SPOILAGE OF OLIVES BY COLON BACILLI'

RALPH L. TRACY
Laboratory of Bacteriology, University of California at Los Angeles

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Received for publication February 20, 1934
Bacterial decomposition of olives during the curing process
is of considerable importance to the olive packing industry.
The spoilage is characterized by the formation of shallow pockets
directly under the epidermis which are filled with soft tissue
detritus, brine and gas. Very often the epidermis is completely
separated from the flesh and is distended so that a blister-like
protuberance is formed. In badly infected olives, the entire
flesh is involved. Occasionally pockets are formed that extend
nearly to the pit of the fruit. These contain gas but seldom any
decomposed material. The flesh is merely split and compressed
sidewise, apparently by the gas pressure. Blistering of the epi-
dermis does not occur with this splitting and usually the only
external evidence of disintegration is the sponginess of the olive
or a slight depression in the fruit directly above the internal gas
pocket. Owing to the gas content, these olives float on the
surface of the storage brines and are known as "floaters."
In early stages of infection only small, white spots under the
epidermis are visible. These seem to be composed of many
thread-like processes radiating into the flesh of the olive from a
central point. Owing to the approximately circular appearance
of these areas, the spoilage has aptly been termed "fish eye."
Figures 4 to 9 in plate 1 show fresh, ripe olives typically affected
by this bacterial spoilage. These were experimentally infected
by purified cultures of bacteria used in this study. Figure 3
1 This problem was begun in the Fruit Products Laboratory, College of Agri-
culture at the University of California, Berkeley, California. During 1933 it
was studied at the Laboratory of Bacteriology, at the University of California
at Los Angeles.
249
2502RALPH L. TRACY

shows the crushed cells parallel to an internal gas pocket. It

was found that the bacteria producing "fish eye," "floaters," and
"internal gas pockets," were not different species.
In general, the curing of ripe olives is divided into three stages:
(1) Recently picked olives are placed in brine of varying degrees
of concentration, known as "holding solutions." (2) After

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several weeks in "holding solution" the olives are treated inter-
mittently with dilute NaOH and exposure to air. Finally the
free NaOH is washed from the olives with water. (3) These
"pickled" olives are then canned and sterilized.
The bacterial decomposition generally occurs in the "holding
solution" or in the "wash" water following the final lye treatment.
Kossowicz (1908) isolated B. coli from green olives that were
putrefying in dilute brine solutions. He found that if he re-
placed these olives in fresh brine solutions (2 per cent) and then
initiated a lactic acid fermentation by adding sour milk the
putrefaction was arrested. Heating the olives to 600 to 700C.
likewise inhibited decomposition.
Cruess and Guthier (1923) were unable to produce decomposi-
tion of sterilized olives by inoculations of B. coli, B. subtilis, or
B. vulgatus. They were able to transmit the infection to steri-
lized fruit by inoculations of juice from infected olives or by the
addition of disintegrating olives to sterilized fruit in weak brine
solutions. A very short rod occurring singly or in pairs was
isolated by them as the probable etiological factor; however, it
was necessary to use this aerobe in combination with an anerobe
also isolated from decomposed fruit before reproduction of typical
spoilage in sterilized olives was possible. They concluded that
the decomposition was caused by several types rather than a
single type of bacteria.
Alvarez (1926) isolated a Gram-negative lactose-fermenting
organism from olives spoiled in "wash" water, and reproduced
typical decomposition in sterilized fresh olives in nutrient broth.
Her organism was pleomorphic and was extremely prolific at
370C. It withstood exposure to 800C. for forty-five minutes
although no spores were demonstrable, but was killed in twenty-
four hours by a 10 per cent solution of NaCl. She concluded
 SPOILAGE OF OLIVES BY COLON BACILLI 251
that the bacterium was not of the true coli group, but was of a
related species and that probably more than one bacterial species
could produce "floaters."
Esty (1930) found that short Gram-positive rods and yeasts
were present both in normally fermented olives and in those
showing gas blisters and softening. By inoculating fresh olives
in 3 per cent brine, he determined that typical gas blisters were

