Microbiology Activity #6 Metabolism of Small Molecules.

Microbiology Activity #6 Metabolism of Small Molecules. Analysis of Carbohydrate Metabolism Organisms that use CO 2 as a carbon source and fix the carbon into biomass are autotrophs, usually obtaining their energy from light or from oxidizing inorganic molecules such as H 2 S or NO 2-. Organisms that use organic compounds as sources of carbon, electrons, and energy are generally referred to as heterotrophs. Microorganisms can show significant variability in their carbon metabolism at different levels. First, bacteria vary in their ability to transport different compounds into the cell and convert them to common metabolic intermediates. Then, organisms may differ in their requirement for oxygen and in fact typically metabolize sugars differently in the presence (aerobic) or absence (anaerobic) of oxygen. Finally, bacteria may release different waste products into their environment. The specific combinations of carbon source utilization and waste product production can often be used to distinguish between microbes. In the absence of oxygen, some organisms will not grow, some will use alternate inorganic electron acceptors, and others will carry out fermentation, producing acids, alcohols and gases such as lactic acid, acetic acid (vinegar), ethanol and H 2. The production of acidic waste products can be detected by including a ph indicator in the medium while gases can be captured with an inverted durham tube. The tubes are NOT shaken (aerated) because fermentation generally only occurs in the absence of oxygen. Phenol red broth base contains a variety of nutrients, the ph indicator phenol red, but no carbohydrate. During an earlier lab period, we prepared phenol red media with a variety of carbohydrates at a concentration of 0.75%. If acids are produced during the fermentation of the carbohydrates, this can be detected by a ph change in the medium. To simplify this process, a ph indicator, phenol red, is incorporated into the medium. The phenol red indicator is red at neutral ph (7.0), and becomes yellow at a slightly acidic ph (6.8). If the medium becomes yellow, we surmise that acid has been produced as an end product in the breakdown of the carbohydrate (sugar) being used. Sometimes, organisms carry out what is called the alkaline swing : They ferment the carbohydrate to acids and then metabolize the amino acids in the medium, forming ammonia, which can remove protons from solution to form an ammonium ion. This results in a magenta color. It is a good idea to check your tubes after 24 hrs to see if acid has been produced, because by 48 hours, the ph may have increased again! In order to detect the production of gases, a small inverted tube called a Durham tube is placed in the fermentation broth. If gas is produced during fermentation, it will displace the medium within the inverted tube and will appear as a bubble. The reactions are scored in the following manner: If acid (yellow) but no gas is produced = a If both acid and gas are produced = ag If there is no color change, but the organism grew well (cloudy) = u No color change or growth = - 2016 Microbiology Laboratory Manual Page 25

Methyl Red-Vogues-Proskauer (MR-VP) test Most heterotrophic bacteria can utilize sugars for their energy demands. Mixed acid fermenters such as Escherichia coli ferment glucose to produce large amounts of acetic, formic, and succinic acids as end products. The large amount of acid produced lowers the ph of the medium to below 5.0. By using the indicator methyl red (MR), the production of these acids as an end product of fermentation can be monitored. If the ph drops below 4.5, the color of the methyl red indicator will be red (a positive result). If the ph is above 6.0, the color will be yellow/orange (negative). It is important not to confuse these color reactions with those of the phenol red indicator that was described above. Some bacteria, rather than producing abundant acid in the fermentation of glucose, produce other products such as alcohols. Enterobacter and Serratia will form products such as ethanol and 2,3-butanediol rather than the large amount of acid, as does Escherichia coli. These reactions can be used to differentiate some common gram-negative rod-shaped organisms. A test for acetylmethylcarbinol (a precursor of 2,3-butanediol that appears in the growth medium) is performed. After Barritt s reagents are added, a pink color developing after a few minutes indicates the presence of acetylmethylcarbinol, and the test is positive. Citrate Utilization Some microorganisms can metabolize citrate (citric acid) as a sole carbon energy source. When testing for citrate utilization with Simmon s Citrate medium, you should be aware that Citrate is transported into the cell in the fully protonated state as citric acid, a tricarboxylic acid. Removal of H + from the medium decreases the acidity and raises the ph. The rise in ph is detected by the color change in the bromothymol blue indicator present in the medium from green to a deep blue. This is indicative of a positive result and verifies that the organism is utilizing citrate. You should be careful to note any growth at all on the Simmons citrate slant, since certain organisms lack a citrate transporter and will not use citrate as a sole carbon source. Score any growth over the original inoculum as a positive reaction also. Using Koser citrate liquid medium, one should observe the development of turbidity to indicate whether growth has occurred. Carbohydrates vs amino acids Litmus milk medium allows one to detect the acid fermentation products of lactose (usually in the anaerobic bottom of the tube) as well as alkaline products of casein (milk protein) digestion (usually at the top of the tube) and formation of rennin (hard) or acid (soft) curd. The litmus ph indicator turns red under acidic conditions and purple under alkaline conditions. Results should be recorded as follows: - = No change Acid = red liquid Acid + curd - red & gelatinous Alkaline = purple near top 2016 Microbiology Laboratory Manual Page 26

