Department of Zoology
Southern Illinois University at Carbondale

ZOOL 409, Lab Week 1

• Week 1 lecture notes
• SYLLABUS (including links to all note pages)
• 409 Homepage (index of course resources)
• Dr. King's School of Medicine histology page
• Slide summary

Objectives for Tuesday lab [also see class participation]:

1. LAB NOTEBOOK:  Establish the practice of documenting your observations.
2. MICROSCOPE:  Learn basic parts and operation of the light microscope.
3. CELLS:  Become acquainted with the appearance of cells as they appear in tissue sections.
4. View slides:  97, 18, 19, 1, 7, 64, 62, 44

Objective for Thursday lab [also see class participation]:

1. View several slides of external and internal body surfaces.
• Notice differences in the shape and texture of cell nuclei.
• Notice differences in the color and texture of cell cytoplasm.
• Identify epithelial tissue and commective tissue.
• Compare and contrast epithelial and connective tissue on several slides.

Complete slide list:
01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100

OUTLINE

Tuesday Objective 1:  Lab Notebook.

Drawing is one of the most powerful study-aids for learning visual information, such as the microscopic appearance of tissues.

Each lab period should receive a notebook entry for each slide that you observe, including:

• Date and time.
• Slide number and slide label for each slide that is examined.
   
• A list of expected observations.
  • What are you looking for?  From text, atlas, and instruction, what features do you expect to see?
     
• Sketches of your actual observations.

  As an aid to sketching, use the the circular field of view in your microscope as a frame.  Imagine a circle drawn halfway between the center of this field and the edge.  Then imagine the circle as a clock-face, with four quarters and each quarter divided into three parts.

  Your sketches might usefully include:
   
  • An overview (or "map") of the specimen, good for relocating specific features.
    • Anything visible by naked eye should be included in this map.
  • Examples of features which you can identify, with appropriate labels.
    • Honestly, from your own observation, what features do you actually see?  (Real specimens do not always display all typical features.)
  • Which of those can you confidently identify?
  • Examples of features which you cannot identify (labelled as "unidentified").

  • A list of features that you expected to find (e.g., based on reference materials) but could not see (and/or could not reliably identify).

If you have a question about some feature that you have found, the best way to ask is by way of a sketch that shows its location in the field of view and its relationship to other features.  Thus, "What is this, that I have drawn here?" works much better than, "What's the red thing we see in the microscope?"  Your instructor may refuse to address your question until you have made such a sketch.

A well-kept notebook, with recognizable and accurately labelled drawings, may be submitted for grade consideration.

 

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Tuesday Objective 2:  Meet your microscope (italics indicates user-adjustable features)

• Basic elements
  • Illuminator (light source)
    • The illuminator may include adjustable field diaphragm.
  • Condenser (light gathering lens)
    • Proper condenser adjustment is important for optimal performance.
    • The condenser includes an adjustable condenser diaphragm (see Kohler illumination).
    • The condenser may include focus adjustment (see Kohler illumination).
  • Stage (platform between condenser and objective)
    • The stage supports the specimen (microscope slide).
    • The stage allows specimen to move in x-y plane.
    • The stage may move up and down for specimen focus.
  • Specimen
    • Note that the specimen mount (slide and coverslip) is part of the optical system.
    • At high magnification, optimal optical adjustment requires that coverslip thickness be appropriate for the objective lens.
  • Objective lens (principal image-forming lens)
    • Functionally, the objective lens is the most critical (and most expensive) element of the microscope.
    • Objectives are interchangeable (usually via a swivelling "nosepiece") to change magnification.
    • Objectives may move up and down for specimen focus.
  • Eyepiece (enlarging lens, paired in binocular m'scopes)
    • If paired, interpupillary distance is adjustable; at least one eyepiece should be independently focussable..
       
• Kohler illumination (follow this link for more information on adjusting illumination for optimal contrast and resolution)
   
• Care of microscope
  • Three rules for carrying a microscope:
    1. Your full attention is on the job, just as if you were carrying a baby.
    2. Use both hands.
    3. Always carry the microscope upright (otherwise the eyepieces can fall out).
        
  • Three rules for cleaning a microscope:
    1. Don't!  It easy to damage a microscope by injudicious cleaning.
    • Never the touch the objective lens surface, unless you have bought and paid for the microscope yourself.
    • Clean eyepieces and other optical surfaces only when absolutely necessary.  It is better to ignore dirt than to cause permanent damage to the microscope. 
    2. If you can see dirt, use logic and experiment to determine where in the optical path the dirt is located.
    • Dirt on a slide moves when you move the slide.
    • Dirt on the condenser will go in and out of focus when you move the condenser up or down.
    • 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 inconspicuous when the diaphragm is opened to the correct setting -- see Kohler illumination.
    • 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 --
      • Begin by simply blowing dust away.
      • Use clean lens paper and moisture from breath.
      • Wipe gently in one direction.  Do not use a circular motion; do not scrub.
      • Do not try to clean an objective lens; ask for help.
         