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produced by certain of the purified yeast cultures within seven
days at 24°C. This yeast was halophilic.
METHOD
Spoiled olives packed in samples of the original "holding
solution" or "wash" water were received at the laboratory sealed
in the usual olive can. In a few instances only was care taken
to steam the can before collecting the samples of brine and de-
composing fruit. In a number of instances, however, collections
were made in sterile wide-mouthed bottles. The usual bacterio-
logical precautions were observed for removing the spoiled
material aseptically from the containers for examination.
Usually 1-ml. portions of the brine were added to tubes of
glucose and lactose broth and to glucose or nutrient agar. In-
cubation was maintained at 320C. for twenty-four to forty-
eight hours. A loopful of broth from each tube was then streaked
upon glucose or nutrient agar plates and incubated at 320C.
Colonies were picked at random from the first set of plates and
planted upon agar slants. After twenty-four hours, incubation
colonies were also picked from the "streak" plates and planted on
agar slants.
Spoiled olives were grasped by forceps, the epidermis covering a
typical decomposed area was seared with a heated needle or
scalpel, and a planting needle, heated to redness, was plunged
directly into the flesh. By a rotary motion of the needle, a hole
was torn in the epidermis and through this a small portion of the
tissue was removed and introduced into lactose and glucose broth
or nutrient agar. The subsequent treatment of these tubes and
plates was identical with the procedure described above.
Cultures obtained in this manner were inoculated into large
252 RALPH L. TRACY

test tubes of sterile nutrient broth containing 3 per cent NaCl

and several sterile, fresh olives. Cultures capable of producing
blisters were kept for further study. In cases of contamination,
isolations were made from the experimentally spoiled fruit and
the process described above repeated until the culture was
purified.

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EXPERIMENTAL
Induced spoilage and identification of bacteria
Cruess (1923) showed that the Sevillano and Ascolano varieties
of olives are far more susceptible to bacterial spoilage than the
Manzanello and Mission varieties, although any one of these types
may become infected. He believed that the presence of carbohy-
drates and other fermentable substances within the olive tissues
was the basis for this susceptibility. Nichols (1930) showed
that on the dry basis the mean sugar content of the Sevillano
and Ascolano varieties was 21.1 and 19.0 per cent respectively
while in the Manzanello and Mission varieties it was 9.4 and
10.6 per cent respectively. For these reasons the Ascolano and
Sevillano varieties were used in the present tests.
Tap water extracts of fresh olives were prepared by mincing
approximately 1 kgm. of olives, softened in hot water, in 3 or 4
liters of water. This material was strained through several
layers of gauze to remove the coarse particles. The solution
was neutralized to approximately pH 6.8 with NaOH and 3 per
cent NaCl was added. The resulting liquid had an average
density of 1.009. For use as a culture medium Andrade's indica-
tor was added.
Purified cultures of bacteria isolated from spoiled fruit and
from other sources were inoculated into test tubes contining
approximately 5 cc. of olive extract. Incubation was at 320C.
for seventy-two hours. From this series inoculations were made
into a second series of tubes containing olive-extract medium.
Usually the inoculum consisted of two or three loops per tube.
In this manner each culture was passed through five different
tubes of olive extract during a period of twenty-one days.
 SPOILAGE OF OLIVES BY COLON BACILLI 253

It was found that a number of the cultures capable of pro-

ducing blisters in sterilized olives suspended in nutrient broth
were unable to grow successfully in this extracted medium after
the second transplanting. The cultures capable of successful
multiplication produced a definite acidity and often showed vigor-
ous gas production. These latter cultures were used in further
tests.

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Attempts to sterilize fresh olives without altering their compo-
sition or texture were unsuccessful. Autoclaving and fractional
sterilization softened the flesh considerably. Chemical steriliza-
tion with NaOH or HCI removed the waxy cuticle of the olive.
TABLE 1
Spoilage of Sevillano olives suspended in olive extract medium by purified cultures*
PER CENT OF PER CENT OF PER CENT OF
EARLIEST TUBES SPOILED TUBES SPOILED CONTROL
SYMPTOMS SPOILED AT
NUMBER STERILZATION OF SPOILAGE
AT E A T DAYS'
AYMTOEALIST INUATITENDY TEN DAYS'
OPTEM OP OLIVIcaSYPTMS NCBATON INCUBATION

Aerobic Anae- Anae- Anae-

robic Aerobic robic Aerobic robic Aerobic Anae-
robic
day. day.
1 Fractional method 1 75 100 0
2 Heated 90°C. in 5 5 76 73 78 78 0 5t
water for 1 min-
ute
* Incubation at 32°C.
t Doubtful bacterial spoilage.

