1. Meet the microbes!
Microscopes are required to see individual microorganisms
2 µm
Escherichia coli
prokaryote
divides every min.
(lab uses nonpathogenic strain)
Saccharomyces cerevisiae
eukaryote
divides every ~90 min.
Phase contrast images X magnification

2. Compound brightfield light microscopes have multiple lenses
Above the specimen on the microscope stage:
series of lenses focuses the image on the viewer’s retina
Below the microscope stage:
condenser lens focuses light reaching the specimen
adjustable iris controls the amount of light passing through the condenser

3. Ocular (eyepiece) lens
Image magnification is a product of the magnification provided by the ocular and objective lenses
Ocular (eyepiece) lens
Oculars lenses provide 10–fold magnification
Four parfocal objective lenses magnify 4x, 10x, 40x and 100x
Objective lenses
Final magnifications are 40x, 100x, 400x, 1000x
Objective lenses are parfocal: lenses focus the images in the same plane
Consequently, lenses can be interchanged without re-focusing the microscope

4. 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?

5. First: adjust the light
Plug in the microscope
Turn on the power
Adjust the amount of light coming from the LED power source using the rheostat
Power On
First let’s talk about some of the other parts and what they’re used for. The power switch and the Rheostat-
We can’t focus or look at our sample if we cant see it! We need to turn the microscope on and then adjust the level of light as appropriate (too much can be as bad as too little)
Rheostat

6. Next, position the specimen in the light path
Place the slide in the slide holder
Use the XY stage controls to center the specimen

7. Choose a lens 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
ALWAYS begin with the 4X or 10X lenses, which have the greatest working distance

8. Diaphragm Light source
Adjust the iris diaphragm to illuminate the specimen
Opening must be wider with higher power lenses
Diaphragm
Leica gives suggestions for iris diaphragm settings for different objective lenses
Light source
You may want to reduce the light for live, unstained cells

9. Next – adjust the focus
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 step
Once we have the light comfortably set we can begin focusing through the use to the two knobs on either side of the base. The inner, larger knob is the course focus- makes big changes gets us in the ball park- we are only going to use this for the 4x (maybe the 10x)
Second is the outer smaller ring which is our fine focus- makes small micro adjustments, its ok for all the lenses, and is the only focusing we should do for the 40x
Fine focus – 2nd Step
Begin by focusing on the specimen, using the 4x or 10x objective and the coarse focus – optimize the image with the fine focus

10. 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?

11. S. cerevisiae stained with iodine
Stains are used to increase contrast
Iodine reacts with starches
S. cerevisiae stained with iodine

12. 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?

13. 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

14. 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
Immersion
Oil

15. 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?

16. Cell division cycle shows distinct differences in the two yeast
S. cerevisiae
S. pombe
size checkpoint
size checkpoint

17. size checkpoint at G1/S boundary
Saccharomyces cerevisiae (5-10 µm)
size checkpoint at G1/S boundary
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

18. size checkpoint at G2/M boundary
Schizosaccharomyces pombe
size checkpoint at G2/M boundary
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 µm