Yonago Acta medica 1999;42:147–152

Survival of Bacteria at a Subfreezing Temperature (–1°C)
Yoshinori Tanaka, Gou Ishino, Takashi Matsuba, Hisao Takayama and Shigeru
Ishida*Department of Bacteriology, Faculty of Medicine, Tottori University, Yonago 683-0826 and*Yonago Public Health Center, Yonago 683-0802, Japan
Preservation of foodstuffs at temperatures around –1°C has attracted special interest
recently. We investigated whether bacteria contaminating foodstuffs, especially contaminating fish, were killed or survived at –1°C compared with 37°C. Survival rates ofEscherichia coli K12 and Staphylococcus aureus IFO12732 in nutrient broth at –1°C for 7
days were 52% and 31%, respectively. However, the survival rate of Vibrio parahaemolyticus in nutrient broth containing 3% NaCl at –1°C for 7days was only 0.03%. Whenthe bacteria were kept in a soy sauce solution containing alcohol and some seasonings(the soy sauce solution) at –1°C, survival rates of E. coli K12 and S. aureus IFO12732
after 2 days were 56% and 54%, respectively, but V. parahaemolyticus was completely
killed after 24 h at –1°C in the soy sauce solution. When E. coli K12 and O157 and V.
parahaemolyticus were incubated at –1°C in the soy sauce solution containing somepieces of raw fish (the improved soy sauce solution), 3 strains of the bacteria were notkilled. These results indicate that bacteria contaminating fish are not killed at –1°C
and that storage of fish at –1°C is not always effective in diminishing food poisoning.
Key words: Escherichia coli; Staphylococcus aureus; subfreezing point; survival rate; Vibrio haemolyticus

During distribution and storage, many foodcommodities need to be kept without loss offood quality. Food spoilage may be caused bybacterial growth and bacterial metabolites,
which lead to pH-changes and formation of
toxic compounds, off-odors and gas, and byoxidation of lipids and pigments, which lead toformation of compounds with undesirable
flavor and discoloration (Barnes, 1994; Huisin't Veld, 1996).
Many methods have been developed formaintaining the quality of foods, such as dehydration of foods, smoking of meats or fish,preservation with salt and cold storage. Thereare 2 main methods for cold storage of foods:
storage in the refrigerator and in the freezer. In
recent years, great interest has been taken in thestorage of foods and aging of processed foodstuffs at a subfreezing temperature (–1°C)(Yamane, 1986, 1987, 1996). For example, a flatfish which was kept for several hours at –1°C

began to swim in warm sea water (Yamane,
1996). In order to keep foods fresh for longer
times, foodstuffs such as viable fish, meats orvegetables, are increasingly stored at –1°C.
However, it is not clear whether microorganisms contaminating the foods are viable at –1°C
or not. In this study we investigated bacterial
survivals at –1°C, and the contaminating bacteria attached to the surface of the fish after
soaking them in the soy sauce, which is a Japanese original cooking addition.

Materials and Methods

Bacterial strains and preparation of bacterial suspension
Escherichia coli K12, Staphylococcus aureus
IFO12732 and a clinically isolated strain ofVibrio parahaemolyticus, all stored in our labo-

Abbreviation: CFU/mL, colony forming units per milliliter

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ratory at –85°C, were used in these experiments. Escherichia coli O157, which was an
isolate from a patient with hemolytic colitis in
Tottori Prefecture in 1996.
E. coli and S. aureus were separately transferred from a stored culture to a nutrient agar
slant, which consisted of 0.5% (w/v) beef extract (Bacto beef extract, Difco Lab., Detroit,
MI), 1% polypeptone (Nihon Seiyaku Co., Ltd.,Tokyo, Japan), 0.5% NaCl and 1.5% agar.
After incubation overnight at 37°C, a loopful ofbacteria was inoculated into fresh nutrient
broth, which consisted of 0.5% beef extract, 1%
polypeptone and 0.5% NaCl. The cultures wereincubated at 37°C overnight with shaking. Bacteria in the stationary phase were collected bycentrifugation at 3,000 revolutions/min for 10
min, resuspended in fresh nutrient broth andprepared at a concentration of approximately
108 organisms/mL. Bacterial suspensions intest tubes were put in ice-water before use.
When V. parahaemolyticus was used for theexperiment, the medium was nutrient brothcontaining 3% NaCl or nutrient agar containing
3% NaCl.

