THE CYTOCHEWCAL EFFECT OF FREEZING ON
ESCHERICHIA COLI {ATCC#11303)

Thesis for the Degree 0! Ph. D.
MWHEGAN S'MTE UNWERSE‘FY
Eciward F. Gritsavage
1965

mags: LIBRARY

Michigan State
University

 

This is to certify that the

thesis entitled

"The Cytochemical Effects of Freezing on
Escherichia coli (ATCC#11303)"

presented by

Edward F. Gritsavage é

has been accepted towards fulfillment
of the requirements for L

Ph.D. degree in Microbiology and
Public ‘Health

24 W i

Major professor

 

Date November 29, 1965

 

0-169

THE CIICCHZLICAL 153:0: CF FREEZIze

C; ESCIEZICXIA CCLI (ATCC§11303)

Edward F. Grits; 35

A; A3ST3ACT CF A THESIS

Submitted to
Kichifen State University
in partial fulfillment of the requirements
for the degree of

DCCTCZ CF PXTLOSCPHY

e

Department of Licrobiology and Public Health

ABSTRACT

THE CZTCCHEIICAL EFFECT OF F” TZI"G
Cu ESCEEAICI‘ IIA CCLI (AT CCfi‘ 11 303)

by Edward F. Gritsavage

When suspensions of microorganisms are frozen
and thawed, different percentages of survivors are
recovered. Survival is affected by the nature of the

uspending fluid and to the th emzi : procedure. Asso-

‘V

U)

ciated vi t1: this loss in number of cells is the re-

lease of intra ellular constituents.

Q.

Kicroorganisns were suspen ed in various menstrua,

J

C+

frozen at -87C *b ected o a fast- and slow-thaw, and

U)
f

.
C.-.)-

the aenstruum analyzed for leakage products.

Solutions of mo ovalent cations afforded poor
recovery whereas solutions of divalent cations gave
med erate recovery. The e: feet f t1".ese cations
appears to be related with tne stabilizing effect they
have on riboson~s. Suspensions that were slow-thawed
gave smaller percentages of recovery than th so that
were fast-thawed.

A relationship between the percent recovery
and the concent :.tion of leakage pr: ducts was estab-
lisned. Analyses showed the presence of a protein-
like material thou: ht to be low molecule r weight
peptides, nentose, and a substance that absorbed

ultraviolet light. The U-V absorbin, substance had

(-
La

”1‘0qu v1

4"
.4GJ;-A.. 1‘

absorption at 57 mu.

heat-stable, dialyzable, an

and recovered from ac: ivated charcoal.
assay .1ith bovine pancreatic ribonucle ase

0-3. v {I

.‘A " 'W 5 ‘ Q 9-. '
the product was not “Mn.

.4.- - '“‘1 - '.
eaterial prOViaea recovery in

O

. Q

I‘ . . \ ‘ '~ '
ADSOTpthfi mailma, Jase

fir "f an! r~-: '5

Lvozdvo 14b1, (lib. tile
‘g r“ A: 1a VA " Qn,

dficateq tize ..C.L€li

:s‘:ar: phosphate gave a

d could

ratio of 1:1:1

Edward F.

The product was
adsorbed by
Ehzymatic

ilidicated

Fre ct ionation of this

a , 3 y
a aarium-soluoe irac—

iv an K ,3
ratios at ;SO:2oO and

“co Jery in t1 is fraction in-

al to be the nucleo ide adenine.

“entose

atin: the ceznaound to be adenos ine- 5' -phosphate.

Gritsavage

THE CYTOCE—ZE-IICAL EFFECT OF FREEZING
ON ESCHERICHIA COLI (ATCC#11303)

BY

\\"
Edward Fl Gritsavage

A THESIS

Submitted to
Michigan State University
in partial qufillment of the requirements
for the degree of

DCCTCR CF PHILOSOPHY
Department of Kicrobiology and Public Health

1965

AC ZZIC'JLEDGEZ-LEITS

ishes to express his sincere

The author 3
pprcciation to Dr. F.R. Peabody for his guidance

and supoort throughout this work.

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0

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Anorecist on

-rce and counsel.

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Dr. W.L. K; mann fo

TABLE OF C”?"I-i5

I‘:rj'3f'\OQC-qlfl‘-
¢L¢LUUL¢ Lb.” 00000000000000000000000000000

LIIEZAIUEE TVVIEN

'9 h 0 r‘ . ‘fl 0 3
Effects of suspending media

fects 0

Effects of
Effects of Desiccation ..
Effects of Freezing .....

HATERIALS AJD EETXCDS

Dacterial Strains ,......
Culture Eaintenance .....

0.0....

Preparation of Cell Suspensions

Assay Of Cells 0000000000
Preparation of Extracts .

mialysis of Extracts ....
Fractionation of Extract
Suspending Fluids .......
FreeZinS and Tm" 1:16 0000

Preparation for Time and Thawing

:cperiment .........

Paper Chroma UO rapfly 0000
Electrophoresis .........
Pervaporation ...........
Heat Stability 0.0.0.0000
Adsorption ..............
zymatic Assay .........
Sonic Rupturing .........

RESULTS

I.

Cccurence of Cell Extracts

1.8at 00000000000000000000

Effects of Ultraviolet Irradiation
Chemical Agents .....

Effects of Freezing and Thawing ....

Relationship Between fiecovery
and Concentration of

v J.
1'18. 9

T3 "Q r“
Let'leC-vce

PTOdUCtS .0000.....00.0.000.00.

iii

page

41

43

page

Effects of Thawing Procedure on
Recovery ....................... 48

II. Characterization of Cell Extract

Effect of Pervaporation on the

Absorption Spectrum of Cell

EZtr‘aCt 000000000000000000000000 53
Effect of Eeating on the Absorption

Spectrum of Cell Extract ....... 55
Effect of Treatment with Horit on

the Absorption Spectrum of

Cell EZItre.Ct 0000000000000000000 55
Elution of Adsorbed Eateriel from

IJOr‘it 0......0......0...‘0...... 58
Fractionation of Cell Extract ....... 63
Effect of Ribonuclease on Ultraviolet

Absorbing haterial ............. 66
Electrophoresis of Cell Extract ..... 66

DISCUSSICI: ....0.....0.................000. 69
SL'I'KKW 00000.0...............0.....0...... 82

LITE‘MATU:LE CITED .00000.00.00.000000.0..0.. 85

iv

LIST F FICUZES

Figure

1.

7.

Absorption Spectrum of Cell Extract
Obtained fro:n F ozen Suspensions of

E: herichia coli (1:5 dilution) .....

Recovery of Escherichia coli
Subjected to Freez in5 in Various
Eenstrua at -87 C for 4 Hours and

Slow-ThaJing at 4C for 12 Hours .....

Concentration of Pentose in Extract
Obtained from Escherichia coli
Subjected to Freez n5 in various
Lenstrua at -87 C for 4 Hours and

Slow-Thawin5 at 4 C for 12 Hours ....

A Comps Irison of the Eumber of
Survivors at Various Time Intervals
During Fast-Ila win5 and Slow-
Thawin5 of Es herichia col; Frozen
'14.

ML: -07 C 0000000000000000000000000000

H

Absorption Sp ectra of Cell Ex tracts
Obtained fr m Frozen Suspensions of

Escherichia coli Subjected to Fast-
and Slo.-Th:.in" Procedures

 

(:5 dilUCiOI’l) 0000000000000000000000

A Go mnper son of Absorption Spectra
of Diluted and PeI rvapor ated Samples
of Cell Extract Obtained from
Frozen Sus>ensions of Es herichia

0011 0.000000000000000.0.0.000......0

A Cor pcrisen of Absorpti on Spectra
of heated and Unheated Salnples of
Cell Exr act Obtained from Frozen
Suspensi ns of Escherichia coli .....

bsorpti on Spectr an of Cell Extract
Obtained from Frozen Suspensions of
Esc cherichia coli after Ireatment

with and Removal of Eorit ...........

Absorption Spectrum of Eaterial
Adsorbed from Cell Extract

Obtained from Frozen Suspensions

of Escherichia coli after Adsorption
witZI and Lqution fI rem EoI it .........

 

45

47

5O

52

54

56

57

59

11.

12.

Page

_ .o A. In I“ - r‘ 4.
Ce parlson oI Absorption opecCra
4" I. r~ 3 — h I ‘2‘ P- I-l " . "-
oI C-trav olet AbsoroInL haterial

1
“orit-Adsorbed fr m Cell Extract
ined from Frozen cuspensions
c

.1 1-1 A1: ”I? ':
»rIcnIa cCII and vuspenaed
r~
LA-

:10 SOl‘bltions ..0.0..0.. 6O

 

Absorption Spectrum of Cell Extract
Cbtained from Frozen SuspensiOns of
a: I

.L
{An-n ‘xrnJ'p'n
V‘ 'Jv-VUV‘

ro'cngs (1:5 dilution .. 62

 

Absorption Soect
Fractions 3 sult nr Ir 5 the

Fractionation of Cell Extract

Obtained ron Frozen Suspensions

Of E:C:fl.er.ic‘i-i:~ Col-j. 0000000000000O0000 64

I o :

ra oI VarIous
0 "I

I

 

. ‘ fl ’-
Electr pnoretorram of Uell Extract

CbCaIned Ire“ :rozen SuspensIons
A“ h" .A .0 Q. n O
O; L..C.LICI'.I.CIIi<J. 0013.. 000000000000000000 68

 

Electroph retorram of Cell Extract

Cbtained from Frozen Suspensions
of Aerobacter aerofenes .............. 68

 

vi

Table

LIST CF TABLE

”he Percentage of Survivors and

&——v

Concentration of Leakage Products

after Freezizg Escherichia coli
in Saline Solution at -b7 C for
4 Hours and Slow-Ihawing at 4 C

M

vii

:13r121—30urs .....00..00.0...000.....

44

I. F‘»?

IKIRCDUCTIUM

Jhether or not a suspension of microorganisms has
been exposed to a stress, losses in viable number occur
and different numbers of survivors are obtained. The
survival of microorganisms under such conditions has
been studied extensively, and many factors have been
incriminated and considered as contributing to this
phenomenon.

When a suspension of microorganisms is introduced
into a new environment it is exposed to a variety of
physical and chemical phenomena not present before-
hand. It is the nature and effect of these phenomena
that are concerned with survival, and, when this
suspension of microorganisms is subjected to a stress,
the proalem of survival becomes more complex.

Under conditions of stress, bi0105ical injury
to the cell results. This injury might be considered
to be due to alterations produced within the cellular
structure and metabolites. Consequently, it mi5ht.be
assumed that some of these alterations could be re-
versed, and if suitable means were applied, the cells
mi5ht survive-- provided that the injury is in a
range of tolerance. To increase survival, the non-

reversal injury should be reduced or eliminated.

Recovery migh apply in principle to all chemical
and physical injuries produced within the cell, and
restoration of viability might constitute a repair
process, regrowth of structural elements, elimination
of toxic materials from within the cell and its en-
vironment, or the formation of new adaptive structures.
Many of the mechanisms involved in killing and pro-
tecting, as well as the responses under various con-
ditions, are strikingly similar, intimating that the
process of survival is related.

Probably the most extensively studied phase of
survival is that of survival after exposure to freezing.
In general, survival in the frozen state implies sur-
vival without growth. Past reviews on the effect of
cold on microorganisms list the following observations:
(1) On freezing, a sudden mortality varying with
species exists, (2) the number of survivors after
freezing is independent of the rate of freezing,

(3) there is a gradual decrease in viability during
frozen storage, (4) the decrease in number during
storage is greater at temperatures near the freezing
point, but less at lower temperatures and is lowest
below -20 C, (5) death, or loss in viability, is
usually greatest during the freezing process but is
less during storage. Despite the volume of literature

on this subject, it is difficult to find agreement.

among the investigators as to the responsible
mechanism.

Associated with freezing is a leakage of intra-
cellular constituents. The purpose of this study is
to characterize the nature of the leakage products

and to determine its correlation with survival.

LITERATUHEE EVIEN

Effects 9: Suspending Media

 

Eicroorgani sms stored in aqueous suspensions

and subjected to no overt stress demonstrated that
survival was ir If _uenced by conditions. Shearer (1917)
eported that physiological saline solution was more
toxic to meningococci than was distilled water or
1.5% EaCl solution. With studies on the mortality

of 'Bacterium coli' and 'Bacterium typhosum"Cohen
(19é2) found that It constant temperature II un-
buffered media like distilled water or tap water,
the mortality was variable and coincident with
apparently insignificant pH variations, but at con—
stant pH, the resistance decreased with a rise in
temperature. The tests of Sherman (1923) demonstrated
that cells from old cultures were more resistant to
brie f exposures to cold, to mild heating, and the
action of phenol. As a result of studies on salt
action, Winslow and Falk (1923a, 1923b) reported that
low concentrations of EaCl and CaCl2 solutions had
no effect 0n.§-.22ll after 24 hours, but higher
concentrations.(o.725; EaCl; O. 435M CaCl2) were toxic.
When both salts were present the effects were additive.
The toxic effect of CaCl2 was attributed to an in-
ability of the microorganism to reduce the alkalinity
of the solution in which it was suspended.

Later, Winslow and Dolloff (1928) reported that an
anta: nistic effect of cations upon bacterial
viability appeared m1.y in a medium held at pH 7.0
and that the effects were additive in acid conditions.
Further work of Winslow and haywood (1931) on the
effects of cations on viability shows that cations
had a "specific poten y" and had a 5eneral influence
on the bacterial cell. fhis influence took the form
of stipulation of viability (associated with in-
creased permeability) when it was present in low con-
centrations and of inhibition (associated with de-
creased perneability) in hi5? er concentrations.
Sherman and Cameron (1934) demonstrated that
young cells of 'Bacterium coli' could be killed by

a’rupt environ ental ha.5es within the natural r an5e

\v

of rowth of the organism. In contrast, Hershey (1939a)

C
found that the LJUlOlO”4C 1 state of the cells did

not influence survival. Harrison (1961) observed that
it did. The role of pH pon survival was considered
when Gale (1943) observed that the enzymatic activities
of a cell could be altered in response to alterations
in the pH of the exterial elvironment. The pro-

duction of Hi 0 ac Cid decarboxylases was stimulated

in ac; 'd pH while the production of deaminases was
suppressed. Pinsky and Stokes (1932) found that

adaptation of enzymes was favored in aging rather

than in whys olcfical youth. Cook and Jills (1958)

showed tha washed ;. coli suspended in phosphate

 

buffer (0.1;; pH 7.0) maintained its via oility setter
than did unwashed or was} led cells suspended in water.

Holaen (1956) reported that incubation of Lactobacillus

 

v'l
$

sinCS‘s in :hosphate buffer at 37 C resulted in

m

(1
CA.

