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Lab Report 1: StainingBIO 235
Eric Weissenborn
Professor Melissa PhilionIntroduction
Since most microorganisms appear virtually colorless, they often need to be stained in order to adequately examine them with a compound light microscope. Staining the bacteria provides a contrast with the background which allows for an examination of their characteristics including, cell size, shape, arrangement, chemical properties, and structures (Alexander & Strete, 2001). There are a variety of staining methods used including, simple staining, acid-fast staining, Gram staining, negative staining, spore staining, and capsule staining. Each staining method provides different information about the microorganism. The microorganisms first need to be fixed to a microscope slide before it can be stained. Fixing kills the microorganism and attaches it to the slide. Fixing involves spreading a thin film of the microorganism over the surface of the slide. This film is called a smear (Tortora, Funke, & Case, 2013).

A simple stain is single basic stain that colors the bacterial cells to observe their size, shape, and arrangement. A basic stain carries a positively charged ion which is attracted to the negatively charged bacterial cell. A chemical called a mordant is sometimes added to intensify the stain. Commonly used simple stains include methylene blue, carbolfuchsin, crystal violet, and safranin (Johnson & Case, 2011).

Another stain used to observe the morphology of a cell is a negative stain. A negative stain uses a single acidic stain that colors the background around the cell instead of the cell itself. This method provides a more accurate determination of the size and shape of cells because fixing is not needed and the cell itself is not stained which can distort the view of the cell. Negative staining is also useful for observing cells that do not readily stain. Acidic stains have a negatively charged ion which is repelled by the negatively charged bacteria surface. This leaves the background instead of the cell colored. Commonly used acidic stains are India ink, nigrosin, and acid fuchsin (Alexander & Strete, 2001).

Differential stains, which include the Gram stain and the acid-fast stain, are used to differentiate bacteria based on the chemical composition of their cell wall (Alexander & Strete, 2001). The Gram stain is a highly important staining method that classifies bacteria as either Gram-positive or Gram-negative. The primary stain in the Gram stain is crystal violet in which all the bacteria are stained. A mordant (iodine) is then used which combines with the crystal violet in the cell to form a crystal violet-iodine complex (CV-I) (Johnson & Case, 2011). A decolorizing agent (either alcohol or alcohol-acetone solution) is added. Some cells are affected, washing out the primary stain (decolorized) while others are unaffected. A counterstain (safranin) is added which stains the decolorized bacteria red. The unaffected cells maintain the purple color of the primary stain. Bacteria that stain purple are classified as Gram-positive, whereas bacteria that stain red are classified as Gram-negative. Bacteria react differently to the Gram stain because of the structure of their cell walls. Gram positive cells have a thicker layer of disaccharides and amino acids called peptidoglycan. The CV-I complex formed by the crystal violet and iodine is larger than the crystal violet molecule and cannot be washed out of the peptidoglycan layer. Gram-negative cell walls have a much thinner layer of peptidoglycan, which results in the CV-I being washed out with the alcohol. Gram-negative bacteria are more resistant to antibiotics than Gram-positive bacteria (Tortora et al., 2013).

The acid-fast stain helps to distinguish different bacteria based on the wax content of their cell walls. Bacteria with a high wax content are called acid-fast bacteria and do not stain with the Gram stain method. These bacteria retain the red primary stain carbolfuchsin. Bacteria that have low wax content in their cell walls are called non-acid-fast. These bacteria lose the primary stain and take up the counterstain, methylene blue. This staining method is used to identify Mycobacterium, which includes Mycobacterium tuberculosis and Mycobacterium leprae (Alexander & Strete, 2001).

Structural stains, which include the capsule and spore stain, are used to examine the structure of bacteria. “Some bacteria release polysaccharides and polypeptides during growth. These substances accumulate around the cell to form a structure called a capsule” (Alexander & Strete, 2001, p. 43). A special staining method referred to as the capsule stain is needed to view the capsules because simple stains to not adhere to the capsule due to their nonionic nature (Johnson & Case, 2011). Capsule stains use a combination of negative and simple stains. Acidic stains such as Congo red and India ink are used to color the background while a simple stain is used to color the bacteria. The capsules do not accept the stain and appear as a halo between the stained cells and background (Alexander & Strete, 2001). “Demonstrating the presence of a capsule is a means of determining the organism’s virulence, the degree to which a pathogen can cause disease” (Tortora et al., 2013, p. 70).