broth medium (final concentration of approximately 105 organisms/mL). The tubes wereplaced in an incubator with a refrigerating machine (Hitachi Incubator CR-14C, Tokyo), andthe bacteria were cultured at –1°C or 37°C withshaking (reciprocating motion at 120 times/min)for up to 7 days. Pure water begins to freeze at
0°C, but bacterial suspension in nutrient broth
did not freeze at –1°C. In order to certify bacterial survival after incubation at –1°C forvarious intervals, 0.2 mL of bacterial culture
kept at –1°C was transferred to a test tube containing fresh nutrient broth and incubated at
37°C with shaking.
In another experiment, 5 µL of bacterial suspensions were transferred into 5 mL of a soy
sauce containing alcohol (mirin) and someseasonings (the soy sauce solution), and incubated at –1°C or 37°C with shaking.
The soy sauce solution itself was further
improved giving it a milder taste by incubating
it at –1°C for 24 h with small pieces of raw fishmeat before adding the bacterial suspension. A
block of fish at a size of approximately 1.5 cm ×
1.5 cm × 2 cm was put into the 17 × 130 mm test
tube and 5 mL soy sauce solution was added(the improved soy sauce solution), and the tube
was kept at –1°C for 24 h. To this suspension 5µL of bacterial suspension was inoculated andincubated at –1°C or 37°C with shaking.

Treatment of bacteria at –1°C and 37°C
Five microlitters of bacterial suspensions wereplaced into tubes with 5 mL of fresh nutrient

A

B

10

9

CFU (log10)/mL

CFU (log10)/mL

9
8
7
6
5

8
7
6
5
4

4
0
3

10

5

10

15

Hour

20

25

30

0

1

3

2

3

4

5

6

7

8

Day

Fig. 1. Survival rate of Escherichia coli K12 in nutrient broth at –1°C. Approximately 1 × 105 E. coli K12 in
5 mL of nutrient broth were incubated at –1°C or 37°C with shaking. At various intervals, 0.2 mL of bacterial
culture was removed and viable cells were counted by colony counting methods using 10-fold serial dilutions.
A: Survival curve of E. coli K12 at –1°C ( ) and 37°C ( ). B: Survival rate of E. coli K12 after incubationat –1°C ( ) for up to 7 days. On days 0, 1, 2, 3, 4 and 7, 0.2-mL aliquots of bacterial culture were put into 5
mL of fresh nutrient broth and incubated at 37°C ( ).

148

Survival of bacteria at –1˚C

10Colony forming units and calculation ofsurvival rate

Results

Survival rates in nutrient broth
Bacterial broth medium in test tubes did notfreeze at –1°C for at least 7 days even in static
cultures. When E. coli K12 was incubated at–1°C for 24 h, viable cell count was 100%, andthe number of organisms did not increase (Fig.
1A). The survival rate of E. coli K12 after
incubation at –1°C for 7 days was 52%. When
0.5 mL of E. coli K12 kept at –1°C for 7 days
was put into fresh nutrient broth medium andincubated at 37°C, the bacteria grew to saturation within 24 h (Fig. 1B).
Viability of S. aureus was also good (31%)
after 7 days in nutrient broth at –1°C (Fig. 2).
By contrast, V. parahaemolyticus was not resistant to the low temperature (–1°C). When V.

A

B

10

CFU (log10)/mL

At various intervals after incubation at –1°C or
37°C, 0.2-mL aliquots of the bacterial suspensions were removed, diluted serially with saline
and plated onto nutrient agar plates with orwithout 3% NaCl for bacterial enumeration bycolony counting. Survival rate was calculatedby division of colony forming units per milliliter (CFU/mL) on fixed day by CFU/mL on
day 0.

