Lab Quiz #3

Through the formation of a black precipitate upon reaction with ferrous sulfate

How does SIM agar (Sulfide indole motility medium) detect the production of hydrogen sulfide gas?

Rotten Eggs

Hydrogen sulfide gas has a characteristic odor that is similar to what smell?

TSA media is enriched. The tested organism is a pigmented bacterium.

An unknown specimen was grown on TSA (Tryptic Soy agar). Based on your observation of this specimen choose the appropriate statements.

The bacterium is unable to ferment lactose and/or sucrose. The bacterium is unable to ferment glucose.

Based on your observation of the pictured TSI (Triple Sugar Iron) medium, select ALL appropriate conclusions.(Recall that an observation and a conclusion are different. An observation is simply what you see; conclusions are statements of what you determine based on your observations.)

Water

Besides urea, what other compound must be available for the urease enzyme to break down urea?

Deep purple in the upper portion of the tube, with a translucent, brown, whey-like appearance in the media.

If organisms are unable to ferment the sugar lactose for energy, milk proteins are available for proteolysis into amino acids. What effect does proteolysis have on the litmus milk media?

The presence of contaminating organisms in the agar

In the middle of running your experiments in lab, the instructor passes around an additional nutrient agar plate. He says that this plate is a control and should not be inoculated, but that it should be incubated and otherwise treated just like the other plates. For what is this controlling?

Yes, the only microbes present will grow on the plates we used.

Is it possible to isolate all the organisms collected in a field sample using culture techniques?

The sample was incubated less than 24 hours, which allowed for only limited fermentation. The test organism ferments sucrose best under reduced oxygen conditions. The organism is limited in its ability to ferment sucrose. Since diffusion is restricted within the Durham tube and it contains only a small volume of media, it turned yellow first.

Observe the pictured Sucrose Fermentation test.

Organisms that use light as their energy source and carbon dioxide (CO2) as their carbon source.

Phototrophs

Amino acids

Pseudomonas aeruginosa cannot utilize any of the carbohydrates used in this experiment, yet growth was still observed in the tube. What alternative nutrient source may have been used to produce energy for this organism?

The VP test is part of the IMViC panel of tests. To develop the Voges-Proskauer, test alpha-naphthol must be added followed by potassium hydroxide. MRVP media is inoculated using an inoculating loop. The media used for the VP test contains glucose (dextrose).

Select ANY & ALL the CORRECT, appropriate statement(s) regarding the Voges-Proskauer (VP) test

The media used for the MR test contains glucose (dextrose) The culture that is growing in the MRVP broth should be split into separate tubes or grown in separate tubes before adding the reagents for the MR portion of the test The MR test is part of the IMViC panel of tests MRVP media is inoculated using an inoculating loop.

Select all the appropriate statement(s) regarding the Methyl Red (MR) test

The test organism is inoculated into the tube by stabbing the agar. During the early stages of incubation, the medium will appear yellow; if the targeted decarboxylase is present, the medium will gradually turn purple. Sterile mineral oil should be added to prevent artificial alkylation of the media's surface.

Select the appropriate statements regarding the decarboxylase tests.

Organisms from different metabolic categories that help each other accomplish catabolism of particular substances.

Syntrophs

Acetoin

The Voges-Proskauer test determines the ability of microorganisms to produce which metabolic end-product?

Peptone, Sodium thiosulfate & Ferrous sulfate

The ability of SIM agar ( Sulfide indole motility medium) to test for hydrogen sulfide production is based on the addition of whichof the following substrates?

Citrate permease facilitates the transport of citrate into the cell.

The ability to utilize citrate as a carbon source depends on the presence of citrate permease. What is the role of this enzyme in organisms that test positive for citrate utilization?

The summer mud would have more bacteria than the winter mud.

You have samples of soil/mud from summer and from winter. Which of the following describes a relationship you would expect to see?

Test organism A ferments lactose Test organism A produced gas Test organism B does not ferment lactose Test organism A lowered the pH of the medium

A Lactose Fermentation test was performed on two different bacterial samples, A and B. Based on your observations what can you conclude?

The organism tested is protected against oxygen radicals. The image shows a positive test result. The organism tested is probably an aerobe or a facultative anaerobe.

