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Laboratory report of exercises 3-5, 3-6, and 3-7 for Microbiology. Taken from the text Microbiology laboratory theory and approach....

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Report 1: Exercises 3-5, 3-6, 3-7 Microbiology Lab 120:335:001 Jessica Aseng 15 October, 2019 TA Alexandra Adams

Objective

Aseng 2 Exercise 3-5 dealt with simple staining in order to view the cell morphologies and arrangements of various organisms. Students used the charges present on the stain and the membranes to color the cells. The objective of exercise 3-6 was to use negative staining techniques to compare the shape and dimensions of three different organisms. In exercise 3-7, students used a differential stain called Gram staining to determine whether organisms are Gram-positive or Gram-negative. With this stain, students can also determine cell morphology, size, and arrangement.

Introduction/Background Exercise 3-5: Simple Stains The stains that are used in this exercise are made up of a solvent and a chromogen, the colored component. A chromophore is the part of the chromogen that gives the stain its color. Students used basic stains which, in this case, are positively charged and are drawn to the negative charges on bacterial cells, which allows the cells to soak up color, leaving the environment transparent. In order to make the bacteria stick to the slide, the smears must be heatfixed; this kills the bacteria as well as coagulates proteins in the cytoplasm to make them easier to view. Popular basic stains include methylene blue, crystal violet, and safranin. This method of staining is used to determine cell morphology, size and arrangement. In order to view these organisms, students must place a drop of water on a slide using the inoculating loop and then mix bacteria into the water after running the loop over the flame. The smear will now air dry and passed through the flame to heat-fix the cells. Once the slide is heatfixed, students placed drops of crystal violet, safranin, or methylene blue on each organism.

Aseng 3 Using the oil immersion lens, the slides can be observed. Looking into the microscope, students can record observations of cell morphology, arrangement, and the size of the cells. No matter the stain, students should see that the individual cells on the slide are colored and not the background. Because of the technique, the cells may be distorted to some degree.

Exercise 3-6: Negative Stains Unlike simple stains, the chromogen in negative stains have a negative charge. This trait allows the negative bacterial cells to repel the negative chromogen, staining the background and leaving the cell untouched. This technique is used on bacteria that are unable to go through heatfixing. The cellular arrangement and morphology of the bacteria can be observed while preventing cell shrinkage. Students will use the negative staining technique on three different specimens: Bacillus cereus, Micrococcus luteus, and Rhodopospirillum rubrum. To carry out the experiment, a drop of acidic stain is placed at one end of a slide and one of the organisms is mixed into the drop with a loop. A second clean slide is then placed against the first slide and dragged across to spread out the stain mixed with bacteria. The stain is left to air dry and then observed under the microscope using the oil immersion lens. When looking through the lens, the bacterial cells should be observed to be unstained against a dark background. Students can determine the cell morphology and arrangement as well as the cell dimensions using the ocular micrometer.

Exercise 3-7: Gram Stain

Aseng 4 The gram stain is a differential stain that allow researchers to observe differences between organisms or differences in the same organism. It works with a decolorization step that occurs between two basic stains. The first, or primary, stain is crystal violet followed by iodine which acts a mordant to make the crystal violet stronger. Decolorization happens next which affects Gram-negative cells and not the Gram-positive cells. This allows the Gram-negative cells to be colored by the red counterstain safranin. In the end. Gram-positive cells look purple and Gram-negative cells are reddish-pink. The colorization of both types of cells is possible because of the lipid content in both Gram-positive and negative cells. Gram-negative cell walls have more lipid content with a thinner peptidoglycan layer than its counterpart. The alcohol component in the decolorizer removes the lipid which lets the crystal-violet stain leak out of the Gram-negative cell walls. The thicker peptidoglycan property of Gram-positive cell walls let these bacteria retain the purple color. In addition to determining the Gram reaction, this staining technique can be used to determine cell morphology size and arrangement. When observing the outcome of the stains, students should see that bacterial smears are either purple or reddish-pink. This can be viewed by looking into the oil immersion lens.

