GRAM STAINS REAGENTS

Technical Data #GRAMa / 98.07.22

The Gram Stain reagents are used to determine the Gram reaction for microorganisms identification. Crystal Violet stains bacterial cell. Iodine, the mordant, bind the stain. Alcohol-acetone solution, the decolorizer, differentiates bacteria by retaining or not crystal violet, wihin their cell wall. Bacteria will be stained by counterstain, when crystal violet is not retained by their cell wall. Basic Fuchsin replaces Safranin as counterstain for some fastidious microorganisms.


FORMULA
in grams per liter

Crystal Violet, Stain

 

Crystal Violet

20,0

Ammonium Oxalate

8,0

Ethanol, Denatured

200,0 ml

Water, deionized

800,0 ml

Iodine, Mordant

 

Iodine

3,3

Potassium Iodine

6,6

Water, deionized

1000,0 ml

Alcohol-Acetone, Decolorizer

 

Ethanol, denatured

500,0 ml

Acetone

500,0 ml

Safranin, Counterstain

 

Safranin O

2,5

Ethanol, denatured

100,0 ml

Water, deionized

900,0 ml

Basic Fuchsin, Counterstain

 

Basic Fuchsine

1,0

Ethanol, denatured

100,0 ml

Water, deionized

900,0 ml


PRECAUTIONS
This medium is for IN VITRO diagnostic use only.


STORAGE
Store in tightly-sealed containers at 15-30ºC in the dark.


SIGN OF DETERIORATION
Stains should not be used if they show signs of deterioration or if the expiration date has passed.


PROCEDURE

  1. Prepare the smears as described in previous standardized methods directly from specimens or from an overnight growth.
  2. Fix smears by passing through a Bunsen burner flame 2-3 times.
  3. Flood slides with Crystal Violet and stain for 60 seconds. Crystal violet may precipitate, before use filter through a filter paper no. 1.
  4. Rinse slides with tap water.
  5. Flood with Gram Iodine and let stand for 60 seconds.
  6. Rinse thouroughly with tap water and decolorize with Alcohol-Acetone until violet color flow disappears.
  7. Rinse with tap water and add counterstain with Safranin or Basic fuchsine for 10 seconds.
  8. Rinse with tap water. Blot dry and examine microscopically.

QUALITY CONTROL

Organisms

ATCC

Color

Morphology

Escherichia coli

25922

red

bacilli

Streptocococcus pyogenes

19615

blue

cocci

LIMITATIONS OF METHOD
A special care should be taken for proper decolorization to obtain the real Gram stain reaction. This is a part of identification. Other methods may be required.


REFERENCES

  1. Finegold, S.M., E.J. Baron 1986 Bailey and Scott's Diagnostic Microbiology 7th ed. C.V. Mosby St. Louis
  2. Lennette, E.H., A. Balows, W.J. Hausler Jr., H.J. Shadomy 1985 Manual of Clinical Microbiology 4th ed. American Society for Microbiology, Washington, D.C.
  3. Lillie, R.D. 1977 H.J. Conn's Biological Stains 9th ed. Williams and Wilkins, Baltimore


CATALOGUE NUMBERS

Crystal Violet 100 g QB-65-1004    
Iodine 100 g QB-65-1007    
Potassium Iodine 500 g QB-39-3708    
Safranin O 100 g QB-65-1016    
         
Prepared solutions

500 ml

1 Liter

4 Liters

6x250 ml

Crystal Violet

8524

8504

8514

8534

Gentian Violet 1% anilin

8570

8469

8468

 

Iodine

8523

8503

8513

8533

Decolorizer

8525

8505

8515

8535

Safranine

8526

8506

8516

8536

Basic fuchsine

8528

8508

8518

 


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