“'9': ..

{

 

 

THE INFLUENCE OF LEAKAGE MATERMLS IN roux
mam-e can. SUPERNATANI «FLUIDS ON, THE -
:RssroRA‘rmN 0F, EswmcmA-ggg (#11303) '

mam: CELLS "

Thesis for Hm Dogréo of M. S.

MICHiGAN smre UNNERsm

Them-as L Roszman '
1963 *

 

 

THESIS.

LIBRARY

Michigan State
Univcrsity

 

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GEL SJEl—I‘;'.r.l&t. T 134...; IMO: Cl! 4.33.. $LLJQTULH§LILJA

CF gscxzazc IA CLI (#11503) F3023 I CELLS

 

By

Thomas L. Roszmon

A THESIS
ubmitted to
Michigsn Stcte University
in partial fulfillment of the requirements

for the degree of
MASTER CF SCIEICE
Department of Microbiology and Public Health

1963

1434?;-

Ax;n\ui

ACIQ‘-IO‘.‘IL’3DCELENTS

The author wishes to express his sircere appre—

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guidance and
support throughout this work.
Appreciation is also extended to Dr. M. L.

Hallmann for his assistance and counsel.

I} .‘TR CDUC T I ON

TABLE OF COHTTNTS

LITERATURE REVIEW

I. General Aspects of the Effect of Freezing
on Microorganisms

Effect
Effect
Effect
Effect
Effect

II. Theories on the Mechanisms

of Sub—Zero Temperature .............
of Storage
of Cooling and Warming Rate .........
of SUSpendins Fluids ................

of Other Factors

Time

OOOIOOOOOOOOOOOCOCOOO

of Freeze Death

Extracellular Ice Theory ...................
Irltracellu1ar Ice Theors’ OOOOOOOOOOOOOOOOOO.
Concentration of Solutes Theory ............

Ibtabolic Damage
Cellular Leakage

MATERIALS AND METHODS

Theory

and Its Consequences ......

Cultivation of Organisms ...................
suspending Fluj-ds O0.0000000000000000000000.
Freer-ng and Storage OOOOOOOOOOOOOOOOOOOOOOO

Preparation of Cells for Oxygen

Uptake Experiments oooooooooooocooooooooo
fianometric TQChniques oooooooooooooooooooooo

Characterization of Frozen Cell

Supernatant .............................
Spectrophotometric Techniques ..............

RESULTS

Cells
Cells
Cells
Cells

Frozen
Frozen
Frozen
Frozen

in
in
in
in

O
O
O
D

i

8
O
l

,5%Saline COO-OOOOOOOOOOO

6 M PhOSphate FUffer .000

5 M Phosphate Buffer ....

tilled Water

111

FLULUUJ

.D$? Uflfl
F‘O orfll-flcn0\

A5
b5

Effect of Adding 0.15 M Th sphate
Puffer and Distilled Water
Frozen Cell Supernatants
to Saline Contrifugod
Cells .........................

Effect of Adding 0.85 E Saline
and Distilled Water Frozen
Cell Supernatants to 0.15 M
Phosphate Buffer Centrifuged
C6118 00......000000

Effect of Addin3 0.87%
and 0.15 M Ph sphate
Buffer Frozen Cell Super-

atants to Distilled We tor
Centrifuzad C; 118 .............
Effect of Dre‘ZWn" on the
Endogenous Rates ..............

Effect Of 0.95% Saline
Frozen Cell Supernatant
on Centrifu3ed Unfrozen
C’:llS ooooooooooooooooooooooooo

CUGrchvriZ tion of the 0.85 T
Saline Frozen Cell Super—
natants from E. coli Cells ....

(T) o

.5011

(1) .

O
8

DISCUSSION 000000000Oloooooooooooooooooo

SUMMARY 0000000..ooooooooooooooooooooooo

LITERATURE CI*-H ooooooooooooooooooooooo

-iv

51

\H
(A

Figure

LIST OF FIGURES

 

CXUgen Uptake of Escherichia coli
Cells Frozen in 0. 51 Saline

CE 115 Froan in O "L" l , ‘ _____

 

 

A77¢n Uptake of Esoteri his 0011
Ce“ lls Frozs n in 0. o Tllnostntte

QUffcr onooooooooooooooooooooooooo

 

Oxygen Uptake of Esc Ulric is coli
Cells Froz en in 0.15 u E? osp eta

EUf fer 000000000000000000000.0000.

 

ny3cn Uptake of EsC'cricnia COll
Cells Fro ozon in Distilled Meter ..

Effects of Addin3 Supernatants
Frorn tie Three Frc:zin3 Methods

to Esch:rickie coli Centrifuged
Cells -rozen in 0.85 % Saline

AS It": asured LU, 0393581”) Uptake ooooo

 

Effects of Adding Supernatants
From Three Freezing Methods to
Escherichia coli Centrifu3ed

 

Cells FFozen in 0.15 M Phos-
phate Puffer as Measured by
XLEJ‘EBI’) Uptalce cocooooooooooooooooo

Effects of Add in3 Supernatants
From Tiree Fre:zin3 liethods to

Esc hericl is coli Ce ntrifu3ed

Cells Frozen in Distilled We.ter

As fie sured by 0x33en Uptake .....

 

Effect of Addin3 Saline Frozen
Cell Supernatant to Unfroz“
Centrifu3ed Cells of Escher-
ichia coli Suspended in 0. 85 n
Saline Es fleasurcd by 0X“3e

Uptake OOOOOOOOOOOOOOOO00.0.0.0...

52

531

ll.

13.

1A.

15.

‘3

Effect Cl Add ; Dial3zed

Saline to Fsc richia coli

Centrifujed Cells Frozen
l

 

In 0.85 Z Sa ine as M3asurcd
b3 Oxygen Uptake .............

(D

Effect of Adding Ha .ted Saline
Frozen Cell Supernatant to
FC‘c‘mric‘cia co].i Centrifuged

 

Cells Frozen in 0.85 Z 83 inc
AS I’lCELSur'Gd 17:7 ORV-”in Uptakp

Effect of Addinc Ctarcoal
Treated 0.85 Z Saline Frozen
C; 11 Supernatant to E crer-
ichia coli Centrifu Cl lls

 

o
Froze n in 0.85 Z Saline As
Measured l3 x;:2n Uptake ....
Effect of Adding ADP, ATP,
and M5019 to Es hsric ia
coli Ccn’rifuigd clls
Frozen in 0.85 Z Saline As
Measured t3 Ox3gen Uptake.....

 

Effect of Adding HAD to
Escrericria coli

 

Centiilubod'cclls Frozen

In 0.85 p Saline As
Measured by Ox33en Uptake ....

Assa3 For MAD #Tn the Saline
Frozen Cell Suoernatant
With Glucose Der dro~enase ...

vi

(:5
C":

(\
f0

(3

U1

70

(L)

Table

LIST OF TABLES

Page

The Effect On the Endogenous ReSpiration

Rate of Escherichia coli Centrifuged

Cells Frozen in Tizree Diluents E3 the

Addition of the hree Frozen Cell

Supernatants As Measured By Ox3gen

Uptake O...OOOOOOOOOOOOOOOOOOOOOO0.0.0.0.... 57

 

Endogenous Respiration Rate of
Escherichia coli Cells, Frozen

 

In Four Susnendin3 A3ents As

 

Pleasured BJ 0X33en Uptake .................. 58
Endogenous ReSpiration Rates For
Escherichia coli Cells Suspended
In‘Differezt Ar:ents Before and
After Fre ezing As Meas ure --d B3
Sen Upta MI 00.000000000000000.00000000000 59

Effect of Adding ADP, ATP, and M301
to scooric ia coli Centrifug d Ce (3183
Frozen in 0.85 Z Saline As n-aStr3d By

OXL-yrQIL-Eh Upta-ke OOOOOOOOOIOOOOOOOOO0.0.0.0.... 67

 

PermJabilit3 and Utilization of Citrate
E3 Fscherichia coli Centrifuged Cells
Frozen In 0.85 Z’Salir e .3 Measured F3
}C:'("“n Ulfltale o.00000000000000.0000...000000 71

 

vii

”"TD hT’flm*h\T
{*LL DL‘Jlik/iv'

H 3 subject of a fireat many papers. The early reports
were primer i1;: concerned With :lie-tcxpcratnwn r la-
tionships and their effect on the per cen

a er freezin3. Only recently have experiments

been designed to elucidate the mechanism of freeze
death and injury. P-s yet, no one mechanism preposed

to account for death and injury, has gained wide accep-
tance. Indications are that the mode of action may be
dependent on xperimental conditions

Cne of the more prominent theories to explain
the action of fr reez in3 and thawing is that of In eta bolic
damage. It has been shown that oreanlsm , after being
subjected to freezing and tnaVir3, are injured
metabolically. This has been found to be manifested

in various ways such as

incre sed nutritional require-
ments, inability to 3row on selective media, and loss

of motility. The reason for this metabolic injury is

not clear. Several investigators, such as Hartsell (1959,
1961), believe that it is caused by the loss of cellular
constituents due to cell leakage. It is the purpose of

this work to determine VJhet her or not cell leaka

H

2

loss of cellular material without lysis, is OCCUPPlHT

.9
v

crzricsga cwli frozen in VPrious

m
L)

with cells Of

 

snapending agents, and if this l;aka;e material can

affect the respiration rate of frozsn E. coli.

LITERATURE REVIEW

I. General Aspects of the Effect of Freezing On Micro-
organisms

 

 

 

 

Microorganisms, when subjected to sub-zero tempera-
tures, give a broad spectra of damage and death, degen-
dent not only on the obvious experimental variants, but
also on more subtle ones. One must not only be concerned
with the Species of organisms, time, temperature and rate
of freezing, suspending agent, thawing time and temperature,
but with the past history of the organism. The method
of culturing the organisms is just as important as
the time and temperature of freezing. Since each in-
vestigator has established his own s;stem to study the
effectsof freezing, care must be taken in comparing the
various experiments and drawing conclusions. To complicate
matters, freezing seems to have a built-in inconsistency,
which invariable effects each experimental trial (Camp-
bell, 1943; Baum, 1943).

Effect of sub—zero temperature. Macfayden and
Rowland (1900 a) observed that a wide variety of micro-
organisms could survive freezing at —190 C for one week.
They made no attempt to determine the number surviving
after freezing. Smith and Swingle (1905), also using

3

Li

Salmonella typhosa suspended in broth, they found a

 

99.5 % reduction in two hours at —17.3 C, and 99.3 %
reduction for the same time of storage at -190 C. On the
basis of this evidence, they concluded that reduction

in viable cell number was as great at -17.8 C as at —190
C. Freezing at ~185 C and using the colon bacillus as
the test organism, Rivers (1927) found approximately the
same percentage reduction. However, he employed several
freezings and thawings, which are known to cause much
greater destruction and damage (Smith and Swingle, 1905;
Hilliard et a1., 1915). Weiser and Osterud (1945), using

‘g. coli suSpended in 1.0 % peptone water, found that

 

freezing at —l95 C, and immediately thawing, gave a 55.1
% reduction.

Other temperature ranges have also been used to de-
termine their effect on microorganisms. Bacteriophage
have sustained no loss in titer, when frozen at —78 C
Sanderson, 1925). Weiser and Osterud (1945) noted better
survival with g. 2911 at —78 C, than at —5, ~15, or -30 C.

Most investigators have found that temperatures above
-30 C are more lethal to microorganisms than below this
temperature. Hilliard et a1. (1915), using ;.g_o_1_i
and Bacillus subtilis, found that ~15 C was more damag-
ing than -2 C. However Haines (1938), also Esing E,

coli, pointed out that storage at -2 C caused more

p‘

.
- o
D
a
g . ..
~ 5
. a
_.
P
,— ,_ fl .
. 1 .
.r—
I
'
‘
.

I‘

5
reduction in viable cell counts than storage at lower
temperatures. Tarn: r and Prayton (1939) found no loss
of activity in various spirochetes and filtcrable
viruses at -78 C, but storage at -10 or —20 C, was followed
the case of the spirochetes by loss of virulence and
death.

More recently, Mazur (19 O a) has stated that

Saccharomyces cerevisiae and Aspergillus flavus, frozen

 

in distilled water, were drastically reduced in number

between the tempera ure ran ge of —10 and -30 C. in the

case of Saccharomyces, this was not in agreement with

 

Goetz and Goetz (1938 . Althoxg h the experimental condi-
tions were quite similar, they found that the greatest
injury occurred in the vicinity of —50 C. They did, how-
ever suSpend their organism for freezing, in Ringergs
solution which would account for the discrepancy.

Effect of storage time. Not only is the tempera-
ture at which microorganisms frozen important, but
also the length of storage. Prudde n (18 87) established
that when bacteria were frozen, a large percentage ex—
hibited an "immediate death," followed then by a more
gradual storage death. This has proven to be one of the
few instances in the liter Htur of low temperature micro—

biology where general agreement was found.

1"

0\

Salmon lla t rhosa, suspended in saline and frozen

1.!

at —l9O C was found by Winchester and Murray (1935) to

‘ u
be reduced from 1x10 to 1x10 viable organi sms in twen-
ty—four hours. But no further drOp was noted for saznples

examined daily for tie next six days. Wei ear and

U)

Osterud (1945) also found simila result usizg E. 0011.

1..
,l

The cells were suSpended in 1.0

3 counts were made on samples thaw;d

Cf‘

-l95 C. Pla
immediately after five hours and ten hours of storaee

at -l95 C. The percentage reductions were the same for
all three samples, s owing clearly that the greatest
reduction occurs immediately after freezing. Reeves ano
Tarrison (1957) again using E, ggli_but suspending in
h.6 M sodium chloride, observed that the greatest per cent

reduction appeared in th e first twent; -four hours of

freezing at -9 and -22 C, followed ty a more gradual de-

The only extensive work done on the effect of storage
time using a wide variety of microorganisms was do one by
Jones and Fabian (1952). Eighty cultures of bacte
representing eleven different genera were sub'ected to
freezing at -1T.8 C. Twen t~-ei;ht of these cultures were
thermOphiles, thirty were mes0philes, and seventeen were

psychrOphiles. Sampling was done before freezing and

7

after, at M, 8, 12, 2M, 48, 72, SC, 120 hours, and after
six weeks. The sharpest drOp in cell counts occurred
during the first twenty-four hours, followed then by a
more gradual reduction as storage time progressed.

