MMBB255 Week 6

Biochemical and Metabolic Properties of Microbes

I. Objective
The goal of this experiment is to characterize an organism based on its ability to produce proteins that allow it to carry out a set of unique reactions, its motility, and oxygen requirements. You will also follow up on the growth curve and unknown two. The Photographic Atlas for the Microbiology Laboratory will be useful.

II. Background
Microorganisms 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. We will examine the following methods for classification: A. Carbohydrate utilization: acid and/or gas production B. Enzyme production C. Aerobic or anaerobic mode of growth D. Motility

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). Other acids include formic and acetic acid. Microbes including Streptococcus and Lactobacillus can produce lactic acid, formic acid, and/or ethanol. Enteric organisms (see below) 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 addition of a small tube, called a Durham tube, which has been inverted in the carbohydrate-containing growth medium to trap gas bubbles. If gas is produced, it typically is CO2 or H2. Carbohydrates that are used in microbial classification include the following classes: a) Monosaccharides are simple sugars with 1 to 6 carbons. For example, tetroses are 4 carbon sugars, such as erythrose. Pentoses are 5 carbon sugars and include ribose, xylose, arabinose, and ribulose. Hexoses are 6 carbon sugars and include glucose (also called dextrose), galactose, mannose, and fructose (also called levulose). b) Polysaccharides are polymers of monosaccharides. For example, a disaccharide (di = two) contains two monosaccharide units. Some examples include sucrose (also called saccharose) composed of the hexoses glucose and fructose, maltose composed of two units of the hexose glucose, and lactose composed of the hexoses glucose and galactose. c) Alcohol sugars are polyhydric alcohols that are reduction products of a monosaccharide. Some examples include adonitol, dulcitol, mannitol, and sorbitol. Carbohydrate utilization is important in distinguishing Enterobacteriaceae (enteric bacteria) are among the members of the family Enterobacteriaceae (the enteric bacteria). The Enterobacteriaceae is a family facultatively anaerobic, oxidase negative, of organisms that are facultatively anaerobic, oxidase Gram negative rods that ferment glucose. 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
Listed below are some brief descriptions of enzymes you might find in a microbe. The ability of an organism to produce or express a particular enzyme can be assayed (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.

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1. Catalase: H2O2 (hydrogen A Few Examples peroxide) is produced as an Catalase Positive Catalase Negative oxidation product during growth Micrococcus and Staphylococcus Streptococcus on carbohydrates in the presence Bacillus Clostridium of oxygen and is toxic if allowed Listeria monocytogenes and some to accumulate. Many organisms Erysipelothrix Corynebacterium sp. make the enzyme catalase to remove hydrogen peroxide. The catalase test The catalase test is useful to differentiate measures the ability of an organism to produce the enzyme catalase that degrades H2O2 to H2O between the Staphylococci/Micrococci and the and O2. Active catalase is a homotetramer (four Streptococci (see the table above). identical subunits) with two heme groups containing trivalent iron (ferric, Fe3+). To see if an organism makes catalase, H2O2 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 H2O2 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 A Few Examples terminal electron acceptor. Oxidase Positive Oxidase Negative Oxidase interacts with the Neisseria, Moraxella Acinetobacter membrane bound Aeromonas, Alcaligenes, Enterobacteriaceae cytochromes and delivers Flavobacterium, Pseudomonas, Vibrio electrons from the cytochromes to O2. As a result, H2O2 or The oxidase test is a useful test for distinguishing H2O is generated. Strict anaerobes do not between the Gram negative rods Pseudomonas use O2 and hence do not possess the oxidase enzyme. Most Gram positive and the Enterobacteriaceae. bacteria are oxidase negative as well as the members of the family Enterobacteriaceae. 3. Gelatinase (an extracellular A Few Examples enzyme): This test determines Gelatinase Positive Gelatinase Negative whether an organism produces a proteolytic-like enzyme that Staphylococcus epidermidis + slow Staphylococcus aureus can liquefy or digest gelatin, a Micrococcus + slow protein produced by the Corynebacterium sp. (most) Listeria monocytogenes hydrolysis of collagen. Proteins are too large to be taken up by microbes, so gelatinases (and other proteases) are secreted into the environment where they break proteins into peptides and amino acids, which can be taken up by the cell. 4. Amylase (an extracellular enzyme): This test measures whether an organism produces amylase, an enzyme that breaks down starch, a glucose polymer. Amylase hydrolyses starch into dextrins (smaller polysaccharides) and maltose (disaccharides), which now can be imported into the cell. 5. Lipase, lecithinase, proteases (extracellular enzymes): Lipases are enzymes that break down triglycerides (lipids) into their constituent glycerol and fatty acids. Lecithinase breaks down lecithin which is a modified phospholipid; one of the fatty acids has a special modification. Cells can use these components as energy or to produce other cellular components. Proteases are enzymes that break down (hydrolyze) proteins and peptides. 6. Urease: The enzyme urease is an A Few Examples amidase produced by certain eukaryotes and prokaryotes. It plays an important Urease Positive Urease Negative role in the decomposition of organic Klebsiella Escherichia compounds. Because not all microbes produce this enzyme, a simple test for Proteus Providencia this enzyme can be used to distinguish Bordetella bronchiseptica Bordetella pertussis between various microbes. For example, urease activity is very characteristic of all Yersinia pseudotuberculosis, Yersinia pestis Proteus spp. This test aids in rapid Y. enterocolitica detection of lactose-nonfermenting enteric organisms such as Proteus vulgaris, an opportunistic pathogen.

