Table of Content:
- Introduction
- Objectives
- Principle
- Requirements
- Procedure
- Result and Interpretation
- Quality Control
- Sulfur Reduction Test Results of Some Common Bacteria
- Precautions
- Applications
- Limitations
Introduction:
The Sulfur Reduction Test, also known as the Hydrogen Sulfide Test, is based on the metabolic ability of certain microorganisms to reduce elemental sulfur and sulfur-containing compounds for energy production. Microorganisms capable of this process are termed sulfur-reducing microorganisms and include various Archaea as well as aerobic, facultative, and anaerobic bacteria. The absence or presence of sulfur reduction ability serves as a valuable criterion for differentiating between bacterial species.
During the test, microorganisms metabolically reduce sulfur to produce hydrogen sulfide (H2S) gas as a byproduct. This gas is detected using an indicator, making the test sensitive to sulfur reduction. Widely employed in medical and non-medical microbiology laboratories, the Sulfur Reduction Test is crucial for diagnostic purposes. It plays a key role in detecting coliforms in water, identifying fecal pathogens, distinguishing enteric pathogenic bacteria, and characterizing various bacterial isolates, whether pathogenic or non-pathogenic.
Objectives of Sulfur Reduction Test
- Evaluate the capacity of bacteria in reducing sulfur compounds.
- Identify the bacterial capability to generate hydrogen sulfide (H2S) gas.
- Biochemically distinguish bacteria to facilitate presumptive identification.
Principle of Sulfur Reduction Test
Bacteria with sulfur-reducing capabilities can metabolize sulfur-containing compounds within the culture medium, converting sulfur into hydrogen sulfide. The liberated hydrogen sulfide interacts with the indicator, typically ferric ions or lead acetate, resulting in the formation of black-colored insoluble components (ferrous sulfide or lead sulfide). This reaction causes the entire medium to turn black. Therefore, the appearance of a black color in the medium following the incubation period signifies the sulfur reduction ability of the test organism.
Requirements for Sulfur Reduction Test:
1. Culture Medium:
- Various sulfur compound-containing culture media are suitable for conducting sulfur reduction tests, with commonly used options including SIM (Sulfide Indole Motility) medium, KIA (Kligler’s Iron Agar), TSI (Triple Sugar Iron) Agar Medium, and Lead Acetate (LA) Agar.
- Sodium thiosulfate is a common sulfur source present in these media.
- SIM medium, preferred for this test, includes sodium thiosulfate as a sulfur source and peptonized iron as an indicator.
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:
- Weigh the appropriate amount of SIM agar powder (36.23 grams per 1000 mL) and mix it with distilled water in a conical flask or glass bottle.
- Stir thoroughly, ensuring complete dissolution of agar in water, and heat the mixture to boiling.
- Dispense around 5 mL of the medium into each test tube, loosely covering the opening with a cap or cotton plug.
- Autoclave the tubes at 121°C and 15 lbs pressure for 15 minutes.
- Allow the medium to cool and solidify in an upright position, forming a butt.
2. Reagents:
- No additional reagents are necessary when using SIM or other sulfur-containing agar.
- Lead acetate paper is required for the lead acetate paper method.
3. Equipment
- Test Tubes
- Incubator
- Weighing Machine
- Inoculating wire
- Bunsen burner
- Autoclave
4. Test Organism (Sample Bacteria)
5. Control Organisms
- Proteus mirabilis ATCC 29906
- Shigella flexneri ATCC 12022
Procedure of Sulfur Reduction Test:
1. SIM Agar Method (Tube Method):
- Using a sterile inoculating wire, collect several colonies of the sample bacteria from a fresh culture (18 to 24 hours old).
- Stab the SIM medium in the tube, penetrating halfway (up to 3 to 5 mm above the base of the tube).
- Incubate the tube aerobically, leaving the cap loose, at 35±2°C for approximately 24 hours.
- Observe the development of black coloration on the medium.
2. Lead Acetate Paper Method:
- Using a sterile inoculating loop or wire, gather several colonies of the sample bacteria from a fresh culture (18 to 24 hours old) and inoculate a nutrient broth or peptone water medium.
- Place a lead acetate paper strip in a way that one end hangs just over the medium, securing the other end at the tube’s neck with a cotton plug or screw cap.
- Incubate the tube aerobically at 35±2°C and observe for blackening of the paper strip after 24 hours.
Result and Interpretation of Sulfur Reduction Test:
- A positive result is indicated by the blackening of the medium or the lead acetate paper turning black.
- A negative result is indicated by the absence of blackening in the medium or no change in the color of the lead acetate paper.
Quality Control:
- A positive result is observed with Proteus mirabilis ATCC 29906, leading to the transformation of the SIM medium and the lead acetate paper into a black color.
- Shigella flexneri ATCC 12022 produces a negative result, causing no color change in the SIM medium and the lead acetate paper.
Sulfur Reduction Test Results of Some Common Bacteria
|
Sulfur Reducing Bacteria |
Sulfur Non-reducing Bacteria |
|
Proteus spp., |
Klebsiella pneumoniae, |
Precautions:
- Avoid using an inoculating loop for inoculating the SIM tube.
- Prior to inoculation, inspect test tubes for any signs of medium cracking.
- Incubate the tubes aerobically with a loose cap.
- Ensure the careful selection of an appropriate medium for the test.
- Avoid direct contact between lead acetate paper and the medium, as lead acetate may exhibit an inhibitory effect.
Applications of Sulfur Reduction Test:
- For the differentiation and presumptive identification of Enterobacteriaceae members.
- To distinguish Salmonella spp. (sulfur-reducing) from Shigella spp. (sulfur non-reducing) and Erysipelothrix spp. (sulfur-reducing) from Lactobacillus spp. (sulfur non-reducing).
- Rapid detection of fecal coliforms in water.
Limitations of Sulfur Reduction Test:
- The test is not confirmatory on its own and necessitates additional biochemical test results for a conclusive bacterial identification.
- The presence of sucrose in the medium may inhibit hydrogen sulfide (H2S) production.
- Inoculum sourced from a liquid medium may extend the required incubation period.
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