Effect of coolin: and warming rate. Little work
has been done on the effect of cooling and warming rates
and their relationship to the freezing and thawing of
microorganisms. With only a few exceptions, have investi-
gators reported the rates of cooling and warming. How-
ever, it is a well-known fact that solutions which are
urapidly frozen, have different physical and thermo-
dynamic characteristics, than solutions which are slowly
frozen. It is entirely possible that these differences
can be translated into the effect they would have on the-
microorganisms suSpended in the solutions.

Mazur, in a series of four papers (Mazur et a1., 1957
a, 1957 b; Mazur, 1960 a, 1950 b), has done the only ex-
tensive study on cooling and warming rates, and their
effect on the freezing microorganisms. Their test
organisms were Saccharomyces cerevisiae, Aspergillus
flavus, and Pasteurella tularensis, suspended in a wide
variety of menstra for freezing. Essentially, they found
a slow cooling rate (1 C/min) favored microbial sur-

lvival, while a rapid cooling rate (50 C/min) was detri—

0"

8
mental. But when thawing was carried out, a rapid
warming rate was better than a slow one. Rapid thawing
also favored survival of spirochetes as Opposed t slow
thawing (Turner and Erayton, 193; .

Effect of suspending fluids. The menstra in which
cells are suSpended for freezing, plays a large part in
the survival of the cells. In fact, the suSpending
fluid is probably more important than any other factor in
the effect of freezing on microorganisms. Depending upon
which suSpending agent is used, per cent recoveries may
range 1 to 99 %.

Various menstra have been used to suspend organisms

for freezing. Freezing E- coli in milk, Keith (1913)

 

found that little destruction took place. However, if

the milk were diluted with water, the reduction became
greater. Tap water proved to be the poorest menstrum
used-~on1y 1 % survival at -20 c for five days. Distilled
water when used as a suSpending agent, gave just the
reverse results-—about 95 % recovery (Clement, 1951;
Harrison, 1956).

Cream containing 30 % butter fat, afforded good pro-
tection to §, ggli_and E, subtilis (Hilliard et a1.,
1915). This is not surprising since cream would act
much like milk. More quantitative results were pre-

sented by Clement (1961), who froze E, coli in skim milk

12% Difco at ~78 C for two minutes. Eighty-six per cent
/

. '1

9
of the organisms could be recovered.

Many other suSpending agents have provided good
protection to organisms upon freezing. Beef—blood serum,
7.5 % glucose, 4.5 % glycerol, beef serum and 7.5%»glu—
cose gave high per cent recoveries, with 2°.Eflil frozen
at -78 C for two minutes (Clement, 1961). Jones and
Fabian (1952) obtained their best survival by freezing
various organisms in vegetable extract and M0 % sucrose
solutions. Postgate and Hunter (1961) also found that
high molarities of glucose and sucrose, favor high sur-
vival rates.

Glycerol, which is a well-known stabilizer of other
biological systems, also has the same effect on micro—
organisms. Gram positive and gram negative organisms
suSpended in Albimi brucella broth containing 15.0 %
glycerol, survived well after five months at ~10 C

(Howard, 1956). The presence of 15 % glycerol, protected

 

 

.E. coli, Diplococcus pneumoniae, and Treponema pallidum

from damage by freezing and thawing (Hollander and Ne11,1959).
SuSpending of microorganisms in saline and buffer

systems has also been reported. fl comparison of saline

and broth suspending media was made by Proom and Hemmons

(19u9). Six organisms were used. Freezing was carried out

at —78 C. In all instances, reCOV€T3 was four to eight

10
times greater in bro h, with the exception of Staphr—

lococcus aureus where 100 % survival was found in both

 

saline and broth. Freezing g, ggli_in saline resulted
in greater injury than those suspended in nutrient
broth (Nakamura and Dawson, 1932). Organisms suspended
for freezing in a high concentration of sodium chloride
decreased more rapidly in number than ones frozen in
distilled water and various concentrations of sugars
(Tanner and Wallace, 1931).

When phosphate buffer at pH 7.0 (0.0003M KHQPOH) was

used as the freeze suspending agent for Salmonella typhi-

 

murflim, Staphylococcus aureus, and Strectoceccus faeca-

 

 

lis, a 90 % reduction in viable count was ob
L.

twenty-four hours at —21 C (Woodburn and Strong, 1950).

Eretz and Hartsell (1050) obtained similar results using

J//

3;. coli frozen in phOSphate buffer (14/15 pH 7.0 13:21:04).

 

After freezing g. 6011 for seven weeks at —9 C, they ob—

reduction in viable count. 0n the other

tained a 99 fl
hand, Mazur (1950 a) found that 0.1 molar solutions of

KHZPOQ, NaCl, and CaC12, when used as suspending mediums,
gave almost identical results as those obtained with dis-

tilled water.

Effect of other factors. Most investigators have not

 

concerned themselves with studying to any extent, the more

ll
subtle manipulations of the experiments which can effect
the freezing and thawing process on microorganisms.
These include such factors as : cell concentration, age
of the culture, cultural conditions, pH of the suspending
fluid, and aeration of the cells before freezing. It
has, however, been shown that factors such as these can
significantly change the results obtained after freezing
and thawing.

Major et a1. (1955) demonstrated with g. 391; and other
bacteria, that initial cell number influenced the survival
after freezing at -22 C. Eretz (1961) substantiated this
work using 3. 391;. He further stated that the greater
the initial cell number, the greater the recovery.

Age of cultures used for freezing can influence the
per cent recovery but this is controversial. Cultures

of E. coli in the log phase of growth, were found to be

 

more susceptible to freeze damage than those twenty-four
hours old (Toyokawa and Hollander, 1956). Jones and Fabian
(1952) found no difference between log phase growth
cultures and older cultures.

Harrison and Cerroni (1956) and Harrison (1956)
aerated suspensions of g, ggli_before freezing at —22 C
for one week and found that the aerated cells were much
more resistant than those not aerated. Bretz and Hartsell
(1959) also using ,3. 39;; and freezing at -9 C for three to
six months, reported that aerated cells were more sen—

sitive to freezing.

12

The hydrogen ion concentration of the freeze sus-
pending menstra is of importance. Less damage occurs
to cells suSpended in a medium around neutrality
whereas alkaline or acid conditions favor greater re-
duction in Viability (Tanner and Wallace, 1931; Mc-
Farlane and Gorsline, 19MB; Arpai, 1962).

Other investigators have stressed the importance of
using diluents of high osmotic strength such as 50 % su-
crose for diluting organisms after freezing (McCleskey
and Christopher, 1941; Bretz and Hartsell, 1959; Hart—
man and Huntsberger, 1961).

II. Theories 9n the Mechanisms 9§_Freeze Death

 

 

A great many theories have been proposed to eXplain
the cause of death and injury to microorganisms subjected
to sub-zero temperatures. Much difficulty has been encoun-
tered, due to the fact that each individual set of ex-
perimental variables results in different per cent sur-
vivals. Any one of these variables could possibly
change the mode of action.

Luyet and Gehenio (19HO) have discussed fully in
their monograph on low temperature, the theories of
death. They presented a list of theories which a number
of workers have used to account for the mechanism of
freezing as follows: (a) A withdrawal of energy,(b) the

attainment of minimal temperature, (c) mechanical injury

 

......
A ' . - . .
—
.—..
‘_/ r , . ,
. .
I I v , 0 ¢ 1
. k g
H
H V
O ’ ' ‘ " ‘
\ \
. . . . . . . - . - , . . . - . - , - . - . , . e ,. . - . . . -
. ..
_ I . ‘ ‘ .
. > v .
‘ ' ‘
_ r a n. l l _ . ,_ . - ” '
I ' l ‘
I ‘ ,' __
, ‘ . , -
u...
- '-
r . .’
M
7* , i ‘ .. _ ‘ e ‘ , .

 

J , . , v i A . . ' J
_ .
e . I ‘ <
v ‘v. I | ' l 9
¢ ‘_
' _ l
‘ 0
g .- . - i , . i . .
\ .A ‘ \ f
, , ‘ ' y g _- A 1..

13

(d) too rapid thawing, (e) dehydration, and (f) various
physiological, physical, and chemical changes. While
most of these theories have been postulated for
biological systems other than microorganisms, they still
cOuld appr.

Belehradek (1935) has also postulated mechanisms
of freeze death and injury. They are listed as follows:
(a) intracellular ice formation with resulting cell wall
rupture by dilation of the freezing water, (b) cell de-
stroyed on thawing, (c) mechanical crushing of cells by
extracellular ide, (d) forming ice withdraws water from
the cell causing dehydration, (e) ice forming outside
the cell destroys the protOplasmic surface-layer, (f)
ice is produced in the interior of the cell with a
simultaneous ice formation around the cell, (g) death
occurs without ice formation by direct action of cold.
Again these theories pertain more to systems other than
microorganisms, but they have at one time or another
found wide acceptance to explain the freezing phenomenon
in microorganisms.

Extracellular ice theory. Kbith (1913) postulated
that the mechanism of freeze death was mechanical destruc—
tion by ice crystal formation. -His evidence came from

the fact that milk and glycerin (5 to 42 %) when used as

 

14

suSpending agents, afforded good protection to bacteria
upon freezing at -20 C. Tap water did not favor sur-
vival. He believe that as the solution froze, the
bacteria and non—aqueous hatter were extruded from the
forming ice crystals. The cells were then protected by
either laying in or among these materials, thus pro—
tecting them from being crushed or otherwise injured.
Tap water, on the other,hand, would have just the Opposite
effect. The bacteria would be crushed by the ice crys-
tals. Conclusions drawn by Hilliard and Davis (1913)
support those of Keith's, that extracellular ice forma-
tion was the mechanism.

Further evidence to support the extracellular ice
theory comes from Goetz and Goetz (1938). They used

Saccharomyces cerevisiae suSpended in Ringer's solution

 

and frozen at different temperatures. Freeze death
in this instance, was termed physical death. Physical
death and ice crystalfization were found to be definite
functions of each other-~by suppressing crystallization,
the death rate could be controlled.

Weiser and Osterud (1945) suggest strongly that the
"immediate death" occurring when microorganisms are fro-
zen is due to extracellular ice formation. By using

freezing times of only eighty seconds with E, coli sus-

15
pended in 1.0 % peptone, pH 7.0, and visually observ-
ing ice formation, they found the greatest decrease
in viable cell counts to occur during formation of ice.
Proom and Hemmons (19M9) stated that extracellular
ice crystals punctured the cell wall and membrane.
Their results revealed that Staphylococcus aurcus, ;.

coli, and Shigella dysenteriae survived much better

 

when in saline and broth at -78 C, than Vibrio comma,
Neisseria intracellularis, and Neisseria gonorrhoeae.
Differences in survival between these two groups of
organisms, they postulated, was due to the strength of
cell wall. In fact, they did find many degenerative
forms on microscopic examination.

The evidence against extracellular ice as the
mechanism of freeze death and injury is just as impressive.
But it must be kept in mind that experimental conditions
are not the same, therefore possibly resulting in
different mechanisms. The most striking evidence that
extracellular ice does not mediate death and injury to
microorganisms,was recently presented by Bretz (1961).

By freezing E. coli at -9 C on ce110phane or membrane

 

filter disks and comparing survivals after freezing to cells
suSpended in M/lS phOSphate buffer and treated in the

same manner, he found that the results were the same.

16
Since extracellular ice would not be present on the
filters, it can be ruled out as the cause of death.
Harrison and Cerroni (1956) presented data corre—
lating physical strength and susceptibility to freez-
ing to diSprove extracellular ice and its crushing
effect. Two test organisms, g, 2211 and Microbacterium
flavum were employed. They made two szspensions of
each organism; one was frozen and thawed a number of
times, the other being blended in a tissue disintegrater.
.2..ggli proved to be more susceptible to freezing and thaw-
ing than_M. flavium, though g, ggli_was physically strong-
er, as shown by the disintegrater experiments. From
these data, they drew the conclusion that there was
no correlation between the physical strength of the cells
and their susceptibility to freezing. On the basis of
this evidence, they further concluded that the lethal
factor of freezing and thawing was not mechanical.
Several investigators, Haines (1938) and Jones
and Fabian (1952), have shown that different Species of
bacteria did not change in size after freezing nor could
they find any broken or distorted cells. If ice crystals
did crush cells, these changes should have been evident.
Weiser and Hargiss (1946) designed an experiment to

prove or disprove the mechanical ice crushing theory.

17

Il— 1'- < . a a . v ~ I“ "'\ 1 1‘ 1 --- ‘
UCSb organism, n. coli, was suSpcnce in a 10 fl su-

C)

The

 

Luv:

crose solution. Only 0.02 ml. 0 the suSpension was

placed between two cover slips and frozen in liquid
nitrogen. This rapid freezing causes tLe formation of
a Vitreous state. Thawing was then carried out in one of
two ways in order to bring about either devitrification or
vitromelting. The control was a sample which was allowed
to crystallize. It was expected that the v trifying
and vitromelting would cause the least damage, and
crystallization and devitrification, the most damage.
However, it was found that crystallization caused the least
damage, indicating that extracellular ice formation was not
as important as previously thought.

It is well known that cold shock can injure micro—
organisms (Hegarty and Weeks, 1940; Meynell, 1958).
This implies that death may be due to the effect of
cold alone, and mediates to an extent, against the mechani-
cal death theory. However, Wieser and Osterud (1945)
found that upon supercooling suspensions of E. 231;, re-
ductions of only about 3 % , as Opposed to reduction
of about 50 % for freezing occurred. Mazur (1960 a,
1960 b,) also found this to be true. Harrison (1956),

using several different organisms suspended in 4.6 M

sodium chloride, got good reductions upon supercooling.

J‘

,4

18

The difference probably lies in the fact that
Weiser and Osterud used a different suspending agent
than Harrison.

Intracellular ice theory, Intracellular ice forma-
tion has often been postulated as being the major factor
in freeze damage to microorganisms. Due to the small
size of the organisms, all evidence to date has been by
necessity, circumstantial.

thur (1960 a, 1960 b) has been the main prOponent
of intracellular ice formation in microorganisms as the
chief cause of death and injury. Evidence for this, he
believes, comes from the fact that the organisms he used
have a definite temperature range, where injury is the
greatest. According to his theory, xtracellular ice is
a prerequisite for intracellular ice formation. He
suggests that water within the cell has a tendency to
supercool, possibly due to the fact that there is little
likelihood for nucleation centers to form in very small
volumes of water found in the cell.n:Eut when extra-
cellular ice appears in the external medium, it enters
the cell, acts as a nucleation center for the super-
cooled water, and effectively seeds it, causing freez-

ing. This explanation requires further hypotheses.

