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thesis entitled

The effect of various low pressures on the per- r
cent livability of Serratia Marcescens and 4

Micrococcua Pyogenes var. anreoua deaiceated
from the frozen state.

presented by
Robert L Christian 1_ ,' .4 4
has been accepted towards fulfillment

' If of the requirements for
V M. 5. degree in Bacteriology

4:424:44:

r / Major profe‘or

 

L1 Date July 31, 1952

 

 

0-169

   

THE EFFECT OF VARIOUS LOW PRESSURES ON THE
PER CENT .LIVABILITY OF SERRATIA MARCESCENS
AND MICROCOCCUS PYOGENES VAR. AUREUS
DESICCATED FROM THE FROZEN STATE

 

 

BY

ROBERT T . CERIS TIAN

A THESIS . 4 ‘4

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Bacteriology

1952

T £515

.44 -. e- ‘45“:—

TABLE OF CONTENTS

INTRODUCTION ..................... _ .......
HISTORICAL REVIEW . ..... ' ............ ; .....
MATERIALSANDMETHODS ......
RESULTS ........................... .. . . .
DISCUSSION ..................... - ........
SUMMARY .................... , ....... .. . . .

REFERENCES ........... ' .................

(3’7“ ’5‘ 5} f T"
'4“... (R15 1; 2.4.1:...) ,3

To Joan

ACKNOWLEDGMENTS

The author wishes to thank Dr. J. J. Stockton, Assistant
Professor of Bacteriology, for suggesting this problem and

guiding it to its conclusion.

INTRODUCTION

The preservation of bacteria in their normal physiological
and antigenic state over long periods of time has for years been
a problem to the bacteriOIOgist. A number of methods of preser-
vation have been developed, many of which have shown some de-
gree of promise. Most were discarded either because they were
not satisfactory for the preservation of the more fastidious organ-
isms or because of the low per cent viability of the preserved
cultures. The most generally satisfactory process developed to
date is the freeze-drying, or ly0philizing, process.

Freeze-drying, with certain variations, has been used for .
the preservation of bacteria since the beginning of the present
century. In the past decade it has achieved prominence as a
means of preserving various biological products, antibiotics, and
blood fractions. However, there still remain many questions in
the mind of the bacteriologist, a few of which are: How many
of the organisms survive? Are there any changes in the bio-
10gica1 characteristics of the organisms? What are the physical

factors to be considered in lyophilization? Some of these

questions have been at least partially answered. The physical
factors to be considered have been reviewed by ROgers (1914),
and the per cent viability by Naylor and Smith (1946).

Various investigators have agreed that the best way to
store ly0philized organisms is in a vacuum, but there have not
been reported quantitative studies of the effect of low pres-
sures on bacteria during storage. Most assuredly, the lower
the pressure at the time of sealing, the more likely are the
desiccated cultures to survive. Laboratory equipment available
in most bi010gica1 laboratories is not of the caliber to allow
drying and sealing at pressures appreciably lower than about 50
microns of mercury. Other factors remaining constant, how
does the per cent viability of a culture sealed at 50 microns
of mercury pressure compare with that of a culture sealed at
100, 200, 500, or 1,000 microns of mercury pressure? What is
the upper limit of sealing pressure at and above which no ap-
preciable viability can be expected to remain during a reason-
able storage period?

Two organisms were chosen in attempts to answer these

two questions. They were Serratia marcescens and Micrococcus

 

pyogenes var. aureus. The former was chosen since it is

3
relatively susceptible to the drying process (Naylor and Smith,
1949), and the latter because of its high resistance to unfavor—

able conditions, including drying.

HISTORICAL REVIEW

That clothes dry and ice slowly "wastes away" on a
very cold day is an age-old observation. The knowledge of
this phenomenon is old in science, too. It was old in 1813,
when William Hyde Wollaston demonstrated freezing by evap-
oration and sublimation before the Royal Society of London.
It is because of the simplicity and clarity with which he dem-
onstrated the relationship between vapor pressure, temperature,
and cooling by evaporation that his experiment is still cited
today.
That a fluid from which a portion is evaporated,
becomes cooler in consequence of the heat absorbed by
that part which assumes the gaseous state; that fluids rise
in the state of vapors at lower temperatures when the
pressure of the atrnosPhere is removed and consequently
may be cooled to a lower degree by evaporation ”in vacuo"
than in Open air are facts too well known to need confirma-
tion before members of this society.
'However, feeling that a new mode of demonstration of an estab-
lished principle deserved attention, he described a little instru-
ment for demonstrating this phenomenon.
Let a glass tube be taken, having its internal diame-

ter about 1/8 of an inch, with a ball at each extremity of
about one inch in diameter and let the tube be bent to a

5
right angle at a distance one-half inch from each ball. One
of these balls should contain a little water (if the ball be
more than half full, it will be liable to burst by the expan—
sion of water in freezing) and the remaining cavity should
be as perfect a vacuum as can be readily obtained . . . .

