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GROWTH‘ DEVELOPEBNI‘, AND T'Hfl
FORMED ELEMENTS C? THE. BLGGD
OF TWENTYWWO ALBiNO RATS
WED VARYING CONCENTRATEONS
OF DRY GARLIC POWDER

fi‘haa’és .90: {in Deg-mo 05 M. S.
MJZCHIGAN STATE COLLEGE
Ruth Mary Nétchsls
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Thisiatooertifgthatthe

thesis entitled

“The Growth. Development and the Formed Elements of the
Blood of Albino Rate Maintained on a Base]. Diet Contain-
ing Dried Garlic Powder.”

presented by

Ruth Mary Nitchale

has been accepted towards fulfillment
of the requirements for

Heater Of_Sa18n.cn—degree inJoodLand Nutrition

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E""

GROWTH, DEVELOPMENT, AND THE FORKED ELEXENTS
OF THE BLOOD OF TWENTY-TWO LBINO RATS FED VARYING

CONCENTRATIONS OF DRY GARLIC POWDER.

by

RUTH MARY ggTCHALs

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
MASTER OF SCIENCE
Department of Foods and Nutrition

1946

THESIS

The author wishes to eXpress her sincere
appreciation to Dr. Margaret A. Ohlson for her interest
in the study and helpful suggestions concerning the
preparation of the paper, to Mrs. Bernice Hess for her
care of the animals, and to Dr. W. D. Baten for his

assistance with the statistical analysis.

TABLE OF CONTEHTS

 

Introduction ...

000000.

Literature .0000. eeeeee eeeeeeeeeeoeeeeeooeee.

Experimental Procedure ............
Results and Dsicussion ..............

Summary .........

Literature Cited

 

49

51

 

10.

LIST gs; TABLES

 

Erythrocyte and Leucocyte Counts and
Hemoglobin Concentrations of Albino
Rats as reported in the Literature ........ l7

EXperimental Diets ........................ 20

Grouping of Experimental Animals
According to Litter Number ................ 21

Average Weight Gains per Gram Food
Consumed .................................. 50

Average Monthly Erythrocyte Counts,

Leucocyte Counts, and Hemoglobin
Concentrations of Eleven Female

Albino Rats Fed Varying Concentrations

of Dry Garlic Powder ...................... 55

Average Monthly Erythrocyte Counts,

Leucocyte Counts, and Hemoglobin
Concentrations of Eleven.Male Albino

Rats Fed Varying Concentrations of

Dry Garlic Powder ......................... 56

Differences in the Monthly Erythrocyte
Counts of the Male and Female Animals
as shown by the "t" Test .................. 59

Differences in the Monthly Hemoglobin
Concentrations of the Female Animals
as shown by the "t" Test .................. 42

The Differential White Blood Cell
Counts of Eleven Female Albino Rats ..... 44—45

The Differential White Blood Cell
Counts of Eleven Male Albino Rats ...... . 46—47

Figure

1.

LI ST 91: FIG THES

Average Weekly Weights of Male
and Female Animals from Weaning
to Forty Weeks of Age ...............

27

INTRODUCTION

Early in the history of bacteriology, certain
antagonistic relationships among various microorganisms
were recognized. DeBary, ('78), attempting to grow
two organisms on one substrate, found that eventually
one would inhibit and completely destroy the other. This
action became known as antibiosis, meaning "against life".
The postulated chemical substance causing the reaction
was called an antibiotic. Pasteur first made practical
application of these phenomena by using microbial
antagonism against the anthrax organism.

In the past decade, renewed emphasis has been
placed upon the search for other antibiotic substances
which may benefit man. Today, because of popular
advertising, the word antibiotic is familiar to the
general public. Literally, the word applies to any
chemical substance of microbial origin that inhibits growth
or metabolic activities of bacteria or other microorganisms.
Recently, however, the term has been eXpanded to include
any chemical substance produced by actinomycetes, bacteria,
algae, or higher plants which will produce the Same results.
Many studies to find the higher plants exhibiting such

properties have been made. Such widely used foods as onions

2
(Walker, Lindegren and Bachman, '25), cabbage (Pederson
and Fisher, '44), rhubarb (Huddleson, Du Frain, Burrows
and Giefel, '44), horseradish (Foter and Goliek, '58),
and garlic (Walton, Herbold and Lindegren, '56) have been
Shown to possess antibiotic properties.

A search of medical literature reveals many
instances of the use of garlic in attempting to cure
disease. Isolated cases are recorded in medical Journals
where treatment of whooping cough (Petrocchi, and Gutman,
'27), tuberculosis (Minchin, '29), dyspepsia (Roos, '25),
flatulence and cholecystitis (J. A. M. A., '54), diabetes
(Mahler and Pasterny '24), and arterial hyertension
(Sunzeri, €26) with large quantities of garlic was made.
Although little of this work has been subjected to
controlled medical research, the possibility that the
cures claimed may have been influenced by the presence of
antibiotic substances in the garlic becomes apparent.

Huddleson and co—workers ('44) suggest possible
prevention of infectious disease by ingestion of garlic
or the garlic components having antibiotic properties. The
adOption of such a suggestion would depend upon the action
of large quantities of garlic on the living body. No
studies have been recorded of a systematic attempt to
determine the effect of large quantities of garlic on

animals. This study was conducted to determine any

3

interference with growth and development which might
result from feeding large quantities of garlic powder to
albino rats.

Since Thorp and Harshfield ('45) found that
in horses a lowered erythrocyte count and anemia resulted
from ingestion of large quantitiies of onion, an
antibiotic producing plant also of the lily family,
studies of the formed elements of the blood were made to
ascertain if garlic feeding would so affect albino rats.

Growth, food consumption, and autopsy records
were kept to show any difference due to feeding dry garlic
powder in the concentration of 2.5 and five percent of
the basic diet. Analysis of the moisture content of garlic
shows it to be 75 percent water. Therefore, the 2.5
percent dry garlic intake is equivalent to 10 perecnt when
calculated on a moist basis. A five percent dry garlic

intake would equal a 20 percent intake of green garlic.

LITERATURE
General Properties of Antibiotics

Microorganisms and products of their metabolism
have been used for the control of disease in man, animals,
and plants with varying degrees of success. As early as
1877, Pasteur was able to repress the production of
anthrax by the simultaneous inoculation of the organism
Bacillus anthracis. Following this early work considerable
attention has been given to the study of bacteria
possessing antibiotic properties. The voluminous literature
'on microbial antagonism which has accumulated since
Pasteur's time is excellently reviewed by Waksman in his
recent book ('45). The following properties of the various
organisms displaying antibiosis, the methods of testing,
and the practical applicationsare taken from various sections
of Waksman's book. No attempt was made to gather the
original material Since the majority of it is recorded in
French.

