k COMPARISON OF- CER’I’AIN SAEIVARY PRQPERTIES
FROM SPECIFIC MAJOR SALIVARY GLANDS 9F
CARIES RESISTANT AND CARIES SUSCEPTIBLE RATS

Thais for flu Dean. of Ph. D.
MICHIGAN STATE UNIVERSITY
CharIes 1. Sylvester
i961

THESIS ’ RARYL

(L ~W L I B
Michigan State

University

 
 
  

“‘1‘:

——-—

This is to certify that the
thesis entitled

”A Comparison of Certain Salivary Properties from
Specific Major Salivary Glands of Caries-
Resistant and Ceries-Susceptible Rats”

presented by

Charles J. Sylvester

has been accepted towards fulfillment
of the requirements for

M..— degree inflamhiology and
Public Health

".
\.—‘ . 7 1 p f: }\ A! .v '
,Iq M, 3-4)- & 71/; J ALI "/I' f' 1/ /

Major professor [/7 \

 

0-169

ABSTRACT
A COMPARISON OF CERTAIN SALIVARY PROPERTIES

FROM SPECIFIC MAJOR SALIVARY GLANDS OF
CARIES RESISTANT AND CARIES SUSCEPTIBLE RATS

By Charles J. Sylvester

Certain physical, biochemical and biological proper-
ties of parotid, submaxillari-sublingual, and whole rat
saliva were studied in order to understand further the fac-
tors that contribute to resistance and susceptibility to
dental caries.

Specific salivary glands were removed to provide parotid
and submaxillari-subllngual salivas. Littermate unoperated
rats were used to supply whole saliva. The secretions*were
collected from anesthetized animals by pilocarpine stimula-
tion.

To study the effect of whole and submaxillari-subllngual
saliva from resistant and susceptible rats on microorganisms,
the growth rates of rat oral lactobacilli and rat oral
streptococci were determined photometrically. Five out of
six strains of lactobacilli were not stimulated by any of
the salivas tested when compared with a saline control. On
the other hand, the four strains of streptococci tested‘were
stimulated by all salivas tested. This stimulation was evi-
dent in the maximum limits of growth, but not in the rate of
growth as compared with a saline control. Whole saliva, but
not submaxillari-sublingual saliva, from susceptible rats
, supported a greater average maximum amount of growth of the

streptococci, than did whole resistant saliva.

Charles J. Sylvester

Amylase activity of rat saliva was expressed as milli-
grams of reducing sugar as glucose fonmed per milliliter of
saliva (using an excess of soluble starch as substrate) at a
constant time and temperature of reaction. More than 99
percent of the amylase activity of saliva originated in the
parotid glands of these animals. Parotid and whole salivas
from susceptible rats showed greater amylase activity than
these salivas from resistant rats. However, no significant
correlation existed between amylase activity and caries ac-
tivity. 5

Relative viscosity was determined by timing the fall
of saliva between twolmarks on a narrow bore glass tubing,
and dividing by the time required for distilled water to
drop the same distance. No difference in relative viscosity
was found in the parotid secretions between the two lines of
rats. Whole and submaxillari-sublingual salivas from re-
sistant rats were more viscous than these salivas from sus-
ceptible rats, when collected at room temperature. However,
when whole saliva was collected in tubes submerged in ice,
the difference between the resistants and susceptibles no
longer existed. Comparison of salivas within the lines of
rats showed parotid saliva to be less viscous than submaxil-
lari-sublingual saliva; whereas, whole saliva gave inter-
mediate values. A correlation analysis of caries experience
and relative viscosity revealed no significant relation

between these two traits.

Charles J. Sylvester

The rate of flow of saliva from pilocarpine-stimulated
glands was recorded on a milliliter per minute basis. There
was no essential difference in the rates of flow in the
three types of saliva from resistant and susceptible rats.
However, within each line of rat, the rate of flow between
salivas differed materially. Whole saliva from unoperated
rats showed the greatest flow rate, parotid saliva was the
slowest, and submaxillari-sublingual saliva was intermediate.

The pH of saliva was measured by a Beckman Glass Elec-
trode pH Meter. The mean pH values of the various salivas
tested fell within a narrow'range, indicating that there
was no significant difference between the salivas.

Buffering capacity was determined as titratable alka-
linity; that is, the number of millilieters required to ad-
just one ml of sallva, diluted one to five, to pH 4.5 3 0.2.
Whole and submaxlllari-sublingual saliva from susceptible
rats had a significantly greater buffering capacity than
these salivas from resistant rats, but this property was not

correlated with caries activity.

A COMPARISON OF CERTAIN SALIVARY PROPERTIES
FROM SPECIFIC MAJOR SALIVARY GLANDS OF
CARIES RESISTANT AND CARIES SUSCEPTIBLE RATS

BY

y‘
at
Charles J?QSylvester

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Microbiology and Public Health

l96l

TABLE OF CONTENTS

INTRODUCT|0N0900000......0.00.0.0...OOOOOOOOOOOOIOOOOO I
GENERAL METHODS AND MATERIALS......................... 8

EFFECT OF SALIVA ON GROWTH OF MICROORGANISMS.......... II
Literature Survey................................. ll
Methws andMateri815000000OOOOOOOOOOOOOOOOOOOOOOO '6

l. Collection and Pretreatment of Saliva...... l6
2. TestOOOOO0......0.0.0.0...OOOOOCOOOOOOOOOOC I6
Resu‘tSOOOOOOCOOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO ‘8

DiscussionOOOOOOOOOOOOOOOOCOOOCOCOCOOOOOOOOOOOO... 22

AMYLASEO0000......0.0OOOOOOOOOOOOOOOOOOOOOO0.0.0.0.... 30
Literature Su rveYo O O O O O C O O O C O C O O O O O O O O O O O O O O O O O O O O 30
Methods and Materia's. O O O O O O O O O O O O O O O O O O I O O O O O O O C O 32
ReSUI ts. 0 O O O O O O O O O O O O O O O O O I O O O O O O O O O O O O O O O O O O O O O O O 3“
DiSCUSSion. O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 O O 37

VISCOSITYOOOOOOOOOOO0.00000000000000000000000000000000 1.0
Literature survey 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 1‘0
MethOds and mterial s O C O O O O O O O O O O O O O O O O O O O O O O O O O 0 O “2
ReSUItSOOOOOOOOOO00......OOOOOOOOOOOOOOOCOOO0.0... “3

DISCUSSimOOOOOOOO0.0...0......OOOOOOOOOOOOOOOOOOO

RATE. OF FLOW.0....00.000000000000000...0.0.0.0000...O. 52
Literature su wey. O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 52
MethOds and Materia‘s. O O O O O O O O O O O O O O O O O O O O O O O O O O O O 55
ReSUNtSOOOOOOOO0.00.0.0000...OOOCOOOOOOOOOOOOOOOOO é“?

DiSCUSSionooeoeooooeeeooeeooooooooooeoooooooeooooo

pHOOOO.O...0......O.0.0.00.0000...OOOOOOOOOOOOOOOO...O
Literature survey 0 O O O O O 0 O O O O O O O C O O O O O O 0 O O O O O O O O O 0 O 63
MethOds and Materials 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 65
ReSUItSOOOOOOOI .0...OOOOOOOOOOOOOOCOOOOO 00.0.00... 65
DiSCUSSImOOOOOOOOOOIOOOOOOOOOOOOCO OOOOOOOOOOOOOO. 66

BUFFER CAPACITY. O O O O O O O O O O C O O C O O O O O O C O O C C O O O O C C O O O O O O O 69
Literature Survey................................. 69
Methws and Materials 0 C O O O O O O O O O O I O O O O O O 0 O C C O C O O O O 73
ReSUItSOOOOCOOOCOOOOO...0..OOOOOOOOOCOOOOOOOOOOOO. 7h
DESCUSSim. C O O O O O O O O O O O O O O O I O O O O C C O O O O O O C O O O O O O O O 0 7“

GENERAL DISCUSSION. ............ ....................... 78
SUMMARY............ ............. ...................... 86
WORKS CITED............. .............. ..... ....... .... 88
APPENDIX.............................. ........ ........ IIO

Table
Number

'0.

II.

LIST OF TABLES

Effect of caries-resistant and caries-
susceptible rat salivas on the mean

slopes and mean maximum limits of

growth of several bacteria......................

Significance of results of the effect
of rat saliva and saline on the growth
response of rat oral streptococci...............

Amylase activity of whole, parotid, and
submaxillari-sublin ual saliva from
caries-resistant an caries-susceptible

ratSOOOOOOOOOOOOOOOOOOOOOOCOOOOO00.000.000.00...

Correlation analysis of rat saliva
properties and caries age.......................

Relative viscosity of whole, parotid, and
submaxillari-sublingual saliva from caries-
resistant and caries-susceptible rats...........

Comparison of differences in relative
viscosity of salivas within the resistant
and susceptible strains of rats.................

Correlation analysis of relative viscosity
of rat whole saliva and caries age..............

Mean rates of flow of whole, parotid and
submaxillari-sublingual saliva from caries-
resistant and caries-susceptible rats...........

The effect of removing the arotid duct from
male and female rats upon t e body weight,

and absolute and relative‘weights of
submaxillari-sublingual glands..................

pH of whole, parotid, and submaxillari-
sublingual saliva from caries-resistant
and caries-susceptible rats.....................

Buffering capacity of whole and submaxillari-
sublingual saliva from caries-resistant and
caries-susceptible rats.........................

Page

19

23

35

36

45

#6

A7

57

59

67

75

LIST OF FIGURES

Figure

Number

I.
2.

3.

Device used for the collection of rat saliva.....

Typical growth curves of a rat oral lacto-
bacillus showing little effect of rat sallva.....

Typical growth curves of rat oral lacto-
bacillus No. ll showing the stimulatory effect

Of var‘ws rat salIvaSCO0.0....OOOOOOOOOOOOOOO...

Typical growth curves of a rat oral strepto-
coccus s owing the stimulatory effect of
var‘ws sa' ivaSOOOOOOOOOOOOOOOOOOOOOOOOOOOOO00.00

Change in relative viscosity in caries-

resistant and caries-susceptible rat

salivas when stored at room temperature

and refrigerator temperature..............,,.,,,,

A typical curve illustrating the buffering

capacity of diluted rat saliva compared to
that Of dTStllled waterOOOOOOOOOOOOOOOOOOOOOOOOO.

iv

Page
IO

20

2|

2%

77

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

5.

IO.

LIST OF APPENDICES

Effect of caries-resistant and caries-
susceptible rat salivas on the growth
of several bacteria.........................

Paired data analysis of the effect of
caries-resistant and caries-susceptible
whole rat salivas on the growth of rat

ora' streptxxCi.OOOOOOOOOOOOOOOOOOO0.0000.

Paired data analysis of the effect of
caries-resistant and caries-susceptible
whole rat salivas on the growth of rat

oral streptococci...........................

Paired data analysis of the effect of
caries-resistant and caries-susceptible
submaxillari-sublingual rat salivas on

the growth of rat oral streptococci.........

Paired data analysis of the effect of
caries-resistant and caries-susceptible
submaxillari-sublingual rat salivas on

the growth of rat oral streptococci.........

Paired data analysis of the effect of
caries-resistant whole saliva and saline
control on the growth of rat oral
streptxxCi.0.000000000000000000000......00

Paired data analysis of the effect of
caries-resistant whole rat saliva and
saline control on the growth of rat

oral streptococci...........................

Paired data analysis of the effect of
caries-resistant submaxillari-sublingual

rat saliva and saline control on the

growth of rat oral streptococci.............

Paired data analysis of the effect of
caries-resistant submaxillari-sublingual

rat saliva and saline control on the

growth of rat oral streptococci.............

Paired data analysis of the effect of caries-
susceptible whole rat saliva and saline
control on the growth of rat oral
streptococci................................

Page

Ill

II2

l13

Ilh

ll5

Il6

II7

I18

ll9

I20

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

II.

I2.

l3.

IA.

I5.

l6.

I7.

I8.

‘90

LIST OF APPENDICES (Cont.)
Page

Paired data analysis of the effect-of
caries-susceptible whole rat saliva and

saline control on the growth of rat oral
strepthCiOOOOOOOOOOIOOOOOOOOOOOO00...... '2'

Paired data anal sis of the effect of
caries-susceptib e submaxillari-sublingual

rat saliva and saline control on the

growth of rat oral streptococci............ 122

Paired data analysis of the effect of
caries-susceptible submaxillari-sublingual

rat saliva and saline control on the

growth of rat oral streptococci............ 123

Data used in the correlation analysis

between amylase activity of whole saliva

from caries-susceptible rats (first

experiment) and caries age................. IZA

Data used in the correlation analysis

between amylase activity of whole saliva

from caries-susceptible rats (second ex-
periment) and caries age................... l25

Data used in the correlation analysis

between amylase activity of whole saliva

from caries-resistant rats (second

experiment) and caries age................. l26

Correlation analysis between amylase

activity and caries age using combined

data of susceptible and resistant rats

used in the second experiment.............. l27

Data used in the correlation analysis

between relative viscosity of whole

saliva from caries-susceptible rats

(first experiment) and caries age.......... l28

Data used in the correlation analysis

between relative viscosity of whole

saliva from caries-susceptible rats

(second experiment) and caries age......... l29

Data used in the correlation anal sis

between relative viscosity of‘who e

saliva from caries-resistant rats

(second experiment) and caries age......... l30

vi

TABLE 20.

