Bacteriological Studies on Freshness of vVhale Meat
(Report No. 1)
Tomoichiro Akiba, Takeshi Tsuchiya,
Makoto Umehara and Yoshiharu Natsume
In using whale meat and organs as food-stuff and material of medica­ments the value of their utilization depends to a large extent· on theirfreshness. Consequently, it is of great· importance to take necessary steps
preventing decrease in freshness, and in other words the factors causing
deterioration must be clear. However,• the research along this line has
been insufficient. In order to study these points it is necessary to inves­tigate the various factors such as whether this is due only to self-digestion
or autolysis after death of whale, or besides what route bacteria take to be
involved in this, or what kind of bacteria invade when and how, if suchbacteria are involved in this. We began this research in the summer of1947 with this object, but our material was very scarce in.1947 due to thepassed fishing season,'and so these results of.1947 and a part of 1948 will
be consolidated and reported as Report No. 1.
Experimental Results

L Collection of Samples. Trips .were made twice, onoe in August, 1947

and another· in July, 1948 to Ayukawa-machi which is located at the tlp of
Ojika Peninsular. The determination of various kinds of bacteria, putre­
faction tests, etc. in this report were carried out on these samples takenfrom one whale which was dissected in August 1947. As materials for our
tests, its blood, muscles, contents of small and large intestines and sea
water were used.. Blood which flowed out from the heart during dissection,
was collected with a sterilized pipette. Consequently, since dissection was
not carried on under sterilized condition, the invasion of bacteria from theenvironments cannot • be absolutely • refuted. However, since a sterilized
pipette was inserted deeply. in a copious flow of blood, it may be assumed
that bacteria contained in the blood itself were collected. As to meat,
about 20 c. cm. of meat lump was cut off, placed in boiling water and boiled.
for about 3 to 5 minutes. After sterilizing the surface of muscle tissue it64

Tomoichiro Akiba, Takeshi Tsuchiya, Makoto Umehara and Yoshiharu Natsume

was cut open with a scapel, and 2 or ,4 gm. of muscle tissue was cut off
from the center. To this, 8 or 2 cc. of physiological saline solution wasadded and stirred well. After standing only the meat juice was taken andused in the experiments. As to the contents of the intestines, these were
cut open and the contents were collected with a sterilized pipette. Thiswas diluted to five times with physiological saline solution and used in theexperiments. The intestines were also ·swollen due to gas produced byfermentation of the contents at that time. Sea water was collected in frontof the works and from the bay.
As culture media sea water agar media, meat infusion agar, Endo's
media and cysteine glucose agar were used. Sea water agar media wereused for the purpose of not losing specific sea water bacteria, cysteine agar
media for anaerobic bacteria, and Endo's media for the isolation of colon.type bacteria. Sea water agar media were made according .to Zo Bell's
method, and 0.5% of peptone, 0.01% of ferric phosphate and 1.5% of agaragar were disolved in 1000 cc. of sea water being aged before the use.
2. Freshness and Number of Bacteria. Number of bacteria detected inblood and muscle was almost proportional to freshness and the number ofbacteria also increased as the time elapsed longer after catching. However,Table 1.

Freshness and Number of Bacteria

Date Whale Caught
Time of elapse after catching (hours)

<Ll
OS

&l::I

'$
~

.c

g
::s

z

Sei
Whale

B 3
Sei
Whale

B 4
Sperm
Whale

Aug. 12·July 25

July 25

July 25

July 27

30

34

26

68

24

Fresh
(80%)

Fresh
(80%)

Satisfactory
(50%)

Fresh
(80%)

70,00012,0009,360,000

23,400

9,400

8,200

5.9
6.0

5.6
6.1

Blood

Meat influsion agar media

per ml.

Sea water agar media

Muscleper gmLarge Intestinecontents
per ml.
Small Intestinecontents
per ml.

