant
What is a mordant?
A substance used to increase the affinity of stain for biological specimen, coats the structure and makes it easier to see
What is the term for the ability of a lens to distinguish fine details and structures?
Resolution
Which 4 lenses do our microscopes use?
4x - scan
10x - low
40x - high
100x - oil immersion
What kind of microscope uses visible light to observe specimen?
Light scopes
What is a dissecting scope?
A scope with no light source under the stage, uses short wavelength (better resolution, less magnification), entire specimen
In what kind of microscopy is the background dark and the specimen light?
Darkfield
Which kind of microscopy can examine live organisms?
Darkfield
What is Brightfield Microscopy?
Microscopes where the background is light and the specimen is colored
Why are darkfield samples left unstained?
It distorts them
Which kind of microscopy gives a detailed examination of living organisms internal organs?
Phase contrast
What kind of microscopy uses fluorescence to study organisms?
Fluorescence
What does fluorescent microscopy use to color the specimen?
Fluorochromes
What kind of fluorescent microscopy assembles 3D images?
Confocal microscopy
How does confocal microscopy work?
fluorchromes are used, then laser, then computer assembles the images
What kind of microscope uses a beam of electrons and electromagnetic lenses instead of light and glass lenses?
Electron microscope
What are electron microscopes used for?
viewing viruses
Which kind of electron microscope makes internal organs visible and is not 3D?
TEM - transmission microscope
What is a Scanning electron microscope?
The electron beam scans the surface of the cell, 3D image, sample doesn’t need to be thin, shows external structure only
In what way do fluorescent microscopes absorb wavelengths of light and reflect them back?
From a short to a long wavelength
What is a disadvantage of the electron microscope?
It cannot be used on live organisms
What are the three overall staining techniques?
Simple
Negative
Special
What stain determine if an organism has a lipid coat?
Acid fast
Fluorescent Antigen-Antibody Technique
Animal injected with antigen, produces antibodies, antibodies removed and combined with flurochrome, fluorescent antibodies added to slide with unknown bacteria, if unknown fluoresces, it is the same bacteria injected into the animal
What is immunofluorescence?
Fluorescent-antibody (FA) technique
What two diagnoses is immunofluorescene especially helpful for?
Syphilis and rabies