 

the degradation of BIA and the subsequent release

9.17

of ultraviolet (by) assorbing substances into the
medium. Similar res'lts were oatained by DeLamater,

F‘

dacock, and Lazzanti (1959) with zacillus mejateri‘n

 

leached in 0.053 ph sphate buffer, pH 7.4, for 2 hours
at room tehperature. Eiguchi and Uemura (1959) ob-
tained the release of nucleotides fr‘m yeast cells.
Harrison (1561) held cells of d'fferent physiological
states in phosphate duff er at 40 C and follo.. ed the
loss 0: viability. Cells from si w log phase cult‘reo
survived better than cells from normal log phase
cultures, and cells from stationary phase cultures

vs

survived best of all. Stranre park and Hess (1961)
a. ’ 9

studied the survival of pooulations of stationary

growth phase Aerosactor aerorenes in non-nutrient

 

 

f5"

builer. They showed that the composition of the

I o ‘ ‘3 ‘. V“ ‘J‘fi A. -'~ r, “‘5 -‘ ' Q 4‘“ F‘ ‘ 4"5 i : 1
growth. mC‘ulu...’ tut, ‘Ihg,.Se C‘ £1‘Os'.7bll, 0.110. bile ’38 ".LOQ

b

A.

oi the stationary phase influenced surviva . Death

‘ " (3AA m—\ 4 ’ ‘. .. 4-: ‘
was preceded .iu acconjaniea 3y degradation and

eicretion of jolymeric cell centents (protein, ribo—
nucleic acid, polysaccharide). Stsange (1961) re-
ported that these reactions degraded the aoility of

tne or anism to form adaptive enzgrnes . The obser-

,..
i.

vations of Postnate and Hunter (1962) showed that

‘u'

continuous cultures of g. aerosenes grown on a

 

limited supply of ilycerol died lin arly Jith tin
without cryptic frowth when aerated in buffered
physiological saline solution at the Optimum pH
and temperature. Death was accelerated in environ-
ments of hirher or 1 r tonicity, in unbuffered
media, at pH values above 7, and temperatures
above 40 C. Death was not attributed to a break-
down of the osmotic barrier. Survival was reported
greater in dense pOpulaticns than in sparser ones.
dyin: pOpulati as also sh wed a rapid breakdown of
intracellular 33A and the release of phesphate and
base fragzehts into the medium; intracellular pro-
t

in reported degr aded but intracellular poly-

(D
S
S)
U)

saccha1ide and DEA scarcely de;1aded at all. A
functional alteration of the cyto1lasnic membrane
was assumed to be the cause for the leakage of
nucleotides from various microorganisms (Chabaya shi,
1962). Later, Ckabayashi, Ioshinoto, and Ide (1963)
noted the excretion of a considerable amount of

ader ine risonucleotide when Brevibacterium was grown

in a medi um con ainin: anino acids as the nitrogen

Several factors infli lence studies on the sur-

0
k

J

mic1oorganist -uspended in non-nutrient
soultions. One factor is the presence of impurities

in buffers that allow li::ited growth of bacteria
Garvie, 19 5a,b). Another factor is the release

of intracellular contents into the medium by dying

populations supplyine nutrients to the surviv01s.

Ryan (1955) teraed this 'cryptic growth.’ Strange

"I

et. al. (1351) called it 'regrowth' and demonstrated

‘-r1 ‘- - ,q. \ van», o. _‘\L a‘ —‘Q' q r\ O . I‘
that it could be p1e.ente1 a” dial§Zin5 tLe sus-

p nsion or renewing the suspending fluid after fil-

 

Licroorganisn: exposed to heat exhibit varied
resn nses. Curran and Ewans (1937) found that the
addition of blood or gluco:e to nutrient agar or
the use of infusion afar usually increased counts of
bacteria exposed to sub-lethal dosage of heat, ultra-
violet light, and mercuric c:1loride. Hershey (1939b)
also ODSGPVGd a related response when he noted that

cultu1“ s of E. coli that were heated to temperatures

 

near the t11ernal death point for 15 minutes Cave

8/
lower survivor counts on nutrient agar than by

11.1.

serial dilu ion in ru t rient broth. A cor 1tinuous

decline in resistance l(.1el in younger cells of
g. coli that were exposed to heat was noticed by
‘ ‘ Fr zier (1938) wl1ile cells in the

Ellic;er and
l rits12ic

and
from broth cultures
ceptible to
nature cultures

jected to

for‘m I‘rOa'ln

pending fluid. otran

311a se sh

te (1959) den

‘. J--‘ r.
1108. ULn‘

The observation that bacteria

1 ‘ .-.
varied resp n
1C‘5—2 177* ’1" r:
'1) i'UAloo nv be“
‘ .7 "‘,'¢’.L'.. D
13895.- ubion O-

the bacteria

owed lower resistance. Lemche

onstrated that cells harvested

0-8 hours old were more sus-
at 55 C than those from more

sub-

suo-lethal heat were more dez1e nding in their

were unheated controls

13:13.11

possible ex; anaticns for

that eXposure to

ribonucleic acid in soluble

t11at dlfiu

be and ”hon (1964) studied the

effects of herua stress on viability and presented
data that take into account many factors. They found
a conjls; ziituLe of substances, one of which ab-
sorbed ultraviolet light, prese1t in th- suspending
fluid after eXposure to heat. 1he addition of tnese
leakage pr ducts to the heated suspension did not

in rease the viaDi‘i ty or recove1y cf the bacte1ia
In the treatnent of their cells, Strange and Shon

(1954) found that

magnesiflm resultin

lutions desor bed

death rate of

1O
bacteria corngai d with bacteria washed in distilled
water. The adiition of magnesium to the diluent in
which the bacteria were heated largel1r eliminated
ferences in tr1er3al me is tanc eresulting
from the pre-wasliing treatment. A relationship be-

w 1

tween tne rates of death and 0 BSA degradation is

'J

i1tinated since magnesium, which stabilized isolated

ribosomes f r he Quillen (19o2 2), decreased the rate

 

cell by reversal of al
cell can be de onstrated when cells are inactivated
with ultraviolet irradiation. Kelner (1949) was

‘. 'p va' _"\_~
one 01 t11e 11ir

(I)

t to show that strains of 1. coli,

 

in; tivated by eznos ur to ultraviolet irradiation,
H: 1: be reactivated by xyosure to nornal light.
About the sage magnitude of reactivation was re—
ported by Anderson (1951) who reactivated irradiated
E. 32;; with heat. Thompson, Hefferd, and fiyss (1951)

failed to recover E. coli from U-V injury by

 

adding pyruvate to the cells. Their intention was
to oxidize the hyd1o; en peroxide formed during
irradiation. However, the enzyme catale as, which

oxidizes hydrOjen peroxide, induced rest01ation

11

 

to cells c- E. coli, straiz X-12, inactivated by

U-V irradiation (tharjet an Cal” 1952).

Heinmets (195") was successful in reactivating

irradiated cells incubated in pvruvate lution.

In later experiments Heinnets and his co-worker
(1954a, 1954b) found that the most effective re-
Hct va .tion :“Cnts for irradiated cells were various

n_inf to

cycle. nino acids, with

the tricarhoxylic acid
the exception of alanine,

re reported to have only a moderate effect. Studies

rar (1.1.8.

F}.

on the effects of

J...

1e] tion to food preserve

r\
L.

and “ac Queen,

 

961) showed that selisitivity was

Lion of microorganisms in

t; on (ELM an, Thatcher,

in-

fluenced by the nature of the suspending medium
durinf irr adiation. In a ifferent area of study,
ctarleto en‘ “ngle (1960) reported that radio-
“es tance (resista1ce to inactivation by x-rays)
of ;. col i strain 2 n, narellels relative reSis-
tancc to t-e C-V irradiation and thermal stress.
Effect s_§ Che: cal Arents

 

 

Permeability defects

have seen re301 ted as

causes for the decrease in survival of orcanisms
tree ted with detergents, chemicals, and dryin .
Slade (19 57) reported losses as great as 99$ when

cells of Strentococcus

nyo~enes we; e treated with a

 

cationic detergent cetyltrimethylanmonium bromide.

12

These losses were accoxzpanied = ith the diffusion of
amino acids, primarily lysine and glutamic acid,
from the cells. Strauss (1961) p1 oduced a similar

effect in cell: of E. coli treated with ethyl sulfate.

 

Once more, there was a decrease in number which was
followed by an excretion of ultraviolet abso 3011:
material into the medium and a decrease in intra-
cellular nucleic acid. The killing and excretion
could be preve ted .iy the addition of magnesium to
the incubation medium. It is interesting to note the
stabilizing effect mafnesiun has on protoplasts
(fieibull, 1956) and on spheroplasts (Lederberg, 1956;
KcQuillen, 1953) suggesting that these ions might
protect bacteria durin
In studies with disinfectants, Wright (1917)
concluded t-at the medium in which the organism was
grown before being acted upon by disinf ctants was
much more iraortar t than he recovery medium in de-
termining ability of organisr s to survive. Jacobs
and Harris (1960) aaintained the opposite. Cells
da ated by phenols continued to decrease in number
after being transferred to nutrient broth but there
was no such decrease if the broth contained Rorit,
an activated charcoal. Later Jacob and Earris (19o1)

reported t11at media, both solid and liquid, when

treated with Ihoait before inoculation with cells

13

da aged by phenol, gave better results than media
that wasn't treated. These findings supported their
view that t e medi m cont ned materials th ant were

toxic to the bacteria.

Effects of Des iccation

 

In the preservation of bacteria by drying,
Sta::1p (1947) fo and that survi vel rates depended upon
t e med ium used and the method of drying. Record and
Taylor (1960) showed titat organis :15 that were dried
in sucrose solutions were subject to internal dif-
fusion pressures on rec: nstitution. Such diffusion
pressures could cause disruption and death of the
organisms. Incree sed p rrme bi lity of the cell wall
of yeast upon dry in; was reported by Ebbutt (1961)
who considered this as of greater significance as
a cause of loss of activity.

The most commonly used method of preserving
microorganisms is lyophilization (freeze-drying)
but his is a drastic procedure for in most in-
stances only 2-35 of the organisms survive the
process. Cultures preserved in this way have re-
mained viable for as long e.s 35 years (Engley, 1956).
The loss in numbei is attributed to altered per—
meebility of the cell me usrane and destruction of

enzymes (basserua. and HOphins, 1958).

14

In studies on the survival of E. coli, Record

.1.

and Taylor (1953) foun that a relationship existed
between the percentage of survivors and the initial
concentration of organisms in the suspension; the
more dilute the suspension, the lower the percentage
of survivors. The dependence of survival was shown to
be due to soluble material derived from the organisms
but protection by the material was afforded in th

dryinr state only. Clement (1961) obtained high

survival in frozen and partly dried speCimens but

4
P
01
(2
P.
}.J
H
(F

k:
p,
(D
O

reased with continued drying and fur-
ther losses occurred when the preparations were
dry. Wagman (1960) presented evidence of cyto-
plasmic injury as the primary effect of drying in
bacteria. Suspensions of g. 99;; were freeze-dried
in water and allowed to stand for 3 hours. A sub-
sequent decrease in numbers was obtained as well as

the release of ultraviolet absorbing aaterial and

no .9 T‘- .2 ,..
«-iects oi rreezln-

The general topic of the loss in viable n ubers
of microorganisms frozen in suspensions has been
reviewed extensively. Despite an extensive study,

diiferences of opinion exist among the investi-

gators as to the cause. As a result, a multitude

15

theories has been presented.

In the early work of Liacfadyen and Ho Iland
(1900) a variety of organisms were frozen at -190 C

7 days and growth was obtained with every type.

However, no quantitative data were reported. Perhaps
the earliest quantitative report was that of Smith
and Swingle (1905) who obtained up to 99.98% kill
of 'Bacterium typhosum' in broth a ter freezing at
-17.8 C. 1310 ir crease in bacteria in milk held at
-9 C was noted by Ravenel, Hastinc s, and Hammar
(1910) whei eas there was a marked increase in milk

held at 0 C. Escherichia coli suspended in a variety

’-

of liouids and frozen a

5

b -20 C yielded different

‘1

ercentages 0: recovery (Keith, 1913). Tap water

'0

was repo: ted to be more letha while milk and
slycerine provided protection. The death in water
was considered due to mechanical crushing. Hilliard,
Gorossian, nd Stone (1915) suspended orra isms in
mi k :-d sucjected them to fluctuations in freezing
temperature and found that -15 C was more fatal
than -2 C. Later Hilliard and Davis (1918) sus-
pended bacteria in various n1enstrua. Some samples
were frozen and refrozen and some were suspended

in a medium that did not freeze but reached sub-

zero temperatures. From their data they concluded

16

that (1) intermittent freezing exerts a more
effective germicidal Hot on than continuous

freezi ng; (2) the reduction is less in milk and
cream than in pure tap water when freezine tem-
peratures are a1,plied; (3) the degree of cold below
freezing is not a very important factor in the des-
truction of bacteria. There is a critical tempera-
ture below freezing where the germ: idal effect is
greatly accelerated; (4) the death rate of E. 32;;

is higher in media which 18 frozen solid than it is

p;

in the same media net soli and at a slightly lower
temperature; (5) crystallization, probably resulting

in mechanical crushing, is an i :portant germicidal

('1’

fee or in causing the death of bacteria at 0 C. The

greatest reduction occurs

’d

romptly upon freezing
and refreezing. Paradoxically, Sanderson (1929)
subjected bacteriophage to repeated freezing and
obtained no loss in titer, and Smart
(1935) observed slight growth in cultures of micro-
organisms stored at about -9 C over a year, indi-

cating that some biological activity is possible.

01
r.

Berry (1933) presented ita showing a decrease in
survivors of :us pensions held at various ten pera-
tures. At -20 C, a 40; decrease {as found; 99¢ at

-10 C, and over 99$ at -2 C. Using a variety of

17

liquid air for one week, Winchester (1936) found
that all remained viable. Turner and Brayton (1939)
noted that storage temperatures of —20 C, -10 C or
warmer decreased virulence for spirochetes but at
a storaje temperature of -78 C there as no demon-
strable loss of virulence. These investicators
maintained tha injury arises from two sets of
factors: the act of freezing and thawing, and the
other with storage. A more extensive study of this
by Jeiser and Csterud (1945) lead them to believe

"immediate"

that death by freezing involves an
death caused by freezilg and tlawing, and a storage
death whi h is a direct function of time and tem-
perature. he mortality due to immediate death by
freezing was marked but did not vary with the in-
tensity of the freezing temperature. This immediate
death occurs at a brief stage during which external
ice is being completed. The rate of storage death
is higher at temperatures above -30 C.

Only two factors were considered by Proom and
hennons (1949) to influence the survival rate after
freezing: (1) the species, and (2) the nature of

the suspending medium. They believed that extra-

18

cellular ice crystals punctured the cell wall and

that different species exhibited differences in the
stren; h of the cell wall. Saline solution was
found to produce the greatest reduction in numbers
while gelatin decreased the loss. The protection
afforded by gelatin was attributed to the formation
of colloids around the cells that protected the
organisms afainst damage from ice crystals.

The protective power provided by glycerol was
lemonstrated by maxy investigators. Lovelock (1953)
obtained protection when red blood cells were frozen
in the presence of ;_ycerol. However, he maintained

hat glycerol was protective only when it had per-
meated the cell before freezing and it did not pro-
tect against osmotic or th rnal shock. Cells im-
permeaole to glycerol ar not likely to survive
even in its presence. Hollander and Hell (1954)
theorized that the protection offered by glycerol
against the effects of freezing could be explained
by its physical characteristics. hey believed

hat deatn resulted from mechanical compression.
Additional reports on the protection by glycerol
come from howard (1956) wzo maintained viability
rozen in 51y erol and stored at -10 C
for up to 5 months, and from Quadling (1960)

who found the glycerol provided protection for

19

Xanthomonas and no differences were found when
hawing was varied. Tanguay (1959) preserved micro-
'r organisms by freezing in glycerol,

T

and Floodgate and hayes (1961) used it to preserve

Increased surVival in other menstrua has also
been reported by various investigators. Squires
and hartsell (1955) indicated that survival was
related to storage nenstruum and that stimulatory

materials accumulated during frozen storage. Sus-

*d

ensions of organisms in lactose-phosphate buffer
solution had remained viable for Head et. al.
(1960) after one year of storage at -23 C.

