Microbiology paper

Microbiology 233

Unknown Bacteria Project

Introduction

According to Brenner et al (1980), E. gergoviae is a gram-negative, rod-shaped, peritrichous when motile bacteria, and found in clinical specimens and the environment. It is also a facultative anaerobe, meaning that they can live in both with or without oxygen in their environments. The bacteria live in the intestinal tract of animals leading to transmission to the outside environment (Rogers, 2017). It is also an opportunistic pathogen that can cause conditions, such as meningitis, bacteremia, pneumonia and urinary tract infections (Rogers, 2017). This is a growing concern as it has become resistant to many antibiotic treatments.

E. faecalis is a gram-positive, cocci in chains, and non-motile (Kau et al, 2005). It is a facultative anaerobe and can survive in temperatures of 60°C for short periods of time and grow in high salt concentrations (Fraser et al, 2017). E. faecalis is found in the gut of all animals and humans and causes over 80% human infections in the US according to the CDC (1998). It is an opportunistic pathogen as it affects many immunocompromised people in the hospital (Leonard, 2017). It is associated with the most severe infections such as sepsis, endocarditis, and meningitis (Leonard, 2017). This bacterium is also a growing concern as it is becoming more resistant to multiple antibiotics including vancomysin (Kau et al, 2005).

Materials and methods

Test 1 for both Samples- Gram Stain

Materials: inoculating loop, Bunsen burner, marking pen, crystal violet, iodine, water, ethanol, safranin, slide, clothespin, microscope, immersion oil

The gram stain test is a differential staining test that distinguishes bacteria in two ways, cell wall and shape. The slide is marked by the marking pen with 2 circles and labeled A and B to distinguish each sample. Water was transferred from the inoculating loop to the center of each circle on the slide. The inoculating loop was sterilized through exposing it to the Bunsen burner flame before, between, and after each use. The bacteria samples were aseptically transferred to the center of the slide and mixed with the water. The slide was air dried, then heat fixed to allow the bacteria to stick to the slide. The clothespin was used to move the slide across the flame of the Bunsen burner. The crystal violet stain was added to the slide and allowed to remain for 1 minute, before rinsing out the excess stain with water. Then iodine stain was added for 1 minute before rinsing out the excess stain with water. The ethanol alcohol was added for 15 seconds. The last counterstain safranin was added for 1 minute. Finally, the slides were examined under the microscope at 100X objective with the use of immersion oil as the bacteria cannot be seen in a lower objective. Gram-positive cells would appear purple, while gram-negative cells would appear pink. The shape of the cell was also observed.

Sample A: Test 2 DNAse test

Materials: DNase test tube, inoculating loop, Bunsen burner

The bacteria were transferred aseptically to the DNase test tube by using the inoculating loop to leave a streak of bacteria on the slant of the broth. A positive result will show a clearing around the growth.

Sample A: Test 3 SIM indole

Materials: Sim Indole broth, Inoculating loop, Bunsen burner, Kovac’s reagent

The bacteria were transferred aseptically to the SIM indole broth in the test tube using the inoculating loop. Then 48 hours later, 3-5 drops of Kovac’s reagent was added to the test tube. A positive result will show the Kovac’s reagent turn red in the presence of indole.

Sample A: Test 4 Casease Test

Materials: Casease plate, Inoculating loop, Bunsen burner

The bacteria were aseptically transferred to the casease plate tube by using the inoculating loop to leave a streak of bacteria on the slant of the broth. The positive results would show a clearing on the plate.

Sample B: Test 2 Methyl Red (MR test)

Materials: Methyl red reagent, inoculating loop, Bunsen burner

The MR test indicates bacteria that perform mixed acid fermentation. The bacteria were aseptically transferred to the MRVP broth in the test tube by using the inoculating loop and left to incubate. Then the prepared sample was split into two and used for the 2 different tests, MR and VP tests. The first prepared test tube was used for the MR test, where 3 drops of methyl red reagent were added. A positive result was an immediate red color change to indicate mixed acid fermentation occurred.

Sample B: Test 3 Voges-Proskauer (MRVP) test

Materials: VP reagent, inoculating loop, Bunsen burner

In the second prepared test tube, 15 drops of VP agent A were added then mixed, then 5 drops of reagent B were added and mixed. The mixture was left for 15-20 minutes to allow time for the fermentation process to occur. A positive result would show a change in the color to red.

Sample B: Test 4 Phenol red broth (PRB) Lactose

Materials: PBR broth, Phenol red, Durham tube, Inoculating loop, Bunsen burner

The last test for Sample B was the PRB lactose test tube with durham tube. The bacteria were transferred aseptically to the PRB lactose test tube and incubated for 48 hours at 37°C. A positive result would show a color change.

Results

The first test conducted for both species of bacteria was the gram stain test. The purpose of this test was to determine the shape of the cell and the type of cell wall present, meaning either gram positive or gram negative. Each step of the test had a purpose to differentiate the type of cell wall. The primary stain was crystal violet, which stained all the cells purple. The iodine acts as a mordant as it improved the adherence of the crystal violet to the cells. All the cells remained purple. The ethanol was added for decolorization. The gram-positive cell walls were not affected and remained purple as gram-positive cell walls are made from the thick layer peptidoglycan. But the gram-negative cell walls consist of an outer membrane and thin layer of peptidoglycan. They were damaged by the ethanol causing the stain to leak out, leaving the cells colorless. The last step was adding safranin, the counterstain, which allowed the gram-negative cells to turn pink. Lastly, the stains are examined under the microscope.

