Lab Report On Unknown Mixed Culture
Introduction
The purpose of this project was to detect which organism we had in our unknown mixed culture tube by running a series of experiments to detect which specific Gram negative organism we had. To detect your gram positive from the mixed culture was given as extra credit points also. A Gram stain was performed and isolation streak plate in order to isolate and observe the unknown organism. Before the series of test, a dichotomous key had to be written up in order to know what steps and tests to run to identify the unknown Gram negative organism. I had to run three experiments in all before I identified my unknown. Identifying unknown organisms is really important, one reason is because it produces benefits for many aspects of …show more content…
the research of microorganisms and helps physicians correctly treat patients.
Materials and Methods
Gram Stain: A Gram stain is always started with a heat fixed smear slide of the unknown. The slide was then covered with the primary stain Crystal Violet for one minute, rinsed with distilled water after. The mordant Iodine was then added for another minute, rinsed with distilled water after. Ethanol was then applied, but it’s only applied rapidly across the slide about 3-4 times from the squeeze bottle, after it’s immediately rinsed with distilled water. The counterstain Safranin was then covered over the slide for a minute and thirty seconds, also rinsed with distilled water. After, the slide was blot dried with bibulous paper and I was able to observe the slide under the microscope. I also had to perform another gram stain after the experiment “Growth Patterns in Broth” from that experiment’s tube.
Streak Plate Method of Isolation: To perform my streak plate method of isolation, two NA plates were obtained and I inoculated my Gram negative unknown into the mediums. They were inoculated into the mediums by streak plate method technique and incubated for 30 degrees Celsius for five days. Afterwards, I observed the growth and the colonies formed.
Growth Patterns in Broth: After the streak plate method was performed I was able to obtain with an inoculating loop two different colonies from the NA mediums and was able to transfer each colony to a BHI broth tube. Afterwards I inoculated both tubes at 37 degrees Celsius for two days and observed the Gram negative unknown growth.
Gram Negative Test
Oxidation-Fermentation Test: Now that the BHI Broth tubes were well grown I was able to perform my O-F test. By using an inoculating needle to obtain the Gram negative unknown from one of my BHI Broth tubes (T2) to make a single stab line inside the O-F medium. I then covered one tube with 5mm of mineral oil and left one as is, incubated for two days at 37 degrees Celsius. After incubation I observed the results.
SIM-Indole Production and Sulfur Reduction: For this experiment I used the inoculating needle to make a single stab of the Gram negative unknown into the SIM medium. Afterwards the medium was incubated at 30 degrees Celsius for two days. For Indole Production results I had to add the Kovacs Reagent to the medium and observe color change results. I also observed the color change results for Sulfur and recorded them. Phenol Red-Arabinose: For this test I had to use an inoculating loop and transfer my Gram negative unknown into a Phenol Red Arabinose Broth. The broth was then incubated at room temperature for two days and after I observed results.
Results
Gram Stain[Fig.1]: The gram stain from the mixed culture was done first to view both the Gram positive and Gram negative cells. While observing under microscopy it showed purple cocci, clusters, and pink bacilli. You can even see diplobacilli, if you look closely at the image.
Streak Plate Method of Isolation[Fig.2]: The two streak plates were incubated for five days 30 degrees Celsius. They both grew isolated colonies. The colonies were round, smooth margin, with a flat elevation. They were also opaque with a tan/yellow color. As you can see I had more well rounded colonies in my second plate than I do in the first plate numbered.
Growth Patterns in Broths[Fig.3]: After picking two colonies from the streak plate I inoculated the BHI broths and there was a successful amount of growth for me in both tubes. T1 and T2 had a cloudy and translucent turbidity. There was little round tan clumps of growth in there also floating around. A tan cloudy pellicle is seen at the top, more in T2 than in T1.
Isolated T2 Gram Stain[Fig.4]: This gram stain was from the isolated T2 broth, it showed results of the Gram negative pink diplobacillus, and bacillus.
O-F Tests[Fig.5]: I had intentions on getting an oxidizer for this test but we cant always get what we want in life. My O-F tubes which were incubated for two days displayed yellow throughout with the mineral oil and without the mineral oil. Meaning it is a fermentor of glucose. It also displayed a nice amount of growth from the stab line in the medium.
SIM Test Indole Production/Sulfur Reduction[Fig.6]: For Indole Production, after adding Kovac’s reagent to the medium there was no color change at the top layer which would make this test negative for Indole production. For sulfur reduction the test was also negative because there was no display of a black color in the medium. There was no color change.
Phenol Red Arabinose Test[Fig.7]: This test was like the other, negative. The test is observed as negative because there was no color change in the tube meaning no fermentation.
Conclusions
While running a series of experiments to see what unknown organism was inside my mixed culture I came to the conclusion that my Gram negative organism is Serratia marcescens.
