Meet the microbes! Microscopes are required to see individual microorganisms 2 µm Escherichia coli prokaryote divides every 20-30 min. (lab uses nonpathogenic strain) Saccharomyces cerevisiae eukaryote divides every ~90 min. Phase contrast images - 400 X magnification

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

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

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?

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

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

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

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

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

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?

S. cerevisiae stained with iodine 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 Immersion Oil

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?

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

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

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 12-15 µm