TABLE 2i.

TABLE 22.

TABLE 24.

TABLE 25.

LIST OF APPENDICES (Cont.)
Page

Correlation analysis between relative

viscosity and caries age using combined

data of susceptible and resistant rats

used in the second experiment.............. l31

Body weights in grams of rats with
(operated) and w thout (control) their
parotid ducts removed...................... l32

Absolute weight in milligrams of the
submaxillari-sublin ual glands from

rats with (operated and without

(control) their parotid ducts removed...... l33

Relative weights in milli rams of the
submaxillari-sublin ual g ands from

rats with (operated and without

(control) their parotid ducts removed...... 13A

Formulae used in testing the significance

of the differences between the body

weights, and absolute and relative

weights of the submaxillari-sublingual

glands in rats with (operated) and

without (control) their parotid ducts
rmvedOOOOOOO0.00.00.000.000.00.0.0000...O '35

Data used in the correlation analysis

between titratable alkalinity of whole

saliva from caries-susceptible rats and

caries ageOOOOOOO0.000000000000000000000000 ‘36

Characteristics of the rat oral lacto-
bacilli used to study the effect of rat
saliva on the growth of microorganisms..... l37

Cultural characteristics of the rat oral

streptococci used to study the effect of
rat saliva on the growth of microorganisms. l38

vii

Acknowtsocsnsms

The author is most appreciative to Dr. Samuel Rosen of
the Department of Zoology for his patient guidance and con-
stant stimulation throughout this investigation and prepara-
tion of this manuscript. Dr. Rosen's comprehensive know-
ledge of the various aspects of experimental dental caries
was an invaluable asset in this study.

The author is indebted to Dr. Harrison R. Hunt of the
Department of Zoology for his kind interest and advice
during my advanced training; to Dr. Carl A. Hoppert of the
Department of Chemistry for the dental caries examination of
the rats used in this study; and to Dr. Harold L. Sadoff of
the Department of Microbiology and Public Health for his
advice and academic guidance.

The use of the caries-resistant and caries-susceptible
rats developed by Drs. Hunt and Hoppert is also gratefully

acknowledged.

viii

INTRODUCTION

Inasmuch as this problem is directly related to den-
tal caries, it would be pertinent to define at the onset,
certain basic tenms relative to this disease. According to
Doriand's American Illustrated Medical Dictionary (20th edi-
tion), caries is defined as "the molecular decay or death
of a bone, in which it becomes softened, discolored and
porous" and the tenm dental caries is the “discoloration and
disintegration of the enamel and dentin by the action of
acid-producing bacteria and their products." The latter
term has been defined in accordance with the predominant
theory for the development of the carious lesion as W. D.
Miller proposed in l895 (Pickerill, l923). He presented
evidence that implicated acidogenic oral microorganisms
and carbohydrate food material. He believed that these
organisms, while attached to the tooth surface, metabolized
the carbohydrate to organic acids, which in turn dissolved
the inorganic constituents of the enamel. Accordingly,den-
tal research has since attempted to elucidate the carbohy-
drate-bacterial relationship and to determine the etiologic
agent of the lesion.

Lactobacilli and streptococci have been associated with
dental caries to a far greater degree than any other groups
of microorganisms studied. However, it has not been defi-
nitely established yet as to whether either or both of these

groups of bacteria constitute the etiologic agent. That

I

2

dental caries is indeed a bacterial disease has been unequi-
vocally established by the experimental evidence reported
by Orland, Blayney, Harrison, Trexler, Wagner, Gordon, and
Luckey (l95h). Their findings indicate that rats reared
under genmfree conditions remained entirely free of even
microscopically demonstrable dental caries. Virtually all
of the conventional control rats, possessing the usual
mixed oral flora, developed caries when maintained on the
same kind of dietary regimen as the germfree animals. It
was concluded, therefore, that dental caries in the rat is
not possible in the absence of microorganisms.

Later, Orland, Blayney, Harrison, Reyniers, Trexler,
Erwin, Gordon and Wagner (I955) inoculated otherwise genu-
free rats with known bacterial cells in which an enterococ-
cus resembling Streptococcus fecalis was the predominating
organism. All these rats developed carious lesions in the
molar teeth. These animals were fed the same standard diet
that was fed to all conventional control rats having an un-
known complex bacterial flora. These control rats regularly
developed caries during the ISO day test period. The above
Studies by Orland, _e_t_ _a_l_,_, were confirmed by Fitzgerald,
Jordan, Stanley, Poole, and Bowler (l960) inoculating the
test animals with an alpha-hemolytic streptococcus isolated
from the oral cavity of the rats receiving the cariogenic
diet.

Kite, Shaw and Sognnaes (I950) eliminated food from in-

tact and desalivated rats by feeding them by stomach tube.

These animals did not develop caries, but the intact and de-
salivated control animals consuming orally the same diet,
did develop caries. These differences were highly signifi-
cant and showed that with all other factors being controlled
and equal, tooth decay is prevented in rats when theldirect
effects of food in the oral cavity are eliminated.

That the carbohydrate must be present locally was con-
firmed by Kamrin (l95h) when he fed dextrose to the right
parabiont of united, genetically similar, pairs of rats. A
high incidence of dental decay was observed in the right
parabiont, but little or no caries in the left parabiont.

One of the major factors contributing to the develop-
ment of dental caries is heredity, as demonstrated in rats
by Hunt and Hoppert (I939.I9AI, l9hh, l9h8b), Hunt Hoppert
and Braunschneider (l9h7) and Hunt, Hoppert and Erwin (l9hh).
As a result of their work, caries-resistant and caries-
susceptible lines of rats (Rattus norvegicug) have been
produced by progeny testing, close inbreeding and selection.

Recently, a report by Keyes (I960) challenged heredity
as a factor to explain caries resistance. He presented
data interpreted as suggesting that caries is an infectious
and transmissible disease. He found caries activity mark-
edly reduced in both hamsters and rats after penicillin-
sensitive oral flora had been depressed prior to feeding a
high cargohydrate diet. Furthermore, hamsters whose peni-
cillin-sensitive oral flora had been depressed, in some

cases, produced several generations of progeny with negli-

gible activity.

a

This suggested that the Hunt-Hoppert rats were caries-
resistant because of the absence of a cariogenic flora. To
test this hypothesis Rosen, Hunt and Hoppert (l96la,b) had
caries-resistant litters, within a day after birth, fostered
by caries-susceptible mothers, and conversely, newly born
caries-susceptible litters fostered by caries-resistant
mothers. In another study, rats from both lines were main-
tained on penicillin until weaned. Littermates from each
line were then orally inoculated with feces from caries-re-
sistant and caries-susceptible rats. In both of the above
experiments, the rats behaved the same as their natural
parents with respect to dental caries.

Like most phenotypic expressions, the carious lesion is
the end result of many prior and interrelated reactions and
processes. Many lines of investigation have been undertaken
to determine any factor that might be associated with re-
sistance or susceptibility to caries in these rats, and
thereby aid in the better understanding of this disease.
For example, sex was found to be of no significance in the
carious process of the animals (Hunt and Hoppert, ISASa).
Slight differences were found between the two strains with
respect to*weight of the teeth, percent ash, and phosphor-
ous content (Hoppert and Shirley, I950). Fissures of the
molar teeth were found to be significantly wider in suscep-
tibles than in resistants, on the average, but the fissure
width of some resistants was wider than those of some sus-

ceptibles (Kifer, Hunt, Hoppert and Nitkop, I956).

Study of the oral flora revealed striking differences
between the two stocks of animals. Using selective culture
media, lactobacilli and Streptococcus salivarius were re-
covered more frequently and in greater numbers from caries-
susceptlble than from caries-resistant rats (Rosen, Be-
narde, Hunt and Hoppert, I955 and Rosen, Hunt, and Hoppert,
I956). No differences in the types of lactobacilli, how-
ever, could be detected in the two strains of rats by Rosen,
Ragheb, Hunt and Hoppert (I956). They found penicillin
very effective in retarding the development of caries in
susceptible rats, and inhibiting the acidogenic bacteria
(lactobacilli and streptococci), However, terramycin also in-
hibited the acidogenic bacteria, but did not appreciably in-
hibit caries (Rosen, Ragheb, Hoppert, Hunt, I956).

Since saliva provides an intimate environment for the
teeth, it was assumed that some of the factors contributing
to resistance or susceptibility to caries may be present in
this secretion. In their study on rat saliva, Rosen, Be-
narde, Fabian, Hunt and Hoppert (l957) could find no real
difference in the saliva of resistants and susceptibles
with regard to antibody titers against lactobacilli, pH,
surface tension, refractive index and specific gravity.
However, relative viscosity‘was about ten percent higher in
saliva from resistant animals. The significance of this
difference‘was minimized by the variability of the values

obtained from one experiment to another.

Willett (l955). extending this study of rat saliva,
assayed for various enzymes. He found protease activity
2.5 to 3 times greater in susceptible saliva than in re-
sistant saliva, but could demonstrate nOrimportant dif-
ferences in the activity of acid and alkaline phosphatase,
Iysozyme, or sulfatase. Hyaluronidase and urease could
not be detected. Since the large difference in protease
activity in the saliva of mature rats was not observed in
young rats of h9-55 days of age, the age when caries de-
velopment has been initiated in susceptible rats, it was
concluded that the protease activity and the degree of
susceptibility are independent traits fixed by the process
of inbreeding.

Rosen, Sreebny, Hoppert, Hunt and Bachem (I958, l959a)
showed that sialoadenectomy reduces resistance. However,
caries deveIOped later in the sialoadenectomized resistants
than in control susceptibles, which indicates that extra-
salivary factors also contribute to caries-susceptibility.

it is generally accepted by others that extirpation of
all of the major salivary glands of certain experimental
animals results in an increase in dental caries. This has
been amply shown by Kondo, Ichikawa and Aral (I938), Cheyne
(I939), Heisberger, Nelson and Boyle (I9h0), Gilda and
Keyes (l9h7), Dale (l9h8), Shaw and Heisberger (I9A9), Kite,
,g£,gl., (I950). Klapper (l953). Klapper and Volker (I953),
Schwartz and Shaw (I953. I955), Fanning, Shaw and Sognnaes
(I954), Bixler, Muhler and Shafer (l95’+, l955). Muhler
and Shafer (l95h) and Blechman, Gupta and Bartels (I960).

When specific glands were removed, however, there was
some disagreement as to the effect upon the development of
caries. Schwartz and Shaw (I955) using a high sucrose
cariogenic diet found that removal of the parotid glands
from rats caused an increase in caries, as was the case when
the sublnaxillary and sublingual glands were removed. But
Keller, Hunt and Hoppert (l95h) using a coarse particle
cariogenic diet reported that interruption of parotid
secretions did not affect the caries incidence.

Since the main difference between the two studies was
the difference in diet, Schwartz, Resnick and Shaw (I958)
repeated the experiment studying the effect of diet on se-
lective sialoadenectomy. They found that caries increased
in rats in the absence of parotid saliva when fed a high
sucrose diet, but not when fed the coarse particle diet,
thus explaining the difference in the above-mentioned re-
sults.

Rosen, g; gfl,, (l959a) found that extirpation of the sub-
maxillary and sublingual glands from rats on the coarse
particle cariogenic diet (also called the Hoppert-Webber-
Canniff diet) caused a decrease in caries-resistance,
whereas removal of the parotid ducts resulted in an increase
in caries resistance.

It was of interest, therefore, to study Specific sali-
vary secretions from physical, biochemical and microbio-
logical points of view to provide more information regarding
-the factors that contribute to resistance and susceptibility

to dental caries.

GENERAL METHODS AND MATERIALS

Caries-resistant and caries-susceptible rats obtained
from the colony of Hunt and Hoppert were used in this study.
The rats were maintained on a modified Hoppert-Webber-Can-
niff coarse particle cariogenic diet (l93l, I932), which
consists of the following ingredients:

Coarsely ground rice so that I to 2 percent

is retained on a ZO-mesh screen 66 percent
Whole powdered milk 30 percent
Alfalfa 3 percent
Sodium chloride I percent

Thirteen-week old rats of each litter had either a por-
tion of their parotid ducts excised and the severed ends
ligated, or their submaxillary and major sublingual glands
removed, and the other rats of the same litter served as
unoperated controls. Approximately four weeks following the
operations, saliva was collected from all rats according to
the method of Benarde, Fabian, Rosen, Hoppert and Hunt (I956).
This involves lightly anesthetizing the animals by injecting
sodium pentabarbital (Nembutal-Abbott) intraperitoneally
followed by a subcutaneous injection of the sialogogue,
pilocarpine hydrochloride (Merck). This procedure does not
elicit a detectable amount of secretion from the minor sali-
vary glands, or fromlthe mucus glands of the oral cavity.

No saliva was obtained from six rats which had their parotid

8

9
ducts, as well as their submaxillary and sublingual glands
removed. Accumulated food and other debris in the mouth
were removed by scrubbing the molars with a number 0 stencil
brush and their mouths flushed with water using a 5 cc
syringe without an attached needle. The residual water was
blotted with cotton swabs. .

