Gram staining

Gram staining

  • Staining is a biochemical technique of coloring specimens.
  • Dyes are the chemical substances which commonly used to stain specimen.

There are three types of staining protocol or procedures:

  1. Simple staining.
  2. Differential staining.
  3. Specialised staining.
  • Gram staining a differential  staining method.
  • It is used to identify and differentiate bacteria into two groups i.e. Gram positive and Gram negative bacteria.
  • This classification is based on the physical properties of the bacterial cell wall.
  • Gram staining procedure is developed in 1884 by Hans Christian gram.

In this staining bacterial smear is of four different reagents:

  1. Crystal Violet it is also known as primary stain.
  2. Iodine it is referred as mordant because it increases the affinity of dyes or a stain for smear.
  3. Alcohol or ethanol it is referred as de-colorizing agent that remove stain from the specimen.
  4. Safranin it is referred as counterstain.

Procedures of Gram staining

  • Prepare a smear of bacteria culture and heat fix the smear.
  • Smear are first stained with Crystal Violet which is a basic dye it imparts purple color to all cells.
  • Washed the slide with distilled water for seconds.
  • Now, smear is treated with iodine (mordant), this permit the stain to retained by forming an insoluble CV-I complex (Crystal Violet-iodine complex).
  • Wash the slide with ethyl alcohol (95%) this is a differential step. At this step some bacteria retain the primary stain i.e. Crystal Violet and appears purple color while some bacteria not retain stain and loose purple color and appears colorless.
  • Bacteria that retain Crystal Violet and appears purple are classified as gram positive bacteria, while that not retain stain and loose the color after decolourisation are classified as Gram negative bacteria.

  • Wash the slide with distilled water and drain it. Now apply counter stain safranin (basic dye), and again wash the slide with distilled water and blot dry with absorbent.
  • Since Gram Positive Bacteria already retain purple color, they are not affected by counterstain but, Gram negative bacteria are colorless so, they directly stained by the safranin. Thus, Gram positive appear purple, and Gram negative appears red or pink.
  • The Gram Positive Bacteria cell walls are thick and chemically simple, in contrast Gram negative bacteria cell wall are thin and complex multi-layered structure.
  • When treated with alcohol gram-positive bacteria are not allowing the loss of CV-I complex and cells remains purple, while Gram negative bacteria have complex structure and have lipopolysaccharide layer which is absent in Gram Positive Bacteria due to which CV-I complex is leaked and resulting in decolorization which later take the counterstain and appears red.

Reference and Sources

  • 2% – https://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=1
  • 1% – https://www.thoughtco.com/gram-stain-procedure-4147683
  • 1% – https://softnotess.blogspot.com/2017/04/mic-210.html
  • 1% – https://www.scribd.com/doc/99585713/Staining-in-Microscopy-Stains-and-Techniques-From-Wikipedia
  • 1% – https://www.microscopemaster.com/gram-positive-and-gram-negative-bacteria.html
  • 2% – https://quizlet.com/156214542/microbiology-lab-exam-1-study-guide-flash-cards/
  • 2% – https://quizlet.com/27554887/spc-citron-micro-lab-practical-1-flash-cards/
  • 2% – https://en.wikipedia.org/wiki/Bacteria
  • 2% – https://www.slideshare.net/zohaibkhan404/microbiology-a-labotory-manual

Gram staining

12 thoughts on “Gram staining”

  1. Amazing!! The first type of staining that I saw in college! And I really liked that flow chart too!
    One doubt: Gram(-) bacteria loses external membrane during this staining and because of this it loses color?? Or the dye is capable to penetrate in the external membrane, but cell wall is too thin that don’t retain it??
    Thanks!!
    😊

    Reply
    • Yess ,gram negative Bacteria have thin peptidoglycan and also contain lipopolysaccharide .the higher amount of lipid is dissolved by alcohol ,resulting in formation of large pores in the cell wall through which CV-I complex is leaked .while in gram positive on treating with alchol it causes closures of cell walls pores and not allowing the loss of CV-I complex.

      Reply
  2. Yess ,gram negative Bacteria have thin peptidoglycan and also contain lipopolysaccharide .the higher amount of lipid is dissolved by alcohol ,resulting in formation of large pores in the cell wall through which CV-I complex is leaked .while in gram positive on treating with alchol it causes closures of cell walls pores and not allowing the loss of CV-I complex.

    Reply
  3. Excellent work ! Never ever got so clear cut idea about gram staining. The flowchart provides quick recap about the whole process & is certainly helpful for us.

    Reply
  4. Thank you so much sir! To provide such an excellent note ,,,, this is a really useful and very clear guideline for practically performance. Now I could clear my confusion. And got good results after that!

    Reply

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