Use of the cryo-replicator
Proper handling and maintenance of the replicator The pins of the replicator are made of a high-quality stainless steel. Nevertheless some maintenance may be required: The surface of the end of the pins may change in the course of time. E.g. improper washing after the transfer of colonies and subsequent heating may cause a thin char layer. Also, excessive heating negatively affects the surface of the pins. A changed surface of the pins -in its turn- may have an influence on i) the heat- transfer during the sampling of the master plates and ii) the extent to which cells stick to end of the pins. Such pin-to-pin variations are undesirable and can be counteracted as follows: If the end of pins are black or damaged, polishing of the end of the pins using sandpaper is adviced: 1. tape a sheet of fine waterproof sand paper (e.g. quality 300) on a flat bench 2. wet the sandpaper with plenty of demineralized water 3. move the replicator over the sandpaper in an orbital way for e.g. one minute: use both hands, press the replicator down at such a force that the pins move up a few mm, and continue until the end of all pins are shiny and flat again Note: If the replicator is accidentally dropped or damaged in another way, it may be necessary to replace one or more of the pins and/or springs. If bent pins can not be bent straight again, replacement pins can be obtained for free. We will also supply advice on the best procedure to replace pins and/or springs.
Sampling a frozen master plate using the cryo-replicator. 1. Place a cryo-replicator (sterilized and allowed to cool down to room temperature as described before) into the replicator-press (in a laminar flow cabinet). 2. Take the MTP with the frozen cultures (master plate) from the –80ºC freezer. 3. Transfer the master plate into the laminar flow cabinet, take the lid of the plate and position the plate under the cryo-replicator. 4. Lower the cryo-replicator into the master plate and press the replicator onto the frozen surfaces of the cultures for three seconds; apply a force sufficient to move up each pin for at least 5 mm relative to the frame of the replicator in which the pins are allowed to travel a). 5. Move the replicator back up to its highest position in the press. 6. Remove the master plate, put the lid on, and immediately transfer the master-plate back into the –80ºC freezer (make sure that the master plate is out of the –80ºC freezer for no longer than 2 minutes: a longer time period may cause partial melting and so affect the future viability of the cells sampled from the frozen master culture). 7. Position a regular PS 96-well MTP (volume wells 0.35 ml) of which each well is filled with 0.18 ml of an appropriate agar -based medium under the replicator). 8. Press the replicator down onto the agar surface at a force sufficient to move up each pin for a few mm relative to the frame of the replicator in which the pins are allowed to travel . 9. Incubate the agar-MTPs for a number of days (dependent on the set of strains used) at an appropriate temperature in order to allow the transferred cells to grow into colonies of a reasonable size (put the plate in a dessicator together with a beaker of water in order to minimize evaporation).
1) Place frozen master- plate umder sterile cryo- replicator
2) Press cryo-replicator down on the frozen surfaces for 3 seconds
3) Inoculate the sampled cells onto agar medium dispensed in an MTP
Sterilization of the cryo-replicator using a hot plate (important note: do not autoclave the replicator: this will cause irreversible damage!!) 1. Preheat a flat metal heating plate to a temperature of 300 ºC (outside the flow cabinet; clearly mark this plate “hot !!” in order to prevent colleagues from burning themselves). 2. Clean the ends of the pins of the replicator: hold the replicator under an angle of approx. 60º above the sink (see photo below) and brush and rinse the ends of the pins of the replicator using demineralized water. Try to prevent that the blue plates and the springs become wet: this may cause deposits of traces of minerals and so gradually lead to more friction of the pins in the guiding plates. 3. Put the cryo-replicator (ends of pins are still wet) on the hot plate for 5 minutes (make sure that all pins touch the surface of the hot plate: if necessary press the replicator down using a stand, see photo below). Important: the replicator may be damaged if it is left on the hot plate for a time period much longer than 5 minutes; use a timer with alarm to prevent this from happening). 4. Transfer the cryo-replicator from the hot plate into the replicator press (or onto one of the two positions on top of the replicator-press) and allow to cool down to room temperature for a minimum time period of 12 minutes before use. (important, it is advisable to use a timer)
Sterilization of the replicator on a hot plate: the aluminium bar presses the grip down and so ensures that all pins are well in contact with the hot plate.
Washing of the replicator using demineralized water: it is important that all cell debris is well removed before sterilizing and using the replicator again. It is advisable to avoid wetting the aluminum plates or the springs during washing.
Footnote: Some microbial strains may be resistant to some extent to the classical method of sterilization of replicators by immersion in ethanol and subsequent flaming. This is particularly relevant if the replicator has been used beforehand to transfer agar-medium grown colonies; not all cells within clumps of biomass that may have remained on the pins may be killed by the ethanol. Subsequent (short) flaming may not heat the pins sufficiently to ensure all pins are sterilized properly either (the flame itself causes some heating of the pins but the evaporation of the ethanol causes some cooling as well). For these reasons we recommend to use the “hot plate method” as described above. Instead, one may also consider heating the pins thoroughly using a flame. This method works well but requires some skill; i) one has to concentrate that all pins are sufficiently heated, and ii) the pins should not become too hot as this may reduce the life span of the replicator (excessive heating affects the quality of the stainless steel and aluminium used).