The saliva from each animal was allowed to flow into a
graduated conical centrifuge tube facilitated by a short
stem funnel (see figure I) . The saliva was collected at
roan temperature for most of the experiments. For the anti-
bacterial study and for the second set of determinations of
anylase activity and viscosity, saliva was collected in
tubes imersed in ice. Care was exercised to prevent nasal
and lacrimal secretions from mingling with the saliva.

Throughout the experimental period, sane of the rats

were observed biweekly for macroscopic caries in the man-

dibular molars.

 

Figure I. Device used for the collection of rat saliva.

EFFECT OF SALIVA ON THE GROWTH OF MICROORGANISMS

Literature Survey

A variety of investigations have been made on the ef-
fect of human saliva on oral microorganisms.

Hugenschmidt (l896) attributed the removal of micro-
organisms to the "washing action" of saliva. This was later
demonstrated by Bloomfield (l920a, b, c).

Sanarelli in l89l first reported a salivary bacteri-
cidal property against certain pathogens using Chamberland
filtrates of saliva. However, Triolo (I897) found that un-
filtered saliva showed bactericidal activity, whereas fil-
tered saliva had no effect. No difference was observed in
parotid and submaxillari-sublingual secretions. Dold and
weigmann (I93A) found that the diphtheria bacillus, among
other microorganisms, died in a few hours when inoculated
into fresh saliva, but grew in the same saliva after it had
been heated to 56° C for 30 minutes. They concluded this
was due to an inherent property of saliva and named this
factor "inhibine." Dold and Weigmann's conclusion was
corroborated by a number of researchers: Clough (I933),
Dold, dehele and Du Dscheng Hsing (I936), Weigmann and
Koehn (I936), Shaefer (I936), Brawley and Sedwick (I936),
Pesch and Damm (I936), Bibby and Ball (I937), Weigmann and
Noeske (I937). Polezhaeva (I938), Dold (I938), Rolleston
(I938), Berg (I938), Mfihlenbach (I939). Skrotskii, Makhlin-

ovskii and Slutskaia (I939). Lande (1939). Dold (Ighz),
‘ ll

I2

Dawson and Blagg (l9h8, I950). and Bonicke, Reif and Arndt
(I953). Many of these workers found the inhibitory princi-
pie to vary greatly in different individuals. They included
a wide range of test microorganisms, and employed _i_;1_ 112.9.
methods to demonstrate this inhibitory activity of saliva.
Appleton (I936, I937) was successful in showing pneumococci
inhibition _i_1_1_ 2.319. by injecting mice with saliva suspensions
and broth suspensions of the pneumococci. On the other
hand, Bezi (I932) could not demonstrate conclusively _i_p_
3112 anti-diphtheria properties in saliva. ‘

Unfortunately the‘workers cited above did not cannulate
the salivary glands in order to obtain sterile saliva. The
antibacterial activity could have been due to the activity
of other microorganisms as shown by Bartels (I933). Besta
and Kuhn (l93h), Clauberg (I935), Appleton and Dietz (I937),
Prica (I937), MUhlenbach (I939). Lande (I939). Weigmann and
H8121 (1940), Thompson and Shebuya (1946), Bethege, Soehring,
and Tschesche (l9h7), Thompson.and Johnson (I947, I95l),
White and Hill (l9li9), Hegeman (I950). Scrivener, Myers,
Moore and Warner (l950a, b, l95l), Scrivener, Warner. Myers,
and Moore (l95l), Lammers (I952), Scrivener (I952), Berger
(I952), and Anmstrong and Jenkins (I953). Some of these
workers have suggested that H202 produced by streptococci is
the agent causing inhibition.

Many investigators have concentrated their studies on

the effects of saliva on microorganisms which have been

suggested as etiologic agents of dental caries.

l3

Miller (l930a) was not successful in demonstrating the ,
inhibition by human saliva of a "caries bacterium." McIntosh,
James and Lazarus-Barlow (I925) could find no appreciable
bactericidal action against L, acidophilus in human saliva.
However, Clough (I934, I935) and Clough, Bibby and Berry
(I938) found virtually all salivas tested to be inhibitory
in varying degrees against L, acidophing, whereas filtered
saliva was not inhibitory. This salivary property was not
correlated with caries. Taylor and Bibby (I935). Thompson
(l94l) and Van Kesteren, Bibby and Berry (I942) confirmed
-the fact that there was an anti-lactobacillus factor in
whole human saliva. Hine (1936) round the inhibitory agent
active against L, acidophilus was present in 9i percent of
saliva samples tested and varied from person to person and
from day to day. Armstrong and Jenkens (I953) demonstrated
an inhibitory substance against "bacteria described as
causing caries" in saliva from virtually all dogs tested.
Williams and Oshtry (I957) presented evidence showing that
sterile, human parotid saliva lacked metabolites for the
multiplication of a homofermentative lactobacillus, among
other organisms. .

Hill (I939). Curotto Devoto (I940) and Hill and Knies-
ner (l94l) indicated that saliva from a caries-susceptible
person supported the growth 0f.L- acidophilus,‘whereas,
caries-resistant saliva did not. However, Rosebury (I930),
failed to detect in saliva from caries-free humans and dogs
growth inhibitory properties against aciduric bacilli iso-

lated from carious teeth.

I4

Grove and Grove (I942), Kesel, O'Donnell, and Kirch
(I945), Kesel, O'Donnell, Kirch and Wach (I946, I947) and
Kesel (I948) attributed the anti-lactobacillus property of
caries-immune saliva to ammonia produced by the oral flora.
Clark and Carter (I927) had previously reported that most
of the ammonia occurring in human saliva was the result of
enzymatic activity other than that elaborated by bacteria,
but Kirchheimer and Douglas (I950) Could not implicate
ammonium ions. Hill, White, Matt and Pearlman (I949) ob-
tained a fraction of saliva that was inhibitory to certain
bacteria and was recovered in larger amounts from the saliva
of persons resistant to caries than from persons suscep-
tible to caries. Green and Dodd (I956) showed an association
of an anti-lactobacillus factor with dental caries in hu-
mans. Green and Dodd (I957). and Green (I958, I959) showed
this anti-lactobacillus factor to be associated with the
globulin fraction of immune saliva, but not susceptible
saliva. Grisamore and Toto (I958) found in the globulin
fraction II of human sera antibodies which inhibit the
growth of L, acidophilus.

Kerr and Widderburn (l958a, b) demonstrated inhibitory
properties in sterile, cannulated, human, parotid and sub-
maxillari-sublingual secretions. Zeldow (I955) found as
much bactericidal activity against L, gglggghjjgs in paro-
tid saliva as in whole saliva, and concluded that, since
the bacterial count of parotid saliva was only one percent
that of whole saliva, this activity could not have a bac-

terial origin. In I959 Zeldow quantitated this bactericidin,

IS

finding levels in the parotid salivas equal to or greater
than in submaxillary salivas. He also showed that this
salivary factor required a dialyzable heat-stable cofactor
for its activity. He not only established the difference
between this agent and lysozyme, but also precluded its simi-
larity to salivary amylase.

After Fleming (I922) demonstrated lysozyme in saliva
and other biological fluids, Bartels (I934), Skrotskii,,gL
‘31., (I939). and Chauncey, Lionetti, Winer and Lisanti (I954)
confirmed the presence of lysozyme in saliva, but failed to
find it correlated with dental caries. Rudinu (I954) found
a correlation of salivary lysozyme with caries in men but
not in women.

By incorporating saliva from a caries-resistant and a
caries-susceptible person into the drinking water of ham-
sters, Granados, Glavind and Dan (I950) demonstrated that
saliva from a caries-resistant person contained a factor
(or factors) which has the ability to decrease dental caries
activity in hamsters. Blechman, 2L gL., (I960) found that
pooled saliva from caries-immune humans added to the drinking
water of sialoadenectomized Sprague-Dawley rats, resulted in
a significant decrease in the average extent of carious
lesions than in sialoadenectomized rats given pooled saliva
from caries-susceptible individuals.

In the only study where saliva from rats was used, Rosen.
gfi,afl,, (I957) was not able to demonstrate an antagonistic
effect against rat oral lactobacilli using whole, stimulated
saliva from Hunt-Happert caries-resistant and caries-suscep-‘

tible rats.

I6

Methods and Materials

I. Collection and Pretreatment of Saliva

For this phase of the study, 25 litters of rats were
used: I3 resistant and l2 susceptible. The size of the lit-
ters ranged from 4 to I6 in number with an average of approx-
imately 8 rats per litter. Since parotid saliva was not
used for this experiment, only parotidectomies were per-
formed. Unoperated littermates were retained to obtain
whole saliva. Thus, only four types of saliva were used.
These were submaxillari-sublingual and whole saliva from
resistant and susceptible lines of rats. The saliva of
Iittenmates receiving the same treatment was pooled, re-
frigerated overnight to allow precipitation of mucin, and
centrifuged. The supernatant liquid was then sterilized
using a Morton Filter Apparatus (Corning). All operations
described thus far were carried out either in an ice bath
or in the refrigerator. One ml of the filtered saliva was
tested for sterility in Bacto Brain Heart Infusion Broth
(Difco), which was incubated three days at 37° C, and the
remainder was stored in the frozen state until needed.

2. Test

To determine the effect of rat saliva upon the various
test organisms the procedure, in brief, was to follow the
growth rates of the organisms photometrically.

Six strains of rat oral lactobacilli and seven strains
of rat oral streptococci as well as Staphylococcus gpggpg,

Escherichia coli, and Bacillus subtilig served as the test

I7

organisms. Cultures of the lactobacilli and streptococci
were kindly supplied by Dr. Samuel Rosen, and also the in-
formation contained in tables 24 and 25 of the Appendix,
which indicates some of their biochemical and cultural
characteristics. , Stock cultures were maintained in Bacto Micro
Assay Culture Agar (Difco). The inoculum for the test was
prepared by subculturing from the stock culture into Bacto
Micro lnoculum Broth (Difco) supplemented with one percent
Bacto Dextrose (Difco) and incubated for 24 hours at 37° C.
From this suspension, another subculture was made into the
same type of broth and incubated under the same conditions.
This resulted in actively growing cultures.

The test medium consisted of 2 ml of double strength
Bacto Micro lnoculum Broth supplemented with one percent
Bacto Dextrose and 2 ml sterile saliva. Each day this ex-
periment was performed, a saline control was used which
consisted of 2 ml of the broth and 2 ml of 0.085 percent
NaCI (the approximate concentration of chloride ion in rat
saliva). This medium was contained in optically matched
I00 mm x l3 mm test tubes. Each tube was inoculated with
0.2 ml of the broth suspension of a test organism. Unino-
culated tubes served as blanks. All tubes were incubated
at 37° C and growth observed in terms of optical density
at zero time and at suitable intervals thereafter, using a
Bausch and Lamb Spectronic 20 at wavelength 620 mu after
setting the instrument at zero optical density with the un-

inoculated blank. Growth cruves were plotted on semilo-

garithmic graph paper.

l8

Results

The results of the effect of rat saliva (i.e., whole
and submaxillari-sublingual from resistant and susceptible
animals) on rat oral lactobacilli and rat oral streptococci,
as well as on Staphylococcus gpggpp, Escherichia gpLL, and
Bacillus subtilis can be seen in table I of the Appendix. A
summary of these data appears In table I on page I9.

All the rat oral lactobacilli (Mos. I, 4, 9, IO and I4),
with one exception (No. II), were not materially affected
on the average by the different saliva samples. That is,
the mean rates and mean maximum limits of growth of these
organisms in the presence of the salivas were virtually the
same as when cultured in the presence of the saline control.
This is graphically illustrated in figure 2, which shows a
typical growth curve of a representative organism from this
group.

The one strain of Iabtobacillus (No. II) that responded
differently from the others, was stimulated by all types of
saliva tested. This stimulation was reflected in both the
rate and maximum limits of growth when compared to the saline
control. Mean values of four tests indicate that essential-
ly no difference existed among the four types of salivas as
to the extent of their stimulatory property for lactobacil-
lus No. II. Figure 3 shows this stimulation in a typical
growth curve. ‘B. subtilis was stimulated in a way similar
to that of lactobacillus No. II.

The rat oral streptococci, when cultured in the pres-

ence of the four types of saliva, showed an increase in the

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20

A—A SALINE CONTROL
o—o RESISTANT SUBMAXILLARI-SUBLINGUAL SALIVA
o——O RESISTANT WHOLE SALIVA

X—X SUSCEPTIBLE SUBMAXILLARl-SUBLINGUAL SALIVA
X--X SUSCEPTIBLE WHOLE SALIVA

0.5..

 

0- 0- IGZOmyl
C
\

I

.0
o
01
l

J

 

 

 

°'°' 1 1 1 1 1 1 1 1 1

8 '2 I6 20
TIME (HOURS)

.5.—

Figure 2. Typical growth curves of a rat oral lactobacillus
. showing small effect of rat saliva.

