Eric Lee Micro 351 Lab Biochemical Test of Microorganisms Lab Report Introduction Biochemical test are used

to differentiate between different microorganisms. Microorganisms are fastidious and respire all many different ways. The purpose of these experiments is to understand and to compare the biochemical activities for taxonomic purposes. In experiment 22, the purpose of the experiment was to understand the function of microbial extracellular enzymes. In addition to understanding this, the secondary purpose was to determine which microorganism has the ability to excrete hydrolytic extracellular enzymes. In experiment 23, the purpose of the experiment was to understand the difference between cellular respiration and fermentative respiration. In addition to understanding this, the secondary purpose was to determine which microorganism has the ability to degrade and ferment carbohydrates with the production of acid and gas. In experiment 24, the purpose of the experiment was to be able to differentiate microorganisms within Enterobacteriaceae. In addition to being able to differentiate microorganisms, the secondary purpose was to distinguish Enterobacteriaceae and other groups of intestinal bacilli. In experiment 25, the purpose of the experiment was an extension of the previous experiment. The extension of this previous experiment was achieved by using the IMViC test. In experiment 27, the purpose of the experiment was to be able to determine which microorganisms have the ability to degrade urea by means of the enzyme urease. In experiment 28, the purpose of the experiment was to be able to determine which microorganisms enzymatically transform different milk substrates into varied metabolic end products. In experiment 29, the purpose of the experiment was to be able to determine which microorganism had the ability to reduce nitrates to nitrites. In experiment 30, the purpose of the experiment was to be able to determine which microorganism had the ability to degrade hydrogen peroxide by producing the enzyme catalase. In experiment 31, the purpose of the experiment was to be able to distinguish among groups of bacteria through experimental testing that tested for cytochrome oxidase activity. In experiment 32, the purpose of the experiment was to be able to differentiate microorganisms by observing which has the ability to enzymatically degrade amino acid substrates.

Methods/Materials Experiment 22: Refer to page 147 of the laboratory manual for materials and methods. Experiment 23: Refer to page 153 of the laboratory manual for materials and methods. Experiment 24: Refer to page 158 of the laboratory manual for materials and methods. Experiment 25: Refer to page 162-5 of the laboratory manual for materials and methods. Experiment 27: Refer to page 175 of the laboratory manual for materials and methods. Experiment 28: Refer to page 180 of the laboratory manual for materials and methods. Experiment 29: Refer to page 186 of the laboratory manual for materials and methods. Experiment 30: Refer to page 189 of the laboratory manual for materials and methods. Experiment 31: Refer to page 193 of the laboratory manual for materials and methods. Experiment 32: Refer to page 198-9 of the laboratory manual for materials and methods. Data Experiment 22 (Extracellular Enzymatic Activities of Microorganisms) Table 1.1 Lipid Hydrolysis
Bacterial Species E. Coli P. Aeruginosa S. Aureus B. Cereus Starch Hydrolysis Appearance of Medium Results (+)(-) Yellow streak Yellow streak Yellow streak Brown yellow + surrounding is undyed Tributyrin Hydrolysis Appearance of Medium Results (+)(-) Streak Green/blue streak + Green/Blue streak + Large streak -

Table 1.2 Gelatin Hydrolysis

Bacterial Species E. Coli P. aeruginosa S. aureus B. cereus Casein Hydrolysis Appearance of Medium white/cloudy Green tinge Green tinge Cloudy white + + + Results (+)(-) -/+/+ -/+ +/+ Gelatin Hydrolysis Liquefacation Rate of Hydrolysis 2days/7Days None Fast Slow Fast

Experiment 23 (Carbohydrate Fermentation) Table 1.3

Bacterial Species E. coli S. typhimurium P. aeruginosa S. aureus Control Lactose Observation Yellow bubble Purple gray Purple, no gas Yellow Purple, no gas Results (A,A/G,-) A/G A/G A Dextrose Observation Yellow bubble Yellow bubble Purple Yellow Purple Results (A,A/G,-) A/G A/G A Sucrose Observation Lavender Lavender Purple, no gas Yellow Purple, no gas Results (A,A/G,-) A/G weak A/G weak A -