Of historical interest is the first antibiotic,
pyocyanase, recognized by Emmerich and Low in 1899. Produced
from Fe. aeruginosa, it was found to have a lytic effect
upon the diphtheria, streptococci, meningococci, pneumococci
and typhoid organisms.

Systematic search has been made to determine which

5

bacteria, acintomycetes, algae, fungi, and higher plants
possess antibiotic properties. Several methods have been
developed to show these antagonistic effects. Most
methods are based upon the growth of a test organism in '
the presence of a living antagonist or the antibiotic
substance produced by it in a liquid or on a solid
nutrient media. Antibiotic action is established by
failure of the test organism to grow. The nature of the
substance must then be determined and quantitatively
measured.

Three general methods for measuring the potency
of an antibiotic are in use. One is the agar plate
dilution method in which the unknown substance is
diluted to various concentrations in liquid agar. The
agar, after solidification, is streaked with several
test bacteria. The highest dilution at which the test
organism fails to grow is recorded as the potency of that
antibiotic. In the serial dilution method, definite
volumes of the media containing the test organism are
placed in test tubes. Various dilutions of the active
substance are added. The highest dilution giving complete
inhibition of growth, Judged by lack of turbidity in the
medium, is considered the end point. A third method is the
agar diffusion method in which the test organism is
inoculated into agar medium, the active agent placed on

the agar, and the rate of diffusion as Judged by absence

6

of bacteria around the antibiotic recorded. The rate of
diffusion is directly proportional to the concentration
of the antibiotic substance.

Antibacterial substances have been classified
chemically as lipoids, pigments, polypeptides,
sulfur-containing compounds, quinone-like substances,
and organic bases. Their mode of action has been studied
by Waksman ('45). By far the greater percentage are
bacteriostatic, arresting growth but not killing the test
organism. Some are bactericidal, bacteriolytic, and even
fungicidal. They are variable in their toxic properties.
Some, such as pencillin, are relatively non-toxic while
the toxicity of others prevents their use in therapeutics
until their toxicity is attenuated. Certain antibiotics
are highly selective in action, displaying their antibiotic
properties on only one test organism; others are active on

either gram positive or gram negative organisms.

Antibiotic Properties Of Allium Sativum (Garlic)

Of more immediate interest to this study are the
reports of the antibiotic properties of garlic. Ten’years
ago interest in garlic as a curative agent was renewed
when Walton, Herbold, and Lindegren , ('36) published their
data of the bactericidal effects of crushed garlic. This
was one of the first studies in which garlic was recognized
as an antibiotic producing plant. Using a modification of

the standard plate exposure method,they exposed crushed

7
garlic vapors to nutrient glycerine agar for varying periods
of time. After treatment with the garlic several bacteria
were streaked upon the plates. Complete arrest of Bacillus
subtilis on the plates eXposed to garlic vapors for four
hours was observed. Further work with the active component
showed it to be bactericidal in action. Its volatility was
less marked at 10°C than at 22°. The heated garlic showed
no bactericidal action. That which was water extracted and
then heated showed no or only slight activity.

Huddleson et al.('44), reporting the results of
serial dilution tests on garlic, found inhibition of growth
in 1:40 to 1:160 dilution of the fresh Juice. They found
that the volatile substance was given off over a long period
of time. Preliminary chemical analysis indicated that the
active substance was not an aldehyde or a carbohydrate.

- Vollrath, Walton,and Lindegren ('57) tested the
effects of substances present in garlic which might cause
the antibiotic effects noted. Allyl polysulfides, compounds
giving garlic its characteristic odor, proved of negligible
bactericidal activity. Aldehydes in garlic such as acrolein
(allyl aldehyde) were found to be powerfully bactericidal.
Further study of acrolein showed it to be the only compound
present in garlic with antibiotic activity comparable to
that of garlic. The results of its ingestion by man are
dizziness, nausea, and diarrhea. Similar symptoms were
observed after eating several ounces of garlic. Proof of the

presence of acrolein in garlic was attributed to a specific

color reaction.

Ingersoll, Vollrath, Scott and Lindegren ('58)
tested the bactericidal properties of crotonaldehyde, a
substance suggested as the antibiotic component of
crushed garlic. They found inhibition of Escherichia coli
in dilution of l:l0,000 when the plates were exposed to
garlic, onion, or the chemically pure aldehyde. Water
soaked garlic was shown to loose its bactericidal
properties. The active principle gave a,characteristic
test for unsaturated aldehydes. When heated for distillation
purposes, the garlic did not yield this test, probably
because the proteins combined with the aldehydes before ‘
distilling off.

Huddleson and co—workers ('44), however, feel
that crotonldehyde is not the antibiotic substance in garlic.
The chemically pure crotonaldehyde which they tested had
antibiotic activity in dilutions up to 1: 4,000. Their garlic
extracts inhibited growth of the same organism in 1: 520,000
to 1: 640,000 dilutions. This wide difference in potency
would indicate that some substance more activethan
crotonaldehyde is responsible for the antibiotic action of -
garlic. In more recent work, Jacobs ('44) has proposed the
active principle to be allylpropyl disulfide plus a

tri-sulfur compound.

At present, the published literature has shown

these properties to be a part of the antibiotic activity of

garlic. The active substance is:

 

9
l. Bactericidal in nature
2. Volatile, having its greatest activity
between 10°and 22°C.

5. Active over a long period of time

4. Destroyed or attenuated by water extraction

~5. Sulfur containing

6. Most active on fungi

7. Destroyed by heat

Work is now being conducted at I'vlichigan State
College to ascertain the active principles of garlic, to
isolate the substances, and to show their effect upon
organisms and higher life. Unpublished data from this
study indicates that water extracts of the garlic bulb
retain their antibiotic properties. Garlic, heated for
distillation purposes under partial vacumm, also retains

its bactericidal properties.
Use of Garlic in Treatment of Disease

Claims of therapeutic properties of the garlic
bud haVe persisted from the time of folklore medicine.
Periodically, the use of garlic in treatment of common
physical disorders has been renewed.

