Guide to the practical teaching
of biology and genetics

Prokaryotes and immersion microscopy

TASK 1: Smear impression of human tongue mucosa

Take a clean slide (from a box labelled "otisk jazyka") and anneal it in a gas burner flame. Let it cool off and then impress the surface of your tongue (after roughing it with your teeth) on the slide. Let the slide dry and then fix it above the flame. Perform Giemsa-Romanowski staining for 10 min. Rinse the dye with distilled water and dry the slide gently using a filtration paper. Observe the stained epithelium cells (polygonal shape, nucleus) and bacteria under immersion objective with immersion oil and measure their size.

Fig.: Epithelium cells of tongue and bacteria.

TASK 2: Bacteriological smear

NP: bacteria (G⁺ and G^- bacteria), PP: stained bacteria

Sterilize the bacteriological loop using the gas burner flame and let the loop cool off (not to kill the bacteria). Take a colony of bacteria from the surface of the agar medium in a Petri dish with the bacteriological loop and make a thin smear in a droplet of water on a slide. Let it dry and fix it over the flame (three times).

Gram-staining:

• Apply the crystal violet (the primary stain) on the slide for 3 min.
• Rinse it with distilled water and apply the Lugol solution (iodine solution) for 2 min.
• Rinse it and add the ethanol (decolorizer) until the stain stops being washed out.
• Apply the carbolfuchsin (counterstain) for 1.5 min.
• Rinse it with distilled water and dry with filter paper.

Observe and draw G⁺ (purplish-blue) and G^- (red) bacteria under immersion objective.

Fig.: Bacteriological smear: A – transfer of bacteria by bacteriological loop, B – homogenisation of bacteria with water, C – bacteriological smear.

TASK 3: Cyanobacteria

NP: "Blue-green algae" (Cyanobacteria; genus Oscillatoria)

Observe the structure of cyanobacteria and compare it with cells of green algae or diatoms (frustules), which can be also present in the sample. Just by the preparing of the sample, you can observe oscillatory motion of cyanobacteria.

Fig.: Comparation of cyanobacteria with green algae and diatom.