Long-term storage of Bacterial Strains

By Acharya Tankeshwar •  Updated: 06/09/22 •  4 min read

Long-term preservation methods permit intervals of months or even years between subcultures. Isolates may be stored indefinitely if they are maintained frozen at -70°C or below; these temperatures can be achieved in an “ultralow freezer” (-70°C) or a liquid nitrogen freezer (-196°C).

Lyophilization or storage at -70°C or below is the best method for long term preservation of bacterial culture and general storage of isolates at -20°C is not recommended.

Freeze Dryer
Freeze Dryer


Most organisms may be successfully stored after lyophilization (freeze-drying). Freeze-drying involves the removal of water from frozen bacterial suspensions by sublimation under reduced pressure. Lyophilized cultures are best maintained at 4°C or lower. Sublimation occurs when a frozen liquid goes directly to a gaseous state without entering a liquid phase.

The freeze-drying process results in a stable readily rehydrated product. This process consists of three steps:

  1. pre-freezing the product in laboratory freezer to form a frozen structure,
  2. primary drying to remove most water,
  3. and secondary drying to remove bound water.

It is recommended using slow rates of cooling as this will result in the formation of vertical ice crystal structures, thus allowing for more efficient water sublimation from the frozen product. Freeze-dried products are hygroscopic and must be protected from moisture during storage.

Preservation in Glycerol at -20 °C

  1. Grow a pure culture on an appropriate solid medium.
  2. When the culture is fully developed, scrape it off with a loop.
  3. Suspend small clumps of the culture in sterile neutral glycerol.
  4. Distribute in quantities of 1–2 ml in screw-capped tubes or vials.
  5. Store at -20 °C. Avoid repeated freezing and thawing. Transfer after 12–18 months.

Mineral oil at room temperature

  1. Prepare tubes of heart infusion agar with a short slant. For fastidious organisms, add fresh native or heated blood.
  2. Sterilize mineral oil (liquid petrolatum) in hot air (170 °C for 1 hour).
  3. Grow a pure culture on the agar slant.
  4. When good growth is seen, add sterile mineral oil to about 1 cm above the tip of the slant.
  5. Subculture when needed by scraping growth from under the oil.
  6. Store at room temperature. Transfer after 6–12 months.

Stab Cultures

Stab cultures at room temperature are used for non-fastidious organisms only, such as staphylococci and Enterobacteriaceae

  1. Prepare tubes with a deep butt of carbohydrate-free agar. Tryptic soy agar is recommended.
  2. Stab the organism into the agar.
  3. Incubate overnight at 35 °C.
  4. Close tube with screw-cap or cork. Dip cap or cork into molten paraffin wax to seal.
  5. Store at room temperature. Transfer after 1 year.

Stab cultures in cystine trypticase agar (CTA) method is recommended for the preservation of  Neisseria and streptococci

  1. Prepare tubes of cystine trypticase agar.
  2. Stab the organism into the medium.
  3. Incubate overnight at 35 °C.
  4. Close tube with screw-cap or cork. Dip cap or cork into molten paraffin wax to seal.
  5. For Neisseria, store at 35 °C, and transfer every 2 weeks. For streptococci, store at room temperature, and transfer every month.

Cooked-meat medium is used for the preservation of anaerobes

  1. Inoculate tubes of cooked meat medium with the isolate.
  2. Incubate overnight at 35 °C.
  3. Close tube with screw-cap or cork.
  4. Store at room temperature. Transfer every 2 months.

Recovery of Bacteria from Lyophilized storage condition

To recover the isolate follow the step wise procedure as mentioned below;

  1. Remove the frozen cultures from the freezer and place them on dry ice or into alcohol and dry-ice bath;
  2.  Transfer to a laboratory safety cabinet or a clean area if a cabinet is not available.
  3. Scrape the top-most portion of the culture using a sterile loop
  4. Transfer to a growth medium without contaminating the top or inside of the vial.
  5. Re-close the vial before the contents completely thaw, and return the vial to the freezer; with careful technique, transfers can be successfully made from the same vial several times.
  6. Incubate 18–24 hours at 35–37°C; perform at least one subculture before using the isolate to inoculate a test.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.