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The urease test measures the ability of a microorganism to split, or hydrolyze, a molecule of urea into two molecules of ammonia using the enzyme urease. A heavy inoculum of a young culture is added to urea broth (this media is designed specifically to differentiate Proteus from other enterics). The critical ingredients are urea and the pH indicator, phenol red. The initial pH of the medium is 6.8 and is slightly buffered (there is less buffer if you use a urease assay like urea agar - designed for organisms other than Proteus). Phenol red is yellow at pH 6.8 (slightly acidic) and red at pH 8.4. Microbes that can make urease, will hydrolyze urea as shown in the box, resulting in a shift in the pH of the medium toward alkaline pH. This H2N changes the phenol red urease indicator to pink (red C O CO2 + H2O + 2 NH3 + 2 HOH or cerise). Urea is a H2N diamide of carbonic acid that is split into Carbon Urea Water Water Ammonia carbon dioxide and Dioxide ammonia by the urease enzyme as shown in the box.

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 O2. Microaerophilic organisms need a small amount of O2 for growth but too much O2 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 O2 is not present. We will test several organisms for their growth pattern in medium to determine if they prefer aerobic or anaerobic conditions. Note that the handling of very strict anaerobes is beyond the scope of this class.

D. Motility: swimming, swarming, twitching, and gliding

Microbes move in a variety of A Few Examples ways that are roughly analogous to Temp. Motile Nonmotile our walking, running, swimming, Vibrio spp. Acintobacillus spp. or crawling movements. Many Enterobacter spp. Klebsiella spp. organisms can swim in liquid (such 37C Aerogenic Escherichia coli Anaerogenic Escherichia coli as lakes or oceans, blood system). Most swimmers move by rotation Other Bacillus spp. Bacillus anthracis of one or more external appendages Pseudomonas spp. Pseudomonas mallei called flagella. This is a complex Corynebacterium spp. Listeria spp. process that is very well studied. (C. aquaticum is positive) 22C The helical shape of the flagellum Yersinia pseudotuberculosis Yersinia pestis is reminiscent of a motorboat Y. enterocolitica 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. Some swimmers can also swarm. Swarming occurs when a swimmer cell enters a viscous environment or a solid surface. The swimmer then increases in length and makes a large number of flagella. Some examples include Proteus mirabilis and Serrratia marcescens. 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. Some organisms, particularly soil organisms, move by gliding. Gliding is a translocation over a solid surface (soil, agar). Gliders do not have flagella or cilia but appear to move by secretion of a material. Some examples of gliders are Myxococcus xanthus and Beggiotoa spp. 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.