Experiment 24 (Triple Sugar-Iron Agar Test) Table 1.4

Bacterial Species E. Coli S. dysenteriae S. typhimurium P. vulgaris P. aeruginosa A. faecalis Control Butt Color and Reaction Acid/Orange Acid/Orange Acid/Orange Acid/Orange Acid/Orange Weak Alkaline/Red Alkaline/Red Carbohydrate Fermentation Slant Color Carbohydrate and Reaction Fermented Acid/Orange Lactose/Sucrose Alkaline/Red Glucose Alkaline/Red Glucose Acid/Orange Lactose/Sucrose Alkaline/Red None Alkaline/Red None Alkaline/Red None Blackening None None Yes None None None None H2S Production H2S (+)or (-) + + + -

Experiment 25 (IMViC Test) Table 1.5 (Voges-Proskauer Test, Methyl Red Test)
Bacterial Species E. coli E. aerogenes K. pneumoniae Control Indole production Test Color of Medium (+) or (-) Red + Yellow Red + Yellow Voges-Proshauer Test Color of Medium (+) or (-) Yellow Pink + Yellow Yellow -

Table 1.6 (Citrate Utilization Test)

Bacterial Species E. coli E. aerogenes K. pneumoniae Control Presence or Absence of Growth (+) or (-) + + Color of Medium Yellow/green Blue Blue Green Citrate Utilization (+) or (-) + + -

Experiment 27 (Urease Test) Table 1.7

Bacterial Species E. coli K. pneumoniae Control Color of Medium Yellow agar color Pink/magenta Yellow Urea Hydrolysis (+) or (-) + -

Experiment 28 (Litmus Milk Reactions) Table 1.8

Bacterial Species E. coli P. aeruginosa A. faecalis L. lactis Appearance of Medium Darker purple on 3-4 cm top,rest is lighter purple 95% lavender,5% white on bottom 1cm pink top, remaining is white 2cm pink top, 6cm white bottom Litmus Milk Reactions Acid with reduction and curd Alkaline Alkaline Acid with reduction and curd

Experiment 29 (Nitrate Reduction Test) Table 1.9

Bacterial Species E. Coli P. aeruginosa A. faecalis Red coloration with Solutions A and B (+) or (-) + Red Coloration with Zinc (+) or (-) + Nitrate Reductions (+) or (-) + + End Products NO2 NH3+/N2 NH3+/N2

Experiment 30 (Catalase Test) Table 2.0

Bacterial Species M. luteus L. lactis S. aureus Presence or Absence or Bubblings + + Catalase Production (+) or (-) + +

Experiment 31 (Oxidase Test) Table 2.1

Bacterial Species E. coli P. vulgaris P. aeruginosa A. faecalis M. Luteus Color of Colonies White/yellow White/yellow No change Purple White/yellow Oxidase Production (+) or (-) + + +

Experiment 32 (Utilization of Amino Acids) Table 2.2

Decarboxylase Test Bacterial Species E. coli P. vulgaris Color of Medium LD + LDPurple+ YellowYellowYellow-

