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M96 Washer Operations

The M96 use twelve 8-way dispensing heads and twelve 8-way aspirating heads to wash all the wells of a microplate simultaneously. The dispense heads are positioned to direct a stream of wash fluid at the center of each well and the aspirate heads are held above the microplate, clear of the fluid, until aspirating begins, thereby avoiding cross-contamination between plates.

Wash fluid volumes, dispense/aspirate modes, number of wash cycles, soak times and mixing times are all programmable. Nine programs can be stored in memory. Each program can include up to four subroutines. This ability to LINK subroutines allows different types of wash cycles to be used within one program.

A system of menus, presented on the alphanumeric display, guides the user through setting up procedures. Programs can be created from scratch, edited and "cloned" from one stored program to others. The program most recently used in a session automatically becomes the default when the machine is switched on for a new session. User communication with the washer is via a numeric keypad. Keys 1 through 4 are context sensitive (sometimes known as "soft" keys), and are used to select menu options.

If a supply of distilled water is kept attached to the washer, rinsing and emptying the washer at the end of a session requires only a single stroke.

PRIMING

To PRIME the washer there must be a wash solution supply line connected to the "Fluid 1" connector, and an extraction system connected to the "Vacuum" connector. The internal logic will not allow a wash program to be executed unless the washer has been primed at least once since power up.

Press the PRIME key and wait about 70 seconds. If you want to check that the washer is really priming, watch the syringe through the window and assure yourself that, by the end of the priming process, it is full of liquid (a small residue bubble is normal). If you are not satisfied that the system is fully primed, you may repeat the PRIME as many times as necessary.

Do not PRIME too early. If you are using a typical saline wash solution you should avoid priming more than about 20 minutes before you are ready to start washing plates.

INITIALIZATION

An automatic system initialization occurs every time the washer is powered up. Initialization causes all moving parts of the washer to actuate as a function check. If all is well, the Main Menu is displayed after about 20 seconds, and the washer is ready for use.

MICROPLATE PROFILE

Microplates are available with various well shapes and sizes. The M96  position dispense and aspirate tubes as precisely as possible and therefore have to be "trained" to know some physical dimensions pertaining to the microplate being used. The profile procedure is a preliminary step in editing a program (and each program has its own unique profile stored along with the other parameters). What profiling does is define where on the "Z" (vertical) axis the top surface of the plate is, where the bottom of the wells are, and where on the "X" axis the centers of the wells are.

It is particularly important to carry out the profiling procedure if you are going to use round bottom, V-bottom or unusually shallow wells to make it unlikely that you will crash the aspirate tubes into the plate if you omit to run profile. If you do not run profile you will probably find your washer leaves too much fluid in the plate typical flat-bottom microplates.

The plate used for profiling must be representative of the plates you will wash with the program concerned. However, do not use a plate that is coated or treated or one you subsequently need to wash. The act of profiling causes the aspirate tubes to touch the plate and material from the plate could contaminate the tubes.

RINSING

The bores of both the dispense tubes and aspirate tubes are rather narrow. This is a necessary feature because achieving even dispensing or aspiration through 96 separate channels requires that no one channel be too "easy" a route for liquid to flow through. A shower head or lawn sprinkler works on the same principle.

As with showers and sprinklers, any buildup of salt deposits in the narrow bores will cause uneven flow and will quickly lead to the complete blockage of one or more tubes. As most plate washing fluids are salt-laden buffers, the M96 has an entirely separate fluid inlet port for distilled water and a one-step rinse procedure to thoroughly displace all salt solutions from the internal plumbing.

We strongly recommend that the rinse procedure be carried out whenever the washer is being shut down. The consequential use of wash fluid for
re-priming is typically worthwhile because stripping the tubes down for cleaning, although easy to do, does take time and, of course, requires re-priming anyway.

7 WASH TYPES

If your application requires the simple removal of a reagent presently in the plate wells, the straightforward dispense/aspirate wash type (type 1) is probably adequate. This is the wash type that is quickest and uses the least amount of wash fluid.

If you are concerned about ensuring that material high up on the walls of the wells is washed away, SUPERWASH (type 2) should be used because it fills the wells to the brim with simultaneous aspiration from the top surface to ensure no well-to-well contamination.

If you need to leave the plate as dry as possible, use SUPERVAC (type 3). This causes the aspirate tubes to traverse the well bottoms extracting more residue liquid. If you only need the extra aspirating efficiency of SUPERVAC for the last cycle, use the linking feature to append a SUPERVAC subroutine to a normal aspiration.

SUPERWASH and SUPERVAC can be used together (type 4), and any permutation of wash types can be run sequentially if the linking feature is used in defining a program.

Dispense only (type 5), aspirate only (type 6), and SUPERVAC only (type 7) are also available. These wash types may be useful for unusual applications, but their main utility is as subroutines linked to the more conventional wash types.

All wash types involving aspiration start with an aspiration when they are used as the first subroutine. The dispense only type does not aspirate. You should be careful to not flood a plate when using the dispense only type (remember to set the "number of washes" to be 1 when doing dispense only).

LINKING

Linking subroutines is a very useful feature, once the concept is understood. Here are some examples to illustrate the applicability:

Imagine you need to leave the plate as empty as possible but also want to process plates as quickly as possible through three wash cycles. A 3 cycle dispense/SUPERVAC would fulfill the first requirement but a 2 cycle dispense/aspirate followed by a single dispense/SUPERVAC would be quicker.

Now imagine that you are concerned about contaminants high up on the wall of the wells but want to minimize consumption of wash fluid, and again need to wash three times. A three cycle SUPERWASH washes the walls but a single SUPERWASH linked to 2 ordinary dispense/aspirates may serve as well, uses less fluid and is also a little quicker.

Finally, what if you want to leave the plates full of wash fluid after, say 2 complete wash cycles? Two dispense/aspirate cycles followed by a dispense only cycle.

 
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