Fred Cross, The Rockefeller University, New York, NY 10065
The ROTOR +was initially designed primarily for use in budding yeast (Saccharomyces cerevisiae) genetics. We are carrying out broad genetic screens in the green alga Chlamydomonas reinhardtii, for which the ROTOR+ technology would be very useful. However, Chlamydomonas differs from budding yeast in cell size, growth rate, colony morphology and adhesiveness, and light responsiveness. Therefore, it was important to test specifically if the ROTOR+ technology would work with Chlamydomonas.
In this Applications Note I address the following issues:
- Are cells reliably picked up from liquid or solid medium onto the RePads replicating pads for the ROTOR+); are the cell numbers/density reasonably reproducible between pins, between pads, and between plates?
Reproducible pickup and dispensing in terms of a number of cells will depend on the surface properties of the cells; how clumpy or sticky they are, how much they stick to (or are repelled by) the transferring surface, etc. Chlamydomonas is different from yeast in these properties. For example, in replica-plating using velvets (standard method for yeast), Chlamydomonas colonies have a strong tendency to either stay entirely on the plate or transfer entirely to the velvet.
- Do cells retain high viability through this procedure?
Independent of a number of cells transferred, it is obviously desirable for high viability to be retained in the transferred cells. It is clear from our initial work that in many respects Chlamydomonas is more susceptible to damage during some operations than yeast or E. coli. For example, when picked from agar onto a glass or stainless steel needle, Chlamydomonas will lose viability almost completely after ~40 seconds in the air.
- Is there significant cross-contamination between pins or across the source or target plates?
Depending again on the properties of the organism, it could easily be imagined that a spray of viable cells could be spread by ROTOR+ replicating operations, resulting in cross-contamination and ruined experiments.
- What density of colonies is the maximum that can reliably be plated and distinguished using the ROTOR+?
The maximum density that the colonies can be plated is dependent on the size and the growth rate of the organism, and will affect the throughput of the screens that can be performed on the organism.
Application: Liquid-to-agar transfer
I used Singer PlusPlates, filled with 50 ml of TAP agar (standard Chlamydomonas medium) supplemented with 50 μg/ml ampicillin to suppress bacterial contamination. Because Chlamydomonas is highly motile in liquid medium and also highly light-responsive, we removed the interior lights from the ROTOR+, and kept the cover closed during operations to the extent possible. Without this precaution, the algae strongly aggregated in different regions of the source plate, obviously confounding the reproducibility of spotting across the plate.
Using an open plate filled with 25 ml of liquid TAP medium, containing wet Chlamydomonas (cc-124 background) at ~10^6 cells/ml, we made liquid-to-agar transfers using the long-pin 384 RePad, in a 384 -> 1536 arraying pattern (revisiting the source between pinnings). Plates were incubated for 3 days at 33°C under strong illumination (Figure 1A), and were examined microscopically at intervals.
Figure 1: Liquid-to-agar transfer
Microscopic examination showed that the pinned spots were approximately 1.5mm in diameter, with fairly random distribution of cells across the spot (Figure 1B). Microscopic counts of cells per spot plated indicated 62 +/- 11 cells per spot (mean +/- sd; n=56). The error is greater than predicted for a Poisson distribution, implying variability in actual volume transferred; a rough calculation suggests a volume distribution of ~60 +/- 10 nl, with occasional outliers.
High viability is evident since essentially every cell plated formed a microcolony by 18 hrs. (Figure 1C). A macroscopic image of the plate demonstrates reproducibility across the pinned area. Good reproducibility between pinned plates was also observed (data not shown).
At this pinning density, there was clear spatial separation between spots, with no evidence of contaminating colonies in between.
In other experiments, we have observed sporadic occurrences of ‘heavy’ pinning events, where a volume of ~120 nl is transferred. When this occurs, it is plate-wide, but specific to an individual pinning event. Because of the sporadic nature of these occurrences, we have been unable to determine how they come about. It is not reproducible for a given RePad™, source or target plate.
The ROTOR+ requires ~5-10 sec between pickup and plating. To determine viability of Chlamydomonas held in air on the RePad™, I paused operation after pickup and before plating for an additional 0, 10, 20, and 40 seconds. The number of cells transferred and their viability on incubation remained essentially unchanged.I tested liquid-to-agar transfer from a Singer plate containing 50 ml of cell suspension at the same density rather than 25. Results were essentially identical, indicating that the effective drop size is largely independent of the depth of liquid into which the pin is inserted.
Application: Agar-to-agar transfer
A lawn of cc-124 background Chlamydomonas on a Singer PlusPlate containing TAP agar was used as a source. A 384 long-pin RePad™ was used to pick up and transfer cells to a fresh plate, pinning 16 consecutive spots without a revisit to the source. This results in 6144 spots, in sets of 16 that progressively dilute the inoculum. These plates were examined microscopically, and incubated to grow up colonies.
Figure 2 shows images of the spots. Spot diameter is ~1 mm. The sequential pinnings result in progressive diminution of the number of cells transferred, although this effect is quantitatively irregular; thus, this method can be used to reduce inoculum density down to isolated single cells, although the dilution factor per strike is variable. Cell viability upon agar-to-agar pinning is generally high, probably comparable to liquid-to-agar pinning.
In general, both individual cells and resulting microcolonies and colonies stay within spatial borders dictated by the pin geometry, because Chlamydomonas is non-motile on plates, and the procedures are accurate with little effective spray or aerosol.
Therefore, these procedures are suitable for effective parallel transfer of large numbers of individual Chlamydomonas cultures, in a very space- and materials-efficient format.