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Microbiology Lab Exam 2
Terms in this set (225)
Inhibit growth of some organisms while encouraging the growth of others. Can be used for isolation of pure cultures from mixed cultures.
Contain indicators to expose differences between organisms. Can be used for isolation of pure cultures from mixed cultures. Distinguish microorganisms from one another based on growth characteristics when grown on a specific media type.
Bile esculin agar (BEA)
Undefined, selective, and differential medium
Bile esculin test purpose
Used to differentiate enterococci and group D streptococci from non-group D streptococci
Ingredients of Bile esculin test
Beef extract, digest of gelatin, esculin, oxgall (bile), ferric citrate, agar
Purpose of esculin in Bile esculin test
Organisms positive for esculin hydrolysis hydrolyze the esculin to esculetin and dextrose. The esculetin reacts with the ferric citrate to form a dark brown or black complex.
Purpose of ferric citrate in Bile esculin test
Ferric citrate reacts with esculetin to form dark brown or black complex if organism can hydrolyze esculetin. It is the source of oxidized oxygen to indicate a positive test.
Purpose of oxgall (bile) in Bile esculin test
Inhibits the gram-positive bacteria, other than enterococci. It is the selective agent.
Purpose of beef extract and enzymatic digest of gelatin in Bile esculin test
Carbon and nitrogen sources used for general growth requirements. Provide nutrients and energy.
Purpose of agar in Bile esculin test
Bile esculin test methods: plate and slant
Plate used for isolation of species from mixed culture, and darkened even slightly indicates bile esculin-positive. Slant used to test esculinase activity in the presence of bile of pure culture. In slants, positive result is when more than half the medium is blackened. Spot inoculation.
Bile esculin test: positive result
If an organism can hydrolyze esculin, then the media will turn dark brown or black. More than half the medium will be dark brown or black after incubation (in slants). Only group D streptococci and enterococci can hydrolyze esculin in the presence of bile salts.
Bile esculin test: negative result
If an organism can't hydrolyze esculin, then there won't be any color change. No blackening will occur, or media will be less than half-blackened.
Bile esculin test: theory
Esculin hydrolysis results in the production of D-glucose and esculetin. Hydrolysis can happen under acidic conditions or be catalyzed by ß-glucosidase enzyme, esculinase. Although many bacteria process esculinase, the number of bacteria that are able to hydrolyze esculin in the presence of bile is much more limited. This feature is what makes this a useful and selective differential medium.
Bile esculin test methods
Spot inoculate with two organisms, incubate for 24-48 hours
Mannitol Salt Agar: Purpose
Isolation and distinction of Staphylococcus aureus from other Staphylococci species. Most Staphylococcus grow on Mannitol Salt Agar but do not ferment mannitol and therefore grow pink or red (medium unchanged). Staphylococcus aureus ferments mannitol, producing yellow colonies surrounded by yellow halo.
Mannitol Salt Agar: Ingredients
Mannitol, NaCl, Phenol Red, Nutrient Agar
Purpose of Mannitol in Mannitol Salt Agar
Carbohydrate, substrate for fermentation. Makes the medium differential.
Purpose of NaCl in Mannitol Salt Agar
Increases selectivity for Staphylococci. Dehydrates or kills most bacteria, but Staphylococci thrive in salty environments, like human skin
Purpose of Phenol Red in Mannitol Salt Agar
pH indicator. Pink at high pH, red between 7.4-8.4, and yellow below 6.8. Indicates whether fermentation with an acid end-product has taken place by changing color as the pH changes.
Components of Nutrient Agar in Mannitol Salt Agar
Beef extract, peptone, agar, distilled water
Mannitol Salt Agar: Methods
Divide nutrient agar and mannitol salt agar plates into three sections. Spot inoculate each section. Bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli
Mannitol Salt Agar: Expected results for Staphylococcus aureus
Yellow growth. Survives NaCl. Produces acid from mannitol fermentation. Gram-positive Staphylococcus fermenting mannitol.
Mannitol Salt Agar: Expected results for Staphylococcus epidermidis
Same as results for any Staphylococcus species other than Staphylococcus aureus. Good growth. Red, not yellow. Red color indicates that no acid is produced and that mannitol is not fermented. Gram-positive Staphylococcus not fermenting mannitol.
Mannitol Salt Agar: Expected results for Escherichia coli
Poor/no growth. Organism is inhibited by high NaCl concentration.
MacConkey Agar Composition
Bile Salts, Crystal Violet, Lactose, neutral red, agar, NaCl, nutrients (pancreatic digest of gelatin and casein, peptic digest of tissue bacterial growth)
Purpose of Bile salts and crystal violet in MacConkey Agar
Inhibit gram-positive bacteria and therefore select for gram-negative bacteria. Gram-negative enteric bacteria can tolerate bile salts because they have a bile-resistant membrane.
Purpose of lactose in MacConkey Agar
Differentiation of gram-negative bacteria based on ability to ferment lactose and make acid end-products that react with pH indicator. Provides carbon and energy.
Purpose of neutral red in MacConkey Agar
pH indicator. When bacteria ferment lactose, acid end-products are formed. pH decreases and the indicator changes color to make a pink color. Pink precipitate forms. Lactose non-fermenters retain the color of the medium.
Purpose of Agar in MacConkey Agar
Purpose of NaCl in MacConkey Agar
Maintains osmotic balance
MacConkey Agar: expected results
Gram-negative lactose fermenter will grow into pink colonies. Gram-negative non-fermenter (noncoliform) will grow but not change the color of the medium. Gram-positive will not grow or will have poor growth.
Selective, differential medium. Contains lactose, bile salts, neutral red, crystal violet. Used to isolate and differentiate members of Enterobacteriaceae based on the ability to ferment lactose.