When an instrument of this description has been suc-
cessfully exhausted, if the ball that is empty be immersed
in a freezing mixture of salt and snow, the other ball,
though at the distance of two or three feet, will be frozen
solid in the course of a very few minutes. The vapor con-
tained in the empty ball is condensed by the cormnon Oper-
ation of cold and vacuum produced by this condensation
gives opportunity for fresh quantity to arise from the 0p-
posite ball, with proportional reduction of its temperature.

Professor John Leslie used sulfuric acid to absorb the vapor
under vacuum in an experiment that obtained to the same end.

These two early investigators demonstrated the princi-
ples of freezing by vacuum and sublimation, but to them it was
merely the demonstration of a physical principle for which they
suggested no practical application.

The modern process of freeze-drying is to a great ex-
tent the work of the biologists who needed a way to preserve
labile biolOgical products. With the prOgress of bacteriolOgy
and serology the deveIOpment of a dependable method for the
preservation of serums and bacterial cultures became impera-
tive. A successful method for preserving serum was described

by Martin, in 1896. He filtered the serum, which was kept at

40 degrees centigrade, through a Chamberland candle and

collected the water vapor in a cooled condenser connected to a
vacuum pump. The stability of this dried serum was recog-
nized by Erlich.

D'Arsonval and Bordas (1906) carried out low-tempera-
ture distillations (15° C.) in a similar manner. Their appa-
ratus approached that now used for freeze-drying. With a
better vacuum pump they could have frozen the serum and dried
from the frozen state. Flosdorf (1949) stated that other workers,
including Noguchi, Friedberg, Massol and Crysezi, Massol and
Nowaczynski, Predtetschensky and Grigorowitsch, described
similar methods for drying serum in 22112. However, none of
these workers dried from the frozen state.

During this time, other investigators showed that bacteria
could be subjected to very low temperatures and still remain
viable. Macfadyen (1900) froze bacteria in liquid air (-190° C.)
and stored them in that medium for 24 hours without destroying
their viability. He later reported that exposure to liquid hydro-
gen at a temperature of -252 degrees centigrade (21° absolute)
did not kill all the cells.

Rogers (1914) stated that Paul and Pratt dried staphylococci

on garnets and kept them in liquid air for one month. They found

no change in the reaction of the cells to disinfectants. Smith
and Swingle (1905) observed that most cells of a bacterial cul-
ture survived freezing, and that the critical temperature was
about 0 degrees centigrade. They felt that those organisms
that could survive 0 degrees centigrade could survive absolute
zero. Further experiments showed that repeated freezing and
thawing slowly reduced the number of viable cells in a culture.
It has since been shown that the death of the organism is due
to the disruption of the cell by ice crystals. Quick freezing
greatly reduces the size of the crystals formed, and therefore
reduces the number of organisms killed.

The above evidence indicates that bacteria (and probably
all biological materials) are not greatly harmed by freezing,
that dried products are more stable than hydrated products,
and that drying from the frozen state is possible.

It comes as no surprise, therefore, that Shackell (1909),
while attempting to preserve glycogen in the liver, minced the
tissue and dried it from the frozen state. After freezing the
material in an ice-salt mixture, he placed it in a desiccator
containing sulfuric acid as the desiccant, and dried the material

_ip_ vacuo. Harris and Shackell (1911), using Shackell's method,

 

8
dried the brains of rabbits infected with rabies virus and found
that the tissue retained its infectivity for 4 months. They be-
lieved that organisms were killed by slow drying because of
the high concentration of salts produced.

Hammer (1911) was the first to use the freeze-dry
method to preserve bacterial cultures. He placed strips of
filter paper, impregnated with bacteria, in test tubes, froze
them in an ice-salt mixture, and put the tubes in a desiccator.
The desiccator, containing sulfuric acid as the desiccant, was
then sealed and evacuated. After 48 hours, the desiccator,
which had been kept in ice, was allowed to come to atmospheric
pressure. The organisms thus preserved were viable for 57
days.

Shattock and Dudgeon (1912) dried films of bacteria on
glass cover slips and stored them in the presence of charcoal
at the temperature of liquid air (-190' C.). Although the re-
sults as to the viability of the cells at this temperature were
inconclusive, they did determine the resistance of the dried
cells to heat and to ultraviolet light. They found dried cultures
to be more resistant to heat, although their resistance to ultra-

violet light was unchanged.