Summary of the early literature in which
mention is made of the use of garlic for medicinal
purposes was made by del Valle Atiles ('20). He quotes
the work of Minchin in which a volatile oil made from

fresh garlic was used successfully to cure typhoid fever,

10
tuberculosis, and infantile diarrhea. The oil was said to
be antiseptic in nature and capable of penetrating
epithelial tissue. A similar oil preparation made from
garlic was used successfully on 96 patients suffering from
diarrhea and dyspepsia by Roos ('25).

An infusion of crushed garlic and milk was
credited by del Valle Atiles ('20) as being an effective
vermicide. R100 ('26) showed that alcohol extracts of garlic
were effective in killing g. Ascaris, a species of worm
including intestinal threadworms and similar parasites. An
invitro study of the effects of a 10 percent extract of
garlic upon these worms showed that the garlic caused a
period of intense irritation in the paraSite which was
followed by paralysis and death in lessthan an hour.

Diabetic patients, fed 10 grams raw garlic daily
by Mahler and Pasterny ('24) reacted with a decrease in
glycosuria and an increase in hyperglycemia. Undesirable
effects were noted in two cases when the patients became
refractory to insulin after two days of garlic treatment.

The possibility that garlic extracts may be
toxic is reported by Petrocchi and Gutman ('27) who fed
two children, ill with whooping cough, large quantities
of garlic. After 24 hours, one child displayed a greatly
increased blood pressure, tachycardia, mydriasis, and
myosis followed by death. Less severe toxic reactions
were experienced by the second child.

On the other hand, garlic has often been

11

recommended for the treatment of arterial hypertension
because of its reported dilating action on the blood
vessels. Several attempts have been made to determine
the effects of garlic upon the circulatory system.
Sunzeri ('26), studying the pharmacology of garlic,
injected aqueous and alcoholic extracts of garlic into
dogs and observed an immediate drop in the blood pressure
due to the stimulation of the vagus nerve. Later, the
blood pressure was lowered by the dilating action of garlic
upon the blood vessels. He suggested the use of garlic in
treating hypertension.

Several studies have Since been recorded in
which this suggestion has been subjected to controlled
medical research. Wakerlin and Gaines ('40) studied plants
and hormones said to be of value in the treatment of
hypertension. They administered two grams desiccated garlic
orally to a dog made hypertensive by bilateral constriction
of the renal arteries. No change in the blood picture was
recorded.

A commercial preparation of garlic was fed to
dogs in doses three times the recommended human allowance
by Goldblatt, Kahn and Lewis ('42). No toxic effects were
noted nor was there any change in the hypertensive level.

Such contradictory studies should be investigated
further using sufficient animals to rule out individual
variation in reaction before any claims for therapeutic

values of garlic are made.

12

The Formed Elements of the Blood and the Hemoglobin

Concentrations of Albino Rate As Recorded in the Literature

Thorp and Harshfield ('45) have shown that anemia
results in horses due to large intakes of onion, an
Iantibiotic producing plant of the lily family. Sebrell ('50)
found that onions fed to dogs in quantities of 15 grams or
over per kilogram of body weight caused severe anemia.
Schlotthauer and Berryman ('45) fed dogs 90 grams fresh
onion daily and observed no effect on the hemoglobin
concentration. When the amount fed was increased to 150 grams
per day, severe anemia followed by death occurred in two
animals. Several dogs withstood 200 grams onion per day but
their erythrocyte counts were reduced from 5.2 to 5.7 million
cells per 0mm. of blood. Garlic is alSo a member of the lily
family producing an antibiotic substance. No studies of the
formed elements and hemoglobin concentrations of the blood of
animals fed garlic are reported. The possibility that garlic
may affect the blood in a way similar to onion can not be.
ignored. This study of the formed elements and the hemoglobin
cdncentrations of theiblood of rats fed garlic powder was
made to show any depressive action of garlic upon these
components-of the blood of the animals. °
A. Erythrogyte counts.The formed elements of the blood of
normal albino rats have been the subject of numerous.

investigations. Chisholm ('11) studied 50 rats weighing from

15
50 to 250 grams and reported an average erythrocyte count
of 8.8 million cells per cmm. of blood. The red blood
cell count rose from 6.5 to 7.6 million cells per cmm.
for animals weighing from 25 to 50 grams to 8.4 to 9.0 for
those whose weights were 200 to 250 grams.

Levy ('26) reports 10.5 million red cells as the
average of the 18 animals studied. Vaughan and Gunn ('29),
using standardized pipettes and counting chambers, found I
the average erythrocyte count of four month old rats to be
10.2 million cells per cmm. of blood while the counts of
the six month old animals averaged 9.6. Kolmer and Boerner
('51) report an average of 9.5 million cells per cmm. of
blood.

A compariSon of the red blood cell counts of one
group of animals fed an all-grain stock diet and a second
group fed an adequate synthetic diet was madely Orten and
Smith ('54). Using only male animals to rule out the
fluctuation caused by pregnancy, they found no essential
difference in the erythrocyte counts of the two groups of
animals. At 200 grams, the animals had an average erythrocyte
count of 7.0 and 7.9 million cells per cmm. of blood for
those fed the stock and synthetic diet respectively.

Possible differences inthe erythrocyte counts of
different strains were investigated by Drabkin and Fitz—
Hugh ('54). Wistar and Pennsylvania strains of rats were

selected for the study. No significant strain difference

14
was observed. The average number of erythrocytes in the
rats weighing 200 grams was 8.8 million cells per cmm. of
blood.

Reich and Dunning ('45) have reported the most
extensive study of the blood of eight species of rats.
Rigidly controlled standard blood counting procedures were
followed. The average red blood cell count for the 5,000
rate they examined was 8.7 million cells per cmm. of
blood.They observed small but significant differences
between the various strains with each exhibiting a
characteristic peripheral blood picture. Unfortunately,

the Wistar Strain was not among those studied.

B. Hemoglobin concentrations. Chisholm ('11) studied the

 

hemoglobin concentration of the blood of 50 rats and
reported an average<af88 percent. Levy ('26) obtained
’94.5 percent as the average of 68 determinations while
Goodall ('10) reports 110 percent. A comparison of these
results is impossible because in no case are the number of
grams of hemoglobin represented by 100 percent given. More
recently, the hemoglobin concentrations of the blood have
been reported as grams per 100 ml. of blood. Drabkin and
Fitz-Hugh ('54) found a range of hemoglobin concentration
in rats of 17.0 to 20.0 grams per 100 m1. of blood. The

hemoglobin concentrations reported by Orten and Smith ('54)

15
for the animals fed the stock diet and synthetic diet
ranged from 15.7 to 16.0 grams per 100 ml. blood. There
were no differences in the dietary groups. Thewlis and
'Meyer ('42) found the hemoglobin concentration of the animals
weighing 125 to 200 grams to be 14.1 grams per m1. of blood;
while that of animals weighing 201 to 450 grams averaged
15.8. Reich and Dunning ('45) report a hemoglobin concentration

of 14.2 as the average of the 5,000 animals studied.