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Tuesdays Procedures:
A. Carbohydrate fermentation and enzymes in carbohydrate catabolism Work in Pairs.
1. You will be assigned E.coli plus one other organism from the list Enterobacter aerogenes to the right. These will be provided as broth cultures. You will Citrobacter freundii need to record the results for the other organisms you do not use. Enterococcus faecalis 2. Obtain and label the following media: (Note that the extra two Escherichia coli tubes are for you and your partner to inoculate your second Klebsiella pneumoniae unknown) Proteus mirabilis 4 tubes of PR-lactose with Durham tubes Pseudomonas aeruginosa 4 tubes of PR-glucose with Durham tubes Salmonella Typhimurium-see note 4 tubes of PR-sucrose with Durham tubes 3. Inoculate each tube aseptically with your loop. Label each tube with the name of the organism, the sugar, the date, your name and lab section. The instructor will keep uninoculated carbohydrate media as negative controls and incubate them along with the inoculated tubes. 4. Incubate all tubes at 37C for 24 hrs. and examine. Note: The nomenclature for the Salmonella species has been changed. There are now only three species of Salmonella, enterica (used to be Salmonella choloraesuis), bongori, and subterranean. Most human pathogens are serovars of S. enterica subspecies enterica (there are 6 subspecies total). For example Salmonella Typhimurium is short for S. enterica subspecies. enterica serovar Typhimurium; similarly there is Salmonella Typhi, Paratyphi, Enteritidis short names for the subsp. enterica. Please note how they are formatted. If you were to hand write them you would underline all italics.

B. Enzymes: Work in Pairs.

i. Extracellular enzyme gelatinase: 1. The bacterial cultures. For these tests, we will use the following microorganisms: Staphylococcus aureus Bacillus cereus Staphylococcus epidermidis Pseudomonas aeruginosa 2. Inoculate. Using an inoculating needle or loop aseptically stab each organism into a labeled nutrient gelatin tube. Incubate at 37C for 48 hrs. ii. Extracellular enzyme amylase: 1. The bacterial cultures. For these tests, you will use the following organisms: (you may also test your second unknown if you want, you will need six sectors) Staphylococcus aureus Bacillus cereus Staphylococcus epidermidis Pseudomonas aeruginosa 2. Inoculate. Using your inoculating loop aseptically make a line in the quadrant of a starch agar plate with each of the test organisms (see the diagram). Label the back and incubate at 37C for 48 hrs. iii. Extracellular enzymes lipase, lecithinase, and protease: 1. The bacterial cultures. For these tests, you will use the following organisms: (you may also test your second unknown if you want, you will need six sectors) Staphylococcus aureus Bacillus cereus Staphylococcus epidermidis Pseudomonas aeruginosa 2. Inoculate. Obtain one plates of egg yolk nutrient agar (EY-NA) from the instructor. The addition of egg yolk at 5-10% (v/v) makes the medium look milky. Aseptically inoculate a single line for each organism on the plate (see the diagram). Incubate at 37C for 24-48 hrs.

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iv. The urease test 1. Work with the following bacterial cultures. Escherichia coli (plate stock) Enterobacter aerogenes (plate stock) Klebsiella pneumoniae (plate stock) Proteus mirabilis (plate stock) Your unknown (plate stock) Your partners unknown (plate stock) 2. Obtain and label: 6 tubes of phenol red urea medium (urea broth). 3. Inoculate. Using your inoculating loop, aseptically transfer a heavy inoculum (loopful) of each organism into a separate tube of urea broth. The lab instructor will keep aside one tube uninoculated and incubate it at 37C as a control. 4. Incubate at 37C. Check your reactions after about 4-6 hours if possible. Fast positive organisms, such as Proteus, may turn pink in a few hours. Check for a positive test at 24 hr. Date and time that tubes were inoculated: _____________ time________________

C. Oxygen requirements Work in Pairs.

1. The bacterial cultures. Escherichia coli Pseudomonas aeruginosa Clostridium sporogenes Streptococcus agalactiae 2. Three methods will be used to monitor the oxygen needs of the four bacterial strains: i. Method 1. Agar deeps. Obtain four tubes of TSA (trypticase soy agar) deeps, tempered at 55C. Remove one tube from the 55C bath and aseptically add two drops of an organism into the tube using a sterile pasteur pipette. Immediately and vigorously roll the tube between the palms of your hands to evenly distribute the cells throughout the agar. Then place the tube into a bucket of ice to solidify the medium. Repeat with the other three cultures. Incubate at 37 C for 48 hrs. ii. Method 2. Thioglycolate broth. Obtain four screw-capped tubes of thioglycolate medium. The medium should be slightly pink or blue in the upper one-fourth of the tube due to the presence of resazurin or methylene blue respectively, both redox indicators (they turn color when there is oxygen). Sodium thioglycolate, the active ingredient, binds to O2. Aseptically inoculate each of the tubes, taking care to stab to the bottom of the tube with your inoculating loop or needle - try not to introduce air. Dont shake or tighten the cap (you want air to get in the tube but not the medium). Incubate at 37C for 18-24 hrs (check Wed). iii. Method 3. Gas-Pak Jar. Obtain two TSA plates from the lab instructor. Aseptically streak a single line of each of the four organisms on both of the plates and your unknowns. [Be sure to label each quadrant correctly]. Immediately place one plate in the Gas-Pak Jar. It will be activated and placed at 37C for 48 hrs. Incubate the other plate at 37C in the air for 48 hours. The Gas-Pak works as follows: To activate the system, water will be added to a reagent in the GasPak to generate CO2 and H2. The jar contains a small amount of palladium that then catalyzes the conversion of O2 and H2 to H2O. This removes sufficient O2 to make an environment favorable for anaerobes. The jar also includes a redox indicator called methylene blue to check for anaerobiasis. It is colorless when reduced and blue when oxidized.

