Phage Spot Lysis Test: Phage detection technique

What is a Spot Test?

A spot test is a simple laboratory method used to determine whether a bacteriophage can infect and kill a specific bacterial strain.

In this method, a small volume of a phage solution is placed (“spotted”) onto a bacterial lawn, which is a uniform layer of bacteria grown on an agar plate. During incubation, the phages diffuse into the bacterial layer and interact with the cells.

• If the phages are active, they infect and lyse the bacteria, producing clear zones called plaques or lysis spots.
• If no lysis is observed, the phage is likely inactive against that bacterial strain.

Because it is fast and easy to perform, the spot test is widely used as a qualitative screening tool in phage research and phage therapy to assess host range and phage activity.

Materials Required

Culture Media and Biological Materials

• LB (Luria–Bertani) agar plates
  Solid growth medium used to culture bacteria. Plates should be prepared and solidified in advance.
• Ready made overlay/soft agar
  This will be pored ontop of solid agar.
• 18-hour bacterial cultures
  Overnight liquid cultures of the target bacterial isolates or reference strains grown in LB broth.
• Phage lysate
  The bacteriophage sample being tested.

Equipment and Consumables

• Micropipette
  Capable of dispensing 500 µL and 10 µL volumes.
• Falcon tube
  Capable of holding 15 ml.
• Sterile pipette tips
  Use a new tip for each transfer to avoid contamination.
• Half-lid cover
  Allows partial airflow while reducing contamination during absorption/you can improvise with a plate lid by partial covering the plate.
• Incubator
  Set to 37 °C for bacterial growth and phage infection (Temperature may change depending on the bacteria you are dealing with).
• Timer or clock
  For monitoring incubation times.

Safety Requirements

• Laboratory coat and gloves
• Sterile working conditions
• Proper disposal of biological waste

Note: All media, solutions, and tools should be prepared and sterilized according to standard laboratory procedures before starting the experiment.

Procedure

Step 1: Preparation of the Bacterial Lawn

1. If the overlay (soft) agar has solidified, re-melt it by heating and maintain it in a water bath at 45–50 °C.
2. Aliquot 4 mL of molten overlay agar into a 15 mL sterile Falcon tube.
3. Add 500 µL of an 18-hour LB bacterial culture to the tube containing the molten overlay agar.
4. Gently swirl or invert the tube to mix thoroughly, avoiding bubble formation.
5. Immediately pour the mixture onto a pre-warmed LB agar plate.
6. Gently and quickly tilt the plate to evenly distribute the overlay before it solidifies.
7. Leave the plate partially covered with a half-lid.
8. Allow the overlay to solidify and absorb for 5–10 minutes at room temperature or 37 °C.

Important:
Bacterial cells are highly active at this stage. To avoid excessive growth and an overly thick lawn, the plate should be used within 1 hour of preparation if kept at room temperature.

• Below ~45 °C → agar may start to solidify too early
• Above ~50 °C → heat stress can kill or injure bacteria, reducing lawn quality

Step 2: Spotting the Phage Solution

1. Using a micropipette, carefully place 10 µL of the phage solution onto the surface of the bacterial lawn.
2. Avoid touching the agar surface with the pipette tip.
3. Multiple phage samples can be tested on the same plate, provided the spots are well separated.

Step 3: Incubation

• Incubate the plates at 37 °C.
• Leave undisturbed for 6–18 hours.

Step 4: Observation and Interpretation

After incubation, examine the plates for clear or turbid zones at the spots where the phage solution was applied.

• Clear plaques or lysis zones
  → Indicates active phage infection and bacterial lysis.
• No visible change
  → Suggests the phage does not infect or lyse the tested bacterial strain.

The presence of a lysis zone confirms phage activity against the target bacterium.

Summary

The spot test is a rapid and qualitative method for detecting bacteriophage activity. It is especially useful for:

• Screening phage host range
• Identifying active phages
• Preliminary testing for phage therapy applications