Rogers (1914) found that the dried ”starter cultures"
used at that time in the dairy industry were very poor products.
While investigating the methods used to produce these cultures,
he found that most of the organisms were killed during the
drying process. His first attempt to find a better method of
preservation consisted in drying the organisms rapidly at a
lowered temperature under reduced pressure. The drying was
hastened by bubbling warmed air through the culture. This
method, while very successful, was deemed too expensive for
the average laboratory. He next investigated drying from the
frozen state. After testing various desiccants, including cal-
cium chloride, sulfuric acid, phosPhorus pentoxide, and a cooled
desiccating chamber, he found that calcium chloride was too slow
and that the temperature he was able to obtain in the cooled
chamber was insufficient to lower the vapor pressure to a prac-
tical level. He was most successful using a chemical desiccant._
In reviewing the conditions necessary for the survival of cells
in the dried state, Regers stated: "The nearer the cell ap-
proaches an absolute dormant condition the longer its actual
death will be postponed." The lower the metabolic activity of

the organism the longer will be its survival. The conditions

10
which control this activity are the moisture content, the tem-
perature at which the culture is held, and the nature of the gas
with which it is in contact. If the moisture content is below 5
per cent, the activity of the cell will be very low, but lowering
the moisture content below 5 per cent does not substantially in-
crease the survival rate. Bacteria succumb to unfavorable con—
ditions most rapidly at the temperature at which they grow most
rapidly. .Rogers observed that the loss of bacteria at 0 degrees
centigrade was within the limit of experimental error, while at
17 degrees centigrade there was a 30 per cent loss, and at 30
degrees centigrade there was a 60 per cent loss. The results
with various gases showed the greatest loss of activity to be in
oxygen or in air, and the least loss of activity to be in a vacuum.

Swift (1921) introduced a method for freeze drying that
remained the standard process for many years. The organisms
were first concentrated, and most of the supernatant fluid
decanted. The tubes were than placed in glycerol in a desic-
cator, the desiccator surrounded with an ice-salt solution, and
.the organisms frozen in the desiccator at -4 to -6 degrees cen-
tigrade. After the cultures were frozen, the desiccator was

sealed and the apparatus placed in an ice box for 12 hours.

11
At the end of this period, the tubes were brought to atmos-
pheric pressure, sealed with paraffin, and stored in the dark
at room temperature.

Brown (1925) published two papers detailing three meth-
ods for the preservation of bacterial cultures. The first meth-
od consisted in simply drying the organisms on glass cover
slips and storing them under vacuum. The second involved
impregnating strips of filter paper with bacterial cultures and
drying them in 2332, after which the papers were sealed in
tubes containing a small amount of calcium chloride. The third
method was a modification to permit storage of larger numbers
of the filter paper strips. Pint milk bottles fitted with ground-
glass covers were substituted for the tubes, They were sealed
by evacuating them in a desiccator, then allowing the atmosPheric
pressure to enter suddenly. This sealed the bottles by forcing
the covers down on the bottles. Cultures preserved in this way
were viable for two years.

Meanwhile, this process was being used for the preserva—
tion of other biolOgics. Sawyer, Lloyd, and Kileken (1929) pre-
served yellow-fever virus by freezing the blood of monkeys that

had the disease. Blood was drawn on the first day of the attack

12
and frozen by Swift's (1921) method. The dried product was
then sealed in tubes containing a small amount of calcium chlo-
ride.

Hartley, Eagleton, and O'Kell (1923) successfully dried
serum and hemolysin under reduced pressure by passing a
stream of warm air through the tubes. Complement, however,
lost 70 per cent of its activity when treated this way. Craig's
(1931) method of drying complement yielded a more satisfactory
product. Fresh guinea pig serum was placed in a desiccator
containing calcium chloride, and the chamber evacuated by means
of a vacuum pump, until the serum froze. The desiccator was
allowed to stand at room temperature for 6 hours, after which
the dried complement was stored at low temperature in 292.3.

Elser, Thomas, and Steffen (1935) reported the investiga-
tions in freeze-drying begun by Elser in 1909. Using inactivated
serum as a suspending medium, he had ly0philized cultures of
such sensitive organisms as pneumococci and meningococci
which, he reported, were viable after 15 years of storage in
121522. These investigators deveIOped the first practical cold
condenser which was the basis of the commercial application of

the freeze-drying process .

