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A amperative Study of In meromologieal
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A COMPARATIVE STUDY OF NEW MICROBIOLOGICAL STABILIZERS

ON IhEAT

by

JAMES BAILEY EMERY

A THESIS
Submitted to the School of Graduate studies of
Kichigan State College of Agriculture and
Applied Science in partial fulfillment of the

requirements for the degree of

MASTER OF SCIENCE
Department of Bacteriology and Public Health

1951

THESIS

ACKNOWLEDGEMENT

The author wishes to express his sincere appreciation
to Dr. F. W. Fabian, Professor of Bacteriology and Public
Health, without whose patient guidance the completion of
this work would have been impossible.

He wishes also to thank Mr. H. Pivnick and Mr. R. C.

Fulde for their helpful suggestions and encouragement.

268377

TABLE OF CON ENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW. . . . . . . . . . . . . . . .
DESCRIPTION OF CHEMICALS USED. . . . . . . . . .

EXPERIJ‘JTENTAL o e e e o o s e o o e e o e o o o 0

@4010

RESULTS ADID DI SCIJSSI ON 0 O O O O O O O O O O O O
smVIMARYO O O 0 O O O O O O O O O 0 O O O O O O O 18

LITERATURE CITED . . . . . . . . . . . . . . . . 19

Introduction

In our present civilization, communities depend on
food being supplied from.distant sections and foreign coun-
tries. This distribution necessitated a means of rapid
. transit and cold storage to preserve perishable products
from excessive shrinkage and chemical and bacteriological
spoilage. Practical methods of meat preservation, some
dating back to antiquity, have been developed. The empiri-
cal methods were: smoking; dehydration; curing with salt,
nitrates, nitrites and sugar; icing and refrigeration; culi-
nary heating; and in recent years packing in metal and glass
containers (16), (26).

At the beginning of the twentieth century, chemical
preservatives were sometimes unscrupulously used to conceal
damaged or inferior products. Through the vigorous came
paigning of Dr. Harvey Wiley, then Chief of the U. 8. Bureau
of Chemistry, the Food and Drug Act of 1906 was enacted to
prohibit the use of substances injurious to public health.
Inhibitors or microbiological stabilizers should not be
misconstrued to be in the same catagory as chemical preser-
vatives. The action of the former was only to arrest
microbial growth for a short time.

To determine the inhibitory properties of dehydroacetic
acid, its sodium salt and sorbic acid on the flora of chilled

beef was the purpose of these experiments.

Literature Review

The microbiological aspects of meat preservation have
been investigated and adequately presented by Jensen (l6),
Empey and Scott (6), Empey and Vickery (7), Tanner (26),
and others. The ubiquity of microorganisms is well known
to the bacteriologist. Their concern has been with the
parasitic and saprOphytic organisms antagonistic to man and
his food supply. Spoilage organisms have been isolated and
identified from both meat and fish by numerous investigators
(l), (6), (7), (10), (ll), (13), (29). Empey and Scott (6)
investigated the sources of contamination of slaughtered
beef. Their investigation showed that the hair and hide,
soil, slaughter floor equipment, contents of the gastro-
intestinal tract, air and water were the sources of infection
of the beef. Haines (10) found Pseudomonas in the latter
three mentioned sources. He concurred with other investi-
gators that infection of the beef was on the slaughter floor.
Jensen and Hess (17) showed that the sticking of hogs
introduced organisms into the blood which caused ham souring.
In the majority of cases of beef and pork spoilage, the

Pseudomonas group played a minor role. Hunter (13) showed

 

that Ps. fluorescens was one of three organisms concerned

 

in the decomposition of salmon. From his investigation, he
concluded that the bacteria responsible for the decomposition

of salmon were the flora whose natural habitat was the sea-

water. The bacteria found within the cannery played no
part in the decomposition of salmon. In experiments on
the bacterial spoilage of crabmeat, Alford gt 2l° (1)

showed that Achromobacter and Pseudomonas constituted the

 

 

majority of flora present at 2° - 40 C.

