Microbiology-microbial Growth 1s1q7
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Vi-An Nguyen BIO 210 Lab report #1 Microbial Growth Background information: In lab, there were four bacteria that were introduced and learned about in class. The first was Staphylococcus epidermidis. This is a non-motile and non-spore cocci shaped bacterium. It showed up purple on a gram stain, and forms grape-like clusters. This bacterium has an optimal temperature range of 26-37°C. S. epidermidis is found in the human body more on the outside skin like wounds. With that, this bacterium can cause urinary tract infections (http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.ht ml). On the other hand, the bacterium Escherichia coli is rod-shaped, Gram-negative, and forms in small clusters or short chains. The bacterium Proteus mirabilis was a unique bacterium because it swarmed, and has an optimal temperature of 37°C (http://www.who.int/mediacentre/factsheets/fs125/en/). This type of bacterium is found and thrives in the gut of humans and overall does not cause any harm to the human body. However, some strands can cause food poisoning (http://www.medicalnewstoday.com/articles/68511.php). It is also possible that some E. coli strands can cause urinary tract infections (RW 743). Proteus mirabilis is a bacterium with a gram-negative reaction and is rod shaped. P. mirabilis is different because it swarms so it is motile. The bacterium has an optimal growth temperature at 37°C (http://www.tgw1916.net/Enterobacteria/Proteus.html). P. mirabilis can replicate and survive in moist reservoirs so can be caused by drinking contaminated water (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122780/).
Pseudomonas fluorescens is a gram-negative rod bacterium. This type of bacterium has an optimal growth temperature of 37° or above for a response (http://www.gutpathogens.com/content/2/1/16). P. fluorescens can be found in food, especially in the form of biofilms. It thrives in spoiled food which can cause sickness in humans when digested (http://www.biomedcentral.com/1472-6750/8/790).
In this lab, both selective and differential media were used to inoculate and observe the bacteria. A selective medium specifically is used only to allow certain types of organisms to grow and prevents any other organisms for growth. For a selective medium to be used in an experiment what is used on the medium or the incubation temperature could be altered. A differential medium differs from selective medium because differential is designed to show how differently the bacteria look when they grow. Differential compares two colonies of bacteria on a growth plate to see their characteristics and growth patterns. This also used oxidase and catalase tests for oxygen utilization. The oxidase test is useful to identify the type of metabolism used by the bacteria because an oxidase tests able to detect if the bacteria contained the oxidized form of cytochrome c and seeing if the colonies of the bacteria form a dark violet color. The oxidase test can also differentiate between Pseudomonas and Aeromonas. The catalase test is beneficial to identify if the bacteria are aerobic which would mean that there would be a formation of bubbles, otherwise they would be facultative anaerobes.
Methods: In the experiment, there were five different types of agar that needed to be inoculated with bacteria to see if there would be growth. The five different type of agar used in the lab were blood agar, MacConkey agar, Mannitol-agar, Hektoen entric agar, and Trypticase soy agar. The
agar is already in the petri dishes and labeled with the type of agar being used. The petri dishes should then be divided in half with a marker on the bottom side of the plate to then label each side with a bacterium to tell them apart. After, there should be dishes containing each bacterium for the experiment needed to be put into the agar plates. With a sterilized inoculation loop, a colony of the bacterium needed for the agar should be taken lightly. Only a small colony should be taken for a sample. Then transfer the bacterium to the appropriate agar plate to match up the bacterium. Then the inoculation loop should streak the plate with a straight line towards the middle of the line, then strokes should be drown coming out of the straight line creating an “E” shape. After, all five plates should be streaked the same way and the petri dishes should be closed right after streaking. The plates should be turned upside down with the label facing up and then be placed in the incubator at 37°C for 24 hours and then should observed for growth. In the oxidase test, the microorganisms E. coli, P. flourescens, and S. epidermidis were being compared to see if they were positive or not. First, a colony of the samples should be transferred to a glass slide from a sterilized inoculating loop. Then, drop one or two drops of the oxidase reagent onto the bacteria. Wait for a one minute or so and examine if there are any color changes. Colonies should start turning into a light color then dark. With that, the results with color changes would be positive. In the catalase test, two slides will be needed because water and S. epidermidis will be used to compare the two. A drop of water is need on one glass slide while a colony of S. epidermidis is needed to be transferred from a sterilized inoculating loop to the glass slide. Then, each slide will need about two to three drops of hydrogen peroxide since that is the reagent. After waiting about a minute the slides should be checked to see if there is bubble formation. If there were bubbles, then the reaction would be positive.