l3

Flosdorf and Mudd (1935) reported work similar to that
begun by Elser (1935). They described ly0philizing units of
capacities suitable for all needs from those of a small labora-
tory to a semicommercial plant. Their publication contained
definite procedures and drawings of the equipment. The ap-
paratus consisted of a manifold which was continuous with the
condensing chamber. The condensing chamber was kept at -78
degrees centigrade by inunersing it in a dry ice-organic solvent
bath. A suitable vacuum pump was connected to the condensing
chamber. The maximum pressure allowable was 0.70 milli-
meter of mercury, but lyophilization should be carried out much
below this, preferably at 0.010 to 0.050 millimeter of mercury.
The material to be ly0philized was shell frozen at the tempera-
ture of dry ice. (The layer of serum should not be more than
3.0 millimeters thick.) The material was kept frozen by the
latent heat of sublimation. This was the first paper on 1y0phi1iza-
tion by the man who was destined to become the authority in this
field. Flosdorf, in his many papers and excellent book, has
covered this field completely. Although he has contributed rela-
tively little to the theoretical knowledge of lyophilization of bac-

teria, the tools he has given the bacteri010gist are invaluable.

14

As so often happens when an adequate tool is placed in
the hands of scientists, there was a great deal of work done
in the next few years. Eagle, Strauss, and Stimer (1935)
showed that complement could be dried by the method of Flos-
dorf and Mudd. They demonstrated that complement processed
in this way and stored for 18 months in the ice box was in-
distinguishable from complement prepared from freshly drawn
guinea pig blood. Rivers and Ward (1935) showed that vaccinia
virus mixed with pure gum acacia and dried from the frozen
state was suitable for intradermal injection in humans after
storage for 1 month at 38 degrees centigrade. Flosdorf and
Kimball (1940) reported that lyophilization preserved cultures

of HemoPhilus pertussis in Phase I for long periods.

 

Flosdorf and Mudd (1938) described the last major change
in the freeze-drying process. Because dry ice is the most ex-
pensive ingredient in the procedure, they attempted to find a
cheaper substitute for it. Anhydrous calcium sulfate was found
to be most satisfactory, because of its low original cost and the
fact that it could be regenerated. The process using this chem-
ical desiccant is called "Cryochem." The cost of drying one

liter of serum by the "Cryochem" method was between forty

15
and seventy-five cents. Drying a like amount of serum using
the ”ly0phil" method cost between three and twenty dollars.

Morton and Pulaski (1938) reviewed the whole field of
pure-culture preservation. They compared freeze-drying
(Swift, 1921) to several other methods. They found that the
freeze-drying process gave good results but was much more
expensive in time and equipment than the other methods tested.

Leifson (1936) dried bacteria on perforated glass beads
and on other substances. The dried cultures were stored in
3.9.9.9. after the method of Brown (1925)..

Swift (1937) reported several modifications of his orig-
inal method. He now froze the organisms in a glycerol—dry
ice mixture and dried them in a desiccator kept in an insulated
box. He repeated his earlier procedure of sealing the organ-
isms under atmospheric pressure with paraffin.

Rahn (1945) reviewed physical methods of sterilization
and discussed many points that apply to this work. While dis-
cussing drying as a means of sterilization, he pointed out that
although bacteria and viruses survive well in the dried state,
there is a steady decrease in viability. At 37 degrees centi-

grade, death was logarithmic, and its cause was found to be

l6

oxidation. Cultures of Vibrio comma slowly lost their viability

 

when kept at warm temperatures in a high vacuum over phos—
phorus pentoxide.

While discussing death by freezing, Rahn (1945) pointed
out that most organisms could not grow at lowered tempera-
tures; in fact, they died slowly, Ninety-five per cent of the

organisms of a young culture of Escherichia coli were killed by

 

cooling them rapidly from 45 to 10 degrees centigrade; however,
gradual cooling did not kill the organisms. Slow freezing formed
ice crystals which punctured the plasma membrane of the bac-
terial cell, killing the organism. Very rapid freezing, on the
other hand, produced a different picture. The water was changed
to a glass-like amorphous mass. Rapid freezing and thawing two
hundred times did not kill the tubercle bacillus.

Lord Stamp (1947) dried bacteria, susPended in gelatin,
ascorbic acid, and other agents, on paraffin discs. He also
ly0philized cultures by the method of Flosdorf (1935). He found
that gelatin was a better suspending medium than blood, serum,
mucin, or polyvinyl alcohol, under the conditions of his test.

The addition of ascorbic acid increased the survival rate, but

the addition of glutathione had no effect. He also tested the

17
relative merits of slow drying versus rapid drying. Slow dry-
ing on paraffin discs in the presence of phosphorus pentoxide
under vacuum gave approximately three times the survival rate
obtained with rapid drying by ly0philization. Viability under
these conditions was reduced to one-half after 2 years of
storage, and to one-quarter after 3 years. No change was
found in chromOgenicity or virulence during this period.

