lab 4: the care and feeding of the microscope lab 5: cell

Lab 4: The Care and Feeding of the Microscope

Lab 5: Cell Structure

Evelyn I. Milian

Instructor

2012

BIOLOGY I - LAB

BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure

OBJECTIVES OF THIS LAB

1. Learn to recognize and use the

parts of a compound light

microscope.

2. Determine magnification power.

3. Focus a microscope.

4. Learn how to make a wet mount.

5. Examine structures of plant and

animal cells.

Evelyn I . Mi l ian - Instructor 2


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FIGURE 3.2

Relationships between the sizes of various specimens and the resolution of the human eye, light microscope, and electron microscope.

It takes a microscope to see most cells and lower levels of biological organization. Cells are visible with the light microscope, but not in much detail. An electron microscope is necessary to see eukaryotic cell organelles (such as the mitochondrion and the nucleus) in detail and to observe viruses (noncellular parasitic agents) and molecules.

In the metric system, each higher unit is ten times greater than the preceding unit. Measurements: 1 centimeter (cm) = 10-2 meter (m) = 0.4 inch; 1 millimeter (mm) = 10-3 m; 1 micrometer (µm) = 10-3 mm = 10-6 m; 1 nanometer = 10-3 µm = 10-9 m.

Evelyn I . Mi l ian - Instructor



FIGURE 3.2 – Relative sizes of objects.

Sizes are shown on a metric scale; names in red are organisms studied in microbiology. Chlamydia and Rickettsia are groups of bacteria that are much smaller in size than other

bacteria. The range of effective use for various instruments is also depicted.


Compound Light Microscope (LM)

• It has multiple lenses and uses visible

light as the source of illumination.

• Visible light is passed through a

specimen and then through glass

lenses that magnify the image.

• The image from the objective lens is

remagnified by the ocular lens.

• Total magnification of a specimen =

magnification of objective lens

magnification of ocular lens.

Example: 10X (objective lens) 10 X

(ocular lens) = 100X (total magnification)

• Maximum resolving power: 0.2 µm.

• Maximum magnification: 1000-2000X.

Evelyn I . Mi l ian - Instructor 5 “X” means “times magnified”.


COMPOUND LIGHT MICROSCOPE (LM)

• When focusing a specimen with the microscope, the objective lenses must be used from the lowest magnification lens to the highest magnification lens.

• Bring the specimen into sharp focus before trying to observe it with the next higher magnification objective lens.

• The sequential steps for the correct use of the objective lenses are:

4X → 10X → 40X → rotate halfway to the next lens and add one drop of immersion oil directly over the area of the microscopic slide to be observed → 100X. *The immersion oil increases the resolution and is used only with the 100X lens, or “oil immersion objective”.




COMPOUND LIGHT MICROSCOPE

• Refractive index is the relative

velocity with which light passes

through a substance (the light-

bending ability of the medium).

• When two substances have

different indexes of refraction,

light will bend as it passes from

one material into the other.

• The light may bend in air so much

that it misses the small highest-

magnification lens.

• Immersion oil is used ONLY with

the oil immersion objective lens

(100X, the highest magnification)

to keep light from bending.


Using and Focusing the Compound Light Microscope

6. Start with the lowest power objective lens in position, by rotating the nosepiece. The lowest objective is usually 4X (the shortest objective).

7. Move the stage all the way up using the coarse focus knob.

8. Look into the ocular lens in the eyepiece, and use the coarse focus knob to clarify the image.

9. Once you have a nearly clear image, use the fine focus knob to get the clearest image.

10. To increase the magnification, put the 10X objective in position. These parfocal microscopes will automatically be in focus or at least be close to in focus.

11. Use only the fine focus knob after this point to sharpen the image, when using 40X and 100X. * Use immersion oil with the 100X.


1. Switch the microscope on (make sure the light works).

2. Lower the mechanical stage as far as it will go, using the coarse focus knob.

3. Place the microscopic slide on the stage, securing it with a stage clamp. Make sure that the slide is not upside down or backwards.

4. Move the slide with the stage adjustment knobs so that the area of interest is in the center of the light beam.

5. Adjust the amount of light with the diaphragm.


LIGHT MICROSCOPY:

Dissecting Microscope (Stereo Microscope)

• It differs from the compound microscope in that it has two (rather than one) objective lenses for each magnification.

• Binocular: It has two oculars.

• Objects can be observed in three dimensions.

• The resolution and magnification capabilities are less than in a compound microscope.

• Magnifications usually range from 4X to 50X.

• It is often used for the microscopic dissection of specimens.



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MICROSCOPY: Magnification, Resolution, Contrast

• Magnification is the ratio of an object’s image size to its real size; magnification simply makes the objects or specimens appear larger.

• Resolution (resolving power) is the ability of an optical instrument to distinguish between two points that are close together, so that they are seen as separate units; it is the ability of the lenses to distinguish fine detail and structure (or a measure of the clarity of the image).

Example: A microscope with a resolving power of 0.4 nm can distinguish between two points if they are at least 0.4 nm apart.

