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THESl\S
This is to certify that the
thesis entitled
"Studies on the Preservation of
Leptoapira ictarohemorrhagiae"
presented by
John M. Shigekatta
has been accepted towards fulfillment
of the requirements for
M. S. degree in Bacteriology
MYW[Major profeissor
Date November 21:, 1951;
0-169
ICTEROHEMORRHAGIAL: BY FREEZING
AND FREE ZE-DR YING
BY
JOHN MASACHIKA SHIGEKAWA
A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of theiequirements
for the degree of
MASTER OF SCIENCE
Department of Bacte riology
1954
THESlS
ACKNOWLEDGMENT
My sincere appreciation to Dr.
Jack J. Stockton for his patient guidance
and counsel during the course of this
study and his assistance in the writing
of this thesis.
ii
34408'?
INTRODUCTION
HISTORICAL REVIEW
TABLE OF CONTENTS
Low Tempe ratu re s ...........................
Freeze -Drying
MATERIALS
METHODS AND RESULTS
Freezing ..................................
FreezeaDrying
DISCUSSION ..................................
SUMMARY..........L ....................... .
BIBLIOGRAPHY
iii
16
21
21
35
39
'43
44
STUDIES ON THE PRESERVATION OF LEPTOSPIRA
ICTEROHEMORRHAGIAE BY FREEZING
AND FREEZE-DRYING
by
JOHN MASACHIKA SHIGEKAWA
This investigation consisted of studying the effects of freez-
ing and freeze-drying on the viability and morphology of Leptospira
icterohemorrhagiae. A comparison was made of various suspending
menstrua and storage temperatures to determine which menstruum and
under what storage conditions the organiSms best survived in the
frozen state and whether the menstrua used could provide suitable
conditions for the successful 1y0philization of the organism.
Six to seven day-old cultures of E. icterohemorrhagiae grown in
Korthoff's medium at 50°C were concentrated by centrifugation and re-
suspended in various menstrua, chosen for this study because of their
colloidal preperty. These were as follows: 1 percent starch, l and 5
percent gelatin, 0.25 percent agar, 1 and 2 percent casein, skim milk,
normal rabbit and horse serum, egg yolk, and fresh and frozen allantoic
fluid. The suspensions of organisms were tubed in "IyOphile" tubes
and following freezing in a dry ice-alcohol mixture at -70°C, were
stored in different deep-freeze units at -27°C, -47°C, and -7§°C. Test
samples were reconstituted after storage of one week, two weeks, one
month, and thereafter at monthly intervals. The contents of the tubes
were inoculated into Korthoff's medium and the viability of the organ-
isms was determined by dark-field microscOpy after one and two weeks'
incubation at 50°C. The criterion used foe determining viability was
motility of the leptospirae.
Leptospirae suspended in the menstrua mentioned above and in
infected tissues of young guinea pigs and hamsters were 1y0philized
according to the technique of Flosdorf and Mudd. Following 1yophi1-
zation, these cultures were checked for viability by incubation in
Korthoff's medium.and subsequent darkbfield microscOpy.
The results obtained in this study have shown that E. 122252-
hemorrhagiae can be frozen and kept stable up to nine months in certain
colloidal substances at subzero temperatures. The most effective of
these substances were skim.milk and slightly hemolyzed normal rabbit
and horse serum. There was very little variation in the results in
the three storage temperatures (-2700, -47°C,,and -7§°O) employed.
Freeze—drying as a method for preserving these organisms met with
unsuccessful results. Although the leptospirae survive the preliminary
freezing, death occurs during the drying process. The reason for this
is still unknown.
LIST OF TABLES
TABLE Page
I. Group I. Longevity of L. icterohemorrhagiag
suspended in various menstrua and stored at
-47°C ................................. 26
II. Group II. Longevity of L. icterohemorrhagiae
suspended in various menstrua and stored at
-47°C ................................. 28
III. Group III. Longevity of L. icterohemorrhagiae
suspended in various menstrua and stored at
—27°C ................................. 30
IV. Group IV. Longevity of 1:; ict‘erohemorrhagiae
suspended in various menstrua and stored at
-73°C ................................. 32
iv
INTRODUCTION
The preservation of bacterial cultures by suitable methods
provides the bacteriologist with readily available cultures of known
characteristics. Satisfactory preservation obviates frequent manipu-
lation of cultures, thereby lessening both the danger of contamina-
tion and the possibility of variations or mutations. Preserved cul-
tures also mean an economy of time, labor, space, and glassware.
The method of preserving bacterial cultures must meet
several criteria if it is to be accepted for use. Preservation should
not only maintain the viability of the organism, but all its natural
characteristics as well. There should be no alteration in morpho-
logical, immunological, and biochemical characteristics, and no
lessening of virulence if a pathogen.
To place it in the realm of the practical and have it adopted
for use in most laboratories, the procedures should be relatively
simple, and the apparatus and equipment employed easy to operate
and economical in cost and operation.
Freezing and freeze-drying have proved efficacious in storage
of many species of bacteria, some surviving for several years with
little adverse effect and no apparent change in characteristics.
However, bacteria vary in their susceptibility to freezing and to
freeze-drying; while some species may be preserved for years by
these methods, others may not withstand the conditions encountered
in these processes.
Preservation of the pathogenic spirochetes has been a prob-
lem to the bacteriologist and one that has not yet been adequately
solved. The importance of spirochete-produced diseases, both in
man and animals, warrants efforts toward a solution to this problem.
The present study was conducted in an attempt to employ
methods previously used in the successful preservation of various
bacteria for preserving one member of the Genus Lgptgspiga,
Lepto spira icte rohemorrhagiae.