Naylor and Smith (1946) dried §_. marcescens in various

 

ways. They first dried the organisms, as did Stamp (1947), on
paraffin discs in the presence of phosphorus pentoxide _i__r_1_ _v_a_<_:_}_I_9_
at 4 degrees centigrade. After 24 hours, the vacuum was re-
leased and the discs stored at room temperature and atmos-
pheric pressure in the presence of phOSphorus pentoxide. Stor-
ing bacteria in this way, they reported, gave less than 0.1 per
cent survival after 10 days of storage. If the cells were sus—
pended in a 1.5 per cent ascorbic acid and 1.0 per cent mannite
solution and dried as above 45 per cent survival was recorded
after the same period. There was no further decrease in via-
bility during a 5-month storage period.

They next investigated ly0philization as a means of pre-

serving cultures. The cells were first concentrated, then

18
resuspended in a solution containing ascorbic acid, thiourea,
ammonium chloride, and dextrin. The optimum concentration

of these substances was found to_ be:

ascorbic acid ........ 0.5 per cent
thiourea . . , ........ 0.5 per cent
ammonium chloride . . . 0.5 per cent
dextrin . . . ......... ' 2.0 per cent

One milliliter of the culture was shell frozen in a 10-
milliliter ampoule by spinning it rapidly in a dry ice-alcohol
mixture.

Investigation of the effect of moisture on viability showed
a decrease in viability as the moisture content of the cells was
reduced from 2.1 per cent to 0.42 per cent.

The following data show their findings regarding the

viability of cells stored in air, nitrogen, and vacuum:

air...., .......... 9percent
nitrogen . . . . ....... 28 per cent
vacuum . . . . . . ; . . . 99 per cent

They dated that 100 per cent counts were obtained under optimum
conditions of drying and storage.

Proom and Hemmons (1949) reported their experience in
preserving between 1,500 and 2,000 strains of bacteria over a
number of years. They used the method of Swift (1937), with a

few modifications. The cultures were grown on agar slopes,

19
then emulsified in nutrient broth. The emulsion was put into
small test tubes and frozen in a dry ice-acetone mixture in a
desiccator containing phosphorus pentoxide. The desiccator was
then evacuated. The following morning the tap of the desiccator
was closed, the vacumn pump turned off, and the unit allowed
to come to room temperature. Dry nitrogen from a cylinder
was run slowly into the desiccator. The tubes were then re-
moved and labeled. This completed the primary drying. For
the secondary drying, the tubes were again placed in the des-
iccator and redried in a high vacuum at room temperature over
phosphorus pentoxide for 7 days. Dry nitrOgen was then run
into the desiccator, and the tubes were removed and sealed.

The organisms tested for resistance to drying fell into
three groups. Typical of the first group is M. pyogenes var.
aureus, which is very resistant and remains viable for many
years. §.£91i_ is representative of the second group. Many
of these. cultures die after a few years of storage. An example

of the third group is Neisseria intracellularis. These cultures

 

begin to die after two years' storage.
In an attempt to simulate long-term storage conditions,

the desiccates were heated to 60, 80, and 100 degrees centigrade

20
for 1 hour. There was evidence that the loss in viability ran
parallel with the storage loss at lower temperatures. Although
they realized that ”it is always possible that heating to high
temperatures introduces factors which would not Operate at
room temperatures . . . in practice it has proved a useful
method Of estimating probable storage loss."

Atkins, Moses, and Gray (1949) found that when yeasts
were ly0philized, only very small numbers survived, and that
variants were found in more than 50 per cent Of the revitalized
cultures.

Hutton, Hilmoe, and Roberts (1951) develOped an elab—
orate apparatus for the control of many factors during the
freeze-drying process, They were able to control the temper-
ature of the ice film before and during drying, to vary the rate
Of drying, and to stop the process of drying at any stage. Using

Brucella abortus as the test organism, they achieved a much

 

higher percentage of viability if the temperature of the frozen
culture was never allowed to rise above 30 degrees centigrade.
They also showed that there is an Optimum rate Of sublimation

for the greatest recovery of cells. Their results showed a

21
direct relationship between viability and the per cent of moisture
remaining in the dried cultures.

The present status of freeze-drying is summed up by
Harris (1951) in his paper reviewing the laboratory application
Of the process. He says, "There seem as yet to be no general
rules for the successful preservation Of all bacterial cultures,
and if the culture alone cannot be dried there is an almost un-
limited choice of protein-containing compounds and mixtures

which may be tested for preservative action."