D. Motility Work in Pairs.

Motility will be tested by examining the distance cells migrate in semisolid medium. Remember motility may also be checked via a hanging drop or wet-mount method. Standard agar medium that we have been using is considered solid medium because it contains 1.5% w/v (1.5 g per 100 ml) agar. The semisolid medium has only 0.5% w/v agar (swarming medium has even less, 0.3 % w/v agar) and contains all the nutrients required for organisms to grow. The semisolid medium may also contain an indicator of cells with active respiration called 2,3,5-tetraphenyltetrazolium chloride (TTC). This allows easier observation of where the organisms are growing since it turns red when reduced by an active electron transport chain (it is colorless when oxidized).

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1. The bacterial cultures. Obtain these strains from your instructor. Escherichia coli Klebsiella pneumoniae Your unknown Your partners unknown 2. Inoculate by stabbing. Label each sectors, then aseptically stab each organism with an inoculating needle into the center of a quadrant of semisolid medium, see the diagram to the right. 3. Incubate. Incubate the plate upright (do not invert) at 30C and examine for motility (movement away from the stab inside the agar not the surface) after 24 and then 48 hrs.

E. Growth curve - Finish

1. Record your statistically valid plate counts (between 25-250), calculate the total viable cell concentration, fill out the table below, and enter the calculated cfu/ml into the computer. time point dilution x dilution by volume calculated # of colonies elapsed min. used factor plated cfu/ml

2. You will get graphing paper and all sections growth curve data next lab period. You should make a growth curve of each growth condition and figure out the doubling time from the graph. Get help if you do not know how to do this.

Thursdays Procedures:
A. Carbohydrate fermentation and -galactosidase activity Work in Pairs.
1. Record Fermentation Data. After 24 hrs, record your results in the table below. Note: if for some reason you do not observe your cultures within about 24 hrs, make a note of the time and date below. The Ferm? heading in the table below is your conclusions that the organism does or does not ferment that sugar- see the background. Date that tubes were inoculated: _____________ time: ________________ Date that observations were taken: _____________ time: ________________
Lactose Glucose Ferm? Growth & Color Gas Ferm? Sucrose Growth & Color Gas Ferm?

Sample Neg. Control E. aerogenes C. freundii E. faecalis E. coli K. pneumoniae P. mirabilis P. aeruginosa S. Typhimurium Your Unknown

Growth & Color

Gas

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2. Assay for -galactosidase activity. The enzyme -galactosidase breaks lactose into its two monosaccharide components, glucose and galactose. Production of the enzyme -galactosidase is regulated by the presence of the substrate, lactose, and the absence of the more favorable substrate, glucose (you may want read about the Lac operon in your text). To demonstrate this, you will determine the amount of -galactosidase produced by Escherichia coli grown in glucose vs. lactose. a. Mix your E. coli lactose and glucose PR tubes to disperse the cells. Label four 1.5 ml eppendorf (eppi) tubes and fill them according to the table below. You do not need to be aseptic. Follow the flow chart below and note the footnotes about the various solutions:
Tube 1 Add 1.5 ml PR-glucose cells to an eppi tube Tube 2 Add 1.5 ml PR-lactose cells to an eppi tube Tube 3 (Blank) Add 1.5 ml PR-glucose cells to an eppi tube Tube 4 (Blank) Add 1.5 ml PR-lactose cells to an eppi tube

Centrifuge @ 12,000 RPMs for 4 min Discard supernatant into originating PR tube. Add 1 ml of 0.1% SDS1 Mix to get all the cells resuspended and then transfer all the liquid to a spectrophotometer tube.
Add 2 ml Z buffer2 and 1 ml ONPG3

Add 2 ml Z buffer and 1 ml ONPG Add 2 ml Z buffer and 1 ml H2O Add 2 ml Z buffer and 1 ml H2O

Incubate at 37C for 5 min.

