Bacteria are often preserved in microbiology laboratories for many purposes such as to re-examine or characterize further or to send to a national reference laboratory for confirmation etc. The selection of a particular storage method depends on the length of time the organisms are to be stored and the laboratory equipment and facilities available. The selection of media for preservation and storage also depends on the type of organism to be preserved.
The preservation of non-fastidious bacteria is comparatively less tedious than that of fastidious bacteria because non-fastidious ones can survive in a minimal nutrient medium with preservatives for a longer period of time. Fastidious pathogens like N. meningitidis, S. pneumoniae, and H. influenzae are fragile bacteria and therefore are easily killed or contaminated. Aseptic techniques should be used at all times during the preparation of isolates for storage and/or transport.
For storage, isolates should be first confirmed as pure cultures. Fresh cultures (i.e., 18-24 hour growth) should always be used for the preparation of isolates to be stored.
Fastidious organisms may not survive more than a few days on an agar plate and may require long-term storage.
N. meningitidis, S. pneumoniae, and H. influenzae only survive for 3-4 days on blood agar plates (BAP) and/or chocolate agar plates (CAP) and do not survive for long periods of time in broth. To overcome the issue, we employ short-term storage methods that are appropriate for bacterial isolates that only need to be stored for several days to a few weeks at a time. These methods include Dorset transport medium, chocolate agar slants, and silica gel packages.
Table of Contents
Dorset Transport medium
Dorset transport medium is best for room temperature (25°C) storage of N. meningitidis, S. pneumoniae, and H. influenzae. On Dorset transport medium, N. meningitidis and H. influenzae can be stored for approximately 3 weeks, whereas S. pneumoniae can be stored for approximately 6 weeks. Dorset transport medium is typically prepared as a 4 ml slant in a 7 ml screw-cap tube. It should be stored at 4°C when not in use and warmed to room temperature (25°C) before use.
How to use:
- Transfer the Fresh cultures into the medium by streaking the slant and incubating the tube overnight at 37°C in 5% CO2.
- After incubation, the Dorset transport slant can be stored at room temperature (25°C).
Chocolate agar slants
In absence of Dorset Transport medium, short-term storage of N. meningitidis, S. pneumoniae, and H. influenzae can also be carried out on chocolate agar slants for up to 1 week. Chocolate agar slants are prepared as a 4 ml slant in a 7 ml screw-cap tube. They should be stored at 4°C when not in use and warmed to room temperature (25°C) before use.
How to use:
- Transfer the Fresh cultures into the medium by streaking the slant and incubating the tube overnight at 37°C in 5% CO2.
- After incubation, store the slants at room temperature (25°C).
Preservation method slightly differs for N.meningitidis isolates. For N. meningitidis, solid screw-caps should be loosened during storage or permeable membrane screw caps (which allow for an exchange of gases and are commercially available) should be used when possible. An overlay of trypticase soy broth (TSB) may also be helpful and might increase viability. N. meningitidis slants SHOULD NOT BE REFRIGERATED.
Slants for S. pneumoniae and H. influenzae are viable longer if they are maintained at 4°C with the cap tightened to avoid drying after incubation.
Silica-gel packages:
N. meningitidis, S. pneumoniae, and H. influenzae can also be stored short-term on swabs stored in silica gel packets, which are typically 1.5 g foil bags, with 75% white gel and 25% blue gel (the blue gel is added to detect moisture). Isolates can survive approximately 2 weeks at 4°C and perhaps slightly shorter at room temperature (25°C). The packets are inexpensive and easy to use but may not be always available from commercial manufacturers. Silica gel packages can be stored at room temperature (25°C) when not in use.
How to use:
- Inspect the contents of the package before use. Blue and white gels should be clearly visible. If only white gels are visible, that indicates that moisture was introduced into the silica gel package and the package should not be used.
- If everything seems fine, cut the silica gel package with sterile scissors.
- Collect the overnight pure culture with a single sterile polyester swab. DO NOT USE cotton swabs as cotton has a bacteriostatic effect that will inhibit bacteria growth.
- Place the swab in the silica gel package with the tip inserted into the silica and the shaft sticking out the top of the package.
- Remove the cover of adhesive tape on the silica gel package and fold down the corners to seal the package.
- Place additional tape around the shaft of the swab and folded corners to secure the swab and seal the package.
References
- Hoefman, S., Pommerening-Röser, A., Samyn, E., De Vos, P., & Heylen, K. (2013). Efficient cryopreservation protocol enables accessibility of a broad range of ammonia-oxidizing bacteria for the scientific community. Research in microbiology, 164(4), 288–292. https://doi.org/10.1016/j.resmic.2013.01.007
- Lagier, J. C., Edouard, S., Pagnier, I., Mediannikov, O., Drancourt, M., & Raoult, D. (2015). Current and past strategies for bacterial culture in clinical microbiology. Clinical microbiology reviews, 28(1), 208–236. https://doi.org/10.1128/CMR.00110-14