19
First it must be assumed that ice crystals can enter
into the cell. This, he feels, is accomplished by
the fact that the cell membrane and wall have small
water-filled channels only angstroms in diameter. Ice
crystals must have a sufficiently small radius of curva-
ture in order to pass through these channels and seed
the intracellular water.

Since it is known that the equilibrium freezing or
melting point of a crystal of such a critical radius
would no longer be 0 C, but at a much lower tempera-
ture, possibly even —30 C (Chambers, 1959), it fits the
data which bhzur has collected. This would explain 1
why rapid cooling is so lethal as Opposed to sl w cool-
ing. Small ice crystals form when rapid cooling takes
place, and these are small enough to pass through the
'pores. Slow cooling would favor much larger crystals
and hinder pasSage.’ Ne (19:0) has reported photo-
graphing intracellular freezing in rapidly cooled yeast
cells but not in slowly cooled ones.

Calorimetric measurements on populations of yeast
cells that allowed determinations to be made on the
fraction of cellular water frozen as a function of
temperature, have been made (Wood and Rosenburg, 1957).

IBterminations showed that normal yeast cells contain

20
69 % total water. As the temperature was lowered
below 0 C, a progressive amount of this was frozen
until at -22 C, about 87¢ of the total water was fro-
zen.

Weiser and Osterud- (1945) presented evidence
against the theory that ice forms within bacterial
cells. Their first finding in support of th's, was that
the intensity of the freezing temperature has no in—
fluence on "immediate death" due to freezing of g,
ggli_in 1.0 % peptone. If intracellular ice were to
contribute a lethal effect, there should be some
temperature where the lethality was more pronounced.
Repeated flucuations of the suSpcnsions between the
temperature ranges of -1.5 C to -195 0, did not cause
any more damage than that in stored controls. Finally
it was observed that marked "immediate death" occurred
at temperatures just below 0 C, where intracellular ice
would not be expected to occur.

More direct evidence against intracellular ice
comes from Haines (1938) and Jones and Fabian (1952).

By staining cells after freezing, neither could find any
distorted cells which would be expected to occur in a
population, if extensive damage due to the intracellular

or extracellular mechanical action of ice had occurred.

21

The withdrawal Of water from cells, due to the
formation of the extracellular ice and the bound water
present in cells (Luyet and Gehenio, 1940), mediates
against intracellular ice formation. The various
eutectic points of the protoplasm of microbial cells
would also offer protection against intracellular freez-
ing at higher temperatures.

Luyet (1951) could find no evidence of intracellu-

 

lar ice formation in frozen cells 0 Streptococcus 3c—
tia examined with the electron microscope. Iis cri-

terion for intracellular freezing was conparison of

ell size and cytoplasmic granule disturbance

(J

frozen cells.

Concentration of solutes theory. As previously
stated, most investigators who have used distilled water
to suspend microorganisms for freezing, have found it
not too detrimental. Recoveries have usually been be—
tween 90 and 99 %, based on viable cell counts. fow-
ever, ii‘certain solutes or electrolytes are added to
distilled water, the per cent survivals are in many
cases , decreaSed. This phenomenon has been attri—

olutes or elec—

Q
\a

U)

U)

tuted to the concentration of the
trolytes around the cells during freezing. As water

freezes it extrudes this foreign matter, leaving it un—

_..._—._. "Wm _.

 

 

22
frozen until the various eutectic points are reached.
Faines (1935), in his classical work on the effect
of freezing on bacteria, found that —2 C was the most
injurious storage temperature. Working with Pseudo-

monas aeruginosa, he isolated the native cellular pro-

 

tein by low temperature extraction methods. Upon

freezing the protein at —2 C, he found that one frac-

tion rapidly coagulated. Such flocculation was negli-
gible at —20 C. This lead him to postulate two factors
being responsible for bacterial freeze death: one unknown
but apparently not mechanical, and the other causing

the flocculation of cellular protein. He thought it was
possible that the flocculation was due to concentra-

tion of solutes or change in pH.

More recent work with E. coli and Lactobacillus

 

fermenti suSpended in broth, broth diluted ten-fold,
one-hundred—fold, and distilled water with freezing at
-22 C, has shown that per cent recoveries are higher,
the more dilute the broth (Harrison, 1956). Undiluted
broth gave the smallest per cent survival, whereas dis-
tilled water gave the highest. By using H.6 M sodium
chloride which remained unsolidified at —22 C, the sur-
vival curves had a continuously negative SlOpe, indi-

cating that solute concentration was probably respon-

n

23
sible. Taking advantage of the fact that glycerol
protects g, ggli_when frozen, he designed an experi-
ment using two different suSpending agents: 4.1 M glyc—
erol, and 1.4 M sodium chloride. Cooling was carried
out at -22 C for one—and-a-half hours without ice
crystal formation. Glycerol alone, caused no reduction.
Sodium dhloride (1.4 M) produced 60 % reduction but the
glycerol-sodium chloride mixture caused only 26 % re-
duction. It therefore seems that glycerol acts as a
solute buffer, protecting cells from high concentra-
tions of solutes since it can counteract much of the
damage induced by the sodium chloride.

Further work along this approach by Reeves and
Farrison (1957) supported their previous work. They
found that cells which are able to survive the rapid
decline in viability at -22 C in 4.6 M NaCl, are able
to maintain themselves in it for long periods, but this
resistance is not heritable.

Both Lovelock (1957) and Mcryman (1955), who have
worked extensively with the effect of freezing on red
blood cells and mammalian cells, believe the damage is
due to concentration of electrolytes.

There are reports in the literature which provide

(W

evidence that high concentration of solutes and their

24
other effects are not Operative under er

stance

O)

A stated before, Pretz (1951) froze bacteria

W 3

CO
C)

on cellophane and membrane filters. Results indicated
that reductions were as low as those for organisms sus-

pended in 0.067 M phosphate buffer. Since organisms

Ho

mp nged on cellOphane and membrane filters are not in
a liquid medium, this eliminates the possibility of
concentration of solutes.

Mazur (1960 a, 1960 b) has Spoken out against
high concentration of solutes as the major cause of
damage in freezing. With Saccharomyces cerevisiae sus—
pended in 0.1 M solutions of KHZPOM, NaCl, and CaCl2,
he found the per cent survivals compared favorably with
those of distilled water suspended organism after freez-
ing. He also stated that if death were the result of
high concentration of solute, one would expect longer
exposures to produce greater damage. He found this to
be just the Opposite.

In an experiment similar to Harrison's (1956),
thur (1960 b) used Asperigillus flavus Spores suSpended
in unfrozen and frozen solutions of 4.0 M calcium chloride
and 3.3 M MgClg. If the media were to be kept in an un-

frozen state, the recoveries would be high. However, if

25
they were frozen, recoveries were very low. This
seems to mediate against high concentration of solutes
in this particular system.

Metabolic damage tbeo y. It is possible that the
freezing process severely stresses microorganisms,
causing injury which is manifested by metabolic damage.
This dam ge could appear in many forms which would
progressively degrade the cell until death resulted.

Currans and Evans (1937) and Nelson (19MB) drew
attention to the fact that bacteria and their spores
are more demanding in their nutritional requirements
after being subjected to extremes of physical or chemi-
cal environment. Similar reports have shown that the
same is true for freezing. Gunderson and Rose (19MB)
found that violet-red bile agar is very inhibitory to colié
form organisms found in foods. Goresline (1946) re-
ported the glucose tryptone agar was superior to other
plating media in the recovery of organisms from frozen
vegetables. Hartsell (1951 a) did further work on this

with g, coli, Shigella dysenteriae, and Micrococcus

 

 

 

aureus suSpended in a preparation of egg-culture mix-
ture and freezing at -9 C and -18 C for up to a year.
He found that E. coli and g, dysenteriae grew better on

yeast extract—veal infusion agar, than on MacConkey agar

 

"I

.~.

26

after freezing. Hi3her recoveries were also found on

YE-VI agar, than on Staphylococcus 110 a3ar waen

., a 4 u \ .- .1 —. 3 .a‘m- °
uh aureus was froze in t1; same manner. loweyer, 1f

3

the defrosteo or3anisms were allowed to stand for
one to six hours, no in”i bition was found upon plating
tiem on tneir respective selective media. The reason
for better recoveries on VE-VI agar, he felt, was due
to certain vitamins an trace e13m3nts, plus the fac
that it contained no solectiVo, toxic a

A similar publication by Yartsell (1951 b) also
showed that such so ective media as desoxfcholate, violet
red bile, MacConk“ and Staphylococcus 110 a are, were in-
hibitory to bacte r1 ia aft:er they have been frozen in beef
and peas. YE-YI agar was again used as a standard
reference medium. He is of the opinion that the freezing
proce' s like the heatin3 process in foods, alters the
nutritional requirements of bacteria. If then, these
cells are given a booster dose of m abo lit es, the;
can be recovered.

Evidence of this nature strongly sugjests that t;
cells are in some way injured or damageC. subos (1937)
showed that pneumococci, living or dead, to be liable

- m m1- - - ~‘AA' ‘L -- . ‘ 1» v
to lgsio, must have their autolytic enzgtes activates.

Freezing accomplished this activation. Haines (1937

O _‘ . 3 ‘
;uru31nosa could be

H)
O
L.
:5
Q1
ct
_)
:1)
cf
(‘1‘
If
(I)
'7')
'1
O
C l
(J
2-“
O
H)
*d
C)
O
")

 

 

de natur-d by freezing, in’icatins possible injury,
depending upon the extent of dama3e. Turner an 1d Tray—
ton (1939) have su33ested that freezing of microor 3ar isms
at temperatures in the vicinity of -10 to -°0 0, could
brin3 about injury mainly be cha 333 iecident to cell
metabolism or to proteol; tic or other enz :mcs.

The injury to microorganisms can be prOCressive,
chan3ing unharmed cells to injured, with varying degie es
(Straka and St okes ,1939). Us in3 this criterion, Straka
and Stokes found that this was true. By using a syn—
thetic medium and a complete medium (trypticase soy

agar), they were able to show that E. coli and several

Species of Pseuddmonas were metabolically damage d after

 

freezing at different temperatures and times. This
metabolic damage was manifested by the fact that the
complete medium could recover more organisms than the
synthetic. The per cent of injured cells in a population
after freezing, was determined quantitatively by the
difference in plate counts on the two media. Injury varied
wit h the time and temperature of stora3e, and wit h

the nature and pH of the suspending fluid. The addi-

tion Of 2 % trypticase to the synthetic agar increased

recoveries after freezing, to those comparable with

28
trypticase soy a3ar. Other substances which did not
prove active included acid hydrolyzed casein enriched
with cystine and tryptOphan, mixtures of Eevitamins,
purines, and pyrimidines. They suggest that the acti-
vity was due to the presence of peptides in the trypti-
cane.

Work with Shi3ella sonnei by Nakamura and Dawson

 

(1952) has shown the same type of freeze injury. Cells
frozen in saline exhibited greater injury than those
frozen in nutrient broth or milk. By addition of meat
extract, peptone, or casamino acids to their synthe—
tic medium, recoveries were again brought to the level
of those on nutrient agar and grain-heart infusion
agar containing whole human or rabbit blood.

Arpai (1052,1963) also using a comparison between
a complete medium and sgnthetic medium to determine
freeze damage, found that as the temperature was lowered
from -7 C to -18 C or ~30 C, the decrease in the number
of unharmed cells was much less rapid. He also found
that the number of injured cells increased as the killing
rate decreased. Using motility as a criterion of freeze
damage, he found that it could be correlated to non—
lethal metabolic freeze injury and further,injury was

effected by the time and temperature of storage as well

29
as the nature of the suSpending fluid.

Moss and Speck (19€3), using treptococcus lactis,
have reported metabolic damage due to freezing, using
techniques similar to those just discussed.

Cellular leaka3e and its conseggences. At the pre-
sent time, it is currently acceptable to speak of
"leaky bacteria" (Hartsell, 1959) and cellular effluent
(Harrison, 1951). Leakage has been found to occur not
only in frozen cells but in cells which have been
stressed in other ways. There also seems to be an
interrelationship between cellular leakage and cell
metabolism. It has been further reported that he
leaka3e material and other substances can stimulate
or restore cellular activity, depending on the cri-
terion used. At the present time, this concept offers
a plausible explanation for freeze injury and death
and perhaps other types of cellular injury and death.

Hartsell (1931 a, 1951 b) and Squire, and Hertsell
(1955) reported that organisms frozen an thawed, ini-
tiated more rapid growth than those which were not
frozen. Tanguay (1979) also found this greater 3:0wt
response with several of his test organisms. He indi—
cated that it was possible that this could be due to

1..

accumulation of stimulating materials within the cell

30
during frozen stora3e.

Further elaboration on this theory came from Hart—
sell (1959) as follows: "Storane at subfreezing
temperatures creates a special type of dormant state
in nonsporulating Species. A synthesis, even at sub-
freezing temperatures, might allow substance A to be
utilized in product C, but the use of product C to form
D, which latter product is essential to cellular divi-
sion, could not occur until the cells were defrosted.
If this conception of events is true, product C should
accumulate as the cells are held in storage and could
be isolated if appropriate techniflues were available."

The isolation of product C was reported by Hartsell
(1951). It is called "Factor 8" — — for stimulating.
It can be isolated from.§, 22;$_suSpended in Sorenson's
buffer for fifty-five days at —9 C; as yet it has not
been identified. The yields of the factor are very low.
It can stimulate other bacteria, and it appears not
to be a vitamin, amino acid, or a carbohydrate. ieh-
ler and Hartsell (1953) deve10ped an assay procedure
for "Factor S" activity by observing spectrOphotomet-
rically, its stimulating influence on the growth rate
of homologous and/Er heterologous broth cultures. Other

organisms which were found to produce "Factor S" were

31
Pseudomonas fragi Sarcina Iqtea Staphylococcus aureus
.3 ’ J A . 3

Pacillus coa3ulans, Bacillus stearot53rm0philus.

 

 

Deotto (194u) also observed a stimulation in cold
shocked 3-.32ll- Using warburg techniques, he found
that if the cells were placed at O C for 20 to 30
minutes, they showed an increase in oxygen uptake after
being placed back in the water bth at 38 C. It was
thought that stimulation was independent of multipli-
cation of the cells and dependent upon the liberation of
a respiratory stimulant from the cold—shocked cells.