Hektoen Enteric Agar
Selective, differential, complex (chemically undefined) medium. Used to isolate Salmonella and Shigella species from other gram-negative enterics. Some can ferment specific carbohydrates and some can reduce sulfur. Inhibits growth of gram-positive organisms, but gram-negatives can grow.
Hektoen Enteric Agar Ingredients
Yeast extract, peptic digest of animal tissue, lactose, sucrose, salicin, bile salts, sodium chloride, sodium thiosulfate, ferric ammonium citrate, bromothymol blue, acid fuchsin, agar, distilled water
Purpose of Ferric ammonium citrate in Hektoen Enteric Agar
Reacts with hydrogen sulfide gas to form a black precipitate. Indicates if sulfur is reduced because if it is, hydrogen sulfide gas will be produced. Used to identify Salmonella.
Purpose of bile salts in Hektoen Enteric Agar
Inhibit growth of gram-positive organisms. They denature proteins between the peptidoglycan layer and the cell wall of gram-positive organisms. Inhibit some nonpathogenic enterics that do not participate in fermentation. Make medium selective.
Purpose of peptic digest of animal tissue in Hektoen Enteric Agar
Promote growth of gram-negative enteric organisms, offsets moderate inhibitory effects of bile salts on them. Digested by Salmonella and Shigella, increasing pH
Purpose of yeast extract in Hektoen Enteric Agar
Promote growth of gram-negative enteric microbes
Purpose of bromothymol blue in Hektoen Enteric Agar
Indicate pH changes. Yellow in acidic solution, blue in basic solutions, and green in neutral solutions
Purpose of Acid Fuchsin dye in Hektoen Enteric Agar
Indicate pH changes. Magenta in acidic or neutral solutions and yellow in basic solutions
Purpose of sodium thiosulfate in Hektoen Enteric Agar
Source of sulfur, which is reduced to hydrogen sulfide gas
Purpose of Lactose, Sucrose, and Salicin in Hektoen Enteric Agar
Can be fermented to form acid end-products
Hektoen Enteric Agar Results: Minimal growth
Organism is inhibited by bile salts or dyes and is likely Gram-positive.
Hektoen Enteric Agar Results: yellow-orange or salmon pink
Bacteria can ferment lactose, sucrose, or salicin to acid end-products. It is neither Salmonella or Shigella. Growth means gram-negative.
Hektoen Enteric Agar Results: blue-green color with black precipitate
Blue-green results from increase in pH when either Salmonella or Shigella digest animal tissue. Bacteria that reduce sulfur to hydrogen sulfide gas will produce a black precipitate when reacted with ferric ammonium citrate. Salmonella. Growth means gram-negative.
Hektoen Enteric Agar Results: blue-green color, no precipitate
Break down animal tissue to raise pH, but do not reduce sulfur to hydrogen sulfide gas. Shigella. Growth means gram-negative.
pH indicator. Turns yellow in acidic conditions, pink in basic conditions. Red at neutral conditions.
Phenol red broth
Differential test medium prepared as a base at pH 7.3. Used to test if a particular microbe can ferment a certain type of carbohydrate. It can be used to distinguish different Enterobacteriaceae and different gram-positive fermenters.
Phenol red broth ingredients
Pancreatic digest of casein (peptone), NaCl, Carbohydrate (glucose, lactose, sucrose), Phenol Red, distilled water, inverted Durham tube.
Phenol Red Broth results: yellow broth
Organism can use particular carbohydrate via fermentation. This produces acid as a by-product. Gas may be produced and will be trapped in inverted Durham tube.
Phenol Red Broth results: pink broth
Organism cannot digest the carbohydrate, but instead uses the peptone. Ammonia is produced as a by-product, making the broth more basic.
Purpose of base broth in Phenol Red Broth?
Base broth contains all ingredients minus a sugar. It is used as another control for color comparison to determine if an organism truly ferments a particular sugar or if it just reacts nonspecifically to some other ingredient in the phenol red broth tubes causing a color change.
Phenol Red Broth results: yellow and bubble
Fermentation with acid and gas end products. A/G.
Phenol Red Broth results: yellow and no bubble
Fermentation with acid end product. No gas production. A/-
Phenol Red Broth results: red and no bubble
No fermentation. -/-
Phenol Red Broth results: pink and no bubble
Degradation of peptone occurs and alkaline end products are formed. K
Purpose of Phenol Red Broth
Fermentation of sugars other than glucose can be used to help differentiate between various fermentative bacteria. Bacteria have various enzymes that allow it to use different sugars. Can determine which organisms can utilize each sugar.
Purpose of inverted Durham tube in Phenol Red Broth?
Indicator of gas production from fermentation
Purpose of peptones in Phenol Red Broth?
Natural source of amino acids, peptides, and proteins in growth media. Can be digested to produce alkaline by-products and pink color.
Methyl Red and Voges-Proskauer Broth
Distinguish between different types of Enterobacteriaceae (IMViC). Determine whether organism performs mixed acid fermentation or 2,3-butanediol fermentation based on the change in pH of the medium.
Methyl Red Test
Determine whether organism performs mixed acid fermentation. It will overcome the phosphate buffer in the medium and lower the pH. Acid-end products are produced, pH lowers, and indicator changes color. The acids of these organisms tend to be stable.
Determine whether bacteria produces acetoin (and 2,3-butanediol) from glucose fermentation. If it does, the reagents interact to produce a red color
MR-VP Test Ingredients
Peptone, phosphate bugger, sugar (dextrose or glucose). Methyl red is a pH indicator containing red dye and ethanol. VP reagent A has alpha-Naphthol, reagent B has KOH.
pH changes in MR-positive organism
pH decreases immediately and levels off. pH is lowered permanently.
pH changes in VP-positive organism
pH initially drops but then goes back up to reach a pH higher than its initial pH. Organisms produce acid and temporarily lower the pH, but because the 2,3-butanediol fermentation end products are neutral, the pH at completion of the test is near neutral.