A Catalase test was done on an unknown specimen. Observe the pictured result and answer the following question. Select ALL appropriate statements regarding the pictured Catalase test result.

yellow

A positive reaction for carbohydrate fermentation is indicated by the production of acidic wastes, which turn the phenol red pH indicator _____.

he cap should be loose when incubating this test to allow for gases to escape The bacterium is unable to ferment glucose The bacterium is unable to ferment lactose and/or sucrose

A specimen was inoculated onto the Triple Sugar Iron (TSI) medium pictured here

The pH of the agar decreased following incubation Hydrogen sulfide (H2S) was produced. The bacterium is able to ferment glucose

A specimen was inoculated onto the Triple Sugar Iron (TSI) medium pictured here...

This is a picture of a positive test result This media is inoculated by stabbing the agar in the tube The organism tested converts tryptophan to indole

An unknown specimen was grown on Indole media. Based on your observation of this specimen choose the appropriate statement(s)

This test is a part of the IMViC panel of tests This media is inoculated by stabbing the agar in the tube This is a picture of a negative test result

An unknown specimen was grown on Indole media. Based on your observation of this specimen choose the appropriate statement(s)

The test organism fermented glucose. The purple surface indicates that there is likely some decarboxylase activity. Andy forgot to add a layer of mineral oil to his sample so the surface of the medium became alkaline from air exposure.

Andy performed an Ornithine Decarboxylase test on his unknown sample. The following image shows what the tube looked like following 24 hours of incubation at 35 ± 2°C. What information can Andy get from this test?

Blue

As citrate is metabolized, carbon dioxide and pyruvic acid are formed. The carbon dioxide combines with sodium and water to form sodium carbonate, which turns the medium _____.

Litmus

As oxidation reactions occur during the process of fermentation, which litmus milk substrate acts as an electron acceptor (getting reduced in the process)?

D. Organisms that use inorganic carbon dioxide (CO2) as a carbon source - regardless of energy source.

Autotrophs

The butt of the medium is red. The bacteria growing on the slant is yellow. The slant of the medium is red

Based on the pictured TSI (Triple Sugar Iron) medium, select ALL appropriate observations.(Recall that an observation and a conclusion are different. An observation is simply what you see; conclusions are statements of what you determine based on your observations.)

To conduct this test the slant must be streaked The bacterium is able to ferment lactose and/or sucrose The bacterium is able to ferment glucose To conduct this test the butt of the tube should be stabbed

Based on your observation of the TSI (Triple Sugar Iron) medium pictured, select ALL appropriate statements

The tested bacterium does not produce urease

Based on your observation of the pictured Urea broth what can you conclude? Based on your observation of the pictured Urease test, select all appropriate conclusions . (Recall that an observation and a conclusion are different. An observation is simply what you see; conclusions are statements of what you learned from your observations.)

The test organism does not produce urease. The media was not inoculated. The media was not incubated long enough. The media was incorrectly prepared. The media was incubated at the incorrect temperature.

Based on your observation of the pictured Urea broth what could have led to the result?

This test detects a fermentation reaction The test contains phenol red which turns yellow under acidic conditions This media is inoculated using an inoculating loop The mouth of the tube should be flamed prior to inoculation and immediately before capping the tube

Below is a Glucose Fermentation test.

The mouth of the tube should be flamed prior to inoculation and immediately before capping the tube An organism must be able to use utilize ammonium dihydrogen phosphate as its sole nitrogen source and sodium citrate as its sole carbon source in order to grow on this medium This media is inoculated by streaking the surface of the slant

Below is a Simmons Citrate slant.

Organism that use electrons from inorganic molecules as an energy source and carbon dioxide (CO2) as their carbon source.

Chemolithotrophs

Organisms that use inorganic or organic chemical reactions (other than fermentation) as energy sources and their source of carbon.

Chemotrophs

Organisms that use pre-formed organic carbon as their energy and carbon sources.

Heterotrophs

Kelsey did not address the motility of the organism As evidenced by the lack of an aqueous layer on the top of the medium, Kelsey forgot to add Kovacs' reagent. As such, she could not truly make a conclusion about whether or not indole was produced

Kelsey inoculated her unknown bacterial sample onto SIM agar and obtained the pictured result. She described her sample as indole negative and hydrogen sulfide (H2S) positive. Unfortunately, Kelsey lost points for her description of her SIM test results. Curious by nature, she turned to you to ask if you could help her determine why she did not earn full credit for her work. Based on your observation of Kelsey's SIM test, select the reason(s) she lost points.