2|

A—A SALINE CONTROL
O—o RESISTANT SUBMAXILLARI-SUBLINGUAL SALIVA

0....0 RESISTANT WHOLE SALIVA
x—x SUSCEPTIBLE SUBMAXILLARI-SUBLINGUAL SALIVA
X——X SUSCEPTIBLE WHOLE SALIVA

 

 

 

0.0. I620my)

 

 

0.02 _
T 1 1 1 ‘1

T r 1: 1 1
4 8 I2 I6 20

TIME (HOURS)

Figure 3. T ical rowth curves of rat oral lactobacillus
M6? ll sgowing the stimulatory effect of various

rat salivas.

22

maximum amount of growth (See table I). This difference is
statistically significant (P‘<. 0.0I) as detenmined by using
the Paired Analysis "t" test (table 2). However, the sali-
vas did not influence the rate of growth (tables I and 2).
Whole susceptible saliva supported greater average
maximum growth of the streptococci than did whole resistant
saliva. This difference is also shown to be statistically
significant (P .( 0.0I). 0n the other hand, submaxillari-
sublingual susceptible saliva did not stimulate growth to
a significantly greater degree than this type of saliva
from resistant animals as indicated in table 2. Tables
2 to l3 of the Appendix show details of these analyses.
Figure 4 is a typical growth curve of a rat oral strepto-
coccus and the stimulatory effect of the salivas.
The maximum.limits of growth of S: gpggpg increased
when cultured in the presence of resistant and susceptible
whole salivas, with no material difference between the two.

E, coli was stimulated only slightly, if at all, by

the resistant and susceptible whole salivas.

Discussion
It has been established by Clough (I935), Dold, pg,§fl,,

(I936), Dold (I938), Skrotskii, g§_§fl,, (I939), Thompson

(l94l), and Van Kesteren, e_t_ 91,, (I942) that human saliva
contains an antibacterial factor other than lysozyme, that
is inhibitory to certain bacteria including those types of
organisms used in this study. Human parotid and submaxil-

Iary secretions have been shown to be active against L,

23

TABLE 2. Statistical significance of results of the effect
of rat saliva and saline on the growth response of
rat oral streptococci

 

MAXIMUM GROWTH GROWTH RATE

t VALUE P t VALUE P

Resistant Whole Saliva with 6.061 (0.01 0.731 >o.5
Susceptible Whole Saliva

Resistant S-S* Saliva with I.5I8 >0.2 0.8II >0.4
Susceptible S-S Saliva

Resistant Whole Saliva with 8.088 (0.01 0.257 >0.5
Saline Control

Resistant S-S Saliva with 10.040 (0.0I 0.302 >0.5
Saline Control

Susceptible Whole Saliva l0.3l2 (0.0] 0.034 >O.5
with Saline Control

Susceptible S-S Saliva with 2I .654 (0.0I 0.855 >0.5
Saline Control

COMPARISON

 

*S-S'SubmaxilIari-SuinnguaI Saliva

24

A—A SALINE CONTROL

o—o RESISTANT SUBMAXILLARI-SUBLINGUAL SALIVA
o-—o RESISTANT WHOLE SALIVA

x—x SUSCEPTIBLE SUBHAXILLARI-SUBLINGUAL SALIVA

X--X SUSCEPTIBLE WHOLE SALIVA

 

 

 

 

 

 

 

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—-0
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E
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:5
to
DJ
0.05-
002 I I I I I I I I r I 1
4 8 I2 I6 20

TIME (HOURS)

Figurg 4. Typical growth curves of a rat oral streptococcus
showing the stimulatory effect of various rat
sa avas.

25

acidophilug by Kerr and Widderburn (I958a, b) and Zeldow
(I955, I959). Others (Granados, 25 $1., I950 and Blechman,
.pL.pL., I960) presented evidence suggesting that only caries-
resistant saliva had this property.

Rosen, _e_t_ 1L” (I957) detected no anti-lactobacillus
factor in whole saliva from caries-resistant and caries-
susceptible rats using the deep well-agar plate method.
However, the possibility that such a factor might be pre-
sent in submaxillari-sublingual saliva from these rats was
suggested when Rosen,‘gLHpL., (l959b) found that caries-
resistance decreased when the submaxillari-sublingual glands
were removed, but that caries-resistance increased when the
parotid duct was removed. Apparently, something in submax-
iIlari-sublingual saliva, not present in parotid saliva,
contributes to caries-resistance.

When the submaxillari-sublingual saliva and whole
saliva from the resistant and susceptible rats was tested
in the experiments described in this report for inhibitory
activity agains lactobacilli, streptococci and other organ-
isms, none was detected with the method employed.

However, the total growth of rat oral streptococci was
decidedly stimulated by salivas from rats. What may be
particularly significant is that whole saliva from the
susceptible rats enhanced the growth of these organisms to
a greater degree than did the whole saliva from resistant
rats. This is especially interesting since streptococci,

which are common inhabitants of the mouth and occur in

26

large numbers, have been implicated recently as etiologic
agents of dental caries in experimental animals, rather
than the lactobacilli.

The initiation of caries by streptococci was clearly
shown by Orland, g§_§l,, (I955) in their work with gnoto-
biotic rats. They succeeded in producing caries in germ-
free animals ingesting a "cariogenic" diet and inoculated
with an enterococcus or an enterococcus plus a proteolytic
bacillus. No lactobacilli were present. This enterococcus
had been isolated from a carious rat tooth and closely
resembled Streptococcus fecalis. Uninoculated animals re-
mained free of even microscopic caries. The authors be-
lieved at the time, that these results did not preclude the
possibility that certain lactobacilli could also produce
dental caries under similar conditions. However, Orland
(I957) decided to confine the germfree-caries studies to
the enterococci, since the lactobacilli studied under simi-
lar conditions failed to produce typical lesions. The
above work of Orland, gL_§l,, (I955) was confirmed by Fitz-
gerald, g; 21,, ,(l960) who also used germfree rats in their
study.

Fitzgerald and Keyes (I960) succeeded in inducing
dental caries in a strain of "caries-inactive" hamsters.
They introduced single or pooled cultures of streptococci
isolated from a carious lesion in a hamster. However, ino-
culation with strains of lactobacilli and diphtheroid or-

ganisms from "caries-active" hamsters and strains of strep-

27

tococci isolated from the oral cavity and feces of "caries-
inactive" hamsters was without effect. I

That the streptococci used in this study were strongly
stimulated by rat saliva, supports the hypothesis that
streptococci rather than the lactobacilli, constitute the
main etiologic agents of dental caries. Relative to the
findings of Rosen, SE 21., (I958, l959a, b), however, this
hypothesis raises a question. When resistant rats were
sialoadenectomized, the incidence of caries and the numbers
of lactobacilli increased, but the numbers of streptococci
did not change materially. If this hypothesis is correct,
why did the streptococci not increase, either along with,
or instead of the lactobacilli? One explanation for this
phenomenon is that a record of the total numbers of strep-
tococci does not give information about the possible change
in the relative types of streptococci occurring in the
mouths of these rats as a result of sialoadenectomy.

It has already been shown that the types of strepto-
cocci occurring in experimental animals vary with regard to
dental caries. Rosen, 2L,§1,, (I955) recovered Streptococcus
salivariup twice as frequently in caries-susceptible rats
than in caries-resistant rats. Further, an unidentified
oral streptococcus producing a rough, crateriform colony
was found in every resistant rat, but only in l8 percent
of the susceptible rats. Fitzgerald and Keyes (I960) found

two types of streptococci in hamsters' oral cavities, some of

‘28
which could produce dental caries when inoculated orally into
caries-inactive hamsters and some that could not.

It would be of interest, then, to compare the cario-
genic potential of the various strains of rat oral strepto-
cocci in a future study. ’Grouped according to whether they
*were stimulated by rat sallva or whether they were not (if
such can be found), these strains would be inoculated
orally into caries-susceptible rats after the rats had been
maintained for a period on a diet containing penicillin.
Such a diet has already been shown to inhibit dental caries
completely in caries-susceptible rats (Rosen, Ragheb, Hop-
pert and Hunt, I956).

Since an antibacterial.factor was not detected in this
study, it is possible that the conditions of the test were
such as to mask any Inhibitory influence exerted by the.
saliva.. That is, the inhibitory effects of the saliva
against the lactobacilli could have been overcome, because
the enriched culture mediumlused supported abundant growth
ofthese organisms. This situation could also be investi-
gated in a future study. If it can be demonstrated that
lactobacilli under minimal growth conditions are in fact
Inhibited by rat saliva, and that the streptococci are
stimulated under the same-conditions, this would be further
evidence that streptococci rather than lactobacilli are the
etiologic agents for dental caries in experimental animals.

The abundant growth supported by the enriched culture

medium could have masked another type of response of the

29

test organisms to rat saliva. Since a greater total growth
of the streptococci was stimulated by susceptible whole
saliva than by resistant whole saliva, one would expect that
the rate of growth of the streptococci would also be sti-
mulated to a greater degree by these salivas. However, no
difference in growth rates was detectable.

Inasmuch as the stimulatory property of only the whole
saliva was significantly greater in the susceptibles than
the resistants, it would be of interest to investigate the
parotid secretion in this regard. One would expect that
the parotid saliva is contributing a stimulatory principle
to whole saliva, at least in part, either additively or

complimentarily. ;

AMYLASE

Literature Survey

There has been some disagreement as to whether amylase
activity of whole human saliva is related directly or in-
versely with caries incidence, or whether any relation
exists at all.

Those investigators who found a direct relation be-
tween amylase activity and caries susceptibility include
Michel (I9I5), Gore (I935), Florestano, Faber and James (I941),
Turner and Crane (l944a, b), Turner and Crowell (I947) and.
Turner, Anders and Becker (I957).

Myers and Adams (I932) and Schneyer (I95l) recognized
that the chloride content of saliva has an important influ-
ence upon the amylolytic index of a given individual. Anders
(I956) and Carter, Englander, Mau and Hoerman (I957) demon-
strated a significantly greater salivary chloride content in
caries-active than in cares-free persons.

0n the other hand, Pickerill (I924a, b) and Day (I934)
found a direct relation between amylase activity and caries
resistance; whereas, Hubbell (I933), Bergeim and Barnfield
(1945), Barany (1947), Hess and Smith (1948) and Ericsson,
Hellstrdm, Jared and Stjernstrbm (I954) found no correlation
between salivary amylase and dental caries in whole human
saliva, and Rosen, 9L,pl., (I957) found none in rat saliva.

Studies were conducted to determine the amylolytic ac-

tivity of saliva secreted from specific major glands, or to
' 30

3l

determine the amylolytic activity of the gland tissue itself.
Gore (I9386), Schneyer (l956b) and KOstIin and Rauch (I957)
found greater.anylase activity in parotid saliva than in
submaxillari-sublingual saliva of humans. The preponder-
ance of amylase activity in the parotid glands was shown to
be the case also in experimental animals. Schneyer and
Schneyer (I956, I960) demonstrated it in the salivary glands
of rats, while Gorden and Utrias (l957) confirmed it in the
salivary glands of rats and mice. Ryan (I909) found that
rabbit parotid saliva showed amylase activity to about the
same degree as human saliva, but that the submaxillary
saliva showed no activity. However, Raynaud and Rebey-
rotte (I950) demonstrated that the amylase activity of the
submaxillary gland of mice exceeded that of the parotid
gland.

The data of McGeachen and Gleason (I956) showed that
although amylase activity varied widely in the saliva of
individual rats, the average is still several times that of
human saliva. Latimer and Warren (l897) found high levels
of amylase in both parotid and submaxillary glands of rats
and mice.

Chittenden and Ely (I883) reported that the variations
in the titratable alkalinity are within too narrow'limits
to exercise any appreciable influence on the amylolytic
action of human saliva. However, Sullivan and Storvick
(l950a) showed a significant positive correlation between

buffer capacity and starch hydrolyzing time.

32

Carlson and Crittenden (l9l0) found parotid amylase to
vary directly with the rate of flow as influenced by various
stimuli. Deakins, Cheyne, Bibby and Van Kesteren (l94l)
working with whole human saliva found these two properties
were not correlated as determined by a statistical analysis

of the data.

Methods and Materials

As soon as possible after collection, usually within

an hour, the assay for amylase activity was carried out.

The six types of saliva under study, i.e., whole, parotid

and submaxillari-sublingual from caries-resistant and caries-
susceptible rats, were collected at room temperature. This
constituted the first experiment. A number of determinations
were made In a second experiment with whole saliva that had
been collected in tubes submerged in ice.

The method used to determine amylase activity was an
adaptation of one by Myers, Free and Rosinski (I944). The
modifications were the substitution of saliva for serum,
and a reduction in the incubation time of the enzyme-sub-
strate reaction mixture from IE to ID minutes. Each saliva
sample from individual rats was tested in duplicate, using
the glucose standard in triplicate.

The test involves the incubation of enzyme (contained
in saliva) with a soluble starch solution as the substrate.
After a specified period of time, the enzymatic reaction
was stopped by the addition of picric acid (2, 4, 6-trinitro-

phenol) in a quantity to yield a saturated solution of the

33
acid. Upon boiling in a solution made basic by sodium
carbonate, picric acid is reduced to picramic acid by the
aldehydic groups produced during the enzymatic hydrolysis

of the starch.

