MCB 301 Lab Manual supplemental

Spring 2008


List of Possible Bacteria
You will each be given one organism to inoculate a set of biochemical tests for
Experiment 11. Organisms that have had their genomes fully sequenced are listed
below with their genome id number. In several cases, a genome sequence is not
available for the bacteria you will grow and test. Alternative genomes are listed in
parentheses. Prior to running the biochemical tests, you will explore the genome of a
given organism and predict the result of the biochemical phenotype on the basis of
Gram Positive Bacilli

Gram Negative Bacilli

Bacillus megaterium (B. clausii 66692.3)
Bacillus subtilis 224308.1
Lactobacillus plantarum 220668.1

Salmonella Arizonae 321314.4 (aka S.
enterica subsp. enterica ser. Choleraesuis)
Proteus vulgaris (P. mirabilis 584.1)
Serratia marcescens (NP) 615.1
Citrobacter freundii (Salmonella
typhimurium 99287.1)
Hafnia alvei (Yersinia pseudotuberculosis
Enterobacter cloacae (Shigella flexneri 2a
str. 301 198214.1)
Morganella morganii (Salmonella enterica
subsp. enterica serovar Typhi Ty2

Gram Positive Cocci
Enterococcus faecium (E. faecalis
Staphylococcus aureus (NP) str. NCTC
8325, 93061.3
Staphylococcus epidermidis str. RP62A,

Experiment 10 a-c: Bioinformatic Predictions, Summary
Predictions will be made from annotated genomes available at the National Microbial
Pathogen Data Resource, Whole genome sequences are available
through a variety of databases, but the NMPDR has many useful tools that will make it
easier for you to predict the biochemical test results. Most importantly, NMPDR
curators have organized the genome annotations into biological subsystems.
In the list above, genome id numbers link to the respective organism's Subsystem
Summary. This summary is a list of genes that have been classified according to
function. Functional roles that meke up a pathway or complex are grouped together into
subsystems. Related subsystems are grouped together under a common heading, for
example, the first set of subsystems is listed under the heading, "Amino Acids and
Derivatives." Subheadings further classify related subsystems. Subsystem names are
linked to a page that describes the subsystem. Proteins listed in each subsystem are
linked to pages that display their genomic context.
The presence or absence of individual genes or groups of genes that form a complete
metabolic pathway will be investigated using the subsystems of NMPDR. These
genotypes will predict the biochemical phenotype that results from each test. If you
have enough information about the genotype to predict the outcome with precision,
record your prediction using the outcome notations listed for each biochemical test. If
you only have an idea of positive or negative, list a plus or minus.

non-fermentative hydrolysis of amino acids in the peptone. However. A: Acid has been produced. The medium has changed color from bluish-green to yellow and a gas bubble has formed in the Durham tube. Gas production. If gas is produced. The medium has turned lime green or yellowish in the bottom of the tube and greenish at the top. (A): A little bit of acid has been produced. like oxygen (aerobic respiration) or some other exogenous source (anaerobic respiration) to generate high yields of ATP through complete oxidation of an organic compound. Lactose. Acid products. can be determined by placing a small. which may be produced from the fermentation of a sugar. The medium has turned yellow. may give an alkaline reaction. Fermentation. sugar fermentation patterns are used in the identification of enteric bacteria. will cause a noticeable color change in the pH indicator included in the medium. only partially oxidizes the substrate and generates a relatively small amount of ATP. The medium is green. Sucrose Bacterial cells are able to generate energy from nutrients through respiration or through fermentation. The determination of a fermentation pattern for a series of different energy/carbon sources (usually sugars) by an unknown bacterial species is often a central part in the identification process. Different bacteria can ferment a wide variety of sugars and other compounds. H2 in particular. which will also cause a color change in the pH indicator. inverted Durham tube in the test medium. Raffinose. Sugar fermentation does not produce alkaline products. present in most fermentation media.MCB 301 Lab Manual supplemental Spring 2008 2 Experiment 10a: Carbohydrate Metabolism 1. Note: reference to gas is for the glucose test only. Sugar Fermentations . Respiration uses an external electron acceptor.Glucose. This change will occur if the amount of alkaline end products made from the utilization of amino acids in the medium exceeds the amount of acidic end products from the sugar. 0 +: No acid or gas has been produced and growth is noted [shake the tube to look for turbidity (cloudiness) indicating bacterial growth]. The medium has turned blue due to an alkaline reaction caused by amino acid utilization as a carbon source. 0 +/B: No acid or gas has been produced and growth is noted. The terminal electron acceptor is usually produced as an intermediate in the pathway and so is internal instead of external. on the other hand. Possible Results A/G: Both acid and gas have been produced. . it is trapped in the Durham tube and can be seen as a bubble. For example.