Image 1: Gram stain, Sample A- E. faecalis

Image 2: Gram stain, Sample B- E. gergoviae

SAMPLE A- Enterococcus faecalis

The next set of tests were run according to the abbreviated flow chart 1 for Sample A (see Chart 1). The second test for sample A was the DNase test. The DNase test is a differential test that looks for the secretion of the DNase enzyme. The purpose of the methyl green in the media is to indicate if DNA is present. A positive result will show a clearing around the growth. The sample A, E. faecalis, showed that the DNA was not able to be broken down as the DNase resulted in no color change.

The third test was SIM indole test. The SIM test is a combination of tests, sulfur reduction, indole production, and motility. Indole is produced as the amino acid tryptophan is broken down from the trytophanase enzyme. The Kovac’s reagent turns red in the presence of indole, which is a positive result. The result showed that the sample A, E. faecalis, for the SIM indole was negative as there was no color change thus indicating the bacteria was not able to break down the tryptophan, so no indole was produced.

The last test for Sample A was the casease test. The positive results would show that the bacteria were able to break down the protein casein to create a clearing. The results from sample A were negative as there was no clearing on the plate meaning that the bacteria were not able to break down the casein. All the test results led to the conclusion that sample A was Enterococcus faecalis.

Image 3: DNAse test, Sample A- E. faecalis

Image 4: SIM indole test, Sample A- E. faecalis

SAMPLE B- Enterobacter gergoviae

The next set of tests were run according to the abbreviated flow chart 2 for Sample B (see Chart 2). The MRVP tests are differential tests that distinguish two different sugar fermentation pathways. The fermentation sugar used in the MRVP was glucose. The MR test displays bacteria that perform mixed acid fermentation. A positive result would be a red color change to show that the mixed acid fermentation had occurred as the acids produced lowered the pH level. The sample B, E. gergoviae, did not result in a color change signifying that mixed acid fermentation did not occur.

The VP test shows bacteria that undergo fermentation to produce acetoin and 2,3-butanediol. A positive result would be the red color change meaning that acetoin was produced. The results showed the sample B, E. gergoviae, had a red color change demonstrating that fermentation had occurred and produced acetoin as the product.

The final test for sample B was the PRB lactose test tube with durham tube. The differential test was used to show if the bacteria ferments the sugar specifically lactose. This means that the bacteria can eat the lactose and produce energy as well as acid waste products. If the bacteria ferments and creates the acid, thereby lowering the pH, then change in the color of the phenol red would occur. The inverted durham tube inside the tube indicated that gas was produced from the lactose fermentation. The results showed that the sample B, E. gergoviae, was not able to ferment the lactose as the color of the tube only changed to a slight orange instead of the yellow, which would indicate a positive result. Also, there was no change to the durham tube showing that since no fermentation occurred, no gas was produced. All the test results led to the conclusion that sample B was E. gergoviae.

Image 6: MR test, Sample B- E. gergoviae

Image 7: VP test, Sample B- E. gergoviae

Image 8: PRB test, Sample B- E. gergoviae

Discussion

My overall results showed that E. gergoviae is gram-negative, rod-shaped bacteria that does not undergo mixed acid or lactose fermentation, but can produce acetoin and 2,3-butanediol through fermentation. In contrast, the results showed that E. faecalis is a gram-positive, cocci-shaped linked in chains. It cannot break down DNA, tryptophan, and casein.

One questionable outcome was that of the PRB lactose test. The test results showed a slight change in color, but not to the extent in which it showed that the fermentation of the lactose had occurred. The color change was a slight orange instead of yellow is indicative for a positive result. Another error was a contaminated sample as a yellow growth appeared in the unused corner of the plate for the casease test, but did not affect the test results. This plate was streaked with 3 different samples, but the growth was not like any of the others, so contamination had occurred. Despite these concerns, it was shown that using the differential tests to isolate specific characteristics can be used to identify unknown species of bacteria.

Chart 1: Abbreviated Flow Chart 1- Sample A

Chart 2: Abbreviated Flow Chart 2- Sample B

References

Brenner et al. (1980). Enterobacter gergoviae sp nov: a New Species of Enterobacteriaceae Found in Clinical Specimens and the Environment.  International Journal of Systematic and Evolutionary Microbiology, 30(1), 1-6.

Cdcgov. (2017).  Cdcgov. Retrieved 14 December, 2017, from http://www.cdc.gov/hai/settings/lab/vreclinical-laboratory.html

Fraser, S. L. (2017, Sept. 5) Enterobacter Infections. Retrieved 14 December, 2017, from http://emedicine.medscape.com/article/216845-overview.

Kau, A. L., Martin, S. M., Lyon, W., Hayes, E., Caparon, M. G., & Hultgren, S. J. (2005).  Enterococcus faecalis Tropism for the Kidneys in the Urinary Tract of C57BL/6J Mice .  Infection and Immunity,  73(4), 2461–2468. http://doi.org/10.1128/IAI.73.4.2461-2468.2005

Leobard, J. (2017, July 12). Enterococcus faecalis: Infections, transmission, and treatment. Retrieved 14 December, 2017, from https://www.medicalnewstoday.com/articles/318337.php

Gram +, cocci

Gram stain

Negative

DNase

Negative

SIM indole

Enterococcus faecalis

Sample A

Negative

Casease

Gram -, rods

Gram stain

Negative

Methyl Red

Postive

VP

Negative

PRB Lactose

Enterobacter gergoviae

Sample B

image3.jpeg

image4.jpeg

image5.jpeg

image6.jpeg

image7.jpeg

image8.jpeg

image1.jpeg

image2.jpeg