8: GRAM STAIN
- Page ID
- 157067
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Interpret Gram stain results to determine bacterial classification.
- Apply the Gram staining technique in a laboratory setting with attention to timing and accuracy.
BACKGROUND
The Gram stain is considered the most essential staining technique in microbiology. Developed by Hans Christian Gram, it originated from his experiments with various dyes and their interactions with bacterial cells. Through this work, he discovered a sequence of staining and rinsing steps that caused some bacteria to retain color while others did not. Today, the method includes a second stain to color the previously unstained cells, allowing both types to be seen under a microscope. As long as the two stains are different colors, the two groups of bacteria can be distinguished. This differential staining became the foundation for classifying bacteria, with most falling into one of two categories: Gram-positive, which retain the primary stain, and Gram-negative, which do not and instead take up the counterstain.
Although the Gram stain was developed before scientists understood bacterial cell wall structure, we now know that the staining outcome is determined by the composition of the cell wall. Gram-positive bacteria have thick layers of a substance called peptidoglycan, which traps the stain. In contrast, Gram-negative bacteria have much thinner peptidoglycan layers, allowing the stain to be washed away more easily. Some bacteria are Gram-variable, meaning their staining results are inconsistent, while others have unusual or even absent cell walls. Because the type of cell wall is so critical to identifying bacteria, the Gram stain is often the first test performed in microbiological analysis and remains essential for accurate classification.
The effectiveness of the Gram stain depends heavily on precise timing and technique. The duration of each staining and rinsing step can significantly affect the final appearance of the cells. For example, if the primary stain and mordant are left on too long, Gram-positive cells may appear nearly black, and Gram-negative cells may look dark blue. On the other hand, excessive rinsing with alcohol can cause Gram-positive cells to lose their color, making them appear pale lavender, while Gram-negative cells may become faintly pink. Because of these variables, it is crucial to include control samples with known Gram-positive and Gram-negative bacteria on the same slide. Without these controls, it is impossible to interpret the results with confidence.
Several factors can interfere with the accuracy of a Gram stain. If bacterial cells are unhealthy or dead, their cell walls may become compromised, causing Gram-positive cells to appear Gram-negative. Clumps of cells can also lead to misleading results, as uneven exposure to stains and rinses may cause Gram-negative cells in the center of a clump to retain the primary stain. This can create the false impression of a mixed population. Similarly, if too few cells are present on the slide, it becomes difficult to distinguish their color or shape, and artifacts such as scratches or stain crystals may be mistaken for bacteria. In many cases, a sparse sample indicates poor bacterial growth, which often correlates with unreliable staining results.
MATERIALS (Supplies Per Group of 4: Each student makes their own slides)
Gram positive control
Gram negative control
Alcohol (95% ethanol)
Microscope slides
Grams iodine
Safranin
Crystal violet
Staining tray
Water wash bottle
METHODS
Make 2-3 slides as follows:
1. Place a slide on top of the diagram below. Smear the Gram positive control in the left circle (+). Smear the Gram negative control in the circle to the right (-). In the center circle (mix) place one loopful of the gram positive control and one loopful of the Gram negative control then use the loop to mix the two controls together. Take care to keep the smears within their designated circles.
2. Place the slides on the slide warmer and allow all smears to dry completely.
3. Heat-fix all slides. Place one slide on the staining tray rack and set the others aside for later use.
DO NOT STAIN ALL SLIDES AT ONCE - ONLY STAIN ONE THEN LOOK AT IT UNDER THE MICROSCOPE
4. Cover the three smears on the slide with crystal violet and let it sit for 30 to 45 seconds.
5. Holding the slide with a clothes pin, tilt the slide and gently rinse with water for 2 to 3 seconds, or until excess stain is no longer visible.
6. Apply Gram’s iodine to the smear and let it sit for 60 seconds.
7. Holding the slide with a clothes pin, tilt the slide again and rinse gently with water for 2 to 3 seconds. Drain thoroughly.
8. Tilt the slide and drip alcohol down its surface until no more visible color is being washed from the smears. Alternatively, you may cover the smears with alcohol for 2 to 5 seconds.
9. Rinse immediately with water. Alcohol will continue to decolorize the smears until it is washed off.
10. Apply safranin to the smears and allow it to sit for approximately 5 minutes. Longer staining time is acceptable.
11. Holding the slide with a clothes pin, tilt the slide and gently rinse with water.
12. Put the slide on top of the diagram and use a sharpie pen to draw circles on the top side of each slide to indicate smear locations.
12. Examine the smears under a microscope. If the color difference between Gram-negative and Gram-positive control cells is unclear, try staining another dried smear using slightly adjusted timing. Consult your instructor if you are unable to achieve a distinct color contrast between the two cell types.
The following is to be completed during lab then turned in on Canvas as a PDF
*If you are using an iPad or tablet you will need to take screen shots of your competed work, save the screenshots as one PDF then submit them on Canvas by the due date designated on Canvas.
*You can also print out the entire exercise to bring to lab with you. If you choose to complete the lab on paper, take pictures of the completed results and conclusions sections only, save them as one PDF, then submit to Canvas by the due date designated on Canvas.
GRAM STAIN
NAME ______________________
EXPECTATIONS
Take a moment to examine your smears without using the microscope. Do they appear too thin or overly clumped? Are cells visible across all smear areas? Can you clearly distinguish between Gram-negative and Gram-positive cells based on the color differences in your staining
RESULTS
After staining one slide, examine the cells under the microscope.
Remember to always begin with the lowest magnification (4x). If the smear is difficult to locate, use the landmark (the circle you drew with the Sharpie) as a guide. When the Sharpe is in focus the cells will be as well. Make sure the area you center in the field of view accurately represents the overall smear, not just an isolated or atypical section.
Once the cells are in focus at 40x slide the objective to the side and apply immersion oil directly to the slide. slide the oil immersion objective (100x) in place and carefully scan across the smear. Take your time and observe thoroughly. Avoid drawing conclusions based on cells at the edges of the smear, as they may be poorly stained. Likewise, be cautious with clumped cells, which can retain excess stain and give misleading results. Scan the slide until you see a region where the cells are spread out and you can see the individual cells clearly. Check the controls first. If the control are the correct colors G+ purple and G- pink then look at the center smear. If the controls are not the correct colors or they are both pink and purple. Stain one of the other slides you have. Think about what could have gone wrong are adjust your timing accordingly.
Sketch what the cells look like to you for both control cultures and the mixed culture smear. Be sure to include the magnification used and the observed colors for each sample.
CONCLUSIONS
1. Describe the colors of Gram negative and positive control cultures on your slide.
2. Are all the Gram positive cells on your slide a single color? Are the Gram negatives all one color? Where on your smear are the colors uneven or inconsistent?
3. If you did all the other steps of a Gram stain perfectly but accidentally left out just one step , WHAT COLOR would you see?
4. Explain why you would see the colors you choose in the table above.
A.
B.
C.
D.
E.