Mercuric chloride and tincture of iodine produced toxic fruit

unless prolonged washing in sterile water was used. Olives
exposed in water to 90°C. for one minute were not injured but
were not always sterilized.
In order to determine which cultures were etiological agents
of the spoilage, the two following tests were made:
(1) Tubes containing several fresh olives suspended in olive
extract medium were fractionally sterilized. Controls for steri-
lization were prepared. These were inoculated with 1-ml.
portions of the final series of extract cultures described above.
Incubation was at 32°C. for ten days.
254 RALPH L. TRACY

(2) Tubes containing olive extract medium were autoclaved.

To each of these were added fresh olives heated in water to 90°C.
for one minute. These were then inoculated as in the above
series and aerobic and anaerobic tubes prepared. Controls
for each were made. Incubation was at 32°C. for ten days.
The data are given in table 1.

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It was found that the cultures producing spoilage in the water-
heated olives were the first to show symptoms of blisters in test
1, and that 22 per cent of the cultures forming blisters in the
sterilized olives were incapable of attacking sound olives in ten
days. All of these cultures, however, grew successfully in olive-
extract medium. This seems to indicate that olive spoilage
bacteria possess a property of invading olive tissues, and that
TABLE 2
Identification of spoilage bacteria by biochemical reactions
BIOCIHUICAL RUACTIONS
GROUPS- _ _ _ _ _ -- _ _ _ _ _-_ _ _ _ _

Lactose Dextrose Sucrose Salicin Acetyl-Methyl

cawbinol
I A.G.* A.G. A.G. A.G. _
II A.G. A.G. A.G. A.G. +
III A.G. A.G. _ - _
* Acid and gas formation. Andrade indicator.

this ability is not common to all bacteria capable of utilizing

olive substances as metabolites. Test 2 likewise indicates that
spoilage may occur equally well under aerobic or anaerobic
conditions.
The cultures producing typical spoilage under the conditions
of test 2 were considered representatives of the causative agents
of bacterial decomposition in olives.
The cultures were divided by biochemical reactions into three
groups. The groups are given in table 2.
These bacteria possessed several common characteristics.
(1) They were Gram-negative rods. Polar bodies stiing more
deeply than the central portions of the cells were often found.
In many instances the cells were extremely short especially in
 SPOILAGE OF OLIVES BY COLON BACILLI 255
olive extract medium. (2) They were capable of fermenting
lactose and glucose. (3) They grew vigorously in olive-extract
medium. (4) They were non-sporulating and (5) were faculta-
tive towards oxygen. (6) Those cultures tested for cellulose
digestion were found to be negative, although not every culture
was studied for this property. (7) A number of these cultures
were found to be killed at 55°C. in ten minutes.

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SOURCE AND CLASSIFICATION OF SPOILAGE BACTERIA
Cruess (1923) was able to show that sterilized olives in 3 per
cent NaCl became infected when material from the following
sources was added to them: (1) fresh olives directly from trees,
(2) olives (uninfected) from "holding solutions," and (3) pieces
of wood or cement from infected vats. He was unable to obtain
infection by addition of well water.
In the present study spoilage bacteria were successfully iso-
lated from the following sources: (1) decomposed olives in
"holding solutions" and "wash" water; (2) normal "holding
solutions" containing only sound olives; (3) water supplies of
several factories; (4) dust at one factory, and (5) in one instance
olives picked under sterile conditions directly from a tree.
These bacteria were classified according to Bergey (1930)
and were found to agree satisfactorily with the biochemical and
cultural reactions of the given type species reported below.
Decomposed olives. The frequency with which spoilage bac-
teria were found is as follows: Group I, 70 per cent; group II,
20 per cent; group III, 10 per cent. The organisms common to
both "holding solutions" and "wash" water were E. neopolitana;
E. pseudocoloides; and A. cloacae. Bacteria found only in "holding
solutions" were E. gastrica, and A. oxytocum. Those found in
"wash" water were E. gritnthali and E. iliaca.
Normal "holding solutions." Isolations were made from brine
samples collected aseptically at two widely separated factories.
The organisms found were A. cloacae, and E. anindolica. The
latter differed from Bergey's type species in being non-motile.
Water supplies. No spoilage bacteria were found in samples
from factories using city water. However, bacteria were isolated
256 RALPH L. TRACY

