Gram Staining Theory and Demonstration ppt
- 2. At the end of the session students will be able to : •Classify staining techniques with examples •Describe the principle and Mechanisms of Gram staining •Describe the procedure of Gram Staining •Perform Gram Staining on the given smear and give the interpretation •Give examples for Gram positive and Gram negative bacteria •Enumerate the uses of Gram staining •Discuss the limitations of Gram staining •Name the modifications of Gram staining and their uses
- 3. Structural details of bacteria cannot be seen under light microscope due to lack of contrast. It is necessary to use staining methods to produce color contrast and thereby increase the visibility
- 5. Simple Staining •A single reagent is used •Basic dyes (methylene blue or basic fuchsin) are used to provide the color contrast •Shape and arrangement of bacteria can be studied . •Imparts the same color to all the bacteria in a smear.
- 6. • Two stains are used . • Impart different colors to different bacteria. • Most commonly used differential stains are: 1. Gram stain: gram-positive and gram-negative groups. 2. Acid-fast stain: acid fast and non acid-fast groups.
- 7. To demonstrate structures like spores, granules, capsule, flagella etc • Albert stain: metachromatic granules in Corynebacterium diphtheriae • Ryu’s stain for flagella • Silver impregnation method or Spirochetes
- 8. Background is stained with an acidic dye such as India Ink or Nigrosin Demonstrates capsule in Cryptococcus neoformans.
- 9. Originally developed by Danish Bacteriologist, Hans Christian Gram (1884). Gram stain still remains the most widely used test in diagnostic bacteriology.
- 10. When bacteria are treated with stains such as crystal violet, methyl violet or gentian violet and then with iodine, a dye- iodine complex is formed. On decolorization , some bacteria retain this complex while some lose the complex. Those which retain the primary stain , appear violet in color and are called Gram positive Those which lose the primary stain and take up the counter stain, appear pink in color and are called Gram Negative .
- 11. Exact mechanism is unknown . Theories have been put forth to describe the mechanism Cell Wall theory Isoelectric pH theory Magnesium ribonucleate theory
- 12. Cell Wall theory •Gram-positive cell wall - thick peptidoglycan layer (50–100 layers thick). •Acts as permeability barrier preventing loss of crystal violet. •Alcohol is thought to shrink the pores of the thick peptidoglycan
- 13. Cell wall theory (contd…) •Gram-negative cell wall - more permeable thus allowing the out flow of crystal violet easily. •Thin peptidoglycan layer in gram-negative cell wall - is not tightly cross linked •Presence of lipopolysaccharide layer in the cell wall of gram-negative bacteria - gets disrupted easily by the decolorizer.
- 14. pH theory: •Cytoplasm of gram-positive bacteria - more acidic -can retain the basic dye (e.g. crystal violet) for longer time. •Iodine serves as mordant - combines with the primary stain to form a dye-iodine complex - gets retained inside the cell
- 15. Magnesium ribonucleate theory •A compound of magnesium ribonucleate and a basic protein concentrated at the cytoplasmic membrane helps the Gram positive bacteria retain the primary stain.
- 16. Fixed smear Reagents Primary stain : Crystal violet Mordant : Gram’s Iodine Decolorizer: Acetone-alcohol Counterstain: Dilute Carbol fuchsin Microscope with oil immersion objective Immersion oil Dilute carbol fuschin
- 17. Most Critical step – Decolorization Alternatives Methyl violet, Gentian violet Acetone-alcohol mixture for 5-10 seconds Acetone , Alcohol Safranin
- 18. Gram-positive bacteria resist decolorization and retain the color of primary stain i.e. violet. Gram-negative bacteria are decolorized and, therefore, take counterstain and appear pink
- 23. Gram positive cocci Gram negative cocci Gram negative bacilli Gram positive bacilli Staphylococcus spp Neisseria spp Escherichia coli Clostridium spp Streptococcus spp Moraxella spp Klebsiella spp Lactobacillus Micrococcus spp Veillonella spp Shigella spp Actinomycete spp Peptostreptococcus Salmonella spp Propionibacterium spp Peptococcus Pseudomonas spp Bifidobacterium
- 24. • To differentiate bacteria into gram-positive and gram negative • Identification of Gram staining from bacterial culture helps in further identification of bacteria • Initiation of empirical treatment with broad spectrum antibiotics - started early before the culture report is available. • Early presumptive identification of fastidious organisms - Haemophilus.
- 25. Gram stain gives a preliminary clue in anaerobic culture (Clostridium) Gram stain is useful for staining certain fungi such as Candida and Cryptococcus (appear gram-positive) Helps in screening the quality of the sputum specimen before processing it for culture.
- 26. Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). When over-decolorized, even Gram positive bacteria may appear pink and when under-decolorized gram negative bacteria may appear Gram positive.
- 27. Small and slender bacteria such as Treponema, Chlamydia, Rickettsia are often difficult to stain by Gram's method Gram positive cells affected by cell wall active agents such as lysozyme or antibiotics may become Gram negative. Bacteria totally devoid of cell wall (Mycoplasma) cannot be stained by Gram’s stain
- 28. Modifications Explanation Use Kopeloff and Beerman’s Primary stain : Methyl violet Counter stain : Basic fuchsin Useful for Anaerobes , diagnosis of Bacterial vaginosis Jensen’s Primary stain : Methyl violet Decolorizer :Absolute alcohol Counter stain: Neutral red Useful for meningococci and gonococci Brown and Brenn Primary stain : Crystal violet Decolorizer :Acetone-alcohol (1:1) Counter stain: Safranin Used for Actinomycetes Weigert’s Primary stain: Carbol gentian violet Decolorizer: Aniline -Xylol Used for staining tissue sections
- 29. 1. What is the function of Gram’s iodine? 2. Describe the mechanism of Gram’s staining? 3. List the uses of Gram stain. 4. Discuss the limitations of Gram staining. 5. Give examples for Gram positive and gram negative bacteria. 6. Enumerate the modifications of Gram staining and their uses