Meat influsion-agar media
Sea water agar media
Meat influsion-agar meiiia

SeveralSea water agar media

0

Meat influsion-agar media

SeveralSea water agar media

Several

·;:::

t)

B 1
Sei
Whale

Satisfactory(50%)
Innumerable
Innumerable
4,575
4,425

Freshness

OS

A 1
Sperm
Whale

B

2

Blood
6.2
6.2
Muscle6.2
5.8
Note : Freshness was expressed by the visual judgement of experts.

pH

I

三
Characteristics
Shape

Size (mirron)Grouping
Gram

Spore

Motility

Meat infusion agar
Turbidity君

Precipitate
Pellicle....
にPotatoGelatin liquefaction
Litmus milk

Indal

比S
Methyl red

Voges.-Proskauer R.

Citrate media

Neutral red Reduct.
Cysteine glucose agar
(ga
Nitrate eduction
Katalase

唸

m
ヽ

コ
ロo ゃ
卜
．窃5 ．笞

0て
.
日も
«
8 u
.←
0

0

3
て

ト切m

品

ヽ

→
Q)

ご

GlucoseLactose

Saccharose
Glycogen
XyloseGalactose

Arabinose

F 19

F

Spherical

Range pH

Optimum salt content
Range

pH

Name of Bacteria

F 65

B 82

Spherical

Spherical

Spherical

single, pairs

single, pairs

single, pairs

pai-rs, clumps

clumpsWhite, roundsmooth glistem嘆Greyish white,round, smooth Canary yellow,
raised drying
glistening

Yellow, roundraisedWhite, round, Greyish white,transparent tur·glistening
bid, round raised＋＋-Greyish W h-ite,poor growth

I.J.-1.5

＋
＋

1.7

＋

+

＋
yellow

Canary yellow,
poor growthWhiteacid coagulation acid coagulation acid coagulation
＋

＋

＋
＋

＋

十
＋・

＋

Yellowish
drying＋
No change

White, poor
growth＋No change

Greyish white,good growth

＋

＋

＋

+ ilt

＋廿
＋廿

27

7.0

5.5-7.73

0.5-12
Micrococcus
sph eroides

+ ttt

＋廿

＋骨

— +

＋廿

＋廿

＋廿
一廿

十廿·

一廿

-

22-43

22-43

-- +
27

7.0

6.6-7.73

0.5-6

M. piltonensis＋

acid coagulation＋

I 45

Sphe1·ical
1.5-1.8

single, pairs
＋

C

32

Spherical
1.3-1.5

single, pairs＋

Reddish yellow,
Yellowish
white, round wrinked,出ying＋+ yellow+ yellow

Greyish white,poor growth
No change

＋

＋

十

＋廿

＋

Characteristic s of Bacteria Exami ned
S 90
Spherical

shrot chains

White, smallcolonies

Transparent,
colorless, smallcolonies, raised+ yellow
Orang e yellow,
wooly
No change