at a factory using water supplied from a ditch, and at a plant

using private well water. These bacteria were found to be
E. neopolitana, E. iliaca, E. alcalescens, and E. pseudocoloides.
One culture of E. iliaca was non-motile, and one culture of E.
neopolitana was incapable of producing indol.
Factory dust. Litmus-lactose-agar plates were exposed to the

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air for approximately fifteen to thirty minutes at two different
packing plants. At one plant no spoilage bacteria were isolated
although 70 per cent of the cultures obtained were capable of
growth in olive extract medium. At the second plant, exposures
were made out of doors on top of a "holding" tank. About 40
per cent of the bacteria isolated fermented lactose, and one of the
cultures was capable of producing typical spoilage in olive-extract
medium. This culture was identified as E. neopolitana. Chem-
ically sterilized olives suspended in olive extract medium de-
veloped typical spoilage after being exposed to the air in the
"pickling" room of a third factory for two weeks. These bacteria
were not classified.
Miscellaneous. Samples of spoiled olives were received from a
factory at which "holding solutions" tests had been made.
The bacteria isolated from them were classified as E. iliaca, E.
neopolitana, E. pseudocoloidis, A. cloacae, and E. griinthali.
One culture of E. iliaca did not liquefy gelatin, and one culture
of E. neopolitana did. E. granthali was non-motile in olive
extract medium, but was motile in nutrient broth.
The above tests indicate that bacterial decomposition of olives
during the curing process is caused by a heterogeneous group of
colon bacilli. These bacteria are widely spread in nature and
may occur in the water supply and dust of an olive factory.
Undoubtedly these latter habitats of the bacteria are sources of
contamination of "holding solutions" and "wash" water. It
was found that both good and spoiled tanks of olives are com-
monly contaminated with these bacteria. This suggests that
probably tanks of olives unaffected by spoilage contain bacterio-
static substances that are absent from affected tanks.
 SPOILAGE OF OLIVES BY COLON BACILLI 257
EFFECT OF SALT AND LACTIC ACID ON SPOILAGE BACTERIA
Cruess (1923 and 1930) has studied the effects of NaCl concen-
trations and lactic acid fermentation on the curing of olives
very thoroughly.
A lactobacillus, capable of good growth in olive extract medium,
was isolated from a normal "holding solution" of Ascolano olives.

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This bacillus produced 0.6 per cent acid in ten days and 0.98
per cent acid in sixty days at room temperature in olive extract
medium enriched with 0.5 per cent glucose. Classification
according to Bergey (1930) indicated that it was closely related
to Lactobacillus beijerincki although differing from the type
species in that it was incapable of acidifying milk.
The antagonistic action of this lactobacillus to spoilage bac-
teria was determined. Two flasks containing 100 cc. of sterile
olive-extract medium enriched with 25 per cent nutrient broth
and 0.5 per cent glucose were inoculated with 0.5-cc. portions
of a lactobacillus culture. An olive-extract culture of E. neo-
politana was then added to one of these flasks in a 0.5-cc. amount
and a similar culture and amount of A. cloacae was added to the
other.
Titration for acidity in grams of lactic acid per 100 cc. of
medium were made with phenolphthalein as the indicator. Por-
tions of 0.1 cc. were immediately removed from these flasks and
inoculated into lactose and glucose broth tubes which were
incubated at 32°C. The tubes were read for acid or acid and gas
production after twenty-four hours. Three other identical
flasks were prepared, each inoculated with one of the above three
cultures as controls. These were treated exactly as were the
two test flasks.
After forty-eight hours' incubation, the titrations were re-
peated and 0.1-cc. samples were removed from the flasks for
inoculation into tubes or glucose and lactose broth. Gas produc-
tion in lactose and glucose broth indicated survival of the spoilage
bacteria. Acid production in glucose broth without gas indicated
survival of the lactobacillus. The data are shown in table 3.
These data show that acid production by the lactobacillus
258 RALPH L. TRACY

was not inhibited in the presence of spoilage bacteria. On the

other hand, the spoilage bacteria were rapidly killed by the
lactobacillus. E. neopolitana was unaffected by 0.38 per cent
lactic acid (pH 4.2); was prevented from utilizing lactose by 0.5
per cent acid; and was killed by 0.6 per cent acid. This re-

