Table of Contents
- Introduction to Hydrogen Sulfide (H2S) Test
- Objectives of Hydrogen Sulfide (H2S) Test
- Principle of Hydrogen Sulfide (H2S) Test
- Requirements for Hydrogen Sulfide (H2S) Test
- Procedure of Hydrogen Sulfide (H2S) Test
- Result and Interpretation of Hydrogen Sulfide (H2S) Test
- Quality Control
- Precautions
- Applications of Hydrogen Sulfide (H2S) Test
- Limitations of Hydrogen Sulfide (H2S) Test
Introduction to Hydrogen Sulfide (H2S) Test
- The Hydrogen Sulfide (H2S) Test is a biochemical method used to determine the ability of bacteria to produce H2S gas.
- Bacteria and archaea can metabolically reduce sulfur-containing compounds to hydrogen sulfide (H2S) and derive energy in the process.
- These sulfur-containing compounds may be:
- Inorganic compounds such as sulfate, sulfite, thiosulfate, or tetrathionates
- Organic sulfur-containing compounds such as sulfur amino acids and proteins
- Elemental sulfur itself
- Sulfur from these compounds is enzymatically reduced and liberated as hydrogen sulfide (H2S) gas.
- A wide range of aerobic, facultative, and anaerobic bacterial species can produce H2S gas, collectively known as hydrogen sulfide-producing bacteria.
- Various chemolithotrophs, autotrophs, and heterotrophs in the environment can:
- Use sulfur compounds to produce metabolic energy
- Decompose organic compounds
- Recycle inorganic compounds
- Several commensal and pathogenic bacterial species can also produce H2S.
- Detecting the ability of bacteria to produce H2S gas is an important biochemical test used in microbiology and diagnostic labs for identifying unknown bacteria.
- The H2S test is crucial for:
- Detecting coliforms in water
- Identifying fecal pathogens
- Differentiating enteric pathogenic bacteria
- Characterizing other pathogenic or non-pathogenic bacterial isolates
- H2S gas is a colorless, toxic, corrosive, and flammable gas with a characteristic rotten egg-like smell.
- Microbial production of H2S is detected by its reaction with ferric ions or lead acetate to produce black-colored ferrous sulfide or lead sulfide, respectively.
- Sodium thiosulfate is mainly used as a sulfur source in the H2S test.
- Some media also contain peptones, cysteine, and sulfites as sulfur sources.
- Indicators used to detect H2S gas production in H2S detecting media include:
- Ferrous sulfate
- Ferric ammonium citrate
- Ferric citrate
- Peptonized iron
- Lead acetate
- Lead acetate is the most sensitive indicator and can detect even trace amounts of H2S.
- However, since most common pathogenic bacteria produce abundant H2S gas, ferrous sulfate is widely used as it is:
- Cheaper
- Less toxic
- Easier to use, though it is less sensitive than lead acetate.
Objectives of Hydrogen Sulfide (H2S) Test
- To detect the ability of bacteria to produce H2S gas.
- To presumptively identify bacterial isolates.
Principle of Hydrogen Sulfide (H2S) Test
- H2S-producing bacteria can metabolize sulfur-containing compounds (mostly sodium thiosulfate) present in the culture media and reduce them, releasing H2S gas.
- The produced H2S gas reacts with ferric ions or lead acetate, forming water-insoluble black-colored ferrous sulfide or lead sulfide, respectively.
- This insoluble black-colored compound turns the culture media black, indicating a positive result for the H2S production test (or H2S production).
Requirements for Hydrogen Sulfide (H2S) Test
a. Culture Media Used for H2S Test
- A wide variety of culture media are available for the detection of hydrogen sulfide production.
- For tube H2S tests, the following media are commonly used:
- SIM (Sulfide Indole Motility) medium
- KIA (Kligler’s Iron Agar)
- TSI (Triple Sugar Iron) Agar Medium
- Lead Acetate (LA) Agar
- For the petri plate method, the following media are commonly used:
- SS (Salmonella-Shigella) Agar
- DCA (Deoxycholate Citrate Agar)
- BS (Bismuth Sulfite) medium
- XLD (Xylose-Lysine Deoxycholate) agar medium
- HE (Hektoen Enteric) Agar
- Nutrient broth, peptone water, tryptic soy broth, or other suitable broth media are used to inoculate bacteria for performing the lead acetate paper method.
- In this article, we will use SIM agar medium. It is a general-purpose medium and is suitable for the detection of H2S production, indole production, and motility of organisms.