0. Bgucocyte gounts. Total leucocyte counts are reported by
Thewlis and Meyer ('42) as 18,819 cells per cmm. of blood;
by Goodall ('10) as 10,600; by Stammer ('26) as 9,800; and by
Kolmer and Boerner ('51) as 15,200 cells per cmm. of blood.
Lotta and Hemderson ('57) present the average leucocyte
count of the blood of rats as 18,000. Reich and Dunning ('45),
averaging the values found in the literature, report a range
of 8,000 to 15,000 cells per cmm. of blood. Initheir own
extensive study, the average leucocyte count was 15,800 cells
per cmm. of blood. The results of the studies cited from the
literature are shown on Table 1.

The average differential white count recorded by
Reich and Dunning ('45) was: neutrophils, 27 percent (range
15 to 40 percent); lymphocytes, 67 percent (range 50 to 80
percent); monocytes, five percent (range two to seven percent);
eosinophils, two percent (range zero to four percent);

and basophils, 0.7 percent ( range zero to 1.5 percent),

16

Growth Studies

'Few laboratories have reported the weights of
albino rate over the entire growth period. Brody ('42)
records the growth of suckling rats of the Wistar Strain
in normal size litters of seven to nine young and in
litters with the young reduced to two or four. The average
weight at 21 days of age when the animals were weaned was
28 grams and 47.5 grams for animals of the large and the
small litters respectively. The author points out that the
difference is undoubtedly due to the more abundant milk
supply available to the young of the smaller litters.

Orten and Smith ('54) found the weight of rats
at 21 days of age to be 42 grams. At 50 days, the young
rats fed an all grain diet averaged 66 grams. At 120 days,
.approximately the time of the first mating inthis
experiment, the animals averaged 590 grams. Animals in their
laboratory fed a synthetic diet, were found to be slightly
lighter, weighing only 545 grams at 120 days of age.

Drabkin and Fitz-Hugh ('54) report the weights
of Wistar rats fed a diet of natural foodstuffs. The
average weight of thesanimals at 50 days of age was 51 grams;
at 120 days it was 258 grams.

Because of the great variation in animal growth
due to differences in diet, climate, temperature, and strain

variation, values reported in the literature are less valuable

 

 

17
TABLE 1

Erythrocyte and leucocyte counts and hemoglobi- concentrations
of albino rats as reported in literature

 

 

 

 

Investigator Erythrocyte Leucocyte Hemoglobin
Count Count Concentration
"~r : " - "1 - - In K" '~», .137" 4‘ C'-
hllilon cells Cells per Grams per 1C0 uv~-.~~w
per cmm. blood cmm. blood ml. blood
Chisholm, '11 8.8 87.8% 27 male, 25 fema-e=
EC to 1:0 gram weight
Levy, '26 10.5 25,400
Vaughan
and Gunn, '29 10.2

0

Four month old rats
Slx month old rats

Kolmer and
Boerner, '51 9.

Cal

15,200

Orten and
Smith, '54

0
<9 G
H
03
..Q
C...
O
(...!
on
I
Z)

200 gram weight; fed all
grain stock diet.

q~d

200 gram weight; fed
synthetic die..

 

Drabkin and

i FitZ*Hush '54 8.8 17.0 to 2

 

(D

K Reich and

Dunning '44 8.7 15,800 14.2 Average of 5,000 animals

Goodall, '10 8.10 10,600 110 T

Thewlis 7.65 2:,072 1a.l 125 to 2'0 gram eigit
and.1¢eyerg '42 £5.55 lEg'llo 1[. 01- 03 4:. Franl vei:-rt
btammer, '25 9,830

Lotta

and

Henderson,'57 18,000

 

18
than the actual growth of the experimental animals compared
with the growth of the stock colony animals from which they
were derived. Such a comparison will be discussed in another

portion of this paper.

19
EXPERIMENTAL PROCEDURE
Plan of Feeding Animals

Eleven male and eleven female rats of the Wistar
Strain reared from stock colony animals in the Foods and
Nutrition Department, Michigan State College, were used for
the experiment. The animals, selected at 28 days of age,
were fed a modification of the Evans diet (Evans and Burr,
'28) shown in Table 2. Three different feeding regimes were
planned. One was a control diet containing no garlic powder;
the other two were diets containing 2.5 and five percent dry
garlic powder, respectively} Four male .and four female rats
were placed upon the diet at weaning. A like number were fed
the diet containing the 2.5 percent concentration of garlic.
Due to a lack of animals of the necessary age, only three
male and three female animals were fed garlic at the five
percent concentration. The grouping of the animals for the
experiment is shown in Table 5.

The diet was mixed weekly with the exception of
the garlic powder and refrigerated until used. The garlic was
added Just prior to feeding in the concentration of five and
2.5 percent, replacing the cornstarch from the control diet.
This represents a concentration 20 and 10 percent fresh

garlic. At the beginning of the experiment, the animals were

 

l. The product was Dehyrated Garlic, manufactured by the
Basic Vegetable Products Company, Vacaville, California.
Acknowledgment is made to Dr. Lucas of the Horticulture
Department, Michigan State College, for testing the
antibiotic potency of the diet.

Ingredient

Cornstarch
Casein

Dried-brewer's
yeast

Osborne—Mendel
Salts

Wesson.0il
Cod Liver Oil

Garlic Powder

20

TABLE 2

Experimental diets

5 percent 2.5 percent
Garlic Garlic
Grams Grams
59.0 61.5
18.0 18.0
8.0 800
4.0 4.0
5.0 5.0
1.0 1.0
5.0 2.5

 

100.0 100.0

Control
No Garlic

Grams

«64.0

18.0

8.0

4.0
5.0
1.0

0.0

100.0

21

TABLE 5

Grouping of experimental animals according
to litter number.*

Experimental Diet 1 Experimental Diet 2 ExperimentalDiet E
5% Garlic 2.5% Garlic No Garlic
Female Male Female Male Female Male
1 l l l l l
2 2 2 2 2 2
5 5 5 5 5 5
4 4 4 4

* All the animals designated by the same number

were litter mates.