Several factors have been suggested as in-
luencing survival. hajor, KcDougal, and harrison
(1955) stored eleven species of bacteria in broth
at various cell concentrations at —22 C and found
that in most cases, survival varied in proportion
to the initial cell concentration. Later, Harrison

F’

(1955) reported that with Lactobacillus, the per-
centage of survival after a series of repeated
freezings and thawings was dependent upon the
initial cell concentaation. Lion (1961) suggested
hat loss of viability occurred during the period

when oxygen was in contact with cells.

20

The growth phase of the culture has also been
mentioned in connection with survival. Horse and
arter (1949) observed a large increment in ERA
during log phase. Cells in this growth phase were
more susceptible to heat, cold, and U-V light.
Uhile inforwation on the connection between these
activities and nucleic acid is limited, a con-
nection is intimated. Toy kawa and Hollander (1956)

noted that g. coli varies in 'ts senSitivity to

 

damage and reduction in freezing and that cultures
in the log phase of growth were more susceptible
than older cultures. Organisms in the stationary
phase of growth were reported to be completely
resistant to freezing in 0.3h sucrose solution
(heynell, 1958) and that susceptibility of growing
organisms changed rapidly during the exponential
growth phase. The response was not due to the
in itself but required a suitable diluent
'as well. The subsequent evidence of Gorrill and
HcIeil (1360) substantiated this view and they
also stated that in eneral the simpler the com-
position of the diluent, the more lethal it was.
During the period of years in which studies
5 i

on freezing were conducted, a number oi theories

appeared which atte pted to explain the mechanisms

21

w: - C P- 'r\- s
AAA—LCL‘OOBL CLa..l-S.T:S

f: z'.' “‘ ") C —. ’\ F
s“ - ‘Q. G ;— .
A ., O 4.. ., ,. - L ‘.. , .L‘, - . -1" ‘ J.” “ -
L.;€‘ C- C 61.6 ".'."L..'_. 6-1. L. L803] OJ. Leflbil DJ
~1Vh‘-:~ r- 1r‘~-7 ‘~ vrr‘c .‘fi :3 (- ‘3‘, \vr ,. 3 ‘n 1(‘17 n-n‘.
C- LAD -4.LA {04-.LCAL Elsi-U ‘31 'O_IO~€LL ‘JJ iLeJ. L‘- J J CL-4.C«

later a" hpinfs ( 938 who f;un no evidence to
sup:o:t this theory. Using direct m asu: nents, no
chanjes in size were discerned in brcteria tha wei e
frozen. haines su°cested that coagulation of protein
van regionsiile for the death of bacterial cells on
ireezing. F‘rt er evidence of harris n and Cerroni
(1:56) discounted the credence of such a theory
beca1:e no relati nship between the cells and sus-
ceptiaility to freeZing was found. The le hal factor

.- :.1 ., , a A.-. . J. a . 'A °- - ...
is consiaeiea iv the; CO .e something 0 ther tha-

Pneunococci to the activity 0i

- 4 -‘. . .__.. \ . “4.1
p ra urea, ”else: and “artiss (19eo) p; opose‘ that

during ice forma ion, mze concentration of the

m

olute surrouza ding the cell was hit gher than within

‘

1e bacterial cell and aehy‘ra tion took place.

Ff

;r results were indicated by Harrison (1956)

U)
M
p.
I...)
c:
)-

who as onstrated siniiali ties oet.reen the behavior

of bacterial cells'in fro: en and unfrozen solute.

1‘ 1 0 A -,. r‘ 4' ‘e (\ 1 ,- J- .‘ .2 P, ‘ 4

“e conclusion was t-at eeati in irozen sus13ersion
‘ ,-. A j q -' J"‘ A,-- . Agfl -_ .‘ J- . A- '0 ' -. ‘ '

has to do x_tn concentration oi the solutes upon

U.-e "IIrOJ-lu

'ain supgort from
of Luyet (1“; ) who studied phase transitions occur-
ring in asueous solutions at low temperatures. He
showed that in dilute solutions, sol. i dification

4.

teen place in two steps: a liTSb in which the sol-

J-‘

vent freezes out while the solute becones Lmo e con-
centr ated; and a second, in which the components of
the cenentwte'1 solution solidify.

.7 Q. ‘,~ A. ‘- 'o '1 ~' :‘A - ~. -‘
however the zesults Cl Mazur anian and
i 9 9

as formation of in‘racellular ice. when

cells f Posteurella tularensis were cooled to tem-

 

peratures between -15 C and —75 C, recoveries after
slow warning depended upon the rate of cooling.
These iinuings, they concluded, we; e co- 1pa tible
with the hypothesis that recovery was related to
the xtent of ir tracellul w. freezing. «00d and
Rosenberg (195 ) reported s‘rvival in yeast even
“cu h as much as 90; of cellular water was frozen.
Intracellular ice was considered to be a pre-re-

-' £4- ” .L‘ ‘ 0., - _'“o _g_w 1
LS... be to (.63. 021 CLIJ.‘ 11.1” 51.017 178.4%”).an ELM-CL tilOCG
V

1 ‘ Kr

"nl " A’ I'- 3 ‘A A” ~ 3 4-‘_,_ g .04.? J. .C‘
lactose whic- lessened the iniurious eiiects oi

slow warming did so by rcduc n" the extent of intra-

L,

23

O
(D
14
*7
f
(7
*4
ya,

. ' .A- ff ._ . - , .-
:eezinj. Later, hasur (1;oO) presented

‘

iniorxaticn strenz'th—nin~ this theory and ileed to
di:c:un the theory of increased conce *ration of
solute as a cause. his data showed tna the drop
in sumiv va was less extensive when the cooling was
slow than when coolin" was rapid. He concluded that
death we not he result of high concentrations of

solute. Ii it were, longer exvo u; es during slow

.. J- u, , A '1
greater dana; and

freez:.n: would have produced

Zecau e linid-protein co slexes rake up cell

c.
,-

"e.iiahcs, Loveloch (1957) proposed an additional

fl-‘

theory. oi 2ice these comilexes ale held to;ether by
weak association forces instead of strong co-valent
oonds, be attributed rupt‘ring of these bonds as

. inis was accomplished in

by (1) increased -le

0
O

trolyte con entration,
(2) han es in pH, pert: cula rly if sparingly soluble
Differing salts crystallize, and (3) removal of
water. "his theory was based on the observation

that mensnanes of red blood cells lost phospho-
lipid when suspended in various aolar concentrations
of HaCl solutions. A loss of solubilit‘r was used as
the criterion of denatuiation a11d human plasma

1i poprotein was dem1 tured when 11 ozen.

ha; r et. al. (1956) detected changes in the
‘ty of ha teria s1spensions with ve. riations
of the osnotic pressure of the suspending medium.
These changes were depend nt upon tne viabilitv of
the cel 8. Since heatin and freezing-thawin
duced doth viaailit an; the onticJ.eiiect, the

redu tion was attributed to osmotic pressure chanfes
consequent to freez ing. Bretz and Hartsell (19 59)
observed tha some bacteria showed osmotic sensi-
ti.ity (' osro"0121tiv t y') after freezing: and

tha.in s since populations gave greater recoveries
viable organis :1s when di uted in 20” sucrose
solution oefore pleti1: than when diluted in or-
dinary buffer solutions. Since materials such as

the diamines of La; er (1959). hich p11otect os-
motically frarile organisms, are likely to be pre-
sent in a rich medium as opposed to a minimal me-
dium, osmosensitivity m ght provide an explan tion
for metabolic iijury. Unfortunately, Postgate and
Hunter (1961, 1963a,b) could not demonstrate this
Optical effect in suspensions of Aerobacter and
their findings were incompatible with the view

that the lethal effects of freezing were connected

iC SliC‘Cli.

.1"
F].
c..-
H‘
t
O
(’1
O
('1’

25

*roaasly the rest tenable theory was that
suggested by liartsell (1C 59). From the assumption
that sore synthe esis is possible even at sub—

freezing teiperatures (Smart, 1935), products

should accumule. e as the cells a1 e held in storage

7

and later iso l1ted. This presented the foundation
for the theory t11at freezing causes "metabolic
injury" to microorganiszz s. St1aaa and Stokes (1959)
supported this theory with the observation that

.0

cold injury was nanilested by m‘. in011ease in 11u-
tritional requirements, and that the active sub-
stance required by injured cells was peptides
ihis was also de1‘1ons trated by Bretz and Basa (1960)
with greater recoveries of cells on complete media
han on minimal media. The mechanism that nigh
xplain this reguirenent was indirectly suggested
by handelstan (1960). In bacteria, cells that have
expended or e suastrate in the growth medium are
engaged in pro due 4 an inducible enzyme for th
utilization of another. This "metabolic injury"
can be explained as a result of protein turnover.
theorv was streng‘hened by the results Bretz
(1931) reported. he disccunted the theories of
I - .w 1. ur .,

- . n r -- N 1‘ --\ r- I"\ . ', ‘- .r
mechanical Crushing and "c:ncentrated solute

as well, when he observed that g. coli frozen on

 

SCZDPC 21e discs or cellophane gave similar responses

4J

as cells in frozen susneneion. There was an inc: -ease
survival of cells regardless of the method of storage
whi h inplied an interaction between cells prior to
freezing.

Arpai (1352) indicated that non-lethal meta-

bolic freezing was related with time, temperature,

-- : 4.x. ,_ J. -_, ,_ -. v? .0 J...‘..l _- ._.,.‘3 .- _ , .011 £ ,1 1‘
(lib; U--€ 11¢. will 0-- .2.1 O; ui.e suspel-ul.fl; .L .LU'..LL'.. Tlle
-— ,f‘ -‘- ‘ A Q e H . q" ~ -. j ‘ 1 1,1 r'" q . ‘- a
ro_e o- tne eds; ending and Lecovery hedium upon sur-

' i "r' I "-"L .‘ "fi . .1 ~ .I‘ t". 1‘, ‘ ‘ml‘j" . " ".1' —
.' l v -. all ten .. ree Z in; was 52 ..C .I.’l Dy ......i-&-..u118. LAC.

A’r) _ _.4_ w J.” '._g' a J- .p_‘ 3. r”
Dawson (1yu2 and suppertea tne View tnat .1eezinC

alters the nutritional requirements of microorganisms.
As - ciated with protein turnov r is a subs quent

-..- - '- ’ "1 ‘fi ’1‘ A r‘ ' o" "'1 z ‘ 1“ 1.1. q 1- I‘ .,
turnover of aha. nanaclstan (19o0) repo.ted tnat tne

4.

instability of KIA seemed to be a consequence of
stepping ;r wth. Under conditions of starvation,

‘

'rate either because there

r
5’

F .
25'.
('9-
O

the ribosomes d's
is a decrease in the concentration of H; ++ or for

. a

some oth»r reason. A solubilization of the ribo-
nucleoerctein occurred and the REA which was pro-
tected in the particulate state became exposed to

attack. The breahdown of LIA was indicated by the

[-10

1 r r1 -.~ 0 r ' a“ r-' W‘. C . . r‘
appearance 0. an ac u- W01 :le . tezial a mo1o1n¢
’/
C- t 2“ V O .“u .

16,: ,.-.-.,,--,, .- .-_,. -9 ‘. g -:- -. - .- 1.
0311.1. 6'... SL1... ::.‘.S.LO..;.. C‘L 116.0,-18 uC.‘ we.e .LOL'IIC.

 

. '34- r a '1 7.. -.‘— PIA, N- ,.-.!l .0 .9
::y stranLe ana uaih (1;os) to ean .ree amino

o" . q ~o v V\ -' . [- ‘--‘- 1' Jun... _.‘ . _
acids, lo. Cl- -la- elihb peptides, a sabstance
1.. p--~..-.r.‘ : 7* 'r '1 : -1 J— 'n .1 . 1 1 151'
that carat)- DEL. u- « .L- -l'o, Jab 110 3.0...CL 80.0.1018 ....A

could ge Cetected. Similar results w~re obs rved

‘1‘" "n ‘n'fi-u’. 1A" 1( '6’.) 1r.” rm“ ' 4‘ '-‘ r11 (1 1r""\ "”1 <1") 3- 4‘"
aJ O'N-rc—c'nofiéc 8 iv; 0-- VLLJJeC Lieu bu 5L4...J_Jel.a¢...o-‘ OJ-
: 4- 1.-.- - .0 4- . - ,. '7 .n _ - ..

llv_ng tuao-cle JCCilli to repeCte: .reczin: and

1., ...v.-- ,..,1 .. 1 ..-,. - v-1 , 1 . -4.-
thawin_ and entzac tea a material that could sensitize
. (x . r- 4- Q- C -. Q N -: ‘_ . ..- ‘ “ 0’ ‘,.‘\ q
"Eli a site to tuieiculin. Biochemical and electro—
A ._,
‘x ‘1. ‘3 ‘ ~,— «A... q ' ‘ ‘o . '
pnoletic studies sLC..ea se1 .1a ploteins, two poly-
3 " 1‘ 1 _-.1 ‘ (5 q :1 ‘.‘\: ’1 0‘,“ r‘ I‘ A, fir) 3‘ V 'n
ECC1A. .. *QeL—v , .L..;.2.L‘~A, L~.L’~.l. k... ;:1 CCA-lcea-t 18 i! 0.51 OJ.
L

.9 a-.. ...° ‘ It-.. ' n“ fl... Pf" ‘
nucleic mat~-ia-. Ambrosini and b.etz (1; 0;) pre-

V" r ‘ C v < ‘c‘ N ' I" I“ n: '1 .. _ n n”. '
ja-ed entracts .rom washed E. 3-; 3y .ree zlng and

t

J-Y.,., :-~r~ --\ . ,.- '- 1114.1 .1 run 4" n \‘1
tnaJlnL, boilin nv, or by auucclavin; tna yielded

fl 0

7e factor for ne increased survival of

1-‘1

J. 4.
Cl. 7‘1“?) uCC u

‘

F 1 ‘2 ‘- 1* v v 'I .q‘ ,1 (1.11», 4‘1‘KH "r 1" 1
I... CC'-1..L u..C.e '..-l"€ -l"CZ€-.1 C...“ u;-C.'JeC1. Survival COT-

 

related with the c1n1o”"d~'te content; Iractionation
gave ar onalous results and Iorit treatment or dia-
rsis increased its activity. Rosznan (1963) pre-
sented evidence that extracts obtain d from frozen

’J'k.

'~ I? ‘wan - A1 . a" r -r7vn~ '|
and tnaved ;. coli stimulated 0. en upt he and

 

observed a decrease in acti'it y when the extract

alvzed or treated with Iorit. Lehler and

d

was di

Hartsell (1963) isol ated a crowth- tinulatinc sub-

 

stance, "?actor S", from frozen :. coli which
appeared to 3 non-specific in its activity. A loss

I W

0 . - ,. «1. - 4.- ,3 n a“..- - , r- ,. -,., . _
i1 Vluaillbj was accenpaniea by a lelease o. —v

:1 ”‘en Lirdeberg and Lode (1363)

loo-vac ~*--
2&4!“ " .2 -( ' D. '
-'.. 4.... 0011 4.4.1 Cw: t... éLCibwl sec;—

. *vwes. Po: t~ate and

O 4' ' J' V" ‘ J‘ ‘ ' 'r\ ‘
Edloef Co El 43 re Ce.u<;A;Cu

-.“er tel.“ (1:53) 8--

‘-o'1 ,: N" .0... _ y ,. ,. ' J.."_ , .-.. -. ,. \- , .,.x_ w
*~leC .J irecz_n an "7 i wnCI QlCCeo on a

‘5..-—

8...