9
8
7
6
5
4
3

0

1

2

3

4

5

6

7

8

Day
Fig. 2. Survival rate of Staphylococcus aureus
IFO12732 after incubation in nutrient broth at –1°C( ) for 7 days. On days 0, 1, 2, 3, 4 and 7, 0.2 mLof bacterial culture was put into the fresh nutrient
broth, and grown at 37°C ( ).

parahaemolyticus was kept in nutrient brothcontaining 3% NaCl at –1°C, viable cell countdecreased gradually, with 15% survival on day
1, 6.2% on day 2, 0.6% on day 4, and 0.03% on
day 7. At each time, transfer of a 0.2-mL
aliquot of V. parahaemolyticus culture to 5 mLof fresh nutrient broth containing 3% NaCl revealed that the bacteria could grow to saturation
within 14 h (Fig. 3).

Survival of bacteria in the soy sauce solution
When E. coli K12 was kept in the soy sauce
solution, the survival rates at –1°C and 37°C
after 2 days, were 56% and 0%, respectively

989CFU (log10)/mL

CFU (log10)/mL

7
8
7
6
5

6
5
4
3
2

4

1

3

0
0

6

12

24

0

1

2

4

Hour

7

Day

Fig. 3. Survival rate of Vibrio parahaemolyticus in nutrient broth with 3% NaCl at –1°C. Symbols are thesame as shown in Fig. 1.

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Y. Tanaka et al.

Table 1. Survival of E. coli, S. aureus and V. parahaemolyticus after incubation at –1°C and
37°C in the soy sauce solution
Bacteria

Temperatureofincubation

CFU (log10)/mL
on day 0

on day 1

on day 2

E. coli K12

–1°C
37°C

6.57
6.57

6.52
0**

6.32
0**

S. aureus IFO12732

–1°C
37°C

5.75
5.75

5.60
0**

5.48
0**

4.12
4.12

0**
0**

0**
0**

V. parahaemolyticus

–1°C
37°C
Data are typical of 2 or 3 experiments.
**P < 0.01 relative to the value on day 0.

(Table 1, from the expression: 106.32/106.57 ×
100).
In contrast to growth in nutrient broth, E. coli
K12 in the soy sauce solution could not grow at
37°C, and was instead killed at 37°C. S. aureus
also survived for 2 days at –1°C (54%), but was
killed at 37°C. V. parahaemolyticus was killedin the soy sauce solution both at –1°C and 37°C.

Survival of bacteria in the improved soy
sauce solution
E. coli K12 was not killed in the improved soy
sauce solution at –1°C, and also retained viability after a 1-day incubation in the improved soy

sauce solution at 37 °C (Table 2). E. coli O157
in the improved soy sauce solution was gradually killed by incubation at 37°C, but was not
killed completely on day 2. Survival rates of V.
parahaemolyticus in the improved soy sauce
solution at –1°C and 37°C on day 1 were 1.1%
and 0.08%, respectively, and those on day 2
further decreased. E. coli K12 and O157 in thesoy sauce solution and the improved soy sauce
solution, and V. parahaemolyticus in the improved soy sauce solution were more easily
killed at 37°C than at –1°C, but when the raw
fish suspended in the soy sauce solution was
incubated at 37° for 2 days it was partially degraded by enzymes of the fish itself.

Table 2. Survival of E. coli K12 and E. coli O157 after incubation at –1°C and 37°C in the soy
sauce solution containing some pieces of raw fish (the improved soy sauce solution)
Bacteria

Temperatureofincubation

CFU (log10)/mL
on day 0

on day 1

on day 2

E. coli K12

–1°C
37°C

6.74
6.74

6.56
3.40

6.79
0**

E. coli O157†

–1°C
37°C

6.96
6.96

7.06
5.58

6.90
2.00**

V. parahaemolyticus

–1°C
37°C

5.25
5.25

3.30
1.86**

2.17
0**†
Clinical isolate from a patient with hemorrhagic colitis in Tottori Prefecture in 1996.
**P < 0.01 relative to the value on day 0.