Fretz (1958), attempting to characterize the pro-
perties of the leakage material from g, gglg_frozen
at —9 C in phOSphate buffer for various periods, found
no release of ultraviolet-adsorbing substances, but
ninhydrin-positive materials were detected. Later,
Eretz and Easa (1950) demonstrated that the lOSS of
this material from g, gpll_in M/15 phosphate buffer
stored at 59 C, protected the survivors. They also
showed that this material adsorbed to the cells.
Characterization of this material was carried out
(Ambrosini and Eretz, 1953) by dialysis, ashing, and
chelation experiments. t was shown that the pro-
tective factor was not a metal, or small molecules.

Norite treatment and dialysis demonstrated that large

32
and small molecular weight compounds were present.

0
..

The large molecular we:

(3

3ht compounds gave protection,

while the smaller molecules were actually 'nhibitory.
Other examples of leakage material from miCrOCrgan-

isms have been reported in the literature. Holden (1958)

found that nucleotides leached out of Lactobacillus

 

arabinosus during incubation in phosphate buffer at

 

37 C. These nucleotides came from the degradation of
intracellular nucleic acids. Ultra-violet absorbing sub-
stances were deteeted in cell leakage material from

Bacillus me3atherum suSpendcd in phOSphate buffer for

 

two hours at 37 C (DeLamater et a1., 1950). Two

J‘d/

‘

fractions were detected one dialvzable and the other
.9 u

NOt dialyzable. Washed cells of Saccharomyces cere-

 

visiae, incubated in .08 M sodium citrate for two

hours with 2 i glucose added, leached material (Higuchi
and Uemura, 1959). The supernatant contained ultra-
violet absorbing substances with a maximum at 258

mu and a minimum at 235 to 2M0 mu. The nucleotides

were probably fragments of ribonucleic acid. By freezing
and thawing the cells, the supernatant was found to con-
tain a greater quantity of material. However, they felt

that not all of this material might be the same since

an increased absorption at 230 to 250 mu was noted.

33

Little work has been done with the leakage material
from frozen cells as far as their effect upon cellular
metabolism and restoration of activity. Work has
been done w th restoring the viability of cells exposed
to ultra-violet light, heat, and chlorine (Hemmets et
a1., 19511 a, and 1954 b), using various metabolites.

Lund (1951) incubated frozen washed yeast cells in
supernatant collected after freezing and in distilled
water in Warburg vessels. With the frozen cell super-
natant, an 86 % increase in cellular nitrogen w s found
as Opposed to only a 5 % increase in distilled water.
Leakage of phOSphorus into the suSpending medium was
detected also.

Eovernick (195T) and Eovarnick and Allen (195M)
found that typhus rickettsiae frozen in isotonic salt
solutions, showed a greatly decreased toxicity for mice,
hemolytic activity, reSpiration, and infectivity for eggs.

Nicotonamide adenine denucleotide could restore most

of this activ ty and coenzyme A, part of the activity.
NAD was detected in the suspending medium after freez—
ing.

It has been postulated that freezing causes an
altered permeability of the cell membrane or wall.

Changes such as these would permit the 1 as of cellular

3A
constituents and perhaps the entry of some previously
impermeable substance.

The term, osmosensitiven ss, has been ap plied to
changes in cell wall and membrane of frozen organisms
upon freezing (Bretz and Hartsell, 1959). Ttey found
That 10 % sucros was a better diluent for frozen
organisms than distilled water or various phOSp hate
buffers. The high esmotic strength of the sucrose
diluent protected the frozen organisms by creating
more favorable enviror ment and perhaps stepped leakage
from these cells even after freezing.

Lipid—protein complexes of cell membranes could
possibly be ruptured upon freezing since they are not
held together by strong covalent bonds, but by weak
association forces (Lovelock, 1957). Causes of this
could be increase in electrolyte concentration changes
in pH, and removal of water. His work with red blood
cell membranes, proved that the cells lose phospholipids
when suSpended in various molar solutions of NaCl.

Lysozyme is know to attack cells which in some

ay have been stressed by heat freezing, etc. Kohn and

Szybalski (19 59) and Kohn (1960), working with E. coli,

 

found that the cells were insensitive to lysozyme in the

growing or resting cell states. However, frozen and

35

thawed organisms were sensitive to lysozyme. They

J

postulated that freezing and thawing tore Op>n the outer

C

layer of the cell wall, which is believed to be a
plastic lipoprotein layer and resistant to lysozyme.
Restoration of lysozyme insensitivity occurred in about
30 minutes after freezing and could not be stOpped by
such inhibitors as chloraphenicol. This implies that smme
type of repair process is going on in these cells due to
the damage caused by freezing.

Other permeability alterations have also been
noted. Faker's yeast, when unfrozen, does not adsorb
or metabolize citrate but once it has been frozen, citrate
becomes freely permeable but still is not metabolized

(Foulkes, 195M).

MATERIALS AHD.METHODS

CUItivation of organism: Eschvrichia coli (Achf

 

11303) obtained from the Division of Laboratories,
Michigan Department of Health, was used throughout the
work. Cells were grown on tryptone glucose extract
agar slants for 18 hours at 37 C and harvested with
the proper diluent. The pooled cells were placed in
sterile Centrifuge.cups, centrifuged at 12,100 x g for
20 minutes, and washed three times with the prOper
diluents. Sufficient diluent was then added to give a

final concentration of about 2.0 x 1010

organism per ml,
with a dry weight of about 6.0 mgm per ml. After the
cells were thoroughly mixed, serial dilutions were

made using Butterfield's Buffer (Butterfield, 1933).
Duplicate TGE agar pour plates were made and incubated

at 37 C for 24 hours before counting.

Suspending fluids. Four different suspending

 

fluids were used to suspend the organisms for freezing:
(l) distilled water, (2) 0.85 % NaCl, (3) 0.15 M phos-

phate buffer (10.65 g. Na HPOM’ 10.25 g. NaH2P04. H20,

2
1000 ml distilled water) pH 6.8 ionicestrength 0.12, and
(h) 0.06 M phosphate buffer (4.26 g. Na2HP04, 4.1% g. Na
12P04 . H2), 1000 ml distilled water) pH 6.8 and ionic

strength 0.30. Hydrogen ion concentration determina-

36

o . - v ‘ - , ., Or- . 1?...
tions were made on the distilled mater and 0.-) T haCl.

In most instances, he pH was about 6.9. Fe. this reason,
# V f
the phosphate buffers were made to pH 3.9.

torafie. Five ml portions of the

 

 

cell suSpensions were pipett—d into sterile 2Cx150

(

mm glass test tubes for freezing. Freezing and storage
of the cell suSpension were carried out in a cooling
bath containing a mixture of 95 % ethanol and water
placed in the freezing compartment of an ordinary
refrigerator. The temperature of the tath was —15 C t,
l. A frequent check was made on the temperature of the
bath with a thermometer, and a Microm X Automatic tempera-
ture recorder (Leeds and Northrup Co., Philadelphia,

Pa.). The glass test tubes were placed in a slanted
position to avoid breakage. From 5‘to 7 minutes was
required for freezing of the 5 m1 portions of the cell
suspensions. torage time was 22 hours.

At the end of a storage period, the tubes were re-
moved from the freeze bath and thawed by immersion with
gentle agitation, in a 50 0 water bath. About 90
seconds were required to thaw the suSpension and reach
room temperature. The cells were then pooled in sterile
centrifuge cups and mixed. Determination of viable cells

was made as previously described.

Preparation of cells for oxggen uptake experiments.

38
In all experiments, 0.5 m1 of the resting cell

suspensions, was added to the Worhurg vessels. Three
different preparations of the cells were used in the
Warburg apparatus, depending upon the exeeriment:

(l) unfrozen cells prepared as previously de-
described

(2) cells frozen and thawed just prior to use
(These will be termed ”frozen cells")

(3) cells frozen and thawed, separated from their
supernatant by centrifugation, and resuspended
in the same type of fresh diluent (These will
be termed "centrifuged cells")

The procedure for the preparation of (2) and (3)
was as follows: From 3 to 5 ml of the pooled frozen
cells in the centrifuge cups were removed. The re-
maining cells were centrifuged in the cold for 30 min-
utes at 22,000 x g. The resulting supernatant was de-
canted and the volume noted. This represents the fro-
zen cell supernatant used in the oxygen uptake stimu-
lation eXperiments. An equal amount of the pr0per
diluent was added to the cells in the centrifuge cups,
followed by thorough mixing. Viable cell determinations
were carried out on these cells to ascertain if the
prOper cell concentration had been maintained.

Manometric techniques. The usual Warturg techniques

were employed to determine the oxygen uptake of the cell

suspensions (Umbreit, et a1., 1557). The contents of t1e

flask

m
g
m
d
(D
I.

0.5 ml of the resting cell suspensions, 0.5
ml of 0.067 M phOSphate buffer pH 7.0, with 0.2 ml of 20 Z

1211-? 1ts ezzce

Ho

KOH in the center well. In all exper
(‘Difco Bacto-dextrose) was placed

0.5 ml of 0.05 M glucose
in the side arm as substrate. To de terzn ine wlethe r or not

the cells had undergone a permeability change , 0.5 m1 of

C]

0.05 M sodium citrate was used as ubstrate, either alone,

or with 0.1 m1 of 0.01 M sodium acetate. The volume of the
vessels was brought up to 3.0 ml by the achi ion of dis-
tilled water.

T.e frozen cell supernatants from ea.ch of the four
freeze suspending agents were tested against their respec-
tive centrifuged cells by adding 1.3 ml of the supernatant
to the vessels in place of distilled water, 0.85Z saline, and

i

0.15 M phOSphate buffer were tested by adlit on Of 1:3 ml to

the vessels as follows:

(1) frozen cell supernatant from distilled water sus-
pended cells, tested with centrifuged cells which
had been frozen in 0. C5Z saline and with cen ri-
fuged cells which had been frozen in 0.15

phosphate buffer.

(2) frozen cell supernatant from 0.C5Z saline sus—
pended cells, tested with centrifufed cells which
had been froze n in distilled water and with centri—
fuged cells which had been frozen in 0.15 H
phosphate buffer.

(3) frozen cell supernata.nt fro Q11 0. 15 H phos phate
buffer suspenc’d ce ls .est ed with eentrifu ;ed
cells which had been frozen in 0.C5 Z saline

40

and with centrifuged cells which had been
frozen in distilled water.

Various cofactors were added to the reaction mix—

1.

ture in the vessels usin~ centrifuged ce ls Wibh no

1»--.

Q

1

frozen cell supernatant added, to determine whethe
or not, they could simulate he stimulating effect.
The cofactors were added to the vessels in 0.1 ml
portions, giving a final concentration of each as
follows:‘ 2 um nicotinamide adenine dinucleotide, 2 um
adenosine triphOSphate, 2 um adenosine diphosphate, and
10 um H3012. These were e ther freshly made for each
experiment or maintained in the frozen state.

The flasks were equilibrated for 15 minutes, and the
oxygen uptake was followed for a period of 50 minutes
at 37 C. All suSpensions were tested in duplicate.
The results reported, represent values after subtracting

the endogenous rates.

Characterization of frozen cell supernatant. In

 

order to determine some of the preperties of the fro-
zen cell supernatant from cells frozen in saline,
various prodedures of characterization were employed.
In all instances the activity of the treated super—

natant was compared against untreated supernatant, using

centrifuged cells for each.

Ml

Dialysis was used to determine the diffusibi-
lity of the active material. Ten ml of the frozen cell
supernatant was placed in cellulose tubing and dia—
lyzed against 0.005 M phOSphate buffer in the cold for
24 hours.

Heat stability was determined by heatinfi 10 ml of
the supernatant at 100 C for 30 minutes. The heated
supernatant was then rapidly cooled under cold tap
water.

An adsorption experiment was performed using an
activated charcoal, Norit A (Pfanstiehl Chemical Co.,
Waukegan, Ill.). One-tenth gm of Norit A was added to
10 m1 of supernatant. After mixing, the supernatant
was incubated at 37 C to increase the adsorption. The
charcoal was removed from the supernatant by gravity
flow filtration.

Spectrophotometric techniques. A Beckman Model D

 

U Spectrophotometer was used for all SpectrOphotometric
work. Three ml of saline frozen cell supernatant was
added to quartz cuvettes and the absorption Spectrum
noted.

Glucose dehydrogenase isolated from Bacillus cereus
(courtesy of Mr. John Kools), was used to assay for nice—

tinamide adenine dinucleotide (NAD) in the saline frozen

12
cell supernatant. The reduction of nicotinamide
adenine dinucleotide was followed spectrOphotometri-
cally at 3H0 mu. One cuvette contained 0.3 ml of
glucose (300 um), 0.3 ml of NAD (Sum), 0.3 ml of glu-
cose dehydrogenase, 1.0 ml of 0.15 M tris (hydroxy-
methyl) aminomethane (tris) buffer pH 8.0, and 1.1 m1
of 0.C5 Z saline to give a final volume of 3.0 ml.
The other cuvette contained the same materials except
that NAD and 0.85 Z saline were excluded and 1.1 m1
of saline frozen cells supernatant added in place of
them, giving a final volume of 3.0 ml. A control w s
also run to determine whether or not the saline frozen
cell supernatant might inhibit the enzyme. This was
done by replacing the 1.1 ml of 0.85 Z saline with an
equal amount of saline frozen cell supernatant. The
change in optical density at 3M0 mu was followed for

180 seconds.

1' .ZSULTS

Cells frozen in saline.

saline always re

cell counts and a large cecrease

Figure 1 shows the decrease in oxyg:
freezing. An oxy3e1 uztake of
fore freezing, as Opposed t
freeZ1nj. 1his represents abou a

was 20. Variation in per cent recoveries
three other trials. They ranged
The removal of th he frozen cell

{1)

the cells caused a further decre

from 132 ul to 110 ul (Fig.

+?“1“\

frozen cell supernatant to the

centrifuged cells, the oxygen upt

158 ul, indicating that the

tained stimulating substances.

natant in the presence of glucose,

uptake, nor was salire alone found
lation This was also fourd to be
frozen cell sup rna mt nts.

The addition of 1.3 ml of supe

h

:1
‘1.)

Freezing cells

299 ul was

frem 11.0 Z to

m.—
3

1). 1‘

g].

