answer key for microscopy quiz

2012 MICROSCOPE REVIEW
by Karen L. Lancour
RELATIVE SIZE OF MICROBES
1000 millimeters (mm) = 1 meter (m)
1000 micrometers (µm or mcm) = 1 millimeter (mm)
1000 nanometers (nm) = 1 micrometer (mcm) Size of Commonly Known Microbes in micrometers (µm or mcm)
| mcm |Type |Name |How known |
|500 |Protozoa |Amoeba |Common in freshwater - have pseudopods |
|250 |Protozoa |Paramecium |Common in freshwater - have cilia |
|200 |Algae |Diatoms |Marine with silicon shells - important plankton |
|130 |Protozoa |Euglena |Common in freshwater - have flagella & |
| | | |chloroplasts |
| 10 |Fungi |Yeast |Baker's yeast - makes dough rise |
| 2 |Bacteria |E. coli |Helps in digestion - one form causes serious |
| | | |food poisoning |
| 2 |Bacteria |Lactobacillus |Used to make yogurt - helps in our digestive |
| | | |system |
| 1 |Bacteria |Cyanobacteria |Has chlorophyll - carries on photosynthesis |
| 1 |Bacteria |Staphylococcus|Causes staph infections |
| 0.3 |Virus |Small pox |Causes small pox |
| | |virus | |
| 0.15|Virus |Rabies virus |Causes rabies |
| 0.10|Virus |Influenza |Causes the flu |
| | |virus | |
| 0.03|Virus |Polio virus |Causes polio |
| 0.03|Virus |Rinovirus |Causes the common cold |
TYPES OF MICROSCOPES Visible Light is the source of illumination: (up to 2000X)
Bright Field Microscope (Lab - Light Compound Microscope)
. Most widely used
. Forms its image when light is transmitted through the specimen
. The specimen produces an image that is darker than the surrounding
illuminated field
. Can be used with live, unstained and preserved, stain specimens Dark Field Microscope
. A bright-field microscope can be adapted to a dark-field microscope by
adding a stop to the condenser
. The stop blocks all light from entering the objective lens except for
peripheral light
. The specimen produces an image that is brightly illuminated against a
dark field
. Effective for visualizing living cells that would be distorted by
drying or heat or that can't be stained with usual methods
. Does not allow for visualization of fine internal details of cells
Phase Contrast Microscope
. Used for live specimens
. Specimen is contrasted against a gray background
. Excellent for internal cell details
Differential - Interference Microscope
. Allows for detailed view of live, unstained specimens
. Includes two prisms that add contrasting colors to the image
. The image is colorful and three-dimensional Ultraviolet rays are the source of illumination: (up to 2000X) Fluorescence Microscope
. Includes a UV radiation source and a filter that protects the viewer's
eyes
. Used with dyes that show fluorescence under UV rays
. Forms a colored image against a black field
. Used in diagnosing infections caused by specific bacteria, protozoa,
and viruses using fluorescent antibodies Confocal Microscope
. Allows for viewing cells at higher magnifications using a laser beam
of light to scan various depths in the specimen
. Most often used on fluorescently stained specimens Electron beam forms image of specimen: . Originally developed for studying nonbiological materials
. Biologists began using it in the early 1930s
. Forms an image with a beam of electrons
o Electrons travel in wavelike patterns 1,000 times shorter than
visible light waves
o This increases the resolving power tremendously
. Magnification can be extremely high (between 5,000X and 1,000,000X for
biological specimens)
. Allows scientists to view the finest structure of cells
Transmission Electron Microscope - TEM (up to 100,000X)
. Often used to view structures of cells and viruses
. Electrons are transmitted through the specimen
. The specimen must be very thin (20-100 nm thick) and stained to
increase image contrast
. dark areas of a TEM image represent thicker or denser parts
TEM of
HIV virus Scanning Electron Microscope - SEM (up to 650,000X)
. Creates an extremely detailed three-dimensional view of all kinds of
objects
. Electrons bombard the surface of a whole metal-coated specimen
. Electrons deflected from the surface are picked up by a sophisticated
detector
. The electron pattern is displayed as an image on a television screen
. Contours of specimens resolved with SEM are very revealing and
surprising
. The images may be computer enhances to give them color
SEM of
HIV virus
Note: Color can be added to the TEM & SEM images by computer MICROSCOPY REVIEW - Light Compound Microscope Parts of the microscope and their function: The number in front of
each part of the microscope represents its number on the diagram. 1. ocular - magnifies the image formed by the objective.
2. nosepiece - holds the objectives.
3. objectives - lenses that receive the light from the field of
view and form the first image.
4. stage - supports the slide and the specimen.
5. stage clips - hold the slide in place.
6. base - foundation which supports the scope & keeps it stable.
7. diaphragm - controls the amount of light reaching the specimen.
8. illuminator - source of light.
9. course adjustment - used for initial or low power adjustment.
10. fine adjustment - used for fine tuning & high power focusing.
11. arm - supports the ocular, objectives and body tube.
12. body tube - tube or barrel between the ocular and the
objectives.
PRINCIPLES OF MICROSCOPY
A. Appearance of objects
1. Inverted and reversed (upside-down & backwards)
2. If an "e" is placed in the stage in its normal
position, it will appear as an " [pic] ".
3. Only a thin layer of the specimen is in focus at any
level (depth of focus).
B. Movement of specimens
1. Actual movement is opposite to appeared direction of
movement.
2. If an organism is actually moving ( ( ), it will
appear to be moving ( ( ).
C. Total magnification
1. Multiply ocular magnification times objective
magnification.
2. Oculars are normally 10X or 12X.
3. Objectives are typically as follows:
a. scanning power - 4X or 5X or 6X
b. low power - 10X or 12X
c. high power - 40X or 43X or 45X
4. Sample problem: If the ocular is 10X and the
objective is 43X, the total
magnification is 430.
D. Changing objectives
1. When changing objectives from scanning power to lower
power to high power, the
following changes will occur:
a. the size of the field of view decreases.
b. the field of view is darker.
c. the size of the image increases..
d. the resolution (ability to separate small
details) increases.
e. the working distance (distance between coverslip
& objective) decreases.
f. the depth of focus (thickness of the specimen
which may be seen in focus) is reduced.
2. The relationships of magnification and the fields of
view diameter and area ratios are
approximately (10 X ocular and listed objective)
| objective | total | diameter | area |
|scanning 5X |magnification |- |- |
|low 10X |50 |1/2 scanning |1/4 scanning |
|high 40X |100 |1/4 low |1/16 low |
| |400 | | | [pic] Principles of advanced microscopy A. Measuring the diameter of the field of view.
1. Place a transparent millimeter ruler on the stage, hold it
down with the stage clips, and observe the
ruler in the desired field of view - scanning or low. (See Fig. 1)
2. Focus on the metric edge of the ruler. Hint: applying
gentle pressure to the free end of the
ruler will help adjust for the thin ruler and allow for better focus.
3. Place the center of one millimeter marking at the left edge
of the field of view (see Figure 2) and measure the
diameter of the field in millimeters. For reference: