Smear Prep and Simple Stain

Smear Prep and Simple Stain

Objectives

• Fix bacteria onto a microscope slide for staining.
• Become familiar with using the light microscope with oil immersion.
• Properly prepare a simple stained bacterial sample.
• Identify the morphology and arrangement of various bacterial species.

Purpose

• Smear Prep: Prepare a sample for microscopic examination.
• Simple Stain: Determine the size, morphology, and arrangement of bacterium.

Background: Smear Prep and Fixation

• Before a simple stain, the microbial sample needs to be attached to a microscope slide; this is called a smear prep.
• Process:
  • A drop of liquid bacteria is placed on a microscope slide using an inoculating needle.
  • Alternatively, a colony of bacteria can be placed on a drop of water on a microscope slide.
  • The sample is then allowed to completely air dry.
• Once the bacteria are dried on the slide, they need to be attached so they will not wash away during the staining process; this is called fixing.
• Attaching or “fixing” the cells to the microscope slide can be achieved by gently heating the slide or by chemical means, such as with methanol.
• By fixing the bacteria to the slide, the proteins denature, metabolic activity is stopped (the bacteria are killed), and the bacteria are physically anchored to the slide.
• Methanol and heat can be used to fix bacteria.
• Once the sample is fixed, it is ready to be stained.

Simple Staining

• Bacteria are normally colorless, cells are stained with a colored dye to make them more visible under the microscope.
• Use of only one dye is called simple staining, and it will make all of the bacterial cells appear as the same color.
• Cell morphology (shape), size, and arrangement may then be determined.
• Stains are solutions consisting of a solvent (usually water or ethanol) and a dye.
• The charged region of the dye molecule that gives it its color is the chromophore.
• Chromophores stain bacteria by making covalent or ionic bonds to the cell.
• Most bacterial cell surfaces are negatively charged, they are most easily colored by basic stains with a positively charged chromophore (see Figure 1).
• Common basic stains include methylene blue, crystal violet, and safranin.

Experiment Overview

• Each pair of students will make 3 bacterial smear preps that will be stained with crystal violet, safranin, and methylene blue.
• All slides will be observed with 100x oil immersion on a light microscope, and observations will be recorded in the lab report.
• Figure 1: Basic stains have a positively charged chromophore (C^+) which forms an ionic bond with the negatively charged bacterial cell, thus colorizing the cell.

Smear Prep Procedure

1. Clean 3 microscope slides with ethanol and wipe dry with lens paper.
2. Label each slide with the sample that you will be putting on it.
   • If you’d like a “target” for your sample, draw a nickel-sized circle on the clean glass slide with the wax pencil.
3. Using aseptic technique, transfer one loopful of the bacterial sample onto its respective slide. Spread the liquid culture over a nickel-sized area of the slide so that the liquid spreads out (which will help it dry faster). Be sure to sterilize your inoculating loop between each transfer.
   • If we were preparing smears from bacterial colonies on a plate, we’d first place a sterile drop of water on the slide, then transfer a “scoop” of bacterial colony into the water, mixing it so that the cells mixed into the water.
4. Let the bacteria air dry completely; be patient!

Fixation Procedure (2 options)

1. Methanol fixation (preferred):
   • Cover the dried culture with methanol for 2 minutes.
   • After 2 minutes, tilt the slide to remove the excess methanol and allow the slide to air dry completely.
2. Heat fixation:
   • Grasp the slide with a clothes pin, and hold it against the opening of the microincinerator for no more than 10 seconds.
   • There is a risk of burning your sample with this method, so be mindful of how long you hold it to the heat.

Troubleshooting

1. If the culture does not air dry before fixation, the cells will be washed away during staining.
2. Dyes will not penetrate the sample if too much bacteria are in one big clump on the slide - too much dye will be concentrated in one area that obscures the morphology and arrangement of the cells.

Staining Procedure

• For your 3 slides, you’ll stain one with crystal violet, one with safranin, and one with methylene blue. It does not matter which sample gets which stain but keep track in your report of which sample gets which dye.
• The general staining procedure below is the same for all simple stains.

1. Place your slide on the wire rack in the metal staining tray. Add just enough dye liquid to your slide to cover your sample of bacteria.
2. Let the stain sit on your slide for 1-2 minutes.
3. Then hold your slide at an angle to let the dye run into the tray, and gently rinse the slide with water (squeeze bottle at the bench) to remove all of the extra methylene blue.
4. DO NOT WASH THE BACTERIA OFF OF YOUR SLIDES! Use a gentle stream of water above your samples (if you hit the bacteria directly, they may wash off).
5. Tap the edge of your slide to remove excess water, and gently BLOT your slide dry with the bibulous paper. Do not wipe your slide dry, as you will remove all of your bacteria and will have to start over.
6. View your slide with a light microscope under 100x oil immersion as you learned previously (start with 10x), and record your observations in the lab report.
   • Once you add oil to your sample, you cannot go back to using the lower objectives (4x, 10x, or 40x). Make sure your sample is in focus before adding oil and moving to the 100x objective! You cannot remove the oil once it is on!

Troubleshooting

1. If the cells do not have any color, the stain may not have been left on long enough.
2. If no cells are visible, they may have been rinsed off or rubbed off with the blotting.
3. If you do not see intact, whole cells, you may have over-fixed the sample (especially if heat was used, which causes the cells to pop or burst open). Alternatively, you may not have transferred enough bacteria onto the slide.

Results and Observations

• Organism name:
• Draw a picture of the morphology of a single cell
• Stain used
• Cell morphology
• Cell arrangement
• Cell color

Questions

1. Why does a thick or dense smear not provide the best results for microscopic viewing?
2. What does fixing do to the bacteria?
3. What is a consequence of over-heating the slide during fixation?
4. How do you prevent cells from clumping in a smear?