It is essential in working with meat and meat foods to
know not merely the number of organisms, but more important
the kind and number of the flora present. Most bacteria
found on meat and meat foods under refrigeration were
psychrOphiles or facultative psychrOphiles. Species of the

genera Achromobacter, Pseudomonas, Serratia, Clostridium,

 

Flavobacterium, Micrococcus, and Streptobacillus have been
isolated and identified (6), (15), (17). Jensen (15) and
Weinzirl (29) have shown that a "high count" of aerobic
bacteria did not indicate putrefaction. Hoffstadt (12)
tried to determine the relationship between the aerobic
bacterial count and aerobic species present in ground beef.
She concluded that a standard method of analysis based upon
this relationship was impractical. The literature did not
agree on what a "normal" plate count for meat and meat
products should be.

From the work which has been done on meat and fish it
appeared that a microbiological stabilizer to supplement
refrigeration would be of economic importance. The use of

carbon dioxide in the storage of chilled beef has been

investigated. Empey and Vickery (7) and Haines (11) showed
that 10 per cent carbon dioxide was required to inhibit
microbial growth. A concentration of two and one-half per
cent carbon dioxide was shown to have a marked antibacterial
activity by Mallmann and Zaikowski (18). Recently Dickinson
and Myers (5) patented a Jellylike coating for meats. The
active ingredient was a metallic pectinate consisting of
pectin and about one per cent of combined nickel.
Dehydroacetic acid and its salts and sorbic acid have
only recently been developed (4), (9). These acids and
salts have been used as mycostatic agents on bread, fruits,
vegetables and dairy products (2), (20), (50). McGowan 33
El. (19) did not find dehydracetic and sorbic acids to
possess as powerful fungicidal prOperties as ethylenic
compounds containing the strong mineral acid group, i. 3.
N02, attached to the ethylenic double bond. Compounds
having ester, ketone, acid or aldehyde groups attached to
the ethylenic double bond showed less activity. Tarr gt 5;.
(27) used the sodium salt of dehydroacetic acid in studying
the effect of antibiotics and food preservatives in retard-
ing the bacterial spoilage of spring salmon. The sodium
salt of dehydroacetic acid exhibited a mild bacteriostatic
action in higher concentrations and a marked pro-oxidant
effect which produced a pigment bleaching and the deve10p-

ment of rancidity in the fish samples.

Description of Chemicals Used

Dehydroacetic acid* (5-acetyl-6-methyl-1,2-pyran-2,4
(5) dione) and the sodium salt are white crystalline powders.
They were designated as DHA and DHA-S, respectively, by The
Dow Chemical Company. The molecular weight of DHA is 168.1.
The acid, DHA, was only slightly soluble in water (0.1 gm.
per 100 gm. solvent at 25° 0.). The sodium salt of DHA was
completely soluble in the concentrations used (55 gm. per
100 gm. solvent at 25° 0.). Its molecular weight is 208.1.
These compounds were found to be stable when heated to
120° - 150° C. for one hour (55). DHA was readily soluble
when autoclaved, however, when the solution cooled the acid
recrystallized to a yellow solid. These compounds were odor-
less and tasteless in all concentrations suitable for use.

Extensive toxicological and pharmacological work has
been done with DHA and its salts (22), (25), (25), (52).
It was shown that DHA and its sodium salt did not irritate
the skin. The LD50 dose of DHA for rats is approximately
0.1 gm. per kg. (25). Normal human subjects tolerate DHA
in doses of 6 -'15 mg. per kg. (given in wine) for 150 days,

carrying plasma levels of 12 - 17 mg. per cent, without any

 

* Chemicals supplied by The Dow Chemical Company, Midland,
Michigan.

evidence of toxicity. There was no selective accumulation
in any particular organ or tissue in the animals tested (52).

Sorbic acid%* is a white crystalline powder Which is
an unsaturated aliphatic mono-carboxylic acid with double
bonds in the alpha and gamma positions. Its molecular
weight is 112.1. The solubility of sorbic acid was similar
to that of DHA in water. The acid was found to be stable
when heated to 121° C. for 20 minutes. However, it recrystal-
lized to a white solid when the solution cooled. The solutions
of higher concentration possessed a faint acrid odor which
was more pronounced when hot.

Smyth and Carpenter (24) determined the LD50 dose of
sorbic acid for rats to be 7.56 gm. per kg. A thirty day
feeding test with rats receiving a maximum daily dose of
0.11 gm. per kg. did not produce depressing or micrOpatho-
logical symptoms.