The choice of this spirochete as the test organism was
prompted by the fact that, among the pathogenic spirochetes, it is
probably the easiest to cultivate in artificial media. Also, the mem-
bers of this genus are the cause of leptospirosis in man and ani-
mals, a disease that has gained increasing attention in recent years
because of growing awareness of its prevalence. Increased interest
will demand more intense study of the disease and its etiological
agents. Therefore, it is desirable that an adequate means of pre-
serving these leptospirae be developed.
This investigation consisted of studying the effects of freezing
and freeze-drying on the viability and morphology of 1:; icterohemorr-
hagiag. A comparison was made of various suspending menstrua and
storage temperatures to determine which menstruum and under what
storage conditions the organisms would best survive in the frozen
state and whether the menstrua used could provide suitable conditions
for the successful lyophilization of the organism.
HISTORICAL REVIEW
Low Temperature 5
Many observations have been presented by investigators on
the effect of low temperatures and freezing on bacteria. Before the
turn of the century, most bacteriologists considered cold a powerful
germicidal agent, but later investigations and observations have shown
that in many instances cold may, in fact, act as a preserver of
microbial life.
One of the earliest observations on the influence of low tem-
peratures on microorganisms was made by Pumpelly in 1882, as cited
by Hilliard and Davis (1918). They stated that when Pumpelly cut
samples from the center of a block of ice and placed them in sterile
beef broth, contamination soon became evident.
Hilliard and Davis (1918) reported that the first extensive
study of the effects of cold on bacteria was made by Prudden in
1887. He subjected Bacillus typhosus to temperatures ranging from
14° to 30°F, and found that. under these conditions, the organisms
remained viable for 103 days.
Ten microorganisms possessing varying degrees of resistance
to environmental conditions were employed by Macfadyen (1900) in
his study of the influence of the temperature of liquid air on bac-
teria. Young cultures were eXposed to the temierature of liquid
air (-190°C) for 24 hours and then thawed and examined. No im-
pairment of vitality or functional activity could be detected. Mac-
fadyen and Rowland (1900) later reported that a number of organisms,
mainly of the Bigllgg group, subjected to the temperature of liquid
hydrogen (-252°C) for 10 hours, remained unchanged in appearance
and vigor of growth. Further studies by Macfadyen and Rowland (1902)
gave similar results. McLean (1918) isolated four species of bac-
teria that had lived dormant in ice, snow, and frozen algae for pro-
longed periods. Luyet and Gehenio (1934) stated that other investi-
gators--Brehme, Citovicz, Gladin, Kasansky, and Pictet--had found,
that various types of bacteria were able to survive for considerable
periods in the frozen state.
Bacteria are known to vary in their resistance to cold and
freezing. Smith and Swingle (1905) demonstrated this using twelve
different bacteria. Represented in this group were saprophytes and
plant and animal pathogens. Cultures 24 to 48 hours old were
suspended in peptonized beef boullion and part of each culture was
frozen in liquid air (-190°C) for 10 minutes to 24 hours and the
remaining cultures in a salt and ice mixture (-17.8°C) for 2 hours.
The cultures varied in their resistance to these temperatures, but
even in case of the most sensitive bacteria, some individuals of
each culture were able to withstand the temperature of liquid air.
Hilliard, Torossian, and Stone (1915) observed a difference in the
susceptibility of Bacillus coli and Bacillus subtilis to freezing in tap
water for 3 hours. The results of other workers--Tanner and Wil-
liamson (1927), Keith (1913), Smart (1935), and Haines (l938)--added
supporting evidence to the findings of previous investigators.
There are a number of variables that appear to have an ef-
fect on the susceptibility of microorganisms to freezing. Among
these are the temperature of the freezing mixture, the duration of
the freezing, the abruptness of the temperature changes, and the
nature of the suspending medium.
To study the effect of the degree of cold used in the freez-
ing mixture, Hilliard, Torossian, and Stone (1915) froze tubes of
cultures and held them for 3 hours for comparison at temperatures
of approximately -15°C and -Z°C. It was observed that the lower
temperature had a considerably greater effect in the reduction of
the number of organisms. Haines (1938) noted that the temperature
or rate of freezing had little effect on the mortality of the cells.
Studies of the effect of. repeated freezings and thawings on
twelve different bacterial cultures by Smith and Swingle (1905) showed
a gradual reduction in the living bacterial populations to zero. Hil-
liard, Torossian, and Stone (1915) stated that freezing and thawing
at intervals was considerably more fatal than continuous freezing.
In contradiction to the findings of the two previously mentioned in—
vestigations, Tanner and Wallace (1931) reported that alternate
freezing and thawing was no more destructive to microorganisms
than continuous freezing.
The findings of Haines (1938) suggested that there is some
correlation between storage temperatures and survival of micro-
organisms. When frozen aqueous suspensions of bacteria were
stored at temperatures ranging from -1°C to -20°C, the most rapid
death rate took place near the highest storage temperature.
A The medium in which the organisms are suspended seems to
have an important bearing on how well they withstand the adverse
effects of the initial freezing and subsequent storage at low tem-
peratures. The formation of ice crystals or crystallization is thought
to be one of the chief factors in the reduction of bacterial populations
undergoing freezing, due to the mechanical disruption of cells. There-
fore, most investigators are of the opinion that those substances
which are colloidal in nature give the greatest degree of protection.
Keith (1913) has compared the survival time of léggiiu_s__c_o_li in dif-
ferent suspending media. One group of cultures was suspended in
milk and the other group in tap water and frozen. Results of this
study proved milk more efficacious in its protection than tap water.
These findings were substantiated by Hilliard and Davis (1918), Prucha
and Brannon (1926), and Tanner and Wallace (1931).
Studies on the effect of freezing on spirochetes have been of
relatively recent date and the number of publications on this subject
have been few. Turner (1938) reported preserving two species of
treponemes in the frozen state at —78°C for as long as one year.
