Introduction to Lab Ex. Differential Stains Gram Staining

Introduction to Lab Ex. Differential Stains Gram Staining Introduction to Lab Ex. Differential Stains Gram Staining Basic classification of bacteria is based on the cell wall structure. There are 2 main groups: Gram positive and Gram negative. Gram staining is a differential staining technique that provides an easy differentiation of bacteria into one of two groups. The staining technique, developed in the late 1700s by Christian Gram classifies the rigid cell walled bacteria into one of two groups based on whether they are able to resist the decolorizing action of an alcoholic solution. Those that resist decolorization by 95% ethanol are arbitrarily termed Gram positive and those that do not are Gram negative

(the terms positive and negative have nothing to do with charges of the cell but based on differences in the cell wall structure of these two groups of bacteria). The characteristic compound found in all true bacterial cell walls is peptidoglycan. The amount of PPG is among one of the differences between the GP and GN cell walls. Gram-positive cell walls walls

Thick peptidoglycan 90% peptidoglycan Teichoic acids 1 layer Not many polysaccharides In acid-fast cells, contains mycolic acid Gram-negative cell

Thin peptidoglycan 5-10% peptidoglycan No teichoic acids 3 layers Outer membrane has lipids, polysaccharides No acid- fast cells (mycolic acid) Figure 4.13b, c The process includes the use of: a primary stain (crystal violet) a mordant (helper) iodine solution,

a decolorizer (95% ethanol), a counterstain (safranin). The Gram stain Thin smear/heat fix Gram stain: a. Flood slide with crystal violet and let stain for 1 minute. b. Drain off crystal violet and rinse off with distilled water; flood slide with Gram's iodine for 1 minute. c. Rinse off Gram's iodine with distilled water.

d. Hold the slide on an angle (preferably with a clothes pin) and drop 95% ethyl alcohol onto it until the alcohol leaving the slide no longer has a purple tint; be sure to drop the alcohol onto the upper portion of the slide so that the smears are subjected to uniform decolorization. Be careful not to "decolorize" dye from the clothes pin!!

Gram positive Gram negativ The crucial step in the staining process is the decolorizing step. The most accepted theory about the rationale for the Gram staining process is the one proposed by Salton. This theory relies on the fact that the PPG is found in layers and the stain molecules are trapped within the many layers of the GP CW when they form the complex with the mordant Iodine molecules. Since the GN CWs lack much PPG the amount of stain captured in those CWs is much lesser.

When the cells are treated with the decolorizer the ethanol this causes denaturation of the proteins in the outer membrane of the GN CWs resulting in gaping holes in these CWs that lead to the removal of the crystal violet-iodine complexes easily, leaving these cells unstained. The counterstain -safranin- thus is used to make these cells visible. There are 4 conditions to be followed for a valid Gram staining procedure: Young cultures - must be young within 18-24hrs old (older cultures lose their Gram staining properties due to changes in the CWs as the cells get older) Thin smear thicker or uneven smears will result in uneven staining and decolorization

Fresh reagents - of proper strength Control cultures - for a known GP bacterium and GN culture (S.aureus & E.coli) Demos: Gram stained slides of Neisseria, Streptococcus, Pseudomonas, Actinomyces species. Pseudomonas Neisseria Streptococcus

Recently Viewed Presentations

  • CIGNA's Healthcare Leadership Program - GKEN

    CIGNA's Healthcare Leadership Program - GKEN

    Global Knowledge Exchange Network Overview * Global Knowledge Exchange Network Vision: The Global Exchange will become the recognized neutral venue for evaluating and promoting better practices in achieving cost-effective health outcomes from across developed and developing nations.
  • Department Of Financial The Navy,Management & Comptroller Financial

    Department Of Financial The Navy,Management & Comptroller Financial

    Improve end-to-end processes allowing for better tracking of property condition throughout itslifecycle. Identify gaps in key controls to streamlineoperations. Ensure items are properly tracked, recorded andvalued.
  • AEDT2120U: Culture and Digital Technologies Course Outline

    AEDT2120U: Culture and Digital Technologies Course Outline

    From the shores of Lac St Jean, these traditional legends of the Pekuakamiulnuatsh - the Montagnais of Lac Saint-Jean - reflect a journey back in time when humans and animals spoke the same language. They share the lessons taught by...
  • Understanding Solid Figures A Mini Lesson by Diana

    Understanding Solid Figures A Mini Lesson by Diana

    A solid figure whose ends are congruent, parallel polygons, and whose sides are rectangles is a prism. These are prisms. Practice counting vertices: Remember to count the vertices that you can't see. 8 0 4 Practice counting faces: 6 Remember...
  • F211 Exchange and Transport Lungs What do animals

    F211 Exchange and Transport Lungs What do animals

    Features of the mammalian lung that enable efficient gas exchange. Individual alveoli are only100-300 micrometers across, very numerous ( about 300-500 million) total surface area = approx 70 m2. Alveoli walls are one cell thick, plasma membranes surround a very...
  • http://miblsi.cenmi.org DIBELS Next Data Interpretation Module 1: Foundations

    http://miblsi.cenmi.org DIBELS Next Data Interpretation Module 1: Foundations

    Doug Carnine Anita Archer. Jan Hasbrouck Gough & Tunmer. ... For example con sider is consider and we can also segment consider into its parts to spell. Because blending and segmenting are so closely related to reading and spelling, these...
  • Drifting Toward Disunion - Weebly

    Drifting Toward Disunion - Weebly

    Amendment shortened this period by 10 weeks. Pres. Buchanan is still the acting president as southern states seceded. Buchanan- respecter of the Constitution did not believe that the South could legally secede nor did it give him authority to stop...
  • The Anglo-Saxon Period

    The Anglo-Saxon Period

    The Anglo-Saxon Chronicles. Tells the story of the history of England from the beginning in 1 A.D. It was ordered to be compiled by King Alfred the Great in 890 A.D. and was written by scholars in monasteries and continuously...