Enterococcus faecalis | Micro Unknown Bacteria Report

UNKNOWN LAB REPORT

Unknown Number 119

Joseph Kartje

Fall Semester

 

INTRODUCTION

Microorganisms are invisible to the naked eye, this can spell disaster when trying to prevent cross contamination necessary to isolate them in a laboratory setting.  Once isolated scientist can identified microorganisms by their cellular structure and function.  Different organisms react in different ways depending on environmental conditions. The recognition of pathogenicity is possible after correct identification and can drastically effect treatment of the infected host.   Not all bacteria in the human body are harmful; many exist in a harmonic balance. Bacteria have beneficial attributes such as organic matter decomposition, creating environments harmful to harmful bacteria, and use some antibiotic drug production, making proper identification a necessity.  The identification of  bacteria with unknown origin in this study was completed by using information learned in the microbiology class BIO203 with techniques and procedures outlined in the April 2011 MCDONALD LAB MANUAL.

 




MATERIALS AND METHODS

An unknown labeled as number 119 was obtained from the lab instructor. A isolation streak was performed using a Nutrient Agar and then left in a incubator at 37 degrees. From the isolation streak two visibly different colonies of bacteria were harvested and placed in separate Nutrient Agars and then incubated at same temperature. After the separate plates were grown one plate was labeled Unknown A and the other Unknown B. Gram staining was performed on both Unknown A and B. Using magnification to determine that the gram stains were done correctly and without cross contamination biochemical tests were then chosen to determine the identity of the unknowns. Unknown A was discovered to be a gram negative rod two different lactose fermentation tests were performed as well as a citrate test. Unknown B was discovered to be a gram positive cocci so a mannitol and nitrate test were performed. All test performed were done to the specifications of the McDonald lab manual.

 

Table 1 list the test, purpose, reagents, observations, and results of Unknown A

 

All the following test were performed on this unknown:

1. Lactose Fermentation test (EMB)
2. Simons Citrate test
3. Lactose Fermentation test (MacConkey)

 

Table 2 list the test, purpose, reagents, observations, and results of Unknown B

All the following test were performed on this unknown:

1. Mannitol test
2. Nitrate test

 

 

 

Table 1: Unknown A TEST	PURPOSE	REAGENTS OR MEDIA	OBSERVATIONS	RESLUTS
Gram Stain	To determine the gram reaction of the bacteria and to observe its shape	Crystal violet, Iodine, Alcohol, Safranin	Red Rods	Gram negative rods
Lactose	To determine if organism can ferment Lactose	Eosin-Methylene Blue Agar (EMB)	Green metallic sheen	Lactose Fermentation with strong acid end products
Citrate	To determine if organism can use citrate as its sole source of carbon	Simmons Citrate Tube	Green Tube	Negative result
Lactose	To determine if organism can ferment Lactose	MacConkey Agar	Colored streak and fermentation around streak	Positive for strong Lactose Fermentation

RESULTS

TEST	PURPOSE	REAGENTS OR MEDIA	OBSERVATIONS	RESLUTS
Gram stain	To determine the Gram reaction of the bacteria	Crystal violet, Iodine, Alcohol, Safranin	Purple cocci	Gram Positve cocci
Mannitol test	To determine if bacteria can grow in a high salt environment and ferment Mannitol	MSA Agar	Medium turned yellow	Positive for Mannitol fermentation
Nitrate test	To determine if bacteria can reduce nitrates to nitrites	Nitrate Tube	No change after reagent dye was added, zinc added and change to red	Negative for nitrate reduction

Table 2: Unknown B

 




DISCUSSION/ CONCLUSION

In this section the process of identifying the Unknown A and B will be discussed separately and then brief background information on Enterococcus faecalis will be presented.  To start Unknown A or Escherichia coli proved easy to separate using the isolation streak method laid out in the lab guide. Several gram stains were then attempted but cross contamination made then unusable. A gram stain was then attempted with the two unknowns on different slides and that proved much more successful. The slide revealed a gram negative made up of rods. This did not narrow down any gram negatives form the unknown chart, because E. coli, Klebsiella pneumonia, Enterobacter aerogenes, Proteus vulgaris, and Pseudomonas aeruginosa are all rods. Using a sterilized inoculation loop a bit of E. coli was taken from the isolated agar plate of Unknown A and placed on a EMB agar plate and a MacConkey agar plate. The plates were then placed in the incubator for 48 hours. The EMB agar had a metallic green growth where the bacterium was spread which means that not only was the lactose in the agar fermented but it also had strong acid end products.  This ruled out P. vulgaris and P. aeruginosa because they cannot ferment lactose.  The results on the MacConkey agar plate matched those of the EMB. Again it was indicated that not only was the lactose fermented but that strong acid end products were in place because the streak was a purple/red and the area surrounding the streak had taken on the same color. The results were recorded and then later the Bactria was placed in a Simmon Citrate tube. The tube was placed in the incubator for 48 hours and there was no change which means that the Bactria could not use citrate as its sole source of carbon. This result ruled out K. pneumoni and, E. aerogenes leaving only E. coli. All techniques and procedures done as laid out in the LAB MANUAL BIO 203 by MCDONLD and results confirmed by teacher.

Unknown B or Enterococcus faecalis was isolated cultured and gram stained in the same fashion and timeline as E. coli. The gram stain was purple and upon magnification was staphylococcus. This allowed the ruling out of Bacills cereus, Bacillus subtilis because they are rod gram positives, leaving Staphylococcus aureus, Staphylococcus epidermidis, and E. faecalis left to rule out. A streak was then made on a MSA agar and after 48 hours of incubation the media turned yellow marking a positive result for mannitol fermentation. This result ruled out S. epidermidis which cannot ferment mannitol leaving only S. aureus and E. faecalis. A Nitrate test was then performed and after the adding of the reagents and zinc the test results show negative for nitrate reduction leaving E. faecalis as Unknown B. All techniques and procedures done as laid out in the LAB MANUAL BIO 203 by MCDONLD and results confirmed by teacher.

E. faecalis is mainly found in hospitals. It is the most common cause of surgical wound infection and is responsible for 10% of total infections (Tortora 416). E. faecalis thrive in environments that are low in oxygen but high in nutrients so they tend to be most prevalent in vaginas, oral cavity’s, and GI tract (Tortora 317). E. faecalis started with a natural resistance to penicillin and quickly deveople resistance after that. It has a total antibiotic resistance of 71% but the more concerning fact is that it is now 90% resistant to vancomycin (Tortora 416, 647). Vancomycin for those that do not know is one of the last lines of defense in a bacterial infection(Tortora 647).

REFERENCES

 

Tortora, Gerard , Berdell Funke, and Christine Case. Microbiology: An Introduction. 11th. Pearson Education, Inc. , 317,416,647. print.