TheNegativeStain Technique

he negative stain technique permits one to see unstainedbacteria on a stained background and determine their mor-phology. Acidic stains, such as nigrosin, congo red, and India

ink, carry a negative charge and therefore are repelled by the negativelycharged bacteria. The technique may be used with samples of bacterialcultures and especially for organisms in the dental plaque because the spi-ral bacteria (spirochetes) in plaque do not stain well with other techniques.Cells appear clear against a dark background.

Precautions must be taken in this technique. Excessive stain is not usedbecause it will make the organisms difficult to locate. Also, the stain must notbe allowed to become contaminated because it will support bacterial growth.

pecial Materials

• Cultures of selected bacterial species• Nigrosin, congo red, or India ink• Toothpicks and dental floss

rocedure

1. Wash a slide until it is scrupulously clean. It is important to remove extraneousdebris and particles from the slide because these may be mistaken for bacteria.Rinse the slide and dry it, preferably in air or with a lint-free cloth.

2. Aseptically obtain a loopful of bacteria according to the procedure describedin Exercise 4A. Place it near the end of the slide.

3. Now place a very small drop of acidic stain in the bacteria drop. Using atoothpick, mix the cells with the stain on the slide.

4. Referring to Figure 5.1A, back the edge of a second slide into the mixture.Now spread the mixture in one smooth movement down the length of theslide surface as shown in Figure 5.1B. Note that the stain is pulled ratherthan pushed across the slide to give a light and even distribution. As the far

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Quick ProcedureNegative Stain

1. Add a loopful of bacteriaat end of slide.

2. Place small drop of acidicdye at end of slide.

3. Smear across face of slide.

4. Air-dry; observe.

PURPOSE: to accuratelymeasure cell size and deter-mine cell shape in an un-distorted manner.

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edge is neared, lift the spreading slide to “feather” the edge of the smear. Thespreading slide should be flamed briefly to destroy any bacteria that havecontaminated it.

5. Allow the slide to air-dry for several minutes on the laboratory bench orwarming tray. Do not heat-fix the slide.

6. Additional slides may be made with different species of bacteria or with asample of dental plaque. To prepare a slide of plaque, use a clean toothpickor piece of dental floss to gather material from between the junction ofteeth and gums. Add this material to a drop of stain on the edge of theslide, then spread as before.

7. Survey the slide with the lower power lens, looking for tiny pinpoints of light(bacteria). Then locate the negative stained bacteria under 40X and oilimmersion by searching for white or clear cells on a colored backgroundas shown in Figure 5.2. Scan the slide for several minutes. Prepare draw-ings in the Results section, being sure to shade the background. Note thesize, shape, and arrangement of the cells. Include the full binomial nameof known organisms together with the total magnification and dye used. Ifrequired, label the slides with a slide label containing your name, thename of the organism, the stain used, and the date. Retain them for laterreference.

8. Another method for preparing a negative stain is as follows: Prepare an air-dried smear of a bacterial species as indicated in Exercise 4A. Do not heat-

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F I G U R E 5 . 2The appearance of bacteria after negative staining.

F I G U R E 5 . 1The procedure for preparing a negative stain.

A BA slide is drawn back into a mixture of stain and bacteria.

The slide pulls the stain-bacteria mixture across the surface.

!Be careful not to touch thestained area of the slide,since the bacteria remainalive until the slide has air-dried.

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fix the smear because the heat will disrupt the bacterial cells. Now take afelt marking pen (black is best) and pass it several times over the smear.The ink will stick to the slide, and the bacteria will appear white against thecolored background. Proceed with your observations as in step 7 above.

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uestions

1. Explain why crystal violet, a basic stain, would not be useful in the nega-tive stain technique.

2. What are the advantages of 1) the negative stain technique over the simplestain technique and 2) the simple stain technique over the negative staintechnique?

3. Suppose a clean slide were used for this exercise and still a number ofunexpected bacteria appeared in the finished smear. What might be theirsource?

4. Why is it a poor idea to heat-fix bacterial smears when performing thenegative stain technique?

5. Compare the size of an organism observed after negative staining withthe size of the same organism stained by the simple stain technique.Explain the reasons for any differences.

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Name

Date Section

Exercise Results

The Negative Stain Technique

Negative-Stained Bacterial Smears

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Organism: ______________________ ______________________ ______________________

Stain: ______________________ ______________________ ______________________

Magnif.: ______________________ ______________________ ______________________

Organism: ______________________ ______________________ ______________________

Stain: ______________________ ______________________ ______________________

Magnif.: ______________________ ______________________ ______________________

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Observations and Conclusions:

Organism: ______________________ ______________________ ______________________

Stain: ______________________ ______________________ ______________________

Magnif.: ______________________ ______________________ ______________________

Organism: ______________________ ______________________ ______________________

Stain: ______________________ ______________________ ______________________

Magnif.: ______________________ ______________________ ______________________

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