and also open the link on page 1 that corresponds to your assigned organism (control-click on this document)." Subheadings could include "Central carbohydrate metabolism. HO mannose H mannitol Possible Results A: Acid has been produced. Subsystems describing the sugar utilization or fermentation will be listed under the major heading. Open the link above for the positive control." .and oligosaccharides. "Carbohydrates. an aldose (sugar aldehyde).1 uses all four sugars as well as mannitol. a sugar alcohol. (A): A little bit of acid may have been produced. from peptone.MCB 301 Lab Manual supplemental Spring 2008 3 2. The test result therefore is dependent on several factors. The medium has changed color from purple to yellow. 0 +: No acid has been produced. Control: Streptococcus mutans 210007. The net reaction observed in the fermentation test is usually the difference between the production of acid from a sugar and the production of alkaline end products. is widespread in plants and algae.Mannitol Mannitol. It is the reduced form of the monosaccharide mannose. such as ammonia." "Fermentations" or "Monosaccharides. how much and at what rate? (2) Will the medium support growth? How highly is it buffered and how much alkaline product will be generated in it? (3) How sensitive is the indicator? We can explore the metabolic capacity of the organisms to predict the answer to (1) above for each of the five separate sugars to be tested. (1) Does the microorganism produce acid? If so. The medium remains purple and growth is noted [shake the tube to look for turbidity (cloudiness) indicating bacterial growth]." "Di. The medium has changed to a grayish purple color (in between yellow and purple). Sugar Fermentation . Mannitol has 14 hydrogens compared to 12 for mannose.

or simply plus or minus? 2. Record your predictions on the answer spreadsheet (last page). . evidence could be "absence of subsystem x" or "presence of gene y. Which functions are missing? Do you predict that this strain will ferment mannitol? Explain. The abbreviations are also listed in the table of functional roles. S. click on the Mannitol Utilization heading (or click here) to open the subsystem. In the Subsystem Summary for the positive control genome. mutans. The page presents a spreadsheet with genomes as rows and functional roles as columns. By looking only at the Subsystem Summary. Numbers with the same background color are in close proximity in the genome. Scroll back up to the subsystem spreadsheet. Genes that perform the listed functions are populated in the cells of the spreadsheet. These notes will explain the variant codes and are frequently very informative. which you must scroll down to find. Notice that there are "missing genes" in one strain of Streptococcus pyogenes." QUESTIONS ON FERMENTATIONS 1. The abbreviated column headers are decoded in pop-up boxes if you point to them. Below the table of functional roles are the curator's notes. Now scroll through the subsystem summary for your assigned genome. is it possible to predict the full range of biochemical test results. and include evidence supporting your prediction in the column labeled "Why?" For example.MCB 301 Lab Manual supplemental Spring 2008 4 Scroll through the subsystem summary for the positive control. Note which subsystems you think are involved in fermentation and utilization of these sugars.