Results: Table 1: Growth of bacteria on selective and differential media Bacterium MacConkey agar Mannitol-Salt agar Uninoculated Dark violet Red S. epidermidis No growth No color change with red zones with no hydrogen sulfide production E. coli Pink colonies Yellow color surrounded with pink surrounding colonies zones with no with no hydrogen hydrogen sulfide sulfide production production P. mirabilis Colorless colonies No color change in with no hydrogen colonies with no sulfide production hydrogen sulfide production
Hektoen enteric agar Green No growth
Salmon-pink colonies with no hydrogen sulfide production
Black colonies with hydrogen sulfide produced
Table 2: Hemolytic reactions of bacteria grown on agar Bacterium Type of hemolysis observed S. epidermidis Gamma hemolysis E. coli Gamma hemolysis P. mirabilis Gamma hemolysis
For the oxidase test, the bacteria E. coli, S. epidermidis, and P. flourescens were tested to see if they were negative or positive. In this case, E. coli demonstrated a negative result with no reaction. On the other hand, both P. flourescens and S. epidermidis bacteria tested positive for the oxidase test.
For the catalase test, water and S. epidermidis were compared to observe if the enzyme catalase were present. When water was tested, the results came out negative. On the other hand, S. epidermidis came out positive because there was a formation of bubbles after adding the reagent.
Discussion: 1. The mediums that were used in the lab were found in the BIO 210 lab manual 1st quarter, 2013, DePaul University, and (October 13, 2012). The first medium that was used in the lab was blood agar. It is a differential medium because it has differential properties due to hemolysis and can be identified with the kind of color zones around the colonies (http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/selectivedifferen tial.htm). Another medium used in the lab was macConkey agar. Since this type of agar uses crystal violet, it is preventing the growth of any gram-positive bacteria. On the other hand, macConkey agar compares the bacteria of their fermentation. In this case, the medium show if the gram-negative bacteria are ferment lactose or lactose-nonfermenting which will differ in colony colors. The use of the red indicator will show that the bacterium is ferment lactose. Mannitol-salt agar is a selective medium because of the sodium chloride that prevents the growth of majority of the other bacteria. It is differential because it is comparing to see if the bacteria are mannitol fermenter or mannitol nonfermenter by the indication of red colonies. Hektoen enteric agar has a selective agent of bile salts that is trying to inhibit the growth of other gram positive organisms. Also, hektoen agar can identify lactose fermenters with the appearance of yellow color from the dyes. 2. If S. epidermidis and E. coli were growing together in a tube on nutrient broth, what can be compared to identify which bacteria is which, the media can help with the color. For example, knowing what the characteristics of E. coli, then that can assist in trying to identify when comparing that bacterium to S. epidermidis. The two organisms would be able to show whether one grows more rapidly than the other because it is possible for one
organism to grow over the other organism. It is essential to use the blood agar medium to first separate and compare between the two bacteria. The blood agar was used as an allpurpose medium because it differentiated the organisms with the reactions on the medium (BIO 210 lab manual, 2013). 3. P. mirabilis is one of the few organisms that are motile that was tested during the lab. Also, it was the only organism to have had colonies with black centers on the Hektoen enteric agar. From observing P. mirabilis, it was a lactose fermenter from the agars which help will help identify what kind of gram reaction it would be because gram-negatives are supposed to grow on the different mediums used in the lab. Since P. mirabilis was a gram-negative bacterium, it was expected to grow on the agars. 4. Since blood agar was used as the all-purpose medium in the lab, it would be beneficial for Staphylococcus aureus and S. epidermidis on blood agar because it will show the color of its colonies to tell them apart because the bacteria are different, so the blood agar would show the their hemolysis, thus the actions on the medium. S. aureus on blood agar should form cream colored colonies, while S. epidermidis should form white colonies (http://www.healthhype.com/lab-tests-for-staph.html). Tryptic soy agar is used as the base for agar plates, so having TSA on an all-purpose agar will help identify between the two organisms. 5. For the oxidase test, only P. flourescens and S. epidermidis had positive reactions which can be concluded that the two organisms must use oxygen to transport their foods during oxidative metabolism (http://www.vumicro.com/vumie/help/VUMICRO/Oxidase_Test.htm). For the catalase test, the organism S. epidermidis tested positive meaning that there is a catalase present
which is an enzyme that breaks down toxic products used in oxygen so the organism is able to tolerate oxygen (http://www.ncbi.nlm.nih.gov/pubmed/1141195). 6. To identify P. mirabilis, lactose would be essential in determining the organism because P. mirabilis was tested as a lactose fermenter and also that P. mirabilis is motile so that differs from E. coli which is another characteristic that could differentiate them apart. Knowing the characteristics of P. mirabilis that it is a gram-negative rod bacterium and that it was the only organism to grow colonies with black centers in the Hektoen agar so it was the only bacterium to have produced hydrogen sulfide.
