Exob.
8-2

PROGRESS REPORT ON WORK AT STANFORD ON EXOBIOLOGY

This will be rather brief although we have hed notific.tion of support from
NASA since about mid-May. Procurement hes been slow and components have only
recently been assembled so that we can begin to check out 4 system of UV Vidicon
microscopy. The new Zeiss achromatic UV objectives have just recently arrived
and these appear to be far superior to the previously avauitable mirror objectives
and catadioptric lenses that were previously available. We nave assembled a
System for microscopy at 2537 R using a low pressure mercury source, interference
and chemical absorption filters (neither of them very satisfactory), the Zeiss
Objectives, and 4 television chain incorporating a UV-sensitive Vidicon tube
(two samples of tie RCA Vidicon and one fram EMI in England). The latter seems
to give a superior picture with better resolution, short lag and, probably,
higher sensitivity. However, both tubes, although gosignated as UV~sensitive, are
rather diszppointing in their performance at <3000 A and probably have not been
designed primarily for such an application. They would be relatively satisfactory
if we could operate at considerably higher intensities of monochromatic UV itlum-
ination but this is difficult to obtain in any event and should probably be
avoided because of its potential damage to viability and to organic materials. The
present microscopic arrangement is, however, already quite satisfactory for photo-
graphic recording. The enswer to our Present problems might lie a) in improvement
of the light source and filters, or perhaps better the incorporation of a simple:
monochromator and 6) the use of the much more sensitive image~intensifying UV
tube, the Ebicon. In any case, this development should be just the first step of
a system in which images can be compared as between the ultra-violet and the
visible. (The flying-spot microscope design is capable of much niigher intrinsic
Sensitivity than the Vidicon image tube, since a photomultiplier can be used as
the detector; however, the Ebicon may give a comparable result. )

AS an alternative to the use of ultra-violet for the visualizetion of trans-
parent objects, the now technically much simpler phase contrast optics might be
employed. Again these would be especially advantageous if a simul taneous
recording could be made of the form of a particle in phase contrast and its
transparency by transmission optics. This might be’converted into color rather
readily by the use of narrow pass filter elements in place of opaque diaphragms
in the phase optics ("color phase contrast!’).

Much more information can then be obtained from UV optical methods by
obtaining an absorption Spectrum, particularly in the range between 2400 and
3000 A. This UV information would be parallel to the image formation, and both
sets of data might be collected with the same optical system, the scan alternating
between wave length and position of the beam.

As far as | can determine, UV spectra of individual bacteria obtained in
such a fashion have not be published, but this partly reflects the limitations
of the microscope optics available until now. The Zeiss achromatic objectives
were designed for just 5uch analytical purposes at the instance of Caspersson in
Stockholm. He believes that it would be technically feasible to obtain such
Spectra, and has agreed to run some representative cultures on his own instrument
during the next few months. Serious problems, e.g. correction of scattering

J. Lederberg
Stanford University
9/4/60
losses, and especially the installation of Sutisfactory light sources must be
dealt with, but this approach is potentially the most powerful that could be
devised for the analysis of micro-organisms by remote technique. If the technical
development is successful, particles could be scanned for their spectra, and

this information then used to monitor the transmission of selected images which
consume more communication time.

