Identification of Unknown Bacteria using a Series of Biochemical Tests

Katie Segal Section 1 Dr. Potter Microbiology 202 December 4, 2008

Introduction: Bacteria are single-celled organisms that are able to exist in any kind of environment (2). Bacteria are found in hot springs, soil, water, etc (2). You are even able to find bacteria in the bodies of humans and other animals (2). In a single day people come into contact with millions of bacteria that are present on surfaces we touch, in the food we eat, and in the air we breath (1). Bacteria can start to cause harm to our bodies when they enter our system (1). Infections caused by bacteria start out as small colonies of bacteria which grow and multiply (1). The bacteria start causing bigger problems when they get into the blood stream (1). In 2000, 5 million people died from diseases that were caused by bacteria (1). Diseases can be caused by bacteria that are known or there could be new unknown bacteria causing problems. The unknown bacteria might not be treated because there are no known treatments for it. Very little information is known about newly discovered bacteria. The purpose of this lab was to use biochemical tests to identify two unknown bacterias. The 13 tests are grouped based on what they are looking for. There are carbohydrate, IMViC, enzymes, and respiration (5). The carbohydrate tests are phenol red, triple sugar iron agar or TSI, and starch agar (5). Indole, methyl red, Voges-Proskauer, and citrate make up the IMViC tests (5). Enzymes tests are urease, gelatinase, and DNase (5). Respiration tests are the catalase, oxidase, and nitrate test (5). Through out the lab aseptic technique was practiced in order to avoid any cross contamination. A Bunsen burner was used to sterilize the opening of any tubes that were opened while obtaining a bacteria sample. An inoculating loop was used to obtain samples from a tube or agar plate. The bench top was cleaned with bleach prior to starting the lab. Gloves were also worn as a precaution. All of the samples were placed into a 37 incubator for a period of time.

Materials and Methods: Thirteen biochemical tests were performed in order to identify the two unknown bacteria. Some of the tests provided immediate results while others had to incubate for a period of time. A gram stain test was performed to find out if the bacteria were gram positive or negative and the shape. A small sample of the bacteria was mixed with a drop of distilled water on a microscope slide and allowed to air dry (5). Once dry, the slide was heat fixed using a Bunsen burner (5). Crystal violet was added to the bacteria and sat for 1 minute then rinsed (5). Iodine was added and sat for another minute and rinsed (5). Alcohol rinsed the slide for about 10 seconds until no more color came off the slide and rinsed with distilled water (5). Safranin was placed on the bacteria for 2 minutes and rinsed again (5). The bacteria were viewed under a microscope to see the shape and to determine if it was positive or negative. Gram positive bacteria are purple after the stain and gram negative bacteria are pink (5). A streak plate was also made to see the color of the bacteria. A small sample of the bacteria was placed on the agar plate using an inoculating loop (5). Three sections were made on the agar plate. The loop was used to spread the bacteria into the different sections so that the third section had single colonies of bacteria. A Phenol red test looks at the bacterias ability to ferment glucose, lactose, or sucrose with the help of a pH indicator (5). A Durham tube was also placed in the tube to collect any CO2 that might have been produced (5). A yellow solution indicated that the bacteria are able to ferment sugars (5). TSI or triple sugar iron agar tube was used to test for the fermentation of only glucose (yellow butt), fermentation of lactose and sucrose (all over yellow), CO2 formed (crack in agar), or ferrous ammonium sulfate produced (black precipitate) (5). The TSI agar had glucose with a 0.1% concentration and lactose and sucrose with a concentration of 1% present (5).

