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Gram stain

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Pranav Patel
Pranav Patel

Gram stain is technique used to differntiate gram positive and gram negative bacteria. If you like this slideshare then please do share and follow me. You can visit my blogs:pranav2705.blogspot.com

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GRAM STAINING. PREPARED BY: PRANAV PATEL. PHARM D, SECOND YEAR. KBIPER,GANDHINAGAR.
INTRODUCTION ? Gram stain or gram staining also called gram's method is method of staining used to distinguish and classify bacterial species into large two groups:Gram positive and Gram negative bacteria. ? The name comes from the danish bacteriologist Hans christian gram ,who developed this technique
OBJECTIVE ? Objective of gram staining is to differntiate the bacteria into two groups:Gram positive and Gram negative,which helps in classification and differntiation of microorganisms ? The gram stain separates bacteria into two groups: 1. Gram positive organisms which retain primary dye(Crsytal violet) 2. Gram negative organisms which take the colour of counterstain(Safranin)
PRINCIPLE ? Gram staining technique is based on differntial strucutre of cellular membrane and cell wall of the organisms. ? Gram positive bacteria contains peptidoglycan thick cell wall,but they do not have an outer membrane.While gram negative bacteria contains peptidoglycan thin wall with an outer
GRAM POSITIVE BACTERIA ? Peptidiglycan layer of gram positive bacteria retains the primary dye,crystal violet(CV),with mordant iodine.Iodine and crystal violet makes a complex within the peptidoglycan.when decolorizer applied to cells CV-I complex remains with cell and it shows dark purple to blue colour. ? Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-coloured when seen through an optical microscope. This is because the thick peptidoglycan layer in the bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the decolorization stage of the test.
GRAM NEGATIVE BACTERIA ? Gram negative bacteria does not have thick peptidoglycan layer.Following the application of crystal violet and iodine,the CV-I complex is not trapped within peptidoglycan.When acid-alcohol is decolorizer dehydrates outer cellular membrane leaving holes in the membrane and effectively washing CV-I complex from cell.The cells appear colourless,so to make it visible a secondary stain safranin is applied leaving gram negative cells pink.
REAGENTS USED IN GRAM STAINNG 1. Primary stain:Crystal violet,95% ethyl alcohol,Ammonium oxalate,Distilled water. 2. Gram's iodine:Potassium iodide,Iodine crystals,Distilled water. 3. Decolorizer:Acetone and ethanol. 4. Counterstain:Safranin,Ethanol,Distilled water.
PROCEDURE ? Prepare and fix the specimen to the microscope slide before staining. ? Cover the smear with crystal violet, the primary stain, for 20 seconds. ? Gently rinse off the stain with water. ? Cover the smear with Gram¡¯s iodine, the mordant, for 1 minute. ? Pour off the excess Gram¡¯s iodine. ? Run the acid-alcohol decolorizer over the smear until the solution appears clear. ? Gently rinse with water. ? Cover the smear with safranin, the secondary or counterstain, for 20 seconds. ? Gently rinse the stain with water.
RESULT ? Gram Positive bacteria:Blue/Purple colour. ? Gram Negative bacteria:Red/Pink colour.
DIFFERENCE BETWEEN GRAM POSITIVE AND GRAM NEGATIVE BACTERIA ? As Gram positive bacteria lack an outer lipid membrane, when correctly referring to their structure rather than staining properties, are termed monoderms. The outer lipid membrane possessed by Gram negative bacteria means that, when referring to their physical structure, they are termed diderms.
KEY CHARACTERS DIFFERNCE S.N. Character Gram positive bacteria Gram negative bacteria. 1 Cell wall thickness Thick(20-80nm) Thin(8-10nm) 2 Peptidoglycan layer Thick Thin 3 Outer membrane Absent Present 4 Porins Absent Present 5 Ratio of RNA:DNA 8:1 Almost 1 6 Pathogens Few pathogenic bacteria belong to gram positive group Most pathogens are gram negative 7 Examples Staphylococcus Streptococcus Bacillus Clostridum Escherichia Salmonella Proteus Helicobacter
THANK YOU. If you have any query you can contact me:pcpatel5102@gmail.com You can also visit my blogs:pranav2705.blogspot.com You can also find me on: @scientifx @scientifx @scientifx05

