Format for Microbiology Lab Paper | Unknown Lab Report

 Microbiology Unknown Lab Report

Unknown Number 111- Gram Negative

By: Allyson Peludat

12/3/2013

Microbiology 203

Fall 2013

Introduction

Microbes are everywhere. Microbes, or bacteria, are in the grass, in the toilet, in Antarctica and even make up the human body. The number one misinterpreted statement is, “Bacteria is bad”. Bacteria allow humans to survive every day by living on top of our skin and in our digestive system. It is not until something out of the ordinary, like a cut on the finger or undercooked food traveling through our intestines, disrupts the bacteria in our body causing a “bad” reaction. If the bacteria cannot be controlled by our own body’s immune system an alternative solution is needed. That is where microbiology comes into play. Microbiology looks at bacterial cells at the molecular level determining new ways of killing potentially harmful bacteria. There are several different classes of bacteria and even the most particular details about a bacteria are needed for the correct treatment. Steps needed to identify specific types of bacteria have been learned thus far in microbiology laboratory and class which will help lead to the identification of two unknown bacteria.




Materials

• Unknown 111
• Unknown Alternative 8- Gram Positive
• Nutrient Agar
• Mannitol Salt Agar
• Gram Staining Technique
• Simmon’s Citrate Agar
• Desoxycholate Agar
• MacConkey Test
• Milk Agar
• Glycerol Test
• Maltose Test
• MR-VP Broth
• Microscope
• Bunsen Burner
• Inoculating loop
• Incubator at 35 degrees Fahrenheit

Methods

Day 1:

• An Unknown Chart was given to each student with the possible 5 gram negative and 5 gram positive’s listed. Each possible bacteria was listed with the correct answers to each test.
• A test tube labeled number 111 with an unknown gram positive and gram negative bacteria was given out by the professor.
• An inoculating loop was sterilized by the flame of a Bunsen burner. A sample of number 111 was taken and placed on a nutrient agar in an isolation streak (1). This was to promote growth and isolation of the two bacteria colonies.
• The nutrient agar was placed in incubator until next class period.

Day 2:

• Nutrient agar with isolation streak was removed from incubator. There was only one colony present in the nutrient agar.
• One colony was sterilely obtained by the inoculating loop and applied to a new nutrient agar to further isolate the colony.

Day 3:

• Only one colony was present on the second attempt to isolate the two unknown colonies.
• The colony from the nutrient agar was used to perform the gram staining.
  • Gram staining technique began with taking a sterile microscope slide and a sterile drop of distilled water and bacteria specimen by an inoculating loop (1). Next, the slide was passed directly through the flame of a Bunsen burner three times to heat fix the specimen. Gram crystal violet was added to the slide for 30 seconds, then gently rinsed with distilled water. Next, Gram iodine was placed on the slide for a mordant for 1 minute, then rinsed gently with distilled water. Next, Gram decolorizer was used to rinse the slide for 10-20 seconds, then rinsed gently with distilled water. Finally, Gram safranin was placed on the slide for 30 seconds, then gently rinsed with distilled water. The slide was then set to the side to air dry. Once the slide was dry it was able to be observed under a microscope.
  • The Gram Staining Technique resulted in Gram negative rods.
  • The Unknown Chart showed all gram negative possibilities were rods. A few tests were chosen to narrow down the possibilities:
    • • Simmon’s Citrate Agar
      • Mannitol Salt Agar
      • A sterile inoculating loop was used to scrape an isolated colony of isolated gram negative bacteria from previous nutrient agar. It was then placed on Simmon’s Citrate Agar slant to see if results showed the bacteria produced citrate permease enzyme. The slant was placed in the incubator for next class period (1).
      • A sterile inoculating loop was used to scrape an isolated colony of isolated gram negative bacteria from previous nutrient agar. It was then placed on a new Mannitol Salt Agar plate to see if results showed the bacteria fermenting mannitol (1).
      • At this point the gram positive bacteria was still not grown or isolated.
      • A sterile inoculating loop with the unknown number 111 was applied to Mannitol Salt Agar because it is supposed to be selective for gram positive bacteria (1). Results from Mannitol Salt Agar from isolated gram negative colony and number 111 plate will be compared next lab period.
      • Unknown number 111 and isolated gram negative colonies in nutrient agar was placed in refrigerator to slow growth of bacteria.