A starch agar plate looks for amylase which breaks down starches (5). The plate was inoculated with a loop, incubated, and then covered with Grams iodine (blue/green color if complex with starch) (5). Indole tests look for the presence or absence of the trytophanase enzyme which breaks down tryptophan (5). A 1% Tryptone broth was used during the test (5). Kovacs reagents are added to the broth and if indole is present then a red coloration forms at the top (5). Methyl red and Voges-Proskauer look for mixed acid and Butanediol fermenters (5). Mixed acid fermenters are able to ferment lactate, acetate, and succinate (5). Butanediol fermenters ferment butanediol, ethanol, and CO2 (5). A MRVP broth was used (5). One tube was used for each test. Half of the broth, once incubated, was removed and placed into a different tube. Methyl red is added to one tube to see if the pH is neutral (yellow) (5). Barritts solution (alphahapthol and potassium hydroxide) is added to the other tube to test the Butanediol fermenters and if the bacteria are Butanediol fermenters then the broth turns red (5). Citrate tests look for the presence of citrate which is the sole source of carbon for bacteria (5). An agar slant with synthetic medium and small amounts of mineral salt was used to perform the test (5). Bromthymol blue (pH indicator) is added to the agar slant and if there is growth (presence of citrate) the agar is blue and if there is no growth the agar is green(5). Urease looks for the breakdown of urea which is a waste product found in a mammal that is formed from the decarboxylation of amino acids (5). A urea broth was used which is a strong buffer that resists alkalination (5). Bacteria that can rapidly breakdown urea are able to change the pH indicator that is present in the broth (5). Pink broth indicates the breakdown of urea (5).

The breakdown of gelatin or collagen is tested using the gelatinase test (5). A nutrient gelatin (peptones, beef extract, gelatin) is inoculated and incubated (5). The tube is put into a refrigerator for 30 minutes and if the agar stays solid then the enzyme is absent (5). DNase or deoxyribonuclease is looking for the breakdown of DNA into smaller fragments (5). The agar plate was made up of peptone, DNA, and methyl green dye (5). A spot streak plate was made. After the plate was incubated if there was a zone of clearing then the enzyme is present that was able to break up the DNA and remove the dye(5). If there was no zone of clearing then the enzyme was absent (5). Catalase tests for the presence of catalase in bacteria with the use of 3% H2O2 (5). A sample of bacteria was spread onto a microscope slide. A few drops of 3% H2O2 were added to the bacteria (5). If catalase was present, the bacteria bubbled and if it was not present then there was no reaction (5). Oxidase tests for the cytochrome oxidase exchange (5). The bacteria were placed onto filter paper using a sterile toothpick and a chromogenic reducing agent was added and if the exchange occurred the bacteria turned dark blue(5). Nitrate tests looked for the presence of nitrite and gas in the Durham tube (5). A broth of beef extract/peptone and KNO3 was used (5). After incubation sulfanilic acid (500ls) and napthylamine (500ls) was added to the tube (5). The broth turned red if nitrite was present and no gas was formed (5). If there was no nitrite then there was no color change and gas was present (5). Any negative tubes had zinc powder added to them to see if there was a complete breakdown (5). If the bacteria were able to completely breakdown, after the zinc powder was added, the broth did not change color (N2 was made) (5). If the broth turned red then there was not a breakdown of NO3 (CO2 was made) (5).

Results: The two unknowns were both identified using a chart that based on the results from the tests gave you a bacteria. Unknown 11 is Enterococcus faecalis and unknown L is Pseudomonas aeruginosa. Figure 1 (attached) shows the results from the tests performed. The name of the test is given along with the time it was incubated and the unknowns result.

Figure 1: Chart showing the tests performed and the results for the two unknown samples. Test Performed Gram stain Color Shape Glucose broth Lactose broth Sucrose broth TSI agar Gelatinase test Time Incubated Immediate 48 hours Immediate 4 days 4 days 4 days 4 days 48 hours, fridge =30 min. Unknown L Negative Clear Rod No CO2 or fermentation No CO2 or fermentation No CO2 or fermentation No change/No change Enzyme absent No amylase Enzyme absent No ammonia was made No nitrite Presence of indole Not positive No change Basic Positive Cytochrome oxidase Unknown 11 Positive White Cocci No CO2, did ferment No CO2, did ferment No CO2, did ferment Ferment lactose or sucrose Enzyme absent No amylase Enzyme absent No ammonia was made No nitrite Presence of indole Positive No change Neutral Negative No cytochrome oxidase

Starch hydrolysis 4 days DNase Urease test Nitrate reduction Indole Methyl red Voges-Proskauer Citrate Catalase Oxidase 48 hours 24 hours 48 hours 48 hours 48 hours 20 minutes 48 hours Immediate Immediate

The above chart shows the results obtained from the tests performed. The tests were listed along with how long the sample was placed in a 37 incubator. The results helped to identify the bacteria.