In addition to carbohydrates, many organisms are able to amino acids as energy sources. Twenty different amino acids occur naturally in proteins. Each amino acid molecule has an amino group ( NH2), a carboxylic acid group ( COOH) and an R group attached to its alpha carbon atom. Removal of the amino group by deaminases produces organic acids that often feed into the central metabolic pathways. Decarboxylases remove CO 2 and produce positively charged amines such as cadaverine that often have unpleasant odors and are associated with decomposition. Other enzymes cleave off the R groups and produce byproducts such as H 2 S or indole Cysteine Desulfhydrase Bacteria that produce the enzyme cysteine desulfhydrase are able to strip the amino acid cysteine of both its sulfhydryl ( SH) and amino ( NH 2 ) groups. The reaction yields hydrogen sulfide (H 2 S), ammonia (NH 3 ), and pyruvic acid. To discover whether a test culture produces the enzyme, a microbiologist checks for hydrogen sulfide (H 2 S), an end product of cysteine desulfhydrase amino acid degradation. Popularly known as rotten egg gas, the end product H 2 S has a distinctly unpleasant and memorable odor. But identification of bacteria by the sniff method has not been standardized. Investigators utilize another chemical property of H 2 S to determine the presence of this malodorous gas. Hydrogen sulfide reacts with heavy metals such as lead or iron to form a visible, black precipitate. This reaction involves metal ions, not bacterial enzymes. But if bacteria produce hydrogen sulfide in the presence of a heavy metal, then visible evidence, the black precipitate, accumulates. The agar you utilize in this test, Sulfide Indole Motility (SIM) Agar, contains a plentiful supply of cysteine in its peptone. The agar also contains charged particles of iron. When bacteria growing in SIM Agar are able to release H 2 S from amino acids, ferrous sulfide (FeS) blackens the medium within 12 hours. The agar in SIM medium is present at a relatively low concentration and therefore motile organisms are able to swim away from a central stab inoculation. Finally, some organisms can be differentiated by their ability or inability to produce the enzyme tryptophanase. This enzyme hydrolyzes the amino acid tryptophan into indole, pyruvic acid, and ammonia. SIM medium is rich in tryptophan and therefore, after scoring the motility and H 2 S results, one can test the medium for the presence of indole using James reagent or Ehrlich s aldehyde, an acidic solution of paradimethylaminobenzaldehyde in alcohol. In the positive reaction, Ehrlich s aldehyde combines with indole forming a red layer that floats on the surface of the medium. In a negative response, Ehrlich s reagent does not become red. 2016 Microbiology Laboratory Manual Page 27

