Your SlideShare is downloading. ×
0
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
How Gram stain works?
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×
Saving this for later? Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime – even offline.
Text the download link to your phone
Standard text messaging rates apply

How Gram stain works?

1,646

Published on

Gram Staining Principle- Step by step procedure with explanation, Reagent Preparation

Gram Staining Principle- Step by step procedure with explanation, Reagent Preparation

Published in: Education
0 Comments
2 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total Views
1,646
On Slideshare
0
From Embeds
0
Number of Embeds
10
Actions
Shares
0
Downloads
0
Comments
0
Likes
2
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 1. How Gram Stain Works?
  • 2. Gram Staining-Principle• Developed by Christian Gram• Classification of bacteria into Grampositive and Gram negative is based on thedifference in the cell wall composition ofGram positive and Gram negative bacteria. http://www.biologyexams4u.com/
  • 3. In Gram positive bacteria -cell wall is made up of 2 layers  A thicker outer layer made of peptidoglycan and the cell membrane Low lipid content In Gram negative bacteria, cell wall has three layers A lipid rich outer wall A middle layer consists of thin layer of peptidoglycan and a cell membrane.http://www.biologyexams4u.com/
  • 4. Step 1:Prepare thin smear of bacteria on a clean slide .;;;.;; ; . ;;;’;;’;’;;’.;;..;;..;;.;. http://www.biologyexams4u.com/
  • 5. Step 2: Cover the smear with few drops of crystal violet for 1 min .;.;;;.;; ; . . ;;;’;;’;’;;’.;;..;;.;.;Step 3: Wash excess stain with water http://www.biologyexams4u.com/
  • 6. Step 4: Add few drops of iodine solution on  Here Iodine solution is a the smear for 1 min mordant.  Mordants are substance  that improves the staining  property of the dye.  Here Iodine favors more  interaction between cell  and the dye (crystal  .;;;.;; ; . violet). So that, the cells  ;;;’;;’;’;;’.;;..;;..;;.;. are more strongly stained.  Step 5: Rinse in water   Now  under  the  microscope,  all  cells  are  violet coloured, we cannot  ;;;;;.;..;,;,;,l, differentiate  Gram  positive  and  Gram  negative bacteria. Under microscope  
  • 7. On washing with ethanol Step 6: Wash with 95% ethanol for a minute. or acetone, the smear  either retains the violet  colour or decolorizes.  Those bacteria that  retain violet colour are  Gram positive, and those  that lose violet colour on  95% alcohol alcohol treatment are the  Gram negative. This difference in  ;;;.;; ; . staining is due to the  ;;;’;;’;’;;’.;;..;;..;;..;. difference in the cell wall  components of Gram  positive and Gram  negative bacteria. http://www.biologyexams4u.com/
  • 8. In  Gram  positive  bacteria,  After ethanol treatment . the  lipid  content  is  very  low  and  has  a  thick  peptidoglycan  wall.  So  ethanol  cannot  remove  the  crystal  violet  stain  bound  to  thick  ;;;;;.;..;,;,;,l, peptidoglycan  wall  of  gram  positive  bacteria.  So  it retains violet colour. Gram positive bacteria Under microscope In Gram negative bacteria On  ethanol  treatment,  lipids  are  dissolved  in  ethanol and peptidoglycan  wall  is  very  thin  to  hold  ;;;;;.;..;,;,;,l, the stain so gram negative  bacteria lose violet colour.Gram negative bacteria Under microscope
  • 9. Step 7: Wash excess stain with water  This is to visualise Gram  negative bacteria. Step 8:  Stain with 1% safranin for 1 minute. After ethanol treatment  in step-6, only Gram  positive bacteria retain  crystal violet.  We need to stain further  using a counter stain like  safranin to visualise Gram  negative bacteria. .; ;;;’;;’ ; .;; .; ;’;;’.;;..;..;;..;; ; ;;;;;.;..;,;,;,l, http://www.biologyexams4u.com/
  • 10. Step 9: Wash stain in excess water. The end result after staining with safranin Gram positive bacteria appear violet or purple (Colour of crystal violet) Gram negative bacteria appear pink to red (colour of safranin). http://www.biologyexams4u.com/
  • 11. Procedure Summary Chart
  • 12. Reagent Preparation Crystal violet solution: 1g crystal violet dissolved in100 ml ethanol. Safranin solution: 1g safranin dissolved in 100 mlH2O. Iodine solution: Dissolve 2g potassium iodide (KI) in100 ml water. To the solution add iodine crystals tilla deep yellow colour develops. http://www.biologyexams4u.com/
  • 13. FOR DETAILED NOTES ON THIS TOPIC VISIThttp://www.biologyexams4u.com/2011/09/microbiology-notes.html http://www.biologyexams4u.com/

×