To provide a standardized method for enumerating viable bacteria in a sample by preparing serial dilutions, plating on a suitable agar medium, incubating under controlled conditions, and counting the resulting colonies.

Total Bacteria Colony Counting Service Procedure

1. Purpose

To provide a standardized method for enumerating viable bacteria in a sample by preparing serial dilutions, plating on a suitable agar medium, incubating under controlled conditions, and counting the resulting colonies.

2. Scope

This procedure applies to all samples submitted for total bacterial enumeration (e.g., water, food, environmental samples) in the laboratory.

3. Responsibilities

• Laboratory Technicians: To perform the procedure in accordance with this SOP, ensuring all steps are properly executed and documented.
• Quality Control Personnel: To review and approve all results and ensure adherence to quality standards.
• Laboratory Manager: To maintain and update the SOP as needed and ensure that all equipment is calibrated and maintained.

4. Materials and Equipment

• Materials:
  • Sample (liquid, solid, or homogenized)
  • Sterile diluents (e.g., 0.1% peptone water or phosphate-buffered saline)
  • Plate Count Agar (or another approved medium)
  • Sterile pipette tips and pipettes/micropipettes
  • Sterile dilution tubes or containers
  • Sterile petri dishes
  • Disposable gloves, lab coat, and safety goggles
• Equipment:
  • Vortex mixer
  • Incubator (set at the required temperature, typically 35°C ± 2°C)
  • Colony counter (manual or automated)
  • Marker pens for labeling

5. Procedure

5.1. Sample Receipt and Preparation

1. Receipt and Documentation:
   • Verify the sample’s identification and record all relevant information (e.g., sample ID, collection date, type of sample) in the laboratory log.
   • Inspect the sample for proper preservation and transport conditions.
2. Sample Homogenization:
   • For liquid samples: Mix the sample thoroughly by inverting or gently shaking.
   • For solid samples: Homogenize using a blender or stomacher in the appropriate volume of sterile diluent.
   • Record the weight (for solids) or volume (for liquids) of the sample.

5.2. Serial Dilution

1. Preparation:
   • Label a series of sterile dilution tubes with the appropriate dilution factors (e.g., 10⁻¹, 10⁻², 10⁻³, etc.).
2. Dilution Process:
   • Transfer 1 mL of the sample into 9 mL of sterile diluent to achieve a 1:10 dilution (10⁻¹).
   • Vortex the tube for 15–30 seconds to mix thoroughly.
   • Continue performing serial dilutions as needed to obtain dilutions that yield countable colonies (typically plates with 30–300 colonies).

5.3. Plating the Dilutions

1. Selecting Dilutions:
   • Choose the dilution(s) expected to provide countable colonies. Often, more than one dilution is plated to ensure at least one plate falls within the countable range.
2. Plating Method:
   • Spread Plate Method:
     • Pipette a measured volume (commonly 0.1 mL) of the selected dilution onto the surface of a pre-poured agar plate.
     • Use a sterile spreader or glass rod to evenly distribute the inoculum over the agar surface.
   • Pour Plate Method (if applicable):
     • Mix the dilution with molten agar (cooled to 45–50°C) and allow the mixture to solidify in a petri dish.
3. Labeling:
   • Clearly label each plate with the sample ID, dilution factor, and date.

5.4. Incubation

1. Incubation Conditions:
   • Invert the plates (to prevent condensation from dripping onto the agar surface).
   • Incubate at the prescribed temperature (e.g., 35°C ± 2°C) for 24–48 hours. Adjust time and temperature if specific organisms or sample types require different conditions.
2. Monitoring:
   • Check for proper incubation conditions periodically.

5.5. Colony Counting

1. Selection:
   • After incubation, select plates that have between 30 and 300 colonies for reliable enumeration.
2. Counting:
   • Count the number of colonies manually or using an automated colony counter.
   • Record the colony counts along with the corresponding dilution factor and plated volume.
3. Quality Check:
   • If colonies are too numerous (i.e., overgrown) or too few (i.e., below 30), note these results and consider re-plating using a different dilution.