Histology Lab I



CRR Histology Lab 1 Introduction to Tissue Microscopy CRR Week 1

Principal objectives for Lab 1

1. Become acquainted with your microscope, your boxed set of study slides, and various print and internet resources.

2. Learn to see cells. Appreciate how all cells share similar structures and how distinct cell types are differentiated from one another.

3. Begin distinguishing basic patterns of tissue organization, particularly epithelial and connective tissues.

Lab guidelines

Scheduled lab periods are optional opportunities for study in the presence of instructors.

Please bring slides and reference materials to labs. Handouts are intended to stimulate, not to educate.

"Check-off boxes" enable your instructor to monitor your learning during the unit..

For additional supporting resources, see back of page.

___________________________________________________________________________________________________________________________________________________________________

LIGHT MICROSCOPY

Scalp, slide 2.

Skin, slide 3.

(EASY) Find an epidermal cell. Question--What kind of tissue is epidermis?

(EASY) Distinguish nucleus from cytoplasm for cells of epidermis. Question--How much of the epidermis consists of cells (nuclei and cytoplasm together)? Question--Can you see any extracellular space in the epidermis?

(EASY) Find the dermis. Question--What kind of tissue is dermis?

(EASY) In dermis, distinguish cells from extracellular material. Question--How much of the dermis consists of cells? How much is extracellular material?

(EASY) Find collagen. Question--Where should you look? (Epidermis or dermis? Within cells or outside cells?)

(HARDER) Try to identify special structures of skin (e.g., hair follicles, sweat glands, blood vessels).

(REPEAT) Find epithelial cells, connective tissue cells, and collagen in cornea (slide 25), tongue (slide 29), and trachea (slides 61, 62).

|□ | |

| |At your discretion, you may notify an instructor for a brief |

| |oral evaluation on this material. |

Blood smear, slide 98.

(EASY) Find a cell without a nucleus. Question--What type of blood cell is this? Find a few hundred more cells of the same type. Question--How much do these cells vary in size?

(EASY) Find a cell with its nucleus pinched into several lobes. Question--What type of blood cell is this? Find a few dozen more cells of the same type. Question--How much do these cells vary in size?

(EASY) Find a cell with a round nucleus. Question--What type of blood cell is this? Find a couple dozen more cells of this type. Question--How much do these cells vary in size?

(HARDER) Find a large cell with a deeply dented nucleus. Question--What kind of cell is this?

(EASY) Find small bits of cytoplasm, without nuclei, much smaller than entire cells. Question--What are these?

Sickle Cell, slide 52. Repeat the exercises above.

Bone marrow smear, slide 99. What is the most striking difference between this and peripheral blood smears?

| |□ |

|At your discretion, you may notify an instructor for a brief oral| |

|evaluation on this material. | |

_____________________________________________________________________________________________________________________________________________________________________

ELECTRON MICROGRAPHS

Use Rhodin's Atlas of Histology (from the Learning Resources Center) or other any other collection of electron micrographs.

Below are approximate sizes (diameter) for some commonly encountered cellular structures.

ribosomes -- about 0.02 µm.

mitochondria -- variable,commonly about 0.5 µm.

"typical" nucleus -- variable, commonly 5-10 µm.

"typical" cell -- variable, commonly 6-20 µm.

red blood cell -- fairly uniform, 6-8 µm.

adipocyte -- variable, commonly about 50 µm.

skeletal muscle fiber -- variable, commonly 30-60 µm.

terminal arterioles -- about 30-50 µm.

(EASY) On an image of an entire cell, identify plasma membrane, cytoplasm, nucleus, and extracellular material. Estimate diameter of cell and nucleus.

(EASY) On an image of an entire cell, identify nuclear membrane, nucleolus, euchromatin and heterochromatin.

(EASY to HARD) On images showing a significant portion of one or more cells, identify mitochondria, rough endoplasmic reticulum, ribosomes, Golgi bodies, secretory vesicles, lysosomes, glycogen granules.

| |□ |

|At your discretion, you may notify an instructor | |

|for a brief oral evaluation on this material. | |

Learning Resources for CRR Histology

Scheduled Activities: siumed.edu/~dking2/crr/CRRsched.htm (or see CRR Unit Calendar).

