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INTRODUCTION

The Gram stain is an extremely important yet basic technique routinely used in the microbiology laboratory for the preliminary identification and classification of bacteria. It was introduced by Danish scientist Hans Christian Gram in 1884. The Gram stain is a differential stain that categorizes most bacteria into two main groups: Gram positive and Gram negative, based on differences in the composition of the cell wall. During the Gram stain procedure, Gram positive organisms retain the crystal violet-iodine complex in their cell wall even after the application of a decolorization agent and are colored purple. Gram negative organisms on the other hand do not retain the crystal violet-iodine complex after decolorization. Therefore safranin is applied as a counter stain which colors the Gram negative cells reddish/pink. The stained specimens are then visually differentiated when viewed with a microscope. In the clinical laboratory, the Gram stain is an important preliminary step for the identification of pathogens that cause infectious disease.

OBJECTIVES

1. To differentiate between the two major categories of bacteria: Gram positive and Gram negative based on chemical and structural differences in their cell walls. 2. To understand the mechanism of the Gram stain and how it affects the cell walls of Gram positive and Gram negative bacteria. OVERVIEW OF STAINING Bacteria are minute living organisms that cannot be seen with the unaided eye. They are present everywhere (ubiquitous) and can be visualized only with the aid of a microscope. The application of stains to bacterial specimens enables investigators to observe the morphology and arrangement of individual cells using a microscope. Stains enhance the contrast in the microscopic image. They are chemical agents that have the ability to adhere strongly to certain molecules present in cells (both prokaryotic and eukaryotic) and tissues. A common feature of stains is the presence of an organic chromophore group which imparts a color to the specimen. Stains are also able to form ionic, covalent or hydrophobic bonds with cellular components. They are classified into two main categories: acidic stains and basic stains. Acidic stains are negatively charged, whereas basic stains are positively charged. Acidic stains have a negative charge due to the presence of a Carboxyl group (– COOH) and hydroxyl group (–OH) which enable them to bind to positively charged cell structures. Eosin, Rose Bengal and Acid Fuchsin are examples of acidic stains. On the other hand, basic stains have a positive charge, due to N+ (Nitrogen) which enables them to bind easily to negatively charged cell structures such as nucleic acids and proteins. Examples of basic stains include Methylene Blue, Basic Fuchsin, Crystal Violet, Safranin and Malachite Green. Differential staining is a technique that uses more than one stain to differentiate between microorganisms as well as cellular and structural components within a single microorganism. The Gram stain is a differential stain using Crystal Violet and Safranin.

THEORY

Gram Positive Cell Wall

The cell wall of Gram positive bacteria contains a thick mesh-like peptidoglycan layer which composes 50% to 90% of the cell wall (Figure 1). It provides strength and rigidity to the cell and also determines the morphology (shape and structure) of the cell. The peptidoglycan layer is located outside the cytoplasmic membrane. Peptidoglycan is a polymer composed of sugars and amino acids. The sugars are N-acetyl glucosamine (NAG) and N-acetylmuramic acid (NAM). The amino acids are D-glutamic acid, D-alanine and mesodiaminopimelic acid. Adjacent layers of peptidoglycan are cross linked and held together by short chains of peptides. The peptidoglycan layer retains the crystal violet-iodine complex of the Gram stain after decolorization in Gram positive organisms, thus staining them purple. Another major component of the Gram positive cell wall is teichioc acids. Teichoic acids are embedded in the peptidoglycan layer and consist of long chains of the polysaccharides glycerol phosphate and rubitol phosphate. They are linked to the peptidoglycan layer via phosphodiester bonds. Some teichoic acids are attached to a lipid which is anchored in the cytoplasmic membrane, and are called lipoteichoic acids. Lipoteichoic acids and teichoic acids provide strength to the bacterial cell wall, may enhance bacterial attachment to substrates, and occasionally act as a virulence factor for pathogenic bacteria. In certain organisms, lipoteichoic acid is responsible for antigenic and immune responses when it is released upon cell death. Examples include Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphtheriae, Clostridium tetani.

Gram Negative Cell Wall

The cell wall of Gram negative bacteria consists of the cytoplasmic membrane, a thin peptidoglycan layer (comprising approximately only 10% of the cell wall) and an outer membrane composed of lipopolysaccharide (Figure 2). The periplasmic space is an area that separates the peptidoglycan layer and the outer membrane. The peptidoglycan layer and lipid layer are attached covalently to each other. Braun’s lipoprotein is a membrane protein tightly associated with the outer membrane and functions to strengthen the outer membrane of Gram negative bacteria. The lipopolysaccharide (LPS) of the outer membrane is composed of Lipid A, Core polysaccharide and O antigen. Lipid A consists of the derivatives of  two glucosamine sugar units and a phosphate group (disaccharide diphosphate) which are attached to three fatty acids. Due to the hydrophobic fatty acid chains, Lipid A is anchored in the phospholipid bilayer of the outer membrane. The toxic effect of LPS in infected hosts is caused by Lipid A. The core polysaccharide is attached to Lipid A and is composed of sugars such as heptoses and 2-keto-3deoxyoctonoic acid. The O antigen is the outer most part of LPS which is attached to the core polysaccharide and consists of a long chain of various sugars. Endotoxin is essentially composed of LPS and is responsible for disease in infected hosts. It is released in large amounts upon bacterial death, causing an immune response in the host. Endotoxin is only present in Gram negative bacteria. Examples of Gram negative pathogens include Neisseria meningitidis, Escherichia coli, Shigella flexneri, and Vibrio cholerae. Other Structures Associated With the Cell Wall In addition to the cell wall, some gram positive and gram negative bacteria possess flagella that aid in their movement, fimbriae for attachment and pili for conjugation (exchange of genetic material). Many bacteria also have capsules, slime layers and S-layers which are outside of the cell wall and inhibit phagocytosis by cells of the host's immune system.

Gram Stain Procedure

A short summary of the Gram stain (performed on a prepared heat-fixed specimen) is given below: 1. Application of the primary stain: Crystal Violet 2. Addition of the mordant: Gram's Iodine (A mordant is a substance that fixes a dye or stain in a particular material.) 3. Decolorization with 95% Ethyl Alcohol 4. Application of the counter-stain: Safranin The thick peptidoglycan layer of the cell wall of Gram positive bacteria traps the color of the primary stain Crystal Violet. On the other hand, the lipid components of the cell wall of Gram negative bacteria easily dissolve upon application of alcohol during the decolorization step, causing the primary stain to leak out of the cell wall. This is due to the large amount of lipid in the Gram negative cell wall and the small amount of peptidoglycan. After decolorizing, Gram negative cells become transparent, and thus require coloring with the counter-stain, Safranin. The principle of this process is discussed in further detail below.