ENVE 301: Environmental Microbiology Laboratory

Differential Staining:

The Gram Stain

The only faith that wears well and holds its color in all weathers, is that which is woven of conviction and set with the sharp mordant of experience.

- My Study Windows. Abraham Lincoln - James Russell Lowell, 1864.

Supplemetal Reading/Web Sites

Pink and Purple Bugs:

The Gram Stain Technique

How to do Gram Stains


Simple staining of bacterial cells (See lab 2) does not provide much information to aid in the identification of bacterial cells. In addition, as early medical microbiologists discovered, all cells, both prokaryotic and eukaryotic will stain with the dyes used in simple staining. The Gram staining method, named after the Danish bacteriologist who originally devised it, Hans Christian Gram, is one of the most important staining techniques in microbiology. In 1883-4 Dr. Gram discovered a method for staining certain bacterial cells purple/blue while staining eukaryotic cells, and many other bacterial cells pink. Essentially, Dr. Gram found that bacteria fell into two distinct categories when stained sequentially with crystal violet followed in sequence by a bath in an iodine solution, a wash with a destaining agent and a counter-stain. One group of cells resisted the removal of the crystal violet when washed with ethanol or acetone (decolorizing agents), whereas the second group was readily decolorized by a brief rinse with these reagents. To visualize the decolorized cells Gram briefly exposed them to a counter-stain, or a stain of a different color from the crystal violet. Gram settled on the red counter stainingdye safranin. Thus cells which resisted decolorization remained deep purple or blue and came to be referred to a Gram positive cells, whereas cells that easily lost the crystal violet dye were red after counter staining. These red cells came to be called Gram negative cells.

Source: http://pubs.acs.org/hotartcl/cenear/960923/gram.html

This type of stain is referred to as a differential stain because one group of organisms reacts to the stain one way and another group of organisms reacts a different way. Gram and others drew several important conclusions from these staining results. First, they realized that differential staining of cells and cell components was possible. Secondly, they recognized that this staining was diagnostic and could be used to identify cells and substances. We now know that Gram positive bacterial cells are very different from Gram negative cells. These differences have proven useful in understanding the physiology of these two categories of bacteria. The gram characteristic is almost as fundamental to a bacterial description as its morphology.

The ability of the Gram stain to differentiate microorganisms into two broad categories is a result of differences in the cell walls of the organisms. The cell walls for gram-negative microorganisms have a higher lipid content than gram-positive cells. Originally, both kinds of cells are penetrated by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed too easily. This step is commonly referred to as fixing the dye. The actual mechanism of decolorization is currently not well understood and remains controversial. However, it is generally agreed that the subsequent treatment with the decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker gram-positive cell wall, closing the pores as the cell wall shrinks during dehydration. As a result, the the diffusion of the stain-iodine complex out from the cell is obstructed, and the cells remain stained.


Stain Step

Gram reagent

What Happens

Color of Gram Positive Cells

Color of Gram Negative Cells

Dyes Present in Cell


Crystal violet

Dye enters cell



Crystal violet



Crystal violet-iodine (CV-I) complex formed in cells





95% alcohol or acetone

Gram positive wall shrinks

Lipids removed from Gram negative walls



Gram positive CV-I

Gram negative: none



Dye enters cell

Purple (the pink of the counterstain is hidden)


Gram positive: all dyes

Gram negative: safranin

Performing a good gram stain is easy, but it does require some experience. For example, the original "bacterial smear" must not be too thick or too thin as this will influence penetration of the staining reagents.

Source: http://www-micro.msb.le.ac.uk/LabWork/gram/gram5.htm

Also the age of the culture is important. Very young and very old cells often produce poor results, whereas mid-log cells that are healthy and growing optimally, tend to give dependable results. The media the cells are grown in and the environmental conditions may also effect the outcome of a gram stain because these ultimately reflect on the chemical nature of the cell.


  1. Prepare a heat fixed smear of the culture you wish to examine
  2. Flood the smear with crystal violet (30 sec. to 2 min)
  3. Quickly and gently wash off excess stain (2 seconds)
  4. Flood the smear with Grams iodine (1 minute)
  5. Decolorize with alcohol (10-20 seconds or until the excess alcohol which flow off the slide is colorless)
  6. Quickly and gently wash off excess stain (2 seconds)
  7. Flood the smear with safranin (30 sec to 2 min.)
  8. Quickly and gently wash off excess stain (2 seconds)
  9. Blot dry with bibulous paper
  10. Examine your slide under the microscope. Record sketches of the organisms, size, color, morphology, and culture identification.

Gram stain and General Microscopy Troubleshooting


Probable Cause

Corrective Action

Slide is almost focused, but is hazy or unclear

Inadequate or no immersion oil

There should be enough oil to make a seal between the slide and the 100X objective

Dirty Objective

Contact instructor to clean the objective

Slide is clear under 10X but cannot be focused under oil

Slide is upside down

Check slide and flip over if necessary

Organisms appear to have a halo around them or cannot be focused

Oil was added before slide was dry

Make a new slide. Allow slide to dry longer or blot more carefully before adding oil

Slide is in focus, then a shadow appears

Air bubble trapped by lens

Rotate objective back and forth

Floating material is seen

Objective scraped the smear

Clean objective; gently remove oil from slide and refocus more carefully

Nothing is visible on the slide

Smear too thin

Remake with a thicker smear

Slide wiped to dry it

Blot slides; never wipe

Smear was not heat fixed

Repeat; heat fix the smear

Excessive deposits of stain crystals on slide

Inadequate rinsing between steps

Repeat; wash off one stain completely before adding the next

Stain was allowed to dry on slide before rinsing

Repeat; watch timing of staining steps more carefully

Gram positive organisms have odd angular shapes

Viewing crystal violet crystals

Repeat: Do not allow dye to dry on the slide. Do not allow crystal violet to remain on slide longer than suggested times

Gram negative organisms look more like needles than rods

Viewing safranin crystals

Specimen appears mixed with large oval cocci when it should be one organism

Yeast growing in the staining reagents

Repeat after the instructor has filtered the reagents

Gram positive cells appear gram negative


Decrease decolorizartion time

Non-viable organisms

Use a fresher culture

Failure to include mordanting step (iodine)

Be careful to follow each step in the proper order

Gram negative organism appears gram positive


Increase decolorization time

Forgot decolorization step

Be careful to follow each step in the proper order


Stain dried on smear

Mixture of gram positive and gram negative organisms from a pure culture

Smear is too thick

Remake smear

Culture is contaminated

Follow proper aseptic technique

Assignment: Upon completion of the laboratory you should be able to:

  1. State why the gram stain is said to be a differential stain
  2. Describe the differences between a gram-positive and a gram-negative cell wall
  3. State the mechanism of the Gram stain and why differential staining of bacterial cells occurs.
  4. Have accurate labeled sketches of all of the specimens you examined. Included with the sketches should be a brief description of the specimen, the magnification under which it was viewed, and an approximation of the size.
  5. Describe the conditions which may result in a gram-positive cell staining gram negative.
  6. State the procedure for the gram stain
  7. Perform a gram stain given all of the necessary materials and reagents
  8. Determine if a bacterium is gram-positive or gram-negative when microscopically viewing the gram stain preparation and state the shape and arrangement of the organism.

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Forward to Lab 4: Pure Culture and Isolation of Organisms