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Mycobacterium Smegmatis & Bacillus Subtilis Endospore Stain: Abstract
Endospore and acid-fast staining are differential staining procedures used to identify bacteria with unique cell properties. The purpose of this lab was to use endospore and acid-fast staining to distinguish between two bacterial species. Old liquid cultures of Bacillus subtilis and Mycobacterium smegmatis were used. Malachite green stain and safranin stains were used for the endospore staining of Bacillus subtilis, whereas carbon fuchsin and methylene blue stains were used for acid-fast staining to distinguish a smear of Mycobacterium smegmatis from non-acid-fast organisms.
For the endospore staining, Bacillus subtilis had a mixture of green and pink staining, indicating the presence of endospores. In contrast, Mycobacterium smegmatis were stained red, whereas Bacillus subtilis were colored blue in the acid-fast staining process, thereby confirming that the former was acid-fast positive while the latter was acid-fast negative.
Acid-fast and Endospore Staining: Introduction
Bacteria are microscopic organisms that can only be seen under a microscope. However, it is difficult to visualize the morphology of bacteria because the cytoplasm is transparent, which makes all parts of the cell appear the same. Staining is a microbiological technique that is used to improve contrast in biological samples thus enabling the visualization of different organelles and parts of the cell. A number of dyes have been developed for this purpose.
Dyes have varying physical and chemical properties that enable them to react with different macro- and macro-molecules found in the cell. The products of the reaction enable the localization of dyes in specific parts of the cell, which lays the basis of bacterial identification because different bacteria have diverse staining patterns.
The most common staining technique is Gram stain, which enables the differentiation of bacteria into two broad groups of Gram-positive and Gram-negative based on the staining of the peptidoglycan layer that is found in bacterial cell walls. Nonetheless, there is a need to further classify bacteria beyond these two groups. Additionally, some bacteria such as the genus Mycobacterium are resistant to Gram staining and cannot be visualized this way. The differential staining of bacteria by various stains is useful in this regard.
Some bacterial genera are can respond to adverse environmental conditions by forming spores that are resistant to heat, desiccation, unfavorable chemicals, and radiation. These spores are known as endospores and are the most resistant forms of life. The endospore stain is a differential stain that is widely used to distinguish between vegetative cells and endospores (BMCC Lab manual). Acid-fast staining was developed by Robert Koch in 1882 and later modified by other scientists.
Koch used the method to observe the tubercle bacillus what we now call Mycobacterium tuberculosis, in sputum samples. While acid-fast and gram staining are both differential stains, the acid-fast stain is much more specific. Many bacteria are either Gram-positive or Gram-negative, but very few are acid-fast (LibreTexts). The acid-fast stain is a laboratory test to use to determine if a sample of sputum, skin tissue, blood, urine, stool, or bone marrow is infected with Mycobacterium. The purpose of this lab is to perform endospore staining of Bacillus subtilis and use the acid-fast stain procedure to differentiate between Mycobacterium smegmatis and Bacillus subtilis.
Endospore & Acid-Fast Staining Methods
Two species of bacteria were used for this lab: Mycobacterium smegmatis and Bacillus subtilis. Cultures of each species were grown in tubes containing solid media. The author performed the endospore staining on Bacillus subtilis and the acid-fast staining procedure on Mycobacterium smegmatis and Bacillus subtilis. For both procedures, latex gloves were worn to protect the skin, and aseptic techniques were involved throughout.
Endospore Staining
Two clean slides were obtained and labeled with the microorganism, date, and stain type. A metal inoculation loop was sterilized in a Bunsen burner flame. One small drop of water was transferred from the cool loop to the slide. The loop was sterilized again and then used to lightly scrape Bacillus subtilis from the tube culture. The sample on the loop was inoculated to the slide and emulsified into the water. The mixture was spread evenly in a smear on the slide.
The loop was sterilized again before air-drying and heat fixing the smear in a beaker of boiling water. The smear was then flooded with malachite green and steamed for 5 minutes. Thereafter, the slide was rinsed thoroughly in two to three changes of cool water, counterstained with safranin for 2 minutes, rinsed thoroughly in 2 to 3 changes of cool water, and air-dried. The slide was then examined under a microscope using the oil immersion lens.