My dichotomous key was the “key” thing to finding my unknown out. After the gram staining of the gram negative, the O-F test was the first test to start my process off; it produced yellow through out both tubes. This meaning it is a fermentor of glucose and can be oxidative of glucose. It is also considered O-F because the mineral oil is added to one to promote an anaerobic growth and fermentation, while the one w/o oil is for oxidation and aerobic growth and it grew well in both. After finding out I had a fermentor the second thing was to do perform an Indole production experiment in order to cut my chances of finding my unknown in half. The Indole production test was recorded as negative. It is observed as negative because after adding the Kovac’s reagent there was no color change. Kovac’s reagent added to the tube reacts with DMABA and with any Indole present and produces a compound that turns the reagent layer pink. No color change for an Indole Production means tryptophan is not broken down into Indole and Pyruvate. After finding out I was negative for Indole, looking at my key my next test was Phenol Red Arabinose which also put me at a four to two ratio for finding out my unknown. After performing this test I found out this test is also negative. I recorded this test as negative because there was no color change in the tube meaning no reaction/no fermentation. Phenol Red Broth is a differential medium which also has a carbohydrate added. Phenol red and peptone are included in the medium as ph indicators; an indicator of gas production called Durham tube is also added. When the pH is raised it turns the tube pink, when lowered it turns it yellow and a bubble would mean gas production. After finding out I was negative for Arabinose test I was really excited because that left me with
two unknown organisms to run one more test for. This test was apart of the SIM test/Sulfur reduction. My Sulfur reduction test came out to be negative. Negative because there was no black in the medium. When the enzyme thiosulfate reductase catalyzes the reduction of sulfur at the end of the anaerobic ETC they both produce H2S, when it occurs in a SIM medium a black cloud would appear and make it positive. Sulfur is not reduced in this case. Since that test was negative that left me with unknown organism number twelve. S. marcescens rank: species. Also named as "Bacillus marcescens” It includes Pantoea sp. NAB7, Enterobacteriaceae bacterium KO4, Serratia sp. UENF-22GI, Serratia sp. NCIM 2919.(1) In medical observation it is the most widespread of that species to cause human infection and it has been found to cause, meningitis, infective endocarditis, pneumonia, wound infection, urinary tract infection and more.(2) Since S. marcescens is moderately easy to classify and identify it was thought to be non-pathogenic, and it was used as tracer organism for studying the movement of particles through air and water.(3) From the 1940s into the 1960s the United States military utilized S. marcescens to conduct experiments on military bases and the general population.(3) It is a facultative anaerobe that uses its flagella to swim. S. marcescens is now documented as an important opportunistic pathogen combining a tendency for healthcare-associated infection and antimicrobial resistance.
The purpose of this project was to detect which organism we had in our unknown mixed culture tube by running a series of experiments to detect which specific Gram negative organism we had. To detect your gram positive from the mixed culture was given as extra credit points also. A Gram stain was performed and isolation streak plate in order to isolate and observe the unknown organism. Before the series of test, a dichotomous key had to be written up in order to know what steps and tests to run to identify the unknown Gram negative organism. I had to run three experiments in all before I identified my unknown. Identifying unknown organisms is really important, one reason is because it produces benefits for many aspects of …show more content…
the research of microorganisms and helps physicians correctly treat patients.
Materials and Methods
Gram Stain: A Gram stain is always started with a heat fixed smear slide of the unknown. The slide was then covered with the primary stain Crystal Violet for one minute, rinsed with distilled water after. The mordant Iodine was then added for another minute, rinsed with distilled water after. Ethanol was then applied, but it’s only applied rapidly across the slide about 3-4 times from the squeeze bottle, after it’s immediately rinsed with distilled water. The counterstain Safranin was then covered over the slide for a minute and thirty seconds, also rinsed with distilled water. After, the slide was blot dried with bibulous paper and I was able to observe the slide under the microscope. I also had to perform another gram stain after the experiment “Growth Patterns in Broth” from that experiment’s tube.
Streak Plate Method of Isolation: To perform my streak plate method of isolation, two NA plates were obtained and I inoculated my Gram negative unknown into the mediums. They were inoculated into the mediums by streak plate method technique and incubated for 30 degrees Celsius for five days. Afterwards, I observed the growth and the colonies formed.
Growth Patterns in Broth: After the streak plate method was performed I was able to obtain with an inoculating loop two different colonies from the NA mediums and was able to transfer each colony to a BHI broth tube. Afterwards I inoculated both tubes at 37 degrees Celsius for two days and observed the Gram negative unknown growth.
Gram Negative Test
Oxidation-Fermentation Test: Now that the BHI Broth tubes were well grown I was able to perform my O-F test. By using an inoculating needle to obtain the Gram negative unknown from one of my BHI Broth tubes (T2) to make a single stab line inside the O-F medium. I then covered one tube with 5mm of mineral oil and left one as is, incubated for two days at 37 degrees Celsius. After incubation I observed the results.