Spherical

1.5-1.6
single, pa如s
short chait1s＋＋＋

White, good
growthNo change
＋

＋

ilt

22

7.0

6.6-7.73

0.5-20M. flavus

`

＋

1.5

＋

十

＋廿

+ 1lt

十 一

＋廿

＋廿

— +

＋廿

27

27

22--:-43

22-30
7.0

7.0

6.6-7.7

6.6-7.733

0.5ー10

＋

＋

一廿

一廿

＋廿

M. subcitreus

＋

I

0.5-6

M. saccatus

＋廿

＋廿

— +
— +

一廿
30

22-43
7.0

十

一

＋ ＋
27

22-27
7.0

十 一

27

22-43
7.0

十

一

十

一

十
-I-

一

十一

十一
十一
十

一

Orange yellow＋
No change＋

I士

Cream yellow,
flat

+

＋
yellow＋

Orange yellow

acid coagulation

＋

十

＋

十

＋

＋

＋

＋榊

＋廿

一

27

22 ― -30

27

22-43

27-43

22-43

22-43

5.5-9-4

6.6-9.4

5.5-8.7

7.0

7.0

6.5-6

0.5-10

0.5-3

0.5-6
Streptococcus
foecalis

3

I M halophilus

3

37

7.0
3

37

7.0
3

＋

single, pairs

十

＋

＋

盆

Uowish Greyish white
Slig ht
wh1＋acid coagulation acid coagulation
＋

＋

＋

ilt

＋

＋

＋

十 一

十一•

廿

一

十 一

No change

廿

＋廿

十 一

single, pairsrod

1.4ー1.5 X 2.1-2.2

＋

一

一

＋

情

＋

十 一

·+

―—ellowish white
White, viscous J!Y＋No change＋rodl.Oxl.8

6

F

＋

＋

＋

＋

97

＋

＋

十

一

rod

S

Greyish white,smooth

十一

十

I 44

＋
Greyish white, Greyish white, Greyish white,
glistening
round
round, smooth一

0.5-10M. luteolusrod

十5.5-7.7

M. pikowskyi

rod

十 一•

6.5-7.7

M. epimetheus

F 15

tft
＋ ・十

6.6-8.7

3＋
yellow一廿

5.5-8.7

3+

No change

6.6-7.7

3

Cream yellow,
glistening

Greyish white

＋

＋

rod

I 43

1.1ー1.3xl.7-l.90.8ー1．りxl.8-2.0 1.0-1.5 x LS-2.0 0.8-1.0 x LS-2.0
single, pairs, sing l e, pairs gra
single, pair!)
single, pairs
short chains n u lar cytoplasm

＋

ttt

tit-

C 33

66

F

土

＋

十榊

＋

＋

士

＋廿

1.4-1.6+ yellow

tit

＋廿

十

＋

tit

— +

十

＋

tit

＋

1.1-1.7

0.8-1.lilt

22- 43

Optimum pH

F 12Spherical

°C

Range

F 17

Spherical

MannitOptimum Tempt.

882.

0.9-1.3
single, pairs,
chains— +- ttt

DextrinTable

十廿- 1l1-

— +

十廿·
＋冊

+ ilt

＋廿

十 一

＋廿

十 一

--

22-43

22-43

22-37

6.6-8.7

6.6-8.7

5.5-7.7

0.5-200.5-20

0.5-6

— +
27

7.0
3

0.5-15
0.5-200.5-15
Achromobacter
Flavobacterium
sewanenseFlav.
|
u ntumubiquitum
fr

27

7.0
3

27

7.0
3

A. halophilum A. Cystinovorum

ヽ

ilt

27

7.0
3

A. lacticum--·

Bacteriological Studies on Freshness of Whale Meat65

it was not clearly recognized that there was some relation between freshness and hydrogen ion concentration in blood and muscle at the time ofdissection, but all the reactions indicate the progressing decomposition of
the sugar.
3. Indentification of Isolated Bacteria. In this paper are reported only
the results of experiments as to the samples collected from a sperm whale
dissected in August 1947. Result of another samples collected in July 1948
shall be reported in the second report. Identification of various bacteria
was attempted as to bacteria isolated at the works and also bacteria isolated after bringing- samples to our laboratory. Even the bactetia obtainedby anaerobic culture grow better by aerobic culture, after successive
transfer on culture media, so all the succeeding examinations of biologicalcharacters were made by aerobic culture.
Microscopic examinations were made as to the pure culture of the 57
strains to determine their shape, grouping, Gram's stain, size, motility, etc.
and furthermore 19 . species were selected according to shape, form andlcolor of colonies on the media such as meat infusion agar, sea water agar
media and cysteine glucose culture, and gas production, liquefaction ofgelatin, hydrogen sulphide production and indol reaction. As to 19 strains
the further examinations were made as following table (Table 2).
Of 19 strains another three were same strains, so that 16 species wereidentified according to Bergey's Manual of Determinative Bacteriology andother literatures. It was possible to determine 9 different Micrococci, 1
Streptococcus foecalis, 2 Flavobacteria and 4 Achromobacters.
These bacteria may generally be found in soil, air;· water, etc., andalthough there may be few cases in which their biological characteristics do
not exactly coincide with those found in references, but the principal ones
coincide exactly and . we could not find new species. In a case as MicrococClJS epimetheu~, there is a question more or less compared with the place
primarily found, but there is not any reason that such bacteria must not
be found in places other than where they were found, so that it may be
assumed that the presence of such bacteria is probable.
4. Putrefaction Tests. When these bacteria were inoculated on pieces ofwhale meat and left standing at 30°C, 8 strains produced rancid irritatingor light deterioration odor after 18 hours, and 14 strains after 48 hours, andthe same time pH trended to alkaline.