MATERIALS AND METHODS

LyOphilization of _S_. marcescens was carried out follow—

 

ing the procedure outlined by Naylor and Smith (1946). The
cells were grown in a liquid medium composed of 1.0 per cent
Difco tryptone, 0.5 per cent Difco yeast extract,_ 0.1 per cent
glucose, 0.2 per cent disodium phosphate, and 0.1 per cent
sodium dihydrogen phosphate at a pH of 6.8 to 7.0. This me-
dium, hereafter referred to as Medium 1, was dispensed in
250-milliliter amounts in 500-milliliter Erlenmeyer flasks.
Stock cultures were maintained on slants of the following com-
position: l.0 per cent Difco tryptone, 0.5 per cent Difco yeast
extract, 0.5 per cent glucose, and 1.5 per cent agar. Here-
after, this will be referred to as Medium 11. TO obtain good
pigment production, stock cultures were incubated at room tem-
perature.

Approximately two standard loopfuls of the stock culture
were transferred to Medium 1. According to Naylor and Smith
(1946), aeration proved to be a very important factor in the

production Of resistant cells in this medium. For this reason,

23
the liquid culture was agitated continuously during incubation
at 30 to 38 degrees centigrade. Agitation was accomplished by
the use of a Burrell Shaker employing amplitudes of 16 milli-
meters and a frequency of 170 cycles per minute. After 18 to
24 hours of continuous shaking, the cells were concentrated by
centrifugation, subjecting the culture to a relative centrifugal
force of approximately 1,400 times gravity for 1 hour. The
supernatant was then decanted, and approximately 3 milliliters
of diluent, composed Of 0.5 per cent ascorbic acid, 0.5 per cent
thiourea, 0.5 per cent ammonium chloride, and 2.0 per cent
dextrin, was added to the sediment.

The cells were resuspended, using a sterile wire loop,
the material pooled, and about 20 milliliters of diluent added.
These cultures were then shaken on the shaking machine for 60
minutes (amplitude 13.5 mm, frequency 150 cycles per minute),
and then plated to determine the initial count.

Serum vials (Kimble, A3-179, 2 ml) were washed with
distilled water, calibrated at 1 milliliter, plugged lightly with
nonabsorbent cotton, and sterilized by dry heat for 2 hours at
160 degrees centigrade. One milliliter of the cell suspension

was introduced into the vials with a l-milliliter tuberculin

24
syringe. The material was shell frozen in a dry ice-alcohol
bath at -70 to -78 degrees centigrade, revolving the vials so
that two-thirds of the wall was covered with an even layer of
the frozen cell suspension. The vials were stored in the
freezing bath until they were placed on the manifold of the
drying apparatus.

The ly0philizing apparatus consisted of a Cenco Megovac
pump, to which was attached a sixteen-outlet manifold which
6 held the vials by means of pressure tubing, and a condenser
filled with Drierite (anhydrous calcium sulphate). A Stokes-
McLeod pressure gauge was connected to the desiccating cham-
ber. The system was evacuated to a pressure of 300 microns
of mercury or less before the vials were attached to the sys-
tem. The vials were exposed to existing room conditions dur-
ing the process Of sublimation, and no attempt was made to
maintain controlled conditions of temperature and relative hu-
midity. LyOphilizatiOn was allowed to proceed for 24 hours,
then the vials were sealed with a cross-fire hand torch. Vials
were sealed at pressures varying from 50 to 1,000 microns of

mercury, as determined by the Stokes-McLeod gauge.

25

The ly0philized cultures were stored in the dark at
room temperature for periods Of from 1 week to 4 months.
They were reconstituted by adding sterile saline (0.9 per cent)
to the l-milliliter mark. Dilutions were made and plated on
Medium 11. The plates were incubated at 30 degrees centigrade,
and counts made after 24 to 48 hours.

The same general procedure was followed in ly0philiz-
ing cultures Of M. pyogenes var. aureus. Stock cultures were
maintained on Difco tryptose agar. .Difco tryptose broth was
inoculated with the organism and incubated without shaking for
48 hours at 35 degrees centigrade. The culture was concen-
trated by centrifugation, resuspended in sterile skim milk, and
then tubed, frozen, dried, and sealed exactly as were cultures

Of §_. marcescens. After approximately 3 months, the cultures

 

were reconstituted, dilutions were made, and they were plated

on Difco tryptose agar.

