61 E 12/14

MULTIVATOR

Proposal for Martner B Experiment - Capsule

May 10, 1961

Submitted by

Joshua Lederberg, Professor of Genetics
Stanford University School of Medicine

In cooperation with George Hobby and Jerry Stewart, Jet Propuiston Laboratory

The multivator is a miniature, multiple purpose ''laboratory" In which a
serles 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 fluld reagent,
and of giving a read-out by simple optical or electrometric measurement. in
addition to a determination of solution or suspension properties (turbidity,
pH, conductivity) the estimation of phosphatase and other enzymes for which

ultramicro-tests are available would be of great help In estimating the possIble
existence of life on Mars.
The multivator Is a miniature, multiple purpose "laboratory In which a
series of simple measurements can be made on samples of atmospheric dust. A
varlety of measurements are proposed, and others are to be considered. They
have the common feature of testing a smal! sample of dust with a flufd reagent,
and of giving a read-out by a simple optical or electrometric measurement. 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 multivator depends on 1) the acquisition of samples
of surface material from atmospheric dust or from the ground, and 2) the choice
of feasf{ble but Informative and sensitive tests to conduct on these samples.
Part one will be the special responsibility of the development group at JPL.
Part two will be the preoccupation of the Exobfology Laboratory at Stanford
University. Our continuing studies are based on the assumption that several
samples of about one milligram of surface material will be avallable (''Clean'’
terrestrial alr contains -— one milligram dust per 10M2: a 100 km column of 1 em
cross section). Plainly thls assumption would most readily be satisfied with
access to the ground environment; however, Mars' atmosphere may be dustfer than
the Earth's. Of the biologicatly oriented experiments proposed hereinbelow,
the determination of phosphatase with the use of substrates that release a
fluorescent chromogen appears to be the most promising gamble fer the detection
of evidence of life.

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 [ts axle to allow each chamber
In turn to intercept a light beam for photometric measurements. Other chambers
wili have built-in electrodes for measurements of electric conductivity or
potential. The JPL will assume responsibillty for and has begun preliminary work
on the problems of mechanical design of the multivator and the collection of the
dust. The Exoblology laboratory at Stanford wlll conduct the calibration of sultable
test reactions and attempt to develop more sensitive and reliable assays.

The followfng measuremants are now proposed or under review to be
conducted on aqueous susponstons of the dust samples. Refined variants are

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

measurements.

A. Solution Properties

1.
2.
3»

Turbidity = for rough calibration of further experiments

pu
Conductivity
oG- polarographic measurement (help to Ident! fy any electrolytes).

B. Enzyme Tests

1.

2.

3

Cc.

2.
3.

Phosphatase =~ colorimetric test by splitting of p-nitrophenyl phosphate
to release nitrophenol
A - fluorimetric test by splitting of fluorescent phosphates
Deoxyribonuclease = conductimetric test by release of dialyzable
nucleotides from DNA

- colorimetric test by splitting of p-nitropheny!l-thymldine-phosphate
Ribonuclease - tests analogous to 82 with RNA substrates
Other esterases (suifatase, acylase) and glycosidases by methods
analogous to 4,

Electron transfer enzymes

Substrates for electron transfer enzymes

Microbial growth on defined media by changes In turbidity, conductivity
or pH of the biochemical tests.

Phosphatases (B44) are among the most ubiquitous enzymes - | know of no

tissue or organism that has failed to show them = and they can be readily

cemonstrated in small samples of soll, dust, sediment from tap water, sea water

(about 3 x 1079 moles of nitropheno! released per milligram soil per hour). This
is just within comfortable detectivity In a laboratory spectrophotometer, and
might also be achieved by differential coloiimetry in a compact device. For
example, a dual light source might consist of two pin lamps with differential
filters, 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 addition, 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 microtests for phosohatase and
other enzymes as well as possible artifacts and improvements. The presence of
phosphatase activity In soil would be presumptive evidence 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 tn my laboratory) has shown that the
detectivity with these substrates is several orders of magnitude higher than
with nitrophenyl compounds: 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 possibility fs to contain the
reaction mixture in a dialysis sac within the multivator 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 thls can be made
sensitive enough depends partly on the electrolyte content of the soll Itself.

The Instrumental aspects of this proposal can be discussed by JPL,

As a preliminary estimate, the design would cost about 5 Ibs (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 1-10 bits

per’ secoid. We are considering further ways of minimizing the welght of the
package, The reagents themselves would, of course, welgh only an ounce altogether;
and the signal outputs are as simple as from any transducer.

lt would, of course, be foolish to delay too long the final deciston 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 wel! be
required for the instrument development work at JPL. It should be possible to
construct at least an elementary version of the multivator with tests and controls
for, say, Items A, 1, 2, 3 and BI, withIn the indicated schedule.