 

OH OH
\\
N02 N02
Picric Acid Picramic Acid

The concentration of picramic acid, a colored com-
pound, was determined photometrically, using a Bausch and
Lamb Spectronic 20 colorimeter. The amount of picramic
acid formed is directly proportional to the number of re-
ducing groups. By comparing the amount of reduction occur-
ing in the system containing saliva and starch with the
reduction produced by a known concentration of glucose, the
amylase activity could thus be expressed as milligrams of
reducing sugar as glucose formed per milliliter of saliva,
after I0 minutes incubation at 40° C. This was calculated
according to the formula

R x D x 0.6 x .l 3 mg glucose/ml saliva
s 9T

where R =Optical density of the saliva assay tube
S=0ptical density of the standard (glucose) tube
D=Dilution factor of saliva
0.6=mg Glucose in the standard tube

9.I=Factor to correct for the fraction of saliva
3 taken from the enzyme-substrate mixture.

34

Results

(The data for amylase activity are given in table 3.
The parotid gland was found to be virtually the sole source
of amylase in rat saliva, since the activity produced by
the submaxillari-sublingual secretion was so slight that
(it might be considered negligible. There was greater ac-
tivity in the saliva from susceptibles than from resistants
(P <( 0.0I). This difference was evident in the parotid
saliva as well as in whole saliva.

Correlation analysis (see table 4) between amylase
activity in whole susceptible saliva and caries age yielded
correlation coefficients (r) of 90.082 and +0.l05 for the
first and second experiments, respectively. A similar
analysis of combined data from resistant and susceptible
whole salivas and caries age indicated r s 00.270. Caries
age is defined as the age in days of‘a rat when a macro-
.scopic carious lesion is first detectable. The aboVe r'
values are not significant at the 5 percent level. How?
ever, significant values (at the 2 percent level) were
obtained in a correlation between amylase activity and
caries age with whole resistant saliva (r : o0.65l).

The data and formulae upon which the values in
. table 4 are based are presented in tables l4 through I6

and page l27 in the Appendix.

35

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36

TABLE 4. Correlation analyses of rat saliva properties and
caries age

 

 

Amylase Activitya and Caries Ageb

Susceptible Whole Saliva +0.082 j>0.05
(first experiment)
Susceptible Whole Saliva +0.l05 )>0.05
(second experiment)
Resistant Whole Saliva +0.65l 4 0.02

(second experiment)

Resistant and Susceptible +0.270 I>0.05
Whole Saliva
(second experiment)

 

a Amylase Activity = mg glucose per ml saliva.

b Caries Age = days of age when a carious lesion first
appeared.

37

Discussion ‘ ‘

The test for amylase activity employed in this study
does not distinguish between alpha or beta amylase. McGea-
chen and Gleason (I956) found that rat salivary amylase was
probably the alpha type, as is also the case in human saliva.
Confirmation of the type of amylase in saliva from the rats
used in this investigation was not considered necessary.

The elaboration by the parotid glands of virtually all
of the salivary amylase in rats, as shown in table 3, sup-
ports the findings of Schneyer and Schneyer (I956, I960),
and Gordon and Utrias (l957). Even though parotid saliva
from susceptible rats showed a significantly greater level
of amylase activity than did parotid saliva from resistant
rats, this difference is not considered to be of great im-
portance, since it does little to explain the caries behav-
ior of the two lines. This can be explained by a consider-
ation of the following facts.

When parotid ducts alone were removed from resistant
animals, thus blocking essentially all salivary amylase,
the effect on caries incidence depended upon whether the
rats were maintained on a diet which included either a
simple carbohydrate (sucrose) or a complex carbohydrate
(coarse particle rice). If the carbohydrate was sucrose,
caries susceptibility increased, but when rice was used, the
susceptibility of the rats to caries either did not change'
or decreased (Keller, gLHgL., I954: Schwartz and Shaw, I955;
Schwartz, gLHg1., I958; and Rosen, 2; p1., l959a). This is

38

what one would expect if greater amylase activity in suscep-
tible rats was interpreted as accounting for susceptibility.
However, the incidence of caries increased when the saliva
from all the major salivary glands was interrupted, thus
blocking the source of salivary amylase in resistant ani-
mals maintained on the rice diet (Rosen,lgpqgl., I958, I959a).
Caries should have decreased if salivary amylase contri-
buted materially to susceptibility.

Furthermore, the level of amylase activity, although
lower in resistant whole saliva than in susceptible1whole
saliva, is still high, and probably sufficient to cata-
lyze the hydrolysis of large quantities of starch into
simple carbohydrates needed by cariogenic bacteria.

Another aspect of amylase activity which minimizes its
importance in accounting for a difference between the two
lines of rats is revealed in the results of correlation
analysis between amylase activity and caries age (table 4).
The correlation coefficients of 6 0.082 and 0 0.I05 for
susceptible rats in two separate experiments, and + 0.270 for
combined data of resistant plus susceptible rats are not
significant at the 5 percent level. Although the caries age
in the susceptible and resistant lines are‘widely different,
the type of analysis carried out with the combined data
corrected for this fact, since the correlation analysis of
two variates assumes a normal distribution.

When the data obtained from the resistant rats alone was

analyzed, a significant positive correlation (r = + 0.65l)

39

was obtained. This might appear somewhat puzzling, since
these animals have a higher mean caries age, but lower mean
amylase activity than the susceptibles. From this, one

might expect to obtain a negative rather than a positive
value for r. It seems apparent that when the resistant

rats were selectively inbred for their low caries activity
(high caries age), the trait corresponding to a high salivary
amylase activity was also selected, even though this trait

probably has no causal relation to caries-resistance.

VISCOSITY

Literature Survey

Lohmann (I904), Rathje and Fr6hlich (l949) and Rathje
(l95l) reported a direct relation between the degree of
viscosity of whole human saliva and susceptibility to caries.
Willsmore (I937) reported no relation existed between vis-
cosity of "resting" saliva (saliva secreted in the absence
of overt stimulation) and general caries susceptibility, but
that a "resting" saliva with a viscous tendency is a pre-
disposing factor in the susceptibility to gingival caries.
Rosen, 2; p1,, (I957) in studying rat saliva found that the
relative viscosity was slightly, but significantly higher
in the saliva of caries-resistant than in caries-susceptible
rats. Shafer, Clark and Muhler (I957) found that higher
levels of thyroxine administered to rats for two months
resulted in a lower incidence of dental caries and a less
viscous saliva. These findings were substantiated by
Shafer, Clark, Bixler and Muhler (I958b) who demonstrated
that a dysfunction of the rat thyroid gland caused by
propylthiouracil and radiothyroidectomy resulted in an in-
crease of salivary viscosity. Thyroxine reversed this effect
and restored the function of the gland.

Lothrop and Gies (l9l0), Rae and Clegg (I949) and Dewar
and Parfitt (l954b) could find no relation between viscosity

and caries activity.

41

Salivary mucin, a glycoprotein, has been investigated
as to its possible role in viscosity of saliva and dental
caries. Willsmore (1937) concluded that mucin is probably
the major factor contributing to the viscosity of saliva.

Gore (I938b) and Simmons (1941) showed that enzymes
in bacteria-free saliva catalyzed the breakdown of mucin in
a way that resulted in its depolymerization, as reflected in'
a decrease in viscosity, and a concomitant release of the
prosthetic carbohydrate group, as evidenced by the liberat-
ion of reducing sugars. Knox (I953b) decreased the viscosity
of salivary mucoid using trypsin, thus depolymerizing the
mucoid, and then liberated a reducing sugar from this tryp-
sinized mucoid using hyaluronidase. Gore (I938b) further
reported that human submaxillarissublingual saliva contained
a greater concentration of carbohydrate than parotid saliva.

Dewar and Parfitt (I954a) demonstrated a highly signi- I
ficant positive correlation between viscosity of saliva
and polymerized mucin. When Shafer, Clark, Bixler and
Muhler (19583) bilaterally ligated the ducts of the submax-
iIlari-sublingual glands of rats, thus blocking the flow of
mucous saliva from the sublingual gland, a significant re-
duction occurred in salivary viscosity. Tests for muco-o
protein in parotid secretion were shown by Bramkamp (1936)
to be negative, but positive for mixed saliva. However,
Bagnell and Young (1930) indicated that the viscosity of

saliva bears no direct relationship to its mucin content.

42

Lohmann (1904) and Miller (1904, 1905) concluded that
saliva containing much mucin was conducive to caries.
Rogers (1948) presented evidence to support this view when
he showed that some groups of organisms in raw saliva are
able to break down and make available the salivary mucins
as a fenmentable carbohydrate source for streptococci, sta-
phylococci and lactobacilli. 0n the other hand, Lothrop and
Gies (1910) and Dewar and Parfitt (1954b) could find no
relation between the concentration of mucin in a given

fraction of saliva and the state of the teeth.

Methods and Materials

Two experiments were conducted for the determination
of viscosity. In the first experiment, whole, parotid and
submaxillari-sublingual salivas from the two lines of rats
‘were collected at room temperature. In the second experi-
ment, only whole saliva from the two lines of rats was
collected in tubes inniersed in an ice bath, then placed in
the refrigerator until tested. These samples were brought
to room temperature quickly and their viscosities measured.
In all cases, saliva from individual rats was used rather
than pooled samples.

Since the purpose of the study was to detect any dif-
ference in viscosity between the various salivas, relative
values instead of absolute values were determined. This
was accomplished by timing the fall of saliva between two
arbitrarily spaced marks on a narrow bore glass tubing, and
dividing by the time required for distilled water to drop

the same distance.

43

Usually within one-half hour after collection, the
salivas were centrifuged to remove any particles, and the
relative viscosity*was determined at room temperature (27° 3
3° C). The viscometer was cleaned for each saliva sample
passing an acid-dichromate solution through it, followed by
a sequence of rinsing solutions. These were distilled water,
70 percent ethanol and acetone. Finally, the viscometer

‘was air-dried before evaluating the next sample of saliva.

Results

The data for relative viscosity are given in table 5.
In the first experiment (saliva collected at room tempera-
ture) the relative viscosity of whole saliva from resistant
rats was 1.92 3 0.037 seconds, whereas that from suscepti-
ble rats was 1.66 3 0.066 seconds. The difference of 0.26 t
0.066 seconds is highly significant (at the 1 percent level).
However, in the second experiment (saliva collected in tubes
immersed in ice), the relative viscosity of whole resistant
saliva was 1.99 s 0.043 seconds, while that from susceptible
rats was 1.89 3 0.033 seconds. This difference (0.10 3 0.054,
seconds) is not significant at the 5 percent level. It can
be seen that the viscosity of resistant whole saliva re-
mained very much the same in both experiments, but the
susceptible whole saliva was more viscous when collected
and kept at a colder temperature.

The relative visCosity of parotid saliva from resistant

rats was 1.29 g 0.035 seconds, and that from the susceptible

Mi

animals was not significantly different with a relative
viscosity of l.33 2 0.03h seconds. Resistant rats produced
a more viscous submaxillari-sublingual saliva than did the
susceptible rats. The relative viscosity of submaxillari-
sublingual saliva from resistant rats was 2.lh and that

from susceptibles was l.94. The difference of 0.20 3 0.08h-
seconds is statistically significant. The saliva from
specific salivary glands used in the viscosity determina-
tions were collected at room temperature.

Comparison of the relative viscosity of parotid, sub-
maxillari-sublingual and whole salivas within each line of
rat (tables 5 and 6) yielded marked differences, which are
significant at the l percent level. Parotid saliva was less
viscous than submaxillari-sublingual saliva, whereas
whole saliva, a natural mixture of the other two types, gave
intermediate values.

Correlation analysis between the relativeviscoslty of
whole saliva and caries age (table 7) did not reveal any
significant relation at the 5 percent level, when these two
traits were considered in resistant animals 29; _s_§ or in
susceptible animals 23; _s__e_. The data and formula used in
these analyses are presented in tables l7, 18 and l9 of the
Appendix..

The correlation coefficient between these two traits
was not significant when the data from the two lines of rats
were combined. The caries age in the susceptible and re-

sistant lines are widely different, but the type of analysis

5
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#6

TABLE 6. Comparison of differences in relative viscosity
of salivas within the resistant and susceptible

strains of rats

 

 

Line of Rats Salivas Compared Difference t P
(Seconds)

Resistant Whole with Parotid 0.63 * 0.050 12.h9 20.01
Resistant Whole with s-s* 0.22 3 0.058 3.82 <0.01
Resistant Parotid with 5-5 0.85 * 0.056 15.08 <0.01
Susceptible Whole with Parotid 0.31 * 0.060 4.82 <0.01
Susceptible Whole with 5-5 0.28 * 0.100 2.79 < 0.01
Susceptible Parotid with 5-5 0.59 * 0.091 6.h9 1<O.OI

 

* 5-5 3 Submaxillarl-Subllngual Saliva.

47

TABLE 7. Correlation analysis of relative viscosity of rat

whole saliva and caries age

 

 

2,r P
Susceptible Whole Saliva «0.251 >0.05
(first experiment)
Susceptible Whole Saliva 40.052 >0.05
(second experiment)
Resistant Whole Saliva -0.0011 > 0.05
(second experiment)
Resistant and Susceptible +0.038 3.0.05

Whole Saliva
(second experiment)

 

48

carried out with the combined data corrected for this fact,
since the correlation analysis of two variates assumes a
normal distribution. See page 131 of the Appendix for

details of this analysis.

