Plating Bacterial Cultures from Freezer Stocks

Recover viable colonies from −80°C glycerol freezer stocks by streak plating onto appropriate agar media.

Plating Bacterial Cultures from Freezer Stocks, v1.0 Kathryn E. Caruso · 0009-0003-2436-1791 Foreman Lab · Center for Biofilm Engineering, Montana State University Updated March 2026

How to cite this protocol Caruso, K.E. (2026). Plating Bacterial Cultures from Freezer Stocks, v1.0. Foreman Lab, Center for Biofilm Engineering, Montana State University. https://kathryncaruso.github.io/methods/plating-from-freezer-stock/

Plating Bacterial Cultures from Freezer Stock

Standard Operating Procedure — Revival by Streak Plating

Purpose

Recover viable colonies from a −80°C glycerol freezer stock by streaking onto an appropriate agar medium. Use this protocol whenever you need isolated colonies from a stored strain — e.g., for re-starting cultures, confirming purity, or creating new working plates.

1 Safety

PPE:

  • Lab coat
  • Nitrile gloves
  • Safety glasses
  • Work in Biosafety Cabinet for all plating steps

2 Materials

  • Agar plates of appropriate medium (e.g., R2A, BHI — see notes below)
  • Sterile inoculating loops
  • Micropipette and sterile tips (for liquid freezer stocks)
  • Access to −80°C freezer
  • Permanent marker or labels
  • Parafilm (optional, for sealing plates)

3 Retrieve freezer stock

  1. Locate the freezer stock vial in the −80°C freezer
  2. Transport on ice or work quickly to minimize thaw/refreeze cycles
  3. Determine which inoculation method to use:
Stock State Method When to Use
Frozen (solid) Scrape with sterile loop Preferred when you want to preserve the stock for many future uses
Thawed (liquid) Pipette a small volume Use when the vial has already thawed or when you need a measured inoculum

Important

If using the frozen/scrape method, work quickly and return the vial to −80°C immediately. Do not allow the entire stock to thaw. Repeated freeze-thaw cycles reduce viability.

4 Streak plating

4.1 From a frozen stock (scrape method)

  1. Open the vial briefly in the Biosafety Cabinet
  2. Using a sterile inoculating loop, scrape a small amount of frozen culture from the surface
  3. Return the vial to −80°C immediately
  4. Streak the loop onto the agar plate using a standard streak-for-isolation pattern:
    • Zone 1: Streak the inoculum back and forth across ~1/3 of the plate
    • Zone 2: Flame/discard loop, rotate plate ~90°, streak through the edge of Zone 1 into a fresh area
    • Zone 3: Flame/discard loop, rotate plate ~90°, streak through the edge of Zone 2 into the remaining area
  5. Replace the plate lid

4.2 From a thawed/liquid stock (pipette method)

  1. Gently vortex or invert the thawed vial to resuspend cells
  2. Pipette 10–50 µL of the freezer stock onto the agar plate near one edge
  3. Using a sterile inoculating loop, spread and streak for isolation:
    • Zone 1: Spread the droplet back and forth across ~1/3 of the plate
    • Zone 2: Flame/discard loop, rotate plate ~90°, streak through the edge of Zone 1 into a fresh area
    • Zone 3: Flame/discard loop, rotate plate ~90°, streak through the edge of Zone 2 into the remaining area
  4. Replace the plate lid
  5. Return the stock vial to −80°C if it will be reused, or discard if fully thawed and no longer needed

5 Incubation

  1. Label the plate with:
    • Organism/strain name
    • Medium type
    • Date
    • Initials
    • Incubation temperature
  2. Optionally seal the edge of the plate with Parafilm to prevent desiccation during long incubations
  3. Incubate plates inverted (agar side up) at the appropriate temperature:
Organism Type Suggested Temperature Expected Time to Colonies
Mesophiles (e.g., S. pasteurii) 30°C 1–2 days
Cold-adapted isolates 15°C or 4°C 3–7+ days
  1. Check plates daily for colony growth

6 After growth

  • Confirm colony morphology matches expected appearance for the strain
  • Plates with isolated colonies can be used to:
    • Pick colonies for liquid cultures or starter cultures
    • Create new freezer stocks
    • Confirm strain purity by visual inspection or 16S sequencing
  • Storage: Store working plates at 4°C, sealed with Parafilm. Use within 2–4 weeks.

7 Medium selection guide

Organism Recommended Agar Notes
Cold-adapted environmental isolates R2A agar Low-nutrient, supports slow growers
Sporosarcina pasteurii BHI agar Rich medium, supports fast growth
General/unknown R2A or TSA Broad-spectrum options

Notes

  • For cold-adapted isolates that grow slowly, allow up to 1–2 weeks before concluding no growth
  • If no isolated colonies appear (lawn growth), re-streak from a single colony area onto a fresh plate to purify
  • When plating multiple strains in one session, change gloves or decontaminate between strains to avoid cross-contamination