Microscopy & Staining – Flashcards

Unlock all answers in this set

Unlock answers
question

Microscopy: The instrument

 

answer
A simple microscope has only one lens
question
Light Microscopy
answer
The use of any kind of microscope that uses visible light to observe specimens.
question

Compound Light Microscope

 

answer
In a Compound Light Microscopy the image from the objective lens is magnified again by ocular lens.
question
Total Magnification =
answer
Objective lens X Ocular lens
question

Compound Light Microscopy

(Resolution)

answer
  • The ability of lenses to distinguish two points.
  • Shorter wave lengths of light provide greater resolution.
  • blue filters increase resolution as blue light has the shortest wavelength of visible light.
  • a microscope with a resolving power of 0.4nm can distinguish two points if they are at least 0.4nm apart.
question

Compound Light Microscope

(Refractive Index)

answer
  • Is a measure of the ligh-bending ability of a medium.
  • The light may bend in air so much that is misses the small high-magnification lens
  • immersion oil is used to prevent this
question
Brightfield Illumination
answer
  • Dark abjects are visible against a bright background.
  • Light reflected off the specimen does not enter the objective lens.
question

Darkfield Illuminaton

 

answer
  • Light objects are visible against a dark background
  • Light reflected off the specimen enters the objective lens.
question
Phase-Contrast Microscopy
answer
  • Accentuates diffractionof the light that passes through a specimen
  • Provides good resolution
question

Diffential Interference Contrast Microscopy

DIC

answer
  • Accentuates diffraction of the light that passes through a specimen; uses two beams of light.
  • makes optical illusion.
  • makes specimen look three dimmensional.
  • used when specimen is colorless and/or dont want to stain it.
  • also to do more detailed internal study.
question
Fluorescence Microscopy
answer
  • Uses UV light.
  • Flourescent substances absorb UV light and emit visible light.
  • Cells may be stained with flourescent dyes(fluorochromes).
  • used to track living organisms (highlights)
question
Confocal Microscopy
answer
  • Cells are stained w/ floorochrome dyes
  • Short wavelength (blue) light is used to excite the dyes.
  • The light illuminates each plane in a specimen to produce a three-dimensional image, up to 100um deep.
  • reconstruct three-dimensional images.
  • are actual images.
question

Two-Photon Microscopy

 

answer
  • Cells are staines w/ fluorochrome dyes
  • 2 photons of long-wavelength (red) light are used to excite the dyes. (red light is less damaging compare to blue light)
  • Used to study organisms attached to a surface. up to 1mm deep.
  • little less resolution
question

Scanning Acoustic Microscopy

SAM

answer
  • Measures sound waves that are reflected back from an object.
  • Used to study cells attached to surfaced
  • resolution 1um
  • good to look for biofilms
  • to visualize molecules (DNA)
question
Atomic Force Microscopy
answer
  • Uses diamonds
  • provides 3-d images
  • good resolution
question

Electron Microscopy

 

answer
  • Uses electrons instead of light
  • The shorter wavelength of electrons the greater resolution.
  • nothing is alive 
  • it uses magnets to focus electron bean 
  • there are 2 types
question

Transmission Electron Microscopy

TEM

answer
  • Ultrathin sections of specimens
  • electron passes thru specimen, then an electromagnetic lens, to a screen or film
  • specimens may be stained w/ a heavy-metal salt
  • used to observe tiny stuff inside cells or molecules
  • resolution 2.5nm
question

Scanning Electron Microscopy

SEM

answer
  • electron gun produces a beam of electrons that scans the surface of a whole specimen
  • secondary electrons emitted from the specimen produce image
  • it sprays gold on specimen (gold is dense so it produces good images)
  • resolutuon 20nm or 1,000-10,000x
question

Sanned-Probe Microscopy

STM

answer
  • uses a metal probe to scan specimen
  • resolution 1/100 of an atom
question

Atomic Force Microscopy

AFM

 

answer
  • uses a metal and diamond probe inserted into the specimen
  • produces 3D images
question
Preparation of Specimens for Light Microscopy
answer
  • Acid dye and basic dye
  • simple staining
  • gram stain,gram posite, gram negative
  • gram stain vs acid fast stain
  • capsule stain, endorspore stain, flagella stain
question

Staining:

 

answer
Coloring the microbe w/ dye that emphasizes certain structures. done to increase contrast.
question
Smear:
answer
A thin film of a solution of microbes on a slide. A smear is usually fixed to attached the microbes to slide and to kill the microbes.
question
Stains consist of:
answer
a Positive and Negative Ion
question
Basic Dye
answer
The Chromophore is a Cation
question
Acid Dye
answer
The chromophore is an anion
question
Negative Staining
answer
Staining the background instead of the cell
question
Some basic dyes used in class are:
answer
  • Methylene blue
  • Crystal Violet
  • Saffranin Red
question
Simple Stain
answer
use of a single basic dye
question
A mordant
answer
may be used to hold the stain or coat the specimen to enlarge it.
question
Differential Stains
answer
  • used to distinguish between bacteria

* gram stain and acid-fast stain

  • Differentiate regions 

  • Require multiple stains 

 

question
Gram Stain
answer
Classifies bacteria into gram-positive or gram-negative
question
Gram positive bacteria
answer
tend to be killed by penicillin and detergents
question
Gram Negative Bacteria
answer
Bacteria is more resistant to antibiotics
question

Whats the color of Gram-Positive cells when stained with Crystal violet,mordant (iodine),decolorizing agent (alcohol-acetone), and counterstain (Safranin)?

 

answer
All 4 result in Purple
question

Whats the color of Gram-Negative cells when stained with Crystal violet,mordant (iodine),decolorizing agent (alcohol-acetone), and counterstain (Safranin)?

 
answer
  • Crystal violet,mordant (iodine) result in purple.
  • decolorizing agent (alcohol-acetone) result in Colorless
  • counterstain (Safranin) result in Red
question
Acid-Fast Stain
answer

One that differentiates bacteria into distinctive groups

binds strongly only to bacteria that have waxy material in the cell wall is not decolorized by acid-alcohol Ex:

  • Mycobacterium
  • Nocardia

 

question
Special Stains
answer

used to distinguish parts of cells in microrganisms.

Capsule Stain

Endospore Stain

Flagella Stain

 

question
Negative Staining for Capsules
answer
  • used to demonstrate the presence of capsules
  • capsules don't accept most satins
  • appear unstained
  • stand out against contrasting background
question
Endospore Staining
answer
  • used to detect presence of endospores in bacteria
  • primary stain machita green with heat
  • decolorazes cells red
  • endospores turn green when machita green with heat is used
  • endospores turn red/pink when counterstain safranin is used
question
Flagella Stain
answer
  • used to demonstrate the presence of flagella
  • mordant is used until it becomes visible
  • then is stained with carbolfuchism
question
Types of Stains
answer
  • Simple (methylene blue,carbolfuschsin,crystal violet and safranin)
  • Differential (Gram, Acid-fast)
  • Special (Negative, Endospore and Flagella)
Get an explanation on any task
Get unstuck with the help of our AI assistant in seconds
New