    Day 4:

    • • Results of Mannital Salt Agar for gram negative plates were negative. The unknown bacteria was a mannitol nonfermentor producing no color change (1).
        • Possibilities in Unknown was: Proteus vulgaris or Pseudomonas aeruginosa
        • Results of Simmon’s Citrate Agar for gram negative slants were negative. The agar had no color change which showed the unknown bacteria did not produce citrate permease enzyme (1).
          • Possibilities in Unknown was: Proteus vulgaris or Escherichia coli
          • Next, tests that were chosen to narrow down the Gram negative bacteria:
            • • Desoxycholate Agar
              • MacConkey Test
              • A sterile inoculating loop was used to scrape an isolated colony of isolated gram negative bacteria from previous nutrient agar. It was then placed on a Desoxycholate agar plate to select for enteric bacteria (1). The plate will be observed next class period to see if results will favor Proteus vulgaris as a large colorless colony.
              • A sterile inoculating loop was used to scrape an isolated colony of isolated gram negative bacteria from previous nutrient agar. It was then placed on MacConkey Agar plate to selects for lactose fermentation (1). The plat will be observed next class period to see if results will favor Proteus vulgaris as a colorless colony.
              • Both Desoxycholate and MacConkey Agar plates were placed in the incubator until next class period.
              • Gram positive results on Mannitol Salt Agar was supposed to be selective for Gram positive bacteria but there was completely no grown on the plate.
              • I discussed with my professor at this point about my negative results for gram positive bacteria. I explained my negative findings with nutrient agar and Mannitol Salt Agar. My professor informed me a new alternative unknown test tube with my isolated gram positive bacteria would be given to me next class period.

            Day 5:

            • • Results of Desoxycholate Agar plate for gram negative bacteria were negative for lactose fermentation (1). The growth on the Desoxycholate Agar was large colorless colonies resulting in Proteus vulgaris as my gram negative bacteria.
              • Results of MacConkey Agar plate for gram negative bacteria was negative for lactose fermentation (1). The growth on MacConkey Agar was large colorless colonies resulting in Proteus vulgaris as my gram negative bacteria.
              • Discussed with my professor my test result findings for my gram negative bacteria. I presented Proteus vulgaris as my gram negative bacteria which was the correct answer.
                • My Gram negative unknown bacteria was Proteus vulgaris.
                • An alternate, isolated, unknown Gram positive number 8 was given to be at this time.
                • Gram Staining Technique was done to figure out the unknown Gram positive shape (1).
                  • Gram staining technique began with taking a sterile microscope slide and a sterile drop of distilled water and bacteria specimen by an inoculating loop. Next, the slide was passed directly through the flame of a Bunsen burner three times to heat fix the specimen. Gram crystal violet was added to the slide for 30 seconds, then gently rinsed with distilled water. Next, Gram iodine was placed on the slide for a mordant for 1 minute, then rinsed gently with distilled water. Next, Gram decolorizer was used to rinse the slide for 10-20 seconds, then rinsed gently with distilled water. Finally, Gram safranin was placed on the slide for 30 seconds, then gently rinsed with distilled water. The slide was then set to the side to air dry. Once the slide was dry it was able to be observed under a microscope.
                  • Gram Staining Technique resulted in Gram positive purple rods.
                  • The Unknown Chart showed two possibilities of gram positive rods:
                    • Bacillus cereus or Bacillus subtilis.
                    • Test chosen to distinguish between the two Gram positive rod options:
                      • Milk Agar Test
                      • A sterile inoculating loop was used to scrape isolated Gram positive bacteria from Alternative number 8. It was then placed on a Milk Agar plate. Milk Agar is made of casein and it will be broken down if the unknown bacteria produces casease (1). If the unknown bacteria is positive for casease then it will be Bacillus cereus and have a clearing around area of growth. If there is no clearing then it will result in Bacillus subtilis.
                      • Milk Agar plate was placed in incubator until next class period.
                      • Alternate number 8 was placed in refrigerator to slow bacteria growth.