Composition of SIM Medium per 1000 mL:
- HM Peptone B (Beef Extract): 3.00 grams
- Peptone: 30.0 grams
- Peptonized Iron: 0.020 grams
- Sodium Thiosulfate: 0.025 grams
- Agar: 3.00 grams
- Final pH: 7.3 ± 0.2 at 25°C
Preparation of SIM Medium
- Measure the appropriate amount of SIM agar powder (or the media components) and mix with the required volume of water in a conical flask (or glass bottle) according to the manufacturer's instructions (36.23 grams per 1000 mL in the above medium).
- Stir well using a magnetic stirrer or manually and heat to boiling so that all components and agar dissolve completely in the water.
- Dispense about 5 mL of the medium into each test tube and loosely place a cap or cotton plug on the opening.
- Autoclave the tubes at 121°C and 15 lbs pressure for 15 minutes.
- Let the medium cool and solidify in an upright position, forming a butt only.
- For the plate method, we will use Hektoen Enteric (HE) agar medium. The plate method is generally used for the isolation of fecal pathogens and enteric bacteria from food, beverage, and water samples.
- HE agar is suitable for almost all enteric pathogenic and non-pathogenic organisms, covering nearly all Enterobacteriaceae members.
- In clinical labs, DCA (Deoxycholate Citrate Agar) and SS (Salmonella-Shigella) agar are preferred over HE agar when the objective is to isolate and differentiate Salmonella spp. and Shigella spp.
- However, DCA and SS agar inhibit several other Enterobacteriaceae, which is why we are using HE agar medium.
Composition of Hektoen Enteric (HE) Agar Medium per 1000 mL:
- Proteose Peptone: 12.0 grams
- Yeast Extract: 3.00 grams
- Lactose: 12.0 grams
- Sucrose: 12.0 grams
- Salicin: 2.00 grams
- Bile Salt Mixtures: 9.00 grams
- Sodium Chloride: 5.00 grams
- Sodium Thiosulfate: 5.00 grams
- Ferric Ammonium Citrate: 1.50 grams
- Acid Fuchsin: 0.10 grams
- Bromothymol Blue: 0.065 grams
- Agar: 15.0 grams
- Final pH: 7.5 ± 0.2 at 25°C
Preparation of HE Agar
- Measure the appropriate amount of HE agar powder (or the media components) and mix with sterile distilled water of the required volume in a sterile conical flask (or glass bottle) according to the manufacturer's instructions (76.67 grams per 1000 mL in the above media).
- Stir well using a sterile magnetic stirrer or manually and heat to boiling so that all components and agar dissolve completely in water.
- DO NOT AUTOCLAVE THE MEDIUM.
- Cool the medium to around 40 – 45°C.
- Dispense the molten medium into sterile petri plates, approximately 25 mL in a 10 cm diameter petri plate.
- Allow the medium to cool and solidify at room temperature.
For performing the lead acetate paper test, we will be using Nutrient broth.
Composition of Nutrient Broth per 1000 mL:
- Peptone: 5.00 grams
- Yeast Extract: 1.50 grams
- Beef Extract (HM Peptone B): 1.50 grams
- Sodium Chloride: 5.00 grams
- Final pH: 7.4 ± 0.2 at 25°C
Preparation of Nutrient Broth
- Measure the appropriate amount of Nutrient broth powder (or the media components) and mix with water of the required volume in a conical flask (or glass bottle) according to the manufacturer's instructions (13.00 grams per 1000 mL in the above media).
- Stir well using a magnetic stirrer or manually and heat to boiling so that all components dissolve completely in water.
- Dispense about 5 mL of the medium into each test tube and loosely place a cap or cotton plug on the opening.
- Autoclave the tubes at 121°C and 15 lbs pressure for 15 minutes.
b. Reagents
- Lead acetate paper, utilized for the lead acetate paper method, is an essential component.
c. Equipment
- Petri Plates
- Test tubes
- Weighing Machine
- Inoculating wire
- Bunsen burner
- Incubator
- Inoculating loop
- Autoclave
- PPE and other general laboratory materials
d. Test Organism (Sample Bacteria)
- Positive control: Proteus mirabilis ATCC 29906
- Negative control: Shigella flexneri ATCC 12022
Procedure of Hydrogen Sulfide (H2S) Test
Tube Method for H2S Test
- The most commonly followed method for detecting H2S production involves several steps.
- Using a sterile inoculating wire, touch a well-isolated colony from a fresh culture (18 to 24 hours old) of the test bacterium.