Methyl Red Test process and results after incubation
Transfer 1 mL of broth to a nonsterile test tube. Add 3 drops of methyl red. MR-positive turn red immediately. MR-negative do not change color (yellow or orange)
Voges-Proskauer Test process and results after incubation
Transfer 1 mL of broth to a nonsterile test tube. Add 15 drops of reagent A, and mix. Add 5 drops of reagent B and mix well to oxygenate. Read the result at 10-minute intervals for 60 minutes. VP-positive tests are red in 60 minutes. VP-negative tests are unchanged after 60 minutes. VP-negative reactions often produce a copper color, and VP-positive often produce a pink color, with the strongest color change at the surface.
SIM, Methyl red, Voges-Proskauer, Citrate tests. Used to distinguish Enterobacteriaceae and differentiate them from Gram-negative rods.
Purpose of peptone in MR-VP Broth
Purpose of glucose in MR-VP Broth
Provides fermentable carbohydrate
Purpose of phosphate buffer in MR-VP Broth
Resists pH changes in the medium
Theory behind using both Methyl Red and Voges-Proskauer tests?
If glucose is fermented, it will either undergo an acidic pathway or neutral pathway. If the pathway is acidic, it produces mixed acids and results in a pH lower than 4.4, which is detectable by Methyl red indicator. If the pathway is neutral, acetoin will be produced and will react with VP reagents A and B to produce a pink color.
Catalase Test purpose
Detects presence of catalase enzyme by addition of hydrogen peroxide. Often related to aerotolerance of bacteria.
Catalase test methods
Transfer small amount of bacteria to microscope slide. Add 1-2 drops of hydrogen peroxide. Cover reaction with petri dish. Watch for bubbling. Don't use metal because it could result in a false positive.
Catalase test results
Bubbling means catalase-positive and the organism produces catalase. No bubbling means catalase-negative and the organism doesn't produce catalase.
Purpose of catalase in organisms
Works with superoxide dismutase to neutralize the effect of dangerous oxygen free radicals. It is present in obligate aerobes, some facultative aerobes, and in small amounts in microaerophiles. It is absent in aerotolerant anaerobes and obligate anaerobes.
Oxidase Test Theory
Cytochrome c oxidase oxidizes cytochrome c. In bacteria, it is located in gram-negative cell periplasm and gram-positive cell membrane. Add Kovac's oxidase reagent (chromogenic reducing agent) to reduce cytochrome c while it is oxidized. If reaction occurs, color changes to deep purple/blue. Color change indicates the presence of cytochrome c oxidase.
Oxidase Test Methods
Place filter paper on paper towel. Aseptically transfer culture onto filter paper. Add few drops of Kovac's oxidase reagent and note any color changes after 20 seconds. Do for 2 different cultures.
Oxidase Test Results: Blue color
Cytochrome c oxidase is present. Positive result.
Oxidase Test Results: no change in color
Negative result. Cytochrome c oxidase is absent.
Nitrate Reduction Test
Undefined medium. Measuring anaerobic respiration in which nitrate is reduced instead of oxygen. Many gram-negative bacteria have nitrate reductase, allowing them to reduce nitrate to nitrite in a single step. Other bacteria undergo denitrification, in which nitrate is converted to molecular nitrogen
Nitrate Broth Ingredients
Beef extract, peptone, Potassium nitrate, inverted Durham tube
Purpose of beef extract in Nitrate Broth
General nutrient medium that supports growth
Purpose of peptone in Nitrate Broth
Nutrient source of carbon, nitrogen, and energy that supports growth
Purpose of potassium nitrate in Nitrate Broth
Source of nitrate for nitrate reduction test
Nitrate Reduction Test Results
- If the Durham contains gas the test is complete and shows that denitrification took place. Gas produced in a nitrate reduction test by an organism capable of fermenting to gas end products is not determinative because the source of the gas is unknown.
- If there is no visual evidence of denitrification, reagents A and B are added to the medium to test for nitrate reduction to nitrite. If present, nitrite will form nitrous acid, which reacts to produce a red compound. So formation of red color after the addition of reagents indicates that the organism reduced nitrate to nitrite. If no color change takes place, nitrate either was not reduced or was reduced to one of the other nitrogenous compounds and we add zinc to determine which occurred.
- Zinc is added to catalyze the reduction of any nitrate to nitrite. If nitrate is present when zinc is added, it will be converted quickly to nitrite, and turn the medium red through the same reaction as before. The red color indicates that nitrate was not reduced by the organism. No color change after adding zinc indicates that the organism reduced the nitrate to a nongaseous nitrogenous compound.
Nitrate Reduction Test Results: Gas produced by non-fermenter
Denitrification occurred. This is the production of nitrogen gas. The test is positive.
Nitrate Reduction Test Results: Gas produced by known fermenter
The source of the gas is unknown, so reagents must be added to determine. Test is inconclusive.
Nitrate Reduction Test Results: red color after addition of reagents A and B
Nitrate was reduced to nitrite. Test is positive.
Nitrate Reduction Test Results: no color change after addition of reagents A and B
Incomplete test that requires addition of zinc dust
Nitrate Reduction Test Results: no color change after addition of zinc dust
Nitrate reduction to nongaseous nitrogenous compounds. Test is positive.