Urea Hydrolysis Urea is a common metabolic waste product that is toxic to most living organisms. The enzyme urease catalyzes the breakdown of urea into ammonia and carbon dioxide. Clinicians, investigating the cause of severe diarrhea or dysentery, routinely check suspect fecal bacteria for urease production. Manufacture of the enzyme distinguishes Proteus from Salmonella or Shigella. All three genera are gram negative, facultatively anaerobic, lactose non fermenting rods. However, Proteus, a member of the normal, intestinal flora of humans, produces the enzyme urease. Salmonella and Shigella, intestinal pathogens, do not. In this exercise you perform a test for urease production. The test medium contains urea and the dye phenol red, a ph indicator. If the bacteria produce urease, there will be a positive test reaction, and the color of the medium will change from yellow to cerise. Nitrate Reduction Many facultatively anaerobic (generally able to utilize an electron transport system to manufacture adenosine triphosphate with or without oxygen) bacteria produce nitrate reductase. This enzyme reduces (adds electrons to or removes oxygen atoms from) nitrate (NO 3 ). By utilizing nitrate reductase, facultative anaerobes can substitute NO 3 for O 2 as the final electron acceptor during their cellular respiration. NO 3 + 2 H + + 2 e NO 2 + H 2 O Testing for Nitrate Reduction To perform the nitrate reduction test, cultivate a pure culture of bacteria in a Nitrate Broth, a medium that contains large amounts of nitrate. After sufficient incubation, test the broth for the presence of nitrite. If you find nitrites, the bacteria have proven themselves positive for nitrate reductase production. But what does an absence of nitrite indicate? If there is no nitrite in the broth, there are at least three explanations for its absence. The bacteria do not reduce nitrate. The bacteria reduce nitrate to nitrite (NO 2 ). With nitrite reductase, they further reduce nitrite to ammonia (NH 3 ). In a process called denitrification, nitrate is reduced to nitrite, the nitrite is reduced to nitrogen gas (N 2 ). As you can see from explanations 2 and 3, simply testing a Nitrate Broth culture for the presence of nitrite is not enough to reveal the presence of nitrate reductase accurately. Bacterial enzymes may have further reduced the nitrite. Yet, the reagents commonly utilized in this procedure, sulfanilic acid and dimethyl alphanaphthylamine, only produce red pigment when they react with nitrite. Microbiologists utilize zinc, an inorganic catalyst that reduces nitrate to nitrite, to confirm the test for nitrate reduction. In this exercise you examine bacteria that produce nitrate reductase, nitrite reductase, and neither enzyme. What is the ecological significance of these reactions? Analysis of genomes in the genus Chryseobacterium has revealed that a cluster within the genus contains nitrite and nitrous oxide reductase, but no nitrate reductase (Clayton & Newman, 2014). We predict that such as organism will be able to carry out denitrification beginning with nitrite. 2015 is our first year testing nitrite reduction. 2016 Microbiology Laboratory Manual Page 28

Procedures: Inoculate media 1. Obtain two tubes of each type of medium on the front bench. Be sure that you can distinguish the different types of media either via their appearance or markings on the tubes. 2. For each type of medium, label one with your initials and K and one with your initials and UK 3. Because you have many tubes to inoculate with each of your organisms, you may find it convenient to use a liquid inoculum. Obtain 2 tubes containing 2 ml sterile water, label with your initials and either UK or K. Inoculate the tubes with the appropriate strain and vortex well. 4. Set a 1000 μl pipettor for 500 μl and draw up 500 μl of the known water suspension. Dispense one drop of suspension into each the media labeled for the known. 5. Repeat step 4 with the unknown organism. 6. Use a flamed inoculating needle to stab down into the SIM, citrate, and nitrite tubes. Incubate all tubes in the room temperature incubator until the next lab period. Data Collection Carbohydrate fermentation tests Phenol red base Red clear medium = for acid production or sugar utilization Red cloudy medium = u for sugar utilization Yellow medium = a for acid production Bubble in durham tube = ag for gas production Methyl Red (MR) Add three drops of the methyl red reagent to one tube of MR VP medium. A reddish color is indicative of a positive methyl red test. Vogues Proskauer (VP) test. Add 0.2 ml of Barritt s solution A (alpha naphthol) and 0.1 ml of Barritt s B solution (KOH). Vortex vigorously, being careful not to spill the contents of the tube. Let the tube stand for 15 20 min. A positive VP test will produce a pink to red color in the medium. Citrate Test Simmons citrate Blue color is +, green is negative Urease Magenta Color is positive, orange is negative 2016 Microbiology Laboratory Manual Page 29

Nitrate Reductase Add 3 drops of Nitrite A reagent (sulfanilic acid) and 3 drops of Nitrite B reagent ( naphthylamine). Do not vortex. If a red color develops, you have a positive result. If the culture does not become red within 5 minutes, use a toothpick to add a small amount of zinc powder to the broth. - A red color at this stage is negative, indicating that the nitrate was still present in the medium, i.e. it was not reduced by the bacteria. - No color development at this stage is positive for denitrification or nitrite reduction the nitrate in the medium had already been reduced to either ammonia or N 2. Nitrite reduction Add 3 drops of Nitrite A reagent (sulfanilic acid) and 3 drops of Nitrite B reagent ( naphthylamine). Do not vortex. If a red color develops, you have a negative result. SIM Cysteine Desulfurylase (sulfur) = + if mediuctionum turns black Tryptophanase (indole) = + if red after adding Ehrlich s aldehyde Motile if cloudiness extends away from central stab 2016 Microbiology Laboratory Manual Page 30

Known Unknown glucose lactose sucrose cellobiose mannitol xylose Methyl Red VP acetoin Sim. citrate Cys desulfurylase Phenylalanine deaminase Indole Motil urease Nitrate reduction denitrification 2016 Microbiology Laboratory Manual Page 31