MCB 301 Lab Manual supplemental Spring 2008 5 3. K12 83333. but differentiates them based on some visible growth characteristic such as colony color. 0 . Your task here is to find a genotype that predicts the phenotype of Gram staining. Bile salts and crystal violet in the medium inhibited growth of the organism. Since this medium is so common. MacConkey agar is a commonly used primary plating medium in many clinical microbiology laboratories. acid products lower the pH below 6. When lactose is fermented. It will be especially helpful in the Unknown Identification lab in the differentiation of your Gram positive and Gramnegative unknowns. If an organism is unable to ferment lactose.1 is Gram negative and does not ferment lactose. MacConkey Agar MacConkey agar is a widely used culture medium that is both selective AND differential.: No growth occurred. You need to find a gene that is present in Gram positives but not Gram negatives. Possible Results A :Growth occurred and all colonies are noticeably pinkish red.8 with the resulting colonial growth turning pinkish-red. while inhibiting the growth of others. it behooves microbiologists to be efficient in interpreting colonial growth. In the case of MacConkey agar. . but some look a little bit pink/red. and another with the opposite distribution. A differential medium does not inhibit the growth of bacteria. and the pH indicator neutral red. the colonies will be colorless or yellow. Scrolling through long lists of categorized genes is getting tedious.1 is Gram positive and ferments lactose. Controls: Streptococcus mutans 210007. Predict the result of MacConkey plating based on lactose fermentation and cell wall characteristics. and because it can provide timely clues as to the identification of some Gram-negative bacilli. You may know of candidate genes from your reading or lecture courses.1 is Gram negative and ferments lactose. or you still may need to explore subsystems to find genes that are diagnostic of cell wall type. 0 + : Growth occurred and colonies are colorless. A selective medium selects for the growth of some organisms. which include potential pathogens. Acid has been produced. No acid has been produced. MacConkey agar contains lactose. a fermentable carbohydrate. Decisive evidence is needed to predict whether your organism will grow on MacConkey agar. You have already predicted lactose fermentation. the presence of bile salts and crystal violet inhibits the growth of most Gram-positive bacteria. The medium thus differentiates between lactose-fermenting bacteria and lactose nonfermenters. Shigella flexneri 198214. You must also realize that the absence of a particular subsystem in these lists may simply mean that the curator has not yet examined the genome you are looking at. Most colonies are colorless. Escherichia coli str. (A) : Growth occurred.

Click on the NMPDR button in the search results table to see protein details. NMPDR data pages always have a search box in the page banner. To limit this search to one genome. Keep in mind that keyword searches look at the labels of sequence data—not the sequence data itself. the spreadsheet will list all organisms included by the curator.11. So." Another strategy is to use the most exact label for an enzyme. the spreadsheet is focused on closely related genomes. click on the link | Subsystems |. do Gram-negative bacteria have a gene for L-alanine aminopeptidase that is absent from Gram-positive genomes? Do a keyword search for this enzyme in your assigned genome as well as in the three controls listed above. simply include the genome id number as a keyword. Gram-negative bacteria tend to have greater activity.4." QUESTION ON Mac CONKEY AGAR 1. and another that indicates Gram negative. You may also use your browser's "find in page" function along with the genome id number to locate your assigned organism. This presents you with a keyword box as well as genomes to select from. but those that NMPDR focuses our curation efforts on are listed first. There are several search boxes available.MCB 301 Lab Manual supplemental Spring 2008 6 In the header of your Subsystem Summary page. such as size and the identity of neighboring genes on the genome." Further expand (click on the plus) the categories for Gram negative and positive cell wall components. This may take a few moments. Within the subsystem summary page there is a search box that will limit your search to the given organism. This will launch a keyword search of all genomes in NMPDR. Keywords are joined by "AND. . Expand the category for "Cell wall and capsule. This presents a collapsed list of all available subsystems. try using "3. To search more than one genome at a time. There are controls below the spreadsheet that allow one to expand or reduce the genomes shown. You may need to explore your search results to be confident that proteins with similar names have similar sequence and function. If you get unexpected results. If "L-alanine aminopeptidase" does not find the match you expect. is this biochemical phenotype explained by genotype? Explain. Choose one gene that indicates Gram positive. Support your test prediction with "presence of gene x AND absence of gene y. Because you are opening them from the subsystems tree rather than from a particular organism's subsystem summary. Please read the curator's notes before deciding on your diagnostic genes.2" as the search term. while Gram-positives have very low activity. use the | Genes | link on the NMPDR home page. A commercial test to replace Gram staning is a colorimetric assay for the function of L-alanine aminopeptidase. Control click to select more than one. then try using "alanine aminopeptidase. When you open a subsystem from an organism summary page. Is this diagnostic phenotype of enzyme activity explained by genotype? That is. as well as to resort their order in the spreadsheet. the EC number. The organization of genomes is mostly alphabetical. Explore any of these subsystems." so it makes no sense to include more than one genome id.