Red; yellow

Methyl red is a pH indicator that turns _____ at a pH of 4, and _____ at a pH of 6.

Organisms that use inorganic compounds for energy and organic compounds as carbon sources.

Mixotrophs

Basic

The citrate utilization test uses a bromthymol blue indicator to detect a pH shift due to the presence of sodium carbonate, which is an _____ end-product.

Tryptophanase

The indole test indicates whether or not bacteria have which enzyme?

E.aerogenes

The inoculation and incubation of which of the following organisms results in a negative methyl red test?

E.coli and E.aerogenes

The methyl red test is particularly useful to separate which two common enteric organisms?

Tryptophan

The test for indole production determines the ability of microorganisms to degrade which of the following essential amino acids?

P.vulgaris

The urease test is especially helpful in the identification of which bacterial species?

Specimen B had a positive result for the VP test Specimen B is fermented glucose and formed neutral end-products

Two different bacterial samples, A and B, were analyzed with the Voges-Poskauer (VP) test. The results are pictured here.

Specimen A fermented glucose. Specimen B fermented glucose. Specimen B converted the amino acid ornithine to the amine putrescine.

Two different bacterial samples, A and B, were grown in Ornithine Decarboxylase media. The following image shows what the tubes looked like following 72 hours of incubation at 35 ± 2°C.

Specimen B did not produce gelatinase Specimen A produced gelatinase Specimen A hydrolyzed gelatin

Two different bacterial samples, A and B, were grown on Gelatin deeps. This is a picture of the test results.

The media was prepared incorrectly The person conducting the test forgot to add Methyl Red to tube A he test was conducted less than 24 hours following inoculation Specimen A fermented glucose and formed low levels of acid end-products

Two different bacterial samples, A and B, were inoculated into MR-VP (Methyl Red + Voges Proskauer) broth. The following image shows what the tubes looked like for the Methyl Red portion of the test.

During prolonged incubation periods, the carbohydrate supply is exhausted and many bacteria will begin growing oxidatively on the peptones in the broth, altering the color of the pH indicator.

Why is it necessary to record the results of carbohydrate fermentation tests no later than 48 hours after inoculation?

Specimen A fermented glucose and formed low levels of acid end-products Specimen B fermented glucose and formed high levels of acid end-products Specimen B can likely convert glucose to lactate, formate, or acetate

Two different bacterial samples, A and B, were inoculated into MRVP (Methyl Red Voges Proskauer) broth. The following image shows what the tubes looked like for the Methyl Red portion of the test.

Specimen A exhibits beta hemolysis Specimen B exhibits gamma hemolysis

Two different bacterial samples, A and B, were inoculated onto Blood agar. What conclusions can you make?

Specimen A is motile Specimen A produced indole Specimen B did not produce hydrogen sulfide (H2S) Specimen B did not produce indole

Two different bacterial samples, A and B, were inoculated onto SIM media. What can you conclude based on your observations?

Specimen B cannot use sodium citrate as a carbon source Specimen A raised the pH of the medium as a result of its metabolic processes Specimen A can use sodium citrate as a carbon source

Two different bacterial samples, A and B, were inoculated onto Simmons Citrate agar. What conclusions can you make? Based on your observation of the pictured citrate media, select ALL appropriate conclusions.

Specimen B most likely has a respiratory chain that uses oxygen as a final electron acceptor. Specimen A shows a negative result Specimen B shows a positive result

Two different bacterial samples, A and B, were tested with an Oxidase dry slide. This is a picture of the test results.

Specimen B produces cytochrome oxidase. Specimen A does not produce cytochrome oxidase.

Two different bacterial samples, A and B, were tested with an Oxidase dry slide. This is a picture of the test results.

Bacterium A converted urea to ammonia Bacterium B decreased the pH of the medium Bacterium B does not produce urease Bacterium A increased the pH of the medium

Two specimens, A and B, were inoculated into Urea broth. What can you conclude from your observations?

Ammonia

What alkaline product is produced when urea is broken down?

The test organism did not produce gas The test organism is unable to ferment glucose

What can you conclude based on your observation of the pictured Glucose (dextrose) Fermentation test?