._.c Q V‘ ‘0 J‘.‘r‘ __\ _ ms. __ . . .1"
.“lCil “4-13.2 U--L..'—l on r’ -JCOI‘ Oil '0 4.116 1336861108 O.L

1.1 x

4.--.) U: .0. ,_ -.. . _ 1-- 'P. i
UVLlc “Cte; ls in aga; as 513' SCed 3d Cacoss

--~ “vwris (1351) was not con riputh C to t e de‘

8...; A.» - L

or ase in numbers. The a dition of FeClq and Horit
3

to the rich recovery rc:di1m to resove toxic materials

J.

ficantlv different f on Chose

on “‘r‘rcl aedia '-.ith:ut Korit.

-..'.A‘...au§'u.h

l-IATEKLXLS ALID IETIiODS

Bacteria strains

Escherichia coli (ATCC #11303) obtained from the

 

Division of Laborato;“ies, Licnigan Department of
Hea 1th and Acre: actor aerogenes obtained fr

Kiss Lisa Ieu, Departnent of Licrobiology and Public
Health, Lichigan State 'niversity, were used through-

out this study.

Culture maintenance

 

all cu res of the test org “leis were main-
t"inrd oy se -;onthly transfer on tryptone glucose

at 4 C. Cells

{J
'"5
A
U
P“
"'3
0
O
V
U)
}._J
fl
:_,
(4"
U)
(D
I 3
i '1
(D
C‘"
0
Fl

(1

cells were reguired. Cultures were incuuated at 37 C
for 22-24 hours. "accine jars were inoculated or
spreaiinc 10 ml of a suspension of cells harvested

r slants over the szrfaoe of the medium.

All cells were ta en from stock cultures and trans-

ferred a least once before beinc used as an inoculum.

L,

£rerration f cell suspensions

 

 

J.

Cells were harveSUed from the a ar by "flooding"
witli sterile p1:"siological saline solution (0.85%

LaCl). Cells were pooled into sterile centrifuge

29

30

ed at room temserature at 22,000 x
for 10-15 minutes. Th liquid was decanted and he

he sterile physiological

cf-

cells resuspended in 10:1 of
l

P
;_,
(D
(D
O
H
C‘
CF
Ho
0
D
O

W
,5,
i.)
(n
' J

roce-dure was repeated three
times. After the final centrifuging, the pellet was

A ‘ ~ o "L 1 wnflfw V ‘. J. I." J— 1
washed sits a shall anount oi tne diluer t and re-

Asse vs 0; viable organ'sms in suspension before
and after testing were deterL;i ned by making 10-fold
iticn: in 0.85% saline solution. Tripli-
cate pour plates of each dilution Hre*e made and in-
cubated at 37 C for 43 hours before counting. Viable

rganisms will be uzed to designate those organisms

on the particular

(4-
R
(H?-
I)
t.)
O
[.J
(.4
O
[‘4
I.)-
(7
‘J
t I
F.)
m
0
0

lion

}.J.
('0

medium used. Pe rcentage survivors were determined
by using the allowing equation:

J # organisms after treatment
fl Survivors : .X 100

# organism before treatment

Ereparation g: extracts

 

 

2

Extracts were prepared by ireezinc a suspension
of organisms, storing at sub-zero te perature, and
thawing. Following tnawing, the suspension was

thoroughly miied by aspiration witn a pipette,

f"

U

w 9 0 ‘~-‘ ‘ ‘p h V ‘- (. '1’ V‘ n '.V f

a volume 0. 1.021 tahcn ior assay, ana the remainder
. 3‘ I‘- ‘ ‘7' I 1' 'r J‘ , A *‘.f‘s ‘l‘ ‘

centriia ea at 22,00 A r at room temperature for

25- 33 mil iutes. Extracts were ireed from ac eri by

stored in sterile, screw-cap tubes at 4 C except

‘ ‘ r- ’0 ' 1‘ V
wnen use| ior analysis.
Analvsis f e‘tracts
#— _

E: ra ts were analyzed for pentose concentration
by th- Bial reaction (Albaum and Umbreit, 1947) with
D-riéose as standard; DXA by 3 rton's (1956) modi-
fication of the diphcnylanine reaction with thymus

‘

- v ‘ N . , J- .‘l . ’ - .-
DMA as staniard. Protein concentration in tne el-

LLined “v the Lowry (1951) modifi—

(3

I:

3

'1
p:

3

tract
cation of tie Folin reaction with bovine serui
ald anin fraction IV as standard. Inorganic and

phate were determined

0
”d
F‘ J
r—J
O
(I)

seven-minute hydrolyzadl-
by the Fis «:e-Suoaarow_(1925) method with KH2P04
(1u mole/ml) as standard. mino acids we: e de-

tected using the modified ninhvdril rea

Zoffat and Lytle (1959) followilg ascendin

chronato ra.sh*. Ultraviolet absorption (U-V) spectra

A U A

O 'I

were ostained w; n a Becknan spectrophotometer,
model DU, with a 1.00m li
deUCfl‘flcb ons were rerfor ea ll matched tubes for

optical cells usin

K)!

‘A ,-, 7" A '4 ". 4' 4' -.~ ‘1 '0 ' g ‘ [- s'v‘ r, J“ .3 -’
,hotoueter. Concentrat_ohs oi reacti1g materials
0 ' ‘ J' "‘-. ‘ ~ ’ qr+' r 'K r‘ p‘. '2

were QQCGPHLECd 3y enurapol Mio1 iron standard

curves which

LY
n

1’16

an”;

n :1.
u--e.b '

‘\L—‘\—&

'“7

foll w

A 4-- -' a. .-
Spectrophotometrl

cehgeun

 

the following e

1

plotted using concentrations

4' ‘M‘ j 1 \ -‘\ v" 4' (N w
the beer- a” rt Law. auantitative
‘ 1 ‘7’ v ‘ ‘ w. -.. ~, ~
ana_ ses 5-1ere only a single

w 1")

present were determined

qu 3

ation

 

C : ......_....
1-!
J5.
where h' is the specific eatinction (absorption)
coefficient, D is the op "cal density of the un known
at the wavelength h', and c is the concentration
:urcssed in soles sen liter, and the light path is
1.0 cm. The eitinc tion coefficieL1t for ALP used in
his studv is 14,900 x'1cm“ at 257 mu.
Fractionstion g; eitraet
Fractionation of the extra t was performed
usinr the nethed for the azalysis of phosp fiiorylated
internediates ou lined by moreit, Burris and Stau ffer
(1959). The following procedure was used: to 10ml of
the cell extra 01 ained fro; concentrated sus-
pensions of froze en ells an equal volume of cold,
10S tri hloroacetic acid (TCA) was added, mixed,
and allowed to stand at 4 C for 30 minutes. The pre-
cipitate was re: cved by cen rifujlllg at 22,000 x g

J N

for 30 minutes at 4 C ar; th-- su ,e rna tant lic aid

A

..

decanted and saved for furth 1 fractionation. The

in 1H LaCH Ior °1ectr01n0tone ric and colorimetric

I‘f'l -
ana ysi 11 necessary.
Wxa ~~fi .hV I: . ‘
1-e superna ant lluh a was e: tracted twic ewitn

10 ml of ethyl ether in a separatory 1u1nel and the
aqueous yhase collected. Excess ether was removed

bv at n n a steam bath “nti 1 no odor of ether

r.
U V

:v. J
(T)

l -10
i-‘o

could be detected. This constituted the TCA-solt 1ble
fraction.

After the removal of TCA, a spectrum Las de-
ternined and the re“‘1nd1r of the ac; d-s oluble
fraction was further separated 33 the use of the
solubilities of the 1arim salts at pH 8.2. Con-
centrated sol tions 0: reahents were used to pre-
vent anoreciable increase in volume of the fractions.

The acid—soluble fraction was adjusted to pH 8.2

:4. 7" T' 1’ .. -- lb ... 1. ,5, . .1._ 4.x .0 _,,
w_tn on nCn and encess Jaridn added in the lord of

- ’.' 4. 1...- .., .-. ... J. 4. , 1, . .1. ., u
c“ s alline sariun acetate un 1 satucat ion wa

reached. The entract after the aa dition of barium
(at pH 8.2) was chilled at 4 C for 15 minutes and
tne precipitate renored 1y centrifugetion at

1-;

1:11;: as U12:- suf ernc tar t fluid ce stituted he b

Q)
"S
Ho

1133

The nrecinitate (oarium insoluble) was dissolved
in 13 E01 and the barium removed “1 m1 a slight excess
of 10K H2504. The recip iteted oarium sulfate was re-
moved by centrifugation. The supernatant liquid was
checked for the presence of barium by the addition
of a rep of 122504 and an absorption spect1°un of the
barium free lieiid was made.

The barium soluble fraction was treate with 10E

U A a . L .. A... .2 ,1 4.x. . .0 ..-_ 1
“2504 to renere tn oarlum as tie sul1ate union was

1

ehotometric a“a_;sis tne barium soluble fra tion

was checLed for tentere end :‘nosphate concentrations.
Blanks for hot; colorimetric and sgeetrophoto-
metric determinations were obtained 33 treat
volume of the Q1 uent used to p: epare the LtPQCt to

L‘.~~ 1w *3 '- V q,
w.e- eaie fraction:.t n groceeure.

~ I. P“ 1Qfi‘l“. .A.‘ n o ‘ C ‘L ‘
Tne lollowin solut1-ns Jere a ed 00 sasp—nd

)

‘Nffi"

the orfanisms for 1reezi 3: (1) 0.53 phos-“ate
buffer (12 ml h 5 IaqHPCA; 460 ml h/S Nam %P04; dis-
L L.
J

tilled water to 1000 ml) pH 5.3, (2) 15 3p glycerol,

(3) 0.2 K magnesium acetate, (4) 0.02 H uagnesium

35

acetate, (5) 0.002 M n".ne-1un acetate, (6) distilled
water, (7) 0. 2H sodiu m acetate, (8) 0.2 H citrate
buffer (2.101 g. citric acid monohydrate; so ml 3/1
nacn; make up to 50 ml and dilute with an eq1al

f distilled water) pH 5.0, (9) 0.2 E sodium

acetate, (10) 0.85fl sodium chloride solution.

Fr »n:-- (a: tch:.1
e'vU*-A-' L44-.'.~ A-‘.rd.‘. ~A’lf4

1 ‘NA- f\"‘l~: ‘ h v ‘, '0 ‘ 3 - 1‘ ‘ 4' Q
Ce1l scsbcns1ons s~re 1rozen in either sterile

D

iuje tubes. G

l...)
f‘ D
(O
03
rt
(0
U)
c+
d“
:1
D
(D
0
F6
*3
(D

a nted position during freezing to
avoid ireanc e. Free21n" and storase of suspensions

K/ V

were done in a freezer chest set a‘ a tempera ture 01

I
C')
fl
0
o
F 3
}
E}
(D
[.4
F )
«15
C)
(D
f h
{'1
'3
E51.
fl"
5‘
F}.
(0
w
0
O
(D
U)
U)
.‘j
C
C
1
(Y)
H
...)
('3
'1)
9.)
F.)
(‘4
<1,

8-12 hours. Thawinr was accomplished either by im-
itation in a 55 C water bath
(fast-thaw) or by standing at room tempere.ture or re-
frigerator (4 C) temperatur (s lo.I-th°v). Fast

3 required about 2-3 minutes and slow thawing
from 15-30 ain‘tes to sev ral hours depending upon

1: suspension. rollow as thawi

the volume 01 t V C,

the sustensions were mixed and viable cells de-

.1.

termined by the ireviouslj,r described method.

.- + ' J" t' - - ‘ L‘. 1. ,‘ - _ .
4P "a": A $1 ‘ - _, rt. .--.v, .v w- ‘v -. Wfi‘V'W/‘fifit
P1P Griz..- Lv U -LU-- ; O.- ._---C Cr" 1Q bl—K-Vil'-n-$1' ea-'-»e.|. -a-Livéb

A washed suspension of cells in saline solution

r diluted in 0.85;.LaCl solution. As many
1 of each dilution were

OcttGi into sterile 202150 as test tubes. Tubes

of each dilution were divided into two sets. One set

(- P . —‘ ’- ‘C‘ «. fa {-1 f‘ L a. 4- - ‘I ‘- J- ‘ n
was placed in a re1r1-e-acor at 4 C and the Ocner

set frozen at -87 C. After various time intervals
two tubes of each dilution were renoved from the

freezer. One set was "fast-thawed" and he other

set "slow-thawed" at room tezierature. Triplicate

A

satples from each dilution of the refliserated and

4)

frozen-thawed suspensions were assays for viable

cells using the previouslv described technique.

into two equal volumes and frozen at ~87 C. One
tube was .c—thswed and the oth- -r slow-thawed at

4 C. Cells «er aiied thoro ou;lily, a suitable volume

J

‘ ‘D '— \VV P-fi, th \ Q, ‘ vfi v , N, "
tahen ior assay, ug1 the entract removed by one

)-

1 -- 1 M ~ w-_ .0 1,, 4.1. 4. .H .
descr1bea method. Lhtracts ire“ been treatments

}.J

.o ution nd

{0

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H.
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(r)
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en aasorotion spectrum noted.

aner chre: :a‘: 0' rs A

H J. ,’

Q ’| s v-\-- ‘ ‘D!
.l'liaa- U'i 'xv‘oob 11d.11*-’€ifl 1 «L

1lter paper was employed
_. ‘LQ “‘0 ‘I _ a 1" r-1.‘ :1
1n the chic to; ra Jhic seas.-ati on 01 the amlflO ac1es

1n the cell e: :tract. Sheets 40 x 57 on w re used to

I - Q —. r.‘ J- n - . . ‘fi g .x._ 1- f“ .. n -‘ ‘ - .O *. A 0 _I H.
JILOJ S11ulcaneous enroiacovraghy 01 20 aaino eeles

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35

:y
d
a volu;

_‘.C‘
lilo

n...
u-‘
‘
L
lfuv ‘firn
Cw..1-JI“
'1‘
J.
1 .
*’

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cted
- ?
.L‘i

C.-
V
fil‘, A.
1.0; .L d. U
C
"s
./
-: ttfl‘jfi
— U‘-
.1

1r

H—n—v

 

det
(

A

I)

r "’ A '1 ‘5 ”'1: --'1 ,1 n . 4.
11 a N sa_-e:a1_c 11c 10-1 :cr 1 mlHUCC stained
2 1.. 3°1-r4-s "fi"’-‘ M
in C301.w e iriliiant clue w- JD 0.“ 1 _n ‘1s-
9 I
'2 ’ vv ‘L " “t. - C: v-~-. J-fi ‘ f... ’1
o1llel .aoe1), 101 J ninttes e1i1 1.11e1ene1ate

“laced in cellu-

Q

J."