Nitrate Reduction Test Results: red color after addition of zinc dust
No nitrate reduction. Test is negative
Simmons Citrate Agar
Differential medium to differentiate between members of Enterobacteriaceae, all of which are facultative anaerobes that have the ability to ferment carbohydrates. Defined medium with exact chemical composition known. Unable to support complex, high-energy yielding respiratory processes, like the citric acid cycle.
Citrate Test ingredients
Sodium citrate, ammonium phosphate, bromothymol blue, sodium chloride, dipotassium phosphate, magnesium sulfate, agar
Purpose of sodium citrate in citrate test
Sole carbon source in the media. Only bacteria containing citrate permease will be able to survive off of this energy source
Purpose of ammonium phosphate in Citrate test
Sole nitrogen source in the media
Purpose of bromothymol blue in citrate test
pH indicator dye. Green at pH 6.9, blue at pH 7.6
Citrate Test Theory
Bacterial species that possess the enzyme citrate permease are able to transport citrate into the cell and perform citrate fermentation. Citrate is converted to oxaloacetate and acetate, and oxaloacetate is converted to pyruvate. Different pHs determine what happens next. The creation of an alkaline pH occurs from the conversion of ammonium phosphate into ammonia and ammonium hydroxide. As media increases in pH, bromothymol blue shifts to blue color. Tests the ability of organism to use citrate as sole carbon source for fermentation.
Citrate Test Methods
Inoculate 2 simmons citrate slants and one control. Inoculate with a needle to avoid confusion between actual growth and a heavy inoculum. Incubate tubes.
Citrate Test Results: no growth, green
Negative result. No utilization of citrate. Citrate cannot be used as sole carbon source. No color change.
Citrate Test Results: growth and blue color
Growth indicates citrate is being used. Citrate can be used as sole carbon source. Color change to blue is positive test.
Purpose of sodium chloride in Simmons Citrate Agar
Maintains osmotic balance
Purpose of dipotassium phosphate in Simmons Citrate Agar
Purpose of magnesium sulfate in Simmons Citrate Agar
Cofactor for metabolic reactions
Purpose of agar in Simmons Citrate Agar
Citrate Test Results: growth and green
Citrate is utilized, positive test. Could be the result of incomplete incubation.
Decarboxylation Test Theory
Differentiate organisms in the family Enterobacteriaceae and distinguish them from other Gram-negative rods. Most members of this family produce one or more enzymes that can break down amino acids. If the bacteria ferments glucose, acidic by-products will be produced and the solution will be turned yellow. Low pH and presence of specific amino acids will induce decarboxylase-positive organisms to produce the enzyme. Decarboxylation results in accumulation of alkaline by-products and the medium turns purple.
Decarboxylation Test Ingredients
Peptone, Beef Extract, Glucose, Bromcresol purple, Cresol red, Pyridoxal phosphate, Lysine/Ornithine/Arginine
Purpose of peptone and beef extract in Decarboxylation Test
Necessary growth nutrients
Removal of carbon dioxide from amino acid with formation of amines
Purpose of Bromcresol purple in Decarboxylation Test
pH indicator. Purple at 6.8 and above, yellow below 5.2.
Purpose of pyridoxal phosphate in Decarboxylation Test
Pyridoxal phosphate is a coenzyme
Purpose of using different amino acids (Lysine, Ornithine, Arginine) in Decarboxylation Test
Decarboxylases are specific for a certain amino acid, so using different ones allow us to identify which decarboxylase it is
Purpose of glucose in Decarboxylation Test
If the bacteria ferments glucose, acidic by-products will be produced and the solution will be turned yellow
Decarboxylation Test: Addition of mineral oil
Creates anaerobic environment that leads to glucose fermentation and creation of acidic environment. This allows the decarboxylation enzymes to be activated. It also decreases the probability of an alkaline shift occurring in the medium as a result of oxidation.
Decarboxylation Test Results: purple
Fermentation occurred, leading to production of specific decarboxylase. Positive test.
Decarboxylation Test Results: yellow
Negative test. Fermentation occurred, but decarboxylation did not.
Decarboxylation Test Results: no change
No fermentation or decarboxylation. Negative test.
Decarboxylation Test Methods
Inoculate 3 of each decarboxylase broth and have 1 control of each. Overlay tubes with sterile mineral oil. Incubate for one week.
Polysaccharide made of alpha-D-glucose in linear and branched configurations. Too large to pass through bacterial cell membranes because of complex network of branching, so it must be broken into smaller fragments or individual glucose molecules first. Organisms secrete extracellular enzymes that can hydrolyze it by breaking the glycosidic linkages. These enzymes are alpha-amylase and oligo-1,6,-glucosidase
Starch Hydrolysis Test
Simple plated medium. When organisms that produce alpha-amylase and oligo-1,6-glucosidase are grown on the plate, they hydrolyze the starch in the surrounding area. Starch and sugar subunits are difficult to see in the medium, so iodine is used to detect whether or not starch is present around the bacterial growth. Iodine reacts with starch to produce blue or dark brown color. So if bacteria hydrolyzes starch, there will be a clear zone without color.
Starch Hydrolysis Test Methods
Inoculate starch agar plate with two organisms. Incubate. Add iodine to plate. Before adding iodine, use sharpie to make a line at margins of growth to prevent confusion between thinning growth at edges with the halo of starch hydrolysis
Starch Hydrolysis Test Results: Clearing around growth
Alpha-amylase and/or oligo-1,6-glucosidase is present because starch is broken down. Positive test result.
Starch Hydrolysis Test Results: No clearing around growth
Neither alpha-amylase nor oligo-1,6-glucosidase are present. Starch is not broken down. Negative test result.
Starch Hydrolysis Test Ingredients
Iodine, Starch Agar plate. Starch Agar plate contains beef extract, soluble starch, agar, deionized water.