This media is inoculated by streaking the surface of the slant The test organism can use sodium citrate as its sole carbon source The test organism can use ammonium dihydrogen phosphate as its sole nitrogen source The test organism raised the pH of the medium

What can you conclude based on your observation of the pictured Simmons Citrate test?

The test organism ferments sucrose The test organism produced gas The test organism lowered the pH of the medium

What can you conclude based on your observation of the pictured Sucrose Fermentation test?

Deep pink

What color does urease broth turn when inoculated with a microorganism positive for the urease enzyme?

Yellow

What color does urease broth turn when inoculated with a urease negative microorganism?

Indole Methyl red Voges-Proskauer Citrate

What does IMViC stand for?

Urease is a hydrolytic enzyme that attacks the nitrogen and carbon bond in urea.

What is the function of the enzyme urease?

Hydrolytic enzyme

What type of molecule is urease?

bromthymol blue

When bacteria break down the citrate in Simmons citrate agar, the medium is alkalized because of the release of ammonia, turning the media blue. The pH indicator used in this reaction is _____.

Ammonia

When the milk protein casein is broken down into its component amino acids, the formation of which end-product produces an alkaline medium?

Indole, pyruvic acid, and ammonia

When the substrate tryptophan is broken down by trytophanase, what products are formed?

pH=9

When urea is broken down, ammonia is produced. Which pH is likely to result from ammonia production?

Loop inoculation

Which inoculation technique is used when testing for urease?

E.aerogenes; rose color change after the addition of Barritt's reagents.

Which of the following associations is correct regarding the Voges-Proskauer test?

You isolated microbes from all of your samples.

Which of the following best explains an experiment in which growth occurred on all of your agar plates?

Gram-negative, non-spore-forming, bacilli

Which of the following correctly describes the cellular morphology for members of the Enterobacteriaceae family?

E.coli and P.vulgaris

Which of the following cultures are indole positive?

E.aerogenes and K.pneumoniae

Which of the following enteric organisms produce acetoin?

Thiosulfate reductase

Which of the following enzymes is critical for the reduction of an inorganic sulfur compounds into sulfite and hydrogen sulfide gas?

All the answers are correct

Which of the following is NOT a reason why sterile technique is important in today's experiments?

The presence of a gas bubble in the inverted Durham tube following incubation indicates the production of acetic acid.

Which of the following is an INCORRECT statement regarding carbohydrate fermentation testing?

L.lactis and E.coli

Which of the following microorganisms is capable of fermenting lactose with the production of lactic acid?

E.aerogenes and K.pneumoniae

Which of the following microorganisms test positive for citrate metabolism?

The formation of tracks or fissures in the solid curds

Which of the following observations indicates that gaseous endproducts have been formed during litmus milk reactions?

A color change to pink

Which of the following observations is indicative of the fermentation of lactose into lactic acid in a litmus milk reaction?

B

Which of the following organisms are capable of lactose fermentation with production of acid?

Staphylococcus aureus and Escherichia coli

Which of the following organisms are capable of lactose fermentation with production of acid?

P. vulgaris & S. typhimurium

Which of the following organisms are positive for hydrogen sulfide production?

The molds grow much more slowly than bacteria

Why must the mold plates be incubated for so much longer than the nutrient agar plates?

L.lactis and E.coli

Which of the following organisms is capable of acid curd formation in litmus milk media?

All three: E. aerogenes, P. vulgaris & S. typhimurium

Which of the following organisms is flagellated and capable of motility?

Upon addition of potassium hydroxide and alphanaphthol, the upper part of the media turns red = positive test for acetoin production

Which of the following represents a correct association regarding the methyl red Voges-Proskauer test?

P.vulgaris and K.pneumoniae

Which of the following species is able to hydrolyze urea?

This nitrogen containing compound is found in the urine of mammals and is widely used in fertilizer.

Which of the following statements best describes urea?

Actinomycetes

Which of the following will grow best on the generic yeast extract plates used in college microbiology labs?

Phenol red

Which pH indicator is used to indicate ammonia production in urea broth?

acid; rennet

While ___________ curds are hard and do not retract from the walls of the test tube, ____________ curds are semisolid and will flow slowly when the test tube is tilted.

Lipase-producing organisms are mesophiles that tolerate a narrow range around room temperatures.

Why are the plates in a lipase assay incubated at room temperature (25°C)?