Media. Difco nutrient agar was used for plating. A nutrient
broth of the following composition was used for transferring

subcultures of the test organisms:

Bacto-Peptone 10 gm.
Bacto-Beef Extract 5 gm.
Salt 5 gm.

Distilled water 1000 m1.
pH after sterilization 6.6

 

** Sorbic acid was supplied by the Carbide and Carbon Chemical
Division of Union Carbide and Carbon Corp., New York 17, N.Y.

Table l. The pH of different percentages of the chemical
solutions.

 

«lb

 

 

 

I Sorbic Acid : DHA : DHA-S :
7% Jpn I pH 7 pH 7
I I I I I
I 1.0 t 5.02 I 5.40 t 7.46 t
I 0.5 t 5.10 t 5.50 t 7.55 t
I 0.2 t 5.10 t 5.61 I 7.60 I
t 0.1 t 5.22 t 5.75 I 7.40 t
t 0.05 i 5.42 t 4.01 t 7.29 t
r
Experimental

Solutions of the respective chemicals were prepared by
dissolving 10 grams of them in one liter of distilled water.
These solutions were autoclaved at 15 pounds pressure for
20 minutes. Solutions of varying percentages by volume,
ranging from 1.0 to 0.05 per cent, were prepared by pipett-
ing the appropriate amount of a one per cent solution of
the chemical into sterile beakers. Sterile distilled water
was added to make 100 ml. The solutions were prepared one
day prior to use and were at room temperature, 20° - 25° 0.,
when the experiment began.

Lean meat, for this experiment, was purchased on the
open market. It was placed in a refrigerator and frozen

before being used. After thawing the beef was cut with the

aid of a sterile scalpel and forceps into pieces of approxi-

mately 0.5 gm. size. The room used was sprayed and the
table surface washed with a 131250 Roccal solution prior
to each days work.

The thawed meat was then inoculated with a 24 hour
broth culture of Pseudomonas fluorescens by smearing a 4 mm.
loopful over the top surface of the meat. The meat was
allowed to set an hour after which it was immersed for one
minute in a 1.0, 0.5, 0.2, 0.1 and 0.05 per cent solution
of DHA, DHA-S and sorbic acid, respectively. After treat-
ment with the chemicals the meat was placed in sterile
Petri dishes. Controls were handled in the same manner
except they were not treated with chemicals. The dishes
containing the meat were then placed in an incubator at a
temperature which fluctuated between -l° and.+4° C.

Tests were made daily for seven days to determine the
inhibiting properties of the three chemicals. 'Bacterial
analysis of the samples were made in duplicate by taking
0.5 gm. samples from each Petri dish, placing them in sepa-
rate dilution blanks with sterile forceps. The bottles
were thoroughly shaken, flamed and decimal dilutions were
made. For each sample duplicate plates from two dilutions
were poured. The Difco nutrient agar was maintained at
42° C. in a circulating warm air incubator prior to pouring.

All plates were incubated at 52° - 550 C. for 48 hours.

The plate counts were made of all organisms, the Pseudomonas

 

fluorescens was used to insure a standard inoculation.

 

Results and Discussion

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2. Bacterial count on meat after inoculation.

I I t I

. Chemical . Bacteria in terms . Log of bacterial ,

, , of millions . count ,

I I I I

I DHA I 6.51 I 7.79029 I

c I 6.05 I I

I T f I

' DHA’S ' 18050 ' 7. 99695 '

v . 1.56 . .

I I r I

' Sorbic v 10.50 1 8.15988 .

