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Gram Negative Unknown Lab Report

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The Unknown Gram Negative bacterium inoculated in a Tryptic Soy broth medium was randomly selected from a group of other unknowns. In order to identify this unknown the seven different types of biochemical tests will be conducted on this unknown bacterium to identify it out of 6 possible bacteria; Escherichia coli(E. coli), Enterobacter aerogenes (E.aerogenes), Klebsiella pneumoniae (K.pneumoniae), Proteus mirabilis(P.miranilis), Pseudomonas aeruginosa(P.aeruginosa), and Salmonella typhimurium (S.typhimurium). The biochemical tests utilized were; Triple Sugar Iron Agar (TSIA) test, Sulfur Indole Motility (SIM) test, Methyl red test and Voges-Proskauer (MR-VP) test, Citrate test, Urea test, and Gelatin test. After conducting each of these tests, the unknown bacteria number 24 was concluded to be Proteus mirabilis. Introduction

This probe purpose was to help identify an unknown gram negative bacterium, distributed by our TA instructor. It is relatively important to identify unknown organisms, those that are unidentified can certainly be harmful and have the potential to cause harm to the public. The use for these several test experiments are very important, mainly because it aids in the identification of unknown and potentially harmful organisms, Along with reflecting how well an organism can grow or react in certain environments.

The T-streak procedure for isolation of unknown was conducted before identifying the morphological properties. A T-streak is performed to isolate bacteria over three regions of a Tryptic Soy Agar plate (TSA), into single colonies that are able to be inoculated for biochemical tests. It is in the third quadrant of the TSA plate is where a pure culture can be found and used for the biochemical tests. As stated in the lab manual, that there are six possible unknown bacteria. The T- streak isolation method will make sure there is no contamination in the unknown.

The bacteria being utilized in this experimentation will be unknown gram negative bacterium number 22 that had been inoculated in a Tryptic Soy Broth. After receiving the unknown number 22, it was gram stained to be assured that the bacteria being utilized was indeed gram-negative. The purpose of the gram stain was to be used to identify the morphological characteristics of the unknown. The gram stain is a differential stain that utilizes a primary stain of crystal violet (i.e. a purple dye), iodine, alcohol decolorization, and safranin (i.e. a tinted red dye); used as counterstain. Standard results of a proper gram stain would show a bacterium to be gram positive if purple, and gram negative if pink.

The results from the Gram Stain of unknown number 24 showed that unknown number 24 has bacillus shaped cell morphology, holding a dark-pink color confirming it to be a gram negative organism indeed. In both gram-positive and gram-negative the cell walls house peptidoglycan, which is a blend of carbohydrates and amino-acids. Gram negative bacteria cell walls have a thin peptidoglycan layer, in which an ample amount of lipids cross throughout the peptidoglycan and cell membrane. During the gram stain, lipids are prevalent in the gram negative bacteria are usually transparent as a result of the alcohol decolorizing agent. This allows the peptidoglycan of gram negative bacteria to retain the counterstain safranin (Leboffe & Pierce, 105)

Specific biochemical tests where implemented to follow, after the gram stain found unknown number 24 to be gram negative. These tests include a Triple Sugar Iron Agar (TSIA) test that uses a rich media that separates bacteria by the fermentation of Lactose, Sucrose, and Glucose, and have the capacity to reduce sulfur (Leboffe & Pierce 206). The objective of the test is to show if the unknown could exploit the carbohydrates in the medium to ferment into gas or reduce the sulfur. Any bacteria that possess the ability to ferment glucose and lactose will also turn the medium yellow throughout (Leboffe and Pierce 206). The absence of fermentation, the entire slant will be red which means that peptone was catabolized both aerobically and anaerobically which produced products that are alkaline. The presence of any sort of black coloring on the slant indicates hydrogen sulfide (H2S). Any kind of air bubbles is an indication that there was gas production.

The sulfur indole motility (SIM) test, determines if bacteria can produce sulfur and indole from tryptophan, and if they are motile, meaning they are flagellated and have the ability to move. Casein and animal tissue are used as sources of amino acids. Sulfur is in the form of thiosulfate and there is also an iron-containing compound. There are two ways in which bacteria can reduce sulfur to hydrogen sulfide: if the enzyme cysteine desulfurase is present cysteine will be catalyzed to pyruvate and if the enzyme thiosulfate reductase is present sulfur will be catalyzed at the end of the electron transport chain (Leboffe & Pierce 202-205).

Similar to the TSIA test, a black coloring within the medium it is an indicator of sulfur reduction. To determine if the bacteria produces indole, Kovacs’ reagent was added to the SIM test shortly after incubation. If the layer where the Kovacs’ reagent was added turns red, this indicates that the organism being tested is an indole producer. This is possible because the casein and animal protein contain tryptophan in which under the presence of a bacteria carrying the enzyme tryptophanse, tryptophan can be hydrolyzed to pyruvate, ammonia, and indole (Leboffe & Pierce 203). Motility is determined by simply examining if there is any radiating growth from a stab line after stab inoculating the bacteria with an inoculating needle.

