Proteins, Amino Acids, and Enzymes I: Hydrogen Sulfide Production and Motility

Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



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24. Proteins, Amino Acids,

& Enzymes I: Hydrogen

Sulfide Production &

Motility



Figure 24.1 Hydrogen Sulfide Production. It should be noted that not all bacteria are either H2S positive with motility or H2S

negative and no motility. Many other possible combinations exist.



Biochemistry within bacteria

SH



CH2

H2O + H2N



H



C



CH3

cysteine

desulfurase



COOH



2–



2S2O3 + 4H



Pyruvic acid



+



thiosulfate

reductase



+



NH3 +



H2S



Ammonia Hydrogen

sulfide gas



2–



2SO3 + 2H2S

Sulfite



Thiosulfate



(b)



O



COOH



Cysteine



(a)



C



Hydrogen

sulfide gas



Biochemistry within tubes

Fe (NH4 )2SO4 + Bacterial acids

H2S + Fe



3+



No H2S + Fe



3+



SIM

medium

Black

precipitate



No black

precipitate



Growth not

restricted to stab line



Growth restricted

to stab line



H2S (+) plus motility



(c)



148



Biochemical Activities of Bacteria



H2S (–) and no motility



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



24. Proteins, Amino Acids,

& Enzymes I: Hydrogen

Sulfide Production &

Motility



In this exercise, the SIM medium (named after

J. S. Simmons in 1926) contains peptones and sodium

thiosulfate as substrates, and ferrous ammonium sulfate, Fe(NH4)SO4, as the H2S indicator. Cysteine is a

component of the peptones used in SIM medium. Sufficient agar is present to make the medium semisolid.

Once H2S is produced, it combines with the ferrous

ammonium sulfate, forming an insoluble, black ferrous

sulfide precipitate that can be seen along the line of the

stab inoculation. If the organism is also motile, the entire tube may turn black. This black line or tube indicates a positive H2S reaction; absence of a black precipitate indicates a negative reaction (figure 24.1c).

SIM agar may also be used to detect the presence

or absence of motility in bacteria as well as indole

production. (See exercise 25 for a discussion of indole

production.) Motility is present when the growth of

the culture is not restricted to the stab line of the inoculation. Growth of nonmotile bacteria is confined to

the line of inoculation.

One can also use semisolid media (motility test

medium deeps) to determine whether a bacterial strain

is motile. During growth, motile bacteria will migrate

from the line of inoculation to form a dense turbidity

in the surrounding medium; nonmotile bacteria will

grow only along the line of the inoculation.



Procedure

First Period

1. Label each of the SIM agar deep tubes with the

name of the bacterium to be inoculated, your

name, and date.

2. Using aseptic technique (see figure 14.3),

inoculate each tube with the appropriate



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bacterium by stabbing the medium f of the way

to the bottom of the tube. Do the same for the

three motility test medium deeps.

3. Incubate the cultures for 24 to 48 hours at 35°C.



Second Period

1. Examine the SIM cultures for the presence or

absence of a black precipitate along the line of the

stab inoculation. A black precipitate of FeS

indicates the presence of H2S.

2. Based on your observations, determine and record

in the report for exercise 27 whether or not each

bacterium was capable of H2S production, and the

presence (+) or absence (–) of motility.

3. If desired, one can also test for indole production

by adding 5 drops of Kovacs’ (named after the

German bacteriologist, Nikolaus Kovacs, in the

early 1900s) reagent to the SIM cultures and

looking for the development of a red color at the

top of the deeps (see exercise 25).



HINTS AND PRECAUTIONS

(1) Be careful when inoculating the deeps to withdraw

the needle from the agar in a line as close as possible to

the line used when entering the agar. (2) Another aid in

visualizing motility is to slowly rotate questionable tubes

containing small amounts of growth around the stab line.

When this is done, the growth appears much wider on

the two opposite sides and narrower on the other two

sides on which the bacteria is not motile. (3) To observe

motility, make sure the outside of your tubes are clean

by wiping them with a Kimwipe. (4) Any blackening of

the medium is considered a positive test for H2S.