SIM-Indole Production and Sulfur Reduction: For this experiment I used the inoculating needle to make a single stab of the Gram negative unknown into the SIM medium. Afterwards the medium was incubated at 30 degrees Celsius for two days. For Indole Production results I had to add the Kovacs Reagent to the medium and observe color change results. I also observed the color change results for Sulfur and recorded them. Phenol Red-Arabinose: For this test I had to use an inoculating loop and transfer my Gram negative unknown into a Phenol Red Arabinose Broth. The broth was then incubated at room temperature for two days and after I observed results.
Results
Gram Stain[Fig.1]: The gram stain from the mixed culture was done first to view both the Gram positive and Gram negative cells. While observing under microscopy it showed purple cocci, clusters, and pink bacilli. You can even see diplobacilli, if you look closely at the image.
Streak Plate Method of Isolation[Fig.2]: The two streak plates were incubated for five days 30 degrees Celsius. They both grew isolated colonies. The colonies were round, smooth margin, with a flat elevation. They were also opaque with a tan/yellow color. As you can see I had more well rounded colonies in my second plate than I do in the first plate numbered.
Growth Patterns in Broths[Fig.3]: After picking two colonies from the streak plate I inoculated the BHI broths and there was a successful amount of growth for me in both tubes. T1 and T2 had a cloudy and translucent turbidity. There was little round tan clumps of growth in there also floating around. A tan cloudy pellicle is seen at the top, more in T2 than in T1.
Isolated T2 Gram Stain[Fig.4]: This gram stain was from the isolated T2 broth, it showed results of the Gram negative pink diplobacillus, and bacillus.
O-F Tests[Fig.5]: I had intentions on getting an oxidizer for this test but we cant always get what we want in life. My O-F tubes which were incubated for two days displayed yellow throughout with the mineral oil and without the mineral oil. Meaning it is a fermentor of glucose. It also displayed a nice amount of growth from the stab line in the medium.
SIM Test Indole Production/Sulfur Reduction[Fig.6]: For Indole Production, after adding Kovac’s reagent to the medium there was no color change at the top layer which would make this test negative for Indole production. For sulfur reduction the test was also negative because there was no display of a black color in the medium. There was no color change.
Phenol Red Arabinose Test[Fig.7]: This test was like the other, negative. The test is observed as negative because there was no color change in the tube meaning no fermentation.
Conclusions
While running a series of experiments to see what unknown organism was inside my mixed culture I came to the conclusion that my Gram negative organism is Serratia marcescens.
My dichotomous key was the “key” thing to finding my unknown out. After the gram staining of the gram negative, the O-F test was the first test to start my process off; it produced yellow through out both tubes. This meaning it is a fermentor of glucose and can be oxidative of glucose. It is also considered O-F because the mineral oil is added to one to promote an anaerobic growth and fermentation, while the one w/o oil is for oxidation and aerobic growth and it grew well in both. After finding out I had a fermentor the second thing was to do perform an Indole production experiment in order to cut my chances of finding my unknown in half. The Indole production test was recorded as negative. It is observed as negative because after adding the Kovac’s reagent there was no color change. Kovac’s reagent added to the tube reacts with DMABA and with any Indole present and produces a compound that turns the reagent layer pink. No color change for an Indole Production means tryptophan is not broken down into Indole and Pyruvate. After finding out I was negative for Indole, looking at my key my next test was Phenol Red Arabinose which also put me at a four to two ratio for finding out my unknown. After performing this test I found out this test is also negative. I recorded this test as negative because there was no color change in the tube meaning no reaction/no fermentation. Phenol Red Broth is a differential medium which also has a carbohydrate added. Phenol red and peptone are included in the medium as ph indicators; an indicator of gas production called Durham tube is also added. When the pH is raised it turns the tube pink, when lowered it turns it yellow and a bubble would mean gas production. After finding out I was negative for Arabinose test I was really excited because that left me with
two unknown organisms to run one more test for. This test was apart of the SIM test/Sulfur reduction. My Sulfur reduction test came out to be negative. Negative because there was no black in the medium. When the enzyme thiosulfate reductase catalyzes the reduction of sulfur at the end of the anaerobic ETC they both produce H2S, when it occurs in a SIM medium a black cloud would appear and make it positive. Sulfur is not reduced in this case. Since that test was negative that left me with unknown organism number twelve. S. marcescens rank: species. Also named as "Bacillus marcescens” It includes Pantoea sp. NAB7, Enterobacteriaceae bacterium KO4, Serratia sp. UENF-22GI, Serratia sp. NCIM 2919.(1) In medical observation it is the most widespread of that species to cause human infection and it has been found to cause, meningitis, infective endocarditis, pneumonia, wound infection, urinary tract infection and more.(2) Since S. marcescens is moderately easy to classify and identify it was thought to be non-pathogenic, and it was used as tracer organism for studying the movement of particles through air and water.(3) From the 1940s into the 1960s the United States military utilized S. marcescens to conduct experiments on military bases and the general population.(3) It is a facultative anaerobe that uses its flagella to swim. S. marcescens is now documented as an important opportunistic pathogen combining a tendency for healthcare-associated infection and antimicrobial resistance.