‘l ..
Wareurg vessels

The

ells was chosen be cause frozen cells,

.2. Or-
.Lf] 0...--.) 7‘:

sulted in a large reduction in viable

in oxygen uptake.

n upta“e ter

observed be—

0 only 132 ul taken up after

r- _ '\ ' - V '-
ca 7 decrease in Lptahe.

experiment

A».
for

found for

.11.
supernatant from

in o::;-'f“e n

ould be raised to

supernatant possibly con-

fr zen cell super-

" A w!- "
snowed no oxygen

:3
Cl‘
C1.
0
r‘.
T
O

rnata

which

.Microliters of oxygen

310

290

270

250

210

190

170

150

130

110

90

70

50

30

10

an

A Unfrozen cells .A
X Frozen cells
0 Centrifuged cells A

El Centrifuged cells with 1.3
ml 0.85% saline frozen cell

supernatant added A

20 Per cent recovery by pour
plate method

A.

10 15 2O 25 30 35 HO #5 50

Time in minutes

Oxygen Uptake of Escherichia coli Cells Frozen
in 0.85% Saline

 

contain about 0.5 ml of frozen cell supernatant, did

not show the bf

C)

4— 1.x. 4. 1. .h-
0 stimulation, In some instances

\

(Fig 2), the stimulation was almost the same. In no

('1‘

instance did he frozen cells have a greater oxygen
uptake than centrifuged cells with 1.3 ml of super-
natant.

Cells frozen in 0.0T M phosphate buffer. The de—

 

crease in oxygen uptake after freezing in 0.05 M phos-

phate buffer was 60 ul (Fig. 3). Removaliof the frozen

(D

cell supernatant caused a further decreas in oxygen
uptake, from 230 ul to 154 ul, a loss of 66 ul. The
addition of 1.3 ml of the 0.06 M phOSphate buffer fro—
zen cell supernatant to the centrifuged cells, restored
the activity to its original level of about 230 ul.
However, the addition of 1.3 ml of supernatant caused
no greater increase than 0.5 ml of supernatant. Two
more subsequent trials give similar data to that in
Figure 3.

The per cent recovery of frozen cells for this
experiment was 36 7. In the other two trials, the per
cent recoveries were 35 and Al.

Cells frozen in 0.l5 M phosphate buffer. Freezing
cells in 0.15 M phOSphate buffer also caused a decrease

in the reSpiration rate. After freezing, only 230 ul

oxygen

q

Microliters of

190~~

l7Q—~

90-;

70——

30“

 

Fig. 2.

l g g I I ' l l
10 15 20 25 3o 35 #0 A5

M6

Unfrozen cells

Frozen cells

Centrifuged cells

Centrifuged cells with 1.3 ml 43
0.85% saline frozen cell

supernatant added

Per cent recovery by pour A
plate method

J l l I
l r

U1
C) “‘

Time in minutes

Oxygen Uptake of Escherichia coli Cells
Frozen in 0.85% Saline

 

 

6%.“.

oxyfi

('5
.1

O

(W

liters

,’\

. _)

Cl”

Elf

1"‘~ i-
.-' g i .
l— . I

FJ
\L)
k.)

F.)
LA)
C)

t “.1
H
C)

 

 

T

r

 

Unfrozen <_Jel s

Frozen cells

Centrifuged cells

Centrifuged cells with 1.3 ml ‘A

0.06 M phOSphate buffer frozen
cell supernatant added

 

36 Per cent recovery by pour
place method A/////%
/A ”a
A» ////
0
47 ////
.A ////’ 0
x ////
/ D /0
.A x//// 0
. a ////
/ o
X ////
U
.A//// 0
x /
U o
A ////
é////'
O
t i t t i t t t t
5 10 15 20 25 3o 5 As 45 91

Time in minutes

Oxygen Uptake of Escherichia coli Cells Frozen
in 0.06 M PhOSphate Buffer

 

of oxygen was taken up as compared to 28? ul for the
unfrozen cells (Fig. h). Th 3 compared well with that
obtained for 0.06 M phOSphate buffer. When the 0.15
M phosphate buffer frozen cell supernatant was re—
moved, a decrease of only 25 ul was noted (Fig. 4), which
could be replaced to the same degree by either 0.5 ml
or 1.3 ml of the 0.1? M phOSphate buffer frozen cell
supernatant. It therefore differed from cells frozen
in 0.06 M phOSphate buffer, which had a decrease in
oxygen uptake of about 2.5 times this much when their
frozen cell supernatant was removed.

The number of cells recovered after freezing in
0.15 M phosphate buffer for this trial, was 60 fl.

Two subsequent trials gave similar results. This was
almost twice the number recovered from 0.06 M phosphate
buffer. '

Cells frozen in distilled water. Cells frozen in
distilled water survived with the least damage as
compared to all other suSpending fluids. A decrease
of only M3 ul of oxygen occurred after freezing (Fig. 5).
More significant was the observation that removal of
the frozen cell supernatant, or addition of 1.3 ml to
centrifuged cells, caused no change in oxygen uptake.
Also 86 T of the cells could be recovered after freez-

ing.

gen

Y] "r

,. -,
J >

of

i
Q

Microliter‘

Pal
\I T .
\—

 

49

Unfrozen cells

Frozen cells

Centrifuged cells

Centrifugted cell with l. 3 sl

of 0.15 M phosphate buf‘fer
frozen cell supernatant added

//
M /
//3/
/’;/

/”x

Per cent recovery by pour
plate method

\\

 

1-
L 1 J l i L 1 1 L L
T I T l T ‘T 'l T 'T T
5 10 15 go 5 30 35 MO MS so
Time in minutes
Fig. 4. Oxygen Uptake of Escherichia coli Cells Fro en

 

in 0.15 M Phosphate Buffer

Microliters of oxygen

50

310 -v-
A: Unfrozen cells
‘A
290"' X Frozen cells
0 Centrifuged cells
270-#
D Centrifuged cells with 1.3 ml 1A
of distilled water frozen
950 “ cell supernatant added X9
/0

86 Per cent recovery by pour

230 “i plate method
210 ~+ :://

O
130 .4, / /

110 ~-

0 4b ////
9 A///
70 «p /X

0
D
. A/

 

r + + 1 4 J. .L
10 15 20 25 30 35 M0 45 50
Time in minutes

Fig. 5. Oxygen Uptake of Escherichia coli Cells Frozen
in Distilled water

 

Two subsequent trials showed no cevia;

that found in Flume 5.

 

Effect of addin :s0.35 phosprate huffgr and dis-
f‘\ v. 7 ~ A v-v r. r\ L J— H
tilled ;a r frozen cell su.crnacancs to saline

 

centr ifuged cells. Results obtained by the addition of

 

frozen cell supernat ants from 0.15 M phosphate buffer and

H.)

distilled water, individually to saline centri u ed cells

is shown in Figure 6.

The phOSphate buffer supernatant caused as much
stimulation of oxygen up ake as the saline frozen cell
supernatant when added to saline centrifuged cells (Fig.
6). The supernatant from cells frozen in distilled
water, however, did not cause as much stimulation in the

cspiration rate as did the saline and phosphate sup er-
natants (Fig. 6).

Two other trials su stant iat ed these flhOln”S with

ittle deviation from

centrifuged cells. When 0.85 T saline and distilled

 

water frozen cell supernatants were added i1 “dizidually
to 0.15 M phOSphate buffer centrifuged cells (FR 7),

an increase in the respiration rate over that of the

Microliters of oxygen

110+

1301-

lEOaL

100%

T

I

804

701

30‘*

20 4—

101-

 

D

Centrifuged cells

Centrifuged cells with l. 3 ml
of O. 85% saline frozen cell
supernatant added

Centrifuged cells with 1.3 ml X

of 0.15 M phosphate buffer 5

frozen cell supernatant added

Centrifuged cells with l. 3 ml

of distilled water frozen cell

supernatant added A

:::::x o

:////x /////
/x ' /°

“/57
/j‘°/

 

~I.
z///fl o
L 1 l I I 1 1 L
1 r q , f v 1 I

L
10 15 so 25 3o 35 'MO 45 50
Time in minutes

Effects of Adding Supernatants From the Three
Freezing Methods to Escherichia coli Centrifuged
Cells Frozen in O. 85% Saline As Measured by
Oxygen Uptake

 

U-a

Microliters of oxygen

310

290

190

170

110

90

7O

30

10

 

O Centrifuged cells

0 Centrifuged cells with 1.3 ml of
0.15 M phOSphate buffer frozen
cell supernatant added

A; Centrifuged cells with 0.85% saline
frozen cell supernatant added

X Centrifuged cells with 1.3 ml of g
distilled water frozen cell A
supernatant added

;/./°
é/f/
ۤ/j;/
V

 

XA

//2

Kg
/A°

AtJX
o
1 1 1 l 1 1 l l l 1
r 1 l I 1 1 r 1 T r
5 10 15 20 25 3o 35 no A5 50
Time in minutes
Fig. 7. Effects of Adding Supernatants From Three

Freezing Methods to Escherichia coli
Centrifuged CelksFrozen in 0.15 M Phosphate
Buffer as Measured by Oxygen Uptake

 

:4

centrifuged cells was noted. The addition of 0.15 M
phOSphate buffer frozen cell supernatant to the centri—
fuged cells gave an oxygen uptake of 23? ul, almost the
same as the 0.;5 fi saline and distilled wete super-

atants (Fi gure 7). Subsequent tria 3 gave data in

:3

agreement with th t of Figure 7.
Effect of adding 0.8557 saline and 0.15 M phos-

ffer frozen cell supernatants to dist‘ illed

 

phateh
water centrifuged cells As shown previously, freezing

 

caused the least amount of damage to cells suspended

in distilled water. Their own frozen cell supernatant
caused no increase in oxygen uptake over that of the
centrifuged cells. Addition of 0.85 fl saline and 0.15 M
phosphate buffer frozen cell supernatant to the distilled
water centrifuged cells, caused no stimulation but

Ether a decrease in oxygen uptake (Fig. 8). There
also wa.s no significant difference in oxygen uptake
between)the 0,85 fl saline, and 0.15 M phosphate buffer
frozen cell supernatants.

The decrease in 0} :ygen uptake due to the pre-

sence of 0.85 s saline and 0.15 M phOSphate super-
natants, can possibly be accounted for on the basis of
the endogenous respiration rate. The endogenous respira-

tion rate for the distilled water centrifuged cells

of

Microliters

‘ijY ’1‘?\‘.
\ 3‘}: 4.;

10‘

 

(:4:
.2.)

C) Centrifuged cells

 

X Centrifuged cells with 1.3 ml of
_ distilled water frozen cell super—
natant added
_ zl Centrifuged cells with 1.3 ml of
.85% saline frozen cell supernatant
added
0
D Centrifused cells with 1.3 m1 sf X
0.155f4 phospfimflx:'tuffer'ilfixux: a
_ cell supernatant added 0
K A
D////
t o
A
X////
D/
O
’(/A
D/
o A
X////
” a
o A
X////
F o
'A
o////
F n
//A
_ o/
X o
/a
_ XO/
0
A
X0
0
A
L.
1 1 l 1 l J L l L 1
fi 1’ T I r 1 I'IT T rt
5 10 15 2o 25 3o 3: to 45 go
Time in minutes
Fig 8. Effects of Adding Supernatants From Three

Freezing Methods to Escherichia coli
Centrifuged Cells Frozen in Fistilled Water
As Measured by Oxygen Uptake

 

'1’
,0

was 39 ul of oxy an, whereas it was increased to 56
and 51 ul when the saline and phosphate supernatants
were added respectively (Table 1).

Effect of freezing on the endogenous rates. The

 

saline frozen cell mtpernatant also seemed to cause
an increase in the end genous respiration rate of 0.15
M phOSphat e centrifuged cells greater than their own
supernatant (Table l). The distilled water and phos-
phat e buffer frozen cell supernatants did not increase
the endogenous rate of saline centrifuged cells over
that of their own supernatant (Table 1).

The endogenous re ppiration rates of saline and
0.15 M phosphate centrifuged cells can be increased by
the addition of their respect tive frozen cell super-
natants. The addition of 1.3 ml of the supernatants in
both cases, gave the greatest increase (Table )
tilled water and 0.06 M phOSphate buffer frozen cells,
did not exhibit this responSE.

Table 3 shows that the endo

were We shed once after removing their suhcrna t ,
deviation we s noted (Table 3, trial 2).

Effect of O 9

n
K 1
do
u)
H
IJ.
Ix‘
L.)
\ )
.s
"S
O
D]
C)
:5
(3
(U
H
[—3
a
r:
( i:
3
0))
3'
Cr
0
L.)

\

Table l. Ihe effect on the endogenous respiration rate of
Escherichia coli centrifuged cells frozen in

ghree diluents bx_the addition of the three fro-
zen cell supernatants as measured by oxygen

 

 

 

 

 

 

uptake.

Centrifuged Frozen Cell Supernatant

cells

frozen
in:
0.85% Saline distilled 0.15 M phosphate
water buffer

0.15 M phos- 576* an 1.1.
phate buffer
0.85%.Saline 53 55 55
Distilled
water 56 35 51

 

* Microliters of oxygen consumed in 50 minutes

. » u
. a
o‘

nu:

. -
v v
I
‘I ~
.. .o
- u
o o

—.

— o I o . u
- - . o a .
o . . - - -
. ._ o - . - -
4- .wt 5 o n -o .

-~-.-Oc-‘-

g-Q. a v...- -~-<

. c -
. . . ..
. a I

1.01..

