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Gram staining procedure

Published on Nov 18, 2015

Learning how to perform a Gram Stain will be the starting step in determining where to start when identifying bacteria for illnesses, disease or infection.


Gram staining procedure

You Will Need:

  • A culture or specimen
  • Gram staining reagents
  • Microscope slides
  • Heat Source (Incinerator)
  • Sterile Swab and Microscope
Photo by Niels_Olson

Label your slide with a Wax Pencil

  • specimen number
  • your name or initials
  • your class period
  • today's date

Using a culture plate:

  • Choose an isolated colony 
  • Lightly touch the colony with the tip of a sterile swab
  • Make sure the swab only touches that colony
  • Do not put the inoculated swab down
Photo by akseabird

place the colony on the slide

  • While still holding the inoculated swab, pick up your slide
  • Gently touch the swab onto the slide
  • Gently spread the bacteria on the slide use a circular motion
Photo by carolyntiry

Heat fix the slide

  • Heatfixing the microorganisms to the slide using the incinerator:
  • Hold the slide with a wooden clothespin so you do not get burned
  • Place underneath side of slide to heat source (bacteria = facing you)
  • Hold the slide against the front circled opening
  • Heat for 1 minute.  This is adhering the bacteria to the slide. 


  • Place slide on staining rack/tray
  • Gently flood the slide with the Crystal Violet 
  • It will cover the slide like this picture 
  • Let sit for 1 minute
  • Gently rinse slide with water 

staining procedure

  • Gently flood the slide with Gram's Iodine
  • Again - the slide will be covered.
  • Let sit for 1 minute
  • Rinse slide with water

staining procedure

  • Gently flood the slide with Gram's Decolorizer
  • Let sit for 30 seconds
  • Rinse with water
  • If you wait too long to rinse, the slide can be over decolorized
Photo by M i x y

staining procedure

  • Gently flood the slide with Safranin Counterstain
  • Let sit for 1 minute
  • Gently rinse with water
  • Now it is time to dry the slide........

Drying and Reading the slide

  • Gently blot the slide with Bibulous Paper 
  • Position slide on microscope stage
  • Proceed with low power first 
  • Locate the bacteria using coarse adjustment
  • Move to dry high power (40X), focus using fine adjustment 

Reading the slide

  • Once focused
  • Move to oil immersion (100X)
  • Apply 1 drop of immersion oil to the slide
  • Focus bacteria into view using fine adjustment
  • Record your results: Gram Positive or Gram Negative
Photo by Key Foster

Recording Results ...Remember That:

  • Gram Positive will appear purple
  • Gram Negative will appear red/pink
  • Coccus = round bacteria
  • Bacillus = rod shape bacteria

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