22
fed daily. Later, assay indicated that the antibiotic
potency of the garlic diets did not change over a 48 hour
period. Thereafter, animals were fed every other day.
Further assay both of the complete diet and the individual
dietary components showed no antibiotic potency in the
basic ration. Therefore, any antibiotic action or variation
of the experimental groups of animals from the control may
be attributed to the effect of the garlic powder used in

the diet.
Animal Grouping

Litter control was maintained by placing a male
and female from each litter upon each of the three diets.
Thus all the animals designated in Table {3 by the number
one (1) were litter mates. The rats were caged separately
on wire racks to enable the accurate collection of food
consumption records. Weighed amounts of food were offered
and the amount not consumed by the next feeding plus any
Spilled was subtracted from that fed to ascertain the food

actually consumed.
Weight Records

Weekly weights were recorded before feeding to
prevent any change in weight due to the recent ingestion of

food.

25
Mating of Animals

The animals were first mated when three months
of age following the brother-sister mating technique. A
male was placed with its sister female of the same
dietary group for ten days. The females were placed on
clean wood shavings when the litters were expected. The
number of young was reduced to six or seven, two days
after birth, and weaned when the smallest animal weighed
40 grams. The animals were mated for the second time 10
weeks after the first mating. The young of the first
litters were discarded. Two male and two female animals
from each of the second litters are being kept upon the
respective diets to determine any long time effects due

to the feeding of dry garlic powder.
Formed Elementsof the Blood.

Examination of the blood was begun when the
animals had been on the diets four weeks. Thereafter, a
study of the formed elements of the blood was made on
each animal approximately every 50 days until a total of
six studies had been completed. Blood from a tail clip
was collected into paraffin cups containing a few
crystals of dry heparin to prevent coagulation. Duplicate

blood samples were taken in red and white Trenner pipettes

24
certified by the U. S. Bureau of Standards. The blood for
the erythrocyte count was diluted 1: 200 with Hayem's
solution. The pipettes were shaken for three minutes to
insure even distribution of cells, a few drops expelled,
and the two chambers of the Neubauer Improved Hemocytometer
with U. S. Bureau of Standard certification, filled.
Chambers which filled rapidly, without overflowing or air
bubbles, were inspected for even distribution of cells. If
the cells were unevenly distributed, the chamber was not
counted. If the chamber was accepted for counting, the
results were recorded regardless of differences in successive
counts. The cells in 80 small squares were counted; the
average of the four chambers was recorded as the erythrocyte
count.

Blood for the leucocyte count was diluted 1:10
in a solution of acetic acid and methylene blue. The cells in
the 400 small Squares of each chamber was counted, totaled,
and divided by two. The average of two such counts was
recorded as the leucocyte count.

The method of Sheard and Sanford ('29) for the
estimation of the hemoglobin concentration of the blood, as
adapted for use in the photo—electric colorimeter, was
followed. Two-tenths of a cubic milliliter of blood was
blown into 20 ml. of a 0.1 percent solution of sodium
carbonate and allowed to stand at least ten minutes to insure
complete laking of the blood and color development. The
solution was read in a Cenco Photolometer against distilled

water set at a reading of 100, using a green filter with

25
maximum transmission at 525eyy. Conversion from the
reading to hemoglobin concentration was made by referring
to the chart supplied by the manufacturer of this
particular instrument. The hemoglobin concentration for
each galvanometer reading had been determined previously
by the oxygen capacity method.

Duplicate blood smears were made upon the glass
slides, quick dried, and stained with Wright's stain. The
percentage of lymphocytes, monocytes, neutrophils,
eosinophils, and baSOphils in 500 consecutive cells was

determined.

Autopsy

Gross autopsies were conducted upon the
chloroformed animals at the end of the eXperimental period.
Particular attention was paid to possible lung infection
since previous work with the stock colony had shown a high
incidence of lung lesions to be present. All abnormal
tissues were saved for histological study, the results of
which will be reported by another worker from this

laboratory.

26
RESULTS AND DISCUSSION

This work is a part of a long time study of the
effects of antibiotic substances when fed in large quantities
to eXperimental animals. The experimental procedure is being
repeated upon another series of rats by another investigator
so that when the data are pooled the experiment will
represent the action of garlic on five male and five female
animals per diet group. No definite conclusions can be drawn
from this study until the data from the second series can be
examined. The results here reported were those shown by the

.first group of animals.
Grcwth and Development

The average weekly weights of the male and female
animals maintained upon the three diets are shown plotted on
Figure 1. The weights at weaning of the animals used in this
study were comparable to those reported by Orten.(”56) and
Drabkin ('54). However, the experimental animals did not
gain the weights following weaning which were reported by
these authors as normal for their colonies. Since the failure
may be due to dietary or species differences in the growth
patterns of the two groups of animals, some other criteria
for Judging the normalcy of weight gains during the growth
period should be adopted. A comparison of the weights of the

N

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28

experimental animals with those of equal age from the stock.
colony from which they had been bred is a more satisfactory
way to Judge growth. Therefore, the average weights of the
female stock colony animals of the same age are plotted with
those of the experimental groups. Similar weight records
were not available for the stock colony males. The average
weekly weight of the stock colony and experimental female
animals were comparable. The general growth pattern of the
stock animals was maintained by those fed the simplified
dietary. Therefore, the experimental diet is as adequate as
the stock diet in promoting good growth. A study of the
weight graphs alone would lead to the assumption that the
simplified control diet sustained better growth than did the
2.5 or five percent garlic diets. The animals made greater
gains in weight when fed garlic at the 2.5 percent
concentration than when fed garlic at the five percent
concentration. Some depression in growth might be attributed
to the presence of garlic in the basic diet.

(However, when the weight gains were calculated as
a gain per gram of food intake (Table 4), it became apparent
that there was no actual depression of growth due to the
feeding of garlic. The lower weights of the animals fed the
ration containing five percent garlic may be attributed to a
smaller intake of food since the weight gain per gram of
food was essentially the same as that made by the heavier

control animals. The weight gain per gram of food consumed

29
was .058, .067, and .057 grams for the female rats fed
five percentgarlic, 2.5 percent garlic, and no garlic
respectively. Male rats gained .O95,.106 and .100 gram
per gram of food on the same three diets. No explanation
of the slightly higher gain per gram of food ingested,
made by both the male and the female animals fed 2.5
percent garlic, is known at present. It is possible that
garlic in some way influences theeconomy with Which the
animals utilize their food.

It is interesting to note that the male animals
gained almost twice the weight per gram of food consumed
'as did the female animals. Autopsy records showed that this
extra gain was not reflected in abnormal fat deposits.