As Haines (1938) had previously noticed with other types of bacteria,
Turner (1938) observed a difference in survival times of treponemes
held at different temperatures. Those stored at -20°C were found
to survive for rnuch shorter periods than those kept at -78°CL
Treponemes held at -10°C had lost their motility and many appeared
shrunken and distorted. In the course of their work, they noted
that the temperature during the initial freezing had little effect on
the organisms. This indicated that the damage to the cells occurred
not at the time of the initial freezing but during the maintenance
period.
In a later publication, Turner and Fleming (1939) stated that
the viability and virulence of various types of spirochetes were main-
tained after storage at -78°C for periods up to 3 years. One strain
of L. icterohemorrhagiae, isolated from rats and propagated in guinea
pigs, remained active after storage for 10 months at -78°C.
Stavitsky (1945) was able to maintain the viability and viru-
lence of L. icterohemorrhagiae in infected whole guinea pig liver
blocks, frozen and kept at -20°C, for 100 days.
Freeze- Drying
Freeze-drying was first described by Shackell (1909), who
suggested several practical applications of this process to biologics.
Martin (1896) had previously reported a simple method of
obtaining dried sterile serum from the liquid state and Morton and
Pulaski (1938) stated that Kitasato, Ficker, Kirstein, and Hein were
successful in preserving several species of bacteria by various
methods of desiccation.
Freeze-drying, as described by Shackell (1909), was the in-
troduction of freezing as a preliminary step to desiccation. His
method consisted of placing the frozen material in a desiccator over
absorbing sulphuric acid which was thoroughly mixed by occasional
10
rotation to facilitate absorption and drying. The vacuum was pro-
duced by the use of a ”Geryk" vacuum pump which was capable of
reducing the air pressure in the desiccator to less than 1 mm. of
mercury in 2 minutes.
Employing Shackell's method of freeze-drying, Harris and
Shackell (1911) successfully dried brains and cords of rabies-infected
rabbits. They observed no destruction of virulence of the virus.
The preservation of bacteria by freeze-drying was first ac-
complished by Hammer (1911). Later, Roger (1914) applied the
principle of freezing and drying to the preservation of bacterial
cultures on a large scale.
A comparative study of the survival of several non-spore—
forming bacteria was made by Shattock and Dudgeon (1912), employ«-
ing two methods of desiccation: desiccation in air and drying in a
liquid air vacuum. In the case of some of the bacteria there was
little difference in viability and survival time, but with others, the
difference was very marked in favor of freeze-drying.
Stock cultures of streptococci and pneumococci were main-
tained by Swift (192.1) for periods from 2 to 4 years after freeze-
drying. He noted that the physical state of the dried material had
much to do with the viability of the organisms. The recovery of
ll
organisms was greatest in those tubes showing a ”dry-foam" con-
dition while in those appearing gummy, recovery was not possible
in most cases. The results of his studies indicated that it was.
necessary to maintain the frozen state until drying was complete.
This led Swift to devise a technique in which the tubes containing
the specimen were immersed in glycerol contained in the lower part
of the desiccator which was then placed in the salt-ice mixture. The
glycerol acted as the medium for the conduction of cold from the
salt-ice mixture to the material which was kept frozen until drying
was complete. Bacteria preserved in this manner were observed to
retain all their desirable characteristics.
The preservation of yellow fever virus by freeze-drying was
reported by Sawyer, Lloyd, and Kitchen (1929). Brown (1932) main-
tained thirty-eight strains of bacteria 4 to 12 years following drying
£1113qu from the frozen state. The efficacy of this method for
preserving the more sensitive bacteria was shown by Rake (1935),
who successfully maintained several strains of meningococci over
a period of many months.
Elser, Thomas, and Steffen (1935) made significant contribu-
tions to the improvement of techniques and apparatus involved in
the freeze-drying process. They described methods and apparatus
12
for freeze-drying biological products and microorganisms which dif-
fered from previously reported procedures in that theirs involved
the use of manifolds and individual containers in the drying process.
Both chemicals and refrigerants were employed as condensers to
dispose of water vapor arising during the high-vacuum desiccation.
The use of refrigerants brought this method into the realm of the
practical for the drying and preservation of large volumes of thera-
peutic products. Chemical desiccants were limited in their capacity
to absorb water and therefore were limited in the volume of product
that could be processed. Therapeutic agents preserved, using the
techniques and apparatus described by these workers, retained
their clarity, porosity of texture, and solubility. They did not de-
teriorate on standing and exhibited no loss in potency even after
long periods of time. Meningococci and gonococci, normally sensi-
tive to adverse conditions, were successfully preserved but attempts
to preserve the spirochete Spirochaeta duttoni by this method proved
discouraging. The organism was recovered only in a very few in-
stances following freeze—drying.
The development of the freeze-drying apparatus to what it is
today was largely due to the efforts of Flosdorf and Mudd (1935).
The increased efficiency of their so-called ”lyophile" apparatus
I
l3
enabled them to preserve large amounts of labile biological products,
bacterial cultures, and viruses without any deleterious effects. These
dried products were porous and reconstituted with ease. Sera so de—
hydrated were designated "lyophile" ("liquid-loving"), a word first
coined by Reichel, as cited by Flosdorf and Mudd (1935).
Flosdorf and Mudd (1938) later described a method by which
biologics could be preserved more conveniently and economically.
This method involved the use of calcium sulfate as the desiccant
in the condenser. The calcium sulfate was specially prepared and
could be easily regenerated by heat for further use. The word
”cryochem" was coined to describe this process.
Swift (1937) stated that colloidal substances, such as serum,
appeared to give greater protection to microorganisms undergoing
drying. Studies by Heller (1941) showed that the death rates of
lyophilized bacteria suspended in crystalline substances were greater
than those of bacteria suSpended in colloids.