‘J.

and ‘e“v”’o1ated in the a

O

,., 1. -7 . 4. 1 2n. ,
€1‘J.ul.‘.;"0 11110.14. 1.1116 V01 1.13116“

J. . .0 .D .1. .L . J.
SCTECLJ O1. 8. 11111 CL. POOL"! L16...

’1', 3

was reduced to 1/10 of the ori: inal volune. The
volune was measuied, brought to the oririn .1 volume

- L‘, I‘f‘r'q":4‘-. - 2‘ hi ‘I' r‘ I'. ’\ ”'1‘". " 1“" . -
oy UHC addition 01 0.05” Saline scluulon, and an

.‘ _' A.) ,_ H .4. . h. ‘3 1.1. - ,,_!. d
3.05.0.3? u..O.1 71-:CC Clad...- LG b63--1-'..11€ o

 

H,

Two 1:5 dilttions o the extract we; e made in

q

0. ES; saline ”11 ' on. Cne eilL tion wts heated in a

C0
0

F ’J
.—
k
(D

boiling water bath at 100 C for 5-20 minute
p31ecipitate was re “v d by centrifugation at
22,000 x g for 15 minutes. An absorption spectrum
of the su Jernatant fluid and t1e unheated dilution

v. r- c- vfifir’. .1
IIWQ AAcb-V. .

Cne-tenth Cram of ficrit (Pfanstiehl Chemical Co.,
Jauhegc n, 111.), autoclaved at 121 C, 15 lb./in2

pressure for 15 minutes an “ct vated by heating

to cherry-red ill a sunsen flame, was added to 10 ml
of the extsact. Cne- -tent11 ml of 95p ethvl alcohol

0 reduce su rface te11sion and the mixture

1:
{‘3
(0
f)
e.
(l)
{W
c}-

~tirrcd at 4 C for 18-24 hours.

‘ - ,.. J. _._
placed on a .CLJCUlC 5
mV ‘ ' 0 J. . v“ 1 ‘A ,1, ,. -3 -- D... " . - .4. _° - .,.‘ ‘-
1ne “orig was TCMOVEQ J3 grav1t3 11itration and tne

supernctant fluid saved for spectrophotometric
analysis.

The Jorit was washed on the filter paper three
tines with O. 85;.LaCl solL. ion. The adsorbed material
was eluted from the cqarc al with 25 ml of a solution
containing absolute ethyl alcohol-cone ntrated
ammonium hydroxide-distilled water (25:1:24 v/v)

An absorption spect um of the eluate was determined.

The eluate was evaporated to dryness at room

4- . \‘ L! -- P‘ +1‘ 'L - 4' ‘ 1 ‘ I N
te13erature and tne resuluant crystals resusucnded

in both 3 £01

J
n.
I?
5
f
O
{3,
O
P’
L)
K 1
O
*3
U
c+
*JO
0
:3

spectra were

plotted and the base ratios at 250x260 and 280:260

Van-ywrrqnt-g neon?
LAMA ..L' - *C L~~u¢k~ Kr

L‘

The presence of ribonucleic acid in tne cell

1 J-

extract was aeternined 5* tie method of Bingham
and Zittl e (1562. The assa;r nixtu1e contained
J

0.65 ml veronal-acetate buffer (pH 7.5), 0.25 ml

.4 L

1N 3ea REA or 0.25 ml of cell extract, and 0.1 ml

of bovine pancreatic riocl iuclease. Increase in

H I
7‘" Vu*v“'~- -.-*V~
.70th lolot-_.-5

 

Cell s‘-‘:'_tc;:"ior.s,

dESCPlDCd manner, were

" 1' c on 7
ULDGH 64-»;

c ntrifuge

A
\—

in a

.1

tested for

exposed

I " f" “‘ . 'n
as C. 4353.29 .96 0.1.

..‘,..- 1 -- .L' a-” s!
prepJPCQ 13 one firevlously

olaced in sterile plastic

A.

DC sonic vibratio.s

Asvtheon conic Oscillator, Kodel 250, for 30

urn o
'0 V" H

removed by centrifu-

2upernatant fluid

fzesence of DEA.

Occurrence 2: Cell Extra cts

 

 

Effects g_ freezin . and tlicwin C

When suspensions of: :icroorge.nisms are frozen
and thawed, diffs: ent numbers of survivors e “e oo-
tained. The d-ath of the microor - a1mis s is accom-
r the degradation an; release of cell con—
medium. These products
consist of pentose, a product that ass oros ultra—
violet light, and protein-like material. Analysis
of the U- V :1a terial showed that maz-zimum aos o; -ption
at ph 7.0 was at 2:0 mu with base ratios at 2503260
and 2803260 of 0.90 and 0.50 respectively (Figure 1).
K0 deoxypentose could be detected, and paper chroma-
tography showed no free amino acids; ninhydrin-posi-
tive materials thought to be low molecular weight
peptides were detected-

Suspensions of coli were ruptured by sonic

m1

 

vibration and the extracts tested for the presence
ofc deoxypento: e. Positive reactions were obtained
for six trials. The abs nce of deoxypen tose in the
extract obtained from frozen cells would discount
the possibility that death was due to rupturing of
the cell membrane and is inco1pat ible with a theory

of death by crushi g.

41

Optical Density

2.

0.5

42

21.0 2 50 260 270 280 290
- Wavelength (In)

Fig. 1. Absorption spectrum of cell extract obtained from
frozen suspensions of Escherichia coli (1: 5 dilution)

43

gelations in between recovery rate and concentration

 

Suspensions of E; coli were subjected to freez ing

 

and slow thawing in saline solution to determine the
percentage of survivors and the concentration of
leakage products. Cne series was analyzed for protein
and pentose and anoth series for absorption at

260 mu. The results are given in Table 1.

ihese results indicate that frozen cells lose
reater rate than chilled (4 C) sus-
nensions and tha the concentration of leakage pro-
ducts obtained fron the frozen cells is greater

than that frcn chilled suzpens ions. From this, it
can be concluded that a relationship exists between
the percentage of survivors and the concentration

of the leahase nreaucts in the suspending medium.

The leakage of cell constituents which occurred

as a reenlt of freezing suggested that a permeability
control mechani: a was affected by freezing. Such a

mechanism would presumably be located in the cvto-
plas.i membrane.

The eifects of t“e nature and concentration of
various menstrua on the survival of E, coli in frozen
suspension a1'1e shown in Figure 2. These data indicate

that :onovalent ions are more lethal than divalent ions.

44

$5.55 was 95300.3 on 3 @3333 one 930: w. you
c 4 as v0.3.3 and .3353 can: 5. consensus on: noveofixfi a.

 

 

 

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I mmnd o.m cow 9..
um: I I mm: m
8.. u .. 8:.” a.
3.06 I I mn.m o
I m.. “.m. o.m m
I m.4 m.cm 00.. a
I a...» 3 R.— n
I «a “Sm 96.. u
I >3 an 9.... . .
A8353 n a: 35533 3.5%"; E2556 papaya
3- Bw ago 5398 838m m it...

 

.952 ~— sou o 4 a. 335 :3. s: 982. e
you c bmc vs .3332. 3.3: 5 «.H..o..o «Evasonoum wan—":96 nevus
$260.5 emexeoa uo cowagaceocoo use 9333.9» no omscoouoa one .. canes

 

45

(I) 0.91 phosphate buffer, pH 5.3
(2; 15% glycerol
(3 0.3! nagnesiu acetate
(1.) 0.024 magnesium acetate
(5 0.0%! nagnesitn acetate
(6 distilled water
(7 0.024 sedit- acetate
(8) 0.3! citrate buffer, #1 5.0
(9) 0.31 eodiun acetate
(IO) 0.851 sodiu- chloride solution

im

    

75

1 Recovery

25

IIIIHHIHIHHHIIHIIIHIIIII

11th and concentration

1'13. 2. Recovery of Escherichia £911 subjected to
freezing in various nenstrua at -87 9 for 1. hours
and slow thawing at l. C for 12 hours.

45

A c 3;:risen of tie two different buffer solutions
;tes the greater effect of the
buffer ion than the pH itself.

Because earlier data (Figure 1) showed that a

relationshi. existed between the proportion of sur-

vivors and the concentration of leaks ge products and
that the natu re 0. the suspendinz medium also was

involved, studies were conducted to determine whether
any relationship existed between survival and concen-
tration in these various menstrua. Horeover, since a

regulatory aechanism seems to be involved and de—

gradation of nuclei acids occurs, the various sus-

pending :enstrua wer- analyzed for the concentration

C:

of pentose. The s”'nrnain" licui s in which high

percentases of s1r1i1.ors were obta“ ined

\r‘

would be
expected to snow low concentrations of pentose,
and vice versa. The results are shown in Figure 3.

The data presented here (Figures 2 and 3) for
091 frozen at -87 C in a variety of
renstrza strongly suggests a relationship between
death and KIA degradation. While the cells that
were suspended in magnesizn solution did not show
the rreatest percentage of survivors, the concen—
tration of pentese was low. hagnesium , which is

Encwn to stabilize Hol ted riboso1x1es, decreased

the amount of pentose; whereas, Citrate, which

Concentration (ups/I1)

‘8

8

8

 

47

0.9! phosphate buffer, xi! 5.3
151 glycerol

0.2! magneuul acetate

0.024 magnesiu- acetate

0.0M! Iagnesiua acetate
distilled rater

0.03! sodium acetate

0.24 citrate buffer, ii! 5.0
(9) 0.21 sodima acetate

0.851 sodiu- chloride solution

AAAAAAAA

O Q Own «P U N ..
VVVVVVVV

(t0)

OOOOOO

1" MINI

la 5 6 ‘7 8 9 '0
Henstruu and concentration

Fig. 3. Concentration of pentose in extract obtained
free scherichia coli subjected to freezing
in various Ion-true at -87 c for 1. hours and
slow thawing at I. 0 for 12 hours.

‘ 1 r e- "h 'r' ‘I. '. rr".: vv‘. fi‘.“l'\ ‘ r v I' ‘
acce-erates rioeeemal oceanaown (undo, 195?; Jade,
611’) 4“'\C f“- fl".
, l-C EQSGCL LI:.'.\/ unhoemibo
Q ~- . 1» A *~‘L" 1" .1 . ‘-‘,-\
rh,sic e ical saline so Helen ,rovided one

recovei; in viable nunbers1while distilled

H
(T)
f)
('1
(F

.4
f)
('f'
O
'3

.. .4. .l: . .0 14 . +.L -
and dilute solutions oi sodium acetate pro-

: f1 3 ‘A 1 V r i f: 1 r“ J' _ In _‘/1" q
Vine; 0;...v.’ .-.C\~ ‘Duue TCCO‘.C- 4.93 Jecause T}

saline solution (0.85; KaCl) produced the highest
concentration of leaha:e p;odacts, it was selected

x'l‘ L'-—.‘ it «(3‘ 0'.“ I, r-fi‘u'.\ ‘ r. e-
-“ this stud; whe-e maximum yield of extracts was

Effects f t aw’ns prcce: U.re on recovery
Since the cell suspensions were subjected to a

ra id freeze and slow thaw, the concentration change

q

in she solute would be rapid and coma eq uentl" would

n

ects. lhis would suggest, then,

manifest no adverse ef
that the Hoce; 3 could exert detrimental

‘
p

t
effects. A slow thaw would subject the cells to
longer exposure to the concentrated solute than

.0

would a rabid thaw. Susjezsions oi bacteria from

he sane pOpulation were frozen at the sage te: perci
ture; duplicate samples were removed at various
intervals of time and subjected to a rapid and a
slow thaw. Cell susoensions from the sane population
stored at 4 C were used as conti Hol . The effects of

‘

the storage time and tne type of thawing are given

49

t-

id Figure 4.

{'1‘
4L--

see data inaicate that no n st drastic re-
duction in numbers occurs durizg the early stages
:18 reduction is
due to the decreas— in temp-rature and the period
of time dur‘n" whi h the organisns ar ezposed to
the cr’t‘cal teggeratures in the range of -10 to
Hhi e th actual freezing process apnears to

‘. o wan-'1“ 5 ~ 4-: ‘x " " .3 ‘ . ‘
oe r sponsio- lor tne decrease in surViVOFS: tne

thaving nrecess can exert effects as well. These

D-J

4—“
51--

t increased

p

“ Q “ Q‘s -—~ ‘- ‘s - .2 .
results Jeald sag,ort tne notio;
--\ -1 fl ‘ - .C‘ y -. l
concentration oi solute influ uences surVival, as
.\ —~. _{‘\_H L -- --- n ‘O W n “‘I" ~—- 1‘ ‘ A.
tnese eliects are earneelj manilest eurin

thaw; n; nrocess.

r-r on 4. -:1 L‘ ,,.-. H ., ,. 1
inc eliects oi bgCJlAV ar- also sh w; in tie

q

oi ff erences in concentration of lea

; J

:age products
resulting from thes rrocedures. Cell extract was

D 4' -! .4: 'n 'w‘ P. ‘5“ ~ .' a -' tlx ‘- f1. -. -
ostainea iron a auSJCMSLOH Ol cells at was civided

procedures, one voluze of cell suspension was fast-
thawed and the other slow—thawed. SlOJ thawing re-
:ireda or0“‘mately 30-45 minutes at room tem-
oerature. The cells were removed by cen trifugation,
the extract diluted 1:5 with sterile 0.85fi saline

solution and an absorption spectrum obtained. Th

50

h

A355 8:.

Ell

3,2,1.on .ll.
255$»: Ylo

H9350 I
voaouowahuom

J.

 

 

1

 

 

 

(0601) 040;qu

O—

results, shown in Figure 5, indicate that the in-

creased reduction in number, a effected by th

(0

thazing axocess, is ccooanicn by an i ncreas in

.. .. ,_ .o . .0 4.x ,fi-h ,,. . w J. m‘.‘ 1
COncehtraticn oi the leahage phoaucts. inis, t.:en,

e is a result of

tration of the solu te C”Tl”" the freezin
but the effects ar 3 re pronounced during that-rimL
These data demonstrate that the loss in viable
nunoers duri ’3 freezins is a consequence of the
nature of the sus
loss of numbers 2 ootained during he early s

0 ‘L‘- .-

o- the freezi;1g piocess, resumaoly during the period

in "hich the orrrnisrs are exposed to the critical

* V“ .f‘
we ,

ratures iron -10 to ~30 C; that the rate or

('5

J- g ', .. A. n.- #1 ‘~ yo -'- fi ‘, _
UgCW-Lp inilaences survival. Tnis decrease

F4)

tvne o

in viable numbers is accompanied by the release of

cell constituents into the me mi”: and he concen-

()4

tration of these leakage products is in direct pro-
portion to the numaer of survivors. The leakage of
the cell constituents is apoarently due to the in-

A

crease; te1Tm‘ility of the cy W0 lasnic membrane

solute durin; freezing.

2.0

Optics]. Density

 

52
H S low-thawed

0—0 Fest-thawed

A

250 0 O O
Henlength (In)

Fig. 5. Absorption sprctre of cell extrects obtained fro-
frosen suspensions of Escherichia 5911 subjected
to fest- end slow-thawing procedures (I: 5 dilution)

53

Characterize tion 9: Cell Extract
revious worh by other invesoigators showed
that leahar Izoducts exhio ited at least two pro-
perties: when added to suspeisions of microorganisms
before stress, it exhibited protective properties
when added to suspensions of microorxanisns after
stress, t exhibited restorative properties. These
Droeerties were determined by ir creased responses of
he cells in suspension. Treatmeit of the extract by
various agents altered these properties but the nature
of the active component was not ascertained.
Physical- -chemical methods were used in thi
study to characterize tie extract and correlate these

data with metabolic responses of the cell suspensions

Effect f pervaporation on the absorption §P€Ctrum

 

Ten milliliters of cell extract, obtained from
freezing and slow thavi;s a concentrated suspension
of E. ggli, were pervaeoratedz ccordin; to the pre-
viou sly described method. A 1:10 dilution of the
same extract was nude with physiological saline
solution and the absorption spectra of both made.

