Gram Staining Technique On Unknown Bacteria Biology Essay

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Morphologically speaking, bacteria come in different shapes. The following bacteria are gram positive and rod shaped: C. diptheriae, B. subtilis, and L. acidophilis. The following bacteria are gram positive and have a coccus shape: M. roseus, S. epidermidis, S. aereus, E. faecalis .The following bacteria are gram negative and rod shaped: P. aeruginosa, B. cepacia, C. freundii, E. aerogenes, E. coli. The following bacteria are gram negative and have a bacillus shape: P. vulgaris, and S. marcescens. In order to use morphology as a means to identify bacteria it crucial to know a bacteria's morphological structure and this includes its shape (Keplit).

Biochemically speaking, gram positive bacteria possess a cytoplasmic lipid membrane and a thick peptidoglycan layer with many cross linkages. Other common characteristics amongst some gram positive species are the presence of capsulated polysaccharides and a flagellum. The aforementioned peptidoglycan layer contains teichoic acids and lipoids; moreover, their presence forms lipoteichoic acids. These lipoteichoic acids are, in essence, chelating agents (Madigan). Identification of gram positive bacteria, through a biochemical perspective, can be achieved by observing the presence of a thick peptidoglycan layer with cross linkages and the presence of teichoic acids.

The 16S RNA is an integral component of the ribosomal RNA. As far as the 16S rRNA is concerned in the identification of bacteria, the 16S rRNA can have many sequences within a bacterial cell, and this allows for the identification of various types of bacteria. Recent advances in the 16S rRNA have indicated that PCR primers have been utilized to increase the 16S rRNA gene in order to supply information that can be used to differentiate between bacterial types. Moreover, primers are used to study the 16S rRNA gene; also, the 16S rRNA contains primer binding sites. PCR is a new technique used to identify bacteria. Scientists are able to do this by identifying the presence of hypervariable regions that exist within the 16S rRNA gene sequence. These hypervariable regions contain explicit sequences that are particular to certain types of bacterial species. As a result, bacterial identification on a species wide basis can be achieved by using PCR technique (Weisburg).

Respiratory organisms obtain their energy through the use of electrons. Respiratory organisms transfer electrons from energy sources to electron acceptors. These electron acceptors are then used for either aerobic or anaerobic respiration (Zillig). According to Dr. Maxwell's laboratory notes on respiratory microorganisms, an example of a respiratory microorganism is Streptococcus pneumonia.

Materials and Methods:

According to Dr. Maxwell's laboratory notes for the respiratory lab, the following materials were used for the respiratory lab: two sterile swabs, one blood agar plate, one tube of saline solution, two tubes of 9 ml sterile water, two liquefied TSA, and two sterile Petri dishes. The following procedure was completed for the respiratory lab: students swabbed the tonsialary region of their lab partner by making a rolling motion up the tonsilary region. Next, the swab was removed and inoculated onto the media. Of the two swabs that were used, the first swab was used to tell if any of the culture was hemolytic. The microorganisms that were collected form the swabs were inoculated onto a blood agar plate using the streaking for isolation method. This blood agar plate was then incubated in a candle jar. After the colonies were grown on the plate and isolated, isolated colonies were identified and checked for hemolytic activity. The isolated colonies were observed and then examined for alpha hemolysis, beta hemolysis, or gamma hemolysis. Gram stains were then made for beta hemolytic or alpha hemolytic colonies. If a beta colony was observed and it was gram positive, then it is likely that the organism is Streptococcus pyogenes. Concerning the second swab, it was used for finding out the population density of the microorganisms from the throat. Aseptic techniques were used when placing the swab into a test tube of sterile water. This test tube was then swirled to get the organisms into the water. Next, dilutions were carried out so that there was one test tube that had a 10-3 dilution and the other test tube had a 10-4 dilution. Afterwards, a pour plate was made for both the 10-3 dilution and the10-4 dilution. After the colonies grew, they were counted to find out the population density present. The lab partners then compared their respective population densities. It is important to note that gram stains of the respiratory organisms were not performed due to limited time and the need to initiate the unknown experiment.