Tomoichiro Akiba, Takeshi Tsuchiya, Makoto Umehara and Yoshiharu Natsume66

Also, in case several bacteria being grouped according to the origines
where they are found, are inoculated on the surface of a piece of meat,
the rancid odor is produced faster than in the previous cases and appears
after 6-12 hours.

~

Before

19
88
15
F 17
F
5
6
F
1
F
F 65
F 66
F 12
c 32
c 33
c 34
B 24
B 82
I 43
I 44
I 45
s 90
s 97
Control

6.2
6.2
6.3
6.2
6.3
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2
- '6.2
6.2
6.2

F
F
F

Note:

I

Table 3.

Deterioration Test, -pH aod Odor

After
4
hrs.

6

6.1
6.2
6.1
6.1
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.1
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2

6.0
6.0
6.0
6.3
6.2
6.0
6.1
5.9
6.0
6.0
6.1
6.3
6.0
6.0
6.0
+6.2
6.2
6.0
6.0
6.0
6.2

I

24

48

6.8
7.0
+5.9
6.4
+7.0
6.4
+6.8
6.4
6.3

6.9
7.4
it-6.66.5+7.4
it-6.8it-6.8±6.6
it-6.3
+6.4
±7.6
6.1
+6.2
+7.4
+7.4
tft7.8
5.8
5.8
±6.7

26

I

6.0
6.0
6.0
6.3
6.2
6.0
'6.1
5,9
6.0
6.0
6.1
6.3
6.0
6.0
6.0

6.3
6.4
+6.0
6.3
±6.4
6.0

it-6°3
6.2
6.0
6.0
6.0
6.2

it-6°6
6.0
6.0
6.0
6.0
6.2

I

±6.4
6.2
6.1
+6.0
6.1
6.1
+6.0

+6.4
6.5
6.0
+5.7
+6.8
7.0
it-6.9
5.8
6.2
5.6
6.3
6.3

±9.4
ti.6

±~.5

6.3

7,7
it-8.2it-8.2it-7.0tft8.0tft7.6it-7.2
+6.8
+6.4
+6.6
tft8.0it-7.0fu6.8
it-8.0it-7.7
tft8.06.6+6.0
I +7.4
I it-7.0
I
I 6.6+-tit indicate the degreee of deterioration odor.

~

Before

After
4
hrs.

F groupC group
B group
I groupS groupControl

6.2
6.2
6.2
6.2
6.2
6.2

6.2
6.2
6.2
6.2
6.2
6.2

B
rial gro ps

I

'

18

12

6

12

18

24

48

Oder

6.4
6.2
6.3

+6.4
6.4
+6.3
+6.3
6.2
6.2

+6.S
6.5
+6.4
+6.5
6.3

it-7.8tft6.2+6.8
fft8.2tft8.0+7.4
6.4

Putrefied odor
Putrefied odor
Ester-like odor
Putrefiedirritating odor
Putrefied odor

+6.6
6.2
6.2

6:2

±6.4

I

it-7.0
+6.8
+6.8
6.2

If 0.2 cc. of bacterial emulsion injected into the p:eces of meat, theproduction of odor is even more conspicuous and als9 pH becomes conspicuously alkaline.
5. Invading Route of Bacteria. The route of entry of bacteria into
.blood and muscle has not been clarified as yet, but 4 routes of their entry