All the females bore first litters well within
the time interval for normal gestation. Only one female
failed to produce a second litter. When she was remated to
another male of the same dietary group but not the same
litter, impregnation resulted. The male with whom she had
first been mated later failed to impregnate another fertile
female. Some loss of fertility must have occurred in the
male between the two matings. No external signs of physical
difficulty were observed nor were any gross abnormalities
seen at autopsy. However, the study of the red blood cell
system of this particular animal at the time he was mated
to the experimental female, showed an extremely low

erythrocyte count and hemoglobin concentration which could

50

TABLE 4
Average weight gains per gram food consumed

Average Average Average

Diet total weight grams food weight gain
gain consumed per gram food
consumed
5% Garlic
Female 162 2,820 .058
Male 285 2,990 .095
2.5% Garlic
Female 197 5,005 .067
Male 526 5,091 .106
Control
Female 192 5,555 .057

Male 547 5,465 .100

'51

not be attributed to poor technique. The lower than average
erythrocyte and hemoglobin concentrations may have resulted
in sterility.

Only four female rats nursed their first litters.
From 59 to 98 percent of the young were lost. This lactation
failure was traced to a manganese d991ciency in the mineral
salts used in the diet. Sufficient manganese had been
supplied to the animals for the first two months after
weaning. During the third, fourth, and fifth months a salt
mixture deficient in manganese was inadvertently used. The
manganese content of the salts was only one tenth that
recommended by Griffith ('42) as necessary for normal
lactation. Manganese deficiencies in rats are characterized
_ by normal pregnancies but failure of the females to nurse
their young followed by a high degree of cannibalism. These
symptoms were those maifested by the experimental animals.
The diet was immediately corrected by the addition of
manganese sulfate. Shils and McCollum ('45), studying
manganese deficiency in rats, reported that addition of
manganese to the diets of deficient animals resulted in
successful mating, lactation, and rearing of young. They
concluded that manganese deficiencies are readily corrected
by the addition of sufficient amounts of manganese in the
diet. In the present study, the animals, supplied with
manganese during a second gestation period, were able to

wean 58 percent of the young of the control group. Seventy—

.32
five percent of the young from females fed 2.5 percent
garlic were weaned while only 47 percent of the offspring
receiving five percent garlic survived. Four of the
eleven females failed to nurse the young. It is interesting
to note that the females fed the 2.5 percent garlic diet
were able to nurse and wean a greater percentage of young
than those fed the other two diets. One female of the
control group bore twelve young but failed to rear them.
This may account for the low percentage of the young of the
control animals weaned.

Twenty of the animals were sacrificed after an
experimental period of 56 weeks. The other two were kept for
further study. Careful macroscopic autopsies were conducted
upon the chloroformed animals to determine if any variation
due to the feeding of garlic powder in 2.5 and five
percent concentrations occurred. Because of the chronic
lung infection in the stock colony animals, particular
attention was paid to determine the extent of lung lesions
present in the experimental animals. The lesions were
characterized by dark spots both on the surface and
throughout the lung lobes which sometimes were full of pus
or serous fluid, sometimes apparently calcified. All degrees
of infection were noted in the eXperimental animals from a
very minimal amount involving only one lobe to an amount
involving every lobe and apparently rendering most of the

lung tissue incapable of action. Respiratory embarrassment

55
characterized by wheezing and difficulty in breathing had
been observed in the stock colony animals. These animals were
seen to have marked lung abnormalities when they were
autopsied. Respiratory difficulty was noticed in only two
experimental animals. One was a female fed the five percent
concentration of garlic; the other a male maintained on the
control diet. Their lungs were found upon examination to be
infected to a moderate degree. Two animals of the six fed
five percent concentration of garlic were infected to a
moderate degree. One was not killed. This represents 40
percent moderate lung infection. Of the eight animals fed the
2.5 concentration of garlic, two were infected to-a marked
degree, one was not killed, and five were free of lung
abnormalities. Only 29 percent of the animals fed 2.5 percent
garlic were infected but the infection was more severe than
that of the animals fed five percent garlic. Autopsy of the
animals fed no garlic showed five animals with extreme lung
abnormalities. This represents 65 percent extensive infection.
Huddleson and others ('44) have suggested that ingestion of
antibiotic substances may have practical use in preventing
infectious disease. The feeding of the antibiotic substance
in garlic did not prevent lung infection. Any protective
effect of garlic would seem to be one’of degree with fewer
animals fed garlic having lesions and the lesions being less

extensive.

54

The Formed Elements of the Blood

 

Wintrobe ('44) studied the hematological system
of the ratzmmd found that the formed elements of the blood
reached adult values by the end of the second month. Since
the erythrocyte and leucocyte counts and the hemoglobin
concentrations reported in this study were first made when
the animals were threemonths of age, adult values may be
assumed. Melcoff ('44) has reported that three weeks are
ample time for both hemoglobin and red blood cell
regeneration after the moderate bleeding necessary to secure
the blood for study. Blood was drawn monthly in this study.

The average monthly erythrocyte and leucocyte

counts and the hemoglobin concentrations of the various
groups of female animals are presented in Table 5, the male

values are shown in Table 6.
Erythrocyte counts

The range of red blood cells of the female
animals fed the higher concentrations of garlic was 6.18 to
8.98 million cells per cmm. of blood, those fed the lower
amount of garlic ranged from 6.92 to 8.89, while the control
animals ranged from 7.59 to 9.20 million cells per cmm. of
blood. The total range of the male animals was much smaller
than that of the female. As was to be eXpected, higher red
blood cellcpunts were recorded for the male animals. The

range of counts was 8.20 to 9.10, 8.54 to 9.44, and 8.55 to

55

TABLE 5

Average monthly erythrocyte counts, leucocyte counts,

and hemoglobin concentrations of eleven female albino

rats fed varying concentrations of dry garlic powder.

Diet Month Erythrocyte Leucocyte Hemoglobin
Counts Counts Concentrations
Million cells Cells per Grams per 100
per cmm. blood cmm. blood ml. blood.
5% Garlic August 7.81 7,067 15.9
September 7.92 7,550 12.8
Oct.-Nov.* 6.18 7,975 11.8
December 8.89 9,858 15.6
January'* 7.42 9,158 15.6
February 8.54 9,658 15.5
2.5% August 8.15 10,925 12.6
Garlic September 8.50 7,415 16.0
Oct.-Nov.* 6.92 10,569 12.1
December 8.89 8,450 15.4
January* 7.99 11,812 14.0
February 7.82 10,505 15.2
Control August 8.80 7,775 16.5
No Garlic September 9.21 6,625 17.0
0ct.—Nov.* 7.77 7,951 12.5
December 9.28 9,456 15.8
January* 7.59 9,981 14.1
February 8.80 9,667 17.5

* Blood examined as near partuition as possible

TABLE 6

Average monthly erythrocyte counts, leucocyte counts and

hemoglobin concentrations of eleven male albino rats fed

varying concentrations of dry garlic powder.