aerogenes is not. a soil organism often found in water. no color change Controls: Escherichia coli str. 6. 8. Possible Results + : growth and/or any blue color change in the medium . 2. K12 83333. 5. 7. Since E. Utilization of citrate (the neutral salt of citric acid) differentiates between some enteric bacteria. It is a practical test for distinguishing between Escherichia coli. Citrate is the sole carbon and energy source present in Simmon’s citrate medium. which oxidizes pyruvate to CO2 during aerobic respiration. oxaloacetate decarboxylase.1 transports citrate. lactate dehydrogenase.: no growth. coli is an indication of fecal contamination and E. pyruvate dehydrogenase complex. diacetyl/acetoin reductase. a fecal organism that cannot use citrate as the sole carbon source. 3. FIG. Citric Acid Utilization Citric acid is an intermediate in the tricarboxylic acid (TCA) cycle.1 does not transport citrate. acetolactate decarboxylase. citrate utilization is a routine test for examining water quality. 4.MCB 301 Lab Manual supplemental Spring 2008 7 4. Schematic pathway showing the metabolic relationships between citrate and glucose. 1. acetate kinase. alcohol dehydrogenase. To use citric acid. which can. Some kinds of bacteria are able to ferment citric acid to acetic or succinic acid. 10. acetolactate synthase. 9. and Enterobacter aerogenes. pyruvate formate lyase. This test is performed to determine whether a bacterium can use citric acid as its sole energy/carbon source. so if an organism is not capable of transporting it across the cell membrane there will be no . 1. Enterococcus faecalis 226185. citrate lyase. a bacterium must be able to transport it across the cell membrane.

Would the presence or absence of a gene for citrate lyase be predictive of the results of the citrate test? Explain 2. Efficient utilization of citrate relies on the activities of citrate lyase and oxaloacetate decarboxylase. "citrate transporter" is highlighted in green.MCB 301 Lab Manual supplemental Spring 2008 8 growth. Click on the button to show compare regions. There is also a table further down the page that lists the identities of the genes shown in the graphic. Which of the genes neighboring the focus gene (E. Arrows of the same color and number share significant sequence similarity. and identities of neighboring genes are shown in pop-ups when pointed to." Predict the results of the citrate test from the presence or absence of genes encoding citrate transporters in your organism. QUESTIONS ON CITRIC ACID 1. The focus gene. This graphic shows genomic neighborhoods in organisms with similar proteins. In the search results for citrate transporter in the positive control genome. click on the NMPDR button to open the protein page. Perform a keyword search of the control genomes as well as your assigned organism for "citrate transporter. faecalis citrate transporter) are also involved in citrate metabolism? Where is the alpha chain of oxaloacetate decarboxylase? (hint: keyword search for EC and genome numbers) .

we will nonenzymatically oxidize any butanediol that is present in the culture back to acetoin (a detectable molecule) by shaking the tube to introduce air (O 2). MR/VP is a very useful classification test for two reasons: (1) There are basically three possible patterns. and in the process helps to determine the species of bacteria that is most likely present. Acetoin is the last intermediate in the butanediol pathway. wk: the medium develops an orange color. The acetoin acts as a terminal electron acceptor with the carbonyl group being reduced to a hydroxyl group. providing good discrimination between bacteria and (2) MR/VP differentiates between E. you may see a positive result for both tests. but it is rare. acetic. In the procedure. MR/VP is actually two tests: The methyl red (MR) test determines whether or not large quantities of acid have been produced from mixed acid fermentation of glucose. acetoin. contains a carbonyl group. The tests are complementary in the sense that often a bacterium will give a positive reaction for one test and a negative reaction for the other. a common soil organism. detected by the VP test. has been produced instead of acid from glucose. outlined below. End products of this pathway include lactic. The three possible patterns of results are: Methyl Red (acid produced from glucose) Voges-Proskauer (acetoin produced from glucose) + - + Occasionally. The Voges-Proskauer (VP) test determines whether a specific neutral metabolic intermediate. Methyl Red / Voges-Proskauer (MR/VP) Test This test enables the microbiologist to determine the pathway being used to ferment glucose. acetoin. The reagents -naphthol and KOH act as oxidizing agents. butanediol. a human intestinal organism and Enterobacter aerogenes. The 4-carbon intermediate that is formed. It is thus useful in sanitary analysis of water supplies. which is a common fermentation pathway in Bacillus (see Figure 1). coli. two molecules of pyruvate condense and two molecules of CO 2 are released. The reduced product. Methyl red test: + : the medium immediately develops a red color. is excreted by the bacteria (see background information on endospore-forming bacteria).: the medium remains yellow. formic and succinic acids. and the addition of creatine intensifies the color change.MCB 301 Lab Manual supplemental Spring 2008 9 5. Voges-Proskauer test: + : a pink-red color develops . In the butanediol fermentation pathway. yellow or black color . .: no color change or turns a brown.

