Gram Stain Protocol

Gram Stain Protocol

Hucker’s Modification

[modified from C.R.C. Manual of Clinical Lab. Proc., 2nd Ed., p.269 & 270 (1970).]


Both Gram-positive (Gm+) and Gram-negative (Gm) organisms form a complex of crystal violet and iodine within the bacterial cell during the Gram-staining procedure. Gm+ organisms are thought to resist decolorization by alcohol or acetone because cell wall permeability is markedly decreased when it is dehydrated by these solvents. Thus, the dye complex is entrapped within the cell, resist being washed out by the solvents, and Gm+ bacteria remain purple following this differential stain.

In contrast, cell wall permeability of Gm- organisms is increased by ethyl alcohol washing because it removes the outer membrane from the Gram-negative cell wall. This allows the removal of the crystal violet-iodine complex from within the cell. The decolorized Gm- cell can then be rendered visible with a suitable counterstain, in this case Safranin O, which stains them pink. Pink which adheres to the Gm+ bacteria is masked by the purple of the crystal violet.

DEMONSTRATION: Prepare and stain three smears on the same slide:

(Do not contaminate original cultures.)

position on slide:) specimen in the smear:                    reaction to Gram stain:
smear 1) thin smear of fresh yogurt only                                        Gm+
smear 2) thin smear of fresh yogurt and fresh E coli culture) both Gm + and Gm-
smear 3) thin smear of E coli culture only                                        Gm-



Prepare thinlyspread smears of fresh bacteria, see Bacterial Smear and Staining Protocol.)
Note that older or low viability cultures may not stain with accurate Gram stain characteristics.

Applying Hucker’s Stain

PRIMARY STAIN: Stain with Hucker’s Stain for 1 minute. (If over-staining results in improper decolorization of known Gram-negative organisms, use less crystal violet.)
Wash in tap water no longer than 2 seconds to remove liquid Hucker’s stain.

MORDANT: Flood the smear with Gram’s iodine. Allow to remain for 1 minute. (Note irridescence when the Gram’s Iodine is of sufficient strength.)

DECOLORIZE with 95% ethyl alcohol over a sink. Continue applying EtOH until the purple dye no longer flows from the smear. (Acetone may with caution be used as a decolorizing agent, since this solvent very rapidly decolorizes the smear.)

Wash in tap water for 2 seconds to remove the EtOH. (The following stain will not take if EtOH remains on the slide.)

Counterstain with Safranin O

COUNTERSTAIN: Flood the smears with safranin 0. Allow to stain for 1 minute.

Wash with tap water.

Blot dry gently between sheets of bibulous paper or lint-free paper towel (do not rub…).

Here is a page of gram stained specimens, including buttermilk, sour cream, sour dough starter, yogurt, yogurt and E. coli, and bacteria in a tongue scraping.
Examine under 1000x oil immersion lens. Illustrate morphologies and staining patterns observed.

Here are three examples:
1)  yogurt alone (Lactobacillus and Streptococcus),
2)  E. coli alone,
3) Mixture of #1 and #2.


1.Crystal violet (Hucker’s Stain
Solution A
crystal violet, certified 2.0 g
ethyl alcohol, 95% 20.0 mL

Solution B
ammonium oxalate 0.8 g
distilled water 80.0 mL

Mix solutions A and B. Store for 24 hours before use. The resulting stain is stable.

2. Gram’s iodine: Dissolve 0.33 g of iodine and 0.66 g of potassium iodide in 100 mL of distilled water Alternately, dilute 0.1 N iodine 1:4. (Gram’s Iodine solution should be fresh. If it has weakened and appears tan it will not work.)
3. Ethyl alcohol (95%)
4. Counterstain stock solution : Dissolve 2.5 g of certified safranin 0 in 100
5. Counterstain working solution:  Dilute stock solution 1:10 with dH2O.


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