7: SMEAR AND SIMPLE STAIN
- Page ID
- 157066
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Prepare bacterial smears from both liquid and solid cultures and apply a simple staining technique to enhance visualization of bacterial cell morphology under the microscope.
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Evaluate the quality of a smear and staining result based on the clarity, distribution, and visibility of bacterial cells when viewed using oil immersion at 1000x magnification.
BACKGROUND
Even under a high-quality light microscope, bacteria appear as extremely small, nearly transparent dots. To distinguish differences in shape and arrangement, bacterial cells must first be stained. In some cases, specific types of bacteria can also be identified based on how they react to particular chemical stains.
The initial step in observing and identifying bacteria microscopically is to prepare a smear (a thin, even layer of bacteria spread onto a glass slide) and apply a staining technique. Accurate interpretation is not possible if the smear is too thick, clumped, or sparsely populated. A well-prepared smear distributes an appropriate number of cells evenly across the slide, allowing for clear observation under the microscope.
Several factors should be considered when preparing smears:
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Smear Size: Avoid making smears that are unnecessarily large. Larger smears may take longer to dry and may spread cells too thinly, making them difficult to locate. Aim to use only the area needed for effective viewing.
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Cell Concentration: The density of the original culture will affect how the smear should be prepared. For dilute liquid cultures, it may be necessary to layer several drops to increase the cell concentration. For samples from solid media, such as agar plates, a small portion should be mixed with water on the slide to prevent overcrowding. It is essential to maintain an appropriate cell density to observe individual bacterial shapes clearly.
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Slide Cleanliness: A clean, smooth glass surface is essential. Oil, dust, or scratches can interfere with smear quality, causing poor adherence or visual artifacts. Slides should be cleaned with 95% ethanol and dried thoroughly prior to use.
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Sample Location: Clearly marking the slide is recommended to help locate the smear during microscopy. It is also helpful to scan the entire smear at low power before switching to higher magnification to avoid clumped or unevenly stained areas.
Because bacteria are transparent, they must be stained to become visible under the microscope. A simple stain involves the use of a single dye that uniformly colors all cells. In today’s exercise, we will use Loeffler’s Methylene Blue, a commonly employed stain that enhances visibility of bacterial morphology. While many different dyes may be used, as long as only one is applied and it stains all cells similarly, the procedure qualifies as a simple stain.
MATERIALS
1 Nutrient broth culture of bacteria
1 Culture of bacteria on a nutrient agar slant
1 Culture of bacteria in nutrient broth
1 Staining tray and rack
1 Bottle Loeffler's methylene blue syain
METHODS
➤ The glass slides are not sterile. Once your loop touches a slide it is contaminated.
You must re-sterilize the loop in the flame every time before inserting it into a culture.
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Label two glass microscope slides with your name, name of the organism you are working with, type of stain, (simple stain) and the date.
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Place the two labeled slides onto a paper towel or white piece of paper. Use a pencil to trace the outline of the slides then remove the slides and place them on the staining rack. Add a line on the diagram that designates the label space. Then draw three circles that represent where you will place the cells. There is an example diagram at the end of the methods section.
➤ Be sure to leave at least a centimeter between the edge of the slide and the third circle
on the diagram or your slide will fall into the opening of the stage when you try to focus.
Preparing a Smear From a Solid Medium (agar plate or slant)
➤ Cell from a solid culture (agar plate or slant) are highly concentrated and
must be diluted in water on the slide in order to see the individual cells.
Always add the water to the slide before the bacteria are added.
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Use the DI water bottle to place 1-2 drops of water on a blank slide.
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Sterilize your inoculating loop by holding it in the blue flame of the Bunsen burner until it glows red-hot. Allow it to cool for 5–10 seconds before touching any materials.
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Using your sterilized loop, transfer two-three loopful of water to each of the circles outlined on the diagram under the slide.
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Using a sterilized loop collect enough of the cells from the solid culture to easily see on the loop and place them in the first circle on the slide. Mix the cells in the water to dilute them. Be sure to stay within the boundaries of the first circle on the diagram.