Some of the most encouraging results obtained so far have been in the
fractionation of organic material from soils. After a considerable search for
Suitable solutions of adequate density, most of which were for one reason or
another rather unsatisfactory, we tested “Ludox'! at the suggestion of Kopec from
NYU. Ludox is . colloidal suspension of silica in water and can be made to 6
density as high as 1.20, which is more than adequate for the flotation of living
cells, while almost all minerals still sediment in it. The silica being chemically
almost inert and having no appreciable osmotic effect is reletively harmless to
the cells and leaves them quite viable. A number of types of soil sumple have |
been fractionated by flotation in Ludox and the method has proven to be startingly
efficient in the detection of micro-organisms even from rather unpromising
sources, for example, volcanic sand collected at 14,000 feet from Mt. Popocatapetl;
old dried mud from a core dredged from the Mariana Trench, and dust that had
settled on the roof of a car and exposed for appreciabie time to California sun-
shine. In each case, the Supernatant fraction was highly enriched for bacteria
and these could readily be seen at the very first glance through the mictoscope.
The same samples had only a marginatly detectable microbial population when
-examined directly. Samples from richer soils also displayed an abundance of
other kinds of organisms, with relatively little interference from residual
adherent soil particles. We ore now seeking even more murginal sumples to see
whether there is any likely sample from the earth's. surface in which we would
hot readily detect microbial life by this method. The flotation-separation is
facilitated by spinning the samples in a centrifuge and larger samples could be
efficiently processed by a continuous flow sedimentation. However, smaller
samples will sediment if merely allowed to stand several hours. te are now
designing a cell which will allow for microscopic observation with no handling
after the sumple is introduced into the cell. The relatively low density of living
particles, having an aqueous base, should be a fairly general principle for the
preliminary separation of them from mineral fragments. More refined criteria are
of course needed for a final conclusion.

(This method of separation promises to have great value as a general
laboratory procedure and may have practical applicetions in other areas of
medicine. )

The basic feasibility of the proposal to grow bacteria on permeable tapes
has been verified; flattened tubes of cellophane dialysis tubing were impregnated
with nutrient broth and would then very sutisfactorily support the growth of
various bacteria and fungi. These were then very informative when examined under
the microscope. The chief problem appears to be the loss of moisture by evaporation
so that the tapes will have to be maintained in a closed humidified chamber. In
view of the long transit time of the space craft, the tapes may have to be flown
dry and filled with water only at the time of the experiment; alternatively
nutrients may be prevented from diffusing from one site to another by mounting the
3.

culture spots as blisters on an impermeable, @.g. quartz, ribbon. We have not
succeeded In obtaining quartz ribbons as might be extremely useful as tapes for
the indicated purpose but they should not be too difficult to fabricate. More
mechanical skill and ingenuity than we can readily muster should be centered on
the production of prototype hardware along these lines for testing and improvement.

Recommendations.

Our experience to date suggests that the basic Strategic principles that had
been formerly enunciated are sound and should be exploited further as the basis of
exobiological detection. However, as we have now reached the stage where con-
siderable new hardware, both electronic and mechanical, must be designed and
built, it is essential that we have the cooperation of an engineering facility.
Needless to say, we would be very happy to maintain a close continuing contact
with such a facility and in particular to assess the results for their biological
usefulness.

In view of the pressure of vehicle schedules, it would be foolhardy to
delay the development of prototype hardware for the exploration of every worthwhile
accessory. New technical advances and new insights into scientific goals would
lead to indefinite postponement of any actual experiment:

Enough information is now at hand to justify the construction of the basic
mechanism of an automatic microscope. This should have provision for the trans-
port of specimens, focus, photoelectric conversion, color discrimination, signal
réduction and telemétry. The first version might be designed to use color phase
contrast, which is technically simpler than UV microscopy. This basic instrument
would be a valuable tool even if time did not permit the addition of the accessories
to improve its sensitivity and precision. Concurrently, design work should
proceed on the technique of collection of samples, and on their centrifugal
fractionation, to furnish theinput to the detector system. Thig much of the system
should lie within the reach of present art. Study work and preliminary trial are
also required for the microspectrophotometer accessory to the microscope, which
does pose serious technical problems, especially the provision of a suitable
light source. A bench model should be constructed for tests on terrestrial
samples as a preliminary to its adaptation to the system for spaceflight. By
concurrent work on the particularized problems thus indicated, we can be sure of
having the best instrument that we had the means to develop in time for schedules
of planetary flight. There are cogent reasons besides the relative scientific
impatience of biologists why such instrumentation should accompany the earliest
planetary explorations. Laboratory work in biology does not now have the benefit
of a well-established tradition of development of instrumentation. Much more than
in the physical sciences, spaceflight research in biology will have to develop new
instruments for which only the principles, and not their technical realization,
are already current in laboratory practice.