References: Bauman, RW. (2012). Microbiology with diseases by body system, third edition. pp 743 http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.ht ml Date accessed: October 12, 2013. Date last updated: September 27, 2004 http://www.scienceprofonline.com/microbiology/differential-selective-bacterial-growthmedia.html Date accessed: October 12, 2013. Date last updated: April 2013 http://www.who.int/mediacentre/factsheets/fs125/en/ Date accessed: October 12, 2013. Date last updated: December 2011 http://www.who.int/topics/escherichia_coli_infections/en/ Date accessed: October 12, 2013. Date last updated: N/A http://www.gutpathogens.com/content/2/1/16 Date accessed: October 13, 2013. Date last updated: November 27, 2010 http://www.gutpathogens.com/content/2/1/16 Date accessed: October 13, 2013. Date last updated: June 2011
http://www.medicalnewstoday.com/articles/68511.php Date accessed: October 14, 2013. Date last updated: May 21, 2013 http://www.biomedcentral.com/1472-6750/8/79 Date accessed: October 14, 2013. Date last updated: October 28, 2008 http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/selectivedifferential.htm Date accessed: October 14, 2013. Date last updated: N/A http://www.healthhype.com/lab-tests-for-staph.html Date accessed: October 14, 2013. Date last updated: June 10, 2013 http://www.vumicro.com/vumie/help/VUMICRO/Oxidase_Test.htm Date accessed: October 14, 2013. Date last updated: N/A http://www.ncbi.nlm.nih.gov/pubmed/1141195 Date accessed: October 14, 2013. Date last updated: July 1975
Pseudomonas fluorescens is a gram-negative rod bacterium. This type of bacterium has an optimal growth temperature of 37° or above for a response (http://www.gutpathogens.com/content/2/1/16). P. fluorescens can be found in food, especially in the form of biofilms. It thrives in spoiled food which can cause sickness in humans when digested (http://www.biomedcentral.com/1472-6750/8/790).
In this lab, both selective and differential media were used to inoculate and observe the bacteria. A selective medium specifically is used only to allow certain types of organisms to grow and prevents any other organisms for growth. For a selective medium to be used in an experiment what is used on the medium or the incubation temperature could be altered. A differential medium differs from selective medium because differential is designed to show how differently the bacteria look when they grow. Differential compares two colonies of bacteria on a growth plate to see their characteristics and growth patterns. This also used oxidase and catalase tests for oxygen utilization. The oxidase test is useful to identify the type of metabolism used by the bacteria because an oxidase tests able to detect if the bacteria contained the oxidized form of cytochrome c and seeing if the colonies of the bacteria form a dark violet color. The oxidase test can also differentiate between Pseudomonas and Aeromonas. The catalase test is beneficial to identify if the bacteria are aerobic which would mean that there would be a formation of bubbles, otherwise they would be facultative anaerobes.