RESULTS

TABLE I

DATA PERTAINING TO EXPERIMENTS WITH S.
MARCESCENS LYOPHILIZED FOR 24 HOURS
AT APPROXIMATELY 100 MICRONS OF

 

 

 

 

MERCURY
Pres-
Date Date Original Final % sure in
Sealed O ened No. Count Count Via- Microns
P bility of Mer-
cury
8 6
2—26 3-5 1 127 x 10 265 x 10 2.0200 60
8 7
2-26 5-13 2 127 x 10 53 x 10 4.3300 60
8 6
2-26 5-13 5 127 x 10 102 x 10 0.7850 135
8 6
2-26 3-5 6 127 x 10 210 x 10 1.5800 135
8 7
2-26 3-5 8 127 x 10 55 x 10 4.3600 190
8 6
2-26 5-13 9 127 x 10 122 x 10 0.9610 400
2-26 3-5 10 127 x 108 100 x 105 0.0788 400
8 5
2-26 3-5 12 127 x 10 113 x 10 0.0890 550
8 6
2-26 3-5 13 127 x 10 470 x 10 0.3700 675

 

 

27
TABLE II
DATA PERTAINING TO EXPERIMENTS WITH PYOG.

VAR. AUREUS LYOPHILIZED FOR 24 HOURS AT
APPROXIMATELY 100 MICRONS OF MERCURY

 

 

 

Pres-
Date Date Original Final % 5‘?" m
S a1 d 0 ed 0' CO t CO t Vla- Microns
e e pen un un bility Of Mer-
cury
7 6
4-11 6-3 1 47 x 10 34 x 10 7.230 100
7 5
4-11 6-3 3 47 x 10 248 x 10 5.280 175
7 5
4-11 6-3 5 47 x 10 197 x 10 4.190 275
7 5
4-11 6-3 7 47 x 10 210 x 10 5.010 475
7 5
4-11 6-3 9 47 x 10 204 x 10 ' 4.340 400
7 5
4-11 6-3 11 47 x 10 45 x 10 0.957 800
7 6
4-11 6-3 15 47 x 10 31 x 10 6.000 1100
7 5
4-11 6-5 2 47 x 10 284 x 10 6.040 100
7 6
4-11 6-5 4 47 x 10 58 x 10 13.000 175
7 5
4-11 6-5 6 47 x 10 97 x 10 2.060 275
7 5
4-11 6-5 8 47 x 10 125 x 10 2.660 475
7 5
4-11 6-5 13 47 x 10 42 x 10 0.892 700
7 4
4-11 6-5 16 47 x 10 266 x 10 0.566 1100

4-18 6-10 1 78 x 10 46 x 107 59.000 70

 

28

TABLE II (Continued)

 

 

 

Pres-
Date Date Original Final 7' 8‘?" 1“
Sealed O ened Count Count Via- Microns
p bility of Mer-
cury
7 7
4-18 6-10 3 78 10 45 10 57.700 150
7 6
4-18 6-10 5 78 10 255 -10 32.700 275
7 6
4-18 6-10 7 78 10 195 10 25.000 350
7 6
4-18 6-10 9 78 10 172 10 22.000 410
7 6
4-18 6-10 11 78 10 140 10 17.800 500
7 7
4-18 6-12. 2 78 10 34 10 43.600 70
7 7
4-18 6-12 6 78 10 82 10 105.000 275
7 7
4-18 6—12 8 78 10 37 10 47.400 350
7 7
4-18 6-12 12 78 10 34 10 43.600 500
7 5
4-18 6-12 13 78 10 62 10 1.260 700
7 6
4-18 6-12 14 78 10 261 10 25.800 700
6 5
4-22 6-13 1 32 10 30 10 9.380 65
6 5
4—22 6-13 2 32 10 66 10 20.600 65
6 4
4-22 6-13 4 32 10 224 10 7.010 100
6 . 4
4-22 6-13 5 32 10 200 10 6.250 275
6 4
4-22 6-13 6 32 10 219 10 6.840 275

 

TABLE II (Continued)

29

 

 

 

Pres-
Date Date Original Final % 5?“? 1n
Sealed O ened 0' Count Count Vla- Microns
P bility of Mer-
cury
6 5
4-22 6-13 7 32 10 33 10 10.300 275
. 6 5
4-22 6-13 8 32 10 130 10 40.600 275
6 4
4-22 6-13 9 32 10 69 10 2.500 425
- 6 4
4-22 6-13 10 32 10 49 10 1.530 425
6 4
4-22 6-13 11 32 10 87 10 2.750 450
6 4
4-22 6-14 12 32 10 89 10 2.780 450
6 4
4-22 6-14 13 32 10 45 10 1.410 750
6 4
4-22 6-14 14 32 10 151 10 4.720 750
6 4
4-22 6-14 15 32 10 114 10 3.560 950

 

 

30

Figure 1. Plot of S. marcescens showing per cent viable cells

 

as per cent determined by plate counts when sealed

and stored at the indicated pressure.

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Figure 2. Plot of Micro. pyo. var. aureus showing per cent
viable cells as per cent determined by plate counts

when sealed and stored at the indicated pressure.

FIGURE 2

 

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0383/10'338 $7733 379171/1 7VN/9/HO

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700 800 900

600

200 300 400

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34

Figure 3. Plot of Micro. pyo. var. aureus showing per cent
viable cells as per cent determined by plate counts

when sealed and stored at the indicated pressure.