Figure 6 sho~ a cospsrison of both spectra. From

.-
‘-1

this comparison, the ace er es of an absorpt on peak

in the region from EO—QoO mu can be seen in the

Optical Density

54

«3—0 Diluted extract

Pervaporated
""" extract

250 250 260 o is u 290
Wavelength (In)

Fig. 6. A oouparison of absorption spectra of diluted and
pemporated samples of cell extract obtained from

frosen suspensions of Escherichia coli.

1.. - ,4. 1 -L. 4.
curve for one perv-,oratee eioaaot.
7-— e
nffect of ne"tins :n t e adsorption spectrum of

cell eit‘

 

 

.,_4.
lat;

 

A
sterile
viou-‘r

“"J

heated)

 

3v
norit ac

After re

of this
thi s plo

the regi

'—

:; dilution of cell extract was plac d in‘
20x150 mm test tubes and heated in the pre-
described manner. A similar dilution (un-

e.

‘ as a control. An absorption spectrum

‘

acte

was plotted and the results given in

. From inspection of the plots, the pre-
an absorption Jean in the region from

nu can be seen in both the heated and un-

‘

cenparin: the curves for both extracts, it
resin: interest to note that the only region

wed any decrease in absorption after heatinr

La

 

novel of the charcoal, the absorption

. '1 . .0 ., ”i.” . J. '1 . "3.2 ,.. . ,.
analfs_s are presentea in :i;ure o. From

t, the absence of the absorption peak in

" “C. A” W.QQ n~ " ',
on ffOL apo-a; as can be seen.

Optical Density

 

56

o-—o Unheated extract

o—o Heated extract

 

Wavelength (In) ‘

Fig. 7. A comparison of absorption spectra of heated and
unheated samples of cell extract obtained from
frozen suspensions of Escherichia 9911.

I

Optical Density

0.5

0.5

0.3

0.2

0.1

 

57

240 250 270 290
Wavelength (mu)

Fig. 8. Absorption spectrum of cell extract obtained from
frozen suspensions of Escherichia coli after
treatment with and removal of Norit.

58

c ..
L".,J—1 fi“ .5 '— nfi \fi-firf r1 ~-r~¢Lrgfi: p1 fi‘flcta Ori t
‘4..- L‘. Ufib‘-- .L Lo My & v L \L ...w- JV‘ .g.»--_'.. .- - --. “ _._

 

The Iorit removed after treating the cell ex-

tract was washed with a mixture con ainins absolute

ethyl alcohol- concentrated annoniuri hydroxid e-

l.‘

.led water 25: :2' v/v). Ho effort was made

‘5

3 J.
QiSu

4. 4L

"' F" .r \
U0 heep t-.e volume of the eluit r

C ‘ ' '.
the volume of the extract that was treated. The ab-

L

‘ ~— A .‘ 'o‘ I‘ ~\(‘ h u.
sorption suec run oi the elutea haterial (gig‘ee 9)

shows the characterist- c abs oration peah in

0

.. acre our. '-,. a M' - .
h _,o-i,0 mu. TAG absence of tLlS peak

res; n fr
V
‘3‘ J“~C'. " ‘0 .‘r‘q ~-J‘“-'V t “5. ~ VA A rm" 3: ‘LO 0“ n
.Li'l Una “Quorruu e..-U.' C~C Fl; sie u stain .sbo ”exace—
w- V 1‘ v-- ‘5 ‘- -. ' 3‘ {‘4’ J"-r\J‘ 4‘.
quen t TGCOvCT’ iron the aorit indicates that unis
erial was removed.

The eluate containin> the ultraviolet absorbing

,-
\1

A‘L ‘3 r- 9 - L q VV 3
material Was.evaporated to or,ness vi

crystalline: aterial was dissolved in 0.1: 501,

pH 1.0 and in 0. 013 ha H, pH 12. Volumes of naterial
Equ unt l the absorption at 300 mu was
about the sa e. An absorpti n spectrum for each
solution was obtained and nresehted in Figure 10.
Laximun absoratioa in acid sol; tion was at 257 mu
and in oasic solution at 260 mu. The base ratios

4'"

~ .1 p r r? ’ -.:- A" m . ,r-v—
oi aosoroance at 2303200 aha -o0z2o0 nu were 0.9;;

Optical Density

59

1.5

0.5

2A0 250 260 O 280 290
Revelength ( nu)

Fig. 9. Absorption spectra of nteriel adsorbed fro-
cell extract obtained fro- froeen suspensions
of Escherichia. coli after adsorption with end
elation from Norit.

Optical Density

60

' Spectrum in
H 0"“ 861’ w '00

1.5
o—o Spectrum in
0.0121 hon, pH 12
/ .
'0.
0.

2&0 250 260 270 280 290
Wavelength (nu)

Fig. 10. A comparison of absorption spectra of ultraviolet
absorbing material Norit—adsorbed from cell
extract obtained from frozen suspensions of
Escherichia coli and suspended in acid and basic
solutions.

.~ ’2 f, 4.; l s. s." '1”: 0 3 s “Cr- .' d
3.11» OQL'Z'Y I'eSfiLC U-LV'e LT $.11 C~C_‘-\l ._)O.L.L'. U1 :1 Col. ‘ OQOUD 89;;

’
~

. V, C _ .L g V' '1 ' e - ‘ e ‘ fi ‘ 3‘ 1' _!
0.eco FCSLCCU_3C;J in )asic solution. Aosorntion
1%r-v-'1 'efi n‘fl'1 -fififi fi+£y fl yy fl AV QHQI-hlq .5 q 44-”,\ + N (5
“.w‘.._- A” Cn--\~ hat-five rL‘- U‘Ou .401 e \JJA AA-) CO VIJ. U$L ULLCL'Je

-‘

. derivatives pre-

'Je
(‘

given in a chart or nucleic ac

J- I

'1 ’ . _" _“ .0
nsrea 33 {1.3100 en Coi‘poratien, Los Angeles, Cali-
9 ‘ ‘ . I. m“ . h ' v 2 sh ' r‘ we: V ‘U 3 ‘A r‘ . I“ 0' 2“ ~\ h ‘
iornia. LMC assorttion malinun in acid and sage

2“ . 1 ‘ ' 3"- #:“l‘ fi-e". .Af-fi-‘e I‘. "r‘ ‘I’ . f‘
coincided ”lb” close presence! ior nucleOSiaes and

nueieotid-s of the purine adenine. However, no

. ‘.. " \J-- - .._ "
s seaween the gas ratios oatainee

" --. N -.. .-- m ~ .1- -. -. ~ 1.. r. .3
lo; t.€ czjetelline gas :12 and those p.’ es er tea

fl - V." A 3-..: ,-- 1 --~- Q
IO.1 3... C‘Ji’l’la CO.-oE3....-l..'.I1. {2064321103.

—~«_ ’ f“ r “" ‘- O
a-€‘ Queue $L U...C

of 0:260

k7]

tIe extract at 2

U)

and 2C0:2o 2 were 0.90 8rd 0.5 respectively and
are in accord with those presented by other inres—
ti ators for their extracts. “0 base ratios for
eitracts obtained from frozen suspensions of

Aerooacter aerOfenes were available or presented.

M ‘ 1- w- ~ °-\ v n : ~-+ n ‘- ‘r l“
lo ~etermine ties values an elt°act was prepared

0
O
: .
O
(U
I
(F
*‘5
P)
c!-
(T)
Q:
I”)
6
'0
( ‘.
:J
I",
.Je
0
f5
['1
O
I
p-
0
*3
O
J
T
O
C
O
‘3

 

1.

aeroeenes. The absorption soectrum for this extract
is given in Figure 11. taxiaum absorption occurred

at 260 mu and base ratios at 250:260 and 280:260 mu

were 0.94 an‘ 0.64 respectivel". agreement exists

:L‘- ‘ V 4": r 1. e v'-" n . ‘ \ .‘
onlv with tne aasorntlon DCleuL; discrepanCies ll

va u—s for base ratios 1 d‘it be

AO;L’--

f)

ttributed to the

assorntion of other leataee products or else to the

d \—V

Optical Density

0.9

0.8

0.7

0.6

0.5

O.‘

0.3

0.2

0.1

622

 

21.0 250 260 270 280 290”
‘Havelength (In)
Fig. :1. Absorption spectrum of cell extract obtained

from frozen suspensions of.Aerobacter aeggggnes
(1:5 dilution)

63

tnat the ultraviolet absorbing r‘teiial in

H,
go
0
Ct

.L‘ .- ,J.. ‘ L _ ‘. .flr- - “4.
0---: CLuI‘CCu -3 C._.L.'.C.."C.'-u.

’3 01\A (‘4‘: ‘ Vrafl- I.“ “N 1‘ 1“ :-
peivaporation s »,ests a low mOlCCulur weight com-
\ 9~ .r‘ 4-. n 9v w a: ’7 n e -r~‘4- ‘. r- -' fi-x v
pound unit would disceane tne pOuSLuilitJ of it

‘ .-_ ~—_-v
361-; ILL.A.

‘1‘
K4

(— i ‘4‘ 1“- -' +5 A ~r+ f‘
Fractionttion oi cell extract

 

Since the act vity of t -e cell extract appears
to be related with the ultraviolet absorbing material,
acti nat:.on was perforaed using the procedure out-

lined in Xanenetric Techniques. Four fractions were

0
U
C‘.
3
He
:3
I“
{L
P)
X J
i
(D
(1
L)
(n
O
*‘3
O
C".
F“
O
L)
*C;
(D
O
C."
E
O
Pb
(D
93
0
LS‘
5.13
*S
{3
O
C"
F‘
O
:3

The presence of he cha acteris tic peak ab-
sorption in the region of 250-270 mu in the TOA-
soluble fraction gives added evidence that the U-V
absorbing material is not REA.

The presence of polysacharide material in
the extract may exhibit colloidal properties and
prevent the precipitation of REA upon tr e addition
f TCA. If tni were the case, then it would seem
likely hat the addition of barium would result in
its precipitation. The absence of any absorption
peak in the barium-insoluole Ira ction a;mpe .rs un-

likely that REA in any form is present.

0ptice1.Density

64

h... TCA soluble
TCA insoluble
O—I.
n Bariul
soluble
o—q Barium
insoluble
1.5 -
1.0
/ .\
0.5 \

2‘0 250 2-0 ' 0 2:0 2°.
‘wewelength (nu)
Fig. 12. Absorption spectra of various fractions resulting

from the fractionation of cell extract obtained
from frozen suspensions of Escherichia coli

65

The U- ‘ material is present in the barium-
soluble fraction. haxinum absorption occurs at 257 mu
with ratios at 250:250 an 2803260 mu at 0.91 and
0.36 respectively. Since the material was in an
acid solution, these ratios most nearly approximate
those for adenosine noncnuosohate (AHP). Colori-
metric analysis for jentose and inor"anic phos-
phate gave mola r retios of 0.04:0.033 micronoles/ml

which is approximately 1:1. There was no in icrea as

c F

in the concentration of the phosphate af er acid

J
)

1ydrolysis '“ich indicates a nonopimo plate. A re-
lationship between color development in the orcinol
test and the gosition of the phosphate linkage has
been noted. This method can be used for differentia-
ting between a 5"linha5e and a3' linkage. A 5'

inha: rives maximum color development in a shorter

9.
period of time than the 3' linlza e. I-Iaximum color
developner t in the pent o: e determination mas a -
taine cin 15 minutes, which would indicate a 5'
linl :age of th e phosphate.

From the data presented in hano etric Techniques
the compound present in tne barium-soluble fraction
that absorbs ultraviolet light is AhP. On the as-

sunption tW1a this was the compound present in the
barium-soluble fraction a tcr iractiona tion, the

oncentration was estina ted from the spectrophoto-

66

metric data. From the optical densitv at the maximum
tion and the extinction coefficient (14,900
A on ), a value of 0.042 micronoles/ml was ob-
tain~d. This value coincided with the concentrations
of pentose and sho a:hate for this fraction and gives

a ratio of 1:1:1 for basezsugarzphosphate-- a model

 

Since 32$ also provi des a model from which a

ratio of 1:1:1 for bas :sugar3p

tCin ed, the ultraviolet absoroin materia was treated

fl

wi n bovine pancreatic rioonuclease. An increase in

‘1

C 3; .—. 1 ,, ,. s 1. J- / .
Optical censio; as 2c0 mu was used as a measur e;'1ent

n

O; ribonucleare activity. ho increase was detected
which would indicate that the ultraviolet absorbing

material is not Du .

“1 An ‘I A": '0 1 -v“' n 4'
Lioctro.horesis o- cell entraco

 

Present in the extract with th- ultraviolet

absorbing 1Ct-rial s a suostance that reacted with

A
- z.
{‘o

the Folin reagent. The subs ta nee was cons dered to
be protein, but the results of the tr-atment of the
cell extract wi h cold 10fl TCA (Figure 10) showed
ion of 280 mu

.N 1 -‘ Q, ‘. “A J_ .g 7 n _‘ L‘,‘
1’10 LCCI‘eCLSG in C.JSO-‘uo.LO.l 1:]. the 1‘6

which is characteristic for protein. Also, the

67

N I"

Q
J tun-.1

precioitate showed no : in this region when it

was resusponded aid the spectrum determined. After

J.

1e ultraviolet material from the extract

h

-. ‘ I - -. ‘- \N I‘“ Q n .. '2 ‘ Q ,_ qy‘l‘ ‘-
w_th “Ofit, no teal Jas oataiaed at 200 mu. thr&CbS

 

. .1

'2 P-«w ‘Q __-.__-‘ “ " Qv-fi ’ 1 1 ' ‘ V m1 "
i.- anj aiiierences e.._suoa bet.-rcen the-i. iae “esults

igures 13a and 13b.

C}-

O

C)
I

Kore bands could be detected in the extrac

“ .‘ ‘- f" v‘ ‘l A‘ V r‘ 4' --~ q .r. -\ L J- . ~' -. sp-o O
tainea iron esczacoer GHQ tne rsteers diiiered also.

 

‘5‘ 3

Tnsse diljerences could Jessialy account for the *is-

.-,. .- 0, .0... '. . n ..-. - i . O .
creuahCles n ted in the ratios at a3‘3260 and 200:260

-. ‘- J- v—, 1" ' ' .3 -. -. 3 . J" ,
9-;0 SCU...C .D;- L) U ~.'1“-. ”01"--101‘. ‘10:;1“ .LlLL‘J. C; U8 ‘1'...“ U

.v m ~q- H ‘. . 1‘ ‘ r‘ _o ‘q
hatciial does a t seen to be tne active snsstance.

68

 

Fig. t3s. Electrophoretogren of cell extract obteined
fro. frozen suspensions of Escherichia coli

 

 

 

Fig. 131:. Electrophoretogre- of cell extract obtained
from frozen suspensions of ‘erobscter eerogene .