DNA Hydrolysis Test
Used to determine the presence of DNase activity, or DNA hydrolysis, and the ability to hydrolyze the phosphodiester linkage in the DNA backbone bond
DNA Hydrolysis Test Ingredients
Tryptose, DNA, Sodium chloride, Agar, Methyl green
Purpose of peptides (from tryptose) in DNA Hydrolysis Test
Source of both carbon and nitrogen
Purpose of sodium chloride in DNA Hydrolysis Test
Regulation of salinity/osmolarity
Purpose of DNA in DNA Hydrolysis Test
Substrate for reaction, further source of carbon and nitrogen
Purpose of methyl green in DNA Hydrolysis Test
Indicator Dye. Binds to DNA if DNA is not split into smaller fragments
DNA Hydrolysis Test Methods
Spot inoculate DNase plate with two organisms, keep one sector as a control. Incubate for 24 hours, and observe the gel for clearing around the bacterial growth.
DNA Hydrolysis Test Theory
If the organism that grows in the medium produces deoxyribonuclease, it breaks down DNA into smaller fragments. Serratia and Staphylococcus DNase enzymes split DNA into molecules with different phosphate group orientations. When the DNA is broken down, it no longer binds to the methyl green, and the green color fades and the colony is surrounded by a colorless zone.
DNA Hydrolysis Test Results: growth and clearing surrounding growth in blue gel
Positive test result. Indicates that deoxyribonuclease broke the DNA down into smaller fragments so it could no longer bind to the methyl green.
DNA Hydrolysis Test Results: growth, no clearing
Negative test result. DNA is not broken down by deoxyribonuclease, so it still binds to the methyl green.
Protein derived from collagen
Family of extracellular enzymes secreted by some microorganisms. Hydrolyzes gelatin to amino acids. Microorganisms can then absorb individual amino acids and use them for metabolic purposes. Their presence can be detected by growing microorganisms on nutrient gelatin.
Gelatin, peptone, beef extract. Solid media but also the substrate for enzymatic activity
Gelatin Hydrolysis Test
When a tube of nutrient gelatin is stab inoculated with gelatinase-positive organisms, the nutrient gelatin will liquefy. Gelatinase-negative organisms do not liquefy the medium. It is used to determine the ability of a microbe to produce gelatinase.
Gelatin Hydrolysis Test Methods
Stab inoculate 2 nutrient gelatin stab tubes. Do not inoculate a control tube. Incubate. Make sure that control is not liquefied, because otherwise gelatin may have melted and test may give false positive. Must ensure that liquefaction is microorganism-related, and not temperature-related.
Gelatin Hydrolysis Test Results
If control tube is solid, test can be read. If it is liquid, all tubes must go in ice bath or refrigerator until control has been re-solidified. If gelatin is liquid, gelatinase is present and test is positive. If gelatin is solid, no gelatinase is present and test is negative.
By-product of decarboxylation of amino acids in land animals. Bacteria use urease to break it down to ammonia and carbon dioxide. Then they use ammonia as a nitrogen source, and can also use it for a buffer to counteract acidic environments.
Urea Agar vs. Urea Broth
Urea broth contains fewer nutrients and more buffer. It can only detect rapid urea hydrolyzers. Slow urea hydrolyzers will not be detected in broth, only in slants.
Urea Hydrolysis Test Methods
Use heavy inoculum with both types. For broth, inoculate with sterilized loop. For agar, cover the whole surface with a heavy inoculum and do not stab the butt of the agar. Both inoculated.
Urea Hydrolysis Test Results: Yellow Broth
Negative result. Hydrolysis of urea has not occurred.
Urea Hydrolysis Test Results: Deep Violet Broth
Positive result. Rapid hydrolysis of urea has occurred.
Urea Hydrolysis Test Results: Yellow Agar Slant
Negative result. Hydrolysis of urea has not occurred.
Urea Hydrolysis Test Results: Violet/Pink Agar Slant
Positive result. Slow hydrolysis of urea has occurred. Slower urease-positive bacteria are detected only via slants
Urea Hydrolysis Test Results: Deep Violet Agar Slant
Positive result. Rapid hydrolysis of urea has occurred.
Urea Agar Ingredients
Urea, peptone, glucose, potassium phosphate, phenol red
Purpose of peptone and glucose in Urea Agar
Provide essential nutrients for many bacteria
Purpose of potassium phosphate in Urea Agar
Mild buffer used to resist alkalinization of the medium from peptone metabolism, so that we only see true urease-positive results
Purpose of phenol red in Urea Agar
Yellow/orange below 8.4 and red or pink above. Pink color in medium in less than 24 hours indicates a rapid urease-positive organism. When ammonia is produced from urea hydrolysis, alkaline environment is created and media turns pink.
How does Urea Broth differ from Urea Agar?
Broth contains a trace amount of nutrients in the form of yeast extract. It contains buffers strong enough to inhibit alkalinization of the medium by all but the rapid urease-positive organisms. Because of the increased amount of buffer, it cannot detect slow urea hydrolyzers. The combination of restrictive amounts of nutrients and pH buffers prevent all but the rapid from making ammonia.
Sulfur reduction, Indole production from Tryptophan, Motility. Enterobacteriaceae differentially reduce sulfur, produce indole, and have motility. Combination differential because it combines various compatible tests into one diagnostic. Semi-solid complex medium made from casein and animal tissue, sodium thiosulfate, ferrous ammonium sulfate, and agar.
