Objectives: 1. To observe the laboratory equipments, their function and operation. 2.

To identify the unknown microorganism through biochemical test. 3. To learn how to do the biochemical test. Introduction: Unknown microorganism identification can be classified, or distinguished from one another, by the ability to (1) grow on different substrates and/or production of different end products, (2) produce specific enzymes, (3) use oxygen, or (4) be motile. For example, certain microbes can use different carbohydrates as sources of energy and/or carbon. Because such variability exists in carbohydrate utilization between different microbes, this can aid in the group, genus, or species identification. A. Carbohydrate utilization Carbohydrates can be fermented or oxidized via aerobic or anaerobic respiration. When a carbohydrate is fermented, acid, and sometimes gas, is produced. The most common end product of carbohydrate fermentation is lactic acid (lactate). Enteric organisms can produce lactic acid, formic acid, succinic acid, as well as ethanol and gasses CO2 and H2. Acid production can be detected by addition of a pH indicator, such as phenol red or brom cresol purple to the medium. Phenol red (PR) turns yellow in acidic conditions (slightly under pH 7.0) while at neutral or basic pH it is red. Gas production can be monitored by which has been inverted in the carbohydratecontaining growth medium to trap gas bubbles. If gas is produced, it typically is CO2 or H2. Carbohydrate utilization is important in distinguishing among the members of the family Enterobacteriaceae (the enteric bacteria). The Enterobacteriaceae is a family of organisms that are facultatively anaerobic, oxidase negative, Gram negative rods that ferment glucose. This family includes the genera Escherichia, Salmonella Proteus, Enterobacter, Serratia, Yersinia, Edwardsiella, Providencia,

Hafnia, Citrobacter, Shigella, and Klebsiella, to name a few. Of the above genera the only lactose fermenters are Escherichia coli, Klebsiella, Citrobacter, and Enterobacter. B. Enzyme production The ability of an organism to produce or express a particular enzyme can be tested in the laboratory using simple biochemical reagents and substrates. Several of these tests require only small amounts of cellular material and may take only a few minutes to interpret. Subsets of microbes can then be classified into groups based on their ability to produce a positive or negative reaction. The results of biochemical tests are used with other data, including the Gram reaction, to identify an organism by name. These tests play critical roles in the swift identification of bacteria causing infection. For example: 1) Catalase: H2O2 (hydrogen peroxide) is produced as an oxidation product during growth on carbohydrates in the presence of oxygen and is toxic if allowed to accumulate. Many organisms make the enzyme catalase to remove hydrogen peroxide. The catalase test measures the ability of an organism to produce the enzyme catalase that degrades H 2O2 to H2O and O2. To see if an organism makes catalase, H 2O2 is added to cells. If catalase is present, dioxygen gas is liberated and bubbles will be produced. Note that two molecules of H2O2 are needed because this is an oxidation-reduction reaction in which one molecule of H 2O2 serves as the substrate and the other serves as a donor. Water is the reduced product and O2 is the oxidized product. 2) Oxidase: The enzyme oxidase is part of the electron transfer system used by some organisms that use molecular oxygen as a terminal electron acceptor. Oxidase interacts with the membrane bound cytochromes and delivers electrons from the cytochromes to O2. As a result, H2O2 or H2O is generated. Strict anaerobes do not use O2 and hence do not possess the oxidase enzyme. Most Gram positive bacteria are oxidase negative as well as the members of the family Enterobacteriaceae.

C. Aerobic or anaerobic mode of growth (Oxygen Requirements) A very important requirement of cellular growth and metabolism is the requirement for oxygen. Microbes can be grouped into five different categories based on their requirement for molecular oxygen. Strict or obligate aerobes must have O2 for growth because O2 is used as the terminal electron acceptor for oxidative phosphorylation. In contrast, strict or obligate anaerobes cannot grow in the presence of O2 and may be killed by trace amounts of O 2. Microaerophilic organisms need a small amount of O 2 for growth but too much O 2 will kill them. A subset of anaerobes can tolerate O2 but they do not use it. They are called aerotolerant anaerobes. Facultative anaerobes grow best when O2 is available, but they can also grow, though not as well, if O 2 is not present. We will test several organisms for their growth pattern in medium to determine if they prefer aerobic or anaerobic conditions. D. Motility: swimming, swarming, twitching, and gliding Microbes move in a variety of ways that are roughly analogous to our walking, running, swimming, or crawling movements. Many organisms can swim in liquid. Most swimmers move by rotation of one or more external appendages called flagella. This is a complex process that is very well studied. The helical shape of the flagellum is reminiscent of a motorboat propeller; movement results from rotating one or more flagella. Flagella can rotate very fast when the cell is in liquid medium, allowing for rapid swimming. When cells with flagella are in more viscous (thicker) medium, such as broth containing some agar, the rate of swimming is reduced. Some examples of swimmers include Escherichia coli, Salmonella Typhimurium, and Bordetella pertussis. Twitching motility is a slow movement of cells over a surface via type IV pili. The cell throws out a pilus which attaches to a surface and is subsequently depolymerized so that the cell moves toward the attachment point. Pathogens, such a Neisseria and Pseudomonas, use twitching motility, as does the soil bacterium Myxococcus xanthus.