Proteins, Amino Acids, and Enzymes I: Hydrogen Sulfide Production and Motility



149



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Laboratory Report



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Companies, 2002



24. Proteins, Amino Acids,

& Enzymes I: Hydrogen

Sulfide Production &

Motility



24



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Proteins, Amino Acids, and Enzymes I:

Hydrogen Sulfide Production and Motility

1. Complete the following table on hydrogen sulfide production and motility.

Motility (+) or (–)

Bacterium



H2S Production (+) or (–)



SIM



Motility Test Medium



K. pneumoniae



________________________



____________



____________



S. typhimurium



________________________



____________



____________



P. vulgaris



________________________



____________



____________



2. Sketch and describe what is happening in each tube with respect to H2S production and motility.



K. pneumoniae



S. typhimurium



P. vulgaris



151



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



24. Proteins, Amino Acids,

& Enzymes I: Hydrogen

Sulfide Production &

Motility



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Companies, 2002



Review Questions

1. Of what use to bacteria is the ability to produce H2S?



2. How is SIM medium used to detect motility?



3. What substrates are acted on in SIM medium in order for H2S to be produced?



4. In addition to H2S production and motility, for what other test can SIM medium be used?



5. How does a black precipitate of FeS indicate the production of H2S?



6. What does cysteine desulfurase catalyze? Show the reaction.



7. What does thiosulfate reductase catalyze? Show the reaction.



152



Biochemical Activities of Bacteria



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



25. Proteins, Amino Acids,

and Enzymes II: The IMViC

Tests



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25



E X E RC I S E

Proteins,Amino Acids, and Enzymes II:

The IMViC Tests

SAFETY CONSIDERATIONS

Be careful with the Bunsen burner flame. No mouth

pipetting. Barritt’s reagent contains naphthol, which is

toxic and may cause peeling of the skin; thus, wear

gloves when using this reagent. Kovacs’ reagent is also

caustic to the skin and mucous membranes due to the

concentrated HCl and p-dimethylaminobenzaldehyde.

In case of contact with either reagent, immediately flush

eyes or skin with plenty of water for at least 15 minutes.

Keep all culture tubes upright in a test-tube rack or can.



Materials per Student

24- to 48-hour tryptic soy broth cultures of

Enterobacter aerogenes (ATCC 13048),

Escherichia coli (ATCC 11229), Klebsiella

oxytoca (ATCC 13182), and Proteus vulgaris

(ATCC 13315)

4 SIM agar deep tubes

Kovacs’ reagent, KEY Indole Test Tablets, or

Difco’s SpotTest Indole Reagent Kovacs

Bunsen burner

inoculating loop and needle

4 MR-VP broth tubes each containing 5 ml of

medium

methyl red indicator

Barritt’s reagent (solutions A and B) or Difco’s

SpotTest Voges-Proskauer reagents A and B

4 Simmons citrate agar slants

4 empty test tubes

4-ml pipettes with pipettor

wax pencil

disposable gloves



Learning Objectives

Each student should be able to



2. Determine the ability of some bacteria to oxidize

glucose with the production of acid end products

3. Differentiate between glucose-fermenting enteric

bacteria

4. Explain the purpose of the Voges-Proskauer test

5. Differentiate among enteric bacteria on the basis

of their ability to ferment citrate

6. Perform the IMViC series of tests



Suggested Reading in Textbook

1. Catabolism of Carbohydrates and Intracellular

Reserve Polymers, section 9.7, see figure 9.10.

2. The Enterobacteriaceae, section 22.3, see

table 22.7.



Pronunciation Guide

Enterobacter aerogenes (en-ter-oh-BAK-ter a-RAHjen-eez)

Escherichia coli (esh-er-I-ke-a KOH-lee)

Klebsiella oxytoca (kleb-se-EL-lah ok-se-TO-se-ah)

Proteus vulgaris (PRO-te-us vul-GA-ris)

Salmonella (sal-mon-EL-ah)

Shigella (shi-GEL-la)

Enterobacteriaceae

Enterobacter aerogenes

Escherichia coli

Klebsiella oxytoca



Lactose

fermenters



Alcaligenes faecalis

Proteus vulgaris

Salmonella typhimurium

Shigella dysentariae



Lactose

nonfermenter



Some

enteric

(intestinal)

bacteria



1. Understand how some bacteria degrade the amino

acid tryptophan



153



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



25. Proteins, Amino Acids,

and Enzymes II: The IMViC

Tests



Why Are the Following Bacteria

Used in This Exercise?

