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  Section: Biotechnology Methods » Microbiology
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Gram Stain and Capsule Stain

  The Microscopy
  The Bright Field Microscope
  Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms
  Cell Size Measurements: Ocular and Stage Micrometers
  Measuring Depth
  Measuring Area
  Cell Count by Hemocytometer or Measuring Volume
  Measurement of Cell Organelles
  Use of Darkfield Illumination
  The Phase Contrast Microscope
  The Inverted Phase Microscope
  Aseptic Technique and Transfer of Microorganisms
  Control of Microorganisms by using Physical Agents
  Control of Microorganisms by using Disinfectants and Antiseptics
  Control of Microorganisms by using Antimicrobial Chemotherapy
  Isolation of Pure Cultures from a Mixed Population
  Bacterial Staining
  Direct Stain and Indirect Stain
  Gram Stain and Capsule Stain
  Endospore Staining and Bacterial Motility
  Enumeration of Microorganisms
  Biochemical Test for Identification of Bacteria
  Triple Sugar Iron Test
  Starch Hydrolysis Test (II Method)
  Gelatin Hydrolysis Test
  Catalase Test
  Oxidase Test
  IMVIC Test
  Extraction of Bacterial DNA
  Medically Significant Gram–Positive Cocci (GPC)
  Protozoans, Fungi, and Animal Parasites
  The Fungi, Part 1–The Yeasts
  Performance Objectives
  The Fungi, Part 2—The Molds
  Viruses: The Bacteriophages
  Serology, Part 1–Direct Serologic Testing
  Serology, Part 2–Indirect Serologic Testing

The Gram Stain
The Gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between Gram-positive and Gram-negative bacteria. Bacteria that stain purple with the Gram-staining procedure are termed Gram-positive; those that stain pink are said to be Gramnegative. The terms positive and negative have nothing to do with electrical charge, but simply designate 2 distinct morphological groups of bacteria. Gram-positive and Gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape, as well as to prevent osmoticlysis. The material in the bacterial cell wall that confers rigidity is peptidoglycan.

In electron micrographs, the Gram-positive cell wall appears as a broad, dense wall 20–80 nm thick and consists of numerous interconnecting layers of peptidoglycan. Chemically, 60% to 90% of the Gram-positive cell wall is peptidoglycan Interwoven in the cell wall of Gram-positive are teichoic acids. Teichoic acids, that extend through and beyond the rest of the cell wall, are composed of polymers of glycerol, phosphates, and the sugar alcohol ribitol. Some have a lipid attached (lipoteichoic acid). The outer surface of the peptidoglycan is studded with proteins that differ with the strain and species of the bacterium.

The Gram-negative cell wall, on the other hand, contains only 2–3 layers of peptidoglycan and is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, lipoprotein, and proteins. Only 10%–20% of the Gram-negative cell wall is peptidoglycan. The phospholipids are located mainly in the inner layer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan. The lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid portion called lipid A embedded in the membrane, and a polysaccharide portion extending outward from the bacterial surface. The outer membrane also contains a number of proteins that differ with the strain and species of the bacterium.

The Gram-staining procedure involves 4 basic steps:
  1. The bacteria are first stained with the basic dye crystal violet. Both Grampositive and Gram-negative bacteria become directly stained and appear purple after this step.
  2. The bacteria are then treated with Gram’s iodine solution. This allows the stain to be retained better by forming an insoluble crystal violet-iodine complex. Both Gram-positive and Gram-negative bacteria remain purple after this step.
  3. Gram’s decolorizer, a mixture of ethyl alcohol and acetone, is then added. This is the differential step. Gram-positive bacteria retain the crystal violetiodine complex, while Gram-negative are decolorized.
  4. Finally, the counterstain safranin (also a basic dye) is applied. Since the Gram-positive bacteria are already stained purple, they are not affected by the counterstain. Gram-negative bacteria, which are now colorless, become directly stained by the safranin. Thus, Gram-positive bacteria appear purple and Gram-negative bacteria appear pink.
With the current theory behind Gram-staining, it is thought that in Grampositive bacteria, the crystal violet and iodine combine to form a larger molecule that precipitates out within the cell. The alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycan, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. In the case of Gram-negative bacteria, the alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan is attached. The single thin layer of peptidoglycan is unable to retain the crystal violetiodine complex and the cell is decolorized.