On-line study guide:

siumed.edu/~dking2/index.htm ../crr/index.htm ../intro/index.htm

Other internet materials:

U. of Utah, The Internet Pathology Laboratory

www-medlib.med.utah.edu/WebPath/webpath.html

U. of Iowa, Virtual Slidebox



Additional resources:



Self-Assessment Questions (SAQs), at the on-line study guide, above.

SAQ slides (unlabelled "unknown" slides), available in the MRC (Medical Resource Center).

Textbooks. All students should read at least one introductory text. All of the following are roughly equivalent in depth and coverage, appropriate for the level of understanding expected in our current year-one curriculum. Your choice may be based on availability or on personal preference (style varies markedly among texts).

Junqueira's Basic Histology: Text & Atlas, 12th ed. (2010), Mescher.

Wheater's Functional Histology: A Text and Colour Atlas, 5th ed. (2006), Young, Lowe, Stevens & Heath (eds.).

Histology: A Text and Atlas, 5th ed. (2006), Ross & Pawlina.

Histology and Cell Biology: An Introduction to Pathology (2002), Kierszenbaum.

Human Histology, 3rd ed. (2005), Stevens & Lowe.

Elsevier’s Integrated Histology (2007), Telser et al.

Atlases. Just as with snapshots of landscapes or persons, single micrographs are seldom sufficient to catch the full character or personality of a tissue. Since any given text typically illustrates each organ/region with only one or two images, students are encouraged to view several sources for multiple images. Histology atlases (some examples are listed below) are quite useful for this purpose, but personal possession of an atlas should not be necessary.

An Atlas of Histology (1976), Rhodin.

Excellent source for electron microscope images. Although out of print, multiple copies are available in the MRC.

This resource is also available online,

Atlas of Descriptive Histology (2009), Ross, Pawlina, Barnash.

Color Atlas of Basic Histology, 3rd ed. (2003), Berman & Milikowski.

Color Atlas of Basic Histopathology (1997), Milikowski & Berman (an excellent resource for images of particular pathologies).

References. The following are much more substantial (heavier, much more detail, ~1200 vs. ~400 pp.), better for reference than for introductory exposure. Students should be aware that such resources exist, but personal ownership is not necessary.

Wheater’s Basic Histopathology, 4th edition (2002), Stevens et al.

Histology for Pathologists, 3rd ed. (2007), Mills; or 2nd ed. (1998), Sternberg.

Robbins and Cotran Pathologic Basis of Disease, 8th ed. (2010), Kumar, et al.

Textbook of Histology, 12th ed. (2001), Fawcett.

KNOW YOUR MICROSCOPE

Basic terminology

Light source

The light source, or "illuminator", should have adjustable brightness and may include an adjustable field diaphragm to minimize scattered light.

Condenser (light-gathering lens)

The condenser is located beneath the stage and includes an adjustable aperture, controlled by a diaphragm within the condenser. Proper condenser adjustment is essential for optimal resolution (see "Kohler illumination" on the other side of this page).

Stage (specimen support platform)

The stage supports the microscope slide and allows specimen movement in the x-y plane. The stage may move up and down for specimen focus.

Specimen

Note that the slide and coverslip are both parts of the optical system. At high magnification, optimal optical function requires that coverslip thickness be appropriate for the objective lens.

Objective lens (image-forming lens)

Functionally, the objective lens is the most critical (and most expensive) element of the microscope. Objectives may move up and down for specimen focus. Never touch the optical surface of the objective lens.

Eyepiece (enlarging lens)

If eyepieces are paired, then interpupillary distance is adjustable. At least one eyepiece should be independently focussable. Eyepieces are removeable; when held upsidedown, an eyepiece can serve as a hand lens.

_______________________________________

Three rules for carrying a microscope:

1. Always carry the microscope upright (otherwise the eyepieces can fall out).

2. Use both hands.

3. Give the job your full attention, just as if you were carrying a baby.

Three rules for cleaning a microscope:

1. Don't! (not without proper training). It is easy to cause permanent damage by injudiciously cleaning a microscope.

▪ Clean eyepieces and other optical surfaces only when absolutely necessary. It is better to ignore dirt than to cause permanent damage to the microscope.

