Principle

"Diff-Quick" is a proprietory brand of a Romanowski stain. The Romanowski group of stains are defined as being the black precipitate formed from the addition of aqueous solutions of methylene blue and eosin, dissolved in methanol.  The variants of the Romanowski group differ in the degree of oxidation (polychroming) of the methylene blue stain prior to the precipitation.

The stain class was originally designed to incorporate cytoplasmic (pink) staining with nuclear (blue) staining and fixation as a single step for smears and thin films of tissue (spread preparations of omentum).  Minor modifications of working stain concentration and staining time have been made over the years for fixed tissue sections.

Technical Points

(step 5) - exposure to alcohol should be as brief as possible to prevent excessive decolourisation.

Method

Paraffin Sections

1.      Bring sections to distilled water

2.      Stain with “Diff Quick” solution II .30 secs

3.      Counterstain (optional) with “Diff Quick” solution I for 30 seconds

4.      Rinse rapidly in tap water

5.      Rapidly dehydrate in absolute alcohol

6.      Clear and mount

Smears/Imprints

1.      Air-dry the smear

2.      Fix in “Diff Quick” Fixative (or methanol) for 30 secs/drain

3.      Stain with “Diff Quick” solution II for 30 secs/drain

4.      Counterstain (optional) with “Diff Quick” solution I for 30 secs/drain

5.      Rinse in tap water to remove excess stain

6.      Rapidly dehydrate in absolute alcohol

7.      Clear and mount

Results

helicobacter........................................dark blue

background.........................................light blue

platelets............................................violet to purple

neutrophils   nucleus……….........……………dark blue

                   cytoplasm....……………………..pale pink

eosinophils   nucleus........…………………….blue

                   cytoplasm.....…………………….blue

                   granules........................red to red/orange

basophils      nucleus.........………………….purple or dark blue

                   granules........…………… ……dark purple/black

monocytes    nucleus(lobated)………… ……violet

                   cytoplasm........……...……..sky blue                          

Reagent Formulae

1.   “Diff Quick” solution I/II or kits can be purchased from IHC Store

      Store Diff-Quik at 5 C to retard growth of organisms.

References

Skipper R and DeStephano D,(1989),J.Histotechnol.,12(4),303

Diff-Quik stain set*64851