Motility can be used to distinguish between certain genera and to aid in species identification. Note that motility can vary as a function of temperature. Therefore it is important to measure motility at the temperature indicated for each organism. Materials: 1. MacConkey agar 2. Wire loop 3. Bunsen burner 4. Unknown microbe in broth 5. Grams stain 6. Slides 7. Microscope 8. Toothpick 9. Oxidase reagent 10. Filter paper 11. Catalase reagent 12. Peptone water 13. MR/VP agar 14. TSI agar 15. Motility test medium 16. Citrate medium 17. Methyl red reagent 18. Voges-proskauer reagent 19. Kovacs reagent (Indole) 20. Dropper

Method: 1. Before starting the experiments, a tour around the lab were done. 2. The students were given explanation regarding equipments inside the lab. The equipments were fume cupboard, incubator, water distiller, autoclave and dryer cupboard. Day 1; 1. The unknown microbes in broth were stain using Grams staining. 2. Then it was observed under the microscope and the shape, color of the stain and the organization of the microbes were observed. 3. Then, the microbes were cultured on the MacConkey agar. Then it was incubate for 24 to 48 hours in the incubator at temperature of 37C. Day 2; 1. After 24 to 48 hours incubation, the cultured microbes were observed it culture characteristics and whether it ferment lactose or not. The numbers of colony in the cultured were calculated.

2. Then, a few biochemical tests were run to observed it reactions. These include oxidase test and catalase test. 3. Other tests were also run but the medium were cultured for 24 hours first before run the tests. The tests were TSI test, motility test, indole test, MR test, VP test and citrate test. The entire tests were run for every colony that was found. 4. For oxidase test, the cultured microbes were put on a litmus paper that contains a few drop of oxidase reagent and the result of color changes on litmus paper was observed and recorded. 5. For catalase test, the cultured microbes were mixed together with the catalase reagent and the result of production of bubbles was observed and recorded. 6. For TSI test, the cultured microbes were stab using straight wire loop until the bottom of the butt of TSI agar and striking at the surface of the slant. Then it was incubated for 24 hours at 37C. 7. For motility test, the cultured microbes were stab about three quarter of the tube. Then it was incubated for 24 hours at 37C. 8. For indole test, the cultured microbes were mixed into the peptone water tube. Then it was incubated for 24 hours at 37C. 9. For MR and VP test, the cultured microbes were mixed with the MR-VP media. Then it was incubated for 24 hours at 37C. 10. For citrate test, the cultured microbes were striking on the surface of citrate agar. Then it was incubated for 24 hours at 37C. Day 3; 1. After 24 hours, the results of TSI test, motility test and citrate test were observed and recorded. 2. For the indole test, MR test and VP test, a few procedures were done to obtain results.

3. For indole test, the cultured media were added with 5 drops of Kovacs reagent. The changes in the tube were observed and recorded. 4. For MR test, the cultured media were added with 5 drops of MR (methyl red) reagent. The changes were observed and results were recorded. 5. For VP test, the cultured media were added with 6 drops of VP 1 and 2 drops of VP 2. It was let mixed for 2 minutes. Then the changes were observed and results were recorded. 6. After all the data were obtained, the results were compared with the biochemical reactions table that was given earlier and the type of microorganisms was identified.

Result:

Tests Lactose TSI Motility Catalase Oxidase Indole MR VP Citrate

Colony 1 (pink) Ferment Slant: Yellow Butt: Yellow (crack) Motile Negative Negative Negative Positive Negative Negative

Colony 2 (green whitish) Non-ferment Slant: Yellow Butt: Yellow Motile Positive Positive Negative Positive Negative Negative

Discussion: From this experiment, the slide that was observed under the microscope contains gram positive cocci bacteria and gram negative bacilli bacteria. However the organization of the bacteria were scattered so the actual organization is unknown. This is due to bad technique when preparing the slide. The cultures of the bacteria on the MacConkey agar showed about growth of 2 to 3 gram negative bacteria that ferment and non-ferment lactose. However due to bad striking techniques, the actual number of colony is unidentified. Also, the striking pattern on the agar also mixed and scattered so the shape of striking cannot be seen. MacConkey's agar is a selective medium that inhibits the growth of Gram-positive bacteria due to the presence of crystal violet and bile salts. Gram-negative bacteria grow well on MacConkeys Agar. MacConkeys is also a differential, meaning that it differentiates or distinguishes between groups of bacteria on the basis of a color change reaction. MacConkeys contains two additives that make it differential; neutral red (a pH indicator) and lactose (a disaccharide).

