We should protect microorganisms for analysis continuity. Use agar plates and stab cultures for short-term storage (weeks) at 4°C. Freeze samples at -80°C for long-term storage (years) and immerse them in liquid nitrogen at -196°C for ultra-long-term storage (many years). Retrieve and tradition saved samples by mild thawing to 37°C adopted by inoculation into progress media.
The fitting preservation methodology could make all of the distinction relating to sustaining the viability of your microbial strains
Given the significance of microorganisms in biology, as protein manufacturing factories, assay targets, and as mannequin organisms, the preservation of microorganisms performs a key function in making certain reproducible outcomes and continuity in analysis.
Sustaining a library of microbial shares additionally allows microorganisms to be simply saved and retrieved, slightly than constantly sub-culturing microbial cultures on plates or in tubes, which might result in the buildup of detrimental mutations.
Relying on the size of storage and kind of microorganism, you’ll have to protect them in a selected approach.
Listed below are the primary methods to protect microorganisms on your analysis.
Why Protect Microbes
The primary purpose we protect microorganisms is to maintain them dormant, free from contamination and genetic modifications, till we’re prepared to make use of them for our experiments. That is conventionally achieved utilizing low temperatures and freeze-drying strategies. These cut back mobile metabolic exercise to nearly nothing with out killing or harming the cells.
The Two Most important Methods to Protect Microorganisms
1. Brief-Time period Storage: Continous Progress
Microbes might be grown constantly for brief durations on an appropriate progress medium blended with agar (which is a setting agent).
Agar plate cultures of microorganisms are viable for just a few weeks when stored within the laboratory fridge at 4°C.
The same however barely longer-term methodology is the stab tradition. Choose a single colony out of your agar plate with a needle and stab it into the middle of a mushy combination of agar and progress media set into an in any other case sterile Falcon™ or Sterlin™ tube.
Most microorganisms (micro organism, fungi, and algae) might be saved utilizing this methodology, and it’s normally one of the best ways to retailer microbes that you just use often. Relying on the species and genus of your microbes, stab cultures can final anyplace from a fortnight to a 12 months.
Utilizing vitamins from the tradition media, these cultures develop constantly, albeit slower at decrease temperatures, enabling them to outlive longer on the accessible vitamins.
Find out how to Reduce Contamination
To reduce contamination and drying of the agar, the plates needs to be securely wrapped with laboratory movie and saved the other way up (agar facet up), to forestall condensation from forming on the plate.
Tubes and vials containing agar stab cultures needs to be capped.
Drawbacks of Brief-Time period Storage
Whereas the benefit of short-term storage is that your microbes are fast and straightforward to recuperate (by streaking them into new plates for brand new progress), the agar will dry over a chronic interval.
Moreover, the vitamins within the tradition media grow to be depleted by the microorganisms, and metabolic waste merchandise additionally accumulate to poisonous ranges. Nutrient hunger and poisonous waste product build-up ultimately trigger the loss of life of the saved microorganisms. Even worse, the microbes could purchase undesirable genetic mutations.
The important thing level right here is to consider the time scale of your experiments and to plan forward earlier than you protect your microbes.
2. Lengthy-Time period Storage: Cryopreservation
For long-term storage, microorganisms are cryopreserved at temperatures under freezing at -20°C or -80°C.
You possibly can both put your cells straight within the freezer or snap-freeze them utilizing liquid nitrogen. Cryopreservation is appropriate for a lot of micro organism, algae, fungi, viruses, and protozoa. Ultralow temperatures drastically cut back enzymatic exercise and, thus, the metabolic processes of microorganisms.
To forestall damaging and killing the cells through ice crystal formation (bear in mind, ice expands when it freezes), resuspended cells in progress media containing cryoprotectants equivalent to ~30 % v/v glycerol, earlier than freezing.
You may as well use skimmed milk as a substitute of glycerol. [1]
To maximise the restoration fee, it’s endorsed to freeze cultures on the stationary part of progress when cell focus is highest.
Cultures saved at -20°C needs to be secure for no less than a 12 months, whereas cultures saved at -80°C or in liquid nitrogen may very well be secure for many years!
Frozen microorganisms might be retrieved by gently thawing them at 37°C and inoculating the cells into contemporary progress medium. [2]
Various Methods to Protect Microbes
Freeze Drying
Freeze drying (lyophilization) removes moisture from frozen samples by way of the sublimation of ice in a vacuum, thus arresting microbial metabolic processes and reworking cells into dry pellets for handy storage and transportation.
Freeze drying has been used to protect micro organism, algae, yeasts, viruses, and sporulating fungi. [3]
Nevertheless, word that lyophilization is unsuitable for sure micro organism and non-sporulating fungi as they could not survive the stresses of dehydration.
The suitability of freeze drying will even rely upon what you wish to do along with your microbes after you’ve retrieved them.
Lyophilized cultures might be recovered by rehydrating the cell pellets in progress media (micro organism and algae), progress medium containing the bacterial hosts (bacteriophages), or water (fungi and yeasts) earlier than inoculating into contemporary tradition media.
Micro-encapsulation
In micro-encapsulation, cells are entrapped in a matrix of some protecting encapsulating materials earlier than storage. [4] It has been proposed as a long-term microbial preservation methodology that doesn’t expose the microorganisms to the cruel stresses of freezing and drying.
Encapsulation matrixes embody:
- Gelatin.
- Ethylcellulose.
- Polyvinyl alcohol (PVA).
The matrix shields the cells and will increase stability throughout storage.
Relating to its advantages to cell viability, micro-encapsulation of probiotic micro organism in calcium alginate has been proven to enhance their viability when saved at -80°C.
Equally, electrospinning and electrospraying, the place microorganisms are trapped in nanofibers and droplets, respectively, have additionally been used to protect the viability of delicate probiotic micro organism. [5]
Microbe Preservation Methods Summarized
Preserving microbes the correct approach ensures they’re in tip-top form on your experiments, in flip saving you time, effort, and cash.
Brief-term steady progress allows you to retrieve your cells immediately, whereas long-term cryopreservation can preserve your cells viable for years. And for actually hardy or actually delicate bugs, there are specialist preservation strategies you possibly can strive!
Have we missed a great way to protect microorganisms? Any ideas for higher pattern survival and simpler retrieval? Tell us within the feedback part under.
Initially printed December 2019. Revised and up to date Might 2023.
References
- Cody WL, Wilson JW, Hendrixson DR, et al. (2008) Skim milk enhances the preservation of thawed -80 levels bacterial shares. J Microbiol Strategies 75(1):135–38
- Tedeschi R and De Paoli P (2011) Assortment and preservation of frozen microorganisms. Strategies Mol Biol 675:313–26
- Prakash O, Nimonkar Y, and Shouche YS (2013) Apply and prospects of microbial preservation. FEMS Microbiol Lett 339(1):1–9
- Rathore S, Mahendrakumar PD, Liew CV, et al. (2013) Microencapsulation of microbial cells. J Meals Eng 116(2) 369–81
- Alonso S (2016) Novel preservation strategies for microbial cultures. In: Novel meals fermentation applied sciences, 1st version. Springer Cham: On-line