D d ‘ -
v

- n .- O

- u c

a " '
4 C C.
. g -
. - n
a . .
. . .

c.—

.-
v
I
O.
i
A..-

51"?
#1.}

mmpsswa Om cw vmsdmcoo smmhxo mo mnmpfiaopofiz *

 

 

 

pampmcmeSm

Hana Cwuoum

mm 4: Hm mm go as m.H

spa: maamo

UmmsmwhpCmo

4m .mn ma 4m maamo smNOhm

a maamo

mm Hm Hm *mm cmusmwnucwo

umwmsn ummmsn Amps:

3.238% s 8.0 wpmnamofi s 3.0 8.3% lento 3:330

_ dowpmasaom

Hawo

pcom< wswvammmsm

 

.mxmpa: :mmmxo an vmusmMms mw mpcmMm wafiucmdmzn
i

know ca cmaonm . madmo “Hoe menofiumSomm mo mush soapmpwamwu mzocmwovcm .N magma

 

 

ill - '1- ‘ girl- ‘3‘- 'I" o',aou-.l'l.u"o,-.‘ ‘, .‘ ‘u’ll‘i‘ "" .3 z- i 1 II-

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Q ‘Iil’I’.’- 4"" Qua- 1. "II‘

coco deans: maamo newsmwupzmo**
Amaamo newsmwppswov um>08mh pampwcthSm Hamo nmuonh *

 

 

 

 

. , Amps:
m4 a¢ mm ma ca mH Umaaflpmfie
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mm mm cm 0H NH ma : mH.o
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wcflnmohm umpm< wzwummhk mnOMmm

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mqwucwmwsm

mopsnas 0m ca mpmpfiaouows ca mxmpms :mmhxo

 

 

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IQ: mehxo hm,vwh5mmms mm «mafimomhm pmpmm new whommn madame pamhowmwu

ca uwvsmdmsn .maamo wHoo mfigowpmnomm pom mmpmh soapwhwdmmh msocmwonnm .m magma

 

 

 

-‘I‘ A! -1.
‘ Ill t o 1 DUI. \ \ Q - - ' iv. 0 a .0 - I I U 0 n a I A? I 1" U
- -‘o- I ’ O l ' U | I I- ‘ l 0' I II 0 - III: 9 I. I c I V t a u 0 ' I -
II.
E - ‘l. I. ..o It- ..-“- 'I.'.A. -l’!‘l‘ I l ' C ‘l'- - O ‘ I I I a. ‘ I I- .I
r. h ' ‘ i ll 0" u v. "I‘ ‘ ‘ Or- . - I. - U f’l..\“- I I "' 0|. 0 ‘0
.ll - -I‘ -1. - ‘ I ‘ ‘ It I - l 4. | I I .0 l..- ‘ Q .0 I‘ll. I! ’ O I. ' I D I
I I
‘ C ‘ CII . ‘ - . -1 O - ll- .- Q Ir J 4 ‘ln II o O O I 0 II‘ v J c I I 1- or! I .I I I (I
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O- o O I ....I u | I 0 lies-.. -..‘-‘--I-"----tv’1.‘ "'I’“OI|'II

. 'l . ,- ,.\ l - J. . ,.
centrifueae unfrozen Cells. Tee results obtained

 

1 ’\ I . a, . r. A -\ I“ 1 2“ ‘
when saline frozen coll sup: r1a tent was auflufl to un—

unfrozen cells without supernatant had an ozyfien up-
take of 285 ul. When 1.3 ml of the frozen cell super—
natant was addet., the oxygen uptake as only 2?5 ul.
The end03enous rate for the cells with the sup: rn tant
was 29 ul as opposed to 19 ul for cells without super—
natant. Similar result US were obtained in two other
trials.

Characterization of the 0.95 d saline frozen cell

super: atant from frozen E. coli cells. Upon dia ysis

 

for 2H hours, the saline frozen cell supernatant failed
to increase the oxV3en uptake of saline centrifuged
cells (Fi3.110).l The oxy3en uptake for the centrifuged
cells with 1.3 ml of the saline frozen cell supernatant
added, was 158 ul, as compared to 123 ul for the centri—
fuged cells and 12M ul for centrifuged cells with 1.3

ml of dialyzed supernatant added.

Heating the saline frozen cell supernatant for 30
minutes at 100 C did not inactivate the stimulatory
preperty or properties of the supernatant (Fi3 11).

The 0: {ygen uptake of the saline centrifu3ed cells for 50
minutes was 60 ul as comp ed to 150 and 1a 5 ul for the

heated and unheated sueernatants res actively for the
t p

,xygen

O

'roliters of

.‘
.L‘v

,4.

*1,” .
1‘1

110

\N
C.)

 

 

Time in minutes

61
t

O Unfrozen cells
b 0

Di Unfrozen cells with 1.3 ml of

0.85% saline frozen cell
_ supernatant 0
a
O
o
r
o
’ a
r.
o
D
L.
o
D
F o
o
r.
O
a
o
a

b-
P %

l L 1 1 L L l 1 1 J

T T ' ,' ,F ‘ T .Z ,‘ _i

5 lo 15 as 35 3o 35 no A; as

Effect of Addin3 Saline Frozen Cell Super—
natant to Unfrozen Centrifuged Cells of

Escherichia coli Suspended in 0.
As leasured by Oxygen Uptake

 

Sf L: (if

' j I";)

Saline

 

 

1601L 62 n
150‘_ O Centrifuged cells
[3 Centrifuged cells with 1.3 ml
luOsr of 0.85% saline frozen cell 0'
supernatant added
130 i, A Centrifuge‘d cells with 1.3 ml
of dialyzed 0.85%‘saline .
' frozen cell sdpernatant.added a 2
120 1'-
~ 0
".1 llO ‘L
& a ////A
z
x
O 100 l» / o
“a a /A
E3 90 “r /
m o
P .A
"—4
O
a
t o
S A
u ,
60s~ ////2
50-~ o
D2///A
40-~ 0
A
u
30 3 /
°A
20 + /
u
o
lO.L
7} § § 3 i: :% 4 i # %
5 10 15 20 25 3O 35 40 45 50
Time in minutes
Fig. 10. Effect of Adding Dialyzed Saline Frozen

Cell Supernatant to Escherichia Coli
Centrifuged Cells Frozen in 0.85% Saline
As Measured By Oxygen Uptake

oxvge‘

{AD

(“1

iters

icrol

l

A

A

 

“\
LU

Centrifuged cells

Centrifuged cells Kl
of saline frozen eel

ad (33 min a5

\

 

l l l l L 1 l l l

I I 1 ‘ 1 t 1 r 1
'1 ' "L " "~ ' ’ ‘ r? 1’ f ." -" F; .3.
l0 1_ . L_’ j 3:]; ‘7 J ‘7 - ‘/, J

Time in minutes

 

Effect of Adding Heated Saline Frozen Cell
Suyernatant to Escherichia coli Centrifuged
Cells Frozen in—CT§?fl Saline A: Measured by
Oxygen Uptake

ON

same period of time.

The absorption spectrum of the saline frozen cell
supernatant showed a strong maximum absorption at 260 mu.

Norit A.,an activated charcoal, was used to deter-
mine whether or not the activity of the saline frozen
cell supe natant could be remO'ed by adsorption. The
centrifuged cells with 1.3 ml of supernatant, exhibited
an oxrgen uptake of 1M8 u1 in 50 minutes, whereas
centrifuged cells alone, had an uptake of only 93 ul (Fig.
12). After treatment of the supernatant with Norit A,
1.3 m1 of the supernatant was added to the centrifuged
cells. A decrease in uptake from 1&8 ul to 105 ul
was noted.

It has been suggested (Halvorson et a1., 1951) that
water soluble cofactors might be leaching out of the
frozen cells, causing injury and death. To ascertain

whether or not this was happening in our freezing
system, certain cofactors were added to the saline
centrifuged cells to determine if they could replace
the activity of the saline frozen cell supernatant.
Figure 13 and Table A show the results obtained

with ATP, ADP, and M301 Table M, which contains the

2.
same data as Figure 13, is presented for the purpose of

clarity. ADP did not increase the oxygen uptake of the

LU
\ 1

I)
(A)

 

,__
7'1 V’ “

<3 Centrifuged cells n

D Centrif1.ed c
oi O.M 2 tall
slpernatan nt a
[3 Centrifuqed cells with l.3 ml
of charcoal trea Eid Q.s;fl
saline frozen cell BRIE?—
natant added 0

 

l 1 1 1 1 1 J L 1 1
1 1 1 T I I 1 ‘ T ‘
L . ‘ ’ ” ‘- ' ’F "7

1 1 1 is: _* 51.- 31, "w 53‘

1*151. ’lP. lVfikfict of“ AéhlirLj ffi1aim3oi1iTieH1te d €1.9'}:
Saline Frozen Cell Supernatant to Escler-
ichia coli Centrifuged Cells Frozen in

f'\

u-L
. 7‘ , ‘7‘ w -
'4 ~ I' (7‘ I‘ ’3 ‘ ‘ 'fl‘ "'7 x ‘ '1 , r1 1 ~ I 1 ‘V‘ r" T r‘ ‘ r
U . x): 7;» :1 1 11163 118 I~l€.:‘1:-.u1’eti by ufl‘y :‘ en Lu 1:.th a

.L

 

of

161°C»

,‘_
k

Microli

7’XVFT'E‘I’1

H

«a

C)
l
I

O\
O\

1601- 9

80d

70‘

60-1

m
c

 

O o Centrifuged cells

D-—-D Centrifuged cells with 1.3ml of
0.85% saline frozen cell super— 0

p natant added /2

)<----—'>< Centrifuged cells with ADI -
added /

r O
a An
Lk—-——Z>Centrifu£ed cells with ATP /
added

1 / A
<D--—C>Centrifuged cells with /%
m_ D

_ MgClg added

/ A X
zg————A Centrifuged cells with /' /////
» ADP, ATP, and MgCl2 2%

mixture added

L U
L\
%
\\
\.
\\::f
><C)

)
\\
b
O
>£

 

 

1 l l I 1 1 x 1 I J
r F n I I 1 n 1 l I 1

— . . , - u I. - -—,»~.
5 1J lb 5U 25 jd 3b 40 Mb by

Time in minutes

 

b1, 13. Ff: st of Adding ADP, ATP, and HgCld to
PSCnerichia coli Uentrifuged Cells Frozen
in 0:8 i Saline As Keasured by Oxygen Uptake

t7

Table A. Effect of Adding_ADP. ATP, and ngl to_
Escherichia coli centrifuged cells, frozen in

QL8§% Saline, as measured by oxygen uptake.

 

 

Cofactor 1Microliters of oxygen
consumed in 50 minutes

A__

ADP 107

ATP 120

Ix’fgCl2 1113

ADP, ATP, MgCl2 139
Centrifuged cells 110

Centrifuged cells

with 1.3 ml of 162
saline frozen cell

supernatant added

 

O

,_O

“‘

 

’ .-
¢ '-
_ u
.
.
.u. ‘
.,

‘ .

Q ~-

. V‘vQ“

.ow'

-a-o

".

._.“

 

.__-
C ..‘ .
- I.‘ .

6 - -

A . ‘

a
-. . ' -‘
o " .

-.- '

- ' V‘
‘ ~

‘ ;

l

- ‘0.

a

,
‘ ,
.

. .

I .~.

 

-Q-

-.Q

9“
o
.D‘.

.

' I

.

I ~ .
-c . .

VV

centrifuged cells and ATP increased it only 10 ul.

the xygen uptake of the centrifu ed cells about HO ul.
The M3012 and the mixture therefore could replace
about half of the activity of the saline frozen cell

supernatant. As would be expected, ADP, ATP, and MgCl2

’2

had no effect on unfrozen cells.

Two micromoles of NAD were also added to the centri-—
fuged cells. The saline frozen cell supernatant, when
added to centrifuged cells, caused a 50 ul increase in
oxygen uptake, whereas the NAD increased the oxygen up-
take MM ul, when added to centrifuged cells (Fig. 1A).

The assay for NAD in the saline frozen cell super-
natant with glucose dehydrogenase is shown in figure
15. When the NAD was replaced by the saline frozen cell
supernatant in the presence of glucose and the enzyme,

no change in Optical densitv was noted (Fig. 15).

(a

)"
Cf)

Using citrate as su trate with unfrozen organ-
isms, no oxygen uptake was noted as would be expecte .
N:ither was citrate metabolized by frozen organisms.

The oxngen uptak; with citrate as substrate for the

.‘J

centrifuged cells, was the same as the endogenous (Table

Ho

5). The addition of acetate along w th the citrate,

showed no greater uptake than with acetate alone

CH

O

Microliters

130

\I/\
l_

 

O\
\0

O Centrifuged cells

CJCentrifueed cells with 1.3
ml of 0.83% salin frozen 0
cell supernatant added

43 Centrifueed cells vith
MAD added 0

 

O
a
/ °
A
D
//// o
A /
”/0
go
0
1 1 1 l 1 4 J I L 1
**t F T TT ,1 ‘ 1 i E l- .i
5 lO 15 -:o :5 3m 3; to up so

F is: -

I
14.

Time in minutes

 

Eflk~ct :Jf ExhiinyfllAT'inj Es é’ri"flxia “$311
Centrifuged Cells Frozen In o.d;fl Saline
As Measured by Oxygen Uptake

:tical density

.1

01

7O

 

 

 

 

‘A NAD
0 NAD and saline frozen cell supernatant
.60a~ g
E Saline frozen cell supernatant ‘///
a
////o
_ L A
.504 /°
A
////o
A
0141,34“ /6
A
////O
A
a
.30+
A
///o
A’
/0
.20«- A
////0
A
/0
A
.lO-/O
D a n 0 a a 52 D. L3 9 C2 £3
,. i 5_ i, +_ ti ._ 1 1_ . L Y.
15 3O 4; CG 75 :3 103 liQ 13; 15; C‘ 153
Time in Seconds
Fig. 15. Assay For NAD In the Saline Frozen Cell

Supernatant With Glucose EehdeOSGHase

71

Table 5. Permeability and utilization of citrate by
Escherichia coli centrifuged cells frozen in
3 % aline, as measured by oxygen uptake.

 

Substrate Microliters of oxygen
Consumed (70 min.)

 

Endogenous _ 51
Citrate 53
Acetate 83
Citrate and 86
Acetate

Glucose lOO

 

 

0
6"
o -

'40-..

‘r -
. .
. .
. -
u
....—o
. a

.'

-d
--
..

C
.-

..-

‘>‘

5
. c
. .
, -
----

0v ‘

,o-- -_
.- I
. --

w
a o
l
t
. n
n I

m
. .
ag- .

- .... o.
o n n
. 'v*
o .
.. o
.
-‘I
- u...
'.--h .r

o».
l C
- .
C .

0 -—~
- c
O -

q - ....---

. n

0 »0

Q o
t O

--...-.'--

 

I I - .0 -' .' . .
. . .. ‘ - .. §
.. .. - .- c - o 0 -°

o. ‘u- -.~

.-\ l'-"

-..-.-.o- -7

‘§. ‘ - .

u.» .,.

u... div-64--

 

' l
Citrate in the presence of acetate was not meta-

bolized by unfrozen cells.

 

 

DISCUSSION

Comparison of the four freeze suSpending agents
showed that distilled water gave the best per cent 0.
viable cells recovered followed then b
phate buffer, 0.05 M phosphate buffer and finally 0.35 f
saline. These results are in general agreement with
those found in the literature.