The following is the dichotomous keys used to determine the unknown. Note, the first test used to identify the unknown was the gram staining test. Based on the results of the gram staining procedure, the bacteria were classified as either a gram positive or gram negative bacteria.

The following tests were completed in order to identify the unknown organism: Gram stain, lactose fermentation, casein digestion, acid released form galactose test, catalase test, indole test, glucose test, citrate test, growth on TSA plate test, MR/ VP test, and manitol test. For the procedures for the aforementioned tests are listed below; in addition, the source for these procedures is from Dr. Maxwell's laboratory notes. The source for the gram staining procedure is Dr. Maxwell's laboratory notes for lab 4. The source for all of the remaining biochemical tests is Dr. Maxwell's laboratory notes for lab 9.

Gram staining procedure:

First a bacterial smear is created. It is important to note that only a small sample of bacteria is needed. In order to get a small amount of bacteria, gently touch the inoculating loop to the bacterial colony. Allow this smear to air dry for about 10-15 minutes. After this, the smear is heat fixed by passing the slide over the flame 2-3 times, and the slide is then cooled. Next, the dyes are used. The smear is first covered with the primary stain- crystal violet for 60 seconds. The slide is then rinsed with water using a rinse bottle; in addition the slide is rinsed until the water is clear or pale lavender. Afterwards, the slide is covered with the mordant, which is gram's iodine, for 60 seconds. The slide is then rinsed with water. Next, the decolorizer is added and kept on the slide for less than 20 seconds, and then the decolorizer is rinsed off with water. Next, the counter stain, which is safranin, is added. Safranin is kept on the slide for 45- 60 seconds. Safranin is then rinsed off with water. Afterwards, the slide is blotted with bibulous paper and observed in through a microscope. Gram positive organisms appear purple in color while gram negative organisms appear red in color.

Lactose Fermentation Test:

Using an inoculating loop, inoculate tubes of media with growth from an 18- 24 hour pure culture. Incubate the tubes with loosened caps at 35 +/- 2 degrees Celsius for 48 hours in either aerobic or anaerobic conditions depending on the type of bacteria. An uninoculated tube will be orange in color. A typical positive reaction with acid and gas will appear to be yellow. A typical negative reaction with positive growth will appear to be red/ pink.

Casein Digestion Test:

Skim milk is used to make the microbiological culture media. The medium is heated in a boiling water bath for 2-5 minutes with the loosened caps. It is then cooled to room temperature with the caps tightened. The tubes are then inoculated by using an inoculating loop. As a side note, if anaerobic bacteria are being investigated, sterile mineral oil is placed over the medium after it has been inoculated. The tubes are then incubated with their caps tightly fixed for clostridia but for all other organisms, the caps are loosened. The incubation temperature is 35 +/- 2 degrees Celsius. Observations are made in intervals of 7 days. Growth and reactions are observed.

Acid released from galactose/ mannose:

Using an inoculating loop, inoculate tubes of media with growth from an 18- 24 hour pure culture. Incubate the tubes with loosened caps at 35 +/- 2 degrees Celsius for 48 hours in either aerobic or anaerobic conditions depending on the type of bacteria. An uninoculated tube will be orange in color. A typical positive reaction with acid and gas will appear to be yellow. A typical negative reaction with positive growth will appear to be red/ pink.

Catalase Test:

The bacterial population is reacted with hydrogen peroxide. An inoculating loop is used to collect a large amount of bacterial cells onto a microscope slide. Next the bacterial cells are smeared onto the microscopic slide. Add one or two drops of hydrogen peroxide on the smear. If bubbles appear, the result is a positive reaction. If bubbles do not appear the reaction is negative.

Indole Test:

Stab the inoculating needle two-thirds of the measured distance to the bottom center of the tube. Do this by using growth from a pure culture. These tubes are then incubated with their caps loosened for 18- 24 hours at 35 +/- 2 degrees Celsius. It is important to note that aerobic conditions are maintained when incubating.

Manitol Test:

It is important to obtain isolated colonies. Once an isolated colony is obtained, plates are incubated for 24- 48 hours at 35 +/- 2 degrees Celsius. It is important to note that aerobic conditions are maintained when incubating.