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TABLE 3
Antagonistic action of lactobacilluw and spoilage bacteria
CULTURES

INCUBATION
AT 32C. E. neopolitana A. cloacae
Acidityt pH Lactose Dextrose Acidity pH Lactose Dextrose
day.8
0 0.0072 6.3 A.G.* A.G. 0.0072 6.3 A.G. A.G.
2 0.0386 4.2 A. A.G. 0.0480 4 A.G. A.G.
4 0.0504 3.7 _ A.G. 0.0570 3.7 - A.
6 0.0594 3.7 - - 0.0648 3.7 - _
A. in A.in
48 hours 48 hours
8 0.0612 3.7 - - 0.0648 3.7 - A.
CONTROLS

INCUBATION E. nwpolitana A. cloacae Lactobacilwus

AT 32 0C .

Acidity Lactose Dextrose Acidity Lactose Dextrose Acidity Lactose Dex-

day.8
0 0.0072 A.G. A.G. 0.0072 A.G. A.G. 0.0072 - A.
2 A.G. A.G. A.G. A.G. - A.
4 A.G. A.G. A.G. A.G. - A.
6 A.G. A.G. A.G. A.G. - A.
8 0.010 A.G. A.G. 0.0090 A.G. A.G. 0.0594 - A.
* A.G. = acid and gas formation.
t Grams of lactic acid per 10 cc. mediums.

quired six days. A. cloacae apparently was killed by 0.5 per

cent acid on the fourth day.
To determine the lethal action of lactic acid, tubes of enriched
olive-extract medium contaning lactic acid (by weight) were
inoculated with spoilage bacteria and the lactobacillus. A loopful
 SPOILAGE OF OLIVES BY COLON BACILLI 259
from each test tube was then streaked onto a glucose agar plate.
These were incubated at 32°C. for forty-eight hours before read-
ing. This procedure was again repeated after twenty-four hours.
The data are shown in table 4.
E. iliaca survived 0.25 per cent lactic acid for twenty-four
hours but was killed in forty-eight hours. E. neopolitana and A.
cloacae showed very feeble resistance to 0.25 per cent acid in twen-

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ty-four hours. The lactobacillus was not affected by 1.0 per
cent acid in this length of time. It is interesting to note that
the germicidal concentrations of lactic acid vary in tables 3 and
4. This is probably because the acidities recorded in table 3
TABLE 4
Lethal action of lactic acid
CONCENTRATION IN PER CENT

I' 0.75 0.5 0.25 0.1

Time of exposure in hours
0O24 48 0 24 48 0 24 48 0 24 48 0 24 48
_-_+-__+_-_++ _-+++
E.iliaca.....+*
E. neopolitana.....+ -+-+-+A+++
A. cloacae.+_ 4 +++
Lactobacillus...... ++ + + + + + + + + + + + +
* + = growth of bacteria.
t 4: = slight growth.

represent not only lactic acid but also other by-products of

bacterial metabolism; however it is possible that an "acquired"
tolerance is produced in spoilage bacteria which have been
grown in the presence of the lactobacillus. This has not been
tested.
The bacteriostatic concentration of lactic acid was found to
parallel closely the germicidal concentration. In this test,
E. iliaca, E. neopolitana, and A. cloacae grew very slowly at room
temperature in enriched olive-extract agar containing 0.1 per
cent lactic acid, but not in media containing higher concentra-
tions of acid. The control cultures, however, grew rapidly,
causing vigorous splitting of the agar.
260 RALPH L. TRACY

These tests show that lactic acid is definitely germicidal to

spoilage bacteria, and indicate that lactobacilli in "holding
solutions" are important factors in preventing spoilage. Like-
wise these tests indicate that solutions with acidities between 0.3"
and 0.5 per cent may contain viable spoilage bacteria.
To determine the lethal action of NaCl on the spoilage organ-

