2 µm meet the microbes! escherichia coli prokaryote divides every 20-30 min. (lab uses...
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2 µm
Meet the microbes!
Escherichia coli prokaryotedivides every 20-30 min.(lab uses nonpathogenic strain)
Saccharomyces cerevisiaeeukaryotedivides every ~90 min.
Microscopes are required to see individual microorganisms
Phase contrast images - 400 X magnification
Above the specimen on the microscope stage:
series of lenses focuses the image on the viewer’s retina
Compound brightfield light microscopes have multiple lenses
Below the microscope stage:
condenser lens focuses light reaching the specimen
adjustable iris controls the amount of light passing through the condenser
Oculars lenses provide 10–fold magnification
Four parfocal objective lenses magnify 4x, 10x, 40x and 100x
Ocular (eyepiece) lens
Objective lenses
Image magnification is a product of the magnification provided by the ocular and objective lenses
Objective lenses are parfocal: lenses focus the images in the same plane
Consequently, lenses can be interchanged without re-focusing the microscope
Final magnifications are 40x, 100x, 400x, 1000x
How do I adjust the light microscope to visualize specimens?
Why are stains used in light microscopy?
What is an oil immersion lens and when is it used?
How can S. cerevisiae and S. pombe be distinguished using light microscopy?
1. Plug in the microscope
2. Turn on the power
3. Adjust the amount of light coming from the LED power source using the rheostat
Power On
Rheostat
First: adjust the light
Place the slide in the slide holder
Use the XY stage controls to center the specimen
Next, position the specimen in the light path
As the magnification of a lens increases,
• the objective becomes longer
• the working distance (distance separating the lens and slide) decreases
• the light-gathering ability of the lens decreases, yielding a smaller field of view and requiring more light
Choose a lens
ALWAYS begin with the 4X or 10X lenses, which have the greatest working distance
Light source
Leica gives suggestions for iris diaphragm settings for different objective lenses
Adjust the iris diaphragm to illuminate the specimen
Opening must be wider with higher power lenses
You may want to reduce the light for live, unstained cells
Diaphragm
Microscope has course and fine focus knobs
Course Focus•Bigger Knob•Big Changes•4x and 10x only
Fine Focus•Smaller Knob•Small Changes•Fine for all lenses
Coarse focus – 1st stepFine focus – 2nd Step
Next – adjust the focus
Begin by focusing on the specimen, using the 4x or 10x objective and the coarse focus – optimize the image with the fine focus
How do I adjust the light microscope to visualize specimens?
Why are stains used in light microscopy?
What is an oil immersion objective and when is it used?
How can S. cerevisiae and S. pombe be distinguished using light microscopy?
Stains are used to increase contrast
Iodine reacts with starches
S. cerevisiae stained with iodine
How do I adjust the light microscope to visualize specimens?
Why are stains used in light microscopy?
What is an oil immersion objective and when is it used?
How can S. cerevisiae and S. pombe be distinguished using light microscopy?
The oil immersion objective is constructed differently than the 4X, 10X and 40X lenses
Dry objectives are destroyed by immersion oil!
Clean them immediately if they encounter oil.
1000X objective MUST be used with immersion oil
Immersion oil has a refractive index similar to that of glass
Prevents bending of light rays as they pass from air into glass
coverslip
sample
slide
specimen
ImmersionOil
How do I adjust the light microscope to visualize specimens?
Why are stains used in light microscopy?
What is an oil immersion objective and when is it used?
How can S. cerevisiae and S. pombe be distinguished using light microscopy?
size checkpoint
S. cerevisiae
size checkpoint
S. pombe
Cell division cycle shows distinct differences in the two yeast
size checkpoint at G1/S boundary
Saccharomyces cerevisiae (5-10 µm)
Buds begin to form in S phase
Buds continue to grow until cells divide
Cells divide before daughter cells reach the size of the mother cell
Prefers a haploid form
G1 is sharply reduced in length
Decision to divide/not divide is made at the G2/M border
Septum forms when cells are 12-15 µm
Schizosaccharomyces pombe
size checkpoint at G2/M
boundary