Methods: In the experiment, there were five different types of agar that needed to be inoculated with bacteria to see if there would be growth. The five different type of agar used in the lab were blood agar, MacConkey agar, Mannitol-agar, Hektoen entric agar, and Trypticase soy agar. The
agar is already in the petri dishes and labeled with the type of agar being used. The petri dishes should then be divided in half with a marker on the bottom side of the plate to then label each side with a bacterium to tell them apart. After, there should be dishes containing each bacterium for the experiment needed to be put into the agar plates. With a sterilized inoculation loop, a colony of the bacterium needed for the agar should be taken lightly. Only a small colony should be taken for a sample. Then transfer the bacterium to the appropriate agar plate to match up the bacterium. Then the inoculation loop should streak the plate with a straight line towards the middle of the line, then strokes should be drown coming out of the straight line creating an “E” shape. After, all five plates should be streaked the same way and the petri dishes should be closed right after streaking. The plates should be turned upside down with the label facing up and then be placed in the incubator at 37°C for 24 hours and then should observed for growth. In the oxidase test, the microorganisms E. coli, P. flourescens, and S. epidermidis were being compared to see if they were positive or not. First, a colony of the samples should be transferred to a glass slide from a sterilized inoculating loop. Then, drop one or two drops of the oxidase reagent onto the bacteria. Wait for a one minute or so and examine if there are any color changes. Colonies should start turning into a light color then dark. With that, the results with color changes would be positive. In the catalase test, two slides will be needed because water and S. epidermidis will be used to compare the two. A drop of water is need on one glass slide while a colony of S. epidermidis is needed to be transferred from a sterilized inoculating loop to the glass slide. Then, each slide will need about two to three drops of hydrogen peroxide since that is the reagent. After waiting about a minute the slides should be checked to see if there is bubble formation. If there were bubbles, then the reaction would be positive.
Results: Table 1: Growth of bacteria on selective and differential media Bacterium MacConkey agar Mannitol-Salt agar Uninoculated Dark violet Red S. epidermidis No growth No color change with red zones with no hydrogen sulfide production E. coli Pink colonies Yellow color surrounded with pink surrounding colonies zones with no with no hydrogen hydrogen sulfide sulfide production production P. mirabilis Colorless colonies No color change in with no hydrogen colonies with no sulfide production hydrogen sulfide production
Hektoen enteric agar Green No growth
Salmon-pink colonies with no hydrogen sulfide production
Black colonies with hydrogen sulfide produced
Table 2: Hemolytic reactions of bacteria grown on agar Bacterium Type of hemolysis observed S. epidermidis Gamma hemolysis E. coli Gamma hemolysis P. mirabilis Gamma hemolysis
For the oxidase test, the bacteria E. coli, S. epidermidis, and P. flourescens were tested to see if they were negative or positive. In this case, E. coli demonstrated a negative result with no reaction. On the other hand, both P. flourescens and S. epidermidis bacteria tested positive for the oxidase test.
For the catalase test, water and S. epidermidis were compared to observe if the enzyme catalase were present. When water was tested, the results came out negative. On the other hand, S. epidermidis came out positive because there was a formation of bubbles after adding the reagent.
