Lab 4 - Lab Report on Acid-Fast Stain, Structural Stain, and Special Media for Isolating PDF

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Lab Report on Acid-Fast Stain, Structural Stain, and Special Media for Isolating Bacteria...

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Introduction: The acid-fast stain is a differential stain that’s used to identify acid-fast organisms like Nocardia and Mycobacterium. The cell walls of acid-fast organisms have mycolic acid that makes the cell wall impermeable to most stains. Due to this, it’s thought to be a factor in the resistance of Mycobacterium in harsh, dry environments. The cell wall is so airtight that before the mycobacteria is cultured, it’s treated with strong sodium hydroxide to remove debris and bacteria that’s contaminating, but the mycobacteria aren’t killed by this procedure. In the Ziehl-Neelsen procedure, the smear is covered with carbolfuchsin, and then heat fixed. The stained smears are then washed with an acid-alcohol mixture that able to easily decolorize most bacteria except for the acid-fast microbes. Methylene blue is then used as a counterstain that’s able to make it possible to observe the non-acid-fast organisms. In the Kinyoun modification—also a cold stain—the concentrations of both the carbolfuchsin and phenol are increased so heating isn’t necessary at all. Structural stains are used to study and identify the structure of bacteria. Endospores are called “resting bodies” because they don’t metabolize, and are resistant to numerous things including heating, various chemicals, and harsh environmental conditions. They can’t reproduce and are only formed when water or other essential nutrients aren’t available. They can be dormant for a long time, but once they form in a cell, the cell will disintegrate. Endospores are impermeable to most stains, so they’re heat-fixed to penetrate the stain, and don’t decolorize once they’re stained. A capsule is a bacterium that secretes chemicals that adhere to their surface, when it’s round/oval shaped. The size of the capsule is determined by the medium on which it’s growing. Most capsules are made of polysaccharides, that are water soluble and uncharged. Due to this, simple stains don’t adhere to it. When bacteria are motile, it means they can move from one place to another. Most of these bacteria have flagella—thin structures that project out from the cell wall and start in the cytoplasm. Flagella

can be stained after being flooded with a mordant, which increases their diameter. There are two main types of flagella: peritrichous (all around the bacteria), and polar (at one/both ends of the cell). One of the biggest limitations of dilution techniques used to isolate bacteria is that organisms that are present in limited amounts may be diluted onto plates. These plates are filled with dominant bacteria. Selective media contains chemicals that prevents the growth of bacteria that’s unwanted, without disturbing the growth of the wanted organism. Enrichment media contains chemicals that increases the growth of the wanted bacteria. Other bacteria will be able to grow, but the growth of the wanted bacteria will be enhanced. Differential media contains nutrients that allows the observer to tell the difference one bacterium from another, and how they’re able to metabolize or change the media with a waste product. Nutrient agar is a solid medium that makes it useful in culture media. Mannitol salt agar is both a selective and differential growth medium that promotes the growth of certain bacteria while restricting the growth of others. EMB agar is a selective stain that’s used for gram-negative bacteria and is both a selective and differential medium. The acid-fast stain purpose is to distinguish between organisms that can resist decolorization with acid alcohol. The purpose of isolating bacteria is to be able to observe the different bacteria without having to worry about contamination. The purpose of this lab was to identify the bacteria in the Mycobacterium and Nocardia genus. Another purpose was to identify and study the structure of bacteria (endospore, capsule, flagella). The final purpose was to differentiate selective and differential media, and to provide an application for selective media. I hypothesized that the general shape for the acid-fast positive stain would be a large clump of dots, the shape of the acid-fast negative stain would also be a large clump of dots, the endospore would be visible because the stain would be able to penetrate it, the capsule would be visible in a shape of cylindrical shapes, and the flagella would be visible in the shape of wiggly lines due to

