Chapter 3 Staining & Microscopy Flashcards
What does ‘Fix’ mean in staining? And what is the function?
Heat sample by Sliding with sample over flame 3 times quickly.
To adhere sample to slide. (But can kill sample)
Why do we stain specimens?
To add color for better view and identify organism
What are the reasons for using Basic stains?
Bacteria are negatively charged, so by using positively charged Basic stains, the stain will stick better to the sample
Opposite charges attract
List some Basic stains.
methylene blue, malachite green, safranin red, crystal violet
List some Acidic stains.
eosin red, India ink purple blue, nigrosine brown
Used primarily to color the Background
What is negative staining, and what is the purpose?
uses an acidic stain to look for capsules
What is the Simple staining technique used for?
Color background of specimen.
Used to identify shape, size and arrangement of bacteria
What is the Differential staining technique used for? And how does it work?
Identify and distinguish between different organisms in a sample
Uses More than 1 dye of contrast colors
What is the Gram Stain used to differentiate between?
Gram + and gram - organisms
Gram +: 40+ layers peptoglycan that make up cell wall
Gram-: 1 layer peptoglycan
What is the Acid Fast Stain used to determine?
What colors should appear?
If lipid coat present outside cell wall
Lipid coat= Acid Fast= RED
No lipid coat= Non acid fast= Blue
What is the Special staining technique used to identify?
Identify cell structures like flagella, endospores and capsules
If I was to do a gram stain what steps do I follow?
- [ ] Stain both purple
- [ ] Use iodine to bond with purple and hold in place
- [ ] Alcohol used to remove outer membrane of gram - and purple color
- [ ] Safranin (red) taken up by both cells but only shown in gram -
Name the special stains
Negative stain
Endospore stain
Flagella stain
What is the purpose of doing Negative Stain?
Color background with acidic (-) stain to identify capsules.
Example sentence: Negative stain is used to visualize the capsules surrounding certain bacteria.
What is the purpose of doing Endospore Stain?
Endospore stain is used to identify bacteria that are capable of forming endospores.
What is the purpose of doing Flagella Stain? Why is this important
Determine if flagella present
# and location of flagella can help determine organism.
Explain the Shafer Folten stain
Form of identifying endospores
green is forced into the endospores by steaming the bacterial emulsion.
Explain the Shafer Folten stain
Form of identifying endospores
green is forced into the endospores by steaming the bacterial emulsion.
What is Mordant (iodine) used for?
Flagella stain- Coat flagella to become visible.
Gram stain - dye adhere better to specimen.
Tell the lens power of each type of magnification:
Scan
Low
High
Oil immersion
Scan: 4X
Low: 10X
High: 40X
Oil immersion: 100X
What is the equation to find total magnification?
Microscope power X 10 (oculars) = total magnification
Light scope
Use long wavelength, gives poor resolution
Dissecting scopes
Use short, wavelengths, better resolution, but worse magnification
Used when dissecting
Describe Brightfield
Background= light
Specimen= dark
Explain Darkfield
Background= dark
Specimen = light
When using darkfield microscopes, what is important to remember about staining?
Do not stain samples because you can kill the organism. Darkfield is used primarily with living organisms.
Phase contrast Microscopy
Studies the internal organs of living organisms
Fluorescence microscopy
Uses Short wave of light to reflect images back at longer wavelengths
Flourochromes used to identify organisms
What are floral chromes and why are they important?
Dyes
Important for identifying certain organisms
Certain florochromes are known to be attracted to specific organisms
What form of microscopy is used to study physiology of a cell?
Confocal microscopy
Gives 3-D clear images by using a laser light to reflect back images that are then put together by a computer
Electron microscope microscopy
Only used to study dead organisms
2 types:
TEM- electron beam trough sample. No 3D image
SEM- scans the surface of the sample and gives a clear 3-D image of the surface only