Bauer and Pickels (1940) reported that yellow fever virus,
ordinarily labile to adverse conditions, could be preserved and kept
active for years if suspended in a medium rich in proteins and prop-
erly desiccated. They observed that it was essential to maintain the
material in the frozen state until desiccation was complete.
l4
Lyophilization has not yet proved to be a satisfactory method
for preserving spirochetes. Results of most workers have been
discouraging. Turner, Bauer, and Kluth (1941), employing the ap-
paratus and techniques used by other investigators in the successful
preservation of bacteria and viruses, were unable to recover two
species of treponemes after freeze-drying. The spirochetes were
suspended in tissue juices and serum and dried for 3 to 5 days
after freezing. Large quantities of this dehydrated material was
then injected into normal rabbits. Results were completely negative,
indicating that the lyophilized organisms had lost viability. Stavitsky
(1945) reported that, after lyophilization of cultures of 1:493:52-
hemorrhagiae and infected guinea pig livers, no leptospirae could be
recovered. Samples of the lyophilized material were checked at
various intervals for viability, variability, and virulence by direct
dark-field examination, subculturing, and inoculation into young
guinea pigs, but in all instances the results were negative.
At variance with reports by earlier workers, Hampp (1947)
reported the successful preservation of spirochetes by freeze-drying.
The test organisms used were Borrelia vincenti and various strains
of Treponemna pallidum which were grown 5 to 7 days in a men-
struum consisting of equal parts of Huntoon's "hormone" broth
15
and 5 percent gastric mucin and enriched with ascitic fluid and
glutathione. ‘This medium was used both for cultivation and suspen-
sion of the organisms for freeze-drying. Lyophilization was done
according to the method of Flosdorf and Mudd, using the "cryochem"
apparatus. The lyophilized cultures were tested for viability both
microscopically and by subcultures The cultures were positive with
only one exception.
Hampp (1951) later reported the maintenance of viability and
pathogenicity of Nichol's rabbit strain of T. pallidum in lyophilized
infected rabbit testes.
Brunner and Meyer (1950) employed lyophilization as a method
to kill leptospirae for preparation of a vaccine for the immunization
of hamsters and dogs against experimental leptospirosis. They found
that lyophilization had no effect on the antigenic quality and the vac-
cine so produced provided excellent protection against the homologous
type of L. icterohemorrha iag.
MA TERIALS
Members of the Genus Leptospira are readily cultivable on
artificial media. Many different media have been used for the cul-
tivation and propagation of these organisms, most of them consisting
of a solution of various salts, enriched with blood, serum, or various
other substances. Babudieri (1943), while studying the survival
times of nineteen strains of various leptospirae as determined on
different blood and serum media, found that serum media were
superior to blood media. Of all the media tested, Korthoff's me-
dium gave the best results.
Serum is an essential ingredient in leptospira culture media.
Rosenfeld and Greene (1941), investigating the effect of growth factors
in stimulating the growth of leptospira species in the presence of se-
rum, observed that no factor or combination of factors was capable
of maintaining leptospiral growth in the absence of serum. Accord-
ing to Babudieri (1943), sera which were slightly hemolyzed gave
much better growth than those that were not hemolyzed. This was
also noted by Ringen and Gillespie (1954), and was true in our own
experience.
16
l7
Korthoff's leptospira medium was used in this study, both for
maintaining the stock cultures and for subculturing organisms taken
out of ”lyophile" and those reconstituted from the frozen state.
This medium consists of one part of 1 percent Bacto-Tryptose to
eight parts of buffer. The buffer was made up of solutions of the
following salts:
NaCl 2.5% ............. 11.2 ml.
NaHCO3 0.1% ........... 4.0 ml.
KCl 0.1% .............. 8.0 ml.
CaClZ 0.1% ............ 8.0 ml.
KHZPO4 2.5% ........... 1.44 ml.
Nazi-1130‘1 2.5% .......... 7.68 ml.
Distilled water q.s ........ 200.00 ml.
After the medium was prepared, the pH was adjusted to 7.4
by addition of approximately 0.1 N NaOH solution drop by drop with
continuous stirring until the desired pH was obtained, as indicated
by a Beckman (model H) pH meter. The medium was then tubed
in 9 ml. amounts in 25 ml. screw-cap test tubes and autoclaved for
15 minutes at 121°C. These were kept at refrigeration temperature
until ready for use. At the time of use 1 ml. of normal serum was
added aseptically to each tube containing 9 ml. of the base medium.
18
The serum used was of rabbit and horse origin. Subsequent
to bleeding, the blood was allowed to clot and the cells were sep-
arated from the serum by centrifugation at approximately 1600 r.p.m.
in an International, Size 2, centrifuge. After collection of the serum,
it was clarified by passing it through clarifying filters and then
sterilized by Seitz-filtering employing No. 3 and No. 6, ST-3, Seitz
type, Hercules sterilizing pads. The’serum was collected in sterile
30 ml. vials, capped, and inactivated in a water bath at 56°C for
30 minutes. This serum was stored under refrigeration till the
time of use.
At the time of bleeding or during the separation of serum
from the cells, some of the red blood cells were purposely lysed
in order to add hemoglobin to the serum.
The test culture employed in this study, L. icterohemorrhaiiae,
strain 871, was obtained from Dr. John P. Newman, Michigan State
College, and was originally isolated from a human case of lepto-
spirosis at Walter Reed Army Hospital, Washington, D. C.