Read Abs (OD) at 420 nm in the spectrophotometer. b. Calculate the units of -galactosidase activity as follows: -gal Units = (A420nm * 213)/Time(min.) Units for glucose-grown cells:____________ Units for lactose-grown cells: _____________ What can you conclude from these results about the regulation of the gene (lacZ) that encodes for -galactosidase?

Add 1ml of 1M Na2CO34

B. More biochemical (enzyme) tests used to identify microbes.

a. The catalase test. This test must be performed on fresh cultures (<24 hr old) to be accurate. 1. Cultures. Use the following organisms from solid (agar) plate medium. Staphylococcus aureus Micrococcus luteus Streptococcus sp. Gp B or Enterococcus faecalis Your unknown from the air grown GasPak experiment plates. 2. Smearing. Using your sterile loop (or sterile toothpicks), scrape off a small section of a colony (enough to see with the unaided eye) and smear it onto a glass slide. 3. Add peroxide. Add a drop of the H2O2 solution and watch for the appearance of bubbles. Any bubble is indicative of gas production and is a positive catalase reaction. Sometimes it takes a minute to see bubbles. 4. Test your unknown. Repeat with your unknown organism.
1 2

To lyse the cells and release the enzyme. 0.1M sodium phosphate, 5mM MgCl2 at pH7.3; supplies buffer and needed salt for the enzyme to work. 3 4 mg/ml o-nitrophenyl--D-galactopyranoside (which is colorless) is hydrolyzed by -galactosidase to o-nitrophenol (which is yellow) and galactose. This is considered a lactose analog since the enzyme thinks it is lactose. 4 This increases the pH to >9, which stops the reaction and maximizes the absorbance at 420 nm.

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Results of the Catalase Test Positive Staphylococcus aureus Micrococcus luteus Streptococcus sp. Gp B or Enterococcus faecalis your unknown b. The Oxidase DrySlideTM test. This test must be performed on fresh cultures (<24 hr) to be accurate. 1. Test the following organisms for oxidase: Escherichia coli Micrococcus luteus Pseudomonas aeruginosa Pseudomonas fluorescens Your unknown from the air grown GasPak experiment plates. 2. Obtain a DrySlideTM Oxidase slide from the lab instructor. This 2x2 inch slide has four plastic film reaction areas that contain N,N,N,N-tetramethyl-para-phenylenediamine dihydrochloride (TMPD), gelatin and ascorbic acid. This is an oxidation-reduction reaction. TMPD is colorless when reduced and purple (blue) when oxidized. Ascorbic acid acts as a stabilizer by being a reducing agent. 3. Apply fresh cells. Scrape up a small glob of cells using a toothpick (do not use any implement containing iron such as your inoculating loop or needle) and rub the cells in one corner of the Oxidase slide film reaction areas and look for a color change from clear to bluish-purple. This reaction must happen within 20 seconds; any change after 20 seconds must be considered negative. You can easily test about 16 different samples on one slide. Pigmented organisms will contribute to the final color and you should take this into account. Also mucoid and heavily pigmented cells will Results of the Oxidase Test delay the reaction somewhat; therefore Positive Negative read within 30 seconds if you have one (M. Escherichia coli luteus is an example). Pseudomonas aeruginosa 4. Test your unknown. Repeat with your Micrococcus luteus unknown organism from the air grown GasPak experiment plates. When you and Pseudomonas fluorescens your partner have completed your tests Your unknown remember to pass the slide to another pair of students in the lab. Negative

C. Extracellular enzyme gelatinase Almost Finished.

If an organism has the enzyme gelatinase, the gelatin in the nutrient gelatin tubes will be broken down and once chilled it will be liquid; it will never set up or solidify like gelatin normally does. 1. Transfer the inoculated gelatin cultures to 4C. This will allow the gelatin to harden unless it has been broken down by the bacteria. After about 30 min, remove the tubes and shake them. If gelatinase has been produced, the samples be fluid. These are considered fast positive reactions. For samples that are not positive after 48 Results of the Gelatinase Test hrs, return them to the After 48 hrs After 5 days incubator for 5 more days (fast positive) (slow positive) and recheck them during Staphylococcus aureus the next lab period; if they become liquefied they are Bacillus cereus considered slow positive Staphylococcus epidermidis reactions. Pseudomonas aeruginosa 2. Record your results in the table.

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D. Extracellular enzyme amylase - Finished.

