6: Gram Stains

Learning Outcomes

By the end of this lab period, you will be able to

1. Define “differential” stains and explain how this general concept is different from a “simple” stain.
2. Explain the purpose of both a “primary” stain and a “counterstain”.
3. Troubleshoot Gram-stains that don’t work properly.

Introduction

We use many different types of staining techniques in microbiology that allow us to visualize these tiny organisms, and also to differentiate between some of the major groups. One of the most common ways to differentiate between two major groups of bacteria is using the Gram stain. The Gram staining technique allows you to quickly categorize bacteria as having either a Gram-positive or a Gram-negative cell wall. You can review the features of both Gram-positive and Gram-negative cell walls in our OpenStax Microbiology textbook in Chapter 3.3, Unique Characteristics of Prokaryotic Cells.

Differential Stains

As we learned about in an earlier lab, a simple stain involves adding one dye to a bacterial smear in order to visualize them. The Gram-stain, however, requires additional steps and results in Gram-positive cells staining a deep purple, while Gram-negative cells stain a lighter red or pink color. Therefore the Gram stain is a “differential stain” because it “differentiates” between Gram-positive and Gram-negative bacteria.

In the gram stain, two dyes are used, crystal violet and safranin. Crystal violet is first applied to the bacterial smear, staining all cells. This first step, if no other steps were completed, would be a simple stain! However, in the Gram stain, the stained smear is then treated with Gram’s iodine. The iodine crosslinks the crystal violet molecules, effectively trapping them within the thick peptidoglycan later of a Gram-positive cell wall. However, the thinner peptidoglycan layer in a Gram-negative cell is not as effective at trapping the dye, and a “decolorizing” step with ethyl alcohol strips the outer Gram-negative cell wall membrane, effectively releasing the crystal violet from those cells. At this stage, Gram-positive cells are deep purple, but Gram-negative cells will be transparent. In order to visualize them, a “counterstain”, safranin is added. Safranin is a much less saturated dye, and it won’t change the deep purple color of a Gram-positive cell, but it will stain the Gram-negative cells so that we can see them. At the end of a Gram-stain, all the Gram-positive cells will be deep purple and the Gram-negative cells will be a light red or pink.

Gram Stain Process

Let's review the 4 main steps in performing a Gram stain (Table 1). Note that after each step, the bacterial smear is rinsed with distilled water in order to remove excess stain or reagent.

Table 1: Gram Staining Process

Number	Process	Effect	Result for G+ cell	Result for G- cell
1.	Stain the smear with Crystal violet	All cells will be stained a deep purple.
2	Immerse stained smear in Gram’s Iodine (mordant)	Iodine crosslinks the crystal violet trapping it inside G+ cells
3	Decolorize with 95% ethyl alcohol	Alcohol strips the outer membrane from G- cells, and the crystal violet is released
4	Counterstain with safranin	Safranin is needed to stain the now-transparent G- cells.

Typical Gram Stain Results (Figure \(\PageIndex{1}\))

Figure \(\PageIndex{1}\): Gram Staining. Left - S. aureus, a Gram-positive coccus. Middle - E. coli, a Gram-negative rod (Dr Graham Beards, CC BY-SA 4.0, via Wikimedia Commons). Right - B. subtilis, a Gram-positive rod exhibiting Gram stain variability

Materials

Per Student

• Compound microscope with oil-immersion lens
• Clean glass microscope slides
• Gram-stain solutions
• Gram crystal violet
• Gram iodine
• 95% ethanol
• Gram safranin
• Squirt bottle with water
• Bibulous paper
• Disposable gloves
• Clothespins as slide holder

Per Student Group

Bacterial culture (these will be your sub-cultures from the Aseptic Technique Lab)

• Staphylococcus aureus
• E. coli
• Bacillus subtilis
• Serratia marcescens
• Chromobacterium violaceum
• Proteus mirabilis

Experiment

1. Using your subcultures from the Aseptic Technique Lab, prepare and heat-fix smears of Gram-positive and Gram-negative bacteria onto a single microscope slide.

Figure \(\PageIndex{2}\): Example of how to organize specimens on the microscope slide prior to staining

2. Because Gram stains require practice, prepare several sets of slides and let them air-dry simultaneously. Then they will be ready when you need them.
3. Using clothespins as slide holders, immerse the slide in the crystal violet staining solution. Stain for one minute.
4. Grasp the slide with the clothespin and hold it at an angle. Gently rinse the slide with distilled water. Aim the water bottle above the smears, let water run down the smears, and rinse. Squirting water directly on the smears will risk losing the bacteria on the slide.
5. Immerse the slide in Gram iodine and stain for one minute.
6. Grasp the slide with the clothespin and hold it at an angle. Gently rinse the slide with distilled water.
7. Immerse the slide in 95% alcohol, and decolorize for 20 seconds. It can help to gently agitate the slide during this step. It is critical that you do not leave the slide in the alcohol for longer than 20 seconds. This can lead to over-decolorization, and the Gram-positive cells appearing to be Gram-negative.
8. Grasp the slide with the clothespin and hold it at an angle. Gently rinse the slide with distilled water.
9. Counterstain the smears by immersing in safranin for one minute.
10. Grasp the slide with the clothespin and hold it at an angle. Gently rinse the slide with distilled water.
11. Gently blot dry with bibulous paper. Do not rub. When dry, observe under oil immersion.
12. Evaluate the quality of the stain result.

Troubleshooting

It’s not trivial to produce a gorgeous Gram stain, but it’s very satisfying when you do! Here are a few tips to help you improve your Gram stains this lab period. Remember, f your gram-positive organism appears purple and your gram-negative organism appears pink, your technique is fine.

Table 2: Troubleshooting the gram stain process

Problem	Solution
The gram-positive organism appears uniformly pink or red	You have over-decolorized your smear. Try again, reducing the decolorization time.
The gram-negative organism appears uniformly purple	You have under-decolorized your smear. Try again, increasing the decolorization time.
A pure culture appears to have both purple and pink/red cells in the same smear	Sections of your smear was too thick. This can trap the crystal violet preventing decolorization from working properly. Prepare another smear before staining, and be sure to make it very, very thin. You want a single layer of cells across much of the smear.
Bacillus subtilis or Bacillus cereus, which are gram-positive, appears pink instead of purple	These species are highly gram-variable. This means that they only reliably retain the crystal violet when the culture is fresh, typically 24 hours old or less. Since most of our lab periods are 48 or more hours apart, we will find that in our hands, these organisms often appear mixed, and sometimes appear mostly gram-negative.

Data

Record your staining results. Sketch the cellular morphology and arrangement of each bacteria. If you prefer, you may capture images using your cell phone. If drawing, use colored pencils to illustrate your staining results, as well as the overall morphology of each organism. Include the total magnification for each sketch. Note: This should be 1000x…it’s not possible to really see these organisms well without using the oil immersion lens. But it’s good practice to record total magnification regardless.