Discussion In experiment 22 table 1.1 and 1.2, the following microorganisms were tested: E. Coli, P. Aeruginosa, S. Aureus, and B. Cereus. These microorganisms were tested for extracellular enzymatic activities. The tests that were done were to check if these microorganisms hydrolyzed the following molecules: Starch, Lipids, Casein (major milk protein), and Gelatins. For the starch hydrolysis plate, the only bacteria that exhibited a positive result was B. Cereus. All the other microorganisms remained with only a streak. This exhibited growth meant that B. Cereus was able to hydrolyze starch which is composed of glucose molecules. For the Tributyrin Hydrolysis plate, the only two microorganisms that exhibited a positive result was P. Aeruginosa, and S. Aureus. This meant that P. Aeruginosa, and S. Aureus, was capable of hydrolyzing lipids. In table 1.2, the microorganisms were then tested for the ability to hydrolyze gelatin and Casein. Casein is a major milk protein that is composed of amino acid subunits. All microorganisms were able to hydrolyze casein except E. Coli. In the Gelatin Hydrolysis, E. Coli was not able to liquefy the gelatin, therefore it did not have any rate of hydrolysis. P. aeruginosa hydrolyzed gelatin at a fast rate, it liquefied in two days. Therefore P. Aeruginosa is able to hydrolyze gelatin at a fast rate. S. aereus hydrolyzed gelatin at a slow rate. The first two days it was not able to hydrolyze gelatin. It was only able to hydrolyze the gelatin after the seventh day. Therefore S. aereus hydrolyzed at a slow rate of hydrolysis. B. cereus hydrolyzed gelatin at a fast rate, it liquefied in two days. Therefore B. cereus had a fast rate of hydrolysis. Compared to the actual data found in page 204, the results in this experiment were accurate. In Experiment 23, table 1.3, the following microorganisms was used to test Carbohydrate fermentation: E. Coli, P. Aeruginosa, S. Aureus, and S. typhimurium. There were three different test medias, they included lactose, dextrose, and sucrose. Under Lactose, Dextrose and Sucrose E. coli exhibited an acidic reaction alongside with production of gasses. In the Sucrose media, E. coli exhibited the acidic reaction with production of gasses weakly. S. typhimurium also exhibited the same results as E. coli. P. aeruginosa exhibited no reaction in all three media, Lactose, Dextrose, and Sucrose. S. aereus in all three media exhibited the same reaction. All the media for S. aereus presented an acidic reaction. The acidic or gaseous reaction helped to identify

microorganisms that are capable of fermenting the carbohydrate substrate with the production of acid or gas. Compared to the actual data found in page 204, the results were accurate. In experiment 24, table 1.4; the following microorganisms were used to test for Carbohydrate fermentation: E. coli, S. dysenteriae, S. typhimurium, P. vulgaris, P. aeruginosa, and A. faecalis. In this test the following observations were recorded: Butt color and Reaction, Slant Color and Reaction, Carbohydrate Fermented, Blackening, and H2S production. This test helps to identify among the members of enterobacteriaceae and their ability to ferment carbohydrates. The first microorganism E. Coli, exhibited an Acid butt and an Acid slant. Blackening was not present, but H2S production was present. The carbohydrate that E. Coli seemed to ferment was Lactose and Sucrose. S. dysenteriae exhibited an acidic butt and alkaline slant. Blackening was not present, but H2S production was present. The carbohydrate that S. dysenteriae seemed to ferment was Glucose. S. typhimurium exhibited an acidic butt and an alkaline slant. Blackening was exhibited, but H2S production did not occur. The carbohydrate that S. typhimurium seem to ferment was glucose. P. vulgaris exhibited an acidic butt and an acidic slant. Blackening did not occur but H2S production was present. The carbohydrate that P. vulgaris seem to ferment was Lactose/Sucrose. P aeruginosa presented an acidic butt and alkaline slant. It did not present blackening or H2S production, it also did not seem to ferment any carbohydrates. A. faecalis presented a weak alkaline butt, and an alkaline slant. Blackening or H2S production did not occur. A. faecalis did not seem to ferment any carbohydrates. These test helped to examine which microorganisms were able to utilize carbohydrate fermentation. In experiment 25, table 1.5 and table 1.6, the following microorganisms went through the IMViC tests, they were: E. coli, E. aerogenes, and K. pneumoniae. The IMViC test consists of four tests. These four tests are the Indole production test, Methyl Red Test, Voges-Proshauer Test, and Citrate Test. All tests were recorded except for the Methyl Red Test. The IMViC test identifies microorganisms that are in the family Enterobacteriaceae. The indole production test, test for a microorganisms ability to hydrolyze tryptophan. The only microorganisms in the Indole production test that exhibited Indole production was E. coli, and K. pneumoniae. The Voges-Proshauer test, test for an microorganisms ability to ferment glucose. The only microorganism that exhibited a positive result to ferment glucose was E. aerogenes. The Citrate utilization test, test for an microorganisms ability to use citrate as its sole source of carbon. The only microorganisms that exhibited growth was E. aerogenes, and K. pneumoniae. Compared to the actual results on page 204 of the laboratory book, the experimental results matched the given results. In experiment 27, table 1.7, the following microorganisms were used to test for the enzyme Urease: E. Coli, and K. pneumoniae. Urease hydrolyzes urea. K. pneumoniae was the only microorganism that tested positive for the urease enzyme. This means that K. pneumoniae is able to hydrolyze urea. Compared to the actual results on page 204 of the laboratory book, the experimental results matched the given results. In experiment 28, table 1.8, the following microorganisms were used to test for their reactions in litmus milk, they are: E. coli, P. aeuginosa, A. faecalis, and L. lactis. The following biochemical changes can result from different microorganisms in litmus milk, they are as follows; Lactose fermentation, Gas production, Litmus reduction, Curd