▪ Never the touch the objective lens surface, unless you own the microscope and have been properly trained.

2. If you see dirt when you look through the microscope, determine by logic and experiment where the dirt is located before trying to eliminate it.

▪ Dirt on a slide moves when you move the slide.

▪ Dirt on the condenser goes in and out of focus when you focus the condenser.

▪ Dirt on an eyepiece will move when you rotate the eyepiece.

▪ Dirt on an objective cannot be seen while viewing through the microscope, but manifests itself as a decrease in image quality.

3. If dirt is objectionable, use the least-intrusive procedure to make it go away.

▪ Some dirt can blown away without touching the surface. Use this procedure freely, but blow gently and be careful not to spit.

▪ Dirt on slides may be wiped off at will, using the same care you would use for eyeglasses.

▪ Dirt on an eyepiece is often accentuated by having the condenser aperture too small; such dirt may become much less conspicuous when the condenser diaphragm is set correctly (see "Kohler illumination" on the other side of this page).

▪ Dirt on the condenser may become inconspicuous if you lower the condenser (i.e., move the dirt out of focus).

▪ If dirt must be removed manually, use clean lens paper and moisture from breath. Wipe gently in one direction. Do not scrub with a circular motion. Do not try to clean an objective lens.

Kohler Illumination

The following instructions apply to most research microscopes, including the multi-viewer microscopes in Lindegren Room 206. Condenser adjustment may be simplified on student microscopes.

Initial set up: Turn on the lamp, place a slide on the stage, focus on the specimen, adjust interocular distance to match that of your own eyes.

1. Focus the condenser.

Close the field diaphragm located on the base of the microscope. This will reduce the illuminated field of view, as seen through the eyepieces, to a small circle of light. Then adjust the substage condenser up or down until the edge of this circle appears sharp.

2. Center the condenser.

Open the field diaphragm until the illuminated circle almost fills the viewing area. Then adjust the screws at either side of the condenser until this circle is precisely centered in your field of view. Now open the field diaphragm completely to restore illumination over the entire field of view.

3. Adjust the condenser diaphragm.

Remove one eyepiece and look into the open tube. You should see an illuminated field of view whose size is controlled by the diaphragm within the condenser. Adjust the condenser diaphragm until its edge just barely encroaches on the illuminated field of view. Then replace the eyepiece.

This adjustment should be repeated whenever the objective lens is changed (i.e., whenever magnification is changed).

4. Adjust illuminator brightness.

Use the voltage regulator on the base of the microscope to adjust image brightness to suit your eyes.

5. Repeat steps 3 and 4 whenever you change objectives.

Congratulations. Your microscope should now be adjusted for proper Kohler illumination.

Magnification

• Magnification is determined in principle by multiplying the power of the Objective Lens by the power of the Eyepiece.

• Magnification is determined in practice by calibration with a specimen of known size.

For research purposes, calibration is done with a reference standard, such as a "calibration slide", engraved with a very finely-calibrated ruler.

An "eyepiece micrometer" (a small, transparent ruler mounted within the eyepiece) can be used to measure specimens, after calibration with a specimen of known size (see preceding item).

• In routine practice, magnification is estimated with reference to familiar objects of known size, such as red blood cells (the diameter of human RBCs is approximately 6-8µm).

CALIBRATE YOUR OWN MICROSCOPE.

• A good calibration standard can be made by using a size-reducing photocopier to make a miniature copy of a millimeter ruler. Measure the actual size of this miniature ruler, so you know the length between marks.

• Put your calibration standard on a microscope slide and view it through the microscope. Measure edge-to-edge across the field of view. Repeat for each magnification (i.e., each different objective lens). Or, measure once with lowest power and use arithmetic to compute the change when magnification is increased. (For example, if magnification is increased from 4x to 40x, which is a factor of 10, the real distance measured across the field of view will decrease by a factor of 10.)

• If your microscope has an eyepiece pointer, you can also measure the thickness of the pointer and use that number as a standard for estimating the size of objects much smaller than the field of view.

___________________________________________________________________________________________________

Additional details may be found at

For yet more information . . .





................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download