. Acid . 17.50 . .
Table 5. The influence of different percentages of DHA

on the bacterial count* of meat.
Y ' g ' ' T I j
. ' , , nys , . . .
I 1 ‘ 2 A 3 4 A 5 l 6 1 7 _j_
t r V 1 r
I I I
:Contr01: 6.40 : 7.56 :54.00 '50.00 '40.50, 40.90 ,57.70 :
. A 9.60 , 7.52 .44-00 '44.00 '47.00,¥58.40,55.50 ,
fi ‘ w 1 fl

I I I I
. 1.0% i 1.58 I 1.00 : 4.72 ' 5.00 z 4.16. 1.68 . 1.50 ,
. g 1.50 , 1.10,: 1.72 ' 4.52 _ 5.84. 2.16 . 2.18 ,
I , v r I l I
. 0.5% : 1.12 , 7.56 : 8.80 ' 5.52 I 5.64. 4.40 . 2.14 .
. _ 0.86 1 1.12%, 4.40,L¥5.15 - 4.24. 5.72 . 9.10 .
I j ' t ' '
. 0.2% : 0.96 I 0.80: 10.00 '59.00 : 4.48. 0.98 .45.40 .
. 4 2.22 , 0.804 10.00 '59.00 A 7.76. 0.92 .45.70 L
I , , r ' I t v
. 0.1% i 1.80 . 5.68. 4.96 : 8.00 :21.10. 65.10 .55.50 .
' , 1088 1 3068 L 50204 8056419000I 57.00 I 32080 1
I ' , ' i I I t
. 0.05% : 1.80 . 0.60. 4.26: 4.40 z 9.80. 19.20 .59.70 .
1 , 1.00 L_0.59. 5.46,, 4.40¥,10.50. 57.20 .57.20 .
* Bacterial count in terms of millions.

-10-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4. The influence of different percentages of DHA-S
on the bacterial count* of meat.
I I I I Days I I I I
' l ' 2 I 3 ' 4 ' 5 ' 6 I '7 '
I 1 I I I I I I I
'Control I1.45 I10.00 I 3.54I 44.00 I55.80 I107.90 I51.60 I
j; I1.40 I 9.92 I 4.76I 44.00I 69.40 I120.50 I42.80 .
f I I I I I I I I
: 1.0% I9.54 I 1.36 I 1.54I 4.40 I37.20 I 58.30 I24.20 I
I7.10 I 1.52 I 0.76I 4.40 I57.20 I 80.60 I24.80 I
t I I I I I I I T—
: 0.5% '1.18 I 0.70 I 3.12I 3.15I 5.54 . 2.94 I42.20 I
1. '0.86 I 0.66 I 1.72I 5.26I 5.56 I 2.40 I32.90 I
' j r— I V fl I I 7
I 0.2% 14.54 I 0.54 I 0.44I 2.52I 31.00 I 4.64I 54.56 I
1, '2.70 I 0.50 I 0.50I 5.78 I52.20 I 9.92 I48.98 I
7 I -I I T" I I . I
1 0.1% '7.28 I 4.24 I 1.72I 3.78 I 3.32 I 12.00 I45.26 I
, {5.25 I 4.00 I 1.46I 4.40I 5.24 I 4.48 I45.84 I
r f I 1 T I— I . F
I
, 0.05% 12.58 I 1.28 I15.44I 5.78 I66.96 I 10.50 I40.92 I
1.72 I 1.14 I12.16' 3.78I 42.16 I 8.70 I45.40 I
* Bacterial count in terms of millions.
Table 5. The influence of different percentages of sorbic
acid on the bacterial count* of meat.
I , I I Days I I I I
I , l I 2 I 3 ' 4 1 5 I 6 I '7 I
I , I I ' I I I I
'Control ,4.58 I18.76 .14.92 ' 8.80. 22.50 . 7.60. 52.08 .
I l 1054 '47050 '16012 ' 5084' 23074 I 4088 I 35096 I
I I I 1 I
I I I I
I 1.0% .2.12 . 0.10 . 0.046I 0.76. 0.12 . 0.15. 0.66 .
I .1.48 I 0.10 . 0.042' 0.70. 0.06 I 0.21 . 1.02 .
' I ' ' I , I ' I I
I 0.5% ,0.40 I 0.60 . 1.70 I 0.70. 4.64 . 2.64. 24.80 .
I 10.46 . 0.50 . 0.58 ' 0.32. 5.12 I 1.88. 16.12 .
r I I ' I I I I
I
I 0.2% .4.06 I 0.116I'O.ll2' 1.52. 4.36 I 0.25. 3.60 .
I 15.16 I 0.288I 0.044I 1.40. 5.02 . 0.22. 4.00 .
T I ' ' v“ I I I I
I 0.1% .1.52I 1.00 I 0.96 I 1.96. 18.76 I59.68 .31.00 .
I .0.52 I 1.14 I 1.02 1 2.64. 21.28 I40.92. 65.24 .
T I ' ' I I ' I I
I 0.05% .5.40 I 4.00 I 5.00 :53.88I 22.52 I59.68I 48.36 .
I .5.48I 4.92 I19.84 26.04. 25.76 I16.12I 45.88 .
* Bacterial count in terms of millions.