Click to open the subsystem diagram.MCB 301 Lab Manual supplemental Spring 2008 10 Controls: MR. ./VP+: Bacillus subtilis 224308. If your test organism does not appear in the list. 6.7-dihydroxycoumarin -D-GLUCOSE Some bacteria are able to perform this hydrolysis with the enzyme beta-glucosidase. then click the button to color the diagram. This complex appears as a black precipitate and denotes a positive esculin test. Predict the results of the test and list the evidence for your prediction. Select the positive control genome from the drop-down list. check the box to show genomes with negative variant codes and wait for the page to redraw. check the box to show genomes with negative variant codes and wait for the page to redraw. If your test organism does not appear in the list. H+ ESCULIN 6. Now color the diagram to show genes present in your test organism. and esculetin which reacts with ferric salts in the media to produce a phenolic iron complex (shown below). -Glucosido-7-hydroxycoumarin + ESCULETIN 6. then click the button to color the diagram.1 MR+ /VP-: Salmonella enterica subsp. It is an acetal derivative of D-glucose. which is hydrolyzed to glucose and esculetin (an acetal moiety) in the presence of acid. Click to open the subsystem diagram. which can be used as a carbon/energy source. The VP test will be positive for organisms that have complete pathways for acetoin. Select the positive control genome from the drop-down list. Esculin Hydrolysis Esculin is a glucoside (sugar derivative). Choleraesuis 321314. enterica ser. resulting in glucose.4 The MR test will be positive for organisms that have complete pathways for mixed acid fermentation. Now color the diagram to show genes present in your test organism. butanediol metabolism.

faecium and E. The bile salts. followed by alphabetical lists of Archaea. the cell moves in a forward direction (smooth swimming). To avoid scrolling. Clearly. 7. such as E. Motility test medium is a semisolid agar that permits the movement of motile bacteria. Thus." Click the go button to launch the search. similarity of the functional names. faecalis. Bacterial flagella are long appendages that propel the cell forward by rotational motion similar to a propeller. then eukaryotes. other Bacteria. will inhibit growth of some Gram positive bacteria. Although the movement is beneficial to the cell. inclusion in subsystems. Copy it.” requiring the pumping of approximately 1000 protons for one rotation of the flagellum. Click on the NMPDR button to open the protein page. This is a rich medium that supports the growth of most non-fastidious organisms. Click the | Blast | link in the banner of the protein page.MCB 301 Lab Manual supplemental Spring 2008 11 Controls: esculin+: Enterococcus faecalis 226185. Select "blastp" in the tool field and select your test genome in the genome field. Scroll to the right to examine the match between the query and subject sequence. Search for " beta glucosidase" in the control genome above. An inoculum of the bacterium is stabbed into the agar.” Although there are different means of microbial locomotion. This movement in response to chemical compounds is referred to as “chemotaxis. if a bacterium is adapted to live in a fixed environment it would not require such an apparatus and could better use its energy supplies in more beneficial ways. Click the button to show the protein sequence. As the cells use up the nutrients in .1 A black precipitate should be present only if the organism is capable of hydrolyzing esculin. Switching the rotation of the flagellum causes the cell to cease lateral movement and “tumble” while it reorients itself. and genetic context when deciding whether your test genome has the capacity to hydrolyze esculin. which were formerly thought to be a subgroup of the streptococci. If the “propeller” is rotated in one direction. This test was designed to differentiate the enterococci (cocci native to the large intestine). Consider the quality of the sequence match. these motors are real “energy hogs. which are present in the bile esculin medium. Recall that the NMPDR core pathogens are listed first in alphabetical order. this becomes another type of differential test to distinguish between species. Paste the control sequence into the sequence box. Motility Test Motility allows microbes to “forage” in different areas of their environment to find essential compounds needed for survival and growth. microbes found in nature are quite often motile in order to allow them to move away from harmful compounds (repellents) and toward nutrients (attractants). Since there are clear differences in motility. the most common method is to move by means of a specialized structure called a flagellum. you can type the id number or part of the name of your test genome into the small text field and click the button labeled "select genomes containing.