Discussion: While doing the tests there were some reactions that did not occur as quickly as they should have. Some of the reactions did not fully complete. For example the oxidase test is supposed to give an immediate answer where as for unknown L after about 5 minutes the color was starting to change. The Voges-Proskauer test took a while to complete after the two solutions were added. The urease test is supposed to produce a pink broth if there is urease present. The broth might not have been a very noticeable pink and could be mistaken for colorless broth giving a different result. I am confident in my decision of what the unknown bacteria really are. I was very careful during the lab so that no cross contamination occurred. When using the chart that was given to me during lab my results were mostly right on with the chart. When looking at my results I was very careful with making sure that I was positive with the result so that mistakes were not made. Unknown 11 produced the same results as what were expected on the given chart. Unknown L had 2 tests that did not match up completely, gelatinase and nitrate. The gelatinase test was supposed to have the enzyme present but when performing the lab the enzyme was not present. The enzyme not being present could be because of the agar or not enough bacteria were present. The sample of bacteria that was inoculated might have been very small so the colonies did not reproduce fast enough to produce a result. The nitrate test also did not produce the result that matched with the chart. In the lab the nitrate test did not show any nitrite present where it should have been. A solution was added to the nitrate tubes after incubation. The tube might not have reacted as fast as expected so it was said to be negative when given more time a positive result might have shown.

Since mistakes are made when doing a lab there are other ways to perform the biochemical tests. An API test only works on gram negative organisms (5). The API test is standardized and is able to perform 21 biochemical tests to identify the family the bacteria is part of (5). Another test that is being used is genetic sequencing. The DNA is extracted from the bacteria and it is compared to other bacteria to find related species. Enterococcus faecalis is a bacterium that is found humans large intestine (3). E. faecalis is not harmful to humans until an infection occurs (3). Most of the infections that occur because of E.faecalis happen after surgery in the abdomen or after any trauma to the stomach area (3). E. faecalis causes infections when IVs and catheters are used in hospitals (3). Urinary tract infections, meningitis, and bacterimia are some of the infections that E. faecalis are known to cause along with wound infections (3). Pseudomonas aeruginosa is found mainly in the soil (4). P. aeruginosa is a pathogen that affects patients in a hospital (4). P. aeruginosa is known to cause infections from the use of catheters, IV infusions, and lumbar punctures (4). In patients who are given radiation treatments or high doses of an antibiotic are more susceptible to an infection caused by P. aeruginosa due to a weakened immune system (4). P. aeruginosa is commonly found in the wounds of surgical patients, burns, abscesses, and ear infections (4). Bacteria are everywhere and there is no way to avoid it. Identification of unknown bacteria could mean that a new antibiotic could be made to treat an illness. The use of an antibiotic could mean that a patients life is saved. Identification is a very important method for microbiology or any scientist. Some of the everyday medications or antibiotics are only available for use today because someone identified a new bacterium that was able to be used.

References:

1)

"Bacteria and Disease." 29 Sept. 2008. 2 Dec. 2008 <http:// www.earthlife.net/prokaryotes/disease.html>.

2)

"Bacteria." Biology Online. 22 July 2008. 2 Dec. 2008 <http:// www.biology-online.org/dictionary/bacteria>.

3)

Glick, Wesley. "Enterococcus faecalis." 1 Dec. 2008

<http://http://web.mst.edu/~microbio/bio221_2005/e_faecalis.htm>.

4)

Madigan, Michael, John Martinko, Paul Dunlap, and David Clark. Brock Biology of

Microorganisms. 12th ed. Boston: Benjamin-Cummings Company, 2008. 414-15.

5)

Potter, Dr. Beth. "Biochemical Tests." Microbiology 202. Penn State Erie, The Behrend

College. 30 Oct. 2008 11 Nov. 2008.