Exercise 6: Gram Stain
Introduction
Differential staining procedures allow a microbiologist to detect differences between organisms in addition to determining morphology (shape and arrangement). A differential stain is a procedure that includes dyes with contrasting colors. Bacterial cells will retain one or the other dye at the end of the procedure based on differences in their cell structure. A trained eye will be able to recognize the difference in color with a light microscope under 1000X (oil immersion) power.
The Gram stain is the most common and useful differential stain. This Gram staining procedure consists of the following steps in order: a primary stain, a mordant, a decolorizer and a counterstain. Crystal violet stain is the primary stain and is a dark purple color. Adding the mordant (iodine) helps the crystal violet adhere to the cell structures. Decolorization occurs after the mordant is added and is the most critical step in the procedure. Ethanol is the decolorizer used to extract the primary dye from the smear. It must be added for a brief period of time only (usually 10 – 15 seconds). During this time some cell types (Gram-negatives) will lose the crystal violet while other cell types (Gram-positives) retain it. The final step is the addition of the counterstain, safranin. The Gram-negative cells that have lost their purple color will be colorized by the red counterstain. The Gram-positive cells retain their purple color and are unchanged by the addition of safranin.
Differences in Gram-positive and Gram-negative wall composition result in the different responses to decolorization. Keep in mind that all cells can be stained with the crystal violet and safranin as simple stains. So, it is not the stains themselves that make this a differential stain. It is the staining process (steps performed in the correct sequence) that determines the outcome. The more consistently you can perform these steps, the more reliable your results will be. Unfortunately, some Gram-positive organisms (especially Bacillus species) break down quickly leading to a Gram-variable result. Variable stains will have a mixture of purple and red cells with identical morphology. Using a young culture (12-18 hours) can usually resolve the issue, so Gram stains with variable results should always be repeated.
In addition to the Gram stain results, students should also note the cellular morphology (shape and arrangement) of the specimens. Each bacterial species that you observe will have a characteristic cellular morphology. Cells may be spheres (cocci, singular coccus), rods (bacilli, singular bacillus), or spiral shaped (spirilla). Additional varieties of these shapes include slightly curved rods (vibrios), short rods (coccobacilli), and flexible spirals (spirochetes). The cell arrangement describes how the cells look in small groups and is determined by planes in which division occurs and whether the cells stay attached after division (see figure 6-1). Common arrangements include strepto– (chains), staphylo– (grape-like clusters), and tetrads (packs of 4).
Course Intended Outcomes
Perform and understand the basis of different types of microbiological staining techniques (e.g., simple stain and gram stain) to characterize and identify bacteria.
Materials Used
Students will work in small groups. Each group will need the following materials for this exercise.
- Bunsen burner
- Inoculation loop
- Glass slides, cleaned
- Test tube rack
Additional materials and cultures will be assigned by your instructor
Procedure
Technique:
- Begin with a freshly prepared smear that has been air dried and heat fixed.
- Place the slide on the slide rack over the sink so that drippings go directly into the sink and not onto the bench top.
- Add crystal violet (primary stain) to the slide and allow to sit for 1 minute. Make sure that you add enough stain to cover the entire smear. Otherwise it will lead to poor results.
- Rinse the slide with DI water.
- Add Gram’s Iodine (mordant) and let sit for 1 minute.
- Rinse with DI water.
- Add ethanol (decolorizer) holding slide at an angle until runoff is clear, 10 – 15 seconds max.
- Rinse with DI water.
- Add safranin to the slide and let sit for 1 minute.
- Rinse with DI water.
- Blot excess stain and water with bibulous paper. Blot gently to avoid wiping off your specimen.
- View with microscope.
Sampling/Inoculation:
- Your instructor will assign the cultures to be used for inoculation by your group.
- Collect the required materials for the lab.
- Label your slides using a wax pencil with the organism initials (e.g., EC = Escherichia coli). This will prevent mixing up your slides and help with slide orientation.
Lab Clean-up
Wash slides with detergent, rinse and dry. Return slides to your slide box and store in the bin labeled for your section.
Return bacteria cultures to the front bench.
Plates are disposable and should be thrown away in the biohazard container.
Disinfect your benchtop and wash your hands before leaving the lab.
Results and Interpretation
Record your results in your lab notebook creating a table as shown.
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Specimen Name / Source |
Color |
Gram +/- |
Shape |
Arrangement |
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Critical Thinking Questions
You have attempted a Gram stain 3 times on an organism and have gotten a variable result (mixture of positive and negative) each time. What should you do to determine if it is the organism or your technique?
What would be the result of a Gram stain if you forgot to add the iodine?
What would be the result of a Gram stain if you did not decolorize long enough?
The image is a table titled “Common Prokaryotic Cell Arrangements.” It has three columns labeled Name, Description, and Illustration. Each row presents a bacterial cell shape or arrangement using pale yellow-orange drawings.
The first row shows coccus, with the plural cocci, described as a single spherical cell. The next row shows diplococcus, with the plural diplococci, illustrated as a pair of two spherical cells. Tetrad, with the plural tetrads, is shown as four spherical cells arranged in a square.
Streptococcus, with the plural streptococci, is illustrated as a curved chain of spherical cells. Staphylococcus, with the plural staphylococci, appears as an irregular, grape-like cluster of spherical cells.
The final two rows show rod-shaped bacteria. Bacillus, with the plural bacilli, is represented by a single rod-shaped cell. Streptobacillus, with the plural streptobacilli, is illustrated as a chain of several connected rod-shaped cells.
The image illustrates the five main steps of the Gram-staining procedure using microscope slides arranged from left to right across the top and then continuing along the bottom.
In the first step, two purple drops are shown above a slide containing a purple bacterial smear. The caption reads, “Add Crystal Violet, 60s, rinse,” indicating that crystal violet is applied for 60 seconds and then rinsed away.
In the second step, two brown-gold drops of iodine are shown above a slide with a dark blue-purple smear. The caption reads, “Add Iodine, 60s, rinse.” Iodine is applied for 60 seconds to help fix the crystal violet stain within the bacterial cells before the slide is rinsed.
The third step shows a tilted slide with clear drops of ethanol above it and pale purple liquid running off the lower edge. The smear on the slide appears light purple. The caption reads, “Add Ethanol, 10–15s, rinse.” This decolorization step removes the primary stain from Gram-negative cells while Gram-positive cells retain the crystal violet–iodine complex.
The fourth step appears at the lower left. Two red drops of safranin are shown above a slide with a red smear. The caption reads, “Add Safranin, 60s, rinse.” Safranin serves as the counterstain, coloring decolorized Gram-negative cells pink or red.
The final step appears at the lower right. It shows a slide with a purple smear and the caption, “Blot dry and view on scope.” After the slide is dried, it is examined under a microscope. Gram-positive cells appear purple because they retain the crystal violet stain, while Gram-negative cells appear pink or red because they take up the safranin counterstain.