The per cent recoveries obtained with 0.05 H and
0.15 M phOSphate buffer suSpending media, were not
those expected. It was expected that 0.15 M phOSphate
buffer would be as damaging, if not more so, than 0.06
M phOSphate buffer. But cells frozen in 0.06 M phos-
phate buffer suffered almost twice as much damage as
those frozen in 0.15 M phOSphate buffer. It does not
seem that ionic strength of the buffers was an im-
portant factor. It is possible that the higher
molarity of the 0.15 M'phosphate buffer in some manner,

protects the cells during the freezing and th.wing pro-

In general, it seems that the effect of freezing
in different diluents is dependent on the electrolytes
sed and not upon the molarity or ionic strength of the
solutions.

73

74

Decreases in oxygen uptake were found after
freezing in each of the four diluents, with 0.35 T
saline showing the greatest decrease. Th; decreases
in uptake after freezing in 0.06 and 0.15 M phosphate
buffers and distilled water were of about the same
magnitude. There seemed to be no correlation between
per cent recovery and decrease in oxygen uptake after
freezing for the four diluents. This is illustrated
by a comparison of 0.06 M phosphate buffer and distilled
water results. has, it is also shown that the oxygen.
uptake is influenced more by the electrolyte used than
by the ionic strength.

Removal of the frozen cell supernatant from cells

frozen in 0.85 “ saline and 0.05 M phOSphate buffe ,

W
caused the greatest decrease in oxygen uptake. 0n the
other hand, only about one-half as much of a decrease
was noted when the frozen cell supernatant was removed
from 0.15 M phOSphatc buffer frozen cells. No decrease
was found for distilled water frozen cells.

If the frozen cell supernatants were added to their
reSpective centrifuged cells, the oxygen uptake could

.

be increased to the level that it was before removal.

The only exception to this was with cells frozen

in 0.85 % saline. Addition of 1.3 ml of the saline

75
frozen cell supernatant to their centrifuged cells,
in most instances, increased the oxygen uptake over
that of the frozen cells. With 0.06 and 0.15 M phos-
phate buffers centrifuged cells, addition of 1.3 ml of
their reSpective frozen cell supernatants raised the
oxygen uptake to the level of the frozen cells, but no
higher.

The difference between the action of the saline
frozen cell supernatant and that of the phOSphate
buffers, might be due to the increased need for the
active material (s) in the saline supernatant by the
saline centrifuged cells. This is very possible since
saline frozen cells sustained the greatest injury and
damage.

The question now arises as to the explanation of
this stimulation of the centrifuged cells by the fro-
zen cell supernatants. Three possibilities seem likely.

hey are: leakage of cellular material, disruption of
the cells, orpa combination of bot

If cell disruption were the major cause of the
st nulation, one would expect to obtain a cell free
preparation. In the case of saline, an 80 to 90 %
cell free preparation could be obtained. Assuming

no loss of cofactors, the frozen cell supernatants

76
then would be expected to metabolize glucose. This
did not occur. However, this does not eliminate the
possibility of some cell lysis—-perhap s at a very low
level. But Grit savage (19 63 ) w s unable to detect any
amino acids or deoxyribonuclcic acid in various frozen
cell supernatants. However, if the cells we re dis-
rupted using ultra-sonic vibration, deoxyribonucleic
acid could be detected.

The other possibility is cell leaka3e. Hartsell
(1951), Lund (1961), and Smith (1954) advocate this
theory. During the freezing and thawing process,
cellular materials leach from the cells. This cause
a metabolic injury to the cells which could be over-
come by re—entry of material from the frozen cell super-
natants. Our results seem to support this occurence.
Further, the results demonstrate a correlation between
freeze dama3e in terms of per cent viability and leak-
age-—the greater t e freeze damage, the greater the
leakage.

Addition of distillel water froz GEL cell super—
natant to saliLe centrifuged cells, demonstrated that
leakage was occurring when cells were frozen in dis-
tilled water. However, the activity of tLe stilled

water supernatant was only two-thirds as creat as that

Ln. '1... . .. M. ‘3 L.° \ . Lunar. 1.1, , a
of bu. saline supernatant, incica l“ eLat tee leaka3e

v. - 4- m 1-

v.4aLl.

771 TV! ’ L y..- -n 4L ”.4. . a 1-1 ,_
lee 0.15 m phes phase supernatant :aVe the same
- 4- L! , n. . - A . 1.. .L .
amount of activity as did ufld saline supernatant whel

added to saline centrifu3>d cells. It is therefore
probable that the leakage from the phosphate frozen
cells is sufficient to supply the leeds of the saline

centrifu3e d cells.

The saline and distilled water frozen cell supernatants,

when added to 0.15 M phosphate buffer centrifu3ed cells,

have the same results as the phosphate supernatant.

This again demonstrates that the 7istilled water frozen
cell supernatant contains an active substance or sub—

stances which can cause stimulation. The sal ne frozen

cell super: Watan was also able to stimulat the

'j)
‘2
(D
0..
O
( D
3.4
H
C)
d-
O
C).
:3.
m
,4
(L
(D
j
*‘S
0
( 3
03
U)
S:
(1)
C1

phOSphate centri13e axe —3

Results with distilled water centrifuged cells and
the three frozen cell supernatants showed that 0.15 M
phosohate and saline frozen cell supernat ants cannot
stimulate the distilled water centrifuged cells. In
fact they seemed to decrease the activity. However, it
was found that the endogenous respir ration rates of the

distilled water centrifuged cells were significantly

78
increased when either the saline or phoSphate frozen
cell supernatants were added. It is possible that
these supernatants contain a utilizable substrate and
the increase in the endogenous rate is due to this.

i
{18

When the endogenous values are substracted from t
exogenous values for the saline and phosphate super-
natants, an error is introduced.

These results demonstrated that the leakage
materials were similar in the sense that they could
stimulate cells frozen in other diluents with the
exception of distilled water centrifuged cells.

(

The reason that the distilled water centrifuged
cells were not stimulated by any of the supernatants
probably can be explained on the basis of cell viability
after freezing. Since between 80 to 90 i of the cells
can be recovered, the percentage of injured cells would
be small. These cells are probably utilizing the material
in the supernataats but to such a small extent that

it cannot be detected with the methods used.

d’

J

Hartsell (1951) has postalated hat his leakage
material is a result of a cellular build—up of an inter-
mediate compound in a sequence of reactions. In other

words, product A goes to product B and this product

combined into C, but the use of C in D cannot occur

79

until the cells are defrosted. Thus, product C
accumulates as the cells are held in storage. Our data
suggests that this might be occurring.

The endOgenous respiration rates for cells frozen
in all the suspending agents were always greater after
freezing had occurred. The esults further showed
that the endogenous respiration rates of saline and
0.15 M phOSphate buffer centrifuged cells were increased
when their respective frozen cell supernatants were
added. This, however, did not seem to be true for cells
frozen in 0.06 M phosphate buffer and distilled water.
Also, distilled water and 0.15 M phosphate buffer
supernatants could increase the endogenous rate of saline
centrifuged cells to the same level as the saline super—
natant. This was true for the addition of saline and
distilled water supernatants to phOSphate centrifuged
cells, except the saline supernatant increased the
uptake more than ph Sphate or distilled water did.

One cehld postulate from these data, that there

ate or intermediates in

H.

is a build up of some intermed

c 1'
U)

a sequence of biochemical even- . The cells once thawed,
could begin to metabolize this intermediate or inter-
mediates, and thus account for the increased endo—

genous respiration rates after freezing and thawing.

80
Wash ing the es firifuged cells once did not lower the
rate, therefore it would seem that the material is

intracellu ar. By addition of the frozen cell super—

natants, more substrate is added and thus an even

4L!

higher rate of oxy3en uptake or the superna ant is

L.
‘u'

('1)
ct

her

15
CL.

'1

actually stimulati ing the endogenous

.« v1
. J.' KAJ.

study would be necessary to establish such sequence of

Ur frozen cells suspended in saline, were not stimu—
lated by the saline frozen cell supernatant. The endog-
enous rate of these unfrozen cells was increased by
addition of the saline frozen cell supernatant, indicating
again, the possibility of a utilizable substrate
being present in low concentrations. Two possible
reasons why unfrozen cells were not stimulated by the
supernatant could be either they have no need for the
substanbe er substances in the supernatant, or that
these substances might be impermeable.

The stimulatory factor or factors, can be emoved
from the saline frozen cell supernatant by dialysis.

The stimulatory effect, therefore seems to reSide in
low molecular weight compounds.

Activit; of the saline frozen cell supernatant

it is beet

C.
Cr
G)
U]
{‘3
Cr
H
O
O
O
O
C}
D
Q.
(I)
*‘S
C?-
(If;
(D
O
O
:5
Cl.
H
d’
H.
O
:3
U)
h

81

able.

ct

s
Treatment of the cells wit1 horit A, removed about

two—thirds of the stimulatorr activit‘ of Ute saline

frozen cell supernatant. Perhaps a lon3er treatment

of the supernatant or incuba

er than 37 C ould have removed more of the acuivity.
Since the saline frozen cell supernatant had a

nwa imum absorption at 250 um, it was thou3ht that ATP

or ADP could pos ly be the active components.

W
CL
(1:

ATP and ADP in the amounts ted, were not able
to simulate the activit; of the frozen cell super-
natant. M3012, on the other hand, was able to increase
the ox;3en uptake about one-half that of he saline

frozen cell supernatant. Adding a mixture of ATP, ADP,

and M3Cl to th 1e salin ecentriftged cells 3eve almost the

2
ame results as M3C12 alone. This was prol Lably due to

m

the EECIQ,C
No effort was made to essa; :or the presence of
M3++ in the frozen cell supernatant, but it is poss ole
hat during freezing and thawing of the cells, M3++
could be lost from the cell, perhaps due to deh3dra-
tion of the cell or an unfavorable cone-e ntra tion 3rad-
ient, surrounding the cell.

Povarnick (1954)‘and Povarnick and Allen (1957

82

(D

th typhus rickettsia , that nico-

Ho

have demonstrated w

Ci‘
0

tinamide adenine dinucleotide can res re respiratory

activity after freezing and thawing of this organism.

11-

The pr sence of NAD was also detected in one froze
cell supernatant.

Addition of NAD to saline centrifuged cells, did
replace the activity of the saline frozen cell super-
natant. To explain the action of NAD stimulation, it must

be assumed that NAD can pass through the cell embrane

in order to cause the stimulation. E, coli, when not

 

frozen, is impermeable to NAD. It is therefore possible

that during freezing and thawing with our system, there

arises an altered permeability of the cell membrane.
Glucose dehydrogenase was used to assay for NAD

in the saline frozen cell supernatant. No NAD could

detected. It was also determined that the frozen

(T
(D

cell supernatant did not contain inhibitors which might
have interferred with the action of the glucose dehy-
drogenase.

Efforts were also made to determine if citrate, which
was impermeable to our strain of E. coli, could be meta-
bolized after freezing. It was found that it could not
he metabolized,whéther alone or in the presence of ace—

tate.

I.
.K. .-
- _, .

0"

r-‘s't

‘

“'0".

SUI: AR V
It was found that the number of viable cells
surviving freezing and thawing for 22 hours at ~15 C
was depen nde nt on the suSpending I‘nublh“. The best
survival was obtained with distil ed water, followed
by 0.15 M phosphate tuffer, 0.05 M phosphate buffer,
and 0.9: Q sa ine.

/ I

Decreases in oxggen upt We ce after freezing, were

crease, while cells frozen in the phosphate buffers

ani distilled water s:~r

Removal of the frozen cell 1supernatants demon—
stated that the 0"“een uptake could he further de-
creased, with the exception of distilled water sus—
pended cells. By re—introducing the supernat nt to
th e centrifuged cells, the oxygen uptake could be
restored. In the case of cells frozen in 0.95 % saline,
the level of oxygen u;a taLe could be 'ncreased over
that of the frozen cells. Evidence indicated hat
cell leakage had occurred during freezing and thawing
with resulting cell injury. There also seem qed to be

a correlation between number of viable oreanisms sur-

83

84

viving freezing, and cellular leakare.

-2

Testin: of 0.85 f saline, 0.15 M phosphate buffer,
and distilled water frozen coll supernatants against
each of the Opposite centrifuged cells revealed (1) that
cellular leakage did occur when cells were frozen in
distilled water but in low concentration, (2) distilled
water centrifuged cell could not be stimulated, (3)
there seemed to be a similarity between the frozen cell
supernatants in regard to the active components.

The endOgenous respiration rates were increased
after freezing cells in the four suspending agents.
Washing the cells once by centrifugation did not remove
this increase.

The addition of the various frozen cell supernatants
to centrifuged cells, caused a further increase in en-
dogenous reSpiration.

Exceptions to this were cells frozen in 0.05 M
phOSphate and distilled water. However, the endogenous
respiration of cells frozen in distilled water could be
increased by the addition of 0.15 M phOSphate buffer and
0.85 % saline frozen cell supernatants.

The active componenuw of the saline frozen cell
supernatant was found to be dialyzable, heat stable for

30 minutes at 100 C, and removable by adsorption to

85
Norit A,

The saline frozen cell supernatant had a maximum
absorption spectrum at 250 um.

Nicotinamide adenine dinucleotide replaced the
activity of 0.85 % saline frozen cell supernatant when
added to the saline centrifuged cells. An assay for
NAD, using glucose dehydrogenase, did not affirm its
presence. The results with NAD indicated that a
possible cellukar permeability change might have
taken place after freezing and thawing.

1

Adenosine diphosphate and ad nosine tripnos-

O

phate did not replace the activity of the saline
frozen cell supernatant when added to saline centri-
fuged cells. M5012 and a mixture of M3012, ADP, and
ATP replaced about one—half of the activity. I

Citrate was not metabolized by cells frozen in
0.85 % saline.

Saline frozen cell supernatant was not able to
increase the oxygen uptake of 0.85 % saline suSpended

unfrozen cells.

I, LZRATURE CITiD

Ambrosini, R.A., and H.W. Drctz. 1963. Survival of
Escherichia coli frozen in cell c
Tacteriol. ‘Proc., p. 6.

 

Arpai, J. 19C2. Ionleth a1 freezi o
bolism and moti ility of PS3 demonas fluore_c~1e

Y. 1

J .1.

 

 

 

an esotericoia coli. J. {,1. factcllo .
10:29{-301.

Arpai, J. 10 C3. Selective effect of fr3ezing as re-
flect ed in growth curves. Folia Microbial._§:lo-
26.

