5 Ways to Get Students Energized about Gram Stains

Grace Chukwuekwu

Bacteria are a broad group of protozoa. They can live in various places on the body and the skin. While some types of bacteria are harmless or even beneficial, others can cause infection and disease. Gram stain helps diagnose harmful bacteria.

Unstained bacteria are too transparent to be seen directly. Bacteria can be easily identified by light microscopy using appropriate staining methods. Various stains are used to visualize and differentiate microorganisms such as the gram staining technique.

The gram stain is a  common laboratory test that can quickly diagnose the presence of a bacterial infection. Gram staining is the definitive method for distinguishing two main groups based on their different cell wall components. The Gram stain procedure distinguishes gram-positive and gram-negative groups staining these cells red or purple.

Read on for some reasons why this can be a daunting topic for teachers and students,  five suggestions for changing it, and thoughts on why virtual labs can make things easier.

Why gram staining can be tricky

There are three reasons in particular why the gram stain technique can be difficult, even for the most diligent of students.

1. It feels abstract

The bacterial cytoplasm is colorless, which makes it tough to detect during diagnosis, mainly in the tissues of people infected with the pathogen. Therefore, stains are used to give them color. The Gram stain is a technique used to detect bacteria from the suspected site of infection, such as the throat, lungs, genitals, or skin sores. Gram stain can also be used to look for bacteria in certain body fluids, such as blood or urine.

2. It’s content-heavy

Gram stain works by distinguishing bacteria based on the chemical and physical properties of their cell walls.

Gram-positive bacteria have cell walls that contain a thick layer of peptidoglycan, the substance that makes up the cell walls of many bacteria. Peptidoglycan makes up about 90% of the cell wall in gram-positive bacteria. As a result, they appear blue to purple on Gram stains. Gram-positive organisms include:

  • Streptococcus species.

  • Staphylococcus species.

  • Clostridium species.

  • Listeria species.

  • Corynebacterium species.

  • Gram-negative bacteria have cell walls with a thin layer of peptidoglycan (10% of the cell wall) and high levels of lipids (fatty acids). As a result, they appear red to pink on Gram stains. Gram-negative organisms include:

  • Neisseria meningitides.

  • Neisseria gonorrheae.

  • Pseudomonas species.

  • Escherichia coli (E. coli)

  • Moraxella species.

  • Klebsiella species.

  • Proteus species.

3. It’s complicated

In the Gram method, One group of bacteria retained the crystal violet-iodine complex even when rinsed with solvent, and another group of bacteria lost the dye on rinsing. The Gram stain process involves four main steps, including:

  • Primary color application (crystal violet).

  • Add a mordant (Gram's iodine).

  • Quick decolorization with acetone, ethanol or a mixture of both.

  • Counterstain with safranin.

  • Gram stain examination.

It depends on the different abilities of the ethanol or ethanol-acetone mixture to extract the crystalline iodine-violet complex from the bacterial cells. This complex is easily extracted from a group of bacteria called Gram-negative, which can then be stained red with an appropriate counter stain while the Gram-positive, is colored purple by the retained crystal violet because it rejects decolorization. The Gram-positive or negative response of the cell reflects which of the two types of walls it has.

Color: In general, gram-positive bacteria appear purple to blue, and gram-negative bacteria appear pink to red.

Shape: The most common shapes are spherical (cocci) or rod-shaped (bacilli). Cocci appear singly, in pairs, in groups of four, in groups, or in chains. Basil that is thick, thin, short, long, or has an enlarged spore at one end.

5 ways to make Gram staining procedure a more approachable topic

With those points in mind, here are five things you can incorporate into the Gram staining process to make it more interesting, accessible, and fun to teach for you and your students.

1. Show the people behind the science

Gram staining is named after the Danish physician and bacteriologist Hans Christian Joachim Gram, born September 13, 1853, in Copenhagen, who in 1883 invented the technique by which all known bacteria can be divided into two categories. He then refined the method and published it in 1884. Christian Gram developed a staining technique that is still used to identify and classify different bacterial species. In the laboratory of the German microbiologist Karl Friedländer, Gram observed that staining a smear of pneumonia bacteria with crystal violet, followed by iodine and organic solvents showed differences in different bacteria cells. "Gram-negative" bacteria have thin cell walls that allow the solvent to partially remove the stain. "Gram-positive" bacteria appear purple under a microscope because their cell walls are thicker, so the primary stain cannot be removed. Currently, the Gram classification is the basis for the identification, classification, taxonomy, and clinical use (drug therapy) of bacteria in medicine. Gram was only 31 years old when he made this accidental discovery.

2. Relate it to the real world

Gram staining is almost always the first step in tentatively identifying a bacterial organism. Gram stain is most often used to determine if a bacterial infection is present. If so, the test will show whether the infection is gram-positive or gram-negative.

It is also useful in deciding how to treat an infection, as some antibiotics are only effective against gram-positive bacteria and others against gram-negative bacteria.

Knowing whether the bacteria is gram-positive or gram-negative can help your doctor identify the type of infection you have and which antibiotics are most effective for treating it.

You may need this technique if you have symptoms of an infection suspected to be bacterial. Fever, pain, and fatigue are common symptoms of many bacterial infections. Other symptoms depend on the type of infection you have and in which part of the body it is.

Your results also contain information about the shape of the bacteria in your sample either spherical ( known as cocci) or rod-shaped ( known as bacilli). The form may contain more information about the nature of your infection.

3. Seeing is believing

Gram staining distinguishes bacteria based on the chemical and physical properties of their cell walls. Gram-positive cells have a thick layer of peptidoglycan in the cell wall which retains the primary color, crystal violet. Gram-negative cells have a thinner layer of peptidoglycan which allows crystal violet to wash away in addition to ethanol. They are colored pink or red by a counterstain,  usually safranine or fuchsine. Lugol's iodine solution is always added after the addition of crystal violet to strengthen the binding of the dye to the cell membrane. Examine the smear slide with an oil immersion objective to look for bacteria. 


. Gram-positive bacteria .Purple 

. Gram-negative bacterium .Pink

Gram stain GIF

Interactive GIF from Labster's Gram Stain: How stains and counterstains work.

4. Make it stick with word-play

The Gram stain process involves four main steps, including:

  • Primary color application (crystal violet).

  • Add a mordant (Gram's iodine).

  • Quick decolorization with acetone, ethanol or a mixture of both.

  • Counterstain with safranin.

Let's pick the initials of each step which are

  • Primary Stain - P

  • Mordant - M

  • Decoloriser - D

  • Counterstain - C

PMDC (Prime Minister Direct Circuit or Friends)

5. Use virtual lab simulations

A unique way to teach gram stains is through a virtual laboratory simulation. At Labster, we’re dedicated to delivering fully interactive advanced laboratory simulations that utilize gamification elements like storytelling and scoring systems, inside an immersive and engaging 3D universe.

Gram Stain GIF 2

GIF from Labster's Building Gram Positive and Gram Negative Cell Walls Virtual Lab.

Check out the Labster Gram Stain simulations that allow students to learn about Gram Stains through active, inquiry-based learning: Gram Stain: How stains and counterstains work, The Gram Stain: Identify and differentiate bacteria, and Using the Gram Stain to Help Diagnose Meningitis.

Learn more about Gram Stains here or get in touch to find out how you can start using virtual labs with your students.

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