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isms and the lactobacillus, tubes of enriched olive extract me-
dium containing varying concentrations of NaCl were inoculated
with E. neopolitana, E. iliaca, A. cloacae, and Lactobacillu8 sp.
Two series, one at pH 5 and one at pH 8.5 were prepared for
each culture. Incubation was made at room temperature.
Test streaks were made onto glucose agar plates.
It was found that each organism survived a concentration of
20 per cent NaCl in twenty-four hours. At pH 8.5 E. iliaca was
TABLE 5
Bacteriostatic action of NaCI on spoilage bacteria
pH or CONCENTRATION
CULTURES TESTED MEDIUM
8 per cent 7 per cent 6 per cent 6 per cent Control

E. iliaca.
E. neopolitana ............. 5.0 _ - A.G. A.G. A.G.
A. cloacae......J. 8.5 A.G. A.G. A.G. A.G.

killed by 15 per cent NaCl in forty-eight hours while E. neopoli-

tana and A. cloacae were killed by 18 and 19 per cent respectively.
However each of these bacteria survived 19 per cent NaCl for
forty-eight hours at pH 5.0. These results corresponded with
those obtained with E. anindolica in glucose broth. At pH 8.5
disinfection occurred at 13 per cent NaCl and at pH 5.0 at 18
per cent in forty-eight hours. The lactobacillus survived a
concentration of 19 per cent NaCl at pH 8.5 and 20 per cent at
pH 5.0 in forty-eight hours.
The bacteriostatic concentration of NaCl was found to be
very much lower, however. This test was conducted exactly
as with lactic acid. The data are given in table 5.
In order to obtain the bacteriostatic concentration of NaCl
 SPOILAGE OF OLIVES BY COLON BACILLI 261
for the lactobacillus flasks with 100 cc. of enriched olive-extract
medium containing varying amounts of salt were seeded with
0.5 cc. of an olive-extract culture of Lactobacillus sp. Titra-
tions for grams of lactic acid per 10 cc. were made immediately.
The flasks were then incubated at room temperature for forty-
eight hours when the acidity was again determined. Two
series of these flasks were prepared, one at pH 5.0 and one at

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pH 8.5. Since acid production was extremely low in the basic
series the data are omitted from table 6.
These tests show that relatively low concentrations of NaCl
are bacteriostatic to the lactobacillus and spoilage bacteria in an
acid medium. This agrees with Winslow and Dolloff (1928)
TABLE 6
Bacteriostatic action of NaCI on lactobacillus
CONCENTRATION IN PER CENT
INCUBATION pH O0 F _ 8 6
AT 32°C. MEDIUM | 9 j 8 | 7 0

Acidity in grams lactic acid per 10 cc.

daysa
0 5.0 0.0072 0.0072 0.0072 0.0072 0.0072 0.0072 0.0072
1 0.0072 0.0072 0.0081 0.0072 0.0081 0.0072 0.0153
2 0.0072 0.0072 0.0072 0.0072 0.0081 0.0099 0.0288
4 0.0072 0.0081 0.0081 0.0117 0.0135 0.0171 0.0450
6 0.0072 0.0081 0.0081 0.0135 0.0153 0.0198 0.0522

who found that 1 molar NaCl was highly toxic for B. coli in
tartrate medium but did not sterilize the solution in twenty
hours. Likewise these tests show that very high concentrations
of NaCl are necessary to exert a rapid germicidal action upon
the microorganisms. This agrees with Hotchkiss (1923) who
found that a 1 per cent peptone solution containing 2 molar
NaCl inhibited growth of B. coli. It is also shown that the
lethal action of NaCl is more effective in a basic medium than
in an acidic one but that the reverse is true, to a slight degree, as
to bacteriostatic effect.
This indicates that NaCl is not an important factor in pre-
venting olive spoilage in "holding solutions" since the concen-
JOURNAL OF BAC?URIOLOGY, VOL. 28, NO. 3
262 RALPH L. TRACY

tration of NaCl necessary to inhibit growth of spoilage bacteria

is beyond the limit packers usually care to utilize.
SUMMARY AND CONCLUSIONS
1. It has been shown that bacterial spoilage of ripe olives in
"holding solutions" and "wash" water is caused by bacteria of