Baum, H.M. 191'3. The resistance to low temperatures
of a oiltur3 of Saccharom gee s c~rev331ae grown
from a single cell. ;iod3 namica. Eg71-7fl.

Eelehradek, J. 1935. emperature and living matter.

Protoplasm Moziograph, Borntraegcr, Berlin.

Eovarnick, M.R. 1954. Reves ible inactivation of ty phu
rickettsiae. I. Inactivation by fro-ezing. J.
Gen. Physiol. .332169-179.

Povarniclc, M.“ n., and B.C.Alle11957.Hev3sib13
inactivation of typhus ricketts ae at O C. J.

Bacteriol. 23556-62.

Pretz, H.W. 1959. Frozen st orare a: d certain life
processes of Escherichia coli. P‘3 .D. thesis,
Purdue University, afayette, Ind.

Eretz, H. W. 19Cl. Death of Tectorict‘a coli on cello-
phane and on membrane filters. ‘Canadl J. Micro—
bial . 11:793—798.

Bretz, H. W., and K.B. Easa. 1960. Protective sub-
stances for frozen Escherichia coli. Bacteriol.
Proc., p. 38.

Pretz, H.W., and 8.2. Hartsell. 1959. Quantitative
evaluation of defroste Escherichia coli. Food
Res. ‘aflz369—37M.

86

m
1L)

87

Putterfield, C. T. 1932. The selection of a dilu—
tion water for bacterio ological examinations. J.
Bacteriol. g3;2$ -368.

Campbell, J. D. 19 93. Resistance of yeast to low
temperatur . Piodynamica. 3565—70.

Chalmers, P. 1959 . How water freezes. Sci. fir erican
200: 111' -l22.

Clement, M.T. 1961. Effects of freezing, freeze-dry-
ine, and storag-e in the freeze-dried and frozen
st t3 on viability of Escherichia coli cells.
Canad. J. Hicrobiol. 739 99- 106.

 

Curr ns, H.R., and F.R. Evans. 1937. The importance of
enrichment in the cultiVation of bacterial spores
previously exposed to lethal agencies. J. Bacter-

iOl. 1317931890

DeLemater,'E.D., K.L. Habcock, and G.R. Ilazz anti. 1959.
On the leakage of cellular material fro om Pacillus
megaterum. J. Pacteriol. ZZ;513—5ll%

 

Deotto, R. 19in. L'azione de fre ddo sulla respira-
zione de 11a cellula bact terica. Arch. Exptl.
Zellforsch. 255101-105.

Dubos, R.J. 1937. Mechanism of the lysis of pneumo-

cocci by freezing and thaJinf, bile and other
agents. J. 3xpt1.fie . 66:101- 112.

Foulkes, E.C. 195M. Citrate met abolism and cell
permeability. J. Bacteriol. 69:505.

Goetz, A., and S. S. Goetz. 1938. Vitrification and
crystallization of photOph3ta at low tcrvcratu
Proc. Am. Philos. Soc. 79:361-393.

Goresline, H.3. 1996. Microbiological examination of

foods: entative methods for microbioloeical
examination of frozen foods. Am. J. Pub. Health.
$332— 355.

~ .A
.1.

‘1

H-

tsavage, E.F. 1963. Persona 1 co.mun1cation.

88

Ctnders on, R.F., and I_.D. R se. 19“?. Survival of
bact -3ria in a prr3 cooceQ fresh froz:n food. Food

9
Res. 13:254—233.
' h A r-v-gum "\ .
Ra aines, R.R. 193;. The effect of fr33ztn ,on bacteria.
,r- /‘
Proc. Roy. Soc. (Lo ndo;). 12URz45l—“33.

 

Ralvors on, H.O., J. Wolf, and V.R. Srinivasan. 1951.
Initiation of growth at low temperaturis Proc.

Low T3mp3ratur3 icrobiol 35y Sympos. Campbell
S 0131? CO.’ CC‘ALC :11, II.'T.

Harr:1.s on, A .P. 1955. Causes of death of bacteria in
frozen suspensions. Antonie Van Leeuwenhoek J.
Microbiol. Serol. 23407-513.

Harrison, A.P. 1961. Aginf and decline of bacteria
in phosphate buffer. Pacteriol. Proc., p. 53.

Farrison, A .P., and R.E. Cerroni. 1956. Fallacy of

”cras hin5o death" in frozen bacterial suspension:.
Proc. Soc. Erptl. Biol. Med. 91:577—579.
Hartman, P.A., and D.V. Huntsber5er. 1951. 111131119310e

of subtle differences in plating procedure on bacterial
counts of prepared frozen foods.- J. Appl. Nicro-
biol. 9:32-3b

Uartsell, S.2. 1951 a. The 5rowth initiation of bacteria
in defrosted 355s. Food Res. 16:96-106.

Iarts3ll, 8.2. 1951 b. The longevity and behavior of
pathogenic bacteria in frozen foods: the influence
pf platine media. Am. J. Pub. He alt: MlleT2-107T.

Symposium on initiation of bacter-

Hartse11,S.:. 19 o
cteriol. Rev. 235250—253.

5
ial growth. E

obio1O5y of frozen foods.

Ra rts ell, 8.3. 1951. The r
1p crobiolO5y Sampo s. Ca mp—
J . \

.mi
Proc. Low Tem erature Mic
bell Soup Co., Camdem, N

r of

‘.-.r

”35arty, C. P., a? d O. B. Weeks. 1990. Sensi’

 

Escherichia coli to cold-shock during 0: 5
growth phase. “J. Bacteriol. 395575-484.

89

Heinmets, F., W. W. Taylor, and J.J. Lehman. 195M a.
The Lse of 33taholit'- in the restoration of viabi-

litv of he at and clemicallv inactivated Escherichia
coli. J. Pacteriol. 57:5- 12. ~

Heinmets F., W.W. Taylor, J. J. L3hman, and R.F. Katha
195i b. The study of factors which influence met
belie reactivation of the ultraviolet inactivated
Escherichia coli. J. Bacteriol. £735ll-522

 

. R31

Higuchi, {., and T. Uemura. 19 3
II ur . l

o
tides from ye st cells. t

ase of nucleo-
2'.

r
3 [.1381—1383.

 

Hilliard, C.K., C. Torossian, and R.P. Stone. 1915.
Notes on the factors involved in the :ermicidal
effects of freezing and low temperature. Science.
33:770—771.

Hilliard, C.M., and M. A. Davis. 1918. The germicidal
action of freezing temp ratures upon bacteria.
J. Eacteriol. 3: L23— L31.

Holden, J.T. 1958. Degradation of intracellular nucleic
acids and lea] {ace of fragments bv Lac tohaeillus
arabinesus. Biochir. EiOphy. Acta. 29506 7-068.

 

Hollander, D.H., and 3.3. Nell. 195H. Improved preser—
vation of Treponema pallidum and other bacteria by
freezing with glycerol. J. Appl. Microbiol.
2:15H—170.

 

Howard, D.H. 1956. The prescflrva ion of bacteria by
freezing in glycerol broth. J. Bacteriol. Z_;625.

Jones, J.H., and F.W. Fabian. 1952. The viability of
microorganisms isOlated from fruits and vegetables 1w en
frozen in different suspending menstrua. Tech. Bull.
Iiich. Agric. Exp. Sta. 229:3 _u2.

Keith, 8.0. 1913. Factors influencing the survival of
bacteria at ter peratures in the vicinitv of the
freezing point of water. Science. 3 i:877— ~879.

Kohn, A; 1960. Lys is of frozen and thaved cells of
Eschericl3 ia coli by lyozyme and their conversion
into Spe roplasts. J. Bacteriol. 79 :697-706.

 

9O

Hohn, A., and If. Szybalsk 1
from thaLed HwCfiericoia 0
Proc., p. 120-121.

590 IJ‘TS‘KJJ“ 18b 8pc Poplas ts
11 cells. Bacteriol.

Lovelock, J.3. 1957. Denatura tion of lipid-protin
omplexe cs as a cause of damaéa bv freezing. Proc.
Roy. Soc. (London). 1L7 2 M27 u33.
Lund, A.J. 1961. Perso a1 communication (Cited from
Halvoson, H.O.,.g§_ l. 19 61. Proc. Low Temperature

ILcrobiolo~v Svmoos Campbell Soup Co., amden,

on
9

1961. Recent dxvelOpnean in or vobiolO3y
and their significance in the study of freezing and
freeze—drying of bacteria. Proc. Low Temperature
Microbiolo3y Synp s. Campbell Soup Co., Camden,
N.J.

1'."
C

(51
(D

H
'"U
H
0

he nio. 1990. Life and death

Luyet, P.I., and P.M. Ge
es. Pi odyn.mica, Normandy, Mo.

at low temperatur

Hacfadyen, A. and S. Rowland. 1900. Furthc r note on
the influence of te"n~rature of 1 dui1d air on
bacteria. Proc. Ho; Soc. (London). 66:339-3H2.

Hajor, C.P., J.D. McDou3al, and A.P. Her son, Jr. 195).

The effect of the initial cell concentration upon
survival of bacteria at ~22 C. J. :acteriol.
60.011.11-2110

— ./ 0‘— ' ' .J 0

Hazur, P. 1960 a. Physical and temporal factors involved
in the death of yeast at sub—zero temperatures.
Biophys. J. lg247-26H.

Nazur, P. 1960 b. Physical factors mplicated in the deat
of microor3anisms at subz ero temperatures. Ann.
N.Y. -cad. Sci. 85 :610—629.

Mazur. P., M.A. Rhian, and P.G. Hahlandt. 1957 a. Sur-
vival of Pasteurella tula.rensis in su3ar solutions
after coolin3 ano warming at sub-zero temperatures.
J. Bacteriol. T3:39“—— 397.

 

Mazur, P., M.A. Rhian, and B.G. Hahlandt. 1957 b.
Survival of Pasteurella tularensis in gelatin-saline
after coolin3 and warming at zero temleratures.
Arch. Eiochim. Eiophvs.'7I;31_51,

91

NeCleskey, C.C., and N.N. ChristOpher. lSMl. Some
Factors influencing t‘e sarvival of patho;enic
bacteria in cold-pac Ir strawberries. Food Res.

§;327-333.

M3 IE crobial

Ib Farlane , V. H., and H.3. Core .
n buff wad sug gar
7.

2501118? r, R.G.’ 8.1,]d 8.3. IIarltsell. 1
a stimulat ry factor from d: :fros ed bacteria.
Facteriol. Proc., p. 7.

Meryman, H.T. 1960. General principles of freezing and
freez n3 ingury in cellular materials. Ann. N.Y.
ACE-id. SCi. 55:503-50/0

Neynell, G.G. 1958 Th
c

effect of sudden chilling on
Escherichia Cc

‘Jn. Licrobiol. 19:330- 3Q“9

 

 

Moss, C.N., ard N.L. Speck.l 1963. Inj ury and death of
Streptococcus lactis due to freezing and frozen
Storage. J. App . I‘I IcrOEiol. ZO-b“

Nakamura, M., and D.A. Dawson. 1952. Role of suspending
and recovery media in the survival of frozen Shieella
sonnei. J. Appl. Nicrobiol. lO:MO-H3.

Nei, T. 1960. Effects of freezing and freeze-drying
on microorganisms, p. 78-86. In Recent research in
freezing and drying. Ed. by A .3. Par Res and A.U.

Smith. Charles C. -Lhom 3, Springfield, Ill.

Nelson, .2. 19MB. Factors which influence the growth

of heat-treated bacteria. I. A comparison of
four agar media. J. Bacteriol. M5¢395—&03.

Postgate, J.R., and J. iR. Iunt er. 1961. On the survival
of froze n bacte a. J. Gen. Microbiol. 2o :367—378.
Proom, H., and L.N. Hemmons. 19M9. flue or"ing and pre-

servation of bacterial cultures. J. Gen. Microbiol.
3:7-18

Prudden, TmM.1887. On bacteria in ice, and their rela-
tions to disease, with special reference to the ice
supply of New York City. Med. Record. 31:391—350,

 

369- 3 8. (Cited from Neiser, R.S. and C.H.
Osterud. 1997. Studies on the death of bacteria
at low t mpc eratures. J. Hacteriol..;Q:Hl3-M39.
Reeves, N.C., and Harrison, A .P., Jr. 1957. Effect of
time and te Imperature upon survival of Esc:ler icbia
coli in sodium chloride. Proc. Soc. pr51. Biol.

_,_1_
Bed. n-2wl—2ca.

Rivers T.H. 1927. Effec’t of repeated freezin3 (-1€=SC)
and thawins on colon bacilli, Virus III, vaccine v1-
rus, Herpes virus, bacteriOpba;:, compl;m3nt, and
trypsin. J. 3xptl.1bd. 22311—21.

Sanderson, 3.8. 1925. Effe ct of freezL33 and thaw1n3
on the bacteriOpha3c. Science. 52:377.

Sm t1, A.U. l95fl. 'Effects of low te: Qeratures on
livin3 cells and tissues, p. 1-32. In Biological
applications of freoZin3 and dryin3. Ed. by
R.J. C. Harris. Academic Press, Inc., H.Y.

Smith,E.F., and D.B. Sw1n3 e. 1905. The effect of
freezing on bacteria. Science. 21+M81—483.

J

U)

L

'\

quire ,R.H., and 8.3. H8 rt sell. 1 55 Survival and
[r wth initiation of defrosted kc orichia coli
as 0effected by frozen storage menstra. J. Appl.
Ilicrobiol. 3. bro-M5.

 

Straka, R.P., and J.L. Stokes. 195 Metabolic injury
to bacteria at low temperatures. J. Pac't eriol.
'70 0 Dr.-
('\J: 101-1.»); .

Tan3uarv, A. E. 1959. Preservation of microbiological
ass av orga iuuu direct freez n3. J. Appl.
IILCr'ObiO-l. ZiglL-x719. ' ‘

Tanne , P.W., and G.I. Wallace. 193 . Effects of

freezin3 on microorjanisms in various nstra.

Proc Soc. Exgtl. Biol. H: . 29: 32—3“.L

 

Toyokawa, K., and D.H. Hollander . 1956. Variation 33
selsitivi7:y of Z'e‘“r~cb3° coli to fre3zin: dame,“
during the frowun c;c13. Proc. Soc. prtl. Hiol
Ned. 92:”99-300.

Turntr, T.E., and H.L. Prayton. 1939 Factors n—
fliencin tLe survival of Spiroctct“3 in fr333n stat3.

 

 

 

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