1

PREPARATION OF H2S KIT

Introduction

H₂S (Hydrogen Sulfide) test kits are widely used for the rapid detection of bacterial contamination in water. These kits work by indicating the presence of H₂S-producing bacteria, which are often associated with fecal contamination. The test is simple, reliable, and suitable for both laboratory and field applications.

  • Common bacteria associated with fecal contamination include E. coli, Salmonella, and H₂S-producing bacteria.
  • Drinking or using water with fecal contamination can cause waterborne diseases like diarrhea, cholera, typhoid.

Materials

  • Whatman Filter Paper No. 41
  • Prepared H₂S media
  • 50 plastic bottles (30 ml each, new)
  • Bottle caps
  • 70% Ethanol

Equipment

  • Laminar Air Flow (LAF) Cabinet
  • UV light (for sterilization in LAF)
  • Oven or incubator (50°C)
  • Forceps (sterile)
  • Sterile gloves
  • Scissors or paper cutter (for cutting strips)
  • Marker (for labeling bottles and strips)

H2S Media Preparation for 50 Bottle :

Name of ChemicalQuantity (gm/ml)
Bacteriological grade peptone20 gm
Di – potassium hydrogen phosphate1.5 gm
Ferric ammonium citrate0.75 gm
Sodium Thiosulphate1.0 gm
L-cysteine HCL0.125 gm
Liquid soap1.0 ml
Distilled water50 ml

Procedure

Step 1: Preparation of Test Strips

  1. Take Whatman Filter Paper No. 41.
  2. Cut the paper into strips of 8 cm × 2.5 cm using scissors or a paper cutter.
    • Ensure cuts are even to fit into the 30 ml bottles easily.
  3. Prepare the H₂S media in a clean, sterile container.
  4. Dip each paper strip completely into the media so it is fully saturated.
    • Handle strips only with sterile forceps to avoid contamination.
  5. Place all dipped strips on a clean tray to dry.
  6. Dry the strips in an oven at 50°C for 40 minutes.
    • This ensures that the media adheres to the paper and excess moisture is removed.
  7. After drying, transfer the strips into the laminar air flow (LAF) cabinet for sterilization.

Step 2: Sterilization of Plastic Bottles

  1. Take 50 new 30 ml plastic bottles.
  2. Clean the outer surface with 70% ethanol before placing them in the LAF.
  3. Place the bottles under UV light in the laminar air flow cabinet for 1 hour.
    • This step sterilizes the bottles and prevents bacterial contamination.
  4. After sterilization, keep bottles inside the LAF until ready for use.

Step 3: Loading Strips into Bottles

  1. Wear sterile gloves before handling strips or bottles.
  2. Use sterile forceps to pick up each dried H₂S strip.
  3. Place one strip per bottle.
    • Avoid touching the strip with your hands or the bottle walls.
  4. Secure the bottle with a tightly fitting cap immediately after placing the strip.
  5. Label each bottle with a unique ID or date for tracking.

Step 4: Post-Preparation Handling

  1. Store the prepared H₂S kits in a clean, dry place away from direct sunlight.
  2. Handle bottles carefully to prevent moisture or contamination from entering.
  3. Inspect bottles for any signs of contamination before use.

Precautions

  • Always work inside a laminar air flow cabinet when handling strips or bottles.
  • Use 70% ethanol to sanitize surfaces, equipment, and hands.
  • Wear sterile gloves at all times during the procedure.
  • Use sterile forceps for transferring strips.
  • Ensure bottle caps are tightly closed to prevent contamination.
  • Avoid prolonged exposure of strips to open air.
  • Regularly check UV lights for functionality to ensure sterilization is effective.

Workflow Summary (Step-by-Step Visual Guide)

  1. Cut Filter Paper → 8×2.5 cm strips
  2. Dip Strips → H₂S media
  3. Dry Strips → Oven 50°C, 40 min
  4. Sterilize Strips → Laminar Air Flow
  5. Sterilize Bottles → UV light, 1 hour in LAF
  6. Load Strips → Forceps, one strip per bottle
  7. Seal & Label → Tight caps, record ID/date

2

Isolation of Bacteria and Fungi from a Decomposed Sample

This activity of isolation of bacteria and fungi from decomposed samples was performed under the guidance of Sayali Mam.

Decomposed organic materials like compost, agricultural waste, and spoiled plant matter contain many microorganisms such as bacteria and fungi. These microbes help in decomposition, nutrient recycling, and improving soil fertility. In this experiment, bacteria and fungi were isolated from a decomposed sample using serial dilution and spread plate techniques.

For bacterial isolation, Nutrient Agar (NA) medium was prepared, while Potato Dextrose Agar (PDA) with Streptomycin was used for fungal isolation. All media, glassware, and instruments were sterilized by autoclaving at 121°C and 15 psi for 15 minutes to avoid contamination.

About 1 gm of decomposed sample was mixed with sterile distilled water and serial dilutions were prepared from 10⁻¹ to 10⁻⁷. From selected dilutions, 0.1 ml sample was spread on NA and PDA plates using a sterile glass spreader.

The plates were incubated in an inverted position to prevent water droplets from disturbing colony growth. Bacterial plates were incubated at 37°C for 24–48 hours, while fungal plates were incubated for 3–5 days.

After incubation, bacterial colonies appeared as small, smooth, shiny colonies, while fungal colonies showed cottony and fluffy growth. Isolated colonies can further be purified and studied for their morphology and characteristics.