Gram Stain Lab Prokaryotic Cell Wall Differentiation


The Gram stain is a very useful stain for identifying and classifying bacteria. The Gram stain is a differential stain that allows you to classify bacteria as either gram positive or gram negative. As a reference figure 27.5 from Campbell can be found at the bottom of page 3 of this lab. The staining technique consists of the following steps:

1. Apply primary stain (crystal violet). All bacteria are stained purple by this basic dye.

2. Apply mordant (Gram's iodine). The iodine combines with the crystal violet in the cell wall to form a crystal

violet-iodine complex called the CV-I.

3. Apply decolorizing agent (95% ethanol or acetone alcohol). The primary stain is washed out (decolorized) of

some of the bacteria, while others are unaffected.

4. Apply secondary stain or counterstain (safranin). This basic dye stains the decolorized bacteria pink.

The most important determining factor in the procedure is that bacteria differ in their rate of decolorization. Those that decolorize easily are referred to as gram-negative and appear pink, whereas those that retain the primary stain are called gram-positive and appear dark blue/purple to almost black.

Bacteria stain differently because of chemical and physical differences in their cell walls. Gram-positive cells consist of many layers of peptidoglycan. The CV-I molecular complex is larger than the crystal violet or iodine molecules that initially entered the cell wall and cannot pass through the thick peptidoglycan. In gram-negative cells, the alcohol dissolves the outer lipopolysaccharide layer, and the CV-I complex washes out through the thin layer of peptidoglycan.  See Fig 8

Examination of Gram-stained organisms usually provides a starting point for classifying, identifying, and characterizing bacteria. Such information helps to determine the source of microbes isolated as contaminants, for example, in the industrial production of foods and pharmaceuticals. The presence of gram-positive cocci indicates that shedding of normal human flora is the likely source of contamination; the presence of gram-positive, endospore-producing bacilli on the other hand suggests environmental sources. A gram stain can steer decisions on how best to remove or destroy the contaminant and how to prevent future contamination by the intruding microbe.

This differential staining technique is also a fundamental step in the diagnosis and treatment of disease. The Gram stain of clinical material taken directly from an infected patient can rapidly provide valuable information about the microorganism(s) causing the disease. This information helps guide the selection of subsequent tests needed to identify the bacteria. For example, identifying gram-positive cocci or gram-positive bacilli requires a different battery of tests than are used for the identification of gram-negative bacilli.

Gram stains of clinical specimens are especially important in determining the most effective antibiotic for critically ill patients who require immediate therapy. Penicillin, for example, is more effective against most gram-positive bacteria than against most gram-negative bacteria


  1. Collect specimen, and smear thin layer onto a new slide.
  2. Prepare a heat-fixed smear of the bacteria to be stained. (Use all fire safety precautions during this step) 
  3. Cover the smear with the Fix for an exposure time of 60 seconds.
  4. Rinse the smear with distilled water.
  5. Cover the smear with Eosinate for an exposure time of 60 seconds.
  6. Rinse the smear with distilled water.
  7. Cover the smear with Polychrome for an exposure time of 60 seconds.
  8. Immediately rinse the smear with distilled water.
  9. Gently blot the slide dry with bibulous paper.
  10. Observe the smear at 1000X (using immersion oil), and identify the bacteria as either gram-positive or gram-negative and identify the shape. Click here for a review on how to use a microscope.

* Some common sources for error:

1. The smear was too thick or too thin..

2. Excessive heat, or not enough heat, was applied during heat fixing.

3. Decolorizing agent was left on the smear too long.



Photo Insert

Identified as

Gram - or Gram +

Identify the shape

Follow-up Questions- Please complete in complete sentences. Use primary evidence from your lab results to support claims and reasoning.

  1. Why do you heat fix a slide? (List two reasons.)

  1. What position should the stage on the microscope be in when starting to focus your slide? What power lens did you see your specimen clearest?

  1. What is the physiological structure difference between a Gram+ bacteria and a Gram - bacteria?

  1. Why is it important to differentiate between Gram + and Gram - bacteria? How does identifying and classifying bacteria prevent antibiotic resistance?

  1. What is the connection of this lab to our core unit, Power Struggle in Latin America?