2 µm meet the microbes! escherichia coli prokaryote divides every 20-30 min. (lab uses...

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