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Gram stain

  • 1. GRAM STAINING. PREPARED BY: PRANAV PATEL. PHARM D, SECOND YEAR. KBIPER,GANDHINAGAR.
  • 2. INTRODUCTION ? Gram stain or gram staining also called gram's method is method of staining used to distinguish and classify bacterial species into large two groups:Gram positive and Gram negative bacteria. ? The name comes from the danish bacteriologist Hans christian gram ,who developed this technique
  • 3. OBJECTIVE ? Objective of gram staining is to differntiate the bacteria into two groups:Gram positive and Gram negative,which helps in classification and differntiation of microorganisms ? The gram stain separates bacteria into two groups: 1. Gram positive organisms which retain primary dye(Crsytal violet) 2. Gram negative organisms which take the colour of counterstain(Safranin)
  • 4. PRINCIPLE ? Gram staining technique is based on differntial strucutre of cellular membrane and cell wall of the organisms. ? Gram positive bacteria contains peptidoglycan thick cell wall,but they do not have an outer membrane.While gram negative bacteria contains peptidoglycan thin wall with an outer
  • 5. GRAM POSITIVE BACTERIA ? Peptidiglycan layer of gram positive bacteria retains the primary dye,crystal violet(CV),with mordant iodine.Iodine and crystal violet makes a complex within the peptidoglycan.when decolorizer applied to cells CV-I complex remains with cell and it shows dark purple to blue colour. ? Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-coloured when seen through an optical microscope. This is because the thick peptidoglycan layer in the bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the decolorization stage of the test.
  • 6. GRAM NEGATIVE BACTERIA ? Gram negative bacteria does not have thick peptidoglycan layer.Following the application of crystal violet and iodine,the CV-I complex is not trapped within peptidoglycan.When acid-alcohol is decolorizer dehydrates outer cellular membrane leaving holes in the membrane and effectively washing CV-I complex from cell.The cells appear colourless,so to make it visible a secondary stain safranin is applied leaving gram negative cells pink.
  • 7. REAGENTS USED IN GRAM STAINNG 1. Primary stain:Crystal violet,95% ethyl alcohol,Ammonium oxalate,Distilled water. 2. Gram's iodine:Potassium iodide,Iodine crystals,Distilled water. 3. Decolorizer:Acetone and ethanol. 4. Counterstain:Safranin,Ethanol,Distilled water.
  • 8. PROCEDURE ? Prepare and fix the specimen to the microscope slide before staining. ? Cover the smear with crystal violet, the primary stain, for 20 seconds. ? Gently rinse off the stain with water. ? Cover the smear with Gram¡¯s iodine, the mordant, for 1 minute. ? Pour off the excess Gram¡¯s iodine. ? Run the acid-alcohol decolorizer over the smear until the solution appears clear. ? Gently rinse with water. ? Cover the smear with safranin, the secondary or counterstain, for 20 seconds. ? Gently rinse the stain with water.
  • 9. RESULT ? Gram Positive bacteria:Blue/Purple colour. ? Gram Negative bacteria:Red/Pink colour.
  • 10. DIFFERENCE BETWEEN GRAM POSITIVE AND GRAM NEGATIVE BACTERIA ? As Gram positive bacteria lack an outer lipid membrane, when correctly referring to their structure rather than staining properties, are termed monoderms. The outer lipid membrane possessed by Gram negative bacteria means that, when referring to their physical structure, they are termed diderms.
  • 11. KEY CHARACTERS DIFFERNCE S.N. Character Gram positive bacteria Gram negative bacteria. 1 Cell wall thickness Thick(20-80nm) Thin(8-10nm) 2 Peptidoglycan layer Thick Thin 3 Outer membrane Absent Present 4 Porins Absent Present 5 Ratio of RNA:DNA 8:1 Almost 1 6 Pathogens Few pathogenic bacteria belong to gram positive group Most pathogens are gram negative 7 Examples Staphylococcus Streptococcus Bacillus Clostridum Escherichia Salmonella Proteus Helicobacter
  • 12. THANK YOU. If you have any query you can contact me:pcpatel5102@gmail.com You can also visit my blogs:pranav2705.blogspot.com You can also find me on: @scientifx @scientifx @scientifx05