Diet

5% Garlic

2.5%
Garlic

Control
No
Garlic

1710 nth

August
September
Oct.—Nov.
December
January
February

August
September
0ct.-Nov.
December
January
February

August
September
Oct.-Nov.
December
January
February

Erythrocyte

Counts

Million cells

per cmm.

8.20
8.42
8.76
8.97
9.10

8.52'

8.66
8.54
8.90
9.90
9.51
9.44

8.55
8.88
9.5

9.15
9.01
8.82

blood cmm.

Leucocyte
Counts

Cells per

8,142
6,592
7,700
7,892
7,850
8,885

6,150
6,550
6,798.
8,786
8,188

10,565

8,175
7,015
8,588
10,094
10,606
10,425

blood

Hemoglobin

Concentrations

Grams per 100
blood.

m1.

14.5
14.5
14.5
14.5
15.2
14.8

15.5
14.8
15.7
16.4
16.1
15.7

17.2
15.2
15.7
15.5
15.5
15.6

57
9.15 million cells per cmm. of blood for the animals fed
five percent, 2.5 percent, and no garlic respectively. The
total range of counts previously reported in the literature
was 6.50 to 10.5 million cells per cmm. of blood. This
close agreement with the values recorded in the literature
would seem to indicate that feeding garlic in the
concentrations used in this study has little effect upon
the total red blood cell count.

However, when the data were subjected to an
analysis of variance in which the interaction of the diets
and months was considered, a highly significant differenCe
was found between the erythrocyte counts of the female rats

fed five percent garlic and the control animals ("t" = 4.16-

with 40 D. F.). A significant difference occurred between
those fed 2.5 percent garlic and the control group (“t" ; 2.9
with 46 D.F.). No difference in the red blood cell count was
found between the animals on the five and 2.5 percent garlic
diets ("t" = 1.42 with 40 D.F.). The lower red blood

cell counts exhibited by the animals fed garlic show a

small depression of the total circulating erythrocytes
associated with the ingestion of garlic.

Male animals show no significant change in the
erythrocyte count between the three diet groups. The "t"
test between the groups ingesting five and 2.5 percent
concentrations of garlic was 1.8 with 40 D.F., between
animals on five percent garlic and control diets, was 1.56

with 40 D.F. and between the groups on 2.5 percent garlic

58
and the control diets, was 0.4 with 46 D. F.

Further analyses of the erythrocyte counts of
the female and the male rats were made to determine between
which months and which feeding regimes significant
variation could be noted. Significant differences in the red
cell counts were observed between the female animals fed the
five percent garlic and the control animals for the first
three months. The erythrocyte counts of the animals fed 2.5
percent dry garlic were significantly lower than those of
the control animals for the first and third months. A
difference was noted between the animals eating the five
and 2.5 percent garlic diets during the third month. Failure
to display a variation in later months may be due to animal
adjustment to the diet fed. There was no apparent reason
for the lack of variation in the second month for those fed
2.5 percent garlic and those fed control diets. The
significant variation between the animals ingesting the two
garlic diets, observed in the third month, may have been
caused by a depressive action upon the red cell system of a
larger total intake of garlic due to increased food
consumption concurrent with pregnancy. The possiblity of
interaction with the effects of the manganese deficiency
should not be overlooked.

At no time was there a significant
difference in the erythrocyte counts of the three groups of
males. The "t" values for monthly comparisons for both

the male and female animals are shown in Table 7.

59

TABLE 7

Differences in the monthly erythrocyte counts of the

male and female animals as shown by the "t" test.

Date

August
September

October—
November

December
January

February

"t" Tests

Between 5% Garlic
and Control Diets

Female
4.95**

5.16*

6.07**

1.70
.67
.48

Male
.91

.56

.78
.50
.16
1.25

Between 5% and
2.5% Garlic

Diets
Female
1.40

'1.46

5.45*
0.0
1.01
1.68

Male
1.14
1.41

.156
1.01

Between 2.5%
Garlic and
Control Diets

Female Male
5.46* .22
1.70 .42.
2.60* .47
1.70 .76
.55 .50
2.16 .55

* One possibility in 20 that results could be attributed to

chance.

** One possibility in 100 that results could be attributed to

u u
t .05

u u
t .01

2.565 with D.F.

chance.

40

B. Hemoglobin concentrations. The average hemoglobin

 

concentrations are presented in Tables 5 and 6. The range
of hemoglobin concentrations of the female rats was 11.8

to 15.5 grams per 100 m1. of blood for those fed five
percent garlic, 12.1 to 15.2 for those fed 2.5 percent
garlic, and 12.5 to 17.5 for the control animals. As with
the red cell counts, the male animals showed less variation
in total range. The ranges of hemoglobin concentrations

of the male animals were 14.5 to 15.2, 14.8 to 16.4, and
15.2 to 17.2 for the five percent garlic, 2.5 percent
garlic and the control diet groups respectively.

The lower range of the hemoglobin concentrations
of the female animals is below that cited from the
literature (15.7 to 20 grams per 100 ml. of blood). In‘
most cases, values below 15 grams were found in studies
made Just after partuition when great drains had been made
upon the blood supply of the animals. iscounting these
pregnancy changes the hemoglobin concentrations of the
female animals are within average ranges reported in the
literature. Male hemoglobin concentrations were well within
the range of average values reported in the literature.

A statistical analysis of the hemoglobin
concentration of the blood of the female animals showed a
significant difference in the concentrations between the

animals fed the five percent garlic and no garlic ("t" 3

5.59 with 40 D.F.) while no significant difference in

41
hemoglobin concentration occurred between the animals

fed 2.5 percent garlic and no garlic ("t" 3 1.72 with 46 D.F)

or the animals fed the five and 2.5 percent garlic diets
("t" : 2.01 with D.F.).