Acid-Fast Staining
Two clean slides were obtained and labeled with the microorganism, date, and stain type. A metal inoculation loop was sterilized in the Bunsen burner flame. One small drop of water was transferred from the cool loop to the slide. The loop was sterilized again and then used to lightly scrape Bacillus subtilis from the tube culture. The sample on the loop was inoculated to the slide and emulsified into the water.
The mixture was spread evenly in a smear on the slide. The loop was sterilized again and put away. The same steps were repeated for another slide with Mycobacterium smegmatis followed by air-drying and heat fixing of both slides. The slides were placed in a beaker containing boiling water before rinsing the slide thoroughly in two to three changes of cool water. The slides were held at an angle, and acid alcohol was run over them. They were then rinsed thoroughly in two to three changes of cool water, counterstained with methylene for 1 minute, and rinsed thoroughly in two to three changes of cool water. The slides were then air-dried and examined using an oil immersion lens under a microscope.
Staining: Microscopy
Both stained slides were examined using a light microscope at 1000X with immersion oil.
Endospore & Acid-Fast Staining: Results & Discussion
The endospore staining and acid-fast stain were performed correctly, leading to the visualization of cell morphology. Bacillus subtilis appeared as pink and greenish rod-shaped (oval) cells. Malachite green, which was the primary stain, penetrated the cell wall during heating and stained the endospores green. The counterstain (safranin) adhered to the vegetative cells and stained them pink, which gave B. subtilis cells a mixture of green and pink appearance.
The endospores were located in the middle of B. subtilis cells and were spherical to oval-shaped. Clinical microbiologists need to identify endospore-forming bacteria because endospores are associated with drug resistance. The presence of an endospore confers bacteria with the ability to resist chemicals such as disinfectants and antibiotics as well as physical methods of sterilization, including drying, boiling, and radiation. Therefore, once endospores have been identified, more intense methods of sterilization need to be employed, for example, prolonged boiling at temperatures exceeding 100oC.
Acid Fast Positive & Negative Results: Conclusion
Conversely, the acid-fast staining revealed red and blue rod-shaped and staphylococcus shapes. The blue, rod-shaped cells were B. subtilis while the red, staphylococci cells were Mycobacterium smegmatis. Therefore, it was noted that B. subtilis was acid-fast negative, whereas M. smegmatis was acid-fast positive. The acid-fast stain is classified under differential staining procedures that are used to pick out acid-fast organisms, including bacteria of the genus Mycobacterium.
These organisms have a thick, waxy almost impervious cell wall made up of mycolic acid and complex lipids. This cell wall cannot be penetrated easily and required specialized staining techniques. The primary stain used in acid-fast staining, carbon fuchsin, which is used as the primary stain, is lipid-soluble. Therefore, it can permeate the cell wall with the aid of heat and stain it. Rinsing with a strong decolorizer removes the stain from all non-acid-fast cells without affecting the acid-fast organisms.
The subsequent addition of a secondary stain (counterstain) colors the de-stained non-acid-fast cells. An acid-fast positive result indicates the presence of a thick lipoidal cell wall in bacteria. Acid-fast staining is important in clinical microbiology because it aids the diagnosis of pathogenic bacteria such as Mycobacterium tuberculosis that causes tuberculosis. Without this technique, it would be difficult to identify these strains because they resist the conventional Gram-staining process.
Acid Fast Staining Procedure: Key Instructions
This is a step-by-step instruction for the Acid-Fast Staining Procedure, a technique used to differentiate acid-fast bacteria from non-acid-fast bacteria based on their staining characteristics. Each step is carefully designed to ensure accurate and reliable results for microbial identification under the microscope. You can use these instructions for your future lab report or research.
References
Anderson, Denise G. NESTERs Microbiology: A Human Perspective. New York: McGraw-Hill, 2016. Print.
Mario Benavides, Sarah Salm, Christopher Thompson, Igor Zaitsev. Microbiology Laboratory Manual BMCCBIO420. Print.
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