It had been shown by several investigators--Hilliard, Toros-
sian, and Stone (1915), Hilliard and Davis (1918), and Heller (194l)--
that the menstrua in which bacteria were suspended during freezing
or freeze-drying had a decided effect on their survival. The results
19
of these investigations indicated that colloidal substances gave greater
protection against mechanical damage than substances which were
crystalline in nature. For this reason, various colloidal materials
were used to suspend the test organisms in preparation for freezing
and lyophilization. These are listed as follows: 1 percent starch,
normal rabbit serum, normal horse serum, egg yolk, fresh allantoic
fluid, frozen allantoic fluid, 1 and 3 percent gelatin, 1 and 2. percent
casein, 0.25 percent agar, and skim milk. With the exception of the
serum, egg yolk, and allantoic fluid, these substances were adjusted
to a pH of 7.4 and then sterilized by autoclaving at 121°C for 15
minutes, prior to use.
Reports by a number of workers--Harris and Shackell (1911),
Hampp (1951), Turner (1938), and Stavitsky (l945)--mentioned that
the freezing and freeze-drying of experimentally infected tissues of
susceptible laboratory animals sometimes resulted in the maintenance
of both the viability and virulence of the infecting microorganism.
Morton (1942) and Larson (1944) had found that young guinea pigs
and young ’golden hamsters were most susceptible to experimentally
produced leptospirosis. The greatest concentration of infective
leptospirae appeared in the kidneys and liver due to the affinity of
these organisms for those particular tissues. An attempt was made
20
in this study to preserve L. icterohemorrhagiae by lyophilizing in-
.0
fected tissues of young guinea pigs and golden hamsters.
METHODS AND RESULTS
Freezing
Stock cultures of L. icterohemorrhagiae were maintained in
Korthoff‘s medium contained in 25 ml.. screw-cap test tubes in 10 ml.
amounts. These were transferred weekly into fresh medium and incu-
bated at 30°C. An inoculum of 0.25 ml. of actively growing culture
was used to seed each tube of fresh medium.
The test cultures that were subjected to "sharp" freezing and
subsequent storage at subzero temperatures were seeded fromthe
stock cultures and incubated for 6 to 7 days at 30°C. At the end
of the incubation period the cultures were observed macroscopically,
using transmitted light from an intense light source, for evidence of
growth as indicated by a slight cloudiness of the medium.
Prior to suspension in the various menstrua, the organisms
_were concentrated by centrifugation in an International, PR-l, refrig-
erated centrifuge (40° to 45°F) at a R.C.F. of approximately 1000 x G
for 45 minutes. The organisms in each suspending menstruum repre-
sented the pooled growth from five tubes.
21
22
The first group of cultures frozen were Suspended in only
eight of the eleven menstrua previously mentioned. These were:
1 percent starch, l and 3 percent gelatin, 0.25 percent agar, l and
2 percent casein, skim milk, and normal rabbit serum. Ten ml. of
each medium was used to suspend the concentrated organisms in each
tube.
For the other groups of test cultures, egg yolk and fresh and
frozen allantoic fluid were used in addition to the suspending men-
strua listed above. In two of the groups, normal horse serum was
used in place of rabbit serum. The suspended organisms in these
groups represented a greater concentration of organisms since only
5 ml. of each menstruum was used for suspension of the organisms.
After the organisms were well suspended in each menstruum,
they were tubed in approximately 0.5 ml. amounts in sterile pyrex
”lyophile” tubes using sterile capillary pipettes, and were then re-
plugged with cotton. Before freezing the tubes were sealed with
“parafilm.”
Some investigators had observed that the rate at which bac-
teria were frozen had a considerable effect on the number of organ-
isms that withstood the freezing process. They observed that the
reduction of bacterial populations was less after ”sharp" freezing
23
at very low temperatures than that of those subjected to slow freez-
ing at 0°C or at temperatures just below the freezing point of water.
For this reason, this work was conducted using a freezing mixture
of solid C02 and 95 percent ethyl alcohol, at a temperature of ap-
proximately -70°C, to assure rapid freezing of the specimens and
decrease the percent reduction due to this factor.
The suspended organisms in each ”lyophile” tube were “bleb”
frozen in the dry ice-alcohol mixture for a period of 5 to 10 minutes,
after which they were placed in suitable containers and stored in
freezing units at different subzero temperatures.
According to Turner (1938), another factor that influenced
the survival of spirochetes in the frozen state was the temperature
at which these organisms were held during the storage period.
To determine the optimum temperature range for the storage
of these leptOSpirae, as indicated by survival times, tubes of sus-5
pended organisms were kept in various freezing cabinets at different
temperatures. These groups of test cultures, held at the various
freezing temperatures, will be designated hereafter as Group I,
Group II, Group III, and Group IV, for convenience.
The suspended cultures in Groups I and II were both stored
in the same deep-freezing unit at a temperature of approximately
24
-47°C. The difference between these two groups, as previously
stated, was that in Group I only eight of the eleven suspending men-
strua were employed, and the suSpensions of organisms were less
concentrated than those in the other three groups.
The tubes in Group III were held in a freezing unit at -27°C,
and those in Group IV in a dry ice chest at approximately -73°C.
To examine the. frozen cultures for viability, tubes were re-
moved from the storage units, thawed at room temperature, and the
contents placed into Korthoff's medium. At the end of one and two
weeks' incubation at 30°C, these subcultures were examined by dark-
field microscopy for viability, with motility of the leptospirae as the
criterion. A rough estimation of the degree of growth was made
and recorded, with one plus indicating little growth, and four plusses
indicating very heavy growth.
Prior to freezing, organisms suspended in each of the sus-
pending menstrua were subcultured in Korthoff's medium to ascer-
tain the viability of the test cultures.
Immediately following the initial freezing in the dry ice-
alcohol mixture, one culture in each of the suspending menstrua
was thawed and inoculated into the cultivation medium to determine
whether or not the organisms survived this initial freezing at -70°C.
