61 E 12/l-4

MULT IVATOR
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Proposal for Mariner B Experiment - Capsule

May 10, 1961

       Submltted by
Joshua Lederberg, Professor of Genetics
Stanford Unlverslty School of Medfctne

In cooperation wtth George hobby and Jerry Stewart, Jet Pr6pulslon Laboratory

  The multlvator is a miniature, multiple purpose ttlaboratory" in \r:hlch a
series of simple measurements can be made on sample; of atmospheric dust. A
vartety of measurements are proposed, and others are to be considered.  They
have the common feature of testing a small sample of dust with a fluid reagent,
and of giving a read-out by simple optical or electrometric measurement.   In
addition to a determlnatlon of solution or suspension properties (turbidity,
pHI conductfvlty) the estlmatlon of phosphatase and other enzymes for which
ultramicro-tests are available would be of great help in estlmatlng the possible
existence of life on Mars.


  The multlvator is a miniature, multfple purpose 181aboratory'In which a
series of simple measurements can be made on samples of atmospheric dust. A
variety of measurements are proposed, and others are to be considered,  They
have the common feature of testing a small sample of dust with a fluid reagents
and of giving a read-out by a simple optical or electrometric measurements  The
device was originally conceived to attempt to cultivate Martian microorganisms
in defined culture media, but brief communication times make it unlikely that
changes based on growth could be observed.

  The effectiveness of the muitivator depends on 1) the acquisltlon of samples
of surface material from atmosphertc dust or from the ground, and 2) the choice
of feasfble but informative and sensitive tests to conduct on these samples.
Part one will be the special responslbillty of the development group at JPL.
Part two ~111 be the preoccupation of the Exobiology Laboratory at Stanford
University.  Our continuing studies are based on the assumption that several
samples of about one ml 11 igram of surface material will be available ("Clean"
terrestrial air contains cv one milligram dust per lOM3: a 100 km column of 1 cm
cross section),  Plainly this assumption would most readily be satisfied with
access to the ground environment; however, Mars' atmosphere may be dustier than
the Earth's.  Of the bfologlca:?y oriented experiments proposed hereinbelow,

the determination of phasphstase with the use of substrates that release a
fluorescent chromogen appears to be the most promising gamble for the detection
of evidence of 1 lfe,

  We propose a device containing 2)+ test chambers (two circular plates with
12 each) containing about one ml of fiuld each and into which the dust sample
can be introduced.  The plate will be rotated on its axle to allow each chamber
ln turn to intercept a ltght beam for photometric measurements. Other chambers
will have built-in electrodes for measurements of electric conductivity or
potential.  The JPL will assume responsibility for and has begun preliminary work
on the problems of mechanical design of the nultlvator and the collection of the
dust.  The Exobiology laboratory at Stanford w!ii conduct the calibration of suitable
test reactions and attempt to develop more sensitive and reliable assays,,

The foliowfng measuremants are now proposed or under review to be


conducted on aqueous srtsponslons of the dust samples.  Ref lned variants are

also indicated.  In scme cases, the same chamber can be used for several parallel

measurements,
A. Solution Properties
  1.  Turbfdi ty  - for rough calibration of further experiments
  2. pH
  3.  Conduct Iv1 ty
    &- polarographic measurement (help to Identify any electrolytes).

B,  Enzyme Tests

  1.





  2,




  3,
  4.



C.

  1,

  2.

  3.

Phosphatase  - calorimetric test by splitting of p-nitrophenyl phosphate
to release nltrophenol
d- fluorimetric test by splitting of fluorescent phosphates
Deoxyribonuciease -  conductimetric test by release of dialyzable
nucleotldes from DNA

- coiorimetrlc test by splitting of p-nityophenyi-thymldine-phosphate
Rlbonuciease  - tests analogous to B2 w!th RNA substrates
Other esterases (sui fbtase, acyiase) and glycosidases by methods
ana iogous to 4,.

Electron transfer enzymes

Substrates for electron transfer enzymes
Mlcrobiai growth on defined media by changes in turbidity, condu,ctlvity
or pH of the biochemical tests.

Phosphatases (84) are among the most ubiquitous enzymes - I kna~ of no

tissue or organism that has failed to show them - and they can be readily
demonstrated in small samples of soil, dust, sedimsnt from tap water, sea water
(about 3 x 10-g moles of nltropheno? released per milligram soil per hour), This


is just within comfortable detectivity in a IaboratQry spectraphotometer, and
might also be achieved by differential coio~imetry in a compact device.  For
example, a dual light source mlghc consist of two pin lamps with differential
fliters, and driven by AC modulated in opposite phase. Differential absorption
of one color would be recognized as an AC output from the detector,
     This list is oriented to the detection of clues to life.  In addltlon, the
measures of part A, together with other microscopic and video reconnaissance
studies will be invaluable preparation for the more ambitious efforts of the
Voyager series.
    We are studying the extension of these mfcrotests for phosphatase and
other enzymes as well as possible artifacts and improvements. The presence of
phosphatase activity in soil would be presumptive evfdence of life related to the
utilization of phosphate and can establish the limits of its prevalence.
    The sensitivity of this test can be augmented by the use of fluorescent
substrates.  Dr. Rotman (now working in my laboratory) has shown that the
detectlvlty with these substrates is several orders of magnitude higher than
with nltrophenyl ~po&tdr,. and he can readily demonstrate the enzyme level of
single bacteria, perhaps even single enzyme molecules.
    Equally important and widespread enzymes are the nucleases. We are
studying ultramicro methods for these.  One posslbi laity 1 s to contain the
reaction mixture in a dialysis sac within the multlvator chamber, with a film
of distilled water in contact with probe electrodes.  The release of low
molecular weight, dlalyzable nucleotides would be signalled by an increased
electrolytic conductivity between the electrodes. Whether this can be made
sensftlve enough depends partly on .the electrolyte content of the soil itself.
    The instrumental aspects of this proposal can be discussed by JPL.
As a prellmlnary estimate, the design would cost about 5 lbs (mostiy the
dust collection which might be integrated into the structure of the capsule)
and <5 watts power.  The information output would be of the order of i-10 blts
pereoccnd. We are considering further ways of mlnlmlrlng the weight of the
package.  The reagents themselves would, of course, weigh only an ounce altogether;
and the signal outputs are as simple as from any transducer.
    It would, of course, be foolish to delay too long the final decision on


test reactions and the ways these can be incorporated into the multivator.
However, the studies on ultramicro assay methods can proceed in parallel with
the mechanical and electronic design of the multivator since the basic output
from the chamber will be the same regardless of the type of reagent used for the
individual test and of the choice between a photometric or more direct electro-
metric estimation.

    The work at Stanford is already adequately funded as an aspect of our
current investigations and we are adding additional staff during the next two
or three months to help to accelerate them,  Additional support may well be
required for the instrument development work at JPL,  It should be possible to
construct at least an elementary version of the muttfvator with tests and controls
for, say, Items A, I, 2, 3 and Bl, withln the indicated schedule.