1. Obtain your inoculated plate and stain with Grams iodine. Iodine will react with intact starch (also called amylose) to produce a blue-black product. However, if the microbe has hydrolyzed the starch due to the enzyme amylase, a clear zone will remain after you flood the area with iodine. 2. Record your results below. Results of the amylase test Positive Negative (clear zone) (blue-black) Staphylococcus aureus Bacillus cereus Staphylococcus epidermidis Pseudomonas aeruginosa

E. Extracellular enzymes lipase, lecithinase, and protease - Finished.

1. Examine the egg yolk nutrient agar plate area around the streaks. If lipase is produced, the triglycerides will be degraded producing an iridescent sheen around and on the lipase-secreting colony; it looks like the oil on water sheen. If lecithinase is produced, you will see a white, opaque zone around the colony. Secretion of proteases will produce a clearing about the streak. An organism can produce all three enzymes, so look for combinations. 2. Record your results below. Results of the lipase, lecithinase, and protease tests Lipase Lecithinase Protease (iridescent) (white, opaque) (clearing) Staphylococcus aureus Bacillus cereus Staphylococcus epidermidis Pseudomonas aeruginosa

F. The urease test - Finished.

1. Examine the color of the urease tubes. At 24 and 48 hrs, record your results in the table below. Peach to pink colors are considered positive reactions; fast positive organisms, such as Proteus, may turn pink in a few hours. 2. Record your results below. Date and time that tubes were inoculated: ___________ time________________ Results of the Urease Test Color (peach to pink is positive) 4-6 hrs. 24 hrs. uninoculated control Escherichia coli Enterobacter aerogenes Klebsiella pneumoniae Proteus mirabilis Your Unknown

48 hrs.

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10

G. Aerobic and anaerobic growth Finished.

1. Results. Record your observations by noting where in the tube it grew and the quantity of growth. From your observations record your conclusions about the type of oxygen requirements. The definitions for the terms are in the background reading material.

Agar Deeps

Thioglycolate

Gas-Pak

vs.

Air

Escherichia coli Pseudomonas aeruginosa Clostridium sporogenes Streptococcus agalactiae Your Unknown

Conclusions* Agar Deeps

Thioglycolate

Gas-Pak

XXXXXXXXXXX

XXXXXXXXXXX

*be sure to use the correct term: obligate aerobe, obligate anaerobe, microaerophile, aerotolerant, or facultative anaerobe (see the background).

H. Motility Finished.
1. Record your results in the diagram. Observe in the agar itself and not the surface; look from the side of the semi-solid agar plate. Did the organism move away from the stab line? 2. Which strains are motile? Organism E. coli K. pneumoniae Your Unknown Non-Motile Motile

I. Unknown Two Finish.

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Study Questions:
1. Be able to determine what is positive and negative for each of the tests. Understand how each test works. 2. Oxidase is very useful to differentiate between which two groups of organisms? What is a positive reaction? 3. Catalase is very useful to differentiate between which two groups of organisms? What is the reaction catalyzed by this enzyme and what do you seen in a positive reaction? 4. What indicates that a sugar is fermented in the PR-sugar test? You may also have what byproduct as seen in the small inverted test tube? What is that small test tube called? 5. What are the purposes of the various solutions in the -galactosidase test? Lactose is a disaccharide comprised of which two monosaccharides? Be able to answer the question given at the end of this experiment. 6. Enzymes are named after the substrates they work on. Name the substrate for each enzyme. For example amylases substrate is amylose or starch. 7. Can you manually plot growth curve data on a piece of semi-log graph paper and determine the doubling time from the plot? What are the four phases of growth - again? 8. Where does a microaerophile grow best in a thioglycollate tube? How about an obligate aerobe, obligate anaerobe, facultative anaerobe, and aerotolerant anaerobe? 9. What kinds of motility are there? 10. How does the Gas-Pak chamber work to give you anaerobic conditions? How do you know if it is working? 11. The first practicum is soon. Here are some typical questions: Practice #1: Describe the cellular morphology and arrangement of this gram stain. What gram reaction is it? What is the difference between Gram positive eubacteria and Gram negative eubacteria? Practice #2: Is this a positive or negative acid-fast stain? Why do we have to use acid-fast stain for Mycobacteria? Practice #3: Determine the O.D. at 420 nm of the yellow sample. This was from the galactosidase assay we did using the ONPG substrate. What operon activity are we detecting and is the above sample from the PR-Glucose or PR-Lactose medium?