25
Subsequently, sets of cultures from each of the four groups were
reconstituted at the end of one and 'two weeks‘ storage, and there-
after at monthly intervals. These were all examined following sim-
ilar procedures as described above.
The results of this study are shown in Tables I to IV.
The cultures in Group I were checked over a period of nine
months. Of the eight menstrua used for suspending the test organ-
isms, five of them still yielded viable organisms at the end of nine
months' storage at a temperature of -47°C. These were: 3 percent
gelatin, 1 and 2 percent casein, skim milk, and normal rabbit serum,
with the rabbit serum appearing to be the most effective. Very good
growth followed thawing and subsequent incubation in Korthoff's me-
dium, and the spirochetes appeared normal and were actively motile.
Casein, l and 2 percent, gave heavy growth, but approximately 50
percent of the organisms were feebly mbtile and those in 2 percent
casein appeared somewhat granulated. Growth of organisms suspended
in skim milk was not quite as heavy as growth of the organisms sus—
pended in l and 2 percent casein and in normal rabbit serum. There
was some granulation of the leptospirae, but they were actively mo-
tile. Growth was slight in 3 percent gelatin, but the organisms were
active and normal in morphology. Starch, 1 percent, supported no
26
TABLE I
GROUP 1. LONGEVITY OF L. ICTEROHEMORRHAGIAE
SUSPENDED IN VARIOUS MENSTRUA
AND STORED AT ~47°C
St a e T' e Starch Gelatin Gelatin
or 1m
g 1% 1% 3%
Not frozen . ++++ ++++ ++++
Reconstituted * - .. ++
immediately ** - +++ ++++
One week * - ++++ ++++
** - ++++ ++++
Two weeks * - - ..
** - - -
One month * - - +
** - + +++
Two months * — - +
** - - ++
Three months * - - -
3101‘ — - -
Four months * - .. -
** - — -
Five months * - .. ..
*3? _ - +
Six months * - - ..
** - + ++
Seven months * - _ _
** - - ++++
Eight months * — - ' +
** - - +
Nine months * - - -
** - - +
*One week incubation; **two weeks incubation; - no growth;
+, ++++ estimated degree of growth.
TABLE I (Continued)
27
. . . N a
Agar Casein Casein Skim R0 2:13
. a 1
o .2507. 1% 2% MilkSe rum
++++ ++++ ++++ ++++ ++++
- ++ - ++ ++++
- ++++ ++++ ++++
- +++ - +++ ++++
+ ++++ - ++++ ++++
’ ++ ++ - ++++
- +++ +++ ++ ++++
- + ++ — ++++
- ++ +++ +++ ++++
_ + + ++++
_ + +++ ++++
- - - - +
- - - - +
- - + + +
- - + ++++ +
- + + - +
- — +++ + +
- - - - +
- + +++ +++
- + - - ++
- ++++ - ++++ +++
- + - ++++
- ++++ ++ +++
- + + - +++
- ++++ ++++ ' ++ ++++
28
TABLE II
GROUP 11. LONGEVITY OF L. ICTEROHEMORRHAGIAE
SUSPENDED IN VARIOUS MENSTRUA
AND STORED AT -47°C
St T' Starch Gelatin Gelatin Agar
ora e 1me
g 1% 1% 3% 0 .2570
Ivotfrozen ++++ ++++ ++++ ++++
Reconstituted * + ++ +++ -
immediately ** ++++ ++++ ++++ -
One week * - — ++ ._
** ++++ - +++ -
Two weeks * - - +++ + ..
** ++++ ++++ —
One month * 1 - .. ++ _
** — - +++ -
Two months * - - + _
** - - +++ -
Three months * - + - _
aw _ + _ —
Six months * - - + _
aunt: - - ++ -
‘—‘_’ J fr
* One week incubation.
** Two weeks incubation.
- No growth.
+, ++++ Estimated degree of growth.
TABLE II (Continued)
29
Fresh Frozen
. . . Normal
Casein Casein Skim Horse Egg Allan- Allan—
10/0 2% Milk Yolk toic toicSe rum . . .
Fluid Fluid
++++ ++++ ++++ ++++ ++++ ++++ ++++
++++ ++ ++ ++++ ++ ++ ++
++++ +++ ++++ ++++ ++++ +++ +++
++ ++ ++ ++++ ++ - -
+++ +++ ++++ ++++ ++++ +++ +
++ ++ +++ ++++ ++ + -
+++ +++ ++++ ++++ +++ ++++ ++++
- - — ++ +++ - _
+ + +++ +++ ++++ +++ -
+ + - - +++ - -
+++ +++ +++ - - +++ ++
+ + ++ ++ +++ - -
++ +++ ++++ - ++++ - +++
- + - ++ ++ - -
++ ++++ ++++ ++++ ++ - -
30
TABLE III
GROUP 111. LONGEVITY OF L. ICTEROHEMORRHAGIAE
SUSPENDED IN VARIOUS MENSTRUA
AND STORED AT -27°C
, Starch Gelatin Gelatin Agar
Storage T1me
1% 1% 3% 0.25%
Not frozen ++++ ++++ ++++ ++++
Reconstituted * +++ ++ H, +
immediately ** +++ +++ ++ +
One week * + + ++ _
** +++ ++++ ++++ -
Two weeks * ++++ ++++ ++++ +
** ++++ ++++ ++++ ++++
One month * + - + _,
** +++ - +++ -
Two months * - +++ + -
** +++ +++ +++ -
Three months * - +++ - _
' ** +++ +++ +++ +++
Four months * + - ++ ..
** +++ +++ +++ -
Five months ’1‘ - - ++ _
** ++ ++++ ++++ -
t
t
* One week incubation.
** Two weeks incubation.
- No growth.
+, 1+++ Estimated degree of growth.