• 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, either a "calibration slide" (e.g.., a glass slide engraved with a very finely-calibrated ruler) or with standardized objects (e.g., microspheres).
    • Some microscopes include an "eyepiece micrometer", essentially a small, transparent ruler mounted within the eyepiece that is used to measure specimens.  Such an eyepiece micrometer must initially be calibrated (at each magnification) using a specimen of known size (see preceding item).
    • Without an eyepiece micrometer, sizes can still be estimated in relation to the field of view or (if there is one) to the thickness of an eyepiece pointer.  Each of these reference standards must be initially calibrated at each magnification.
    • For the microscopes used in this course:
      

      With this objective:	The field-of-view diameter is:	The pointer thickness is:
      4 X	4.5 mm  /  4500 µm	50 µm
      10 X	1.8 mm  /  1800 µm	20 µm
      40 X	0.45 mm  /  450 µm	5 µm

    • You may check these values with the stage micrometer available from your instructor.
    • Use these values to estimate the size of the objects provided.
       
  • Even with an uncalibrated microscope, sizes of tissue features can also be estimated with reference to familiar objects of consistent size, such as red blood cells (6-8µm, in humans).
     
• Resolution and numerical aperture
  • "Resolution" refers to the "resolving power" of the microscope, i.e., the 'scope's ability to "resolve", or distinguish as separate, two nearby objects.  Resolution is at least as important as magnification.  An increase in magnification that is not accompanied by an increase in resolution is termed empty magnification, with the implication that nothing is really gained (i.e., you cannot see any additional detail by an empty increase in magnification.
  • Resolution is determined by optical quality and fundamentally limited by the wavelength of light and by the numerical aperture of the objective/condenser combination.
     
• Outside links to more information on microscopes and microscopy.
  • Using the microscope [micrographia.com]
  • Microscopy primer [fsu.edu]
     

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Tuesday Objective 3:  Meet some cells.

All living things are composed of cells and cell products.  This is one of the greatest unifying principles of biology.

However, recognizing this unity among the diversity of animal tissues represents a major accomplishment in the history of scientific observation.

Find cells on slide 97; recognize nuclei and cytoplasm, estimate cell size
Find cells on slide 1; recognize nuclei and cytoplasm, estimate cell size
Find cells on slide 7; recognize nuclei and cytoplasm, recognize extracellular substance, estimate cell size
Find cells on slide 64; recognize nuclei and cytoplasm, estimate size of cell and nucleus,	liver
Find cells on slide 62; recognize nuclei and cytoplasm, estimate size of cell and nucleus, recognize extracellular substance, find Golgi apparatus within cytoplasm, estimate size of Golgi apparatus.
Find cells on slide 44; recognize nuclei and cytoplasm, estimate diameter of cell and of nucleus, notice Golgi apparatus within cytoplasm.

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Thursday Objective 1:  Compare cells and tissues on several different specimens.

1. View several slides of external and internal body surfaces.
• Notice differences in the size, shape, and texture of cell nuclei.
• Notice differences in the amount, color, and texture of cell cytoplasm.
• Identify epithelial tissue and commective tissue.
• Compare and contrast epithelial and connective tissue on several slides.

Examine slide 04 [trachea]; Find at least two different types of cells, with markedly differing appearances. Note size, shape, and texture of nucleus. Note amount, color, and texture of cytoplasm. Find epithelium. Find connective tissue. Compare and contrast with other slides in this exercise.
Examine slide 07 [esophagus]; Find at least two different types of cells, with markedly differing appearances. Note size, shape, and texture of nucleus. Note amount, color, and texture of cytoplasm. Find epithelium. Find connective tissue. Compare and contrast with other slides in this exercise.
Examine slide 8 [bladder]; Find at least two different types of cells, with markedly differing appearances. Note size, shape, and texture of nucleus. Note amount, color, and texture of cytoplasm. Find epithelium. Find connective tissue. Compare and contrast with other slides in this exercise.
Examine slide 38 [colon]; Find at least two different types of cells, with markedly differing appearances. Note size, shape, and texture of nucleus. Note amount, color, and texture of cytoplasm. Find epithelium. Find connective tissue. Compare and contrast with other slides in this exercise.
Examine slide 43 [scalp]; Find at least two different types of cells, with markedly differing appearances. Note size, shape, and texture of nucleus. Note amount, color, and texture of cytoplasm. Find epithelium. Find connective tissue. Compare and contrast with other slides in this exercise.
Examine slide 46 [tongue]; Find at least two different types of cells, with markedly differing appearances. Note size, shape, and texture of nucleus. Note amount, color, and texture of cytoplasm. Find epithelium. Find connective tissue. Compare and contrast with other slides in this exercise.

Introduction to basic tissue types.

More:  see Ed (The Path Guy)'s Basic Histology Gallery.

Checklist of cell structures / functions

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Comments and questions: dgking@siu.edu
Department of Zoology e-mail: zoology@zoology.siu.edu
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