SURVIVAL OF BACTERIA UPON REPEATED FREEZING AND THAWING'
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SURVIVAL OF BACTERIA UPON REPEATED FREEZING AND THAWING'
ARTHUR P. HARRISON, JR.
Department of Biology, Vanderbilt University, Nashville, Tennessee
Received for publication June 9, 1955
The purpose of the research presented in this solidified, which required about 5 min, the tubes
paper has been to determine the effect of certain were removed from the alcohol bath but kept
factors upon survival of bacteria after successive within the cold chest at -22 C. Thawing was ac-
freezings. These factors include length of storage complished by quickly transferring a tube from
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between each successive freezing, initial cell the cold chest to a 35 C water bath and required
concentration, suspending medium, and the ef- from 2 to 3 min.
fect of aeration. No critical study of these fac- To facilitate discussion of the protocol em-
tors appears in the literature. ployed, the experiments may be divided into
three types. In the first, a batch of tubes were
MATERIALS AND METHODS frozen and stored at -22 C as outlined above.
Periodically, one tube was thawed and a 1-ml
The following bacteria were employed as test aliquot diluted and plated; this allowed the col-
organisms: Lactobacillus fermenti strain 69L-3, lection of data for the construction of a survival
Escherichia coli strain 69L-15, and Serratia mar- curve resulting from a single freezing-thawing.
cescens strain ATCC 274. They were cultivated (One tube was used for one plating only; after
as outlined previously (Major et al., 1955). removal of the 1-ml aliquot the tube was dis-
Broth cultures of the organisms were cen- carded.) Concurrently, after various storage in-
trifuged, the spent broth pipetted off, the cells tervals, a set of three to five tubes was thawed,
resuspended in a minimal amount of fresh broth, frozen a second time and again stored at -22 C.
and then the suspension diluted with additional Periodically, one tube was thawed and a 1-mi
broth to the desired cell concentration. The aliquot diluted and plated; this then permitted
broth used as the suspending medium had the the construction of "secondary" survival curves.
following percentage composition: yeast extract These curves, of course, have their points of
(B.B.L., dehydrated), 1.0; K2HP04, 0.2; MgSO4. origin on the primary survival curve and will be
7H20, 0.01; and "tween 80" (Atlas Powder Co.), called "pendent" curves. Two such series of
0.1. In some experiments with L. fermenti the curves are plotted in figure 1 where the curve
suspending medium contained 1 per cent trypti- representing survival after a single freezing-
case (B.B.L., dehydrated) and 2 per cent glucose thawing is heavily drawn and the pendent curves
as additional ingredients. In experiments where are drawn more lightly. In all figures, the point
water was used as the suspending medium the at 0 weeks represents the initial cell count of the
cells from the centrifuged culture were resus- test suspension, that is, the cell count per ml im-
pended in an equal volume of distilled water and mediately prior to the first freezing.
again centrifuged, the supernatant pipetted off, In the second type of experiment, there may
the cells resuspended in a minimal amount of be three or four pendent curves, each having its
distilled water, and then the suspension diluted origin at a point on the preceding curve. Such a
with additional water to the desired cell concen- series of curves represents survival after a num-
tration. ber of alternate freezings and thawings (figures
The suspension was dispensed in 5-ml aliquots 2 and 3).
into test tubes (15 mm by 150 mm) fitted loosely In the third type of experiment, the storage in-
with aluminum caps. Freezing was accomplished terval between successive freezings is too short
by immersion of the tubes in an alcohol bath at to permit storage counts to be made. Experiments
-22 C. After the contents of the tubes had of this sort are simpler, since a single tube may be
1 This research was supported in part by funds repeatedly frozen and thawed if 0.1-ml aliquots
provided by the Vanderbilt University Research are removed for the platings, thereby not chang-
Council. ing appreciably the volume of the test suspension
711712 HARRI ISON, JR. [VOL. 70
-4
4'
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Figure S. Survival of Lactobacillus fermenti
in broth to successive freezings at storage inter-
t I ' vals of 1 week and 4 weeks.
Figure 1. Survival of Lactobacillus fermenti in V
broth to a single freezing and to a second freezing
STORACE Z#TERYAL
after storage intervals of 1 week, 4 weeks, and 8 0
weeks. 8 BErWEEN FREEZINGCS
I
7' 0 WP
\; O
' "0 'O
'' * sO
%%% .. %
I')
!6
a b-.O *--_
-4 dlb
2
NI_-
XP. N.
#0. A -O
AXP O.