Bacteria, known as lactose fermenters, eat the medias lactose, and, in the process, create an acidic end product that causes the pH indicator, neutral red, to turn pink. With MacConkeys, it is not the media that changes color, but rather the actual colonies of lactose fermenting bacteria that appear pink. This can be seen in colony 1 in the culture media. Non-lactose fermenting bacteria will be colorless or, if they have any color, will be their natural color rather than pink. This can be seen in colony 2 of the culture media. These pink colonies are typically coliform bacteria in the family Enterobacteriaceae, inlcuding the genera Escherichia, Klebsiella, Enterobacter, Hafnia and Citrobacter. Non-lactose fermenting, non-coliform members of Enterboacteriaceae include the genera Proteus, Morganella, Providencia, Edwardsiella, Salmonella, Shigella andYersenia (plague bacteria). Biochemical tests were done to confirm the species of the Gram negative bacteria. The results from these tests were listed on the table above. In the TSI test, Grams negative bacteria that ferment any of the three sugars (lactose, sucrose and glucose) in the medium will produce byproducts. These byproducts are usually acids, which will change the color of the red pH-sensitive dye (phenol red) to a yellow color. Position of the color change distinguishes the acid production associated with glucose fermentation from the acidic byproducts of lactose or sucrose fermentation. All lactose fermenters result in yellow slant/yellow butt (acid/acid reaction), whereas non-lactose fermenters may result in pink/yellow (alkaline/acid reaction). Both colonies from this experiment had acid/acid reaction which indicates that they are lactose fermenters. Under anaerobic conditions some bacteria use H+ as an electron acceptor and reduce it to hydrogen gas. This is not very soluble and may accumulate as bubbles along the inoculation track, between the agar and the glass, or in the fluid which accumulates at the bottom of the slant. Hydrogen production may lift the agar from the butt of the tube or fracture the agar. Carbon dioxide, if produced, may not show as bubbles because it is far more soluble in the medium. This can be seen in colony 1 of the culture media.

For the motility tests, both colonies are motile bacteria. This is because on both motility medium, the bacteria growth along the stab line towards the surface of agar. For catalase test, colony 1 showed negative result while colony 2 showed positive results by production of bubbles when the colony sample was mixed with catalase reagent. For oxidase test, colony 1 showed negative result. Meanwhile, colony 2 showed positive result by changing color to pink when the sample colony was put on the wet filter paper (using oxidase reagent). For IMViC test, which are indole tests, Methyl Red test, Voges-Proskauer test and citrate test, both colonies showed negative results in most test except for Methyl Red test. Both colonies showed positive results in this test. For indole test, bacteria that posses the enzyme tryptophanase can break down the amino acid tryptophan to indole. When indole reacts with para-dimethylaminobenzaldehye (Kovacs reagent) a pink-colored complex is produced. For both colonies, no pink-colored complex was produced. For MR test, some bacteria produce acid from the metabolism of glucose in a sufficient quantity to produce a pH of 4.4 in the media. These acids are not further metabolized and are said to be stable acids. At a pH of 4.4 or less the pH indicator methyl red is a bright cherry red. pH indicator for both colonies showed bright cherry red. For VP test, some bacteria initially produce acid from glucose metabolism but further metabolize the acid produced to neutral end products. Initially the pH may drop to 4.4 but the neutral end products raise the pH so the methyl red test will be negative. These neutral end products under alkaline conditions will react with alpha-naphthol to produce a mahogany red color. But in this experiment, both colonies do not produce any color changes. For citrate test, citrate contains carbon. If an organism can use citrate as its only source of carbon the citrate in the media will be metabolized. Bromthymol blue is incorporated into the media as an indicator. Under alkaline conditions this indicator turns from green to blue. The utilization of citrate in the media releases alkaline bicarbonate ions that cause the media pH to increase above 7.4. The indicator for both colonies remains green. The biochemical test of the cultured showed many fake results due to mix of bacteria colony when it was taken from the culture plate to run the test. Also, it may be due to the different colony sample was taken when running the biochemical test.

Conclusion: As a conclusion, from this experiment it is confirmed that the colony 1 which are ferment lactose is Escherichia coli. Meanwhile, colony 2 which are non-ferment lactose are either Salmonella sp. or Pseudomonas aeruginosa.