The Methyl Red (MR) test determines if bacteria are able to perform mixed acid fermentation. Methyl red is a combination medium containing peptone, glucose, and a phosphate buffer in the form of potassium phosphate. In order to find out if bacteria are capable of mixed acid fermentation, methyl red indicator dye is added after incubation of the broth. Methyl red is red at pH 4.4 and yellow at pH 6.2. Between these two pH values, it is various shades of orange (Leboffe and Pierce 161). Any other color produced other than red means the organism is not capable of mixed acid fermentation.

The Voges-Proskauer (VP) test is conducted using a similar broth of that used in the methyl red test. Both methyl red and Voges-Proskauer make up a combination medium. The Voges-Proskauer test was designed for organisms that are able to ferment glucose, but quickly convert their acid products to acetoin and 2,3-butandiol (Leboffe and Pierce 161). After incubation, the Vogues Proskauer reagent A and reagent B are added to the broth. VP broth changes colors due to the oxidizing of reagents the acetoin to diacetyl which react with guanidine nuclei that turns the broth red if it is positive. If the color of the broth turns red, this means it is a positive result, and if there is no color change at all or the color is copper, this is an indication of a negative result.

The citrate test determines if a bacterium possess citrate-permease as citrate is the only source of carbon in the medium. The medium used in this test is the Simmons Citrate Agar, which is a type of utilization media. Utilization media are highly defined formulations designed to differentiate organisms based on their ability to grown when an essential nutrient is strictly limited (Leboffe and Pierce 175). If bacteria have the enzyme citrate-permease they can transport molecules that will be converted to pyruvate within the cell. Once the molecules are converted to pyruvate many products can be made all depending on the pH of the environment. The actual Simmons Citrate Agar has sodium citrate as the carbon source and ammonium phosphate as the nitrogen source. In order to tell whether or not an organism tests positive for citrate utilization, Bromthymol blue dye is added to the agar. If the pH is above 7.6, then the dye will be blue which is indicative of a positive test result. If there is no color change, meaning the Simmons Citrate slant stayed green after incubation, there a negative result meaning citrate is not utilized.

The urea hydrolysis test determines which organisms are able or unable to hydrolyze urea with the enzyme urease. The urea hydrolysis test can yield four possible results: rapid urease-strong urease production-positive, slow urease-weak urease production- weak positive, and urease absent-no urease hydrolysis-negative bacteria. The actual broth is made with urea, peptone, potassium phosphate, glucose, and phenol red. Within the urea broth all of the essential nutrients that bacteria would need are provided by peptone and glucose and the potassium phosphate is used as a mild buffer that resists alkalinization due to peptone metabolism. A positive urease bacteria will turn the broth pink in approximately 24 hours to 6 days for a rapid urease-positive organism and can take up to 8 days for a slow urease-positive organism. Urea hydrolysis to ammonia by urease-positive organisms will overcome the buffer in the medium and change it from orange to pink (Leboffe and Pierce 187).

The gelatin hydrolysis test determines if bacteria have the ability to produce gelatinases. Some microorganisms that are able to hydrolyze gelatin secrete the enzymes known as gelatinases, which break down gelatin (Leboffe & Pierce 192). A simple test medium known as nutrient gelatin was used to determine if a certain bacterium can produce gelatinases. Nutrient gelatin is made up of gelatin, peptone, and beef extract. A point worth noting is that nutrient gelatin is very different when compared to other media because the actual solidifying agent is also the substrate (Leboffe & Pierce 192). There are two possible results yielded from this test: a gelatinase-positive organism which breaks down the Gelatin medium into liquid, and a gelatinase-negative organism that does not liquefy the medium therefore meaning that it does not secrete gelatinases. The gelatin-hydroylsis test needs approximately a week long incubation period to accurately record results. Methods

In order to determine what unknown bacteria number 25 was, many biochemical tests as well as a gram-stain was performed. All aseptic techniques were carefully followed with each experiment. Before any tests could be performed the bacteria had to be isolated into a pure colony. This was achieved by a T-streak. Biochemical tests were started and incubated, after the T-streak results showed no contamination or mixture of organisms, for 24 hours in a hot room of 37 degrees celcius. All of the bacteria that were used in the biochemical tests came from the TSA plate used to T-streak unknown number 25. In order to determine the morphology of the bacteria a gram-stain followed the T-streak. Yielding results that showed the unknown number 25 to be a gram negative bacillus. After identifying the morphology of the bacteria, the biochemical tests came next.

The TSIA test was performed by obtaining a sample of bacteria from the T-streak plate with an inoculating needle.. The needle was flamed and then a sample from the Streak plate was gathered, and then inoculated by stabbing the agar butt and then streaking the slant. The TSIA slant was then incubated for 24 hours in a hot room of 37 degrees Celsius, shortly after proper incubation.

The SIM test was performed by stab inoculating the provided SIM tube. The needle was flamed and bacteria samples were collected from the streak plate containing the unknown. The stab-inoculation method followed and the needl was inserted only within 1cm of the bottom of the SIM tube, shortly afterwards inoculation a 24 hours incubation period in a hot room of 37 degrees Celsius.

