Gram Stain / Gram Twort

Classification: microorganism stain  

Mechanism of staining: ionic bonding and differential staining   

Purpose: stain Gram negative(red) and positive(purple) organisms   

Control tissue: appendix and tissues known to contain microorganisms  

Gram Twort

Gram positive bacteria in upper third of esophagus

Crystal violet precipitations

Gram Twort

Fungi stain Gram postive in the lung

Dark circles are RBC’s due to hemorrhage 

Gram Stain

Gram positive and negative microorganism in the appendix






Crystal Violet

Primary stain (regressive)

Suppresses sulphydryl and tryrosine groups and exposes aldehyde groups.

Omitted: No demonstration of Gram positive organisms.

Too short: Some Gram positive organisms may be missed.

Too long: No effect.

Lugol’s iodine

Trapping agent

Bonds with Crystal Violet

Omitted: No CV-I complex and no demonstration of Gram positive organisms.

Too short: Bond for CV-I complex might be weak.

Too long: No effect.

Acetone Alcohol



Alcohol concentration: 50-70% aq. Alcohol is desirable to decolorize cells more rapidly than a higher concentration.


Lipids on the outside of Gram negative organisms are lost due to larger pores that allow CV-I to be removed.

Peptidoglycans present in Gram positive organisms are dehydrated and pore size is reduced so CV-I complex is retained.

Omitted: Primary stain obscure tissue.

Too short: Precipitation as if not added.

Too long: Over decolorize tissue and Gram positive organisms not demonstrated.

Neutral Red or Safranin

Counterstain (progressive)


Ion bonding



Omitted: No demonstration of Gram negative organisms.

Too short: If few Gram negative organisms are present, they may be missed.

Too long: No effect.

Twort solution


Counterstain for cytoplasm and collagen

(dH2O, 0.2% NR in 100% alcohol, 0.2% Fast Green FCF in 100% alcohol)

Acid solution that stains negatively charged tissue components

Omitted: No counterstain of tissues.

Too short: Light counterstain, more difficult to visualization of Gram negative organisms.

Too long: Dark counterstain, could obscure some microorganisms.




Gram Twort

No Gram positive or Gram negative microorganisms seen in this appendix. 

Possible sources of error include: no counterstain added, over decolourized, or organisms washed away.

Gram Twort


red- Gram negative microorganisms

purple - Gram positive microorganisms

Gram Twort

Gram negative bacteria in an appendix

Gram Twort

Gram positive bacteria in an appendix

Gram Stain

Appendix: without counterstain

Special Considerations

Any formalin pigments should be removed but mercury pigments will be removed during the iodine step.

A final rinse of acetic acid can be added to remove excess Twort solution.


Officer B. HIML 251 Lecture notes: Microorganism stains: Gram and Gram Twort, February 4, 2009