Microbes of the Body: The Enterics

Question 1

Enteric bacteria are phenotypically similar to several other genera of Gram-negative rod-shaped bacteria, such as Pseudomonas and Vibrio. However, how do these bacteria differ in terms of glucose?

Answer 1

Enteric bacteria are phenotypically similar to several other genera of Gram-negative rod-shaped bacteria, such as Pseudomonas and Vibrio. However, how do these bacteria differ in terms of glucose?

• Generally, a distinction can be made on the ability to ferment glucose; enteric bacteria all ferment glucose to acid end products while similar Gram-negative bacteria (e.g. Pseudomonas) cannot ferment glucose.

Question 2

Describe Coliforms

• Gram
• shape
• spore
• Oxygen sensitivity
• oxidase presence
• lactose fermentation

Answer 2

Describe Coliforms

• Gram
  
  • (-)
• shape
  
  • rod
• spore
  
  • no spores
• Oxygen sensitivity
  
  • facultative
• oxidase presence
  
  • oxidase negative
• lactose fermentation
  
  • ferment lactose

Question 3

True or False

All enterics are coliforms; however, not all coliforms are enterics.

Answer 3

False

All coliforms are enterics; however, not all enterics are coliforms.

Question 4

Where, besides the body, can coliforms reside?

Answer 4

C​oliforms can be colon-dwelling bacteria but can also be found in soil or on plants and decaying vegetation.

Question 5

What enzyme is specific to fecal coliforms?

Answer 5

β-glucuronidase.

Question 6

What does the enzyme oxidase do?

Answer 6

• Oxidase enzymes play a vital role in the electron transport system during aerobic respiration.
• Cytochrome oxidase catalyzes the oxidation of a reduced cytochrome by O2, resulting in the formation of usually H2O or sometimes H2O2.

Question 7

What kind of bacteria exhibit oxidase activity?

Answer 7

• Aerobic bacteria, as well as some facultative anerobes and microaerophiles, exhibit oxidase activity.

Question 8

What is the actual agent that detects cytochrome c oxidase in the oxidase test? How does it work?

Answer 8

• tetramethyl-p-phenylenediamine
  
  • The dye changes color upon oxidation.
  • It only reacts with cytochrome c oxidase. (Remeber, all bacteria have terminal electron acceptors and some may not have Cytochrome C.
  • Thus, only bacteria that use this particular protein will react with tetramethyl- p-phenylenediamine and cause a color change.

Question 9

Describe the following as oxidase negative or oxidase positive

• Enterobacteriaceae
• Pseudomonas

Answer 9

Describe the following as oxidase positive or oxidase negative

• Enterobacteriaceae
  
  • ​​oxidase-negative​
• Pseudomonas
  
  • ​​oxidase-positive​

Question 10

IMViC tests are used primarily for the differeniation between which two coliforms?

Answer 10

• IMViC tests are used primarily for the differeniation of the two coliforms Escherichia coli and Enterobacter aerogenes

Question 11

What does the indole test determine?

Answer 11

Tryptophan degradation

Question 12

What is Kovac’s reagent made of?

Answer 12

mixture of of p- dimethylaminobenzaldehyde, butanol and HCl.

Question 13

What does indole bond to in a positive indole test? What does it form?

Answer 13

• Indole is extracted from the medium (tryptone broth) into the reagent layer by the acidified alcohol component.
• This forms a cherry red complex with the p-dimethylaminobenzaldehyde.

Question 14

What bacteria species are differentiated with the indole test?

Answer 14

• E. coli
  
  • is indole-positive;
• Enterobacter aerogenes
  
  • indole negative.

Question 15

What is the most important ingredient in MRVP broth?

Answer 15

Glucose, bitch

Question 16

• When _____________(E. aerogenes/E. coli) is grown in glucose broth, mixed acids (lactic, formic and acetic) are formed until a pH of 4-5 is reached.
• In contrast, _____________(E. aerogenes /E. coli) ​ converts the organic acids into non-acidic end products such as 2,3-butanediol.

Answer 16

• When E. coli is grown in glucose broth, mixed acids (lactic, formic and acetic) are formed until a pH of 4-5 is reached.
• In contrast, E. aerogenes converts the organic acids into non-acidic end products such as 2,3-butanediol.

Question 17

The initial pH of the MRVP broth is approximately ____

Answer 17

The initial pH of the MRVP broth is approximately 6

Question 18

• The methyl red pH indicator turns red between pH 4.4 and 5, and remains yellow between pH 6 and 7.
  
  • A red color in the medium indicates a _______ MR test (mixed acid fermentation; E. coli).
  • A yellow color indicates a ______ MR test (E. aerogenes).

Answer 18

• The methyl red pH indicator turns red between pH 4.4 and 5, and remains yellow between pH 6 and 7.
  
  • A red color in the medium indicates a positive MR test (mixed acid fermentation; E. coli).
  • A yellow color indicates a negative MR test (E. aerogenes).

Question 19

In the Vogues-Proskauer Test, how is 2,3-butanediol fermentation detected?

Answer 19

The VP test detects the pathway intermediate, acetylmethylcarbinol (a.k.a. acetoin).

Question 20

What kind of biochemistry (chemicals, reactions, etc) occurs in the Vogues-Proskauer Test?

Answer 20

• Acetoin is oxidized by α-napthol (VP reagent A) to a diacetyl compound in the presence of KOH (VP reagent B) and O2.
• The diacetyl compound then reacts with a guanidine group that is present in the peptone of the MR-VP medium to form a pink complex.

Question 21

In the VP test, label each as acetoin positive or acetoin negative

• E. aerogenes yields a positive test (pink)
• E. coli is VP negative (yellow)

Question 22

In the absence of fermentable sucrose, glucose, or lactose, some enteric bacteria are capable of using ______ as a carbon source.