Bacteriological Studies on Freshness of Whale Meat

can be assumed, that is, explosion wound .caused. by spear, compressed air
pumped into the whale after death in order to keep it afloat, respiratory
tract such as lung and the intestinal tract.
In the case of entry through the explosion wound, there is cons:derable
negative pressure in the severed vein on account of the bleeding, therefore
it is understandable that some bacteria sticking to the part affected will beabsorbed and reach to the heart. When pig is slaughtered and bacteriacoated on the wound (in such cases, it is obvious that easily detectablebacteria are used), it is reported that the same bacteria can be found in
the bone marrow, and so these facts support this theory. Whales being
speared are especially different from animals being slaughtered, because
the former try to escape with strenuous activity for more than one hour.
More opportunity may be given for bacterial invasion due to the laboured
breathing during that time. Next, in regards to pumping in high pressure
air, special efforts to prevent entry of bacteria are not taken, so it can beassumed that there is a possibility of bacterial invasion. Also the cutting
open the abdomen to cool can give the path of entry for bacteria. Thirdly,in the case of respiratory tract, bacteria can be discovered in the lung of
human being and slaughtered pig, in which even the scraps of food arealso occasionally found, so there are some scholars who say -contam:nationof blood in heart is caused by passing through veins from the lung. If
the detection of principally soil bacteria in lung of pig by Htilphers is taken
into consideration, this path of entry can be naturally considered. Even
with the :present experiments, Micrococcus halophilus, which is a specificsea water bacteria, was detected in the sea water and muscle ; also
bacteria detected in blood and muscle· belong chiefly to soil bacteria; andsince a long time has elapsed between the time of catching and thedissection, it is considered that the above three routes of entry play considerably important parts.
However, since whales are animals living in the sea, there isn't any
reason why the entry of bacteria into blood and muscle by way of intestinal
tract cannot be considered. Even in the case of drowned or poisoned
human beings or animals, it can be assumed that perhaps the large numberof bacteria which originarily live in the intestines, enter into the blood vessels through the mucus membrane of the intestine due to the bacterial
permeability of the intestines after death and to the extraordinary increase. 68

Tomoichiro Akiba, Takeshi Tsuchiya, Makoto Umehara and Yoshiharu Natsumeof bacteria. In the case of whale this mode of entry maY be accelerated
by the air forced into the abdomen, by which the bacteria invade into theblood, and then they will circulate throughout the body and extend from theend of blood vessels into the muscle. ·Furthermore, from long experience,the staleness of fishes is prevented by removal of the intestine as soon as
possible after death, so it may be assumed that intestinal route plays a. major role. Also, from our experiments, the facts that 5 kinds of bacteria,
i. e. Micr6coccus piltonensis, , M. epimetheus, Flavobacterium sewanense,
Achromobacter ubiquitum and Achromoba'cter halophilum were found in
the contents of the intestine, muscle and blood, and that Streptococcus
faecalis which is always found in the intestine was detected in muscles,
supports the theory of the intestinal tract route. We were not able todiscover Bacterium coli in samples of 1947. This may be due to the factthat Bacter:um coli cannot exist for long in the blood as whale is a warm
blooded animal similar to pig. However, this requires further study in the
future to settle this point.
6. Putrefaction and Bacteria. It was proven that the number of bacteria
will increase in blood and muscle as degree of freshness decreases. Next
is that three strains of the detected bacteria do not grow at temperatures
above 35°C. The majority has optimum temperatures from 27-30°C, so if
the temperature of the body of a whale from the time of catching to dissection is within this range, it will be the optimum condition for increase ofbacteria and their putrefaction will occur faster.
Furthermore, these bacteria grow even in a media of low salt content(1%), but growth will be better at about 3%. If the concentration isincreased to 15%, growth will be greatly reduced, only 4 strains will exist
at 20% and growth is checked at 25%. Therefore, putrefaction can beprevented by maintaining salt contents above 25%.
Of the 57 strains of bact.eria, 38 decomposed glucose and produced large
quantity of gas. The large quantity of foam observed on stale whale meat..is probably due to such .bacteria. If such bacteria are inoculated on apiece of whale meat and its pH is exam:ined, at first it is about 6.2 andwill become lower after 24 hours, but after 48 hours it will become 6.48.2 and will give off rancid, irritating or at times a mild putrified odor.
These phenomena will become very marked when several strains are inoculated together, but even then the odor is not as foul as in the case of