flagella typically being shaped like that. I also hypothesized that the MSA agar would be selective towards the gram negative bacteria, the NA agar would be selective towards the bacteria, and the EMB agar would be selective towards the gram positive bacteria. Materials and Methods: The materials and methods used were in the lab manual. Results: The acid-fast positive stain was stained a very bright pink and appeared to be in clumps There were large groups of the bacteria, and it had the general shape of swirls and dots outlining it. Most of the bacteria was concentrated towards the middle, and then spread out. The length of one “clump” of bacteria appeared to be about 4 mm. Even though the acid-fast negative stain couldn’t be seen, it generally is blue and kind of pink in color. A picture taken from a secondary website through Google was used to observe. There are medium-sized dots usually covering the entire slide, and a lot of bacteria is present. There is concentration in some places, and not any concentration in others. The length of a large area of bacteria appeared to be about 8 mm. The endospore was blue in color, but was stained pink is some places as well. The shape consisted of swirls and a variety of lines that were u-shaped, and small dots towards the ends. There were clumps of bacteria in some areas but was spread out evenly otherwise. The length of a large area of the endospore appeared to be 2 mm. The capsule was stained pink itself, and the entirety of the background was also stained pink. The area that was surrounding the capsule was white. The capsules shape was like an elongated-oval. There weren’t any areas where there was a large “clump” of bacteria present—it seems like they were all evenly distributed. The length of one capsule appeared to be about 0.75 mm.

The flagella itself was stained pink, and the background was white. The shape of the flagella seemed to be “squiggly-lines.” Throughout the flagella, however, there appeared to also be airbubbles present, but it’s unclear if they’re meant to be present or if it was a mistake that was made. There wasn’t a large area of bacteria that was present, as it seems like it was evenly spread out. The length of one flagellum appeared to be about 3 mm. For this lab, the broth A mixture was used. For the NA agar, there was growth on all the plates containing Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas fluorescens as well as the unknown mix. There seemed to be consistent color of yellowish-white on all the plates as well. For the MSA agar, there was no growth on the E. coli and P. fluorescens plates. The color was mostly consistent throughout the plates, with S. aureus and S. epidermidis being yellow, and the rest of the plates being a mixture of red and yellow. For the EMB agar, there wasn’t any growth on the S. aureus and S. epidermidis plates, but there was growth on the other plates. There also was no color on S. aureus and S. epidermidis, but the rest of the plates had a dark purple color. Discussion: Acid-fast organisms have mycolic acid in their cell walls, which helps with adhering a stain into those cells that are resistant to decolorization. The purpose of heating during the staining procedure using the Ziehl-Neelsen method was to “melt” the cell wall, and the wax present in it, to allow the stain to enter the cell. The Kinyoun method doesn’t require heat in the staining process. However, none of these methods were used, because a prepared slide was already provided. Mycobacterium that was observed under the microscope with a 1000x magnification and immersion oil showed an acid-fast positive reaction, because the color was reddish-purple. This meant that the mycobacterium had mycolic acids in their cell walls, because the carbolfuchsin stain was able to enter the cell and stain it.

A negative stain uses a dye that “repels” certain bacteria—which results in the bacteria being unstained and the background being stained. In this case, only the capsule was negatively stained. Endospores are usually a dormant form of bacteria that’s resistant to harsh and dry environments. Since endospores are impermeable to most stains, the slide is heat-fixed for the stain to actually penetrate into the endospore. Once stained, they don’t decolorize so it’s easy to observe under a microscope. Capsules have a nonionic nature, so simple staining doesn’t work. Most of the techniques that are used to stain capsules, stain only the bacteria and the background so it’s easier to see the unstained capsules. Flagella is a common way for motility. Due to their fragility, they’re not visible with a light microscope. However, they can be stained with mordant, which increases their diameter and makes it kind of easier to see. Flagella usually is stained a crystal-violet color, which makes it more visible to see. To identify the two organisms in the “unknown mix,” which was E. coli and S. aureus, E. coli would have a green color growth on the EMB agar and the S. aureus would have a yellow color growth on the MSA agar. It was found that EMB is selective for gram-bacteria, and MSA is selective for gram- and gram+ bacteria—meaning gram negative bacteria grows well on the EMB agar, and gram-positive bacteria grows well on the MSA agar. Due to this, E. coli will use lactose as an energy source, and S. aureus uses mannitol. Both EMB and MSA agar are selective medias, and the purpose of using agar in the media was to use it as a solidifying agent. It was found that the MSA plate was selective against all the bacteria except for S. aureus and S. epidermidis. It was also found that there’s peptone present in the agar. Due to this, the peptone can provide nutrients and appropriate agar to the bacteria—it then becomes their main source of carbon and nitrogen, and it helps the culture to grow more. It’s possible to use acid-fast staining in a variety of ways in the health-care field. It’s mainly used for lab tests that helps to determine if tissue, blood, or any other bodily substance is infected

