Gram staining procedure

You Will Need:

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

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

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

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. 

STAINING PROCEDURE

• 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

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

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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