Ch 2 Flashcards
Define magnification
How many times larger the image is than the actual object
Define resolution
The ability to distinguish individual objects as separate entities
Light Microscope
-how does it work? Explain.
light is sent from light source, through a specimen.
Image of the specimen is magnified by the objective lens, and this image is magnified again by the eyepiece lens
Light microscope
-advantages and disadvantages
Inexpensive to buy & operate Small so portable Simple(er) sample prep (= less artefacts) Can view living specimen Colour is seen X 2D image X Lower magnification (up to 2000X) X Lower Resolution (resolving power 200nm)
Laser Scanning Confocal Microscope
A beam of light is used to create image;
Laser is used to illuminate specimen (point illumination)
Specimen must have been treated with fluorescent dye - fluorescent components emit light. This light is filtered through a pin hole aperture.
Only light radiated from close to the focal plane is detected.
What is diffraction
Diffraction is the tendency for light to spread as it passes close to physical structures
Electron Microscopy
A beam of electrons with a wavelength less than 1nm is used to illuminate a specimen.
More detail of cell ultra structure can be seen because electrons have a smaller wavelength than light.
+higher magnification & resolution (resolution is no longer a limiting factor)
Transmission Electron Microscope
Electron beam penetrates the cell providing detail of a cell’s internal structures. \+high resolution (0.5nm) \+see internal structure of organelles -2D image -need v. thin specimen
Scanning Electron Microscope
Beam of electrons moves back n forth across a cell’s surface, creating details of the cell surface characteristics. \+3D image \+specimen doesn’t need to be as thin - Lower resolution than TEM (3-10nm) -no colour
What is an Artefact
An artefact is a visible structural detail caused by processing the specimen and is not a feature of the specimen, e.g., air bubble
Cytoskeleton components
Microfilaments
Microtubules
Intermediate fibres
Microfilaments
Contractile fibres
Made of actin
Responsible for cell movement and cell contraction in cyokinesis
Microtubules
Scaffold-like structure : Globular tubular proteins polymerise to form tubes.
Determines cell shape
Act as tracks for organelle movement
Spindle fibres are composed of microtubules
Intermediate fibres
Give mechanical strength to cells
Help to maintain cell integrity
State Methods of Sample Preparation
Dry Mount
Wet Mount
- Squash Slides
- Smear Slides
How to prepare a Dry Mount
Solid specimen - whole or thinly sliced (sectioning) Specimen placed in centre of slide Place Cover Slip over whole - hair, pollon, dust sectioned - muscle tissue, plants
How to prepare a Wet Mount
Specimen suspended in liquid such as Water or Immersion Oil
Place Cover Slip on from an Angle
Aquatic sample vied this way
How to prepare a Squash Slide
Prepare Wet Mount: specimen suspended in liquid
Use Lense Tissue to Gently Press Down Cover Slip
Avoid Damge by Squashing sample between 2 microscope slides
! take care in ensuring cover slip is not broken when pressed !
Root tip squashed are used to look at Cell Division
How to prepare a Smear Slide
Edge of slide used to Smear sample, creating Thin, Even Coating on another slide.
Cover Slip placed over sample
Blood Sample to view blood cells
Why is Staining used?
to Increase in Contrast, making Components Visable for identification
- Stains Increase Contrast as Different Components within a cell Take up stains to Different Degrees.
How to Prepare Sample for Staining
Place Sample on Slide & allow to Air Dry
Pass through a Flame - Heat Fix = Specimen Adheres to microscope Slide and will take up stain
What is Differential Staining?
technique that enables 2 types of Organism to be distinguished
It can differentiate between different organelles of a single organism within a tissue sample
E.G. Gram staining and Acid-Fast
What is Gram Staining technique of Differential Staining?
separates bacteria into Gram-positive (penicillin susceptible) and Gram-negative bacteria
Crystal Violet is first applied to specimen on a slide, then Iodine (fixes the dye)
Wash Slide with Alcohol -
Gram-positive Retain crystal violet stain = Blue/Purple
Gram-negative Lose stain (have thin walls). Then stained with Safrinin dye, a Counterstain = appear Red
What is Acid-Fast technique of Differential Staining?
Used to Differentiate species of Myobacterium from other bacteria.
Lipid Solvent is used to carry Carbolfuchsin Dye into the cells.
Cells washed with Dilute Acid-Alcohol solution.
Myobacteriam retain Carbolfuchsin stain as not affected by acid-alcohol solution (Red)
Other bacteria Lose the stain and are Exposed to Methylene Blue Stain
Rules for Scientific Drawings:
Title State Magnification Sharp Pencil Use as much paper as possible; at least half space Smooth, Continuous Lines Do not Shade Correct Proportions Label lines drawn with Ruler, Parallel to Top of Page
Using a Graticule to Calibrate a Light Microscope
Calibrate Eyepiece Graticule for magnification using Stage Micrometer
What is an Eyepiece Graticule?
Glass Disc with a scale marked 1 - 100 (No Units)
Scale remains Unchanged by objective lens
Relative Size of Divisions Increase with Increased Magnification
Scale on Graticule is Calibrated using Stage Micrometer
Used to Measure size of Sample under Microscope
What is a Stage Micrometer?
a Microscope Slide with a very Accurate Scale in Micrometers engraved.
Scale is usually 100 divisions = 1mm (1 division = 10um)
Used to Calibrate Eyepiece Graticule for specific Magnification
Golgi Apparatus
series of flattened membranous sacs
Transport vesicles fuse with Golgi Apparatus - contents emptied into Golgi. Contents (proteins/lipids) travel through the Golgi; sorted, packaged, tagged, so sent to right place.
(Golgi) Vesicles
Store and Transport modified proteins/lipids from Golgi Apparatus.
Lysosomes contain Lysozymes
Endoplasmic Reticulum
a series of interconnected membranous sacs and tubules.
phospholipid bilayer embedded with proteins
RER - process & fold proteins
SER - make & process lipids
Centrioles
Cylindrical - made of parallel Microtubules that surround a central cavity
Key component of centromeres
Flagella
eukaryotic - 9 + 2 microtubules (tubulin)
used for locomotion
whipping motion
Cilia
central core = axoneme
Primary Cilia have 9 + 0
Motile Cilia = 9 + 2
beat rhythmically
Emulsion Test
for Lipids
place sample in boiling tube Add Ethanol Shake Well. leeave for 5 Add distilled Water White, Milky Layers forms if lipid present
Biuret Test
for Proteins
Add sample to Distilled water + Biuret Solution
Shake Well, leave for 5
Observe Colour change (from blue)
Violet = protein present
Benedict’s Test for non-reducing Sugars
Boil in Dilute HCl
Add Sodium bicarbonate
Benedicts test - positive if non-reducing sugar present
Test for Starch
Add few drops of Iodine to sample
Observe colour change:
Orange to blue/black
Chromatography
separates substances based on their interactions with a Mobile and Stationary phase