31
TABLE III (Continued)
Fresh Frozen
. . . No rmalCasein Casein Skim Rabbit Egg Allan- Allan-
l% 2% Milk Se rum Yolk toic toic
Fluid Fluid
++++ ++++ ++++ ++++ ++++ ++++ ++++
++ ++ +++ ++++ + + +
++ +++ ++++ ++++ + - +++
+ ++ ++ +++ + - -
- +++ +++ ++++ +++ - -
+++ +++ +++ + + - -
++++ ++++ ++++ ++++ + - -
_ .. + .. - .. -
+ + ++++ - +++ - -
- _ + _ + - -
++++ ++++ +++ - + - -
+ - ++++ + — - -
+ .. _ .. - - _
++++ + +++ - - - -
- +++ ++++ — - - -
32
TABLE IV
GROUP IV. LONGEVITY OF L. ICTEROHEMORRHAGIAE
SUSPENDED IN VARIOUS MENSTRUA
AND STORED AT -73°C
‘—:
mr‘t— T
St a Ti Starch Gelatin Gelatin Agar
or e me
g 1% 1% 3% 0.25%
liotfrozen ++++ ++++ ++++ ++++
Reconstituted * + + ++ -
immediately ** ++ + ++ ..
Two weeks * ' — - .. -
** + - - -
One month * - + + ..
** + ++++ ++++ -
Two months * - - _ _
** ++++ - - -
Three months * - - - ..
** +++ +++ -
Four months * - - .. -
#31: .. - - ..
f
* One week incubation.
** Two weeks incubation.
- No growth.
+, ++++ Estimated degree of growth.
TABLE IV (Continued)
33
Fresh Frozen
. . . Normal
Case1n Case1n Skim H 5 Egg Allan- Allan-
, or e . .
1% 2% Milk Ser m Yolk tom tom
u . .
Fluid Fluid
++++ ++++ ++++ ++++ ++++ ++++ ++++
++ ++ +++ ++ +++ + +++
+++ +++ ++++ +++ +++ - +++
_ — ++ ++++ +++ - +++
+ + ++++ ++++ ++++ - ++++
+++ + +++ ++++ +++ +++ ++++
++++ ++++ +++ ++++ ++++ ++++ ++++
+++ +++ +++ ++++ ++ - -
++++ +++ +++ +++ - - -
++ ++++ ++ ++++ - - +
+++ ++++ ++++ ++++ + - +++
_ .. - + .. .. _
_ +++ ++++ ++++ - - -
34
life at all, and 0.25 percent agar gave negative results after one
week. Gelatin, 1 percent, gave erratic results, all being negative
with the exception of those cultures reconstituted after one and six
months. These cultures showed very little growth.
Of the eleven menstrua used in Group 11, normal horse serum,
skim milk, and 2 percent casein showed heavy, active growth after
storage of six months at -47°C. Egg yolk and 3 percent gelatin gave
moderate growth, and the leptospirae were actively motile. Growth
was observed in cultures suspended in fresh allantoic fluid and
frozen for two months, in frozen allantoic fluid for three months,
and in 1 percent gelatin after two weeks; however, one culture, sus-
pended in 1 percent gelatin, reconstituted after three months' storage
showed actively motile leptOSpirae. The organisms in 1 percent
starch were sluggishly motile after a month in the frozen state.
It was noted that in each case where growth was observed, a con-
taminating mold-was present. Cultures suspended in 0.25 percent
agar were completely negative.
The Group III cultures were stored at -27°C. The latest set
of cultures reconstituted were frozen for five months. The greatest
amount of growth was found from test samples suspended in skim
milk and l and 3 percent gelatin. Gelatin, 3 percent, showed a
35
number of feebly motile leptospirae, whereas those from skim milk
and 1 percent gelatin appeared actively motile. Starch, 1 percent,
and 2 percent casein also supported viability of the leptospirae.
Organisms that were suspended in these menstrua were active and
normal in appearance.
Leptospirae kept in a dry ice chest at approximately -73°C
were observed for viability and morphological changes over a four-
month period. After four months in the frozen state, only those
organisms suspended in 2 percent casein, skim milk, and normal
horse serum were still living. However, after storage for three
months at this temperature, the organisms suspended in all menstrua
but 0.25 percent agar and fresh allantoic fluid gave growth after
being subcultured in Korthoff's medium.
Freeze-Drying
s that were subjected to freeze-dryingwere
The organism
first frozen following similar procedures as described in the section
‘ ‘'
sed in
on freezing. In addition to the eleven suspending menstrua u
the study of the effects of freezing and storage at various subzero
temperatures, infected tissues of young guinea pigs and young golden
hamste rs we re lyophilized.
36
Different groups of animals were inoculated intraperitoneally
with l to 2 ml. of an actively growing eight- to fourteen-day-old cul-
ture of L. icterohemorrhagiag. The animals were observed daily for
symptoms of leptospirosis and death. If the animals did not die
within a week's time, they were sacrificed and the tissues harvested.
As soon as possible after death, an autopsy was performed, and the
kidneys, liver, and, in some cases, the spleen, were removed. The
tissues were removed aseptically and placed in a sterile mortar.
Sterile allundum was added as an abrasive and the tissues were
emulsified with a pestle, taking care to avoid contamination. Just
enough normal horse serum was added as a diluent to facilitate
pipetting the ground tissues into lyophile tubes for freezing and
drying.
To make certain that the tissues to be lyophilized were in-
fected with the leptospirae,.dark—field examinations of saline suspen—
sions of the minced tissues were done and some of the same material
was also inoculated into Korthoff's medium, incubated, and observed
for evidence of growth.
In many instances, the leptospirae could not be isolated
from either the animals that died, or from those that were sacri-
ficed. Only those tissues that were infected, as determined by dark:
field microscopy. were used for lyophilization.