DISCUSSICK

He
I ’J
U)

F'I‘. \ s . - 4n, . J- u m .. 0 H.--
J..‘-C in r: that resul s we llCl” oorcan
.. .0- ,_, , s. . a . in: 2.0 J. 1 .
are lfCuC“ and snag d is haniiesoed by losses in

. w . ., a 1 , w- s all
viaole nu 363 and the leanape oi intracel 1‘ ar con—

stituents. Such results can se ettribut ble to two

ca“ses: lvsis or ru turinr of tfle cell wall, which

in the cell :egbrane, the structure responsible for

hi, --+r—-’~--\..-,. L‘a- 4' ~15'4—1v ‘-
" 'lue " u--k, -1-u€ 3-9.} OJ. u

n

.l a. ‘ - P 1 D - "A... “5 . 0 eat . ' .
Direct osservation o- irosen cells under 011 inn fSlOfl-

and electron icrograghs of frozen and ‘nfirozen cells
(heyhnell, 1330) present indirect evidence elimi-

e I J‘ I-
y‘ r~_ " - y} P. 'Pfi' f: f: ‘ ’1 r‘ . .
ofiL-[—'.~A-\_’ l 1 Dig GS be: .LKI’C UCI‘.

ma ~ n .-. .r‘ L L.-.‘ C :2 q , ....L r. -.
-ie ass;ncs e- a r oeuCCbuDlG ceoiygohto e in

““4
L‘ 1 ,_.L- 4. ,..'\,‘, 4&3: 4. «L‘fl + "
the cell -itraco we la als ihai a. ciao lysis did
ens ‘- 11 ne-AJ- ‘ ‘ve- 'A’ .‘
not occur. Cells rapsurea 3, scale Vioration "ed

the fresence of tlis caroohydrate whic
outed to D? . If ‘ no on cells are lye-d or ruptured,
they too should liberate DIA. Ions was detected
in this studV. As a result, such results would refute
theory of death as a result of crushing.
frees in: asparently lie in some
alters tion of the cell meznor:.ne. The cell m mbrane
not or 1" prevents leakage of the cytpp asnic con-

.1.

stituents into tie environncnt b o is also concerned

69

70

with the transport of ;-utri e -1ts into tne cell. Any
es this scai-permeable
no crane by alterin~ its 3 ye ico- chemi al structure
will ause disorranization o- cellular function.
when its selective jroperties are lost, comionents
leak out o; the Call res ltinf in injury or death.
Evidence to suowort this view can so obtai11eed
iron the data pr--sented on freezing. A rel ati nsl ip
has been estaolishe‘ between the viable number of
and the loss of
internal diifus ib lc cellular constituents. This
feet is considered by hitchell and Hoyle (195’)

7
f

ferences int:

0
b

c int anal osmotic

‘.,‘F\ - VV' L 1 q' 1 4’ + "'3 4' '
sh,ula ve c-ertca tie is: b e ouno 0 stress aid
.3 .- .‘r‘ ... ,4. ~4. _._ .. J. a - 1 w .1. 1
;iven the LBQQUCQU CMOLflu oi recovery. he data oo-

f indicate the distilled water

These observ tions are also in conflict with those
recorded by Harrison (1956) and Cleicnt (1901) who

found is fll ed water a relatively innocuous en—

H)

or frees ing. Csnotic effects can also

c}-

vircnnen
oe eliminated on the basis of recovery after the
organisms were frozen in other menstrua. Higher

rolarit es 0

’le

1e solute should have pH‘ov ided the

‘ .‘A ~ ,‘ . 4- . 4‘ 5.1 u.‘ .- ‘ V
nignest eeatn rates ll osnetic 3“€SShP€ rere re-

t was noted only with sodium

Uhen the cells were susoended in the various
li:ible period of tine (ape oproxinately
5 minute 3) elapsed before freezing. This would elimi-

gossib ilitv OI incubation in the solute or

U

nate any
penetration of the agent. Under such conditions,
these results re inconpat ible with a view that any
protection provided during freezing requires (a)
penetration o; the agent and (b) osmotic dehydration.
Postgate and Hunter (1361) obtained equally e fective
protection from substances tiat did and did not pene-

,iJ

The va ious responses that were ob ained in this
study must be attributed to the effect that he par-
ticular solute has on the microorga ism.

node-ately higli recoveries were obtained with
s lycerol. mhe protective action of glycerol was de-
monstrated to be conestible with the results obtained
by oth r investigators. The protective action is
attributed to tne ability of the glycerol to stabi-
lize permeability.

The losses in number in mens+ rua that should
have provided protection nig'

process in breparin; the cells for suszension. In this

.

’dy, saline soluti ns were used. Hashing Wibfl
aline solution Aas been reported to desoro ma
e and Snon, 1964) which
increased sensitivity to t1- er: 1al stress. T
of magne sium reversed these effects.

”Al ’le the cells that were suspended in magnesium
olutions did not show the greatest percents e of sur-
vivors A, the conceAtration of pentose was low. The ac-

tivity of magnesium lies in its staoiliza tion of th

:gcgo;c and ribonucleo-protein (Lederberg, 1956;

*Jo

Neioull, 1:53). Xhen these ons are replaced or re-
moved by uonovalent cations, the ribonucleic particles
undergo a se“ies of dissociations-— first reversible,
then irreversible (Elson and Tal, 1959). This pro-
cess could occur either during the wasl ing process
or in the final suspension. The degree of dissociation
could account for the ability of some a: ents to in-
crease the recovery of survivors—- providing the
essential nutrients to aid recovery are present.

uch an effect on the staoility of the ribo-
some can not only be seen witA ma nesium, which de-
creased tne amount of pentose, out also with citrate,
which accelerates riaosonal brea1fi morn and increased

the amount of pentose. Similar effects with citrate

were repel ted oy Chao (19 57) and Wade (1961).

73

now an the extreme toxicity of odium be ex-
p ained? First of all, the cell monorane
fouid to be gerneable to the sodi un ion (hitchell

and Loyle, 1959). This would eXplain the breakdown
tein and the leakage of the
intracellular prolucts. A_s -n (19 So) obtained similar

results. The addition of sodium chloride to isolated

cule. Secondly, sodium is inoortant in water metabo-
lism of cells. It controls the movement of water
and sodium intake effects or controls hydration.
Replacement of lost electrol"tes dur'n3 the washing

procedure with saline solution leads to migration

into the cells which would produce profound alteration.

.‘J
H.
H
(D
c!-
(71
7
(x

hypotheses might explain the effects

on VlCDllit", they do not oxnlain why

-

1"

'__1

t

1")

fl
Law

arious

m

.1
O

ab e numbers when cells

1*

tnere should be reduction in v
arJ frozen on menoaanes in the absence of any sus-
nending medium. The cells were reported to behave
Aini arly to cell: frozen in a menstruum (31etz,1961)
in that there was a nutritiolal demand. However, no
data were available concernin: leaLa ~.

The ensnasis on the behavior of cells that have
been xno; ed to sue—zero ten: era In es has been on the
freezin3 process. Truly,it should, but he data pre-

sented indicates that thawing plays a role in this

74

loss in ;;u-..3ers and l alza3e. L13. ortuna tel;r there is

no way to deternine whether the thawing nrocess is in

('3
H.
H
D-
<:
i".
5::
I

essence tAe facgor r s nonsiole for tne los

(J
1'-
[J
(f-
.‘
'4
b
(D
x:
F'
f:
..
r"
O
O
Q)
ct-
H
0
f)
()
cf-
Q
0
C)
(1‘
U)
cf-
§
a
|_Jo
(1"
F)-

I

H

PJ-

:3
I

N" I‘ 1 v . 3A A w. 1‘ v' ~ J J.‘ .9. J- .1 v i .
Lartial_1 iAvAlv~u, GVCA i1 As is e1ij aecause

y

.f'i 4.:

. . . ... L .. :.. ‘.' t .L‘ , ,
creases eAe a sane 01 A_Ae durins3 wAicA tAe cells are
.-‘ ~~> ! . r0 ‘- ‘.\ I“. -. C ‘ - q ‘ ~ A" ’.

AurAA3 tAe ireenin3 process, b4“ meet ~raseAc
Q‘ 1". - "f‘ y ' R ‘q -. ~ *"- v
red sci A i1 nuAserA ei viagle cells occurs 1ithin

’3 "-"1‘ "‘- “'1 'T'fi" 31 PF‘.
LA! -m‘r'_ -A- “A. La; U6... QV

- 1".- ,. J. - ". ‘. .L .0 .,.
Alicaeion. in AA tiAe studg,
a ' ‘ ‘ _' fl! . '"b- '_' ‘3' -fi ‘1 r ~- -' ‘3

tAe cell sea; AAi ns JCPG iztnmn1zuni sampleA reaoved
_.,A +~n~r '- he: cc~n~rrs .w v I‘LCNA tire in“‘Nvfls
$0.. Uo-‘r‘.'*o-: LV-od ‘v-u»,\¢v" M; ‘10; Cat L) Ugh“ .1... ALIC (Na-.0.
AAe suspensions were 0.3 served durin: l eezin” and

,.. .."1 .A , , ‘1 -. 0.. . 4.:
samsies rer ed iAAeeiAee v upOA solidi; cation.

-1‘~’ A ~"““ 1' P- L! -* ‘. In ‘L“ .7 ‘ ’4'3'" *~ ‘4' 1‘
AAA poiAe Ire-A; WACA as eAe Aero—tiAe ior tne stuej.
m“

.Ae results showed AAat tAere was no decrease in

’ " .th -
Viaule nun

k)

*-- ‘ '0' \ fl .«rx 1 n
ers aA; AA- tJUC oi tAaw Aad no ac1ditional

O
r?‘
o

effe It ..ould s-eA, then, that the act1m process

.AAA'- .L‘- ~ ‘1 .-- . .4. av
fl ;.\.’Q UALC QC-LC Men: 113 C—:€:lb0 isle

*Jo
C

C‘I‘. 1e. gust/-1

O
p.
P.
O
(D
F 'J

lsrsest reducti n in nun ers occurred within 30

g:

Ainutes fter solidific at; on wi h no signiLican
reduction afterwards durin3 stora3e. It appears that
the reduction occurs during tAe time that the micro-
rganisms were engoeed to t ;e critical ten:_ erature
between -10 and —33 C. Why this ee““e”'eure range

Acal has not seen ascertained. In as much as

*Jo
(7
O
’3
[J-
(’9'

L ‘4 : .I“. ~ I‘“,\ '2 ’- ‘3 . 3 :2 J‘ :_ r‘ '1 ‘. .
w

.- w. l- * a «:1 i. i 4 . - .
enienc studied, and t;:t external ice nas alieady

formed, it would lot seen nlikely hat this is the

I‘ ‘ .J-agJ-QQ > 4' ~ -'-.-' .
eeiel onetltdents co comprise a

\’ “‘1 .‘ '__ o - ~ “- _"‘n 4‘ .3 V .. _o 9 .V 1 “1 ‘
conglen linense 0; material JHLCJ surelj rust nave a

n ,-. .- ,_ .fl - . 4. -.,. 4. - up .0 4.1- .‘
Ireezln_ goinc tint 1: lower then most oi tne sto-
o"'.-‘ r ‘c‘ r: Pa v ‘~‘—-~.~o-‘--n ~q
unculceo Lin...€»; (we: ScLS )ElLC 111:..1LL.LJUlL.uo

PW" ‘

lne iornetion of intracellular ice 7. uld explcin
*ne deterior of cell: frozen on memdrenes. Internal
freezin< culd ceuzc the alterations in cell mem-
branes since tlese would 30 in direct 001 act with
LC frozen mgteriel, whereas they would not in tn-
case of eiternel ice formation. The fornletion of
intracellular ice would not occur p n initial
freezing of the suspending liquid Nh1011 vould account
for the high proportion of survivors that were re-

covered Eur in: the iirst lornac ion of ice.

-I
n.

The data presented in this stu‘y on the effects
of freezing and the r en nses given by the cells in

susyension stron“lv su“fest hat the cause of the
wienonena is an increase in cell perm aoili ty. Sub-
stances that steailizc ~6P'C”blli‘J provided pro-
tection while those tLet did not crovi*ed a decree
in recovery and an increase in tn~ a cunt of pentose

released. Altlou'n there is tuis release of materie

H
O
f )
(7)
P)
(7
(‘3
OJ
L)
(I)
U)
C 3
‘L
O
I‘"
F*
b
H
’3'
’t
L
:3:-
f)
:5
Q:
{'1
C"
}-o
}_J
*4 H
3'
(T)
C 3
f)
H
L)

J.

The leakage material prese-t in the suspendir

ed: u; has been reported to possess protectiw and

restorative prepe rti»s . Protecti n by the extract ias
been denonstrated by Strec ;e a11d anon (1964) with in-

.L J.

creased resistance of cells that were exposed to
heatirg, and to a lesser xcent by Po wtvate and
Hunter ( :61) .ho obtained some jrotection f: on free-

J.‘

zin; wflen the extra t as added to tne sue

diun. Bretz and Ambrosini (1563) obtained a pro-

 

tective factor for the increased s*rvi val of E; coli
that were frozen and thEMZGd Survival correlated
the extract.
Restoration, in the form of stimulation of ac-
tivity, has been de: en. trated by DeLamater et. al.
(1956) with extract obtained by leaching cell
ghospnote buffer solution. The material stir ulated
01:":en uxta:e in tne presence of Cl ucos e. Sinilar
(

1963) who 0‘-

results were obtained by Roszman

*4
(I)

added to suspezsions f frozen and thaaed cel
Henler and “artsell (1963) isolated a growth stimu-

latin" substance fro; ;._coli that oney termed

"Factor 5." The sti ula inn activity of the extract
seen: to be related to tLe ultraviolet aosorbing

‘- ‘3‘ 3’, 4- 1- Q J- . o a
material aui spectropnctenetric analvses indicates

that tle presence of a tea; in the regi n from 250-

g

270 mu ccrrespon as with activity. The results ob—
tained in tLis study are in direct agreement "ith
zese obtained in this stud" by R szman. When the

cell extract is treated by the same technioue he

" fl

used, the presence or essence 0: absorption peaks

es in activity of

Tao character stics of tne U- Va osorbin: material
obtained in this Stud:r differ from those reported by

rd Hunter (1962) iso-

f‘;
C’-
O
*‘5
m
o
"d
O
(D
C’-
(7
9)
c!-
(D
f.)

arved Aerooacter aerosenes

'4
('3
c!-
(T)
Q.
(4
I
*1
f)
c P'
C)
’3
F”
{‘1
F.)
F
"S
P
x")
c+

 

w th an absorption peak near 260 mu. The compound

('1

was considered to be adenosire triplmo pr ate (ATP).
However, this material could not account for all of
the U-V adsorption. Str nge and Shon (19 64) ootained
a comp un‘ assor sing ra::inally at 255 mu from heated
Aerowacter aergjenes that posses.ed characteristics

of deaninated hypoxanthine. DeLsmater et. al. (1958)

leached a susst:.nce fro: suspensions of sacillus

 

" ”'. n4. 4. , _’ _~.\, g< * '
ph 7.0 case ratios at 23'3200 and 2o0:2c0 nu mere

3
0.89 and O. 50 respectively. Ko loss of -bsorption

{a

78

occurred upon dialysis and gyilol' is and cT:1roma-
ocrophy yielded the four :.ucleo tides present in
KIA. flourish and Xorr (1 957) oatain d extracts from

a -I

" al.- 11- Q A ‘n4' qr’.‘ -
emotical_j disrupted “sot osacter tluu u u strong

.‘ l h . . .n. , .,- . .. - . .0
A suzstlnce was dEb ected in lPOZEA suspCASions oi

E. 001i. ‘Dfir L122? 8:8.6570 61.26; 1405.8 (1963) t1-8.t slad ..¢a::imum

 

3

adsorption Lear 260 mu. The 3; se rtm tics o: the con-

/ -~- f‘. < \ 9 \‘N‘
pound at 250:_oO add 2oO:2cO mu were O.o9 add 0.49

1 ' K. .,.I r- '5 ‘1.
T;e Jase ratios at 250:2o0 and 380:260 01 tde

J-

L-V mote iol obtained from g. coli in this study

.1.

were 0. SO ord 0.50 respectivel". While these are in
agrecmcgt 11th tlcse once inc; by DeLamater for

1'1

3:c_i us mobstcritza and Lir -deberg for‘ oli,

up

 

 

4.1“... .~‘ - ' »~- "tn” ‘- n ‘. o. ,2 .o 4.
tier do act a ree mica tuose ootsincu ior noterial

“resent in extracts IFOL Aorooacter thut were used in

 

“ . § 1 ,'- 5" \‘H L v ‘A .1, ’- . ‘ ‘- \ r
t;-is stucy. A reeszielw seems to .Je on 5' witn tne Lani—
mum sasorotion wavelenctn.