• Contrast is the effect of a striking difference, as in color or tone, of adjacent parts (for example, in a photograph or image). It is based on the differential absorption of light by parts of the specimen. Contrast accentuates differences in parts of the sample and is also an important parameter in microscopy.


Preparation of Specimens for Light Microscopy


• Staining increases contrast—a striking difference, as in color or tone, of adjacent parts (for example, in a photograph or image) based on the differential absorption of light by parts of the specimen:

Microscopists improve contrast by coloring specimens with stains (dyes) that bind to cellular structures and absorb light to provide contrast.

Live or unstained cells have little contrast with the surrounding medium. However, researchers do make discoveries about cell behavior looking at live specimens.


Preparation of Specimens for Light Microscopy


• Wet Mounts

Technique in which a drop of medium containing the organisms or cells is placed on a microscope slide and covered with a coverslip; it can be used to view living microorganisms or to stain specimens to observe them.

1) Take a small amount of the sample to be studied.

2) Place a drop on a

microscope slide.

3) Place one edge of the coverslip on

the drop and carefully pull the drop

along. When the coverslip is in

position, gently let it drop.

4) Sample is ready to observe

under the microscope.


Place a drop of water on the slide.

Place your sample in the water. In

some cases you may need to spread it.

Gently lower cover slip onto liquid; start with

it upright touching one side of the liquid. If not staining, view

under the scope

If staining, place a drop of stain on

one side of the cover slip and a piece

of absorbent paper on the other side

(to suck the stain through).

View under the microscope .

Preparation of Specimens for Light Microscopy:

Wet Mount with Staining

14 Evelyn I . Mi l ian - Instructor



Bacteria Microscopic Algae Protozoa Fungi: Molds

Parasitic Microscopic Worms

Fungi: Yeasts



PROTOZOA: Unpigmented, eukaryotic

microoorganisms; larger than bacteria.

ALGAE: Greenish or golden-

brown eukaryotic microorganisms;

larger than bacteria.

CYANOBACTERIA:

Blue-green, prokaryotic

microorganisms; smaller

than eukaryotic

microorganisms.

Protozoa, Algae, and Cyanobacteria

* To observe the

cyanobacteria, you need

patience!!! Remember

that bacteria are

prokaryotic and are

smaller than eukaryotic

microorganisms.


COMPARISON OF ANIMAL CELLS AND PLANT CELLS


ELODEA PLANT: Leaf cells in Elodea, an

aquatic plant (weed), showing chloroplasts inside

ONION: Onion cells stained

with iodine (cell walls and

some nuclei are visible)

CHEEK CELLS: Some nuclei are visible inside the cheek cells.


Similarities and differences to pay attention to:

• Different types of cells have different features, shapes, and sizes.

Those differences usually enable cells to function

differently from one another.

Form follows function!

• In this lab, you will be viewing different types of cells and structures.

• Particularly, you should pay attention to similaries and differences between:

Animal cells, plant cells, protist cells



Common Mistakes in Microscopy

• Common mistakes that lead to problems:

1. Too little or too much light—check your condenser and diaphragm.

2. Not in the correct focal plane—keep adjusting coarse and fine focus.

3. In a bad field—not all visual fields will contain specimen. Once

focused properly, keep moving the stage side-to-side until you find

your sample.

4. Slide upside down—when preparing your own slides, make sure to

mark the side of the slide that was inoculated with the sample (such as

bacteria or plant cells).

5. Dirty lens will make everything look blurry—Wipe the lens with

alcohol and lens paper. * Do not use regular paper towel or drying

paper for the lens!!!



References

• Audesirk, Teresa; Audesirk, Gerald & Byers, Bruce E. (2005). Biology: Life on Earth.

Seventh Edition. Pearson Education, Inc.-Prentice Hall. NJ, USA.

• Campbell, Neil A.; Reece, Jane B., et al. (2011). Campbell Biology. Ninth Edition. Pearson

Education, Inc.-Pearson Benjamin Cummings. CA, USA.

• Mader, Sylvia S. (2010). Biology. Tenth Edition. The McGraw-Hill Companies, Inc. NY, USA.

• Presson, Joelle & Jenner, Jan. (2008). Biology, Dimensions of Life. The McGraw-Hill

Companies, Inc. NY, USA.

• Solomon, Eldra; Berg, Linda; Martin, Diana W. (2008). Biology. Eighth Edition. Cengage

Learning. OH, USA.

• Starr, Cecie. (2008). Biology: Concepts and Applications Volume I. Thompson

Brooks/Cole. OH, USA.

• Tortora, Gerard J.; Derrickson, Bryan. (2006). Principles of Anatomy and Physiology.

Eleventh Edition. John Wiley & Sons, Inc. NJ, USA. www.wiley.com/college/apcentral.

• Tortora, Gerard J.; Funke, Berdell R.; Case, Christine L. (2010). Microbiology An

Introduction. Tenth Edition. Pearson Education, Inc.-Benjamin Cummings; CA, USA.

www.microbiologyplace.com.

• Trefil, James & Hazen, Robert M. (2011). The Sciences An Integrated Approach. Sixth

Edition. John Wiley & Sons, Inc. NJ, USA.


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