Gram Stain Microbio Test

Steps

1. Prepare slide via heat fixing

2. Flood slide with crystal violet (simple stain), both gram + and - will be purple

3. wash with water

4. add gram iodine, acts as mordant, intensify purple color, solidified crystal violet in thick peptidoglycan

5. wash with water

6. add decolorizer (alcohol, ethanol)

   -add drop by drop until corner of slide almost runs clear
   -gram + remains purple, gram - clear

7. wash with water

8. counter stain with safranin
   -gram + remain purple, gram - pink

9. wash with water

10. blot dry with bulboneous paper

Forgot Iodine?

gram +: turns pink, clear originally, clear with decolorizer, turns pink

gram -: stays pink, still undergoes decolorizer

Primary stain

gives color to cells

crystal violet

mordant

intensifies color of the primary stain

killing against, fixes primary dye to cell wall

decolorizing agent

may remove the primary stain from the entire cell or only from certain cell structures

outer layer on gram - is easily removed bc of thin peptidoglycan, therefore can be decolorized

most important step, over application might even take layer off of gram +

Counterstain

has a contrasting color to that of the primary stain