formation, Proteolysis, and Alkaline reaction. E. coli had an acidic reaction with reduction and curd. P. aeruginosa had an alkalytic reaction. A. faecalis also presented an alkalytic reaction. L. lactis presented an acidic reaction with reduction and curd. Compared to the actual results on page 204 of the laboratory book, the experimental results matched the given results except for P. aeruginosa. P. aeruginosa was supposed to exhibit rapid peptonization. A possibility for a difference in reaction was that there could have not been enough microorganisms present to have rapid peptonization. Another possibility could have been a not long enough incubation time. In experiment 29, table 1.9, the following microorganisms were tested for the ability to reduce nitrates: E. coli, P. aeruginosa, and A. faecalis. These test work by testing if these microorganisms have the ability to reduce nitrates to nitrites. P. aeruginosa did not develop any red coloration when solutions A and B were added, and also when Zinc was added. This could possibly mean that P. aeruginosa had nitrate reductase enzymes. Therefore P. aeruginosa was able to reduce the nitrates to nitrites with nitrate reductase enzymes. It did not appear that E. Coli had nitrate reductase since red coloration stayed after solution A and B was added. Therefore, the determination factor of E. colis ability to reduce nitrates was when zinc was added. Coloration did not appear when zinc was added. Therefore nitrate reduction occurs in E. Coli. A. faecalis was not able to reduce nitrates. This was determined by red coloration appearing when zinc was added to the media. It appeared that the end product of E. coli was NO2 and for P. aeruginosa and A. faecalis it was NH3+/N2. Compared to the actual results on page 204 of the laboratory book, the experimental results matched the given results. In experiment 30, table 2.0, the following microorganisms were tested for the enzyme catalase: M. luteus, L. lactis, and S. aureus. The catalase test determines which microorganisms are capable of producing catalase to degrade hydrogen peroxide. The presence of bubbling when hydrogen peroxide is added signifies that the enzyme catalse is present in the microorganism. The two microorganisms that presented catalase production and bubbled in the presence of hydrogen peroxide was M. luteus, and S. aureus. Compared to the actual results on page 204 of the laboratory book, the experimental results matched the given results. In experiment 31, table 2.1, the following microorganisms were tested for the enzyme cytochrome oxidase: E. coli, P. vulgaris, P. aeruginosa, A. faecalis, and M. luteus. Cytochrome oxidase catalyzes the oxidation of cytochrome. P. vulgaris, A. faecalis, and M. luteus presented a positive reaction for oxidase production. This means that these three microorganisms contained the enzyme cytochrome oxidase. Compared to the actual results on page 204 of the laboratory book, the experimental results matched the given results. In experiment 32, table 2.2, the following microorganisms were tested for their ability to degrade amino acid substrates: E. coli, and P. vulgaris. The Decararboxylase test, determines which microorganisms can enzymatically degrade amino acid substrates by determining which microorganism has the decarboxylase enzyme. If the microorganism is able to use L-lysine, it will change the pH color to purple. This indicates that L-lysine has been decarboxylated. The only microorganism that had the decarboxylase enzyme and was able to degrade L-lysine was E. coli. Compared to the actual results on page 204 of the laboratory book, the experimental results matched the given results.

Conclusion I felt that this lab was very important. It gave us the ability to learn how to identify organisms by their different enzymatic activities. There were a lot of tests, but it helped to narrow down the list of microorganisms dramatically. I think these experiments were not that hard compared to the others. Much of it was just inoculating with a lot of different media. I didnt imagine that there would be so much media, and I am guessing that there is a lot more types of different media to do different types of tests.