Results Exercise 3-5: Table 1. Observations and Interpretations of Simple Stains Organism Bacillus cereus

Stain Safranin

Cellular Morphology and Arrangement ● Rod-shaped with square

Cell Dimensions (micrometers) Width = 0.025 Length = 0.1

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ends ● Observed in small clumps and short chains Micrococcus luteus

Safranin

● Spherical ● Found in clusters

Diameter = 0.0375

Escherichia coli

Methylene blue

● Rod-shaped ● Some are seen individually, others are found in clumps

Length = 0.25 Width = 0.075

Vibrio fischeri

Methylene blue

● Rod-shaped

Length = 0.01 Width = 0.005

Rhodospirillum rubrum

Crystal violet

● Spiral shape ● Flagellated

Length = 0.4 Width = 0.005

Staphylococcus saprophyticus

Crystal violet

● Globular shaped

Diameter = 0.15

Table 1 shows six different bacterial organisms undergoing the simple staining technique in order for student to observe morphology and measure the cellular dimensions.

Exercise 3-6: Table 2. Observations and Interpretations of Negative Stains Organism

Micrococcus luteus

Stain

Nigrosin

Cellular Morphology and Arrangement

● Spherical ● Cells are

Cell Dimensions from Negative Stain (micrometers)

Cell Dimensions from Simple Stains

Diameter = 0.05

(see Table1)

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bunched together Bacillus cereus

Nigrosin

● Rod-shaped ● Small clumps and chains

Length = 0.05 Width = 0.015

(see Table1)

Rhodospirillum rubrum

Nigrosin

● Spiral shape

Length = 0.25 Width = 0.005

(see Table1)

Table 2 depicts the observations from negative staining of bacterial cells. These organisms were used to compare to the same organisms that were used in the basic staining technique in Table 1. The cell dimensions measured in both techniques are similar.

Exercise 3-7: Table 3. Observation and Interpretations of Gram Stains Organism of Source

Cellular Morphology and Arrangement

Color

Gram Reaction (+/-)

Escherichia coli

(see Tables above)

Pink

Negative

Staphylococcus saprophyticus

(see Tables above)

Purple

Positive

Escherichia coli

(see Tables above)

Pink

Negative

Bacillus cereus

(see Tables above)

Purple

Positive

Table 3 shows the effects of Gram-staining various types of bacteria. If the individual bacterium were stained pink, then the organism is Gramnegative. If the cells were stained purple, then the organism is Gram-positive.

Discussion/Conclusion Exercise 3-5 After students used the oil immersion lens to observe the bacteria that were covered by simple stains, the cellular morphology and arrangement were recorded in Table 1. As shown, the six organisms were also measured for cell dimensions. Bacillus cereus was found to be rodshaped and attached to many other cells (chains). Researchers may be looking for this bacteria if

Aseng 7 a patient presents with abdominal pain and associate diarrhea; B. cereus is commonly found in the soil and vegetation and can be easily contracted (Tajkarimi, 2007). Micrococcus luteus was stained in Safranin, like B. cereus, and cells were found to be spherical. Finding these cells in the cultures of patients can be a little more unnerving because they can be the cause of pneumonia or meningitis (Hetem et. al, 2017). I used Methylene blue on the next two organisms Escherichia coli and Vibrio fischeri. Both were observed to be rod-shaped in the simple stain. The third and final stain used was Crystal violet, staining Rhodospirillum rubrum and Staphylococcus saprophyticus. R. rubrum was observed to be spiral-shaped with a singular flagella attached to each cell, meaning that the cells had motility. S. saprophyticus was globular-shaped, or spherical, and found in clumps.

Exercise 3-6 Negative stains are different from simple stains in that the bacteria are not heat-fixed to the slide. This technique provides a more accurate size of the observed cells because the lack of heat minimizes cell shrinkage. Students should have seen slightly larger cell sizes in the negative stain. Negative stains are also different in that Nigrosin stains the background and leaves the cells themselves untouched; though the staining technique is different, the morphology of the cells should not be different than the simple stain observations. In addition, ocular units were used when initially measuring the cells of the organisms but were later converted to micrometers using the equation 1 OU = 0.5 micrometers. Only three organisms were observed with Nigrosin: Micrococcus luteus, Bacillus cereus, and Rhodospirillum rubrum. M. luteus was examined to be spherical and clumped together, similar to the basic stain, and its cell dimensions was 0.05 in diameter. The average cell