It is important to note that Gram-positivity (the ability to retain the purple crystal violet-iodine complex) is not an all-or-nothing phenomenon, but a matter of degree. There are several factors that could result in a Gram-positive organism staining Gram-negatively:
  1. The method and techniques used: Overheating during heat fixation, overdecolorization with alcohol, and even too much washing with water between steps may result in Gram-positive bacteria losing the crystal violetiodine complex.
  2. The age of the culture: Cultures more than 24 hours old may lose their ability to retain the crystal violet-iodine complex.
  3. The organism itself: Some Gram-positive bacteria are more able to retain the crystal violet-iodine complex than others. Therefore, one must use very precise techniques in Gram staining and interpret the results with discretion.

Trypticase soy agar plate cultures of Escherichia coli (a small, Gram-negative rod) and Staphylococcus epidermidis (a Gram-positive coccus in irregular, often grapelike clusters).

  1. Heat-fix a smear of a mixture of Escherichia coli and Staphylococcus epidermidis as follows:
    1. Using the dropper bottle of distilled water found in your staining rack, place a small drop of water on a clean slide by touching the dropper to the slide.
    2. Aseptically remove a small amount of Staphylococcus epidermidis from the agar surface and mix it generously with the water. Flame the loop and let it cool. Now, aseptically remove a small amount of Escherichia coli and sparingly add it to the water. Flame the loop and let it cool.
    3. Using the loop, spread the mixture over the entire slide to form a thin film.
    4. Allow this thin suspension to completely air dry.
    5. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix.
  2. Stain with Hucker’s crystal violet for 1 minute. Gently wash with water. Shake off the excess water, but do not blot dry between steps.
  3. Stain with Gram’s iodine solution for 1 minute and gently wash with water.
  4. Decolorize by adding Gram’s decolorizer drop by drop until the purple stops flowing. Wash immediately with water.
  5. Stain with safranin for 1 minute and wash with water.
  6. Blot dry and observe using oil immersion microscopy.

The Capsule Stain

Many bacteria secrete a slimy, viscous covering called a capsule or glycocalyx. This is usually composed of polysaccharide, polypeptide, or both.

The ability to produce a capsule is an inherited property of the organism, but the capsule is not an absolutely essential cellular component. Capsules are often produced only under specific growth conditions. Even though not essential for life, capsules probably help bacteria survive in nature. Capsules help many pathogenic and normal flora bacteria to initially resist phagocytosis by the host’s phagocytic cells. In soil and water, capsules help prevent bacteria from being engulfed by protozoans. Capsules also help many bacteria adhere to surfaces and, thus, resist flushing.

Skim milk broth culture of Enterobacter aerogenes—the skim milk supplies essential nutrients for capsule production and also provides a slightly stainable background.

  1. Stir up the skim milk broth culture with your loop and place 2–3 loops of Enterobacter aerogenes on a slide.
  2. Using your loop, spread it out over the entire slide to form a thin film.
  3. Let it completely air dry. Do not heat-fix. Capsules stick well to glass, and heat may destroy the capsule.
  4. Stain with crystal violet for 1 minute.
  5. Wash off the excess stain with copper sulfate solution. Do not use water!
  6. Blot dry and observe using oil immersion microscopy. The organism and the milk dried on the slide will pick up the purple dye, while the capsule will remain colorless.
  7. Observe the demonstration capsule stain of Streptococcus pneumoniae (the pneumococcus), an encapsulated bacterium that often has a diplococcus arrangement.

The Capsule Stain
Make a drawing of your capsule stain preparation of Enterobacter aerogenes and the demonstration capsule stain of Streptococcus pneumoniae.

Performance Objectives
The Gram Stain
  1. Explain why the Gram stain is a differential stain.
  2. Describe the differences between a Gram-positive and Gram-negative cell wall.
  3. Explain the theory as to why Gram-positive bacteria retain the crystal violet-iodine complex, while Gram-negatives become decolorized.
  4. Describe 3 conditions that may result in a Gram-positive organism staining Gram-negatively.

  1. Describe the procedure for the gram stain.
  2. Perform a Gram stain with the necessary materials.
Determine if a bacterium is Gram-positive or Gram-negative when microscopically viewing a Gram stain preparation, and describe the shape and arrangement of the organism.

The Capsule Stain
Describe the chemical nature and major functions of bacterial capsules.

Recognize capsules as the structures observed when microscopically viewing a capsule stain preparation.


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