I* '2 ' *+ Ia I I
IS I
*/O ' *B2
NUMBER OF SUCCESSIVE REEZIMNGA WINS
Figure 4. Survival of Lactobacillus fermenti at
different initial cell concentrations in broth to
successive freezings after storage intervals of
M hour and 24 hours.
A 'S by a broken line as an aid in showing the relation-
Figure S. Survival of bacteria to successive ship of the points to one another. In like manner,
freezings after storage intervals of 1 week. A, the counts resulting after each successive freez-
Lactobacillus fermenti in broth. B, Escherichia coli ing-thawing in the case of experiments of the
in water. second type are also connected by broken lines.
Before plating or refreezing, the thawed sus-
within the tube during the course of the experi- pensions were always gently agitated to ensure
ment. Curves from experiments of this type are homogeneity. Dilutions for plating were pre-
seen in figures 4 and 5, where the counts after pared by transferring the 1.0- or the 0.1-ml ali-
each successive freezing-thawing are connected quot to 99 ml of diluent in a dilution bottle.1955]REPEATED FREEZING AND THAWING OF BACTERIA
1955] 713
regard is whether a single freezing-thawing or
repeated freezing-thawings are required to demon-
strate this dependence.
-'l In the case of a single freezing-thawing with
E. coli the percentage survival varies in propor-
A~~~~ZA tion to the initial cell concentration. The survivals
resulting from a second freezing are somewhat
erratic, but in general show the same behavior as
in the case of L. fermenti. The dependence of
survival upon the initial cell concentration may
0'0
be seen also when washed cells of E. coli have
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N.WIS
Z-WO r A I-
been suspended in water and subjected to suc-
cessive freezing-thawings (figure 2B).
With water as the suspending medium and at
initial cell counts much below 107 per ml the
Figure 5. Survival of Escherichia coli at three storage counts of E. coli are unexpectedly low and
different initial cell concentrations in broth to erratic. Upon extensive dilution of a water sus-
successive freezings after storage intervals of Mi pension of E. coli some character of the suspen-
hour and 24 hours. sion is so altered that the cells become unusually
sensitive to freezing-thawing and it is not possible
Serial dilutions were made using 99 ml of diluent. to plot smooth survival curves. For this reason
The diluent contained 0.5 g of trypticase (B.B.L., the data obtained in this experiment at the lower
dehydrated) and 0.6 ml of buffer solution initial cell concentrations have not been included
(2.8 g KH2PO4, and 13.7 g K2HP04, per 200 ml in figure 2B. The phenomenon has been noted in
of water) per liter of distilled water. Plating was replicate experiments and at about the same de-
carried out as already outlined (Major et al., gree of dilution. This observation explains why
1955). contradictory conclusions regarding the com-
parative merit of broth and water as suspending
RESUJLTS AD DISCIJSSION media occur in the literature. Water may prove
It will be noted in figure 1 that in the case of a superior or inferior to broth as menstruum, de-
single freezing-thawing (the two heavily drawn pending upon the initial cell concentration and
curves), L. fementi manifests a percentage sur- perhaps also upon the degree of washing of the
vival which is constant and independent of the cells prior to their final suspension and freezing.
initial cell concentration. However, when the The death resulting from each successive freez-
suspensions are subjected to a second freezing- ing is not of the same magnitude; with L. fer-
menti, the second freezing is always more lethal
thawing it becomes apparent that the initial than either the first freezing or any of the sub-
cell concentration does, after all, exert an effect sequent freezings. This is true whether the length
upon the suspensions, for careful examination of storage between successive freezings be at
shows that the percentage death represented by intervals of 4 weeks (figure 3), 1 week (figures
the three lower pendent curves is in every case 2A and 3), 24 hours, or Y hour (figure 4). In all
somewbat greater than the percentage death cases, after the first three or four successive freez-
represented by the three corresponding curves ings, additional freezings cause relatively little
directly above. The effect of the cell concentra- death. With E. coli, when the interval between
tion upon survival becomes more noticeable with
yet additional successive freezing-thawings, as successive freezings is reduced to % hour, each
may be seen in figure 2A where L. femetri at successive freezing causes the same degree of
two initial cell concentrations has been succes- death (figure 5), and the broken curve connect-
sively frozen and thawed four times. ing the counts after each freezing-thawing may
It is probable that the percentage survival of now be considered a straight line. At storage in-
all bacteria is dependent upon the initial cell tervals of 1 day and greater, however, this or-
concentration. The difference in bacteria in this ganism behaves more like L. fermenti, at least714 HARRISON, JR. [voL. 70
9 logarithm of survivors versus the number of
.