Glucose Test:

Using an inoculating loop, inoculate tubes of media with growth from an 18- 24 hour pure culture. Incubate the tubes with loosened caps at 35 +/- 2 degrees Celsius for 48 hours in either aerobic or anaerobic conditions depending on the type of bacteria. An uninoculated tube will be orange in color. A typical positive reaction with acid and gas will appear to be yellow. A typical negative reaction with positive growth will appear to be red/ pink.

Citrate Test:

Slants are inoculated with pure culture. This is done by using a light inoculum. Tubes are incubated for 4 days at 35 +/- 2 degrees Celsius. It is important to note that aerobic conditions are maintained when incubating.

Growth on TSA plate:

This is done merely by observing the color change and odor released from the growth of bacteria onto the TSA plate. Bacteria are grown onto a TSA plate. Once the bacteria is grown onto the plate, the color and odor are examined. A green color and grape-like scent indicate the presence of P. aeruginosa. If there is a clear growth with no green color and consequently no grape-like scent, then this indicates the presence of B. cepacia.

MR-VP Test:

Tubes of MR-VP media are inoculated by using a light inoculum. These tubes are inoculated with 18-24 hour pure cultures; in addition, the tubes are held under aerobic conditions when they are incubated at 35 +/- 2 degrees Celsius for at least 48 hours. The methyl red indicator is made by dissolving 0.1g of methyl red in 300 ml of 95% ethyl alcohol. Purified water is then added to make 500 ml. After incubation has taken place, the aliquots are aseptically removed. For the methyl red test, 5 drops of methyl red indicator are added to an aliquot of broth. The color is then examined. For the Voges- Proskauer Test (VP), 15 drops from the reagent A dropper and 5 drops from the reagent B dropper are placed into 1 ml of broth culture. It is important to shake after each reagent is added, and this is done to aerate the sample.

Results

For the respiratory lab

Two plates were inoculated with bacteria; moreover, these two plates contained dilutions from the original swabs. These two dilutions included one plate that contained a 10-3 dilution and the other plate contained a 10-4 dilution. The plate with the 10-3 dilution had a "too numerous to count" number of colony forming units. The plate with the 10-4 dilution had 228 colonies. Each colony, from both plates, appeared to be very small and white.

For the unknown lab

The unknown number was 11. When the two bacteria were separated from each other and pure isolated colonies were grown, the results of the gram stain indicated that one was gram positive (purple in color) and the other was gram negative (red in color). 11A will be used to describe the gram positive bacteria, and 11B will be used to describe the gram negative bacteria. The gram positive bacteria, 11A, had a creamy white color and the sizes of the bacterial colonies were medium sized, but not very large. The gram negative bacteria, 11B, had very small colonies and the color was pink. The gram positive bacterium was coccus shaped and the gram negative bacterium was bacillus shaped.

UNKNOWN 11A

Test

Result

Gram staining

Purple color= gram positive.

Lactose Fermentation

Positive, yellow color means typical positive reaction with acid and gas.

Catalase Test

Positive, bubbles formed.

Manitol Test

Yellow zones in the middle and orange zones on the sides.

Observation of color

Very white

UNKNOWN 11B

Test

Result

Lactose Fermentation

Negative, red color means typical negative reaction with positive growth.

Glucose Test

Positive, yellow color means typical positive reaction with acid and gas.

Indole Test

Negative, clear and no red color

UNKNOWN 3, unknown number 113

Test

Result

Gram stain

Gram negative rods

Lactose test

At first the color appeared to be orange, but later the color changed to yellow, and this is a typical positive reaction with acid and gas. As a result of this error and lack of time it was not possible to continue experiments to determine the identity of the unknown.

Conclusion:

For the respiratory organism, since there was no clear zone around the bacterial colonies and not any green pigmented byproduct, the respiratory organism had a gamma hemolysis characteristic.