Cadaverine is a toxic diamine. which is linked in the beginning of this document. Lysine Decarboxylase Some sugar-fermenting bacteria also produce decarboxylases." then input your test genome id number as a search word. like the acidic reactions in fermentation. They are degraded to a variety of end products. which turns pinkish-red when reduced. synthesis of lysine decarboxylase is turned on and utilization of lysine begins. Experiment 10b: Organic Nitrogen Metabolism (individual experiments) Amino acids and proteins can be used by bacteria as nitrogen and energy/carbon sources. You may also use the keyword search on the NMPDR home page. or click the | Subsystems | link. which remove the carboxyl group from specific amino acids. This change usually occurs in the first 10 to 12 hours of incubation. click the radio button for "Motility and Chemotasis.MCB 301 Lab Manual supplemental Spring 2008 12 the area of the initial stab line. Initially. to browse or search the subsystems tree. The test medium also contains a redox indicator. As the pH of the medium drops. The presence of amino groups in these products causes an alkaline reaction that. oxidizing the carbon compounds and reducing the indicator to produce a pink color wherever it goes. The BCP eventually turns the medium purple in response to the accumulation of cadaverine. Cadaverine is alkaline and its accumulation causes a pH increase in the medium. Lysine decarboxylase removes the carboxyl group from the amino acid lysine producing carbon dioxide and cadaverine. The indicator brom cresol purple (BCP) that is used in this test is purple at alkaline pH and yellow at acidic pH. and click Go. located both on the home page and in the banner of all data pages. which is similar to putrescine -. Do a keyword search or browse subsystems to determine whether you test genome is motile. Cadaverine is a foul-smelling polyamine produced by protein hydrolysis during putrefaction of animal tissue. 1. is a return to the original purple color.a compound produced by ornithine decarboxylation. BCP turns the medium yellow in response to fermentation of glucose to acidic end products. they must either cease growth (non-motile) or move to areas of the medium that still contain nutrients (motile). are readily detectable and useful for identification. Remember that you can start from the subsystem summary for your organism. . then. A motile bacterium will move throughout the medium. triphenyltetrazolium chloride (TTC). A positive test. A nonmotile organism will be unable to move so that the pink color will be evident only along the stab line. To search the tree.

also known as L-tryptophan indole-lyase. 93061. 2. Controls: indole+: indole-: Shigella flexneri 2a str. pyruvic acid and ammonia. Use the controls to make sure you are looking for the right thing. One specific use of the indole test is to aid in distinguishing between Shigella and Salmonella.3 Use a combination of keyword searching and subsystems browsing to determine the pattern for your test organism. tryptophanase + + NH3 Ammonia Not all bacteria are capable of hydrolyzing tryptophan. Use the controls to make sure you are looking for the right thing. two genera that contain human pathogenic species. . Indole Production from Tryptophan Hydrolysis of the amino acid tryptophan by tryptophanase. Controls: Salmonella typhi S. typhimurium Lysine + + Ornithine + Arginine + + Use a combination of keyword searching and subsystems browsing to determine the pattern for your test organism. so this test is useful in identifying bacteria. results in the production of indole (a putrefactive compound). 301 198214. NCTC 8325. The indole may accumulate as an end product.MCB 301 Lab Manual supplemental Spring 2008 13 H H H H H O H H H H H H 2 N­C­C­C­C­C­C­OH H H H H NH 2 lysine decarboxylase Lysine H 2 N­C­C­C­C­C­ NH 2 + CO 2 H H H H H Cadaverine The pattern of decarboxylation of each of the three amino acids given below is useful for the identification of enteric bacteria. and use the control sequence to blast against the test genome if necessary.1 Staphylococcus aureus (NP) str. and use the control sequence to blast against the test genome if necessary.