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Using a sterilized loop collect a tiny pinpoint of cells from the same solid culture then place the cells in the middle circle on the slide. Mix the cells in the water to spread them out but remain within the boundaries of the middle circle on the diagram.
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Using a sterilized loop barely touch the surface of the culture (you should not be able to see any cells on the loop) then place the cells in the third circle on the slide. Mix the cells in the water to spread them out but remain within the boundaries of the third circle on the diagram.
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Allow the smear to air dry on the slide warmer until completely before moving on to heat-fixing and staining.
Preparing a Smear From a Liquid (broth)
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Wipe any remaining water off the slide from above Place the slide on the diagram you drew.
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Using a sterilized loop add one loopful of the culture in the nutrient broth (liquid medium) to the first circle on the diagram. use care when spreading the cells in the circle – stay within the boundaries.
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Using a sterilized loop add 3 loopful of the culture in the nutrient broth (liquid medium) to the middle circle on the diagram. Remember to use care when spreading the cells in the circle – stay within the boundaries.
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Using a sterilized loop add 5 loopful of the culture in the nutrient broth (liquid medium) to the first circle on the diagram. Remember to use care when spreading the cells in the circle – stay within the boundaries.
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Allow the smear to air dry on the slide warmer until completely before moving on to heat-fixing and staining.
Example
Heat Fixing Smears:
Heat fixing ensures the cells adhere to the slide and are no longer viable (alive).
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Using a clothes pin, hold the slide at an angle and pass it slowly through the blue cone of the Bunsen burner flame for 2-3 seconds.
⚠️ If you detect a burning odor, the slide is too hot.
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Allow the slide to cool before staining to prevent altering the chemical properties of the stain.
Staining Smears:
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Once the slide is cool, cover each smear completely with Loeffler’s Methylene Blue stain.
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Let the stain sit for 30–60 seconds.
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Pick up the slide by the frosted end using your clothes pin and tilt it slightly.
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Gently rinse off the stain using the distilled water wash bottle until no excess dye remains.
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Dispose of used stain and rinse water from your staining tray in the sink.
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Rinse and clean both your tray and the drying rack thoroughly. Dried stain on the rack can transfer to future slides and affect staining results.
Viewing the Cells:
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Place the dried, stained slide back on the diagram you drew.
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Using a medium sharpie pen, trace the landmark circles onto the top of the glass. The circles should surround the inoculate on the slide. These reference marks will help you locate samples under the microscope.
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Place the slide in the stage clip on the microscope stage. Focus on the sharpie circle under the 4x objective. Progress through the objectives (10x then 40x) centering the sharpie edge in the center of the field of view as you go up in magnification.
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Once the pen mark is focused under the 40x objective, rotate the objectives to the open space between the 100x and 4x objectives. Place a drop or two of oil directly on the slide in the middle of your field of view where the light source is shinning through. Use the the black ring to rotate the 100x (oil immersion lens) in place.
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Once you have the cells on the slide in focus at 100x (oil immersion lens) do not lower the stage to view other areas on the slide. You can place oil anywhere on the slide then scan over to view it without lowering the stage and starting over.
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Draw what you see for all three smears on each slide under oil in the results section.
➤ Bacteria can only clearly be viewed under 1000x with immersion oil, however, always start with the 4x objective then progress through each objective focusing as you go. Since bacteria cannot be seen at lower magnification always focus on the inner edge of the landmark (sharpie circle)
then scan the area inside the circle to find the cells. If the sharpie is in focus the bacteria will be too.
EXERCISE 7 Smear and Simple Stain
NAME ______________________
EXPECTATIONS
How many loopful of the broth do you think are necessary in order to see any cells on the slide?
RESULTS
CONCLUSIONS
- Compare your results from the liquid (broth) culture and the solid (agar slant) culture.
Explain which technique gave you the clearest, most useful smear and why.
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Why do bacteria have to be stained before viewing under a microscope?
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Are simple stains always blue?
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List two important reasons why heat fixation is necessary before staining.
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Explain two reasons a sharpie is used to draw a circle on the slide?
6. Why should you not use a sharpie to label the slide?