The MR and VP tests were performed by inoculating the MR and VP tubes with the pure culture of unknown number 25. Using a flamed inoculating loop, the pure culture of unknown number 25 was added to the MR and VP tubes. Afterwards the proper reagents were added to each tube. Three drops of methyl red reagent were added to the methyl red tube where the results were observed immediately and properly recorded. Fifteen drops of VP reagent A were added to the VP tube and mixed properly. Then five drops of VP reagent B were added and mixed properly. The VP tube was observed 10 minutes, and results were recorded.

The citrate test was performed by streaking a Simmons Citrate slant with an inoculating loop. The loop was flamed and a collection of bacteria from the pure culture of unknown number 25 followed. The citrate slant was then inoculated by lightly moving the tube in a swivel pattern to help the loop not touch the walls of the container. The Citrate slant was then incubated for 48 hours in a hot room of 37 degrees Celsius. Observations of any change or growth were properly recoded afterwards. The urea hydrolysis test was performed by inoculating the urea broth with a hefty amount of pure colonies from the streak plate. An inoculating loop was used for proper inoculation of the bacteria. After properly inoculating the urea broth with the unknown bacteria, the urea broth tube was incubated for 24 hours in a hot room of 37 degrees Celsius, they were checked after 24 hours and then incubated again for several more days to get proper results from the test. Any observation of color change were recorded.

The gelatin hydrolysis test was performed by stab-inoculating a tube of Nutrient Gelatin tube with an inoculating needle. An incubation period of a week in the hot room of 37 degrees Celsius. After the week long incubation the Nutrient Gelatin tube was placed in the cold room for one hour to allow it to solidify. The nutrient gelatin tube was taken out of the cold room and carefully observed for any melting or liquefaction. Any observations were then recorded. Results

After conducting the TSIA test, it was observed that the slant had turned a yellow and had a yellow butt. This proves that unknown number 25 is a fermenter of glucose, lactose and possibly sucrose. Also the bottom of the slant had crack and abrasions in it, signifying that there was also gas production. There was no black precipitate in the TSIA slant, proving that the unknown organism does not reduce sulfur.

After the SIM test, it was observed that no black coloring had come about the medium, further showing that the unknown number 25 does not reduce sulfur. When testing for indole production the Kovac’s reagent was added to the medium and there was a production of a red precipitate at the top of the medium after a few minutes proving this organism ability to produce indole. The results of the motility test were easiest to determine. The organisms showed weak motility, proving that the unknown organism was flagellated and can produce movement.

After conducting the MR test it was confirmed that the unknown bacteria tested positive. After five drops of the methyl red reagent were added a red color began to form which was an immediate indication of a positive test. This positive test means that the unknown bacteria is capable of performing mixed acid fermentation. The Voges-Proskauer test came back with a negative result. After adding 15 drops of VP reagent A and 5 drops of VP reagent B there was no color changes evident and were observed for 10 minutes. This negative result indicated that unknown bacteria 25 did not produce any acetoin and was not a fermenter of 2,3-buteandiol.

The citrate test showed a negative result with no color change or growth of the unknown bacterium. This proves that the unknown number 25 did not utilize the citrate in the medium, also meaning the unknown did not posses citrate-permease, which is necessary to transport the molecules of the bacteria into the cell converting it to pyruvate (Leboff & Pierce 175).With Escherichia coli being the only bacteria that had a negative result for this test, for the remaining of the lab report the unknown will be simply referred to as E.coli.

After 8 days of incubation time in a 37°C hot room the Urea test show a negative result of no color change. This proves the inability of E.coli to hydrolyze urea, and possibly does not produce urease.

The Gelatin test also yielded a negative result. The gelatin medium did not breakdown obviously showing that E.coli does not have or produce Gelatinase.

This experiment required many different biochemical tests to be conducted for a few reasons; the first being to identify which bacteria was the one that was randomly selected and secondly for educational purposes so that students will become familiar and gain experience about the different types of media and laboratory techniques. The unknown bacteria which I selected was number 25. The citrate test truly helped confirm that the unknown bacteria 25 was in fact Escherichia coli. After these experimentation my original guess was correct in saying I believed the unknown bacteria I had was E.coli, and indeed it was.

E.coli is a member of the Enterobacteriaceae family which is also where Salmonella typhinurium is also classified. E.coli lives in the intestines of humans and can cause many infections ranging in severity. It doesn’t even require any growth factors, and can synthesize all essential purines, pyrimidines, amino acids and vitamins, starting with their carbon source, as part of their own intermediary metabolism (Todar). I was nervous about working with E.coli and bacteria because in general before starting this lab because of some of the symptoms they can cause. Especially intestinal swelling (MedLineplus). Even with that stated I have grown to enjoy this experiment and have learned so much valuable information that will benefit me in my nursing career.


Todar, Kenneth. “Nutrition and growth of Bacteria.” Online Textbook of Bacteriology. Web. 30 Mar. 2012. .

Leboffe, M. J. and B.E. Pierce. 2010. Microbiology Laboratory Theory and Application. 3rd ed. Morton Publishing Company

“E. Coli Enteritis.” Medline Plus (n.d.): n. pag. 10 Jan. 2011. Web. 28 Mar. 2013.

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