BASIC MICROSCOPY AND ANIMAL CELL STRUCTURE

INTRODUCTION Cells, as the basic unit of life, form the basic units of all living organisms. All animals are composed of cells, a principle that was elucidated by Theodor Schwann in 1839. Most of the cells that compose animal tissues and organs (with the exception of some egg cells) are small and invisible to the naked eye. However, with the aid of a microscope, one can observe the basic animal cell structure. Cell parts that can be observed using a compound light microscope include the cell membrane, nucleus, and cytoplasm. Microscopy, therefore, is a very important laboratory technique in the study of animal specimens. OBJECTIVES At the end of the laboratory activity, the students should be able to:

1. Determine the magnification of a microscope depending on the specific objective in place; 2. Estimate the size of a microscopic object; 3. Prepare a stained smear of animal cells; and 4. Identify an animal cell and their cell parts.

MATERIALS Microscope Transparent ruler Ocular micrometer disc Glass slide Methylene blue

Dropper Toothpick Alcohol lamp Matches/lighter Prepared slide of a nervous tissue

METHODS A. Magnification, Field of Vision, and Calibration Constant

1. Magnification: Calculate the total magnification of your microscope when using the a) low

power objective (LPO) and b) high power objective (HPO).

2. Diameter field of vision: Place a transparent millimeter (mm) ruler under the LPO. Estimate the diameter of the field of vision in mm by counting the number of millimeter divisions you see through the microscope. Record this measurement and convert to micrometer units (1 mm = 1000 m).

Calculate the field of vision with the high power objective using the following formula: Diameter of field at high power = Magnification at low power x Diameter of field at low power Magnification at high power

3. Calibration Constant. Replace your ocular with a micrometer eyepiece or insert a

micrometer disc inside the ocular. Place a ruler with millimeter divisions on the stage and determine the number of micrometer spaces within one millimeter division when the LPO is used. Make three measurements and get the mean number of micrometer spaces per millimeter. Compute for the calibration constant, C, for that micrometer eyepiece using the following formula:

C = 1/X, where X = mean number of micrometer spaces per millimeter B. Preparation of a Stained Smear

1. Put a drop of methylene blue on a clean glass slide.

2. Obtain squamous cells from the walls of your oral cavity using the blunt tip of an unused toothpick. Mix the tip of the toothpick into the drop of methylene blue on the glass slide.

3. Pass the slide through the flame until the drop dries completely.

4. Observe squamous cells under LPO and HPO. Draw your observation. Label the following

structures: cell membrane, nucleus, and cytoplasm. C. Estimating the Size of a Microscopic Object

1. Place the stained smear of squamous cells under the microscope.

2. Estimate the size of a cell by counting the number of micrometer spaces that span its entire

diameter. Multiply this by the calibration constant. This will give the size in millimeters. Convert this to micrometers.

3. Repeat the measurements thrice and get the average.

D. Observation of Animal Cells

1. View a prepared slide of nervous tissue under the microscope. Observe the slides under both LPO and HPO. Draw and label the following representative parts of the neuron as seen under the microscope: cell membrane, cytoplasm, nucleus, dendrites, and axon.

RESULTS A. Magnification and Field of Vision Table 1. Magnification of Microscope No. _____

Objective Lens Ocular Lens Magnification

Objective Lens Magnification

Total Magnification

Scanning power objective Low power objective (LPO) High power objective (HPO) Table 2. Diameter Field of Vision of Microscope No. _____

Objective Lens

Diameter Field of Vision (in m)

Low power objective (LPO) High power objective (HPO)

Table 3. Calibration Constant of Microscope No. _____

Trial Number Number of Micrometer Spaces per mm 1 2 3

Mean number of micrometer spaces per mm: Calibration constant, C:

B. Preparation of Stained Smear

Specimen: _______________ Specimen: _______________ Magnification: x ___________ Magnification: x ___________

C. Estimating the Size of a Microscopic Object

Table 4. Size of a Squamous Cell

Trial Number Calibration Constant

Number of Micrometer Spaces

Size (in mm)

Size (in m)

1 2 3

Specimen: _______________ Magnification: x ___________ Average size (in m): _______

D. Observation of Animal Cells

Specimen: _______________ Specimen: _______________ Magnification: x ___________ Magnification: x ___________

QUESTIONS

1. How would an eyepiece of 5X change total magnification in relation with the two objectives lenses (i.e., LPO and HPO)?

2. Which objective lens covers a wider field, LPO or HPO? Explain.

3. What cells parts did you observe in the stained smear of squamous cells? In the prepared slide of nervous tissue? Describe these parts as seen under the microscope.

REFERENCES Angtuaco SP, Carumbana EE, Pena-Muralla RM, Yago RE. 1994. Zoology: A Laboratory Manual.

Quezon City: Ateneo de Manila University Office of Research and Publications. 109 p. Hickman CP, Roberts LS, Keen SL, Eisenhour DJ, Larson A, Anson H. 2011. Integrated Principles of

Zoology. 15th ed. New York (NY): McGraw-Hill. 842 p.