3

FIGURE

200 300 400

450

 

 

 

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e a 2 8 8 ‘2
asuaAogau 57730 379le '1th9180

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MICRONS Hg

36

Figure 4. Plot of Micro. pyo. var. aureus showing per cent

 

viable cells as per cent determined by plate counts

when sealed and stored at the indicated pressure.

4

FIGU RE

 

1000

300 400 500 600 700 800 900
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100 200

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MICRONS

DISCUSSION

The above data show a definite correlation between the
pressure under which a dried culture is stored and the per

cent survival of the organisms. This relationship is more evi-

 

dent with M. pyOgenes var. aureus than with S. marcescens,
probably because a larger number of cultures of M. pyogenes
were plated at the same time, and the methods had been better

develOped. Even considering this, S. marcescens showed the

 

same pressure-survival relationship.

The theoretical pressure above which M. pyogenes could
not be stored is approximately 835 microns of mercury, a fig-
ure obtained by extrapolating to the abcissa (Figures 2, 3, 4).
The experimental values are very close to this value. It is
evident that some organisms survived the theoretically maximum
pressure, but the per cent is very low. This is not surprising,
since Swift (1921) and Hammer (1911) preserved organisms by
drying and storing them at atmospheric pressure and room tem-
perature. The fact that the data from three experiments fall in

a relatively narrow range gives support to these conclusions.

39

During the early experiments using S. marcescens, more

 

than 100 per cent of the organisms were recovered on plating.
Proom and Hemmons (1949) reported a similar condition, and
concluded that it was due to a more explicit disaggregation Of
the clumps of bacteria. In these experiments, the concentrated
culture was thoroughly shaken to break up the clumps before
plating. Thus, there were no clumps to be broken up during
freezing, and the viability was consistently below 100 per cent.
The data in Figure 1 suggest that a higher per cent re-
covery could have been obtained if the pressure under which
the tubes were sealed had been lower. It seems that if a pres-
sure of l or 2 microns of mercury could have been obtained,
as reported by Naylor and Smith (1946), viability would have
been 100 per cent. It is unfortunate that pressures below 50
microns of mercury could not be Obtained on the apparatus used.
Figure 1 shows an attempt to determine the effect Of

storage time on the viability of S. marcescens. Cultures were

 

plated after 1 week and after 3 months of storage, and the re-
sults were plotted on the same graph. There seems to be

little, if any, difference in the number of organisms surviving.

SUMMARY

A correlation was found to exist between the pressure
under which a culture was stored and the per cent viability Of

ly0philized cultures Of S. marcescens and M. pyo. var. aureus.

 

Eight hundred thirty-five microns Of mercury was found to be
the highest pressure under which M. pyo. var. aureus could be

stored, under the conditions Of drying as described herein.

40

The fact that there was little batch-tO-batch reproduci-

bility is not of great importance. It must be remembered that

only the pressure was controlled, and that such factors as rate
of sublimation and per cent Of residual moisture, reported as
being of great importance by Hutton e_t 31. (1951), were vari-

able .

REFERENCES

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BROWN, J. H. 1925. The preservation of bacteria in vacuo.
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D'ARSONVAL and BORDAS, F. "Les basses temperatures at
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EAGLE, H., STRAUSS, H., and STEINER, R. 1935. The use
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ELSER, E., THOMAS, R. A., and STEFFEN, G. L. 1935. The
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FLOSDORF, E. W., HULL, L. W., and MUDD, S. 1945. Drying
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FLOSDORF, E. W., and KIMBALL, A. C. 1940. Studies with
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43

FLOSDORF, E. W., and MUDD, S. 1935. Procedure and ap-
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HUTTON, R. S., HELMOE, R. J., and ROBERTS, J. L. 1951.
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LEIFSON, E. 1936. The preservation Of bacteria by drying in
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MACFADYEN, A. 1899. On the influence of the temperature
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44

MORTON, H. E., and PULASKI. 1938. The preservation of
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NAYLOR, H. B., and SMITH, P. A. 1946. Factors affecting
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PROOM, H., and HEMMONS. 1949. The drying and preserva-
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RAHN, O. 1945. Physical methods Of sterilization of micro-
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RIVERS, T. M., and WARD, S. M. 1935. Further observa-
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ROGERS, L. A. 1914. The preservation of dried cultures. J.
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SAWYER, W. A., LLOYD, W. D. M., and KITCHEN, S. F. 1929.
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SHACKELL, L. F. 1909. An improved method Of desiccation
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45

SWIFT, H. F. 1921. Preservation of stock cultures of bacteria
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SWIFT, H. F. 1937. A simple method for preserving bacterial
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421.

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