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the genera Escherichia and Aerobacter.
2. The bacteria were found as common contaminants of the
"holding solutions" and in factory dust. Also they were ob-
served as contaminants of the water supply of two factories.
3. These colon bacteria were markedly inhibited by growth of a
lactobacillus isolated from a normal "holding solution." Olive-
extract medium containing heavy inoculums of spoilage bacteria
was sterilized within four to six days by this lactobacillus at 32°C.
It was also determined that 0.25 per cent lactic acid was germi-
cidal for spoilage bacteria in twenty-four hours.
4. These colon bacilli were resistant to 20 per cent NaCl for
twenty-four hours, but were killed by 19 to 15 per cent in forty-
eight hours. The lethal action was increased in a basic medium.
A 7 per cent concentration of NaCl in an acidic medium was
found to be bacteriostatic to the microorganisms.
5. It is concluded that lactobacilli are the natural agents con-
trolling bacterial spoilage of olives in "holding solutions."
Sincere thanks are extended to Dr. W. V. Cruess for the sug-
gestion of this problem and for his valuable aid during its study.
Many thanks are due Dr. T. D. Beckwith for his helpful advice
and criticisms. Gratitude is expressed to Elizabeth E. Phillips
for histological preparations and to Boris Krichesky and Taki
A. Shima for photographs.
REFERENCES
ALVAREZ, R. S. 1926 Jour. Bact., 12, 359.
BERGEY, D. H. 1930 Manual of Determinative Bacteriology. Williams &
Wilkins Co., Baltimore.
CnUmSs, W. V., AND GUTHIER, E. H. 1923 Agri. Experi. St. Bull. 368, U. C.,
Berkeley.
 SPOILAGE OF OLIVES BY COLON BACILLI 263

CRUiss, W. V. 1930 Agri. Exp. St. Bull. no. 498, U. C., Berkeley.
ESTY, J. R. 1930 Proc. Ann. Tech. Conf. Cal. Olive Assoc., 9, 41.
HOTCHKISS, M. 1923 Jour. Bact., 8, 141
KossowIcz, A. 1908 Ztschr. Landw. Versuch, Osterr., 11, 725. Chem. Abst.,
1909, 1654.
NICHOLS, P. F. 1930 Jour. Agri. Res., 41, 89.
WINSLOW, C.-E. A., AND DOLLOFF, A. F. 1928 Jour. Bact., 15, 67.

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264 RALPH L. TRACY

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PLATE 1
FIG. 1. Tissue of a normal, sound olive, Sevillano variety, showing cuticle
covering the regular, flattened epidermal cells. X 100. Mallory hematoxylin
and light green stain.
FIG. 2. Tissue of a typically infected olive showing intact cuticle and heavily
infected epidermal and parenchymal cells. The epidermis is still attached to
the flesh. Note that the infection radiates from the central point of decomposi-
tion along the epidermis. The bacteria stained with hematoxylin and the tissue
with light green. Natural infection in olive from "wash" water. X 100.
FIG. 3. Tissue of a typically infected olive with gas pockets. The paren-
chymal cells are crushed, but do not appear disintegrated. See figure 9. This
section is well below the epidermis and parallel to the internal gas pocket. Bac-
teria were found scattered throughout cells surrounding area when examined
under oil immersion. Experimental infection. X 100. Mallory hematoxylin
and light green stain.
FIG. 4. Olive showing incipient "fish eye" spots. Experimental infection.
Natural size.
FIG. 5. Large "fish eye" spots showing radiating lines of infection from central
point of decomposition. Experimental infection. Natural size.
FIG. 6. Typical "floater" with epidermis loosened from flesh. Note the ex-
tensive area involved. Experimental infection. Natural size.
FIG. 7. Blister formation showing a small gas bubble under epidermis. Ex-
perimental infection. Natural size.
FIG. 8 AND 9. Typical "floater" with internal gas pockets. Note signs of
depressed epidermis in figure 8. No disintegration is visible. Experimental
infection. Natural size.
JOURNAL OF BACTERIOLOGY, VOL. XXVIII PLATE t

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I

4'

7- .

9
(Ralph L. Tracy: Spoilage of olives by colon bacilli)