COLECCIÓN ESPAÑOLA DE CULTIVOS TIPO

Parc Científic Universitat de València

C/ Catedrático Agustín Escardino, 9
46980 Paterna (Valencia), España
www.uv.es/cect

Instrucions: Opening of lyophilic

Before we begin...
1. The ampoules should be stored protected from light and at controlled temperatura
(between 4 and 24ºC, preferably 18ºC). Do not freeze
CECT guarantees the viability of strains for 1 month from shipmen. Many freeze-dried strains are viable
for long periods of time if kept under optimal conditions. This period varies from strain to strain and
CECT cannot guarantee viability beyond the guarantee period.

2. Check that you have the recommended culture medium for each strain and that
you can control the specified physicochemical parameters (incubation
temperature, anaerobiosis conditions, etc.).
To do so, please refer to the strain data sheet in our catalog (www.uv.es/cect).
In addition to the liquid medium necessary for the reconstitution of the lyophil, in most cases it is desirable
to have solid medium as well.
The media to be used must be freshly prepared or preserved in good conditions (not dried out or with
excessive humidity, without contaminants or precipitates, not expired).

3. Make sure you have the appropriate basic equipment (containers for disposal of
glass fragments, sterile water, sterile Pasteur pipettes and metal forceps ) and that
your laboratory infrastructure allows you to work in a microbiologically safe
environment.

Blister opening
1. Heating of the tip to the flame
Depending on the intensity of the combustion it may require between 5
and 15 seconds (slightly more if the flame is very weak).
Make sure that the heat cone only affects the narrow tip of the ampoule
so as not to damage the lyophilus..
The inner cotton plug should not darken (as this would be a sign of
overheating).

2. Cracking of the glass with sterile water

All these operations must be carried out with the due care required for
handling broken glass (e.g. protect your eyes, do not remove fragments
with your fingers...).
Drop 1-4 drops (drop by drop, not in a stream) of sterile distilled water. If
no cracking occurs repeat the previous step by slightly extending the
heating time.

Ed_12_20220214
 COLECCIÓN ESPAÑOLA DE CULTIVOS TIPO
Parc Científic Universitat de València
C/ Catedrático Agustín Escardino, 9
46980 Paterna (Valencia), España
www.uv.es/cect

If at the moment of cracking the cotton shoots inward, it is a sign that

the cotton has been overheated (it is displaced by the violent entry of
air). In such a case, use a stinging handle to pull it out to the end. For
more information, we recommend watching the video with opening
instructions available on our website.

Lyophil resuspension and seeding

1. Resuspension
Using a Pasteur pipette add 0.2-0.3 ml of the sterile liquid medium
recommended for growth of the microorganism to the opened glass vial.
Carefully resuspend the lyophil using the Pasteur pipette to aspirate and expel
the suspension. Do this gently avoiding the formation of air bubbles, especially
if it is an anaerobic or microaerophilic microorganism. As long as sterile
conditions can be maintained, leave the suspension for 20-30 minutes until
complete rehydration is achieved.

2. Seeding
Use the entire suspension to inoculate a solid medium (agar slant tube or Petri
dish) and a tube with 5-10 ml of liquid medium which should be incubated until
growth is observed before scaling up to larger volumes.
Do not keep part of the suspension in the ampoule itself as a reserve.
In most of our batches you will find a cellulose filter rectangle. You can also transfer it if you wish, as
many cells adhere to it. However, it is an operation that requires some dexterity (it is not easy to
manipulate without contaminating it), so try to do it without compromising the axenic state of the
whole suspension.

3. Incubation
Incubate at the optimum temperature for the microorganism strictly following the indications of the
strain data sheet in our catalog, (e.g. incubation in anaerobiosis, exposure to light, etc.).
Some strains have a long dormancy period. Incubate for up to two weeks before considering the culture
unviable.

Important
Subculture at least once after activation and prior to use as a working strain.

https://youtu.be/S4K0xAapD0Y

Instructions as taken pictures on the phone

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