Bacteriological Studies on Freshness of Whale Meat

69

actually putrefied fish. Also, in case of actual putrefaction proteus bacteria
are sometimes detected, but in our cases there was no such ad.vanced caseof putrefaction, and the Micrococcus flavus, M. pikowskyi, M. halophilus,
Strept. faecalis, Flavobacterium sewanense and Achromobacter halophilum
found in our examinations coincided with those detected by other researchers.
Table 4.

Species of Bacteria· detected

Name of Bacteria

Number ofstrains

Blood

Micrococcus epimetheus
Achromobacter ubiquitum2
6

rubber tree
·soil, water

3
2
1
1

MuscleMicrococcus sphero,ides
Micrococcus piltonensis
Micrococcus flavus
Micrococcus subcitreus
Micrococcus saccatus
Micrococcus halophilus
Streptococcus faecalis
Flavobacterium sewanense
Achromobacter ubiquitum
Achromobacter halophilum
Achromobacter lacticum

8
1
1

manure, soil
manure, soil
air, milk
air, water
mucous of nosesea water
animal faeces
sea watersoil, watersea water
cow's milk

1
1
1
1
1
1
7
1

.manure, soil
rubber tree
sea water
cheesehalibut
sea water•soil, watersea water

2
6

sea water
soil

Samples
examined

Intestines

Sea watet

Micrococcus piltonensis
Micrococcus epimetheus
Micrococcus pikowskyi
Micrococcus luteolus
Flavobacterium fucatum
Flavobacterium sewanense
Achromobacter ubiquitum
Achromobacter halophilum

1
3
5
.1

I
I
I

I
I

I
I

Micrococcus halophilum)
Achromobacter cystinovorum

Place ordinarily found,according to references

Conclusions. 1. Freshness is decreased in proportion to the time elapsed from catching
to dissection and to the number of bacteria detected in blood and muscle.
2. As shown in Table 4, 15 species of bacteria were detected in whale's
blood, muscle and intestines and 2 species were found in sea water. Of
the above, 6 species coincides with those reported by other re~earchers.
3. There are 4 possible routes for entry of bacteria into blood and'·muscle and it may be assumed that the intestinal tract is the most impor-70

Tomoichiro Akiba, Takeshi Tsuchiya, Makoto Umehara and Yoshiharu Katsume

tant route.
4. Although the bacteria detected may not play the .leading role inputrefaction, it is obvious that they contribute to the decrease in freshness.
5. Therefore, the most effective method to prevent decrease in freshness
is to dissect and dispose of it as soon as possible after catching.
6. ·That the previous method of salting and freezing after dissection iseffective for prevention of putrefaction, can be proven by our experimental
results on arresting increase of isolated bacteria.
References
Hunter, A. C.: Bacterial groups in decomposing salmon, J. of Bact. Vol. 5, 1920.
Jensen, L. B.: Microbiological problems in the preservation of meats. Bact. Review, Vol. 8, Nr.
3, Sept. 1944.
Sanborn, J. R. : Certain relationships of marine bacteria to the decomposition of fish. J. of Bact.
Vol. 19, 1930.
Takarada Michinori : Research on intestinal bacteria of fresh water fish, J. of Chiba Medical
Society, Vol. 20, No. 4.
Takayama Naokiyo: Bacteriological research on decomposition of lobster, J. of Chiba Medical
Society, Vol. 21, No. 9, 1943.
Weinzirl, J. : Concerning the relation of the bacterial count to the putrefaction of meat, Amer,

J. of Public Health, Vol. 14, 1924.
ZoBell, c. E.: Studies on marine bacteria. J. of Marine Research. Vol. 4. Nr. 1. 1941.