Microscopy and Staining: Gram, Capsule, and Endospore Staining

1. Gram Staining

1. Set-up
   1. Wear gloves and a non-flammable lab coat, as dyes will stain hands and clothing.
   2. A Bunsen burner is used to heat-fix the bacteria. Use care when working with flame; tie back long hair.
   3. Commercially available Gram stain reagents will be used.
   4. Clean microscope slides with laboratory wipes.
2. Protocol
   1. Pipet 10 µL phosphate-buffered saline or culture broth onto the slide.
   2. Smear a bacterial colony into the liquid to produce a thin, even layer.
      Note: Do not use cultures older than 24 hours, as bacteria too old could have changes in their cell wall, which will affect the Gram stain results (1, 4).
   3. Completely air-dry slide.
   4. Once dried, heat-fix bacteria by passing slide through the flame (bacteria side up) 4-5 times.
      Note: Do not hold the slide in the flame too long or you might distort the bacterial cells (1).
   5. Working over the sink, hold the slide level and apply Gram's Crystal Violet to completely cover the heat-fixed bacteria, allow to stand 45 seconds.
   6. Rinse excess Crystal Violet by holding the slide at an angle and squirting a gentle, indirect stream of water onto the slide and letting it run down over the stained bacteria. Do not squirt water directly onto the bacteria.
   7. Holding slide level again, apply Gram's Iodine Solution to completely cover the stained bacteria, allow to stand 45 seconds.
   8. Rinse excess Iodine as in step 1.2.6 above.
   9. While holding the slide at an angle, add a few drops of Decolorizer onto the slide, letting it trickle down over the stained bacteria just until the runoff is clear; typically, about 5 seconds. Immediately rinse with water as in step 1.2.6 above.
      Note: This is a critical step in the protocol. Allowing Decolorizer to trickle too long or not long enough will result in false Gram-staining (4).
   10. Holding the slide level again, apply Gram's Safranin to completely cover the bacteria, allow to stand 45 seconds.
   11. Rinse excess Safranin as in step 1.2.6 above.
   12. Blot, do not rub, excess water from slide using paper towels.
   13. Examine slide on the microscope using oil-immersion with a 100X objective.
3. Results and Data Analysis
   1. Gram-positive bacteria will stain purple.
   2. Gram-negative bacteria will stain red.
   3. Shape (cocci, bacilli, curved rods, spirals) of bacteria will be visible.
   4. Arrangement of bacterial cells (single cells, paired cells, chains of cells, clusters, groupings) will be visible.

2. Capsule Staining

1. Set-up
   1. Wear gloves and a lab coat as dyes stain hands and clothing.
   2. To prepare 1% Crystal Violet solution, mix 0.25 gram Crystal Violet with 25 mL distilled water until dissolved.
   3. To prepare 1% Congo Red solution, mix 0.25 gram Congo Red with 25 mL distilled water until dissolved.
   4. Clean slides with laboratory wipes.
2. Protocol
   1. Place 10 µL Congo Red on slide.
   2. Using a pipet tip, smear a bacterial colony into the dye to produce a thin even layer.
   3. Completely air-dry slide with dye/cell mixture, 5-7 minutes.
      Note: Do not heat fix as heating can dehydrate or distort the capsule.
   4. Flood the smear with 1% Crystal Violet for 1 minute.
   5. Rinse excess stain by holding the slide at an angle and squirting a gentle, indirect stream of water onto the slide and letting it run down over the stained bacteria. Do not squirt water directly onto the bacteria.
   6. Hold the slide at a 45-degree angle until completely air-dried.
   7. Examine smear on the microscope under oil immersion with a 100X objective.
3. Results and Data Analysis
   1. Bacterial cells will stain purple.
   2. The background of the slide will stain dark.
   3. Capsules will be a clear halo around cells against a dark background.

3. Endospore Staining (Schaeffer-Fulton method)

1. Set-up
   1. Wear gloves and a non-flammable lab coat to protect hands and clothing from dyes and flame.
   2. A Bunsen burner is used to heat fix the bacteria. Use care when working with flame; tie back long hair.
   3. To prepare 0.5% Malachite Green solution, mix 0.125 grams Malachite Green with 25 mL distilled water until dissolved.
   4. Use commercially available Gram's Safranin reagent solution.
   5. Clean slides with laboratory wipes.
2. Protocol
   1. Pipet 10 µl phosphate buffered saline (PBS) or culture broth onto slide.
   2. Using aseptic technique, smear a bacterial colony into the liquid to produce a thin, even layer.
      Note: Endospores generally do not form in young cells; therefore, the culture is recommended to be between 18 and 36 hours old (9).
   3. Completely air-dry slide.
   4. Heat fix by passing slide (bacteria side up) through flame 4-5 times.
   5. To help contain the dye, place a piece of lens paper (cut to fit the bacterial smear) over the heat-fixed smear.
   6. Saturate lens paper with Malachite Green solution.
   7. Place slide on top of the beaker of boiling water on a hot plate, and steam slide for 5 minutes, keeping lens paper moist by adding more dye a drop at a time as needed.
      Note: Avoid overheating and drying out the dye solution.
   8. Remove slide from beaker, remove and discard lens paper, allow slide to cool 2 minutes.
   9. Holding slide at an angle, rinse thoroughly by squirting a gentle, indirect stream of water onto slide, allowing it to drain down over smear.
   10. Holding slide level, flood smear with Safranin, allow to stand 1 minute.
   11. Rinse excess Safranin as in step 3.2.9 above.
   12. Allow to air-dry.
   13. Examine slide on microscope under oil-immersion with a 100X objective.
3. Results and Data Analysis
   1. Spores will stain green.
   2. Vegetative cells will stain red.
   3. Some vegetative cells will contain spores; the cells will stain red, while the endospores will stain green.