introduction to cell biology

1. Tips for Today’s Experiment
• Prepare the mung bean seeds first since it needs
to stand for 30 minutes. Transfer to a test tube
using a dropper.
• The Molisch’s Test will form two or three layers if
done properly. Please do not shake the mixture
after adding H2SO4 so the layers will form.
• Keep shaking in the Crouzel-Dupin’s Test because
it may take a while for the reaction to occur.
• If the experiment requires performing in the
fume hood, please do so for safety purposes.

2. Quiz
1. Stain A stains dicot stems red and monocot stems
pink. Cite the steps from start to finish to prepare a
slide of monocot stem using stain A.
2. Cite the steps from start to finish to prepare a slide of
dicot stem using silver particles.
3. Devise a method from start to finish to determine if
protein B is located in the plasma membrane using a
fluorescence microscope.
4. Devise a method from start to finish to determine if
protein C is located in a plant cell using an electron
microscope.
5. In a given microscope, the ocular is 15x, the LPO is
10x, the HPO is 40x, and the OIO is 100x. Find the
linear magnification of the (a) LPO (b) HPO (c) OIO.

3. Introduction to Cell Biology

4. Types of Prokaryotic Cells Based on
Shape
1. Coccus are spherical in shape.
– Diplococci occur in pairs.
– Tetrads occur in groups of four.
– Sarcinae occur in groups of eight.
– Staphylococci occur in grapelike clusters.
– Streptococci occur in chainlike patterns.
2. Bacillus are rod-shaped.
– Diplobacilli occur in pairs.
– Streptobacilli occur in chainlike patterns.
– Coccobacilli are oval in shape.
3. Spiral
– Vibrios look like curved rods.
– Spirilla have a helical shape and fairly rigid bodies.
– Spirochetes are helical and flexible.

5. Inclusions
1. Metachromatic granules
– Large inclusions that stain red with certain blue dyes
– Collectively known as volutin
– Found in prokaryotes, fungi, and protists
– Formed by cells that grow in phosphate-rich environments
– Reserves of phosphate groups that can be used in the
synthesis of ATP
2. Polysaccharide granules
– Consist of glycogen and starch
– Glycogen appears reddish brown and starch appears blue in
the presence of iodine
– Found in prokaryotic cells
– Glycogen is found in muscle and liver cells of animals and
fungal cells and starch is found in plants.
– Appears magenta with the periodic acid-Schiff’s reaction
(PAS)

6. PAS Staining
• Stains polysaccharides such as glycogen and cellulose
• Recommended fixatives are 10% formalin, methanol, or
Carnoy’s fixative (a mixture of alcohol, chloroform, and
acetic acid).
• The enzymes diastase and amylase breaks down
polysaccharides.
1. The cells are treated with periodic acid.
– Periodic acid oxidizes two hydroxyl groups in
carbohydrates into two carbonyl groups.
2. The periodic acid is washed off.
3. The cells are treated with Schiff’s reagent.
– Schiff’s reagent reacts with the two carbonyl groups
to form a colorless compound, which is then
transformed into a pink-colored product.

7. PAS Staining
4. The Schiff’s reagent is washed off with water.
– Water acts as a mordant because it intensifies the
pink color into magenta color.
5. The cells are treated with a blue dye known as
hematoxylin.
– Hematoxylin acts as a counterstain because it
provides a contrasting color to the positive stain.
– Hematoxylin stains the nucleus of all cells blue.
6. The hematoxylin is washed off.
7. The slide is viewed under the microscope.

8. Question
• Create a positive control and a negative
control for PAS staining.
• Solution: Since glycogen is found in muscle
cells, stain muscle cells with PAS for positive
control.
• Since amylase digests polysaccharides, treat
muscle cells with amylase first, and then stain
with PAS to create a negative control.

9. Inclusions
3. Lipid inclusions
– Revealed by fat-soluble stains like Sudan dyes (Sudan II,
Sudan III, Sudan IV, Oil Red O, Sudan Black B)
– Composed of poly-β-hydroxybutyric acid, a common lipid-
storage material found only in prokaryotes
– Found also in fat and liver cells of animals
4. Sulfur granules
– Serve as energy reserve for prokaryotes that oxidize sulfur
and sulfur-containing compounds
5. Carboxysomes
– Inclusions that contain the enzyme ribulose 1,5-diphosphate
carboxylase
– Required for carbon dioxide fixation during photosynthesis
– Found only in photosynthetic prokaryotes and prokaryotes
that use carbon dioxide as their sole source of carbon

10. Lipid Staining
• Recommended fixative is 10% formalin.
A. Oil Red O staining
1. The cells are treated with Oil Red O.
• Oil Red O acts as a primary stain because it stains all cells
red.
2. The Oil Red O is washed off with isopropanol.
• Isopropanol acts as a decolorizing agent because it
removes the red stain from some cells but not others.
3. The cells are treated with hematoxylin.
• Hematoxylin acts as a counterstain because it provides a
contrasting color to the primary stain.
4. The hematoxylin is washed off.
5. The slide is viewed under the microscope.

11. Lipid Staining
B. Sudan Black B staining
1. The cells are treated with Sudan Black B.
• Sudan Black B acts as a primary stain because it stains all cells
black.
2. The Sudan Black B is washed off with propylene glycol, ethanol,
or xylene.
• Propylene glycol, ethanol, or xylene acts as a decolorizing
agent because it removes the red stain from some cells but
not others.
3. The cells are treated with nuclear fast red or safranin (also a red
dye).
• Nuclear fast red or safranin acts as a counterstain because it
provides a contrasting color to the primary stain.
• Nuclear fast red or safranin stains the nucleus of all cells red.
4. The nuclear fast red or safranin is washed off.
5. The slide is viewed under the microscope.

12. Inclusions
6. Magnetosomes
– Inclusions of iron oxide formed by Gram-negative bacteria
that act like magnets
– Can decompose hydrogen peroxide
– Helps bacterial cells to move downward until a suitable
attachment site is found
7. Crystals
– Found in Sertoli and Leydig cells of the testis and
macrophages
– Made of calcium carbonate around a cellulose core
(cystolith), calcium oxalate, or silicon dioxide (phytolith) in
plants
– Calcium oxalate may be found as clusters (druse), needle-like
bundles (raphide), solitary (styloid), prisms (prismatic), star-
shaped (sphaeraphide), or powdery (crystal sand)

13. Inclusions
8. Pigments
– Hemoglobin
• A red pigment found in red blood cells
• Helps transport oxygen from the air to the body
– Melanin
• A brown pigment produced by melanocytes in the skin,
pigment cells in the retina, and specialized nerve cells in
the brain
• Has protective functions in the skin and aids in the sense
of sight in the retina
• May be detected using the Fontana-Masson stain
– Lipofuscin
• A yellow to brown pigment found in cardiac tissue,
macrophages, fat cells, and nerve cells.
• Formed by the fusion of a lysosome and a phagosome
• May be detected using the Kluver-Barrera stain

14. Fontana-Masson Staining
• Recommended fixative is sodium thiosulfate (Na2S2O3) and
10% formalin.
1. The cells are treated with a silver nitrate-ammonia solution.
– Melanin reduces silver nitrate to a black metallic silver
precipitate.
2. The silver nitrate-ammonia solution is washed off.
3. The cells are treated with a gold chloride solution.
– Gold chloride acts as a mordant because it intensifies the
black stain.
4. The gold chloride solution is washed off.
5. The cells are treated with nuclear fast red solution.
– Nuclear fast red acts as a counterstain because it provides a
contrasting color to the positive stain.
6. The nuclear fast red is washed off.
7. The slide is viewed under the microscope.