- Inoculate the SIM medium tube by stabbing the medium more than halfway (up to 3 to 5 mm above the base of the tube) with the inoculating wire.
- (When inoculating TSI, KIA, or other media with slant and butt, stab the butt and streak the slant surface using the inoculating wire.)
- Incubate the tube aerobically (with a loose cap) at 35±22°C for about 24 hours. (If suspected of Campylobacter, incubate for 72 hours.)
- Examine the formation of a black-colored precipitate in the medium, turning the medium black.
Plate Method for H2S Test
- Using a sterile inoculating loop, touch a well-isolated colony from a fresh culture of the test bacterium.
- Streak the culture over the agar plate to obtain well-isolated colonies.
- Incubate aerobically at 35±22°C for about 24 hours.
- Observe the color of the developed colonies.
Lead Acetate Paper Method for H2S Test
- Using a sterile inoculating loop, touch a well-isolated colony from a fresh culture of the test bacterium and inoculate the nutrient broth tube.
- Place a lead acetate paper strip so that it hangs with one end just above the medium and the other end stuck at the neck of the tube by the screw cap or cotton plug.
- Incubate the tube aerobically at 35±22°C and observe for blackening of the paper strip after 24 hours. (For slow growers and Campylobacter, incubate for at least 3 days and observe for color change daily.)
Result and Interpretation of Hydrogen Sulfide (H2S) Test
Positive H2S Test:
- Tube Method: Blackening of the culture media. (The entire media may turn black or the junction of slant and butt or any part of butt or slant may turn black. A black patch at any inoculated area is considered positive.)
- Plate Method: Presence of black colonies and/or colorless or colored colonies with a black center.
- Lead Acetate Paper Method: Blackening (brownish-black color formation) of the lead acetate paper.
Negative H2S Test:
- Tube Method: No blackening of the media in any area.
- Plate Method: Absence of black-colored colonies and/or absence of black-centered colonies.
- Lead Acetate Paper Method: No change in color of the lead acetate paper strip.
Quality Control
- Proteus mirabilis ATCC 29906 will turn the medium/lead acetate paper black (and produce black colonies in the plate method).
- Shigella flexneri ATCC 12022 will not turn/produce black color in the test tube, agar plate, and/or lead acetate paper.
H2S Test Positive Bacteria:
- Proteus spp., Citrobacter spp., Salmonella spp., Staphylococcus saprophyticus, Campylobacter spp., etc.
H2S Test Negative Bacteria:
- Klebsiella pneumoniae, Shigella spp., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Vibrio cholerae, Yersinia pestis, etc.
Precautions
- Use the appropriate medium; avoid DCA, SS agar, and XLD medium if testing for Enterobacterales other than Salmonella and Shigella.
- Before inoculation, check for cracking of the medium in test tubes; do not use if the medium is cracked.
- Avoid using an inoculating loop for stabbing tubes.
- Do not tightly cap the test tubes while incubating.
- Do not autoclave HE, DCA, and BS medium; carefully follow instructions before preparing the medium.
- While preparing a medium that must not be autoclaved, use sterile equipment and work in a sterile zone.
- When streaking a plate, use a small amount of inoculum and ensure there is some distance between streaks to obtain well-isolated colonies.
- When using lead acetate paper, ensure it does not touch the medium; hang it slightly above the medium to prevent inhibition of bacterial growth.
- If the test organism is a sucrose fermenter, avoid using a sucrose-containing medium.
Applications of Hydrogen Sulfide (H2S) Test
- For the presumptive identification of bacteria.
- Differentiation of Salmonella spp. (H2S test positive) from Shigella spp. (H2S test negative).
- Characterizing and identifying Enterobacterales.
- Performing fecal culture and characterization of fecal pathogens.
- Rapid fecal coliform detection in water.
- Separation of Lactobacilli (H2S test negative) from Erysipelothrix spp. (H2S test positive).
Limitations of Hydrogen Sulfide (H2S) Test
- The H2S test is not a confirmatory test; therefore, other biochemical test results are necessary for complete identification of the bacterium.
- Sucrose in the medium may inhibit H2S production.
- Some fastidious organisms may not grow in SIM, NB, DCA, and other basic mediums.
- Lead acetate is the most sensitive in detecting H2S but is toxic to bacteria.
- Some bacteria may require long incubation periods, such as 3 days for Campylobacter spp., and even longer for other slow-growing bacteria.
- There may be confusion while choosing media for the test.