with the bacteria that causes TB and other illness. The role of structural staining is also useful in healthcare because it plays an important role in the control of bacterial infections, isolation of bacteria, and the identification of microorganisms. Information that’s provided by structural staining will help to identify the microorganism and help determine what the next step in care is. Selective media helps to isolate one type of bacteria to help it grow and be able to be observed. Differential media helps to differentiate bacteria due to change in color or growth that helps to distinguish between the groups of organism’s present. Enriched media helps to grow organisms quicker than they normally do so it’s easier to distinguish between certain diseases.

LITERATURE CITED

Johnson, & Case. (2018). Microbiology Lab Manual (11th Edition). Boston: Pearson Collections. Amazon Kindle. Bacteria Staining (Gram, Acid-fast, Endospore). (2016, February). Retrieved October 24, 2018, from https://www.scienceprofonline.com/sciene-image-libr/sci-image/libr-bacterialstaining.html

PICTURES AND FIGURES Figure 1.1—Capsule at 100x magnification w/ immersion oil

Figure 2.2—endospore at 100x magnification w/ immersion oil

Figure 3.1— Acid-Fast Positive at 100x magnification w/ immersion oil

Figure 4.1— Acid-Fast Negative at 100x magnification w/ immersion oil ¹

_________________________________________________________________________________ ¹ Bacteria Staining (Gram, Acid-fast, Endospore). (2016, February). Retrieved October 24, 2018, from https://www.scienceprofonline.com/sciene-image-libr/sci-image/libr-bacterialstaining.html Figure 5.1—Flagella at 100x magnification w/ immersion oil

Figure 6.1—Special Media for Isolating Bacteria Table

Control Plates

Strain Expected Gram Classification Observed on slide (Gram +/-) NA Growt Agar h (Y or N) Appear ance/ color MSA Growt agar h (Y or N) Color media EM Growt B h (Y or agar N) Colony Color

Staphylococcus aureus Gram positive

Staphylococcus epidermidis Gram positive

Experimental

Escherichia coli Gram negative

Pseudomonas fluorescens Gram negative

Plate Unknown mix Both +/-

Yes

Yes

Yes

Yes

Yes

Yellowishwhite

Yellowish-white

Yellowishwhite

Yellowishwhite

Yellowishwhite

Yes

Yes

No

No

Yes

Yellow

Yellow

Red + yellow

Red + yellow

No

No

Red + yellow Yes

Yes

Yes

None

None

Dark purple

Dark purple

Dark purple

Figure 7.1—Experimental Plate (NA agar) [broth]

Figure 7.2—Control Plate (NA agar) [S. aureus, S. epidermidis, E. coli, P. fluorescens]

Figure 8.1—Experimental Plate (MSA agar) [broth]

Figure 8.2—Control Plate (MSA agar) [S. aureus, S. epidermidis, E. coli, P. fluorescens]

Figure 9.1—Experimental Plate (EMB agar) [broth]

Figure 8.2—Control Plate (EMB agar) [S. aureus, S. epidermidis, E. coli, P. fluorescens]...