37
Following the preliminary "sharp" freezing in the freezing
mixture, the ”lyophile" tubes containing the frozen‘, suspended organ-
isms were placed in a jar which was stoppered with a one-hole
rubber stopper. By means of glass and rubber tubing, the bottle
was connected to one of the outlets in the manifold of the lyophile
apparatus and dried according to the technique of Flosdorf and Mudd
(1938).
The lyophile apparatus consisted of a manifold with sixteen
outlets which was connected to the condenser containing "Drierite"
(anhydrous calcium sulphate) as the desiccant. The vacuum was
produced by a Cenco ”megavac" vacuum pump which was capable
of producing a vacuum of approximately 100 1microns as measured
by a Stokes-McLeod vacuum gauge.
The cultures were allowed to dry for approximately seventy-
two hours under reduced pressure. At the end of this period, the
tubes were taken from the jar and attached to the outlets of the
manifold by means of pressure tubing. The vacuum was applied
again and when the pressure was sufficiently low, the tubes were
hermetically sealed, using a cross-fire oxygen torch. After seal-
ing, the tubes were kept at refrigeration temperature (40° to 45°F)
until they were reconstituted.
38
The dried cultures were reconstituted by inoculation into
K0 rthoff's medium and incubating them at 30°C. These subcultures
we re examined at the end of one and two weeks' incubation by dark-
field microscopy for evidence of active leptospirae. In no instance
could the organisms be recovered after lyophilization.
DISCUSSION
Freezing and subsequent storage at subzero temperatures shows
promise as a means of preserving members of the Genus Lgptospire
for periods from several months to possibly several years.
Frozen cultures of L. icterohemorrhagiae suspended in various
colloidal substances and stored in a deep freeze at -47°C for nine
months were thawed and, after inoculation into Korthoff's medium
and subsequent incubation, were observed using dark-field micro-
scopy. The leptOSpirae from several of these suspending menstrua
appeared actively motile and normal in morphology. From these
observations, it appeared that, if the original leptospiral population
were great enough and a suitable storage temperature employed,
the organisms would survive for considerable periods of time in the
frozen state.
The ability of the leptospirae to survive freezing temperatures
seemed to vary somewhat with the type of menstruum in which they
were suspended and the storage temperature at which they were held.
Certain menstrua were consistent in supporting viability, while
others consistently gave negative results regardless of the tempera-
ture at which they were kept. .
39
40
The effectiveness of a few of the suspending menstrua varied
with the storage temperature; for example, 1 percent starch at a
temperature of -47°C did not support viability after a storage period
of three months, whereas those leptospirae kept at -27°C and —73°C
were observed to be actively motile after incubation in Korthoff's
medium.
Although there was some variation of results in the cultures
held at different temperatures, the difference did not seem great
enough to be significant, especially in those suspending menstrua
that consistently gave positive results.
The suspending menstrua that gave the most consistent results
in supporting leptospiral life during initial freezing and subsequent
cold storage were 3 percent gelatin, 1 and 2 percent casein, skim
milk, and slightly hemolyzed normal rabbit or horse serum. It was
observed during this study that the leptospirae that had been growing
well in Korthoff's medium containing normal horse serum grew very
poorly when subcultured into medium containing normal rabbit serum
and that it took several series of transfers before good growth was
obtained. The same was true with organisms growing in medium
with normal rabbit serum subcultured into medium containing normal
I
horse serum. The poor maintenance of leptOSpirae suspended in
41
normal rabbit serum in Group 111 may possibly have been due to
this factor since they had been grown in Korthoff‘s medium contain-
ing horse serum prior to suspension and freezing.
Skim milk and serum gave the most consistent results of
heavy growth and motility with no apparent adverse effects on the
morphological characteristics of the organisms. Therefore, these
would be the suspending menstrua of choice.
Freezing presents a practical method of preserving cultures.
The procedure is not only simple as far as operation and materials
are concerned, but is economical, representing economy of time,
labor, glassware, and space. This method must be investigated
further, to prove its worth for the leptospirae, but the results of
this study showed that there are possibilities.
Since Shackell (1900) first introduced freeze-drying and sug-
gested a number of applications of its use for biologics, Elser,
Thomas, and Steffen (1935), Flosdorf and Mudd (1935), and other
investigators have developed the techniques and apparatus to such a
Point of efficiency and practicality that it has probably become the
most effective method for preserving bacterialcultures.
Attempts to lyophilize cultures of 1:. icterohemorrhagiae,
however, have resulted in death of the organism. Stavitsky (1945)
42
could not recover the organisms from infected guinea pig livers
that were subjected to freeze-drying. Similar work by other inves-
tigators also gave discouraging results.
This study was conducted using procedures and apparatus
similar to those described by Flosdorf and Mudd (1935), and the
same suspending menstrua as those used in the experiments on freez-
ing. Infected animal tissues were also lyophilized, but the organisms
could not be recovered in any instance.
Under specified conditions these organisms can withstand
freezing, but attempts at freeze-drying have thus far been unsuccess-
ful. The factor, or factors, involved are still unknown, so there is
need for further research.
SUMMARY
This study has shown that L. icterohemorrhagiae can be frozen
and kept viable without too great adverse effects as to activity and
morphology for periods up to nine months or longer when suspended
in certain colloidal substances. The most effective of these sub—
stances were skim milk and slightly hemolyzed, normal rabbit or
horse serum. There was very little variation in the three storage
temperatures (-27°C, -47°C, and -73°C) employed.
Freeze-drying as a method of preservation has thus far met
with unsuccessful results. Although the organisms survive the pre-
liminary freezing, death occurs sometime during the drying process.
Why this occurs is not yet known.
43
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