Discussion: 1. The mediums that were used in the lab were found in the BIO 210 lab manual 1st quarter, 2013, DePaul University, and (October 13, 2012). The first medium that was used in the lab was blood agar. It is a differential medium because it has differential properties due to hemolysis and can be identified with the kind of color zones around the colonies (http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/selectivedifferen tial.htm). Another medium used in the lab was macConkey agar. Since this type of agar uses crystal violet, it is preventing the growth of any gram-positive bacteria. On the other hand, macConkey agar compares the bacteria of their fermentation. In this case, the medium show if the gram-negative bacteria are ferment lactose or lactose-nonfermenting which will differ in colony colors. The use of the red indicator will show that the bacterium is ferment lactose. Mannitol-salt agar is a selective medium because of the sodium chloride that prevents the growth of majority of the other bacteria. It is differential because it is comparing to see if the bacteria are mannitol fermenter or mannitol nonfermenter by the indication of red colonies. Hektoen enteric agar has a selective agent of bile salts that is trying to inhibit the growth of other gram positive organisms. Also, hektoen agar can identify lactose fermenters with the appearance of yellow color from the dyes. 2. If S. epidermidis and E. coli were growing together in a tube on nutrient broth, what can be compared to identify which bacteria is which, the media can help with the color. For example, knowing what the characteristics of E. coli, then that can assist in trying to identify when comparing that bacterium to S. epidermidis. The two organisms would be able to show whether one grows more rapidly than the other because it is possible for one
organism to grow over the other organism. It is essential to use the blood agar medium to first separate and compare between the two bacteria. The blood agar was used as an allpurpose medium because it differentiated the organisms with the reactions on the medium (BIO 210 lab manual, 2013). 3. P. mirabilis is one of the few organisms that are motile that was tested during the lab. Also, it was the only organism to have had colonies with black centers on the Hektoen enteric agar. From observing P. mirabilis, it was a lactose fermenter from the agars which help will help identify what kind of gram reaction it would be because gram-negatives are supposed to grow on the different mediums used in the lab. Since P. mirabilis was a gram-negative bacterium, it was expected to grow on the agars. 4. Since blood agar was used as the all-purpose medium in the lab, it would be beneficial for Staphylococcus aureus and S. epidermidis on blood agar because it will show the color of its colonies to tell them apart because the bacteria are different, so the blood agar would show the their hemolysis, thus the actions on the medium. S. aureus on blood agar should form cream colored colonies, while S. epidermidis should form white colonies (http://www.healthhype.com/lab-tests-for-staph.html). Tryptic soy agar is used as the base for agar plates, so having TSA on an all-purpose agar will help identify between the two organisms. 5. For the oxidase test, only P. flourescens and S. epidermidis had positive reactions which can be concluded that the two organisms must use oxygen to transport their foods during oxidative metabolism (http://www.vumicro.com/vumie/help/VUMICRO/Oxidase_Test.htm). For the catalase test, the organism S. epidermidis tested positive meaning that there is a catalase present
which is an enzyme that breaks down toxic products used in oxygen so the organism is able to tolerate oxygen (http://www.ncbi.nlm.nih.gov/pubmed/1141195). 6. To identify P. mirabilis, lactose would be essential in determining the organism because P. mirabilis was tested as a lactose fermenter and also that P. mirabilis is motile so that differs from E. coli which is another characteristic that could differentiate them apart. Knowing the characteristics of P. mirabilis that it is a gram-negative rod bacterium and that it was the only organism to grow colonies with black centers in the Hektoen agar so it was the only bacterium to have produced hydrogen sulfide.
References: Bauman, RW. (2012). Microbiology with diseases by body system, third edition. pp 743 http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.ht ml Date accessed: October 12, 2013. Date last updated: September 27, 2004 http://www.scienceprofonline.com/microbiology/differential-selective-bacterial-growthmedia.html Date accessed: October 12, 2013. Date last updated: April 2013 http://www.who.int/mediacentre/factsheets/fs125/en/ Date accessed: October 12, 2013. Date last updated: December 2011 http://www.who.int/topics/escherichia_coli_infections/en/ Date accessed: October 12, 2013. Date last updated: N/A http://www.gutpathogens.com/content/2/1/16 Date accessed: October 13, 2013. Date last updated: November 27, 2010 http://www.gutpathogens.com/content/2/1/16 Date accessed: October 13, 2013. Date last updated: June 2011
http://www.medicalnewstoday.com/articles/68511.php Date accessed: October 14, 2013. Date last updated: May 21, 2013 http://www.biomedcentral.com/1472-6750/8/79 Date accessed: October 14, 2013. Date last updated: October 28, 2008 http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/selectivedifferential.htm Date accessed: October 14, 2013. Date last updated: N/A http://www.healthhype.com/lab-tests-for-staph.html Date accessed: October 14, 2013. Date last updated: June 10, 2013 http://www.vumicro.com/vumie/help/VUMICRO/Oxidase_Test.htm Date accessed: October 14, 2013. Date last updated: N/A http://www.ncbi.nlm.nih.gov/pubmed/1141195 Date accessed: October 14, 2013. Date last updated: July 1975
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