Like the compare regions graphic. in numerical order. cysteine and methionine. an insoluble black precipitate. What subsystem does tryptophanase belong to in the positive control genome? 2. The central "pin" is the focus gene. The agar contains high levels of peptones (sources of cysteine and methionine) and ferrous sulfate as an indicator. The most frequently co-localized similar neighbor is labeled 2. which is red medium remains orange. A new window will open with a display of homologous regions in other genomes.MCB 301 Lab Manual supplemental Spring 2008 14 QUESTIONS ON INDOLE 1. H2S hydrogen sulfide + FeSO4 ferrous sulfate  FeS ferrous sulfide + H2SO4 sulfuric acid The Kligler's Iron Agar test can also be used to examine glucose and lactose metabolism by the production of acid from one or the other sugar. Carbohydrate utilization glucose+ lactose+: glucose+ lactose-: glucose. Under anaerobic conditions the sulfhydryl (-SH) group on cysteine is reduced by cysteine desulfurase. slant/air interface may turn red . and click on the Pins button for the focus protein. and so on. The pH indicator phenol red is included in the medium (see the sugar fermentation discussion). Pinned regions shows genes with similar protein sequences in the same color. Scroll down to the context table.lactose-: medium is entirely yellow including slant medium turns yellow except for slant/air interface. Hydrogen sulfide is released as a byproduct when carbon and nitrogen atoms in the amino acids are consumed as nutrients by the cells. Which protein is most often clustered with Tryptophanase? 3. When H2S is produced. the ferrous ion reacts with it to give ferrous sulfide. The Kligler's Iron test is used to detect liberation of H2S gas by bacteria growing on an excess of these sulfur-containing amino acids. We will not be using the sugar metabolism component of Kligler's agar for identification. Open the NMPDR page for the control result. Hydrogen Sulfide Production (Kligler's Test) Hydrogen sulfide (H2S) is produced by bacterial anaerobic degradation of the two sulfurcontaining amino acids. lagbeled 1.

oxygen. The last electron carrier in the electron transport chain. Use a combination of keyword searching and subsystems browsing to predict the result for your test organism. The ammonia is a nitrogen source for amino acid biosynthesis as well as for synthesis of other nitrogen-containing molecules in the cell.1) Serratia marcescens (NP) 615. H2N \ C = O + 2 H2O  CO2 + H2O + 2 NH3  (NH4)2CO3 / H2N urea ammonium carbonate The production of ammonia raises the pH of the medium. Hence. cytochrome oxidase. Experiment 10c: Electron Transport (individual experiments) In this section. and turns bright pinkishred at pH >8. coupled oxidation-reduction reactions and electron carriers are often part of what is called an electron transport chain. The diffusible electron carriers NADH and FADH 2 carry hydrogen atoms (protons and electrons) from substrates in exergonic catabolic pathways such as glycolysis and the citric acid cycle to other electron carriers that are embedded in membranes. Controls: urease+: urease-: Proteus vulgaris (P.1 The urease test was devised to distinguish Proteus species from other enterics. The indicator phenol red is present in the broth.1. transfers the electrons to the terminal electron acceptor.8. Use the controls to make sure you are looking for the right thing. and cytochromes. 4. In the case of anaerobic respiration. a positive urea test is denoted by the change of medium color from yellow to pinkish-red (cerise). The medium described here is buffered enough so that weak urease producers appear negative. Some bacteria can cleave it to produce carbon dioxide and ammonia. we shall use two tests that detect activities involved in the transport of electrons generated by respiration. Urease Urea is a nitrogenous waste product of animals. These membrane-associated electron carriers include flavoproteins. During the process of aerobic respiration.MCB 301 Lab Manual supplemental Spring 2008 15 Use your previous investigation of carbohydrate utilization along with a search for cysteine desulfurase to predict the color and presence of precipitate. quinones. a series of electron carriers that eventually transfers electrons from NADH and FADH2 to oxygen. Phenol red is orange-yellow at pH <6. iron-sulfur proteins. the last . mirabilis 584. and use the control sequence to blast against the test genome if necessary.

