Introduction to Cell BiologyPart 2

Kluver-Barrera Staining1. The cells are treated with Luxol fast blue solution.

– Luxol fast blue acts as a primary stain because it stains all cells blue.

2. The Luxol fast blue is washed off with alcohol.– Alcohol acts as a decolorizing agent because it removes the blue

stain from some cells but not others.3. The cells are treated with lithium carbonate (LiCO3) solution.

– LiCO3 also acts as a decolorizing agent because it removes the blue stain from some cells but not others.

4. The cells are treated with alcohol.5. The alcohol is washed off.6. The cells are treated with cresyl violet.

– Cresyl violet acts as a counterstain because it provides a contrasting color to the primary stain.

– Cresyl violet stains the nucleus of all cells violet.7. The cresyl violet is washed off with alcohol.8. The slide is examined under the microscope.

Inclusions8. Pigments

– Hemosiderin • A pigment produced by the breakdown of old red blood

cells• Found in the spleen, liver cells, and macrophages• May be detected using the Prussian blue stain

– Found in plants as red or blue anthocyanins and yellow anthoxanthins

9. Aleurone grains– Storage proteins found in aleuroplasts in plants– May be amorphous, crystalloid (with carbohydrate), or

globloid (with lipid and phytin).

10. Excretory and secretory products– Mucus, gums, tannins, resins, alkaloids, latex, glucosides,

essential oils, nectar, etc.

Prussian Blue Staining• Recommended fixative is 10% formalin.

1. The cells are treated with hydrochloric acid (HCl) and potassium ferrocyanide (K4Fe(CN)6).– Iron in hemosiderin in the form of ferric ions reacts with

ferrocyanide to form potassium ferric ferrocyanide or Prussian blue.

2. The hydrochloric acid and potassium ferrocyanide are washed off.

3. The cells are treated with nuclear fast red.– Nuclear fast red acts as a counterstain because it provides a

contrasting color to the positive cells.

4. The nuclear fast red is washed off.

5. The slide is examined under the microscope.

Parts of the Cell2. Periplasmic space

– Space between the cell wall and the plasma membrane– Composed of proteins

3. Cell wall– Rigid structure outside the cell membrane– Composed mainly of polysaccharides and proteins– Protects the plasma membrane– Prevents cells from rupturing when the cells swell due to

water uptake – Helps maintain the shape of the cell– Serves as a point of anchorage for flagella (when present)– When the cell wall is completely damaged, the remaining

cell is called protoplast.– When the cell wall is partially damaged, the remaining

cell is called spheroplast.

The Prokaryotic Cell Wall• Composed mainly of peptidoglycan or murein, which is

a network of disaccharides attached to proteins• Gram-positive bacteria consists of many layers of

peptidoglycan, while Gram-negative bacteria contain only a thin layer of peptidoglycan.

• Gram-positive bacteria may contain teichoic acids (in the form of lipoteichoic acid or wall teichoic acid) or mycolic acid.

• Gram-negative bacteria contain an extra outer membrane composed of lipopolysaccharides (LPS) and phospholipids.– Helps evade the immune system– Provides a barrier to certain antibiotics

• Porins are proteins in the outer membrane that form channels to allow passage of nutrients into the cell.

Gram Staining• Simple stain is an aqueous or alcohol solution of a single dye.

• Differential stain is a stain that reacts differently with different kinds of bacteria.

• Special stain is used to color specific parts of the cell.

• Recommended fixative is heating the slide with microorganisms over a Bunsen burner.

1. The cells are treated with crystal violet.

– Crystal violet acts as a primary stain because it stains all cells purple.

2. The crystal violet is washed off.

3. The cells are treated with iodine.

– Iodine forms a complex with crystal violet and acts as a mordant because it intensifies the purple stain.

4. The iodine is washed off.

Gram Staining5. The cells are washed with alcohol or alcohol-acetone solution.

– The alcohol or alcohol-acetone solution acts as a decolorizing agent because it removes the purple stain from some bacteria but not from others.

– Gram-positive bacteria retain the purple color, while Gram-negative bacteria lose the color.

– In Gram-positive bacteria, the complex cannot be washed out due to the peptidoglycan layer.

– In Gram-negative bacteria, the alcohol or alcohol-acetone solution disrupts the outer membrane, and the thin layer of peptidoglycan is unable to retain the complex.

6. The alcohol is washed off.7. The cells are stained with a red dye called safranin.

– Safranin acts a counterstain because it has a contrasting color to the primary stain.

– Safranin turns the colorless Gram-negative bacteria pink.8. The safranin is washed off.9. The slide is examined under the microscope.

Ziehl-Nielsen or Acid-Fast Staining• Recommended fixative is heating the slide with

microorganisms over a Bunsen burner.1. The cells are treated with the red dye carbolfuchsin.

– Carbolfuchsin acts as the primary stain.2. The slide is gently heated for several minutes.

– Heating enhances penetration and retention of the dye. 3. The slide is cooled and washed with water.4. The cells are treated with acid-alcohol.

– Acid-alcohol acts as a decolorizing agent because carbolfuchsin is slightly soluble in acid-alcohol and will leave bacteria that are not acid-fast.

– Carbolfuchsin is more soluble in mycolic acid and will not leave bacteria that are acid-fast.

