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
REAGENT |
PURPOSE |
MECHANISM OF STAINING |
SOURCE OF ERROR |
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 |
Decolorizer 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 (optional) |
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
Appendix
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.
References
Officer B. HIML 251 Lecture notes: Microorganism stains: Gram and Gram Twort, February 4, 2009