7: Acid Fast and Endospore Stains

Learning Outcomes

By the end of this lab period you will be able to:

• Distinguish between acid-fast and non-acid-fast bacteria in a stained sample.
• Identify vegetative cells and endospores in a stained sample.
• Employ the use of a steam bath to assist penetration of the primary stain into resistant cells and structures.
• Describe the clinical relevance of both acid-fast and spore-forming microorganisms.

Introduction

Today we are going to learn how to perform two new differential stains. The stains themselves are unrelated to each other, but both stains require heat to drive the primary stain into the bacteria, so it makes sense for us to learn about them on the same day.

Acid-Fast Stains

The Acid-Fast staining technique allows us to differentiate between “acid-fast” and “non-acid-fast” bacteria. There is an important clinical reason for us to learn about this technique, and that’s because it’s used to identify Mycobacterium tuberculosis in sputum smears. Mycobacterium tuberculosis is an incredibly serious and prevalent pathogen, and is the 13th leading cause of death worldwide, according to the WHO. A cousin of Mycobacterium tuberculosis, Mycobacterium leprae, causes leprosy. Prior to the availability of effective treatments, individuals with leprosy were banished to leper colonies, a practice with a long and tragic history. Today, we will work with the non-pathogenic Mycobacterium smegmatis.

Mycobacteria can be difficult to stain because their outer cell wall is rich in mycolic acids which are very hydrophobic (waxy), making it difficult for stain to penetrate. Common aqueous (water-based) stains such as methylene blue or crystal violet are repelled by the hydrophobic nature of the mycolic acids. In order to stain these cells, a primary stain called carbolfuchsin is used. This dye is hydrophobic just like the mycolic acids, and it is able to penetrate the cell walls of acid-fast organisms. We also help the process along by staining acid-fast organisms in a steam bath. The higher temperature further facilitates the entry of the dye into the cells.

Non-acid fast organisms can be decolorized using an acid-alcohol decolorizer and counterstained with methylene blue.

Table 1: Acid-Fast Staining Process

Number	Process	Effect	Result for acid-fast cell	Result for non-acid-fast cell
1.	Stain the smear with carbolfuchsin for 10 minutes over a steam bath	All cells will be stained bright fuchsia color.
2.	Decolorize with acid alcohol (3% HCl in 95% ethyl alcohol)	Acid-alcohol strips the color from non-acid-fast cells
3.	Counterstain with methylene blue	Methylene blue is needed to stain the now-transparent non-acid-fast cells.

Figure \(\PageIndex{1}\): Acid Fast Stain of Mycobacterium tuberculosis. CDC/Dr. George P. Kubica, Public domain, via Wikimedia Commons

Endospore Stains

Another differential staining technique, which also requires heat to assist penetration of the primary stain, is the endospore stain. Endospores are structures made by some (not all!) Gram-positive bacteria to withstand long periods of harsh conditions. Endospores consist of bacterial DNA in a tough protective coat of protein (keratin) and allow the organism to remain dormant until conditions become favorable for bacterial metabolism and growth. You can review endospores in our OpenStax Microbiology textbook in Chapter 3.3, Unique Characteristics of Prokaryotic Cells.

Endospores that form in the center of a cell are called central endospores and are characteristic of the Bacillus genus. Members of the Clostridium genus produce terminal endospores.

Spore-forming organisms are responsible for some very significant pathogens. Bacillus cereus and Clostridium botulinum are associated with foodborne illness. Clostridium tetani causes tetanus and Clostridium perfringens is associated with both foodborne illness as well as wound infections that can lead to gas gangrene.

Like the acid-fast cell wall, the keratin in the spore coat is very resistant to staining, so it is difficult for biological dyes to penetrate spores. Heat (in the form of a steam bath) is used to force the primary stain, malachite green, into the spore. Vegetative cells can be decolorized with distilled water and counterstained with safranin.

Table 2: Endospore Staining Process

Number	Process	Effect	Result for spores and vegetative cells
1.	Stain the smear with malachite green for 10 minutes over a steam bath	All cells and spores will be stained green
2.	Decolorize with distilled water	Water strips the color from vegetative cells
3.	Counterstain with safranin	Safranin is needed to stain the now-transparent vegetative cells

Figure \(\PageIndex{2}\): Endospore stains. Left - B. subtilis showing central endospores. Y tambe (original uploader), CC BY-SA 3.0, via Wikimedia Commons Right - Clostridium botulinum showing terminal endospores. CDC/ Courtesy of Larry Stauffer, Oregon State Public Health Laboratory, public domain.