5. The cells are treated with methylene blue.– Methylene blue acts as a counterstain.

6. The slide is examined under the microscope.

The Eukaryotic Cell Wall• Some protists replace the cell wall with a flexible outer

covering known as pellicle. • The fungal cell wall polysaccharides include chitin, glucan,

and mannan.• The plant cell wall polysaccharides include cellulose,

hemicellulose, xyloglucan, xylan, and pectin.• Cellulose is synthesized in the plasma membrane by the

enzyme cellulose synthase (CESA genes).• Callose is synthesized in the plasma membrane by the

enzyme callose synthase (GSL genes) as response to injury.• Other polysaccharides are synthesized in the Golgi apparatus.• Proteins either expand the cell wall during water uptake

(expansins and XTH enzymes) or disassemble the cell wall during fruit ripening (pectinase, cellulase, and hemicellulaseenzymes).

Questions

• Suppose Protein A from the cell wall is hard to crystallize. What technique can be used to determine its three-dimensional structure?

• Suppose the hardness or softness of the cell wall is to be determined during water uptake. What technique can be used?

Procedure: Analyzing Total Carbohydrates/Proteins

1. Extract the total carbohydrates/proteins in the sample.2. Quantify the total carbohydrates/proteins using a

spectrophotometer.3. Run a gel electrophoresis to separate the

carbohydrates/proteins from each other.4. Identify the carbohydrates/proteins a. By position in the gel b. By cutting portions of the gel, extracting the specific

carbohydrate/protein band out of the gel, and analyzing the carbohydrate/protein through NMR spectroscopy, X-ray crystallography, or mass spectrometry

c. By transferring the carbohydrates/proteins in the gel into a membrane and analyzing the specific carbohydrate/protein using Western blotting

Procedure: Analyzing Specific Carbohydrates/Proteins

1. Extract the specific carbohydrate/protein from the sample.2. Purify the specific carbohydrate/protein.3. Quantify the specific carbohydrate/protein using a

spectrophotometer.4. Run a gel electrophoresis to determine the purity of the

carbohydrate/protein.5. Reaffirm the identity of the carbohydrate/protein a. By position in the gel b. By cutting the band out of the gel, extracting the specific

carbohydrate/protein out of the gel, and analyzing the carbohydrate/protein through NMR spectroscopy, X-ray crystallography, or mass spectrometry

c. By transferring the carbohydrates/proteins in the gel into a membrane and analyzing the specific carbohydrate/protein using Western blotting

Technique: Extraction of Total Carbohydrates

A. Enzyme Extraction (most efficient method but expensive)

1. The sample is first boiled for gelatinization.2. The sample is incubated with enzyme for several hours.3. Protein is removed by addition of lead acetate.4. The sample is filtered to remove insoluble impurities.

B. Dilute Acid Extraction1. The sample is first boiled in dilute H2SO4.2. The sample is filtered to remove insoluble impurities.3. The sample is neutralized with NaOH.

C. Water Extraction (least efficient method but easiest)1. The sample is either boiled in water or shaken in cold water.2. The sample is filtered to remove insoluble impurities.

Technique: Extraction of Total Proteins1. The sample is incubated in cell lysis buffer on ice.

– The cold temperature reduces the rate of undesirable reactions.

2. The sample is centrifuged at 4°C to remove insoluble impurities.

• Components of the cell lysis buffer:– Metal ions like Na+, K+, Mg2+, Ca2+, or Fe3+ stabilize proteins.

– Dithiothreitol (DTT) or β-mercaptoethanol prevents oxidation of proteins.

– Phenylmethylsulfonyl fluoride (PMSF), leupeptin, or aprotininprevent the degradation of proteins.

– Sodium orthovanadate (Na3VO4) prevents the degradation of the phosphate groups of proteins.

Technique: Extraction of Specific Proteins

A. Salting out1. The salt is added at a concentration just below that

necessary to precipitate the protein of interest.• The most commonly used salt for this procedure is ammonium

sulfate, (NH4)2SO4.• The purpose of this step is to precipitate the proteins that are

less soluble than the protein of interest.• Water forms interactions with the salt and is unable to hydrate

the proteins, causing the precipitation of the proteins.

2. The insoluble impurities are filtered out of the solution.3. The salt is increased to a concentration just above that

necessary to precipitate the protein of interest.• The proteins that are more soluble than the protein of interest

remain in the solution.• Problem: The protein is contaminated with large amounts of

salt, which may interfere with succeeding steps.

4. The protein of interest is filtered out of the solution.

Technique: Extraction of Specific Proteins

B. Isoelectric precipitation• The isoelectric point (pI) is the pH in which the net charge of

the protein is zero.1. The pH of the solution is adjusted to the pI of the protein of

interest.– The lack of charge causes the protein of interest to aggregate and

precipitate, while the other proteins remain in solution.

2. The protein of interest is filtered out of the solution.C. Solvent fractionation1. A water-soluble organic solvent like ethanol is added into

the solution at low temperatures.– The mixture of organic solvents with water causes temperature

increases that may lead to protein denaturation.– Organic solvents decrease the dielectric constant of the solution,

causing less ionized molecules like proteins to precipitate.

2. The protein of interest is filtered out of the solution.

Technique: Extraction of Specific Proteins

D. Heat treatment• This is only applicable to proteins that are stable at high

temperatures.1. The solution is heated at a high temperature, causing the

precipitation of other proteins, while the protein of interest remain in solution.

2. The insoluble impurities are filtered out of the solution.E. Addition of acidic or basic precipitants• This is only applicable to proteins that are stable at extreme

pH.1. An acid or a base is added to the solution, causing the

solution to become extremely acidic or extremely basic.– The other proteins will precipitate, but the protein of interest

will remain in solution.2. The insoluble impurities are filtered out of the solution.