Answer 22

In the absence of fermentable glucose or lactose, some enteric bacteria are capable of using citrate as a carbon source.

Question 23

In citrate agar what is:

• the sole nitrogen source
• the sole carbon source
• the pH indicator

Answer 23

In citrate agar what is:

• the sole nitrogen source
  
  • ammonium
• the sole carbon source
  
  • citrate
• the pH indicator
  
  • bromthymol blue indicator
    
    • green at pH 6.8
    • blue at pH > 7.6.

Question 24

Out of E. coli and E. aerogenes, which utilizes citrate as a sole carbon source and grows on citrate medium, changing the color of the agar from green to deep “Prussian blue.”

Answer 24

E. aerogenes

Question 25

How does citrate enter cells, to be used as a carbon source?

Answer 25

The ability to use citrate as a carbon source depends on the presence of a citrate permease that facilitates the transport of citrate into the cell.

Question 26

What is triple sugar iron (TSI) used for?

Answer 26

Triple sugar iron (TSI) is a differential medium used for the identification of Gram- negative enteric pathogens

Question 27

What does TSI medium indicate?

Answer 27

• The medium indicates ability to
  
  • ferment lactose,
  • ferment sucrose
  • ferment glucose
  • produce acid and gas,
  • ability to produce H2S.
• These are the characteristics which are used to identify the Salmonella-Shigella group of organisms.

Question 28

How does TSI allow for the sole detection of glucose fermentation?

Answer 28

• TSI agar slants contain lactose and sucrose in 1% concentrations and glucose in a concentration of 0.1%,
  
  • The concentration difference allows detection of glucose fermentation alone.

Question 29

• What pH indicator is used in TSI?
• Describe what changes in color show

Answer 29

• The pH indicator phenol red is also incorporated in the medium.
• The color of phenol red changes gradually with pH.
  • From pH 6.6-7.4 it is yellow
  • From 7.5- 8.0, red.
  • Above pH 8.1, phenol red turns a bright pink color.

Question 30

How is carbohydrate fermentation indicated TSI agar?

Answer 30

Carbohydrate fermentation is indicated by a change in color of the medium from orange-red to yellow in the presence of acids.

Question 31

Interpret these TSI Results

• Alkaline slant (red) and acid butt (yellow) with or without gas production (breaks in the agar butt)

Answer 31

• Only glucose fermentation has occurred.
• The organisms preferentially degrade glucose first.
• Since glucose is present in minimal concentration, the small amount of acid produced on the slant surface is oxidized rapidly.
• The acid produced by glucose fermentation in the butt of the tube will be maintained because of the low oxygen tension and slower growth of the organisms.

Question 32

Interpret these TSI Results

• Acid slant (yellow) and acid butt (yellow) with or without gas production

Answer 32

• Lactose and/or sucrose fermentation has occurred.
• As these sugars are present in higher concentrations, they serve as substrates for continued fermentative activities and maintain an acid reaction in both slant and butt.

Question 33

Interpret these TSI Results

• Alkaline slant (red) and alkaline butt (red) or no change (orange-red)

Answer 33

Either

• No carbohydrate fermentation has occurred.
• OR
• Peptones in the medium may have been catabolized;
  
  • the pH may be alkaline due to the production of ammonia.

Question 34

TSI agar contains:

• sodium thiosulfate.
  
  • This is a substrate for ___ production
• ferrous sulfate
  
  • for detection of the colorless ___.
• Organisms capable of producing ___ will show an extensive blackening in the butt because of the precipitation of the insoluble _________.

Answer 34

TSI agar contains:

• sodium thiosulfate.
  
  • This is a substrate for H2S production
• ferrous sulfate
  
  • for detection of the colorless H2S.
• Organisms capable of producing H2S will show an extensive blackening in the butt because of the precipitation of the insoluble ferrous sulfide.

Question 35

Why must TSI agar slants be analyzed within 18 hours?

Answer 35

• It is absolutely essential to observe the cultures and record the reactions within 18 hours.
• Doing so will ensure that the carbohydrate substrates have not been depleted and that degradation of peptones yielding alkaline end products has not taken place.

Question 36

What is the swarming agar test used for?

Answer 36

This is a definitive test for the highly motile enteric bacterium, Proteus mirabilis.

Question 37

What does a positive test look like on swarming agar? A negative one?

Answer 37

Positive

• yields concentric circles in a series of rings on agar plates.

Negative

• no concentric circles in a series of rings on agar plates.

Question 38

What is the urease test used for?

Answer 38

This tests for the ability to hydrolyze urea with the enzyme urease, an ability seen in Proteus.

Question 39

Only Proteus genus can rapidly metabolize urea to ammonia with the urease enzyme. What does this look like in urea agar, and what indicator dye is used?

Answer 39

• The urea agar contains a pH indicator dye, phenol red.
• At pH 8.0 or above, bright pink color is seen. This is considered a positive test.
• Orange or yellow negative.

Question 40

You perform the methyl red test with E. coli and see a lighter (pinkish) color instead of red. What could be the reason for this?

Answer 40

• A red color in the broth indicates the presence of acid. This should be the case for E. coli.
• This color may be lighter (pinkish) if the culture was not very old and didn’t have a chance to ferment profusely. This means the older the culture, the redder the test.

Question 41

In the VP test, you accidentally add VP reagent B (KOH) before adding VP reagent A (α-napthol). What could happen as a result of this?

Answer 41

• You need to add VP reagent A (α-napthol) before adding VP reagent B (KOH).
• Reversing the order of reagents may result in a false negative result.