Materials

Per Student Group

• Carbolfuchsin stain
• Methylene blue stain
• Acid alcohol (95% ethanol + 3% HCl)
• Malachite green stain
• Safranin stain
• Squirt bottle with water
• 1 L beaker to boil water
• Bibulous paper
• Clothespins as slide holders
• Disposable gloves
• Compound microscope with oil-immersion lens
• Immersion oil
• Lens paper
• Bacterial cultures
  • Mycobacterium smegmatis
  • Staphylococcus aureus
  • Bacillus subtilis (> 72 hours)
  • Clostridium sporogenes (> 72 hours)

Experiment

1. Both the acid-fast and endospore stains require the use of steam to drive the dyes into bacterial cells (Figure below TBD). Due to time constraints, each student will perform one of the two staining methods. Decide with your lab partner who is to perform the acid-fast stain, and who is to perform the endospore stain.
2. The student performing the acid-fast stain should collect Mycobacterium smegmatis and Staphylococcus aureus, and the student performing the endospore stain should collect Bacillus subtilis and Clostridium sporogenes.
3. To prepare the smear for the acid-fast stain, add a drop of water on the slide, and prepare a smear of S. aureus, dry and heat-fix the slide on the hot plate. Take a small amount of M. smegmatis and put it directly on top of the S. aureus smear. Because Mycobacterium cells tend to stick together in clumps, use the inoculation loop to chop up the M. smegmatis clumps, as if you were mincing garlic. The finer the mincing, the better your staining result will be.
4. To prepare the smears for the endospore stain, add two drops of water on the same slide, and make smears of B. subtilis and C. sporogenes separately. Dry and heat-fix on the hot plate.
5. Set up the heating device with the Bunsen burner, metal stand, mesh, and 1-liter beaker with ~ 500 ml water in it. Heat up the water until boiling, and reduce the heat to maintain a low rolling boil.

Figure \(\PageIndex{3}\): Setting up the acid-fast and endospore stains. A. Steam-bath apparatus. B. Cut bibulous paper or paper towel to fit the microscope slide. C. Place the bibulous paper over the dried/fixed smear to help keep the smear moist with stain during primary staining.

For the acid-fast stain:

6. Cover the smear with a strip of bibulous paper cut slightly smaller than the slide. Place the slide over the steaming device (beaker containing boiling water).
7. Saturate the bibulous paper with carbolfuchsin stain. Heat steam for 10 minutes. Be sure to keep the paper moist with stain. If the bibulous paper looks dry, add a few drops of the stain.
8. Remove the bibulous paper. Hold the slide on an angle over the sink and gently rinse both sides with distilled water until the runoff is clear.
9. Continue holding the slide with a slide holder. Decolorize with acid-alcohol until the run-off is clear. Use caution when handling the acid-alcohol.
10. Still holding the slide on an angle, gently rinse with distilled water into the sink.
11. Place the slide on the staining rack and counterstain with methylene blue for one minute.
12. Gently rinse the slide with distilled water into the sink.
13. Carefully blot the slide dry with fresh bibulous paper. Do not rub.

For the endospore stain:

1. Cover the smears with a strip of bibulous paper cut slightly smaller than the slide. Place the slide over the steaming device (beaker containing boiling water).
2. Saturate the bibulous paper with malachite green stain. Heat steam for 10 minutes. Be sure to keep the paper moist with stain. If the bibulous paper looks dry, add a few drops of the stain.
3. Remove the bibulous paper. Hold the slide on an angle over the sink and gently rinse both sides with distilled water until the runoff is clear.
4. Place the slide on the staining rack and counterstain with safranin for one minute.
5. Gently rinse the slide with distilled water into the sink.
6. Carefully blot the slide dry with fresh bibulous paper. Do not rub.
7. Observe using the oil-immersion lens. Record your observations of cell morphology and arrangement, acid-fast reaction, endospore presence, position, and shape.

Data

Record your staining results. Sketch the cellular morphology and arrangement of each bacteria. Use colored pencils to indicate staining results or take photographs to record what you saw. Include the total magnification for each sketch.

Questions

1. What is the role of steaming the smear during primary staining for both the endospore and acid-fast staining techniques? What might the results look like if the primary staining was done at room temperature?
2. Why does the endospore stain exercise call for an older culture of Bacillus? What would you expect the difference in the results to look like between a very fresh culture and an older culture?
3. Describe the clinical relevance of organisms that are acid-fast. What common pathogens are acid-fast organisms?
4. Describe the clinical relevance of some spore-forming organisms.