 

- 11 -

Table 6. Logarithm of mean of bacterial counts for DHA.

 

1b

D818 ' Control ' 1.0%7 ' 0.5% ' 0.2%7 ' O.l%r '0.05% '
I I I I

j I

7.60206 '7.18752 '7.29667 '7.20140 '7.26482'7.l4613'
I 1 3

7.87157 '7.03745 '7.62757 '6.90309 '7.56585'6.77452'
1 I I I

8.59106 '7.50786 '7.81954 '8.00000 '7.70586'7.58659'
I I I T T '

8.56820 '7.67761 '7.52244 '7.59106 '7.91803'7.64545'
T I v I

V I

8.64048 37.69897 '7.59550 '7.78675 '8.50105'8.00860'
W T T T T r

 

 

 

 

dr-d‘1-d’1-d

8.59879 '7.28550 '7.50855 '5.97772 '8.78604'8.45025'
T T I I I j—

8.74429 '7.24055 '7.74974 '8.64955 '8.52114'8.58453'

+

‘ «0 I.

QGCfifiCflMI-J

 

Table 7. Logarithm of mean of bacterial counts for DHA-S.

 

 

 

 

 

 

 

 

 

:Days; Control 1 1.0% I 0.5g_;; 0.2%“<: 0.1%” : 0.05%“:
i 1 : 7.15229_:7.92012;7.00860 :7.95551 :7.79557 :7.85451:
;*2 : 7.99828_;7.15858‘:8.852517:6.71500fi;7.81490 :7.08279:
: 5 L7.61805fi:7.06070:7.38382‘L6.67210 :7.20140 :8.10721:
L 4 : 8.84545 :7.64345_:7.50651:7.49851‘;7.61172 :7.57749:
;_5 : 8.79657 £8.57054:7.55023_:8.49969_:7.65144 :8.73687E
;:5 : 9.05729 :8.84136:7.42651 :7.86215;7.91593 :7.98227:
' 7 ' 8.57045 '8.58917 '8.57405 '8.71408 '8.65849 '8.52490

 

Table 8. Logarithm of mean of bacterial counts for Sorbic Acid.

 

'Days ’ Control ' 1.0%9:f 0.5% ' 0.2% v 0.1%wglf'0.05%_

I 1 I '
1 ' 7.48572 '7.2552738.85547 '7.55751. 5.95579' 7.55656
I ' f T ' T
' 8.52022 '5.00000L5.74058 '5.50555. 7.02958j;7.54955
v 1 I I ' 1

I
I
I
I
I
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8.19089 '5.54545 17.05745 15.89209: 8.99584j,8.05767:
—‘ fi ‘1 1
I
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7.85451 '6.863524L6.70757 '7.15554u 7.55175j_8.47554
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7.58885!7.67117: 8.501461,8.55458
. ' ,

7.55411I6.58175: 8.55547!»8.44580:
_TV ' .4

8.51091 '7.57978v 8.87521' 8.67521'

8.56399 '5.95424
1

7.79518 '6.25045
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-15-

The curves for the controls (Figs. 1, 2 and 5) reflect
only'the effect of thermal change on the growth trend of
the organisms. Microorganisms can buffer their ionic en-
vironment but the ability of mesophilic characters to buffer
their thermal environment and to grow at low temperatures
in the free water of meat, if given a preliminary incubation
at their optimum temperature must also be considered (16).
The time elapsing between inoculation and immersion of the
meat samples was long enough to permit adjustment of the
organisms to the new environment. There was no moisture
in the Petri dishes containing the control samples. A dry-
ing of the samples was noted after three or four days.
However, those samples dipped in the respective chemical
solutions retained a considerable residue of the solutions.
It was shown by Jensen (16) that excessive moisture on meats
offsets, by several degrees, the good effects of refrigera-
tion. Therefore, the ability of the chemical to inhibit
microbial growth must be proportionately greater.