Aseng 8 dimension from the simple stain was 0.0375; as expected, the dimensions of the simple stain are smaller than the negative stain because of the heat applied. The dimensions are similar enough in size though that the measurements appear accurate. B. cereus had similar cell morphology to the basic staining technique as well as the cell dimensions: in the simple stain, the length 0.025 and width = 0.1. The length of the negative stained cells was 0.05 and width = 0.15. However, the measurement differences themselves were not as expected. The simple stain dimensions should have been smaller than the negative stained cells. Issues may have come up in the simple stain when heat-fixing bacteria, the bacteria may not have been held against the flame long enough to adhere to the slide which would not have let them shrink. In the negative stain, I may have made a mistake during the staining process; perhaps I measured the cells incorrectly using the ocular measuring tool. Observations of R. rubrum revealed spiral-shaped cells with an average length of 0.25 and width of 0.005. The cell dimensions of the same bacteria from the simple stain were 0.4 and 0.005 width. Again, the dimensions were not fully expected, though they are similar in size, the basic stained cells were predicted to be smaller than the negatively stained R. rubrum. As stated above, mistakes may have been made when heat-fixing the slides in the simple stain or measurements that were taken both during the basic stains and negative stains. Exercise 3-7 Gram staining is one of the most important techniques in microbiology and medicine. It is termed a differential stain because it is used to differentiate between Gram-positive and Gramnegative organisms which are categorized based on their respective cell walls. Because of their differences in cell wall structure, it affects how the cells absorb and retain stains. The Grampositive bacteria will stain purple as a result of the thick peptidoglycan layer in the cell wall. The ethyl alcohol that is poured over the slide after the crystal violet shrinks the peptidoglycan layer

Aseng 9 which causes the stain to stay trapped in the cell wall of Gram-positive bacteria (Gram Stain). Adversely, Gram-negative bacteria will not be able to retain the crystal violet of the primary stain because of its thin peptidoglycan layer. Safranin is added to the slides because it is a lighter stain and will not affect the colorization in the Gram-positive cells, but will stain Gram-negative cells pink. Gram staining helps scientists by establishing a bacterial cell’s wall and membrane which has an affect on the pathogenicity and level of virulence of the bacteria (Gram Stain). It allows scientists to determine if a patient has a bacterial infection and what kind of bacteria is causing the infection. Figuring out the category of bacteria that is infecting a patient is important because physicians can determine what kind of antibiotics to use to treat the illness. When performing the experiment, two slides were used with two organisms on each slide, one Gram-positive and one Gram-negative to make the differences in stain colors apparent. The first slide was emulsified with E.coli and S. saprophyticus; the former was observed to be pink indicating that it is Gram-negative and the latter was purple or Gram-positive. E.coli was ruled to have a thin peptidoglycan layer which only picked up the color of the counterstain while S. saprophyticus has a thicker peptidoglycan layer in its cell walls so it easily retained the purple stain. The second slide was emulsified with E.coli and B. cereus. staining revealed that B. cereus is Gram-positive as opposed to the pink cells of E. coli. B. cereus has a thicker peptidoglycan layer in its cell wall and can retain the Crystal violet stain much better than E. coli. Gram-negative bacteria are generally believed to be more dangerous as diseases because the outer membrane is kept hidden by a capsule. This camouflages the antigens of the cells so the human immune system cannot recognize it; the bacteria spread throughout the body undetected by the defense of humans to cause infection. Thus, Gram-staining is important because it

Aseng 10 determines what bacteria is present and how best to treat it.

References “Gram Stain - Purpose, Procedure and Preparation.” MicroscopeMaster, www.microscopemaster.com/gram-stain.html. Hetem, David, and Miquel Ekkelenkamp. “Micrococcus.” Micrococcus, 2017, www.sciencedirect.com/topics/immunology-and-microbiology/micrococcus. Leboffe, Michael J., and Burton E. Pierce. “Bacterial Structure and Simple Stains, Differential and Structural Stains.” Microbiology: Lab Theory and Application, Fourth ed., MORTON Publishing COMPANY., 2015, pp. 185–202. Tajkarimi, Mehrdad. “Bacillus Cereus.” California Department of Food and Agriculture, 25 Apr. 2007, www.cdfa.ca.gov/ahfss/Animal_Health/PHR250/2007/25007BcerMH__2_.pdf....