Purpose of casein and amino acids in SIM Medium
Provide tryptophan, which is needed for the indole test
Sulfur Reduction of SIM Test
Sulfur is contained in the medium as sodium thiosulfate, or is contained in the amino acid cysteine, provided by animal tissue and casein in the medium. Bacteria that contain the enzyme cysteine desulfurase or thiosulfate reductase can reduce sulfur into hydrogen sulfide. Ferrous sulfate reacts with hydrogen sulfide to form black precipitate. Bacteria that are positive for sulfur reduction will produce this black precipitate.
Indole Production of SIM Test
Indole is produced from the hydrolysis of another amino acid, tryptophan, which is provided by the animal tissue and casein in the medium. It is hydrolyzed by tryptophanase to produce indole. Bacteria that contain this enzyme will be positive for the indole production test. Tryptophan hydrolysis/indole production is detected by the addition of Kovac's reagent to the medium. It contains DMABA which reacts with indole if present to form a red layer at the top of the medium.
Motility of SIM Test
SIM medium is semisolid, meaning it only contains a small concentration of agar, allowing motile bacteria to move around. If growth radiates off of the stab line in all directions, the bacteria are motile. Indeterminate for organisms positive for sulfur reduction.
SIM Test Methods
Stab inoculate medium with 3 organisms. Incubate tubes. Determine sulfide reduction and motility status before adding Kovac's reagent. Add Kovac's reagent to a depth of 2-3 mm. Red layer formation shows within several minutes.
Triple Sugar Iron Agar
Rich medium designed to differentiate bacteria on the basis of glucose fermentation, lactose fermentation, sucrose fermentation, and sulfur reduction. Prepared as a shallow agar slant with a deep butt, providing aerobic and anaerobic growth environment
Triple Sugar Iron Agar ingredients
Lactose, sucrose, dextrose, animal proteins, ferrous sulfate and sodium thiosulfate, phenol red, ferrous sulfate, sodium chloride, agar, deionized water
Relative concentrations of lactose, sucrose, and glucose in triple sugar iron agar
Glucose concentration is 1/10th of the concentration of lactose and sucrose to facilitate the detection of organisms that only ferment glucose
Purpose of animal proteins in Triple Sugar Iron Agar
Source of carbon and nitrogen. Supply amino acids for ammonia production after glucose is gone via reversion. Contains cysteine, another source of sulfur.
Purpose of ferrous sulfate and sodium thiosulfate in Triple Sugar Iron Agar
Source of oxidized sulfur
Purpose of phenol red in Triple Sugar Iron Agar
pH indicator. Turns yellow in acidic conditions.
Purpose of ferrous sulfate in Triple Sugar Iron Agar
Iron in ferrous sulfate used as hydrogen sulfide indicator
Triple Sugar Iron Agar Methods
Use straight inoculation needle to stab through center of medium to bottom of tube, then streak the surface of the agar slant. Incubate. Read too early and could appear to be false fermenter of two or more sugars. Read results too late and could appear to be false glucose-only fermenter
Triple Sugar Iron Agar Results: Red slant and yellow butt
Glucose only fermenter. Acid products from fermentation will lower the pH and turn the entire medium yellow within a few hours. But glucose is in short supply and will be exhausted within 12 hours. The organisms in the slant (aerobic region) will break down available amino acids and produce ammonia and raise the pH via reversion. This only occurs in the slant because the butt is anaerobic. Increase in pH changes slant color to red after 24 hours. K/A
Triple Sugar Iron Agar Results: Yellow slant and butt
Fermenter of glucose and lactose and/or sucrose. Lactose and sucrose concentrations were ten times higher than glucose, resulting in greater acid production for a longer period of time. This makes both the slant and the butt yellow after 24 hours. A/A
Triple Sugar Iron Agar Results: Black precipitate
Reduction of thiosulfate occurs in the medium. Ferrous sulfate reacts with hydrogen sulfide to form black precipitate, usually in butt. Acid conditions must exist for this reaction, so black precipitate in the medium is an indication of sulfur reduction and fermentation.
Triple Sugar Iron Agar Results: Cracked or lifted agar
Gas production (G)
Triple Sugar Iron Agar Results: Red slant and red butt
No fermentation occurred. Peptone catabolized aerobically and anaerobically with alkaline products. K/K
Triple Sugar Iron Agar Results: red slant and no change in butt
No fermentation. Peptone catabolized aerobically with alkaline products. K/NC.
Triple Sugar Iron Agar Results: no change in slant or butt
Organism is growing slowly or not at all. NC/NC
Tryptic Soy Agar and Sheep blood. Contains beef heart for infusion, pancreatic digest of casein, sodium chloride, yeast extract, agar, and sheep blood. Differential for gram-positive cocci, not selective. Based on ability to hemolyze red blood cells with hemolysins.
Partial destruction of red blood cells. Typical. No clearing. Green color around growth.
Complete destruction of red blood cells. Potentially pathogenic. Clearing around growth. Agar is clear/yellowish.
No destruction of red blood cells. Appears as normal growth. Typical. No clearing. No color change (red).
Hemolysin produced by streptococci. Type O is not oxygen stable, type S is oxygen stable. Both grow optimally in anaerobic environment.
Blood Agar Methods
Swab throat, quadrant streak plate, stab agar 2-3 places in first streak and 2-3 places not inoculated. Streak-stab technique provides a favorable anaerobic environment for streptolysins to grow on blood agar plates.
A positive result for the DNA hydrolysis test does not distinguish between Staphylococcus DNase and the DNase produced by Serratia. If you had the expertise to correct this weakness in the system, would you be improving the test's sensitivity or specificity?
Specificity. It can already detect both species so it's sufficiently sensitive, but the test is not specific enough to distinguish the two
Suggest a reason why the DNA hydrolysis test is read after only 24 hours while other tests may take a week. What would be the likely consequences of incubating DNase Agar for a week?