Discussion

That saliva from submaxillari-sublingual glands is
decidedly more viscous than that from the parotid glands,
confirms similar findings by Shafer, g£_§l,, (l958a). They
reported a significant reduction in viscosity of rat saliva
when the submaxillari-sublingual ducts were ligated, which
blocked the flow of mucous saliva from the sublingual gland.
Related to these findings also is the work of Gore (1938b),
who reported that the "thick" human mandibular (submaxillari-
sublingual) saliva has much more mucin than parotid saliva.

The greater degree of relative viscosity of whole re-
sistant saliva than of susceptible whole saliva confirms
the findings of Rosen, gt_§l,, (1957). Little, if any,
importance can be attached to this difference, however.

When it was noticed that the relative viscosity of saliva
decreased on standing at room temperature, an experiment was
conducted to determine the rate of decrease in whole saliva
from a resistant rat and from a susceptible rat when these
salivas were stored for a number of hours at refrigerator
and at room temperature. As can be seen in figure 5, the
decrease was considerably greater at room temperature with

both salivas, especially with susceptible saliva.

#0

Other workers reported similar findings. Gore (1935,
1938b) observed the spontaneous autolysis of human salivary
mucin at 37° 0, 00° 0 - u5° c, and 75° 0, which had the
effect of decreasing the viscosity of saliva and simultane-
ously increasing reducing sugars. He attributed this auto-
lysis to the hydrolytic activity of salivary amylase on the
carbohydrate component of mucin. Although, Ericsson and
Stjernstram (1951) also noticed that the viscosity of hu-
man whole saliva decreased upon standing at room temperature,
they showed that neither alpha amylase nor salivary bacteria
had any effect. instead, ascorbic acid in the presence of
hydrogen peroxide, which bacteria are known to elaborate,
or in the presence of traces of cupric ion, reduced the
viscosity rapidly. Mandibular saliva was stable or changed
slowly. Dewar and Parfitt (1950a) noted that a 30 percent
destruction (depolymerization) of mucin present in saliva
occurred within 30 minutes, and 50 percent within two hours
at room temperature, but that only slight destruction
occurred if the sample was placed on ice immediately after
collection. Even in the cold, however, they observed a
30 percent destruction within two hours.

Knox (1953a, b) attributed the decrease in viscosity
in saliva upon standing to mucinase, an enzyme having the
ability to depolymerize mucin, and occurring normally in
the mouth. He found that several microorganisms with
mucolytic activity were included among the human oral flora.

Not only did Knox demonstrate the ability of mucinase to

50

SUSCEPTIBLE SALIVA STORED AT ROOM TEMP.
SUSCEPTIBLE SALIVA STORED AT REFRIG. TEMP.
RESISTANT SALIVA STORED AT ROOM TEMP.
RESISTANT SALIVA STORED AT REFRIG. TEMP.

 

 

3.00
2.80 ax~

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TIME (HOURS)
m5. Change in relative viscosity in caries-resistant

and caries-susceptible rat salivas when stored
at room temperature and refrigerator temperature.

51

affect a reduction in salivary mucoid viscosity, but that
trypsin (a protease) was similarly active. It is perhaps a
protease that causes the observed change in relative vis-
cosity at room temperature in saliva from the two lines of
rats used in this study, particularly from susceptible rat
saliva. Hillett, (1955) reported a 2% to 3 times greater
protease activity in whole susceptible saliva than in whole

resistant saliva.

RATE OF FLOW

Literature Survey

There has not been complete agreement as to whether
there exists a correlation between the rate of salivary
flow and the incidence of dental caries. Rigolet (1901),
Pickerill (192kb), Trimble, Etherington and Losch (1938),
Gurley (1939). Losch and Heisberger (19h0), Cushman, Ether-
ington and Thompson (1940, 1981), Rathje and Frohlich (19h9),
Rathje (1951), Rovelstad (1957) and Rovelstad, Geller and
Cohen (1958) concluded that there is a direct relationship
between rate of flow and resistance to caries.‘

In their study with rats, Muhler, Bixler and Shafer
(1957) reported that the salivary flow-dental caries rela-
tionship is not a simple one. Although daily administrations
of 2 mg of pilocarpine, a sialogogue, significantly reduced
caries, 6 and 12 mg did not.

Shafer, Clark and Huhler (1957) found that higher
levels of thyroxine injected into rats for two months
resulted in a greater rate of flow of pilocarpine-stimulated
saliva and a lower incidence of dental caries. Shafer,
Clark, Bixler and Muhler (l958b) demonstrated that a dys-
function of the rat thyroid gland caused by propylthiouracil
and radiothroidectomy resulted in a reduced salivary flow
and a greater incidence of dental caries. Thyroxine re-

versed this effect.

52

53

Miller (1903b, 190h) indicated that the rate of salivary
flow was directly related to dental caries only in extreme
cases; that is, when the flow was very strong or very scant.
Even in cases where the flow was strong, he did not feel this
would affect the incidence of caries that originate in deep
fissures "where saliva scarcely penetrates." Burrill and
Fosdick (l9hh) and Sarany (19k7) concluded that the“: has
only a tendency t. a larger quantity of saliva in persons
with relatively little caries.

0n the other hand, Becks, Wainwright and Young (l9h1,
19A3) and Karshan (l9h2) found no significant difference
between the rates of flow of whole human saliva in caries-
active and caries-free groups. This was confirmed by
Englander, Mau, Hoerman and Chauncey (1958) who studied the
flow rates of human parotid saliva.

Becks and Wainwright (1939) thought that resting (un-
stimulated) saliva is better for routine analytical purposes,
because they found that subjects with originally low or
high rates of flow of resting saliva arrive at approxi-
mately the same rate when the salivary glands are stimu-
lated. This would disguise the true secretory capacity of
the resting gland, which differs greatly among individuals.

Little agreement can be found in the literature re-
garding the rates of flow of the individual salivary glands.
Gore (1938a) found no difference in the rates of flow of the
parotid and submaxillari-sublingual "resting" glands. When

these glands were stimulated, however, the rate of flow of

5#

the parotid secretion was more than three times greater.
Zipkin and Soban (1957) likewise found that stimulated paro-
tid glands contributed more saliva (60 percent of whole
saliva) than did the submaxillari-sublingual glands. Later,
Gore (1956) pointed out that this rate difference was less
in persons on a high carbohydrate diet, especially during
the nocturnal hours.

This was not confirmed by Henriques and Chauncey (1958)
who found no difference in rates of flow between stimulated
parotid and submaxillary glands. Chauncey, Weiss and Lisanti
(1956) detected no difference in rates of flow in the left
and right parotid glands either before or after eating.

Schneyer and Levin (1955a) in their study with essentlah-
ly resting glands of humans, calculated that the parotid
glands secreted 26 percent, submaxillary glands 69 percent,
and sublingual glands 5 percent. The volume of whole saliva
collected from the "resting" glands was 42 percent greater
than the combined volume of the individual secretions. When
the glands from the same subjects were stimulated (1955b),
the submaxillary secretion still accounted for the largest
fraction, the sublingual secretion the smallest fraction,
and the parotid secretion for an intermediate portion of
the combined volume. However, with increased stimulation,
the relative proportion contributed by the parotid glands
increased. Here again, whole saliva collected from these
stimulated glands was #7 percent greater than the combined

volume of the individual secretions.

55

Schneyer (l956a) attributed the discrepancy of the
greater volume of whole saliva secreted to the method used
for collection of whole saliva, and not to the secretion of
mucosal glands.

1n the attempt to explain the wide variation that
occurs in the rates of flow of specific salivary glands,
Schneyer, Pigman, Hanahan and Gilmore (1956) concluded,
as a result of their work, that saliva collected routinely
in the laboratory as "resting" saliva is, in fact, stimu-
lated or activated secretion.' Gross variations in the
rate of secretion, then, are due to fluctuations in the

intensity and frequency of internal stimulation.

Methods and Materials

Selectively desalivated and intact caries-resistant
and caries-susceptible rats were lightly anesthetized with
Nembutal, then injected with pilocarpine to stimulate the
flow of saliva. Each animal was allowed to salivate into
a graduated 15 m1 conical centrifuge tube. The collection
was continued for 20 minutes, unless the rat died before
this time, or the effects of anesthesia diminished. The
number of minutes correSponding to the period of collection
was noted. Accordingly, the rate of flow was calculated on
a milliliter per minute basis.

The rats that had their submaxillari-sublingual glands
removed yielded essentially only parotid saliva. Those
that had a section of their parotid ducts removed and the

cut ends ligated yielded essentially only submaxillari-

56

sublingual saliva, and the unoperated (intact) animals
secreted whole saliva. The method employed for collecting
saliva does not elicit a detectable amount of secretion
from the minor salivary glands, or from the mucous glands
of the oral cavity. No saliva could be collected from six
rats which had their parotid ducts and submaxillari-sub-
lingual glands removed.

In order to ascertain what effect the removal of the
parotid ducts might have on the weight of the submaxillari-
sublingual glands, both previously desalivated rats and
littermate controls were sacrificed when they were an
average of 31 weeks of age (an average of 18 weeks following
desalivation). Their submaxillari-sublingual glands were
removed and weighed on an analytical balance. Prior to
removing the glands for weighing, their total body weights

were noted.

Results

The data pertaining to the salivary rates of flow are
presented in table 8. The mean rate of flow of the various
salivas was not essentially different in resistant and
susceptible rats, but the rate of flow of the various salivas
within the lines of rats differed materially. Whole saliva
from unoperated (intact) rats showed the greatest flow rates
(0.102 and 0.103 ml/min.); those of submaxillari-sublingual
secretion were intermediate (0.082 and 0.084 ml/min.); and

parotid secretion showed the slowest rate (0.040 and 0.034

ml/min.).

57

TABLE 8. Mean rates of flow of whole, parotid, and submaxil-
lari-sublingual saliva from caries-resistant and
caries-susceptible rats

 

 

Type of Saliva Strain of Rat Number Rate of Flow
of Rats (ml/minute)
Resistant 27 0.102
Whole
Susceptible 27 0.103
Resistant 14 0.040
Parotid
Susceptible 14 0.034
Resistant 18 0.082

Submaxillari-
Sublingual Susceptible 21 0.084

 

58

Whole saliva is made up of a natural mixture of parotid,
submaxillary and sublingual salivas, and their combined rates
of flow contribute to the observed rate of whole saliva.
However, when the separate rates of flow of parotid and sub-
maxillari-subllngual saliva were added together, their sum
was 15 percent greater than the rate for whole saliva. There-
fore, it was of interest to see if removal of one set of
glands affected the size of the remaining set of glands.

The body weights, and the absolute and relative sub-
maxillari-sublingual gland weights were analyzed statisti-
cally using the "t" test, comparing rats that had their
parotid ducts intact with those that did not. The results
of the analyses are presented in table 9. The data and
formulae used in the analyses are found in tables 20 through
22, and page 135 of the Appendix, respectively.

The total body weight of male rats with parotid ducts
intact was 405 9 49.2 9, whereas those with parotid ducts
removed was 401 3 45.7 g. In these two groups of male rats,
the absolute weight of the submaxillari-sublingual glands
was 682 3 65.5 mg and 676 3 96.4 mg, respectively. The
relative weights (mg of gland weight per 100 gm of body
weight) of the submexillari-sublingual glands in male animals
with parotid ducts intact *weighed 170 3 17.0 mg, and those
with parotid ducts removed had glands that weighed 169 3
20.4 mg. The differences observed above, whether for total
body weight, absolute gland weight or for relative gland

weight, are not significant at the 5 percent level.

9
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60

In female rats, the total body weight with parotid
ducts intact was 244 3 35.4 g, and 237 3 24.3 g with the
parotid ducts removed. The difference of 7 grams is not
significant. Similarly, no significant difference occurred
in the absolute weight of the submaxillari-subllngual glands
obtained from these two groups of female rats (535 5 60.7 mg
and 549 3 52.4 mg, respectively). However, when considering
the relative weights of the submaxillari-sublingual glands in
these females, those with parotid ducts intact had signifi-
cantly lighter glands (220 3 19.6 mg) than those with paro-
tid ducts removed (233 3 28.3 mg). This difference is sig-

nificant at a level of 5 percent.

Discussion

Since the values for the rate of flow of stimulated
saliva was essentially the same in resistants and suscepti-
bles, indicating that probably no direct relation exists
between rate of flow and incidence of caries, no additional
information would be exPected by studying the relation of
these two characteristics in rats by correlation analysis.

There may still be a difference in the rate of flow of
unstimulated saliva between the resistant and susceptible
rats, even though there does not exist a difference in the
rate of flow of stimulated saliva. As Becks and Wainwright
(1939) pointed out, the wide variation in the secretory ca-

pacity of the resting salivary gland among human individuals

61

' That is, the subjects

was masked by stimulating the glands.
with originally low or high rates of flow of resting (unsti-
mulated) saliva arrived at about the same rate when the
salivary glands were stimulated. Perhaps, this phenomenon
is also operative in the salivary glands of rats. However,
the detenmination of the rate of flow of resting saliva in
the rat would be very difficult, if possible at all. In the
first place, the conscious animal has swallowing reflexes that
would interfere with collections. Secondly, tongue and cheek
muscles are active in the conscious animal, which stimulates
the salivary glands in varying degrees. To overcome the
swallowing and other mouth movements by anesthesia, would
introduce a factor that would very possibly affect the
secretory activity of their salivary glands.

