Author: Kelsey Goldman, HTL (ASCP)

Last Update: 06 January 2024

Copyright: 2024,

Cite this Page! -> Goldman, Kelsey. “Gram.” HistologyOutlines.Com, 06 January 2024. Accessed (today) 

Special Stain

  • To determine the presence of gram positive or negative bacteria. The differentiation of these differnet bacterial subtypes can be very helpful in the clinical setting (Jones)
Control Tissues:
  • Control tissue is often from lungs or  placentae. The “spongy” nature of these tissues allows for bacteria to be cultured throughout the sample. With the aid of a microbiologist, swab and incubate both known gram positive and gram negative cultures onto the sample. Some labs obtain grocery bough chicken livers for the tissue to grow the bacteria on. Controls can be purchased. Purchased controls must demonstrate both Gram positive and Gram negative bacteria (Vaughan et al.).
Staining Pattern:
  • Gram Positive Bacteria as well as fibrin, some fungus, Paneth cell granulates, kerato-hyaline, and keratin- Blue
  • Gram negative and dead bacteria- Red
  • Nuclei- Red
  • Background – Yellow (Suvarna et al.)
Biochemistry Theory:
  • The crystal violet interacts with the iodine to form a complex.  This complex is made it is not easily washed out. This combination of crystal violet and iodine is called “fixing” the dye. The fixed dye that is not bound to cells walls of bacteria are washed out in the ethanol and acetone steps. The most important aspect of gram positive bacteria is that these bacteria retain the fixed dye even after washing.All bacteria take up the crystal violet fixed dye when first applied. The gram negative bacteria do not hold the crystal violet because the acetone solvent removed lipids from their cell walls and with this removal the crystal violet is also removed. The basic fushsin is added to give the gram negative bacteria a red color so that they can be identified. (Tripathi and Sapra)
  • Uneven decoloration is often caused by letting the slides dry out in early stages of staining. It is critical that slides are not allowed to dry out at any point in staining (Suvarna et al.).
Additional References:
  • Jones, Glida. The Gram Stain : A New Look at an Old Tool. Rev. March 1986, 1986.
  • Suvarna, S. Kim, et al., editors. Bancroft’s Theory and Practice of Histological Techniques. Eighth edition, Elsevier, 2019.
  • Tripathi, Nishant, and Amit Sapra. “Gram Staining.” StatPearls, StatPearls Publishing, 2023,
  • Vaughan, Kristine J., et al. “An Efficient, Inexpensive Method for Making Gram Stain Tissue Control Blocks.” Journal of Histotechnology, vol. 30, no. 1, 2007, pp. 11–13,
    Sample Protocol:
Modified Brown- Brown protocol for Gram staining in FFPE sections


Crystal violet

  • 10% alcoholic crystal violet 2mL

  • distilled water 18mL  

  • 1% Ammonium Oxalate 80 mL

Modified Gram Iodine

  • iodine 2 g

  • Potassium Iodine 4g

  • Distilled water 400 mL

Ethyl alcohol

  • Absolute Ethyl Alcohol 50 mL

  • Acetone 50 mL

0.5% Basic Fuchsin Stock

  • Basic fuchsin or pararosaniline 0.5g

  • Distilled Water  100mL

  • dissolve under heat and mix well

Working solution of Basic Fushsin

  • Stock Basic Fushsin 10 mL

  • Distilled water 40 mL

Picric Acid – Acetone Solution

  • picric Acid 0.1g ( Be advised of best practices when using picric acid the dry product is very explosive)

  • Acetone 100mL


  • Deparaffinize and rehydrate to distilled water

  • Filter the crystal violet working solution and stain slide for 1 minute

  • rinse well in distilled water

  • Place slide into iodine solution for 1 minute

  • Rinse the slide in distilled water

  • Blot the slide dry. Be careful not to touch or blot the tissue on the slide

  • Decolorize the slide by dipping into the alcohol- acetone solution. Dip until the blue dye stops running off of the slide. This will be very quick; you should only need to dip it once or twice. It may be helpful to quickly check under a microscope to make sure that not all of the staining has been removed. When checking a slide under a scope that is not yet cover-slipped, be carful not to dry out the sample under the bulb.  

  • Counterstain in the working basic fushsin solution for 1 minute.

  • Rinse in distilled water. Once again blot the slide, being careful to avoid touching or blotting the tissue section on the slide.

  • Place the slide in a single quick dip of acetone

  • Place the slide into picric acid acetone until the sections have a pink/ yellow color.

  • Dip into the a half and half mixture of acetone/ xylene. This may require several dips. Check again under the microscope to ensure the tissue still looks properly stained.

  • Clear in Xylene and coverslip. You should not dehydrate with alcohols as some of the staining on the slide is alcohol soluble and may wash out. There is no need to use alcohols to remove water prior to Xylene. The removal of the water is accomplished in the acetone and acetone xylene dips.


Modified from

Suvarna, S. Kim, et al., editors. Bancroft’s Theory and Practice of Histological Techniques. Eighth edition, Elsevier, 2019.