' . successive freezing-thawings resulted in a linear
N --x- relationship.
8 \ I'x " -X,
\ \x *'x*, It has already been reported from this labora-
.+ ;'a\O
\ '0 \ 'I
tory (Major et al., 1955) that aerated cells of such
bacteria as E. coli and S. marcescens are more
4. "X resistant to the lethal effect of a single freezing-
('S
4. 6 N x
\ " thawing than are nonaerated cells. In the present
\
\
' study experiments were undertaken to compare
cS' survival of aerated and nonaerated cells of E.
3. \" o X N coli after repeated freezing and thawing; the re-
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'
0
3. sults are plotted in figure 6. With water as men-
4. struum, survival of aerated cells is far superior to
'33 \ "-- that of nonaerated celLs during the first six
successive freezings, but with additional freez-
a BAO/TH, Al AAFrED X- -X
X---x
AArED x-x
WArfAAfX owATff
0 'Zo~'N\ ings this superiority is less pronounced. Likewise,
OROrT, NOAVAIAATf 0---4
WA7-EdR, NOAWXAAre-D .0 a
\\ 0 aerated cells in broth are more resistant for the
0 first six successive freezings; after additional
0.* 'z ,
# 6 'a3 ' '1 freezings, however, the difference is less pro-
UMBER Or SUC7CE.:VYJEZ i-/EJZIf#V-rWAW1NGS
N
nounced. In this figure a comparison may also
Figure 6. Comparison of the survival of aerated be made between survival in broth and in dis-
and nonaerated cells of Escherichia coli in broth tied water. During the first few successive
and in water to successive freezings after storage freezings survival is better in water. The dif-
intervals of 24 hours. ference is less pronounced after additional suc-
cessive freezings, and eventually survival appears
at the lower cell concentrations (figure 5). It is better in broth.
admitted that the difference in the death occur-
ring after the first and after the second freezing
would be expected if the cells were clumped. In- Although Lactobacillm fementi upon a single
deed, this may be a factor, as it is difficult to freezing-thawing manifests a survival which is
completely disperse all clumped cells even with constant and independent of the initial cell
agitation and incorporation of "tween 80" in the concentration, the percentage survival which re-
medium. However, another factor may possibly sults after a series of repeated freezings and
be involved, because the amount of clumping ob- thawings varies in proportion to the initial cell
served microscopically in the suspensions prior concentration.
to freezing is negligible, and because diluting the With L. fementi, regardless of the length of
suspension prior to freezing and lengthening the the storage interval between successive freezings,
storage interval between successive freezings the lethal effect of the second freezing is greater
(with E. coli) increases the bend in the curve than the lethal effect of any subsequent freezing
(figure 5). With Serratia marcescens, where ab- or of the first freezing. Therefore, when logarithms
solutely no clumping was observed, the results of survivors are plotted versus the number of
are the same; the curve is sigmoidal when the successive freezings a sigmoidal curve is obtained.
storage interval is 1 day, but approaches a The configuration of the sigmoidal curve is in-
straight line when the storage interval is reduced fluenced by the length of the storage interval
to 6i hour. between the successive freezings and also by the
Hollander and Nell (1954) alternately froze menstruum, the initial cell concentration, and
and thawed suspensions of E. coli and Diplococcus conditions under which the test organism is cul-
pneumoniae and made platings after one, two, tivated. With Escherchia coli and Serratia mar-
four, and eight such freezing-thawings. There was cescenm the curve may also be sigmoidal, except
no storage interval between each successive when the storage interval is short; then the curve
freezing. They also observed that plotting becomes a straight line.1955] REPEATED FREEZING AND THAWING OF BACTERIA 715
At moderately high cell concentrations, sur- REFERENCES
vival of E. coli to successive freezings is, at first, HOLLANDER, D. H. AND NELL, E. E. 1954 Im-
greater in water than in broth, but after addi- proved preservation of Treponema pallidium
tional successive freezings the converse may be and other bacteria by freezing with glycerol.
true. Aerated cells of E. coli, whether suspended Appl. Microbiol., 2, 164-170.
in water or broth, at first survive much better MAJOR, C. P., MCDOUGAL, J. D., AND HARRISON,
A. P., JR. 1955 The effect of the initial cell
than nonaerated cells, but upon additional concentration upon survival of bacteria at
freezings the difference becomes less pronounced. -22 C. J. Bacteriol., 69, 244-249.
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