The results obtained in this study pertaining to the
moderately significant increase in the relative weight of
the submaxillary and sublingual glands when the parotid ducts
were tied and severed in the female rats (see table 9) are
in agreement with those of Schwartz and Weisberger (1955).
They found that a significant hypertrophy of the remaining
salivary glands took place in partially sialoadenectomized

rats. They, too, measured the increase in terms of the

ratio of gland weight to body weight.

 

' The saliva of rats was stimulated by pilocarpine. This
is a different kind of stimulation than that caused by the
senses of smell or taste and by chewing. Human subjects
are usually stimulated by chewing.

62

Schwartz and Shaw (1955) found a highly significant in-
crease in the weight of submaxillary glands of rats when the
parotid glands were removed. This was confirmed by Schwartz,
Resnick and Shaw (1958) who found a moderately significant
increase in the weight of the submaxillary glands of rats
when either the parotid ducts were tied and severed, or when

the parotid glands were extirpated.

pH

Literature Survey

Conflicting reports have appeared which have attempted
to discern a relation between salivary pH and incidence of
dental caries.

Those who concluded that a direct relationship exists
between a low pH in whole human saliva and caries-suscepti-
bility, and conversely, a high pH and caries-resistance have
been R3$e (I905), McIntosh, 5; 91., (1925), Entin (1927),
Entin and Stark (1928), Skosovsky (1935), Staz (1938),
Krasnow (1938), Hanke (1939). Belding and Raiding (1939)
and Sullivan and Storvick (1950b). In addition to these,
Karshan, Krasnow and Krejci (l93la,b) and Krasnow (1932,
1936) noticed a tendency for such a correlation. Focusing
their attention on children, who seem to develop caries at a
more rapid rate than adults, Turner, Scribner and Bell (1953.
1954) also found a high salivary pH tended to be associated
with caries-immunity, and a low salivary pH with a high
caries incidence.

However, many more investigators believe that no con-
sistent relation exists between salivary pH and dental caries
activity. These investigators include: Miller (1904), Loth-
rop and Gies (1910), Marshall (1916a), D'Alise (1921), Cans
(1926), Kallhart (1928), White and Bunting (1935), Grove and
Grove (1935), Ziskin and Hotelling (1937), Swerdiove (1942),

63

64

Whyte (1943), Mackenzie (1945), Rovelstad (1957), Rovelstad,
_t_§l,, (1958), Muracciole and Castro (1959), and Englander,
Mau, Hoerman and Chauncey (1958) . ’

Furthenmore, the following researchers concluded that
there was no consistent pH difference in saliva of caries-
resistant and caries-susceptible children: McKeag (1928),
Roskin (1928), Magee, Drain and Boyd (1929), Stern (1931),
Brodsky (1933), Hubbell (1933), White and Bunting (1935,
1936), Stones, Lawton, Bransby and Hartley (1950) and Muracci-
ole and Castro (1959).

Rosen, gt gL., (1957) found no significant difference
in salivary pH between caries-resistant and caries-susceptible
rats.

In a study of parotid saliva from caries-active and
caries-resistant persons, Englander, Mau, Hoerman and Chaun-
cey (1958) could not correlate pH with caries activity, al-
though they did find that pH varied directly with the rate
of flow of stimulated parotid saliva. This correlation was
confinmed by Chauncey, Lisanti and Winer (1958). Not only
was there no significant difference between the secretions
of the left and right parotid glands of humans, as shown by
Chauncey, gthl., (1956), but also Shafer, gt al., (1958a)
and Henriques and Chauncey (1958) found no marked difference
in pH between the parotid and submaxillari-sublingual rat
salivas.

The pH of the isoelectrlc zone of saliva protein-was

demonstrated by Arnold and McClure (1941) to be a relatively

65

constant characteristic of each person‘s saliva, but varies
among individuals. This variation was not, however, corre-
lated with caries activity.

Krasnow and Oblatt (1933) found a greater interdependence
of pH and titratable alkalinity in caries-susceptible cases
. than in caries-resistant cases. Similarly, Sullivan and
Storvick (1950a) showed a significantly positive correlation
between pH and buffer capacity, although they made no attempt

to relate this to caries activity.

Methods and Materials

As soon as possible (usually within an hour) after<
collecting parotid, submaxillari-sublingual, and whole saliva
from caries-resistant and caries-susceptible rats, the pH
of the samples was determined using a Beckman Glass Elec-
trode pH Meter, model H2. Each saliva sample was from an
individual rat, and kept at room temperature until the pH

determination was made.

Results

The pH of the various salivas listed in table 10 ranged
from 8.61 to 8.77. The constant pH observed in these sali-
vas indicates that probably there is no significant differ-
ence between the pH of salivas from resistant and susceptible
rats, or between the salivas within each line of rat. 1

The pH of whole resistant and whole susceptible salivas
were8.65 and 8.68, respectively. Parotid saliva showed a

similar pH (8.77). on the average, in both lines of rats.

66

Submaxillari-sublingual saliva from the resistants showed a
pH of 8.75, and that from the susceptibres was 8.61.. Thus,

the various salivas were within 0.2 pH units of each other.

Discussion

The data of table 10 do not show any relation between
the pH of the various pilocarpine-stimulated salivas (i.e.,
parotid, submaxillari-sublingual and whole) and caries
activity, as this activity relates to resistance or suscep-
tibility to caries in the rats used in this study. This
confirms the findings of Rosen, 95 al,, (1957) who studied
whole saliva from these two lines of rats. Furthenmore,
Shafer, st 21., (l958a) found that the pH of parotid and
submaxillari-sublingual rat salivas ranged from 8.1 to 8.2.
Henriques and Chauncey (1958) found no significant differ-
ence in the pH of parotid and submaxillari-sublingual human
salivas.

The average pH of all salivas in table 10 is 8.70. This
is close to the pH of 8.3 of pilocarpine-stimulated whole
rat saliva reported by Shafer,.et,g1., (1958a). There is
some indication that the pH of rat saliva is as high 15,

vivo as it was shown to be by the in vitro method employed

 

in this investigation. Indicator paper was inserted in the
mouths of 12 nonmal rats equally represented by males and
females, susceptibles and resistants, and the pH color
change was roughly in the 8.5 zone in all rats tested.

One might expect this slightly alkaline saliva to neu-

tralize, to some extent, the acids produced by the acidogenic

67

TABLE 10. pH of whole, parotid, and submaxillari-sublingual
saliva from caries-resistant and caries-susceptible

 

 

rats -
Type of Saliva Strain of Rat Number pH
of Rats

Resistant . 24 8.65
Whole

Susceptible 30 8.68

Resistant 7 8.77
Parotid

Susceptible 12 8.77

Resistant 16 8.75

Submaxillari-
Sublingual Susceptible 22 8.61

68

microorganisms on the teeth and, thus, prevent the deminer-
alization of the tooth enamel (inhibition of the cariogenic
process). This would be true provided that the saliva, and
the ions contained in it, had free access to the acids pro-
duced by the bacteria. This does not always seem to be the
case, however, as studies with humans indicate. That is, at
the onset of carious lesion formation, many acidogenic
microorganisms are enmeshed in an amorphous, mucinous mat,
called the plague, which is attached to the tooth surface.
When fermentable carbohydrates are ingested, they diffuse
into the plague to supply the acidogenic microorganisms with
the necessary substrate. The rate of acid production with-
in the plague is rapid.

Perhaps the caries-resistant and caries-susceptible rats
differ, therefore, in the degree and quality of plague for-
mation on the teeth, and thereby, in the extent to which the
microorganisms are encouraged to form acids. This could

possibly be checked in a future study.

BUFFER CAPACITY

Literature Survey

Although there has been some controversy concerning
the question of whether the buffering action of saliva was
related to caries activity, a large majority of investigators
have shown in various ways, but mainly by titratable alka-
linity, that caries-resistance is associated with a high
buffering action than is caries-susceptibility. These in-
Vestigators include Rbse (1905), Marshall (1917 a, b), Pick-
erill (1924a, b) Hubbell (1933). Grove and Grove (1934), Core
(1935), Hanke (1937, 1939), Hawkins (1939), Fosdick, Campaigne
and Faucher (1941), Dreizen, Mann, Cline and Spies (1946),
Fosdick (1947), Muracciole (1955), Rovelstad, gt g1., (1958)
and Englander, Shklair and Fosdick (1958).

Turner, Scribner and Bell (1953, 1954) reported that
titratable alkalinity of children's saliva was associated
statistically with caries incidence, but that titratable
acidity was less consistently associated. Turner and Anders
(1956) indicated that caries-free children as a group show
lower titratable acidity and higher titratable alkalinity

than those cases with extensive decay, which show higher

titratable acidity and lower titratable alkalinity. Forbes
and Gurley (1932) and Burrill and Fosdick (1944) were able
to demonstrate only a tendency toward a relationship be-

tween buffer capacity and caries incidence.

69

70

Dreizen, gt g1., (1946) found that the lactobacillus
count of saliva varied inversely with its buffer capacity;
however, this was not confirmed by Roe and Clegg (1949).

Although Marshall (1915, 1916a, b, 1917a, b) reported
that no relation existed between dental caries and titratable
alkalinity or titratable acidity in either stimulated or "un-
stimulated" saliva, he maintained that his "salivary factor"
appeared to be indicative of immunity or susceptibility to
caries. He defined the "salivary factor" as the quotient
of the neutralizing power of "nonmal resting" saliva divided
by that of stimulated saliva multiplied by 100. The neutra-
lizing power was a function of the titratable alkalinity
plus the titratable acidity. He obtained a salivary factor
of 43 to 80 for caries-immune saliva and a factor of 80 to
132 for caries-susceptible saliva. Bunting and Wixon (1917)
confirmed these findings when applied to caries-immune sali-
va, but not when applied to caries-susceptible saliva. Shep-
ard and Gies (1916), Gies (1916a, b, 1917) and Gies, Lowen-
stein, Heft and Noland (1917), on the other hand, vigor-
ously criticized Marshall's "salivary factor" as invalid and
not associated with dental caries. Other workers who were
unable to demonstrate a relation between dental caries and
buffer action of saliva were Karshan, g3 g1., (1931a, b),
Belding and Belding (1939) and Rovelstad (l957).

Rése (1905) reported that carbonates contributed in
part to the alkalinity of saliva, but thought organic con-

stituents of saliva were also important in this regard.

71

Marshall (1917a, b) thought the inorganic constituents of
saliva rather than its organic ones were the source of the
buffering capacity of saliva. Later it was amply demonstrated
by Sellman (1949), Wah Leung (1951), Dreizen, Reed, Neider-
meier and Spies (1953) and Lilienthal (1953. 1955a, b) that
the buffer system mainly responsible for the buffering ca-
pacity of stimulated saliva at "physiologic" pH is the car-
bonate-bicarbonate system. They also found that phosphates
constitute the next most important buffer system while sali-
vary proteins, as well as bacteria, contribute little, if
any, to the total buffering capacity of saliva. Knox and
Still (1953) also found mucoid, the major salivary protein,
to be ineffective as a buffer _i_n_ 31533.

Related to the carbonate-bicarbonate system is what has
been referred to as the C02 capacity of saliva, which is the
ability of saliva to absorb C02, and a measure of its abil-
ity to neutralize acid. Carbon dioxide capacity has been
shown to be higher in saliva of caries-free persons than in
caries-active persons by Hubbell (1933), Karshan (1936, 1939),
Karshan, Rosebury and Waugh (1939), and Karshan, Siegel and
Waugh (I940).

Dreizen,($pies, Dreizen and Spies (1957) found that the
salivary buffers acting to protect against dental caries
originate in the gland rather than the serum.

Krasnow and Oblatt (1933) demonstrated a greater inter-
dependence of pH and titratable alkalinity in caries-suscep-

tible saliva that was particularly marked in "unstimulated"

72

morning saliva. Similarly, Sullivan and Storvick (1950a, b)
and Dreizen, gt al., (1953) reported a significant positive
correlation between salivary pH and buffer capacity, but made
no attempt to relate this to caries activity.

Deakins, g§,§l,, (1941) studied the acid neutralizing
power, base neutralizing power, and total buffer capacity of
saliva from 20 subjects. No definite correlation was found
between any of these properties and rate of flow, either on
an amount-per-cc basis, or on an amount-per-cc-per-minute
basis. Englander, Mau, Hoerman and Chauncey (1958) detected
' no difference in titratable alkalinity in parotid saliva of
caries-free and caries-rampant males, but found titratable
alkalinity to vary directly with rate of flow. Chauncey, Li-
santi and Winer (1958) indicated a significant positive
correlation between the rate of flow of parotid saliva and
its bicarbonate content.

