Guide to the practical teaching
of biology and genetics

Tasks - Microscopic technique - list

Dry objectives, centring the objects, iris diaphragm function

TASK 1: Arranging the object in the visual field

Permanent preparation (PP) of a letter - "písmeno"

Put the permanent preparation with a block letter on the stage of microscope to be able to read the word (not reversed). Draw one asymmetrical letter (a, e, y, k...) using the smaller magnification of the objective (4×). What image can you see in the microscope?

TASK 2: Arranging the object using different magnifications 4×, 10×, 40×

PP: insect wing - "křídlo hmyzu" (or letter)

Observe the specimen under different magnifications. How does the free working distance, visual field size and resolution change based on magnification used?

Fig.: Different magnifications of objective (A - 4×, B - 10×, C - 40×) and visual field (insect wing).

TASK 3: The function of iris diaphragm

PP: feather - "prachové peří"

Observe the feather under different objectives (4×, 10× a 40×) with an opened and closed iris diaphragm. What is the difference and what principle arises from this observation?

TASK 4: Centring the object

PP: stained wool - "vlna barvená"

Place the stained wool (cross of fibers or other characteristic point) in the centre of the visual field under the smallest magnification (4×). Then use the 10× magnifying objective without drifting the object and observe the wool. Can you see some change? (Do the same with 40× magnifying objective). What principle arises from this observation?

Fig.: Centring the object (stained wool) and different magnification of objectives (A - 4×, B - 10×, C - 40×).

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Permanent preparations

TASK 1: Blood smear

Drop the anticoagulant-treated blood onto the slide glass. Use a skewed glass at an angle of 45° C - to smear the blood drop on the slide glass by pulling the drop behind the skewed glass. Otherwise you will damage and change the shape of the erythrocytes (they are extremely fragile). Please avoid these mistakes: small or too large drop of blood, oily slide glass, unequal smear, accumulation of blood at the end of smear.

TASK 2: Blood smear and "Panoptic" staining after Pappenheim (May-Grünwald, Giemsa-Romanowski)

Prepare the blood smear. Apply the May-Grünwald (5 min.); add distilled water (5 min.) and pour off dye. Apply the Giemsa-Romanowski (10 min.), pour off dye and rinse it with distilled water. Dry it by the careful by using a filtration paper. Observe the red blood cells in the thin layer, measure the diameter of a mammal erythrocyte. Compare the mammal blood smear with a permanent preparation of a bird blood smear. Measure the size of bird erythrocyte.

Fig.: Procedure: A – put drop of blood on the slide, B – attach a glass to the drop at an angle of 45° C, C – make blood smear. Comparison of erythrocytes: D – bird (oval, with nucleus), E – mammal (round, without nucleus).

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Optical planes, measuring the size and thickness of microscopic objects

TASK 1: Optical planes and measuring of the thickness of the objects

PP: insect wing - "křídlo hmyzu"

Rotate the fine focus adjustment knob in one direction and note the first sharp image (a hair), medium (a major branch) and the last (a hair) = upper, middle and lower optical plane. Note which unit of the fine focus adjustment knob corresponds to the first optical plane and which corresponds to the last one (measure it against a fixed point on the coarse knob) and calculate the difference. What is the thickness of the object in µm?

Fig.: Optical planes (A - upper, B - middle, C - lower).

TASK 2: Measuring the length of microscopic objects

PP: bird red blood cells - "ptačí krev/krvinky"

Put the objective micrometer on the stage and observe it using 4×, 10×, 40× and 100× magnifying objectives. The left margins of both scales must be aligned. Read the corresponding numbers of units on both scales where they meet and calculate the micrometric coefficient for each objective. When changing to the next magnification, readjust the centering, brightness, focus and realign the left margins of both scales. Note these coefficients for future measuring of microscopic objects. Measure the length of the red blood cells.

Fig.: Comparison of the length of different cells.

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Native preparation, phase contrast

TASK 1: Ciliates (Infusoria)

Native preparation (NP): hay infusion (or ciliates from soil)

Take a water drop from the hay infusion surface, place it on a slide glass and cover with a cover glass so as not to form air bubbles. Observe the fast moving protozoans, especially ciliates, flagellates and also bacteria, vinegar worms or flatworms. Mistakes that can be made: too much water results in vibrations or drifting of the objects away from visual field, a wet lower side of the slide (and thus wet stage) leads to the impaired control of the specimen movement; lack of water results in air bubbles forming and drying of the sample.

Infusoria are named after hay infusion (water, hay, decomposing plants and leaves, soil) in which they can be found. In hay there are cysts of infusorians that give rise to active ciliates.

Fig.: Soil organisms.

TASK 2: Phase contrast microscopy

NP: ciliates or pollen grains

Prepare microscope for observation in the phase contrast and then observe ciliates (or pollen grains). Observation in phase contrast increases contrast of colourless structures.

TASK 3: Digestion and contractile vacuoles of protozoans

NP: hay infusion - water surface with ciliates (infusorians) and numerous bacteria

Take a water drop from the hay infusion surface; put it on a slide glass, stain with 0.1 % neutral red (possibly causing death of protozoans after some time) and cover with a cover glass. Observe the contractile vacuole (for osmoregulation), colorless round structures near a pole of a ciliate alternately increasing size and disappearing after emptying to the outer environment. Fast motion of ciliates can be slowed down by soaking off the excessive water using a filter paper.

TASK 4: Digestive vacuoles

PP: digestive vacuoles containing charcoal – "potravní vakuoly"

The ciliate Paramecium (Paramecium sp.) was cultured in a medium containing charcoal. The digestive vacuoles are stained black due to charcoal contained in them.

Fig.: Ciliate with digestive and contractile vacuoles. A – native staining, B – stained with charcoal.