Analysis of the monthly concentrations showed
a significant variation between the hemoglobin
concentrations of the animals fed five percent garlic and
control animals for the first and fourth months. No
other significant differences were noted. These values are
recorded in Table 8. The lower concentration during the
first month may be attributed to some difficulty in
adjusting to the garlic in the diet but no reason is
apparent for the change seen in the fourth month. The
blood study of the fourth month was done approximately 50
days after the birth of the first litters which should
allow ample time for regeneration of the blood. Since
few females nursed their young, depletion of the hemoglobin
forming materials due to lactation cannot be assumed.

Ohlson et a1 ('44) showed the red blood cell

count to be the least changing and therefore probably
the most accurate measure of the normalcy of the red blood
cell system. Since no significant differences were noted
in theerythrocyte counts of thenmle animals and since
inspection of the hemoglobin concentrations showed

homogenity of data, statistical analysis did not appear warranted.

42
TABLE 8

Differences in the monthly hemoglobin concentrations of

the female animals as shown by the "t" test.

"t" Tests

Date Between 5% Garlic Between 5% and Between 2.5%

and Control Diets 2.5% Garlic Garlic and

Diet. Control Diets

August 5.50* 1.60 1.60
September 2010 lo 64 046
October—
November 1.27 .54 .75
December 2.71* 2.22 .49
January .40 .52 .18
February 1.60 .55 2.28

One possiblity in 20 that the results could be attributed

to chance.

"t" .05 : 2.565 with 8 D. F.

"t" Cfl.= 5.499 with 8 D. F.

45

C. Leucocyte counts. The average total leucocyte

 

counts of these animals are shown in Tables 5 and 6.
The range for the female rats fed the five percent
garlic diet was 7,067 to 9,858 cells per cmm. of
blood; for those fed 2.5 percent garlic 7,415 to 11,812;
and for the control animals 6,625 to 9,981. Male animals
had leucocyte counts ranging from 6,592 to 8,885; 6,150
to 10,565; and 7,015 to 10,606 cells per cmm. of blood.
The range in the,literature cited was 8,000 to 19,000.
The average leucocyte count of each dietary group
studied is lower than those reported in the literature.
The significance of, or the importance of, a lower than
average white count is still unknown. It is possible
that higher than average values resulting from counting
techniques less strictly standardized than those used
in this study are recorded in the studies cited. Because
there was no trend shown in the white cell count either
by the individual animal or in averages of the various
groups, no statistical analysis of the data was made.
The differential white cell counts are recorded
in Tables 9 and_10.Due to the.wide individual variation,
the results are presented for each animal instead of as
the average of groups. The differential counts failed
to show any trends dueto the feeding of garlic. All the

values were within the range found as average by Reich ('45).

TABLE 9

The differential white blood cell counts of eleven female albino rats

 

October-November

September

August

__A

Month

 

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lttqdossg

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Percent of 300 cells

Percent of 300 cells

Percent of 300 cells

5%
Garlic

 

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000
MON
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8
89
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2.54 Garlic

 

 

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HOHN

r~r~cnj

r—uwouw
N\FVGN\

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N\O MM

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0000
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Control

 

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MMMN
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‘ 100 cells counted

# 400 cells counted

 

February

 

January

TABLE 9- cont'd

 

The differential white blood cell counts of eleven female albino rats
Decem er

 

 

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Animal

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Percent of 300 cells

Percent of 300 cells

Percent of 300 cells

Garlic

 

 

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September

TABLE 10

August

The differential white blood cell counts of eleven male albinorats

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TABLE 10 -cont'd

The differential white blood cell counts of eleven male albino rats
December

 

 

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48

While studying the differential blood slides an
unidentified protozoa—like organism which stained blue
with Wright's Stain was observed on some of the slides.
Attempts to identify the organism have failed. No
mention has been found in any available literature of
such an abnormality in rat blood. Attempts were made to
correlate its presence with the total white blood cell
count but no correlation could be found (r 3- .058)
Studies to isolate, grow, and identify the organism

are now in progress. The results of this work will be
reported by another worker from this laboratory. There
is no group difference in the prevalance of the organism
on the slides and presumably in the blood stream of the
animals. The antibiotic effect of garlic did not prevent
infestation with the organism. However, antibiotic
substances have been shown to be highly selective in
action. Garlic is more active against fungi than other
forms of microorganisms. Failure to prevent this infection
must not be considered as invalidating the antibiotic

claims made for garlic.

49
SUL’iMARY

The growth, development, and the formed elements
of the blood of albino rats fed varying concentrations of
dry garlic powder have been studied and general trends
recorded.

Growth of the female animals on the experimental
diets was shown to be comparable to that of the stock
colony females. Weight gains per gram food consumed were
greater for the male and female animals fed 2.5 percent
garlic.

Normal gestation periods were observed. The
failure to wean the first litters was traced to a manganese
deficiency in the diet. Second litters, given the recommended
manganese allowances, were more successfully reared.

Autopsy records showed the only abnormality to be
a chronic lung infection. It is possible that the antibiotic
effect of garlic was responsible for the lesser prevalence
and less severe cases of lung infection noted in the animals
fed garlic.

The total red blood cell count and hemoglobin
concentrations reportadare within the average range found in
the literature. No permanent change in the red blood cell
system was produced by the addition of garlic in the diet.

Significantlowering of the red blood cell count for the first

50

three months observed in the female animals may have been
caused by the action of garlic upon the animals. The lowered
hemoglobin concentration of the female animals fed five
percent garlic may also have been due to some depressive
effect of garlic.upon red blood cell formation.

The white blood cell system was apparently
unaffected by the inclusion of garlic in the eXperimental
diets.

The presence of an unidentified protozoa—like
substance on the Stained differential slides was noted.
Further study is being conducted to determine the characteristics
of this organism.

The eXperimental procedure has been duplicated upon an
additional group of animals. Results of both studies will be
pooled when the second study is completed. Until this is done
and the trends recorded in this paper are either sustained or
changedgno definite results of the feeding of dry garlic

powder to albino rats can be claimed.

51
LITERATURE CITED

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52

Question and Answer section. 1934 J.Am. Med. Assn.. vol. 3.

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Kolmer, John A. and Fred Boerner 1931 Approved Laboratory
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Levy. Margarita 1926 Zur Hamatologue der weissen Mans und
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53

Roos. E. 1925 Garlic Treatment of Dyspepsia. Munchener
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54

Walker. J.C.. Carl C. Lindegren and Freda M. Bachmann 1925
Further studies on the toxicity of juice extracted
from succulent onion scales. J. Agr. Res..v01. 30.

pp. 175-187-

Walton. Lucile. Milton Herbold. and Carl C. Lindegren 1936
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J. Anatomy. vol. 58. pp. 313-328.

 

 

 

 

 

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