It is read after only 24 hours because the DNA could be broken down in other ways over the course of a longer timeframe. This would result in a false positive because clearing would be observed but not necessarily as a result of DNase presence.
If the control for gelatin hydrolysis is solid and the inoculated tube is liquid, is it acceptable to read the result before the complete incubation time has elapsed?
Yes. If the control is solid, then the temperature of incubation doesn't account for the liquefaction of the inoculated tube. Therefore, we are able to determine that the gelatinase is the reason for the liquefaction.
Why is it acceptable to record positive urea hydrolysis test results before the suggested incubation time is completed, but not acceptable to record a negative test result early?
The urease-positive bacteria can undergo either rapid or slow hydrolysis. Even if the test appears negative after a certain length of time, it may later on yield a positive result. Once it yields a positive result, it will stay that way and not turn negative after, so there is no danger in reporting a positive result early.
Is the uninoculated SIM tube a positive or negative control?
What purpose does the uninoculated SIM tube serve in the sulfur reduction test?
It shows whether an organism can reduce sulfur to produce hydrogen sulfide by having something to compare to. Any black precipitate is a positive test.
What purpose does the uninoculated SIM tube serve in the indole test?
The indole test shows whether an organism produces tryptophanase and hydrolyzes tryptophan into indole and pyruvate. This is observed with the red color from the reaction of indole with DMABA. It ensures that the red color is not produced from another reaction but from the indole alone.
What purpose does the uninoculated SIM tube serve in the motility test?
It ensures that there is motility because growth along the stab line can appear to be motile when in reality is not radiating from the stab line.
What are the consequences for Triple Sugar Iron Agar (TSIA) if 1% glucose is added instead of the amount specified in the recipe?
If too much glucose is present, it would not be exhausted as quickly. The organisms in the aerobic area will continue to breakdown glucose and not resort to amino acids, so ammonia would not be produced and pH would not be raised. This would make the slant appear yellow and be a false positive for lactose or sucrose fermentation.
What are the consequences for Triple Sugar Iron Agar (TSIA) if ferrous ammonium sulfate is omitted?
This is the source of oxidized sulfur. Without this, there would be no reaction with hydrogen sulfide to form black precipitate. Bacteria that reduce sulfur may be misidentified as not having this capacity.
What are the consequences for Triple Sugar Iron Agar (TSIA) if casein and animal tissue are omitted?
These are the sources of carbon and nitrogen. When broken down, ammonia is produced, which happens when only glucose can be fermented, and not sucrose or lactose. Without these, ammonia can't be produced to raise the pH and indicate that the bacteria is a glucose-only fermenter.
What are the consequences for Triple Sugar Iron Agar (TSIA) if sodium thiosulfate is omitted?
This is what is reduced to produce hydrogen sulfide. Without it, no black precipitate would be produced by any organisms because no hydrogen sulfide would be produced. This would result in the false conclusion that an organism cannot reduce sulfur.
What are the consequences for Triple Sugar Iron Agar (TSIA) if phenol red is omitted?
Phenol red is the pH indicator. Without it, we won't be able to distinguish fermentation occurs because we could not detect its acid end-products. No fermentation would be indicated, and this would result in misinterpretation of results.
What are the consequences for Triple Sugar Iron Agar (TSIA) if the initial pH is 8.2?
Phenol red is bright pink at pHs above 8.1, so starting at such a high pH could skew the results by making decreases in pH harder to observe, as the indicator changes from yellow to red over pH of 6.6 to 8.1.
What are the consequences for Triple Sugar Iron Agar (TSIA) if the agar butt is shallow rather than deep?
The entire tube will show the results of only aerobic conditions, as anaerobic conditions can't be established. This may lead to misinterpretation.
Why is the streak plate preferred over the spot inoculations in this procedure?
The streak plate allows for isolation of colonies from a dense culture. This is useful for a throat culture because different species of bacteria and different types of hemolysis may be present. This would be hard to see in a spot inoculation only.
What is the application of Bile Esculin Agar (BEA) as a plated medium?
It is used to differentiate between bacteria that can hydrolyze esculin in bile and bacteria that cannot. The ones that can hydrolyze esculin are bile esculin-positive and include enterococci and the bovis group of Streptococci. Non-Group D Streptococci cannot hydrolyze esculin and produce a negative result.
Is Bile Esculin Agar a defined or undefined medium? Why is this desirable?
Undefined (exact composition unknown). This is desirable because they contain more nutrients and support growth of bacteria.
What is the role of sodium chloride in Mannitol Salt Agar (MSA) and how does it work?
It is responsible for the selectivity of the media. Its high concentrations are dehydrating and make survival difficult for bacteria other than Staphylococcus. This means that any growth on the media should be Staphylococci bacteria.
What are the roles of mannitol and phenol red in Mannitol Salt Agar (MSA)?
Mannitol is the carbohydrate that is fermented by bacteria to an acid end-product, changing the pH of the media. This is detected by the phenol red, which is pink/red in the presence of the acid (Staphylococcus aureus is not present). Phenol red is yellow when acid-end products are produced by Staphylococcus aureus in the fermentation of mannitol.
What is the application of MacConkey agar?
It allows for differentiation of gram-positive and gram-negative organisms. Gram-positive will not grow. Within the gram-negative category, Enterobacteriaceae are further differentiated into those that can ferment lactose and produce pink/red color, and those that do not ferment lactose and do not change the color of the media.
Growth on the MacConkey agar and NA plates was recorded as good, poor, or none. These are qualitative and subjective. How were they measured?
NA is a control plate to compare growth.
Why wouldn't it be advisable to compare growth of the organisms on each plate to each other in the MacConkey Agar?
Original cell densities could have been different, so one may seem to have grown more but in reality was just plated in a higher amount
What is the role of bile salts in the Hektoen Enteric Agar?