150Survival of bacteria at –1˚C

Discussion
The relationship between bacterial growth andtemperature has been investigated in detail(Ratkowsky, 1982; Heitzer, 1991; Zwietering,1991). Theoretical models of bacterial growth
were developed for temperatures between 5 and
50°C. However, it is well known that bacterial
growth is inhibited at temperatures below 4°C(Davey, 1994). As an exception, psychrophilic
bacteria, yeasts and moulds can grow at temperatures below 4°C (Huis in't Veld, 1996; Berry,1997). Preservation of foods by low temperature
is one of the best strategies for keeping foods
fresh (Yamane, 1986).
There are many studies on the bacterial
growth in food (Majeed, 1990; Nerbrink, 1993;
Baranyi, 1994; Barnes, 1994). Deterioration offood quality is partially due to spoilage bybacteria. Furthermore, up to this time in experimental laboratories, bacteria in stab media
have been stored in the refrigerator (4°C) for
short terms, and bacteria in an appropriate suspending media have been preserved in thefreezer (–30°C or –85°C) or in liquid nitrogen(–196°C) for longer terms. So, it is important toknow whether food-contaminating bacteria cansurvive at low temperatures, especially at –1°C,
or not.
In our study, when E. coli, S. aureus and V.
parahaemolyticus were suspended in nutrient
broth with or without 3% NaCl and incubated at–1°C or at 37°C, the 3 species of bacteria usedgrew at 37°C, but did not grow at –1°C. However, E. coli and S. aureus in the soy sauce solution were killed at 37°C and only partially killed at –1°C. Because the soy sauce solution contains high concentrations of NaCl and alcohol,
E. coli and S. aureus with higher metabolic
activity (i.e., at 37°C) might have been moreeasily killed than resting bacteria (i.e., at –1°C).
However, V. parahaemolyticus did not survivewell at –1°C. Species of Vibrio vary with respect to the temperatures at which growthoccurs. V. parahaemolyticus grows at temperatures above 20°C, but viability decreases gradually at temperatures below 15°C (Baumann,
1984). V. parahaemolyticus was completely

killed in the soy sauce solution at –1°C and at
37°C during a 24-h period (Table 1). Killing of
V. parahaemolyticus at –1°C might be due tothe low temperature and inhibitory components
in the soy sauce solution, such as NaCl andalcohol, and killing at 37°C might be due tometabolic activities of bacteria and the inhibitory components in the soy sauce solution.
In Japan and other countries in South-East
Asia, a soy sauce solution containing alcoholand seasoning (the soy sauce solution) is often
used for marinating raw fish before cooking.
The soy sauce attaches and sinks into the fish,
which makes it taste good. It is important toknow whether raw fish containing E. coli in thesoy sauce solution might be killed or not. We
intended to test bacterial survival in the soy
sauce solution following the cooking procedure
as closely as possible. Another reason for
testing the survival rate of E. coli in the soy
sauce solution is an outbreak of food poisoningby E. coli O157 all over Japan in 1996 (Infectious Disease Surveillance Center, 1997). As
people in Japan often eat raw fish, they wereafraid that fish were contaminated withpathogenic microorganisms such as E. coliO157, Salmonella, V. parahaemolyticus, etc.
In contrast to our expectation that survival
rates of E. coli K12 and O157 and V. parahaemolyticus in the improved soy sauce solution
were the same as those in the soy sauce solution, 3 strains of the bacteria suspended in the
improved soy sauce solution survived at –1°C
for 2 days and also at 37°C for at least 1 day(Table 2). The improved soy sauce solution
may contain some components of relatively low
molecular weight, which oozed from the fishand might have neutralized the toxic substancesin the soy sauce solution against bacteria. Another possibility is that the fish meat may
absorb the unsuitable substances for bacteria
surviving in the soy sauce solution, such as
NaCl, alcohol and other ingredients. We used
codfish in our experiment, and as we get
codfish commercially, the condition of the fish
was different with the change of season. Sometimes the fish was contaminated, and grampositive rods and fungi were multiplied after 1or 2-day culture. Thus, the details of the151

Y. Tanaka et al.

experimental methods exhibit difficulties, suchas the components of soy sauce and alcohol(mirin), the kinds and conditions of fish (frozen
and thawed, or fresh raw); in these cases, we
could only show general tendencies.
From these data, we conclude that whenfoods are preserved at –1°C, most contaminating bacteria can survive for a long time, just as
the bacteria on the food can survive (or grow) at
4°C. Heating of the fish stored at –1°C may benecessary for complete killing of bacteria. Although the quality of foodstuffs might bemaintained at –1°C for a relatively long term,
bacteria on the foods might also survive at thistemperature. Consequently, the storage offoodstuffs, especially fish at –1°C, can not always prevent food poisoning.

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(Received March 18, Accepted March 31, 1998)

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