The spore stain used to view the endospore formation of bacteria. This is a characteristic that can aide in identifying some bacteria. Endospores are highly resistant dormant structures that form within some vegetative bacteria cells such as Bacillus subtilis and Clostridium botulinum in a process called sporogenesis. Free spores are released after the cell disintegrates (Alexander & Strete, 2001). Endospores protect bacteria from many adverse environmental conditions. A special spore staining method is used because basic stains cannot penetrate the endospore walls. The Schaeffer-Fulton endospore stain is a commonly used method which uses malachite green as the primary stain and applying heat to penetrate the spore wall. The malachite green is then washed off which removes the stain from all part of the cell except the endospores. Safranin is then used as the counterstain to stain the other portions of the cell. The endospores will appear as green and oval-shaped and the rest of the cell will appear red (Tortora et al., 2013).

The following staining methods will be carried out on various bacterial cells: Simple stain, Gram stain, negative stain, spore stain, and capsule stain. The stained bacteria will then be examined through a compound light microscope. A prepared slide of acid-fast stained bacteria will be also be viewed. Various characteristics of the bacterial cells will be then be studied and noted.

Materials and Methods
The following bacteria cultures were used: Staphylococcus epidermidis, Escherichia coli, Bacillus subtilis, Enterobacter aerogenes, and a prepared slide of Mycobacterium. Unknown bacteria cultures were also used. The following equipment was used: Compound light microscope, slides, a ceramic heater, inoculating loops, wash bottle of distilled water, methylene blue, crystal violet, Gram’s iodine, safranin, ethyl alcohol, India ink, acid-alcohol, malachite green Congo red, acid fuchsin, carbolfuchsin, beaker. Separate slides were used for each culture.

The smears were prepared using the method in the lab book listed on page 24 from a solid medium. Instead of using 2 loopfuls of water, only 1 was used. A ceramic heater was used in place of the Bunsen burner.
The simple stain was carried out following the procedure on page 26 of the lab book except that the bacteria used were Staphylococcus epidermidis, Escherichia coli, and unknown bacteria. The Gram stain was performed following the procedure on page 30 of the lab book except that unknown bacteria were used in place of the Bacillus subtilis. The negative stain was performed following the procedure in the lab book on pages 35 and 36 except that Bacillus subtilis was not used and unknown bacteria were used in place of the mouth bacteria. India ink was used in place of nigrosine. Broth culture and water were not used. The acid-fast stain was not performed. A prepared slide of Mycobacterium was viewed instead. The capsule stain was performed following the procedure on page 49 of the lab book except S. epidermidis was used in place of S. salivarius. Unknown bacteria were also used. Instead of drawing two circles on the slide, separate slides were used for each of the bacteria. The spore stain was performed following the procedure on pages 47 and 48 in the lab book except that heat fixing was not done to the smear and a separate slide was used for each bacterium. E. coli was used instead of Bacillus megaterium and only one Bacillus subtilis was used. Unknown bacteria were used as well. All the slides were labeled with a permanent marker and the inoculating loops were sterilized using the ceramic heater after each smear. All slides were observed through the oil immersion objective with 1000x magnification.

Results
Using the simple stain method, the E. coli appeared blue and could clearly be observed as rod shaped (Fig.1). The S. epidermidis appeared blue and could clearly be observed as spherical shaped (Fig. 2). The unknown bacteria appeared blue and could clearly be observed as rod shaped (Fig. 3). It also contained colorless oval-shaped objects within the rods.

Using the Gram stain method, the S. epidermidis could be observed as red with a spherical shape (Fig. 5). The E. coli could be observed as red and rod shaped (Fig. 4). The unknown bacterium was observed as red and rod shaped (Fig. 6). The prepared slide of Mycobacterium showed thin red rods that were faint in appearance (Fig. 7).

Using the negative stain method, the S. epidermidis was observed as colorless spheres within a black background (Fig. 8). The unknown bacteria were observed as colorless spheres and rods within a black background (Fig. 9).
Using the capsule stain method, the Enterobacter aerogenes was observed as red rods against a blue background (Fig.10). Colorless halos were observed around the red rods. The S. epidermidis was observed as cocci against as sphere-shaped objects against a blue background (Fig. 11). It contained colorless spots throughout the slide. The unknown bacteria were observed as red rods with white halos and white oval-shaped objects (Fig. 12).
Using the spore stain method, the Bacillus subtilis was clearly observed as red rods with green oval-shaped objects within the rods (Fig. 13). There were also green oval-shaped objects observed by themselves. The E. coli was clearly observed as red rods (Fig. 14). The rods did not contain any oval or spherical-shaped objects. There was however, a green shaded area between the rods. The unknown bacterium was clearly observed as red rods with green oval-shaped objects within the rods (Fig. 15).

