Hydrogen Sulfide Test: Principle, Procedure And Results Interpretation

By Prof Daniel Asrat

Introduction

The objective of Hydrogen Sulphide Test is to determine whether the microbe reduces sulfur-containing compounds to sulfides to produce hydrogen sulfide gas.Some bacteria have the ability to reduce sulphur to hydrogen sulphide. It is a colorless gas, which reacts with iron (ferrous salts) to produce black precipitates of ferrous sulphide.It also reacts with lead acetate to produce black precipitates of lead sulphide.

Principle

An iron compound and a sulfur compound are included in the test medium to test for the production of hydrogen sulfide gas.  Hydrogen sulfide is produced if the sulfur compound is reduced by the bacterial strain. This test thus determines whether the microbe reduces sulfur-containing compounds to sulfides during the process of metabolism.  H₂S is produced by certain bacteria through reduction of sulphur containing amino acids like cystine,  methionine or through the reduction of inorganic sulphur compounds such as thiosulfates, sulfates or sulfites during protein degradation or when anaerobic respiration shuttles the electrons to sulfur instead of to oxygen.

In either case H₂S is produced (hydrogen sulfide gas) which reacts with the iron compound to form the black precipitate of ferric sulfide.  The black color acts as an indicator for the presence of hydrogen sulfide. The detection of hydrogen sulphide (H2S) gas produced by an organism. is used mainly to assist in the identification of that particular organism.

Hydrogen sulphide test (H, S test) can be done in two ways, based on the source of sulphur, that is:

1. H₂S test using inorganic source of sulphur
2. H₂S test using organic source of sulphur

Experiment 1

(i) H₂S Test Using Inorganic Source of Sulphur

In this test, the test bacteria is grown on triple sugar iron agar slants (TSI agar slants), which contain glucose, sucrose, lactose, phenol red, sodium thiosulphate and ferrous sulphate. If the bacteria utilises the inorganic sulphur (sodium thiosulphate) used in the medium, H₂S is produced, which combines with the ferrous sulphate in the medium to form black precipitates of ferrous sulphide resulting in a change in the colour of the butt to black.

Besides utilising inorganic sulphur, if the bacteria can utilise any of the three sugars (glucose, sucrose or lactose), acid is produced, which reduces the pH of the medium. As a result, the colour of the slant changes from red to yellow.

Reagents And Materials Required

• Test tubes
• Conical flask
• Cotton plugs
• Inoculating needle
• Autoclave
• Bunsen burner
• Laminar flow chamber
• Dispose jar
• Incubator
• Triple sugar iron agar (TSI agar) isolated colonies or pure cultures of bacteria

Procedure

1. The ingredients of TSI agar medium (containing the 3 sugars and iron as the main components) or its ready-made powder required for 100 ml of the medium is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling.
2. Its pH is determined using a pH paper or pH meter and adjusted to 7.4 using 0.1N HCI if it is more or using 0.1N NaOH if it is less.
3. The flask is heated to dissolve the agar in the medium completely.
4. Before it solidifies, the medium in warm molten condition is distributed into 5 test tubes (approximately 20 ml each).
5. The test tubes are cotton-plugged, covered with craft paper and tied with thread or rubber band.
6. They are sterilised at 121 °C (15 psi pressure) for 15 minutes in an autoclave.
7. After sterilisation, they are removed from the autoclave and kept in a slanting position to cool and solidify the medium, so as to get TSI agar slants.
8. The test bacteria is inoculated aseptically, preferably in a laminar flow chamber, into the slants by stabbing into the butt and streaking on the surface of the slants with the help of a flame- sterilised needle. The needle is sterilised after each inoculation.
9. The inoculated slants are incubated at 37°C for 24 hours in an incubator.

Observations

1. Colour of butt changes to black: H₂S positive.
2. Colour of butt does not change to black: H₂S negative.

Experiment 2

(ii) H₂S Test Using Organic Source of Sulphur

In this test, the test bacteria is grown in cysteine broth, which contains cysteine as the organic source of sulphur. If the bacteria utilizes the organic sulphur (cysteine) used in the medium with the help of its enzyme ‘cysteine desulphurase’, H₂S is produced. This H₂S combines with the lead acetate soaked in a paper to form black precipitates of lead sulphide resulting in a change in the color of the paper strip to black.

Reagents And Materials Required

• Test tubes
• Conical flask
• Cotton plugs
• Inoculating loop
• Autoclave
• Bunsen burner
• Laminar flow chamber
• Dispose jar
• Incubator
• Cysteine broth
• Whatman filter paper strips
• Lead acetate solution (saturated)
• Isolated colonies or pure cultures of bacteria

Procedure

1.  The ingredients of cysteine broth medium (containing cysteine as the main component) or its ready-made powder required for 100 ml of the broth is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling.
2.  Its pH is determined using a pH paper or pH meter and adjusted to 7.5 using 0.1N HCI if it is more or using 0.1N NaOH if it is less. The flask is heated, if required, to dissolve the ingredients completely.
3. The broth is distributed into five test tubes (approximately 10 ml each), cotton-plugged, covered with craft paper and tied with thread or rubber band.
4. The broth tubes are sterilized at 121°C (15 psi pressure) for 15 minutes in an autoclave.
5. The broth tubes are allowed to cool to room temperature.
6. The test bacteria is inoculated aseptically, preferably in a laminar flow chamber, into the broth with the help of an inoculating loop sterilized over bunsen flame. The loop is sterilized after each inoculation.
7. A small strip of paper soaked in lead acetate solution (saturated) is fixed at the mouth of each test tube in such a way that a long portion of it remains inside the test tube and a small portion remains outside.
8. The inoculated broth tubes are incubated at 37°C for 48 hours in an incubator.

Observations

Color of lead acetate strip changes to black: H₂S positive.

2. Color of lead acetate strip does not change to black: H₂S negative.