The bacteriostatic action of the three compounds was
shown by Figs. 1, 2 and 3. Sorbic acid exhibited the great-
est inhibition (Fig. 5), while DHA-S gave slightly better
results (Figs. 1 and 2) than DHA. The graphs for the
percentage solutions of sorbic acid, with the exception of
the 0.05 per cent solution, showed greater inhibition for

a longer time when compared with the graphs for DHA and

DHA" S o

The inhibitory prOperties of sorbic acid are based on
the action of an unsaturated aliphatic mono-carboxylic acid
which possesses at least one double bond in the alpha
position (9). When the molecular weights of the respective
chemicals were compared it was noted that sorbic acid had
the lowest, which allowed a greater number of molecules of
the acid to be actively present for a given weight of the
chemical. In aqueous solution the acid was strongly ionized.
The pH of the five solutions ranged from 3.02 to 5.42 (See
Table 1). However, some of the effectiveness of the acidity
was possibly neutralized by the proteins in the meat. No
correlation was made between the pH and the amount of
bacteriostasis.

Cheeseworth and COOper (3) stated that unsaturated
compounds tended to have a greater bactericidal and prot-
ozoacidal action than the corresponding saturated compounds.
The greater precipitating action of the unsaturated hydroxy-
compounds on proteins and lecitho-proteins suggested that
the superior germicidal efficiency of the unsaturated com-
pounds was associated with their greater capacity to induce
precipitation or denaturation in colloid suspensions.

Phillips and Mundt (20) stated that a concentration of
0.1 per cent sorbic acid inhibited the growth of scum yeasts

on pickle fermentation. The lactic acid production by the

- 17 -

Gram—positive Leuconostoc and Lactobacillus was slow but

 

eventually exceeded that of the control.

The sodium salt of dehydroacetic acid exhibited slightly
greater inhibition than DHA. Only the one per cent solution
of DHA showed more inhibition than the corresponding solution
of DHA-S. The solubility of DHA-S in water was greater than
that of the acid, but the pH of DHA-S was above neutrality.
The action of DHA and its salts as develOped by Coleman and
Wolf (4) was based on the relationship between chemical
structure and antimicrobial activity and not hydrogen ion
concentration. It was observed (8), (21) that certain
antibiotics were alpha, beta unsaturated ketones, 2.5.
clavicin, penicillic acid and kojic acid. The structural
feature common to the previously mentioned antibiotics,
namely the -CH:0—é:0 group, is also found in the structural
formula of DHA and its salts. Geiger and Conn (8) pointed
out that this structural feature was probably responsible
for the antibacterial activity of clavicin and penicillic
acid.

To explain the action of microbiological stabilizers
on microorganisms, the interference of an enzyme system or
the destruction of an essential metabolite must be considered.
The present theories of the nature and specificity of enzymes

have been founded, to a considerable extent, on the effects

- 18 -

of inhibitors. According to these theories, enzymes contain
active centers or groups which combine with the substrates.
The active centers are presumed to be oriented so as to
confer a high degree of specificity (21). The mode of action
of the inhibitor may be any of the following: (a) salt or
complex formation, (b) chemical reaction forming an inactive

product, (0) oxidation of metabolic substance which must be

 

reduced in the process of bacterial nutrition or vice versa,
(d) direct inhibition of cellular oxidations, particularly
those involving nitrogenous compounds, (8) competitive in-
hibition of an enzyme or tissue receptor associated with a
metabolite by a structurally related antagonist (21), (28),
(51).

Summary

1. Sorbic acid exhibited the greatest amount of
inhibition of the three chemicals tested.

2. The 0.2 per cent solution of sorbic acid was the
minimum concentration of the acid which would inhibit
growth for seven days.

3. The one per cent and 0.5 per cent solutions of DHA
inhibited growth sufficiently to maintain bacterial counts
below the initial count for seven days.

4. The majority of curves indicate an increase in
the number of organisms after seven days.

5. No correlation of results could be made on the

basis of pH.

(1)

(2)

(5)

(6)

(7)

(8)

(9)

(10)

_ 19 -

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Summary of toxicological studies on DHA.

 

The Dow Chemical Company, Midland, Michigan.

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