since H2O2 is a toxic by-product of aerobic growth. and use the control sequence to blast against the test genome if necessary. Catalase Activity One of the by-products of oxidation-reduction in the presence of O 2 during aerobic respiration is hydrogen peroxide (H2O2). particularly Bacteroides. Go to the | Genes | search page. Catalase production is generally associated with aerobic organisms. although some strains of Enterococcus faecalis may be positive..3 . e. Most aerobes synthesize the enzyme catalase. It can be spread by inhalation (referred to as pneumonic plague). This compound is highly reactive and must be degraded in the cytoplasm of the cell producing it.g. It is a major method of distinguishing between Staphylococcus (catalase positive). which breaks down H2O2 into water and oxygen (see background information on aerobic spore-formers). so the catalase test. Controls: + : Staphylococcus epidermidis str. a gut organism. RP62A. All of the Gram negative bacteria we use in this lab are catalase positive. which is a very infectious organism causing a deadly disease. Use the control genomes to make sure you are looking for the right thing. and Enterococcus (catalase negative). is dangerous. especially if they are exposed to air.1 Use a combination of keyword searching and subsystems browsing to predict the result for your test organism. 2 H2O2 catalase 2 H2O + O2 The O2 gas is identified by the production of bubbles from a concentrated cell suspension. A variation of the test is used in identification of the plague bacillus Yersinia pestis. Based on this result. produce catalase.: Enterococcus faecalis 226185. . nitrate (NO3-). enter the keyword and select all available strains and species of Yersinia from the scrolling list.g. Streptococcus (catalase negative). 176279. It can be especially damaging to molecules of DNA. Some anaerobes. nitrate reductase) transfers electrons to an oxygen-containing compound other than O2. The catalase test can be done in such a way that the bubbles produced do not make an aerosol. QUESTION ON CATALASE 1. The test for catalase is simple and usually very reliable. Use the NMPDR resources to predict the results of this test in several strains of Yersinia pestis.MCB 301 Lab Manual supplemental Spring 2008 16 electron carrier (e. which forms bubbles. 1. but not always.. is the diagnostic value of the catalase test in Yersinia worth the potential risk? Explain.

+ 2 H+ + 2 e- nitrate reductase NO2. The standard test assays for the appearance of one of the products. Since some of the possible products of NO 3. A medium that will support growth must be used and the cells must be grown anaerobically (no shaking). The initial step is the reduction of nitrate (NO 3-) to nitrite (NO2-). Nitrate is an oxidized compound and there are several steps possible in its reduction. reacts with the iodide in the reagent to produce iodine. NO3. Bacteria vary in their ability to perform these reactions. The test we use relies on the production of nitrous acid from the nitrite. Summary of Nitrate test results and interpretations RESULT INTERPRETATION Inky Blue after addition of Trommsdorf I & II Nitrate was reduced to nitrite No color after addition of zinc dust Nitrate was reduced to nitrite and then further reduced to another compound such as NH3 SYMBOL +1 Blue color after addition of zinc Nitrate is still present. some bacteria are able to use nitrate (NO 3-) as an external terminal electron acceptor. ammonia. and hydroxylamine. nitrogen gas (N2). a useful characteristic for identification. it is important to pre-test the medium to ensure no detectable nitrite is . Possible reduced end products include the following: N 2 (molecular nitrogen. a gas). One could either assay for disappearance of nitrate or the appearance of the end products. This kind of metabolism is analogous to the use of oxygen as a terminal electron acceptor by aerobic organisms and is called anaerobic respiration. This. N2O (nitrous oxide). NH3 (ammonia). The dust bacteria being tested did not reduce the nitrate +2 _ There are many possible end products of nitrate reduction such as nitrite. a Durham tube is sometimes inverted in the culture tube to trap gases. nitrite.+ H2O Several possible products can be made from further reduction of nitrite. The iodine then reacts with the starch in the reagent to produce a blue color. This being the case. Growth in the presence of oxygen will decrease or eliminate nitrate reduction.reduction are gaseous. The Trommsdorf test is used to detect nitrite. nitrous oxides.MCB 301 Lab Manual supplemental Spring 2008 17 2. Nitrate Reduction Under anaerobic conditions. in turn.

to reduce any nitrate to nitrite to determine whether the nitrite was also reduced. It is this reaction that is responsible for the color of meats. . such as hot dogs. in the case of a negative test. Use a combination of keyword searching and subsystems browsing to predict the result for your test organism. and. An interesting variation of this test is the use of blood agar containing nitrate. it reacts with hemoglobin to give a bright red color.MCB 301 Lab Manual supplemental Spring 2008 18 present at the beginning. The blood agar test has the advantage of no color change occurring if the nitrite is further reduced. Use the control genomes to make sure you are looking for the right thing. which are preserved with sodium nitrite. instead of the dark red color of hemoglobin. If nitrite is produced. and use the control sequence to blast against the test genome if necessary.

MCB 301 Lab Manual supplemental Spring 2008 19 Experiment 10: Phenotype prediction Worksheet 20 points total. due February 11/12 Student's Name: __________________________________ Name of Test Predicted Result Why? provide evidence .