‘ 4. '

H' v.a r- _ .2 _ .
AOSZJCH founl not dialysis re sulted ii a loo

[0

P1?

.0 1, J.'..." n .0 ,... 0 -o J.‘ .4. ,. .4.
Ci tne Solulhldtln. acti‘v 'iuy Oi @163 G.-.bl"c.Cu. 4.36

L

socctrophotonetric result: of 2crvs> rated extracts

'5

in this study sho”ed the essence c: any absorption
peak in th U-V region. These data are in oo‘osition
1..

with the results of DeLamater nhO reported no loss

7'."

the extract.

It ausears the n that the d- V material is not tne

sane. Since DeLamater obtained the four nucleotides

( "J
01

ext 111 23A upon L"drolysis, it

V

eems unlikely
that the product present in the extract obtained from
E. coli that absorbs U-V is RIA.

diitiozlal evidence ajafnst tne froduct being
33A is feund in the fractionation procedure. Io
U—V material was present in tLe TCA-insoluole frac-
tion. Althoujh LIA is insoluole in cold TCA,
log'f llen (1:33) resorted an acid-soluble material
absorsin; at 260 mu released from E; coli when peni—
cillin was added to growing cultures. However,
roduct was never identified.
Le characterization of the U-Va sorainc material
as adenOCine re :oph sphate (AR?) was made on the basis
of its solugility in oar i‘un and th ratio 01 the

39.363511

-ar:3nosuuase. The phospliat e concentration

-as determ ned oy a seven-minute hydrolvsis in HCl

U
which we Jld liberate the phosphate groups. .ny in—
crease during this hylrolysis would be attributed

to the pres enc~ Since no

( D
O
D- l)
p.
' _Jo
I
O
H
0
d
*‘5
H
I
J
.3”
O
'J
(‘3
d‘
(D
D)
C

e was concluded

(4‘-

increase was obtained, a monopnospne

as seine present.

(

Tne position of tne uh senate group on the
pentose nae seen sLown b0 affect the speed of color

develOpment in tr e orcinol reaction. Aloaum and

59:!

Unbreit (1347) used this as a method for aiiferenti-

3 - —.- ..
' and 3 linkages. Jith 3' linkages,

I

ating between 5
maxinun color developnent is attained after approxi—
rttely 1 hour a 100 C, whereas wit
maximum color devel paent is attained after 15 minutes.
Lo increase was evident upon continued neatin of the
tubes duriny this deternin cztion. Consequently, it was
concluC.ed that th nLospnate was linked to the pentose
in a 5' position. From the spectrophotometric data,
concentration of pen‘ose and phosphate, and the rate
of color development, t1-e corr ound was considered

to be adenosine nonsphosphate (ALP).

‘n otner data SUCfeSt that REA is not pre-
sent as the U-V ass orbin; material, it does provide a
model that fits a 131:1 “atio of base:su
phate. Ho ever, the results obtained wi
zyne ribonuclea se strongly indi ate tha
not present.

rm,

in material that is apparently considered to
be protein might be pep ide. ince no absorption
peaks were noticed in the region of 280 mu, which
is specific for proteins, it is possible that no
protein is present or el se tne protein lacks the
amino acids that ive the char elis tic a.3sorption

peak-- tyrosine and tryptOphane..The Folin reagent

81

is 10-20 times more sensitive then absorption at
2C0 nu which could so ount lor ne detection colori-

v-v- .- ‘-9 ‘fiA q.“ r‘ '1‘:
etri sally dd net of absorption.

vim, -. ~‘, - 1 —. j v-- ‘. _.4-
gnoretic psworn ostlinee -ron the entracts was al-

5.3
:3 I.
:3]

’. . 1 . ' ~,—~ ‘1 .J- ,A "_.
ways tne sale. lizis uld l-lCl._LCS.L.G tnat t--e mate

" f‘, 4' l‘ ‘V‘ f‘ l .1 .6 D n -. "A 4 A .~~ _ w“
tnat leans as a result Cl ireesin; comes from sene

cell structure that has a dc fin nite composition. If

‘ '1 r... n 4m. . .. a1- '1 a... ..
tne lealuge 0. use protein-line materi ai a...e from

1 -,.£‘ .'. .— .--¢- 1 J-‘ «A . ~ y. .
ihQiCCli:.inube sites, tnen an array of patterns

would have been obtained. The ribosome would be one

. ‘ 4... J... .2‘- 4. . " .,‘ .. 1A0. :4.
secs cell structure that would possess a delinitc

. 4L: ., ..': ‘ .1 4.‘ . , w,
cospotitlon “lion :lVCS furtner evidence tnat this

In L.-‘ 9-1" V‘ $‘V‘r‘ ’- v w
is u“: stru0nnx:iltolvcd.

From these data, it is concluded that the
fie ts cf freezin" are due to an increased per-
meability of the cell membran and the lea age of
material from the cell as a result of the dis-
sociation of some cell structure with a defin to

composition.

SUMHARX

Differen percentages of viable cells were ob-

tained when suspensions of hieroor anisms were frozen

C
at ~67 C for four hours and thawed. Survival depended
upon the nature of the suspen 113 menstruum. Cells
suspended in phosphate buffer solutions (0.53) sur-

vived better than cells suspended in 0.85% saline

A

solution.

Crushing as a result of ice formation was elimi-
nated as a factor since very few cells were lost upon
solidification of the menstruum. The most drastic re-

duction in numbers occurred after 1 hour of freezing
with no appreciable loss obtained during subsequent

The rate of thawing frozen suspensions contri-
buted to the decrease in viable numbers. A slow thaw
was more detrimental tha a fast thaw.

Death of the microorganisns was accompanied by
the release of cell constituents into the suspending
medium. These products consisted of a protein-like
substance, pentose, and a substance that absorbed
ultraviolet light. 30 deoxypentose could be detected
and paper chromatography showed no free amino acids.
Leakare of cell constituents indicated that a per-

\a

meability control mechanism was affected. A corre-

82

83

lation existed between the perc n age of survivors
and the concentration of tne leakage products in
the suspending medium..

Absorption spectra of extracts obtained from
frozen suspensions of microorganisms showed that the
ultraviolet absorbing material had a maximum absorp-
tion at 25 mu. Additional spectra of treated extracts
showed that the ultraviolet absorbing material was
heat-stable, dialyzable, could be adsorbed from
solution on Iorit, and could be eluted from tn- Eorit.
The reported activity of the cell extract appears to
be related with the ultraviolet absorbing material.

Fractionation of cell extracts yielded four
fractions and absorption spectra of these fractions
demonstrated that the ultraviolet absorbing material
was recovered in the barium soluble fraction. The
behavior of the ultraviolet absorbing material
during fractionation lead to the conclusion that

this material :as not REA. Later tests with ribo-

.54

nuclease confirmed this conclusion.
The ratio of the basezsugarzphosphate, obtained
by physical and chemical means, most nearly approxi-

1

.ated tnose for adenosine monophosphate. Tie relation-

2

ship between the rate of color development of the

pentose in the orcinol test and the Losition of the

phosphate linhare indicated that the phosphate was

k.

81+

linked to the pentose in the 5' position.
aiectrophor ”l of cell extracts obtained from

.e*orenes presented patterns

Lg

0)
*3

both E. coli and g.
that differed in both the n‘aber and position of

bands. However, the same pattern was obtained for
each extract in every instance intimatin

J-

- 4-. 1 up, bro 1 0 ° .. =5
-n the cell was aiiected by ireesin . Tn

U)

( .7

(Q'-
J.

protection afforded by magnesium salts and tne de-
tr Liienta l e fect of citrate and sodium paralleled

the behavior reported for isolate d ribosomes.

LITERATURE CITE

Albaum, 3.3., an .J. Umbreit. 1947. Differentiation
between ri ose- -3- phosphate and ribose-S-phos—
phate by means of the orcinol-pentose reaction.
J. Biol. C

hen. flg36c—3760

Ambros ini, R..A., and H.W. Bretz. 1963. Survival of
Esc;ericr is coli frozen in cell extracts.
Bacteriol. Proc., p.6.

 

Anderson, 3.3. 1951. Heat reactivation of ultra-
violet-inactivated bacteria. J. Bacteriol.
61:3o9-394.

Arpai, J. 1962. Lonlethal freezing injury to meta-
"n and motility of Pseudomonas fluorescens

 

 

 

0011:)“ q
and Esc‘weri hia coli. J. Appl. Bacteriol.
10:297-301.

Ber y, J. A. 19 33. Destruction and our ival of micro-
- or'; nisms in frozen pa a foods. J. Bacteriol.

Binrham, E.W., and C.A. Zittle. 1962. Ribonuclease
in bovine milk. Bioch m and Biophys. Res.
Comm. 1:408-413.

Bretz, 3.1. 1961. Death of Escherichia coli frozen
on cellopl ne and on “tibiane filters
Canad. J. Kicrooiol. 1.793— -798.

 

 

. ., and K.B. Base. 1960. Protective sub-
stances for frozen Esch ieri chia coli.
Bacteriol. Prec., p.3o.

Bretz, H.W., and 5.7. Hartsell. 1959. Quantitative
evalreti on of defrosted Escherichia coli.
Food 2 s. 24:369—374
Burton], K. 19 56. A study of the conditions and
echaniszas of the diohenvlamine reaction for
the colorimetric estimation of deoxyribonu—
cleic acid. Biochem. J. _g:315-323.

Califano L. 1952. Liberation d'acide nucleique par

les cellules bacteriennes sous 1' action de la
C-i&l€llI‘ o 3111].. o :{Orld Iltllo org; 0 _:19"540

85

86

. 1957. Dissociation of macroniolecular
onuoleopretein of veast. Arch. Biochem.

J

and Biophys. 19:426- 431.

Chao, F.C
fl

Clement, K.T. 1961. Effects of freezing, freeze-
drying, and storage in the freeze-dried and
fro? en state on 'iability of Escherichia coli
cells. Canad. J. Kicrooiol. 1399—106.

 

Cohen, 3. 1922. Disinfection studies. The effect of
temperature and hydrogen ion concentration upon
the viability of Bacterium coli and Bacterium

vb: osum in water. J. 3acteri ol. 1:183- 231.

{

Cook, A.K., and 3.A. Hills. 1958. The use of stored
uspensions of Escherichia coli l in the evalu-
Stion of bactericidal action. J. Appl. Bacteriol.
21:180-187.

Curran, 3.3., and F.R. Evans 1937. The importance of
enriczmer ts in the cultivation of bacterial
spores ,r viously exposed to lethal agencies.
J. Bacteriol. 35:179-189.

DeLaaater, E. D., K.L. Babcock, and G.R. Kazzanti.
1959. On the leakage of cellular material from
Bacillus moratorium. J. Bacteriol. 11:513-514.

Du30s, R.J. 19 37. Kechanism on the lysis of pneumo-
cocci by freezing and hawing, bile, and other
agents. J. Exptl. fled. §§:101-112.

Ebbutt, L.J.K. 1961. The relationship between activity
and cell-w all permeability in dried ba1:er' s
yeast. J. Gen. Iicro iol. _5:87-95.

Ellicker, P.R., and 3.0. Frazier. 1938. Influence
of time and temperature of incubation on heat
resistance of Esc -richia coli. J. Bacteriol.
$383-$383 .

Elson, D. 1958. Latent ribonuclease acti ivity in a
ribonucleoprotein. Biochim. Biophys. Acta

21:217-228.

Elson, D., and‘ n. Tal. 1959. Biochemical differences
in ribonucle protein. Biochim. Biophys. Acta

n-Cr)

2_6_:281-c.uo

87

\

Ehrley, F.3. 19'56. The pcrs istence (survival) of
microorganism s. III. In culture media.
Texas Kepts. Biol. Ked.ll&:114-203.

.E., F.S. Tlatcher, and K.F. K acQueen.

1. Studies on the irradiation of micro-
anisms in relation to food preservation.
I. The comparative sensitivities of specific
bacteria of public health significance.
Canad. J. Kicrobiol. 13199-205

The colori-

FisKe , C.K., and I. Subbarow. .
phorus. J. Biol.

1
metric determination of ph
Chemo §§:375—4000

Floodgate, G.D., and P.R. Kayes.1961. The pre-
servation of marine bacteria. J. Appl.
Bacteriol. g3:87-93.

Gale, E.F. 1943. Factors controlling the enzyme
activiti s of bacteria. Bacteriol. Rev.

1:139-1730

Garvie E. I. 1955a. The growth of Escherichia coli
, v :—
in buffer su as trate and distilled water.

Jo BaCteriOlo 6?:3% ‘3980

Garvie, E.I. 19550. The resistance of two strains

of B: cteriu:n coli type I to dr ying under at-
ospheric con-ditions. J. Appl. Bacteriol.
8:78 ~09.

At}

Gorrill, 5.5., and F.K. KcKeil. 1960. The effect of
cold diluent on the viable count of Pseudomonas
pyocyanea. J. Gen. Kicrobiol. ggz437—442.

 

Haines, 3.3. 1938. The effect of freezing on oacteria.
Proc. Roy. Soc. (London). 1243. 451— 463.

rrison, A.P. 1955. Survival of bacteria on repeated
freezing and tliawinc. J. Bacteriol.1_:711-715.

Harrison, A.P. 1956. Causes of death of bacteria in
frozen suspensions. Antonie Van Leeuwenhoek J.
Kicrobiol. Serol. gg;407-418.

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

88

r rrison, A.P., and R.E. Cerroni. 1956. Fallacy of
”cru hing death" in frozen bacterial suspensions.
Proc. Soc. Eiptl. Biol. Ked. 91. 577- 579.

Kartsell, S.E. 1959. Symposium on initiation of
bacterial growth.: Bac teriol. Rev. 23:250-253.

J

Kein.ets, F. 1953. neactivat ion of ultraviolet in-
activated Es heri hia coli by pyruvate.
Jo 3&CCEP101. fig 455- :7.

 

Q.

Keinmets, F., W.W. Taylor, and J.J. Lehman. 1954a.
The use of metabolites in the restoration of
viability of heat and chemically inactivated
Escherichia coli. J. Bacteriol. 6155-12.

Keinmets, F., W.W..Taylor, J.J. Lehman, and R. K.
Kat? n. 1954b. The study of factors which in-
fluence: metabolic reactivation of the ultra-
violet inactive ted Escherichia coli.

J. Bacteriol. _1:511-522.

ers1“.ey, A.D. 939a Factors limiting bacterial
growth. If. The age of the parent cul cure and
the rate of growth of trans plants of Escherichia
oli. J. Bacteriol. 21;285-299.

 

Ker s--ey , A. D. 191339 F
rro‘.th. VII. yes
of mczerichia c
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