                      Day 6:

                      • Results of Milk Agar for Gram positive bacteria results showed a clearing around the bacteria colony which means the bacteria was positive for producing casease enzyme. Results point to unknown Gram positive bacteria as Bacillus cereus.
                      • Next tests to prove Bacillus cereus is the Gram positive unknown:
                        • Glycerol Fermentation Test
                        • Maltose Fermentation Test
                        • Both Glycerol and Maltose Tests are to test for bacteria fermentation of carbohydrates (1). In the test tubes, there is a broth medium with specific sugar and phenol red dye that changes colors to yellow when the pH is changed to an acid. There is also a Durham tube present in the liquid which shows if a gas is produced from bacteria fermentation. If an air bubble is present in the Durham tube then it is positive for fermentation.
                        • A sterile inoculating loop was used to scrape isolated Gram positive bacteria from Alternative number 8. It was then placed in Glycerol Fermentation test tube. If the liquid changes color from red to yellow it indicates fermentation of glycerol and a positive result (1). If there is an air bubble in the Durham tube it indicates fermentation of glycerol and a positive results as well.
                        • A sterile inoculating loop was used to scrape isolated Gram positive bacteria from Alternative number 8. It was then placed in Maltose Fermentation test tube. If the liquid changes color from red to yellow it indicates fermentation of maltose and a positive result (1). If there is an air bubble in the Durham tube it indicates fermentation of maltose and a positive results as well.
                        • Both test tubes were placed in incubator until next class period.

                      Day 7:

                      • Results for glycerol fermentation was negative. The liquid was red in color and no air bubble present in the Durham Tube. This test result shows Bacillus subtilis was my Gram positive unknown bacteria.
                      • Results for maltose fermentation was positive. The liquid was yellow, but there was no air bubble in the Durham Tube. This test results shows Bacillus cereus as my Gram positive unknown bacteria.
                      • These two tests did not help in deciding which Gram positive bacteria was present. I then tried the same two tests again and also tried a new test:
                        • Methyl Red Test
                        • A sterile inoculating loop was used to scrape isolated Gram positive bacteria from Alternative number 8. It was then placed in MR-VP Broth which is made up of glucose, peptone and pH buffer (1). This test is done to show if the unknown bacteria is able to ferment glucose. Methyl red indicator is added to the MR-VP Broth after several days of incubation. If the broth turns red immediately then it shows a positive test for glucose fermentation.
                        • A sterile inoculating loop was used to scrape isolated Gram positive bacteria from Alternative number 8. It was then placed in Glycerol Fermentation test tube. If the liquid changes color from red to yellow it indicates fermentation of glycerol and a positive result (1). If there is an air bubble in the Durham tube it indicates fermentation of glycerol and a positive results as well.
                        • A sterile inoculating loop was used to scrape isolated Gram positive bacteria from Alternative number 8. It was then placed in Maltose Fermentation test tube. If the liquid changes color from red to yellow it indicates fermentation of maltose and a positive result (1). If there is an air bubble in the Durham tube it indicates fermentation of maltose and a positive results as well.
                        • All three test tubes were placed in the incubator until next class period.

                      Day 8:

                      • Results for glycerol fermentation was negative. The liquid was red in color and no air bubble present in the Durham Tube. Which were the same results as last time.
                      • Results for maltose fermentation was positive. The liquid was yellow, but there was no air bubble in the Durham Tube. Which were the same results as last time.
                      • The MR-VP Broth was removed from the incubator and several drops of methyl red was added. After only two drops the entire broth became bright red (1). This resulted in a positive glucose fermentation test. The only Gram positive rod that should result in a positive methyl red test would be Bacillus cereus.
                      • Discussed findings with each Gram positive test with my professor and came to the conclusion that Bacillus cereus was my Gram positive unknown. My professor agreed with my decision.
                        • My Gram positive bacteria is Bacillus cereus.

                      Results

                      Unknown number 111 was used to determine Gram negative bacteria. In table 1, there is a list of all biochemical tests done to come to the conclusion Proteus vulgaris was my Gram negative bacteria. I was given an Alternative unknown 8 for my Gram positive bacteria because I was unable to grow my Gram positive bacteria on nutrient agar or mannitol salt agar. In table 2, there is a list of all biochemical tests done to come to the conclusion Bacillus cereus was my Gram positive bacteria.

                       

                      Table 1: Gram Negative Table of Tests = Proteus vulgaris (1)

                      Tests	Purpose	Reagents	Observations	Results
                      Nutrient Agar	To promote growth and isolate individual colonies	None	Individual clear colonies	Isolated colonies for further testing
                      Gram Stain	To determine the Gram reaction of the bacterium	Crystal violet, Iodine, Alcohol, Safranin	Pink Rods	Gram negative rods
                      Simmon’s Citrate Test	To determine if bacteria produce citrate permease.	None	Remained green. No color change	Negative for production of citrate permease
                      Mannitol Salt Agar	To determine if bacteria will ferment mannitol	None	No color change to agar plate.	Negative for mannitol fermentation.
                      Desoxycholate Agar	To determine if the bacteria ferments lactose	None	Clear, colorless growth.	Negative for lactose fermentation.
                      MacConkey Test	To determine if bacteria ferments lactose.	None	Large, colorless colonies.	Negative for lactose fermentation.