For the gram positive bacterium, the Lactose Fermentation test was performed. The result for the gram positive organism was that it appeared yellow after the Lactose Fermentation test, and this means that it is a typical positive reaction with acid and gas. Next, the Catalase test was performed for the gram positive organism. The result of the Ctalase test was that bubbles appeared and this indicates a positive result. Next the Manitol test was performed on the gram positive organism, and the result is that there were yellow zones and an orange zone and this indicates a positive result for this test. This indicated that the gram positive organism is S. aereus; in addition, since the bacterium is very white, it can be concluded that the gram positive unknown is S. aerues white.

For the gram negative bacterium, the Lactose Fermentation test was performed. The gram negative organism appeared red after the Lactose Fermentation test, and this means that it is a typical negative reaction with positive growth. The Glucose test was performed after the Lactose Fermentation test. The results of the Glucose Test were that the color observed was yellow and this indicated a typical positive reaction with acid and gas. Next the Indole test was performed for the gram negative bacterium. The results of the Indole test show that the tube was clear and no red color was present and this means that the result is negative for indole formation. Therefore, it can be concluded that the gram negative organism is S. marcescens.

For the third unknown, it was a gram negative organism that was rod shaped. After performing the Lactose Fermentation test, the initial result was that it appeared orange (a negative result) and the TA confirmed this result. However, one day later the color changed to yellow. This yellow result indicates that the result is in fact positive with the presence of acid and gas. Due to this error and not enough time due to final exams, the identity of the third unknown could not be determined. However, the following illustrates steps that should have been carried out. Since the Lactose Fermentation test had a positive result, the next test to be performed should have been the Citrate test. Once the Citrate test is completed, a negative result would indicate that the unknown bacterium is E. coli. A positive result for the Citrate test would indicate that the unknown bacterium is either C. freundii or E. aerogenes. If the result was positive for the Citrate test, then the MR-VP test would have to be conducted in order to differentiate between C. freundii and E. aerogenes. A negative result for the MR-VP test indicates that the unknown is C. freundii. A positive result for the MR-VP test indicates that the unknown is E. aerogenes.

Information on S. aerues white

The following is the taxonomy: Bacteria; Firmicutes; Bacilli; Bacillales; Staphylococcaceae; Staphylococcus; Staphylococcus aureus. S. aerues white is a gram positive bacterium that has a coccus shape; moreover its structure includes a thick peptidoglycan layer, cross bridges, and no flagella. As far as physiology is concerned, S. aerues white is resistant to penicillin due to the production of penicillinase which is an enzyme that breaks down the beta-lactam ring of the penicillin molecule. S. aerues white is a facultative anaerobe which means it makes ATP by aerobic respiration but can also obtain energy from fermentation. Its habitat includes soil and mucous membranes of mammals. S. aerues white's use as a pathogen is only effective if the skin barrier has already been broken, and one disease that it can cause is known as "furuncles" ("Staphylococcus aureus and S. epidermis").

Information on S. marcescens

The following is the taxonomy: Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Serratia; Serratia marcescens. As far as structure is concerned, S. marcescens is bacillus shaped and is a gram negative bacterium with a thin peptidoglycan layer. As far as physiology is concerned, S. marcescens can secrete the heme-binding protein HasA. S. marcescens is a facultative anaerobe which means it makes ATP by aerobic respiration but can also obtain energy from fermentation. Its habitat includes living in the soil, water, and intestines of mammals. Its use as a pathogen includes its role in nocosomial infection; moreover it is known to cause urinary and respiratory tract infections ("Serratia marcescens").

Sources:

Keplit, E. (n.d.). Bacteria: more on morphology. Retrieved from http://www.ucmp.berkeley.edu/bacteria/bacteriamm.html

Madigan M; Martinko J (editors). (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall.

Serratia marcescens. (n.d.). Retrieved from http://www.sunysccc.edu/academic/mst/microbes/23smarc.htm

Staphylococcus aureus and S. epidermis. (n.d.). Retrieved from http://www.sunysccc.edu/academic/mst/microbes/13saure.htm

Weisburg, WG., Barns, SM., Pelletier, DA., & Lane, DJ. (1991). 16s ribosomal dna amplification for phylogenetic study. Journal of Bacteriology, 2, 697-703.

Zillig W (1991). "Comparative biochemistry of Archaea and Bacteria". Curr Opin Genet Dev 1 (4): 544-51.

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