They inhibit growth of gram-positive bacteria by destroying their membrane integrity. We are only interested in looking at gram-negative bacteria, and gram-positive won't grow in the presence of bile salts.
What are the roles of lactose, sucrose, and salicin in Hektoen Enteric Agar?
They are the carbohydrates that are fermented to acid end-products. The acid lowers pH and is indicated by color change to orange/pink. Neither Salmonella nor Shigella ferment these, so an organism that results in orange/pink is neither of these organisms.
What are the roles of acid fuchsin and bromothymol blue in Hektoen Enteric Agar?
They are pH indicators. When orange/pink, the pH has decreased in response to the acid end-products of fermentation. When blue-green, the bacteria have digested animal tissue, resulting in pH increase
Were the uninoculated controls for the phenol red broth positive or negative, and what purpose did they serve?
Negative because no response is expected. They are controls for color comparison to determine the color of the broth with the sugar but without an organism to ferment anything. No fermentation should occur here.
What purpose did the phenol red base broths serve?
They are a reference point for color comparison. They have all ingredients except the sugar. They help determine whether an organism ferments a specific sugar or reacts nonspecifically to a different ingredient in the broth, giving a color change.
Why were you told to shake the VP tubes after the reagents were added?
Mixing well oxygenates the medium, giving the chance for acetoin to be oxidized to diacetyl, and for diacetyl to react with guanidine nuclei from peptone to produce a red color. Without oxygen, the acetoin may not be fully oxidized and a false negative could result.
Why is the methyl red test read immediately after addition of methyl red reagent and Voges-Proskauer is read up to 60 minutes after the addition of VP reagents A and B?
Acids that participate in mixed acid fermentation and give a positive MR result are stable. If VP is read too quickly, it will initially show a lowered pH, but the end pH goes back up to neutral. The color of a positive VP test is the result of a chain reaction following oxidation of acetoin. MR positive lowers the pH permanently and results will not change later, but time is more sensitive for VP-positive.
What happens to the oxidase reagent after 20 seconds? Does this only happen after 20 seconds? Why a 20-second time limit?
The reagents will oxidize with the environment due to instability. Oxygen in the environment will change the reagent color to blue as time passes. The reaction should occur quickly with the fresh cultures, so by 20 seconds, the reaction we are interested in should be complete. The oxygen in the environment will not substantially alter the results before the end of this time period, but the effects add up as time passes.
Is testing a known catalase-positive organism along with an unknown a positive or negative control? What information is provided?
It is a positive control because we are expecting a reaction. The control should bubble. If it doesn't, then when we test the unknown, we may get a false negative. If there are no bubbles when hydrogen peroxide is added to the catalase-positive, the experiment will not give the correct results. If it bubbles as expected, we can expect our results to be accurate and correctly bubble if catalase is present.
Why is gas production not recognized as nitrate reduction when the organism is a known fermenter?
The source of the gas would not be known. If the organism is a known fermenter, the gas may result from that but could also result from nitrate reduction.
A known fermenter has a gas bubble in the Durham tube of the nitrate reduction test. Knowing that fermenters frequently produce gas, you ignore the bubble and proceed. Adding reagents produces no change, and neither does adding zinc. Is this consistent with what we know about the test?
Yes. This shows that the fermenter reduced nitrate to a nongaseous nitrogenous compound because there was no color change. The gas produced was not N2, which is still possible since other gases can be produced.
Is the uninoculated tube in the citrate utilization test a positive or negative control? What information is provided by it?
Negative. It is an example of a negative result. We can determine if the color is more yellow or if there is growth, which would be difficult to tell without the control. If the color and growth are different, this could indicate citrate utilization and pH change and therefore a positive result. If there is contamination, we should be able to tell by looking at the control to avoid false positives.
Many bacteria that are able to metabolize citrate in the citric acid cycle produce negative results in the citrate utilization test. Why?
This test shows us whether an organism would be able to use citrate as its only source of carbon to perform citrate fermentation. The complex citric acid cycle is not supported here, so even if an organism uses the citric acid cycle, it may produce a negative if it doesn't specifically have citrate permease. Also, bacteria using this need to survive in the presence of ammonia, which may not be possible for all.
In the starch hydrolysis test, there are clearings in the uninoculated area when pouring iodine. What are some possible explanations?
The plate may be contaminated because iodine alone would not create a clearing.
In the starch hydrolysis test, there are clearings in the uninoculated area when pouring iodine. Was the integrity of the test compromised?
Yes. The plate may be contaminated. There may be hydrolysis in organismal areas where the responsible enzymes are not present.
In the starch hydrolysis test, there are clearings in the uninoculated area when pouring iodine. What measures could be taken to avoid this problem in the future?
Why isn't a different base broth required for each decarboxylase test?
It doesn't have any amino acids in it. The amino acids are the differential part of the test, and since the rest of the base can be generalized, each organism can be tested in it without needing more broths. The results would be the same for each organism.
Is base broth a positive or negative control for decarboxylation tests?
Negative. We don't expect a reaction to occur in it.
What information is provided by the base broth in the decarboxylation tests?
The reaction of the organism in the base broth shows the interaction without the amino acids. Fermentation and yellow color should occur here if the organism is a fermenter, but decarboxylation will not because that requires amino acids.
How can no color change in the broth and a conversion to a yellow color both be considered negative results for the decarboxylation test?
The negative vs. positive distinction refers to decarboxylation. A yellow color only indicates that fermentation occurred, but since this is separate from decarboxylation, the organism that produces yellow can still be negative and not be able to decarboxylate the amino acids present. Yellow means the organism can ferment glucose but not necessarily decarboxylate.
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