                       

                      Table 2: Gram Positive Table of Tests = Bacillus cereus  (1)

                      Tests	Purpose	Reagents	Observations	Results
                      Nutrient Agar with original 111 unknown	To promote growth and isolate individual colonies.	None	No growth.	No bacterial growth.
                      Mannitol Salt Agar with original 111 unknown	To promote Gram positive growth.	None	No growth.	No bacterial growth.
                      Gram Stain with Alternative 8	To determine the Gram reaction of the bacterium	Crystal violet, Iodine, Alcohol, Safranin	Purple Rods	Gram positive rods
                      Casein Test with Alternative 8	To determine if bacteria produce casease enzyme.	None	Clearing around area of growth.	Production of casease enzyme
                      Glycerol Test with Alternative 8	To determine if bacteria ferments glycerol.	None	Red color with no gas present in Durham tube	Negative for glycerol fermentation.
                      Maltose Test with Alternative 8	To determine if bacteria ferments maltose.	None	Yellow color with no gas present in Durham tube.	Positive for maltose fermentation.
                      Methyl Red Test with Alternative 8	To determine if bacteria ferments glucose.	Methyl Red	Red color when the pH indicator methyl red was added	Positive for glucose fermentation.

                       

                      Flowcharts ** Removed because of formatting issues

                      
                      
                      
                      
                      Discussion/Conclusion

                      For my Gram negative bacteria, I was able to come to the conclusion that Proteus vulgaris was my unknown fairly easily. I was first able to isolate my Gram negative colony on a nutrient agar. Then, my Gram Staining Technique resulted in the correct red rods. Finally, throughout all 4 of my tests, Simmon’s Citrate, Mannitol, Desoxycholate, and MacConkey, I had no errors in my results and every test pointed towards my Gram negative bacteria being Proteus vulgaris. When explaining my step by step process to my professor I came to the correct answer on my first try.

                      For my Gram positive bacteria, I had a few issues with initial isolation and with false test results. My initial issue with my Gram positive came when I was unable to have any type of growth on nutrient agar or mannitol salt agar. After discussing this major issue with my professor I was given an Alternative number 8 tube that had my Gram positive already isolated. My Gram Staining resulted in purple rods narrowing down my options to only 2 bacterial types. There were only a few tests to differentiate between the 2 Gram positive rods: glycerol, maltose and methyl red. After the first day, I prepared glycerol and maltose tubes for incubation assuming both would either be positive or negative and would easily point to the correct Gram positive. When I returned the next class period and glycerol was negative and maltose was positive I was thrown off. I forced myself to retry both glycerol and maltose test to search for an error that could have occurred. I also started MR-VP Broth to test for methyl red. The last class, I received a positive result for methyl red test, but again my glycerol was negative and maltose was positive. There must have been an error with how my Gram positive bacteria was reaction with the two fermentation tubes or one of the fermentation tubes was not prepared correctly. I had to disregard the positive maltose result to come to the conclusion Bacillus cereus was my Gram positive bacteria. My professor informed me that indeed Bacillus cereus was my correct Gram positive unknown bacteria.

                      Background Information on Proteus vulgaris

                      The genus Proteus is classified as an enteric bacteria, which means it is found in the digestive system of most animals (2). It is in the same family as E. coli or Salmonella which are main components in most food poisonings. Enteric bacteria are gram negative rods and are facultative anaerobes which means they are able to thrive in both anaerobic and aerobic conditions. Proteus in particular are mainly found in soil and decomposing organic matter. In humans, Proteus can cause urinary tract infections.

                       

                      References                                                                                     

                      1. McDonald, V., Thoele, M., Salsgiver, B., Gero, S., Lab Manual for General Microbiology, Revised April 2011.
                      2. Deacon, J. (1997, Oct 8). The microbial world: Proteus vulgaris and clinical diagnostics. Retrieved from http://archive.bio.ed.ac.uk/jdeacon/microbes/proteus.htm.