In this exercise the student will learn how to perform the

IMViC series of tests that distinguish between different enteric (pertaining to the small intestine) bacteria. To illustrate

the various IMViC reactions, the authors have chosen four

enteric bacteria. Enterobacter aerogenes (Gr. aer, air) is a

facultatively anaerobic gram-negative rod that has peritrichous flagella. It is a motile lactose fermenter. E. aerogenes

is widely distributed in nature, occurring in fresh water, soil,

sewage, plants, vegetables, and animal and human feces. It is

indole negative, MR negative, VP positive, and Simmons citrate positive. Escherichia coli (Gr. colon, large intestine) is

a facultatively anaerobic gram-negative rod that is motile

with peritrichous flagella or nonmotile. It is a lactose fermenter. E. coli occurs as normal flora in the lower part of the

intestine of warm-blooded animals. It is indole positive, MR

positive, VP negative, and Simmons citrate negative. Klebsiella oxytoca is a facultatively anaerobic gram-negative rod.

It is nonmotile and a lactose fermenter. K. oxytoca occurs in

human feces and clinical specimens, soil, water, grain, fruits,

and vegetables. It is indole positive, often MR negative, VP

positive, and Simmons citrate positive. Proteus vulgaris (L.

vulgaris, common) is a gram-negative facultatively anaerobic rod that occurs in the intestines of humans and a wide variety of animals, in manure, and in polluted waters. It has

peritrichous flagella, is motile, and does not ferment lactose.

P. vulgaris is indole positive, MR positive, VP negative, and

sometimes Simmons citrate positive.



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found in the intestines of humans and other mammals

belong to the family Enterobacteriaceae. These bacteria are short, gram-negative, nonsporing bacilli. They

can be subdivided into lactose fermenters and nonfermenters. Examples include pathogens (Salmonella and

Shigella, lactose nonfermenters), occasional pathogens

(Klebsiella and Escherichia, lactose fermenters; and

Proteus, lactose nonfermenter), and normal intestinal

microbiota (Enterobacter, lactose fermenter).

The differentiation and identification of these enteric bacteria can be accomplished by using the

IMViC test (indole, methyl red, Voges-Proskauer,

and citrate; the “i” is for ease of pronunciation).



Indole Production

The amino acid tryptophan is found in nearly all proteins. Bacteria that contain the enzyme tryptophanase

can hydrolyze tryptophan to its metabolic products,

namely, indole, pyruvic acid, and ammonia. The bacteria use the pyruvic acid and ammonia to satisfy nutritional needs; indole is not used and accumulates in the

medium. The presence of indole can be detected by the

addition of Kovacs’ reagent. Kovacs’ reagent reacts

with the indole, producing a bright red compound on

the surface of the medium (figures 25.1, 25.2). Bacteria

producing a red layer following addition of Kovacs’

reagent are indole positive; the absence of a red color

indicates tryptophan was not hydrolyzed, and the bacteria are indole negative.



Methyl Red Test

Medical Application

The following medically important bacteria are MRϩ: Escherichia coli (opportunistic urinary tract infections), Salmonella typhi (typhoid fever), Shigella dysenteriae (bacterial dysentery), and Yersinia pestis (plague). The following

is MRϪ: Enterobacter aerogenes (urinary tract infections).

Bordetella pertussis (whooping cough) is citrate negative whereas all other Bordetella species are citrate positive. The enteric bacteria such as Klebsiella pneumoniae

(pneumonia) and Enterobacter are citrate positive and can

be distinguished in the clinical laboratory from the opportunistic pathogen Escherichia coli (urinary tract infections)

which is citrate negative.



Principles

The identification of enteric (intestinal) bacteria is of

prime importance in determining certain food-borne

and waterborne diseases. Many of the bacteria that are



154



Biochemical Activities of Bacteria



All enteric bacteria catabolize glucose for their energy

needs; however, the end products vary depending on

the enzyme pathways present in the bacteria. The pH

indicator methyl red (see appendix E) detects a pH

change to the acid range as a result of acidic end products such as lactic, acetic, and formic acids. This test is

of value in distinguishing between E. coli (a mixed

acid fermenter) and E. aerogenes (a butanediol fermenter). Mixed acid fermenters such as E. coli produce a mixture of fermentation acids and thus acidify

the medium. Butanediol fermenters such as E. aerogenes form butanediol, acetoin, and fewer organic

acids. The pH of the medium does not fall as low as

during mixed acid fermentation. As illustrated in figure 25.3, at a pH of 4, the methyl red indicator turns

red—a positive methyl red test. At a pH of 6, the indicator turns yellow—a negative methyl red test.



Voges-Proskauer Test

The Voges-Proskauer test (named after Daniel Voges,

German physician, and Bernhard Proskauer, German