Discussion
The simple stain made it possible to clearly observe the shapes of the bacteria. The methylene blue provided a clear contrast with the background making it possible to observe E. coli as rod shaped, which was the expected result (Alexander & Strete, 2001). The S. epidermidis was observed as coccus shaped, which was the expected result (Alexander & Strete, 2001). The unknown bacterium was observed as rod shaped. The uncolored oval-shaped objects in the center of the rods may be spores, which means the bacteria entered a vegetative state and the spores formed to protect the cell in harsh environmental conditions.

The Gram stain made it possible to observe the shape of the bacteria as well as to classify them as Gram-positive or Gram-negative. The S. epidermidis was observed as coccus shaped and red, which indicates a Gram-negative result. This was not the expected result since S. epidermidis is Gram-positive (Alexander & Strete, 2001). The culture being used could have been older, making it not retain the primary stain, giving an inaccurate result (Johnson & Case, 2011). The E. coli was observed as rod shaped and red, indicating a Gram-negative result. This was the expected result (Alexander & Strete, 2001). The unknown bacterium was observed as rod shaped and red, indicating a Gram-negative result. The Gram-negative bacteria had the crystal violet stain washed out because they have a thin layer of peptidoglycan in their cell walls. They will also be more resistant to antibiotics than Gram-positive bacteria. The Gram-positive bacteria would have a thicker layer of peptidoglycan in their cell walls, which prevents the primary stain from washing out. The prepared slide of Mycobacterium tuberculosis was rod shaped and red in appearance, indicating an acid-fast result, which was the expected result (Alexander & Strete, 2001). This indicates that it has a high wax content in its cell wall and would not stain with the Gram stain.

The negative stain made it possible to observe the shape of the S. epidermidis and the unknown bacteria. The S. epidermidis was coccus shaped, which was the expected result (Alexander & Strete, 2001). The unknown bacteria were mixed coccus and bacillus. The negative stain was not able to more accurately observe the shape than with the simple stain. The black background was inconsistent, making it hard to distinguish the colorless shapes. This was not the expected result since the negative stain is supposed to give a more accurate view of the shapes than the other staining methods. This could be due to the nigrosin not being evenly distributed on the slide.

The spore stain made it possible to observe spores in the bacteria. The Bacillus subtilis was observed as red and rod shaped. Endospores were present as green oval-shaped objects within the rods. This was the expected result since Bacillus subtilis is spore-forming-bacteria (Alexander & Strete, 2001). This indicated that the cells of the bacteria are now more resistant to harsh environmental conditions. There were also free spores observed which means some of the cells disintegrated. This suggests that the bacteria were from an older culture. This was the expected result since older cultures are more likely to contain more spores (Alexander & Strete, 2001). The E. coli appeared as red and rod shaped. The spores were not present inside the rods. This was the expected result since E. coli is non-spore-forming bacteria (Alexander & Strete, 2001). There was however, a shaded green area which was not the expected result since E. coli is non-spore-forming bacteria. This could be from not rinsing the malachite green adequately enough. The unknown bacteria appeared as red and bacillus shaped. Spores were present which means the bacteria is more resistant to the harsh environment.

The capsule stain helped observe the presence of capsules around the Enterobacter aerogenes. The capsules appeared as a white halo around the rod-shaped bacteria because capsules do not accept most stains. This was the expected result (Alexander & Strete, 2001). The presence of capsules indicates the bacteria has an enhanced ability to cause disease. It was difficult to observe the presence of capsules in the S. epidermidis. The colorless spots were hard to distinguish as halos. The presence of capsules appearing as colorless halos around the bacteria was the expected result (Alexander & Strete, 2001). The unknown bacteria contained colorless halos around the bacteria, indicating the presence of capsules. It also contained colorless oval-shaped objects. They may have been capsules as well, or spore formation.

The various staining methods allowed an examination of different characteristics of the bacteria. Each staining method emphasized certain structures. The characteristics examined provided important clues about the nature of the bacteria. This can be helpful in classifying the bacteria. It can also give important information on the severity of a disease or how to treat various diseases. It is also important to perform the staining methods multiple times and not rely on initial results. Various factors can skew the results, leading to false conclusions.

References
Alexander, S. K., & Strete, D. (2001). Microbiology: A photographic atlas for the laboratory. San Francisco: Benjamin Cummings.

Johnson, T. R, & Case, C. L. (2011). The pearson custom library for the biological sciences. Boston: Pearson.

Tortora, G. J., Funke, B. R, & Case, C. L. (2013). Microbiology: An introduction (11th ed). Boston: Pearson.

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