Grove and Grove (1934) did not believe that titratable
alkalinity estimations in themselves were indicative of either
susceptibility or resistance, but are of value as an indica-
tion of the amount of alkaline salts in saliva, including
salts of ammonia. Ammonia is a solvent for dental plaques
and salivary mucin, so that in mouths where pH is above 7.
plaque fonmation is obstructed by the action of ammonia,
and caries thereby retarded. However, ammonia must be in

the form of its alkaline salts to be effective.

73

Methods and Materials

In order to establish the degree of buffering capacity
of rat saliva, the titratable alkalinity was detenmined. This
was accomplished by diluting submaxillari-sublingual and whole
saliva 1-5 with distilled water, then titrating the diluted
saliva sample with 0.0235N hydrochloric acid until the pH
reached 4.5 3 0.2. As may be seen in figure 6, rat saliva
diluted 1-5 has definite buffering capacity in the approxi-
mate pH range of 7.5 to 6.0, whereas, distilled water does
not impart any buffering action to the saliva. Adding only
0.05 ml of 0.02N HCl to 4 ml distilled water resulted in a
precipitous drop in pH.

The pH was measured at room temperature with a Beckman
Glass Electrode pH Meter (Model H2), using saliva that had
been stored in the refrigerator (5° 3 1° C) up to 7 days.

It was found that no significant change in buffering capa-
city of rat saliva occurs if it is held at a low tempera-
ture for this period of time.

The number of parotid saliva samples that were of suf-
ficient volume for use in this test, was inadequate for pre-
sentation in this study. Consequently, the titratable alka-
linity of only submaxillari-sublingual and whole saliva
will be considered.

A correlation analysis was made between the titratable

alkalinity of whole saliva from susceptible rats and caries

age in these same rats.

74

Results

The data for buffering capacity is presented in table
11. Saliva from caries-susceptible rats had a greater
capacity to buffer hydrogen ions than did saliva from
caries-resistant rats. This was evident in both whole and
submaxillari-sublingual saliva. Whole saliva from resis-
tant rats had a titratable alkalinity of 2.05 3 0.042 ml,
and that from susceptible saliva was 2.25 3 0.037 ml. The
difference of 0.20 3 0.056 ml between these two is highly
Significant (P < 0.01). The titratable alkalinity of sub-
maxillari-sublingual saliva from susceptible rats was 2.25
3 0.052 ml, which was 0.41 3 0.150 ml greater than that
from resistant animals. This difference is significant at
a level of 2 percent.

When a correlation analysis was made of the titratable
alkalinity of whole susceptible saliva and caries age of
these same rats, the correlation coefficient (r) was found
to be 90.192, which is not significant (P,> 0.05). The
data and fommula used in this analysis is presented in

table 23 of the Appendix.

Discussion
The difference of 0.20 3 0.056 ml between resistant and

susceptible whole saliva, and the difference of 0.41 3 0.150
ml between resistant and susceptible submaxillari-sublingual
saliva is statistically significant (table 11). These

differences are meaningful not only statistically, but when

75

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76

small increments (0.05 ml) of 0.02N HCl were added to saliva
at a pH below 6.0, the pH decreased by about 0.3 (figure 6).
Therefore, differences of 0.2 or 0.4 m1 of 0.02M HCl are
probably meaningful.

The greater buffering capacity of saliva from
susceptible rats than of saliva from resistant rats was
rather unexpected, since this would suggest that this sali-
vary property contributes to caries-susceptibility, when
almost all reports based on human saliva correlated buffer-
Iing capacity with caries resistance (no reports based on
animal saliva were found). But the correlation analysis of
the data from these rats indicates that probably no im-
portant relation exists between this property and incidence
of caries, and therefore, caries susceptibility. This may
be another instance of two unrelated traits (i.e., caries-
susceptibility and buffer capacity) being selected for as

a result of inbreeding.

77

 

 

 

 

o — 1 ml RAT SALIVA + 4 ml DISTILLED WATER
9—
X - 4 ml DISTILLED WATER
q
0
i
q
0
z; \\\\\\\\\
O
—1 \0\
£6. %\
-1 °\
5... °\
0
\.
4-1
X
T T U f 1 I l r T T
0.5 IO 1.5 2.0 2.5

MILLILITERS 0.0235 N HCl

Figure 6. A typical curve illustrating the buffering
capacity of diluted rat saliva compared to
that of distilled water.

GENERAL DISCUSSION

The saliva from caries-susceptible rats stimulated the
growth of rat oral streptococci to a greater extent than the
saliva from caries-resistant rats. The saliva from both
strains of rats supported, but did not stimulate, the growth
of five out of six strains of rat oral lactobacilli.

The stimulation of the streptococci could have been
due to a supply of nutrients made specifically available
to these organisms. A carbon and nitrogen source could be
provided, for example, by the large quantity of mucin, which
is a glycoprotein and an important component of saliva.

Knox (1953a, b) demonstrated a lysis of mucin in human
saliva, which he attributed to the partially purified enzyme,
mucinase. This may actually be the expression of more than
one enzyme, as the following discussion will suggest. Knox
found several salivary microorganisms capable of elaborating
an exocellular mucolytic enzyme. He concluded that the mode
of action is one of a primary depolymerization of salivary
mucin by the activity of a proteolytic enzyme, and sub-
sequently the liberation of the components of the muco-
polysaccharide by a hyaluronidase-like enzyme. This con-
clusion is based on his experiments, which showed that
trypsin (an endopeptidase) decreased the viscosity of
salivary mucin, and hyaluronidase liberated reducing sugar
from mucinase or trypsin depolymerized mucin. If hyaluron-

idase does, indeed, mediate in the breakdown of the
‘ 78

79

polysaccharide moiety of mucin (hyaluronic acid), acetly-
glucosamine and glucuronic acid would be fonmed. He further
suggests that other enzymes are probably required before
complete depolymerization results. Simmons (1941) had
previously found a similar two-stage mucolytic activity in

human saliva.
The saliva of rats used in this study seems to be

equipped for such mucolytic activity. Willett (1955) found
that the salivary glands of these rats, particularly sus-
ceptibles, elaborate a protease; and hyaluronidase is ela-
borated by microorganisms contained in the oral cavity of
these animals.

Growth factors other than those possibly made available
by the mucolytic activity in saliva, may be present to a
greater extent in susceptible saliva than in resistant sa-
liva. These growth factors could be in the form of metallic
ions, as well as other inorganic ions, or vitamins, or the
purines and pyrimidines, for example. These may be pre-
sent in the rat's parotid saliva, and act in a complimentary
manner with other nutrients found in the submaxillari-sub-
lingual saliva, since the latter saliva did not stimulate
the streptococci but whole saliva did.

Another possible source of nutrients usable by the
streptococci is the food'ingested by the rats. Although all
rats were on the same diet, their teeth brushed, their mouths
rinsed, and finally the saliva filter sterilized, there

could still have been sufficient residual food not rinsed

80

out before saliva collection to influence the growth of the
streptococci. The residual material might then be washed
out of the mouth during active sallvation. Since susceptible
rats contain 2% to 3 times more protease in their saliva
than do the resistants, it is conceivable that more protein
(casein) ingested as food would be hydrolyzed to simpler
compounds in susceptible rats. Thus, the growth of strep-
tococci would be favored more in the mouths of susceptible
rats if the hydrolyzed protein would be a more readily
utilizable form for these organisms.

Another possibility might account for the difference
in the degree to which the susceptible and resistant whole
saliva stimulated the growth of streptococci. Instead of the
susceptible saliva containing more of the stimulatory sub-
stance than the resistant saliva, perhaps they both had
equal quantities of the stimulatory substance, but that the
resistant saliva contains, in addition, something else acting
as an inhibitor to this substance. The resistant saliva
would then show a diminished stimulation of the streptococci.

It might be desirable to investigate rat saliva further
for the presence of an inhibitory agent active against rat
oral streptococci and lactobacilli, with the technique used
in this study modified to employ a minimal growth medium
that would be less apt to mask inhibition by saliva, rather
than employing a maximal growth medium. *1. I

Still to be established is whether the streptococci,

whose growth is stimulated jg vitro, are also favored jg vivo.

81

Furthermore, it remains for a future study to determine
whether some strains of rat oral streptococci are stimulated
in the mouths of susceptible rats, but not in the mouths of
resistant rats; and if so, to detenmine their relative
numbers and cariogenic potential.

This greater stimulation of streptococci but not lacto-
bacilli by whole saliva from susceptible than from resistant
rats supports the concept that streptococci rather than the
lactobacilli are primary etiologic agents in dental caries
in rats. Evidence contributed by others suggesting this
hypothesis is as follows: Dental caries are induced in
genm-free rats by an enterococcus, but not by lactobacilli
(Orland, et 51., 1955 and Fitzgerald, g§_§l,, 1960). Den-
tal caries are induced in "caries-inactive" hamsters by
single or pooled cultures of streptococci from a carious
hamster; not so by strains of lactobacilli from "caries-
active" hamsters, or strains of streptococci from “caries-
inactive" hamsters (Fitzgerald and Keyes, 1960). There are
different strains of streptococci in caries-resistant and
caries-susceptible rats (Rosen, gt,§1., 1955), as well as
in "caries-active" and "caries-inactive" hamsters (Fitz-
gerald and Keyes, 1960). I

if this greater stimulation of the streptococci in
susceptible whole saliva is to be interpreted as supporting
the hypothesis of the streptococci being the etiologic
agent for dental caries in rats, certain findings relating

to sialoadenectomized rats should be explained. When saliva

82

from all the major glands (i.e., parotid, submaxillary,

and major sublingual) was interrupted, the incidence of car-
ies increased in the resistant rats. Since the stimulatory
effect of the saliva for the streptococci was absent, why
did the incidence of caries not diminish, instead? Stimu-
lation of the streptococci is only one of the many inter-
related factors which may account for the difference between
the resistant and susceptible rats. The adverse effects, that
are introduced when saliva is prevented from entering the
oral cavity and thereby inducing a greater degeree of

caries development, far outweigh the beneficial effect of
depriving the streptococci of their original stimulatory
source (saliva).

Some adverse effects that are possibly involved by
removal of the major glands are: the accumulation of in-
gested food in the rat's mouth; lack of buffering action
by saliva of acid produced by microorganisms; and a possible
symbiotic or commensalistic relationship between the strep-
tococci and the lactobacilli (the population of the latter
increases significantly in a sialoadenectomized rat, as
shown by Rosen, gt_§l,, 1953, 1959a, b).

The amylase study demonstrated that both resistant and
susceptible rats have adequate stores of this enzyme to
account for the hydrolysis of starch that might be lodged
in the tooth fissures. This would provide large amounts of
fermentable carbohydrates for acidogenic organisms. How-

ever, even though whole saliva of susceptible rats has

83

significantly more amylase activity than resistant rats, both
have high levels. In addition, amylase activity was not
correlated with caries age in susceptibles alone, or when
susceptible and resistant rats were considered together.
Finally, rats deprived of the secretion from all major
salivary glands, and therefore also amylase from these
glands, show a greater incidence of caries. Thus, it seems
that salivary amylase activity does not play a major role

in the etiology of dental caries in rats.

Viscosity and rate of flow are related salivary charac-
teristics, since they both regulate the movement of saliva
over the teeth. The greater this movement, the more the
various other salivary factors can influence the state of
dental health, such as the washing and lubricating action
of saliva, its stimulatory properties for certain bacteria,
and its buffering action. Relative viscosity and rate of
flow of whole, parotid, and submaxillari-sublingual salivas
do not appear to be significant factors in accounting for
the difference between the two lines of rats. The signi-
ficant difference in viscosity noted between resistant and
susceptible whole saliva when coilected at room temperature
no longer existed when the saliva was collected at a lower

temperature.

This is not to say that these two salivary properties
have no influence on the state of dental health in rats.
Rather, if the values for viscosity increased and the rate

of flow decreased significantly, an adverse effect upon the

84

teeth could be expected. 0n the other hand, a beneficial
effect could be expected if the values for viscosity de-
creased and the rate of flow increased significantly.

Of interest in this regard is the work of Muhler,
Bixler and Shafer (1957), who reduced caries significantly
in rats with daily administrations of 2 mg of pilocarpine
(a sialogogue), although 6 and 12 mg did not. Moreover,
Shafer, Clark and Muhler (1957) found that higher levels
of thyroxine adminstered to rats for two months resulted in
a lower incidence of caries and a less viscous saliva that
was stimulated by pilocarpine.

Selye, Vielleux and Cantin (1961) were able to induce
selective growth of rat's salivary glands to about five
times their nonnal size, by chronic treatment with iso-
proterenol. This suggests a study to detenmine whether the
increased rate of flow resulting from mitotic prolifera-
tion and hypertrophy of the parenchymatous cells will in-
fluence significantly the caries incidence in susceptible
and resistant rats. Caries inhibition due to increased
size and function of the salivary glands should be evident
to a greater degree with the susceptible rats.

Buffering capacity and pH are two other salivary charac-
teristics closely related to one another. Together they
tend to minimize the harmful effect that the acid produced
by microorganisms has on the inorganic constituents of the
teeth. According to the acidogenic theory of dental caries,

the factors determining whether a tooth will be eroded are,

85

ultimately, the rate of acid formation and the rate of
acid neutralization.

In this study, the buffering capacity was found to be
greater in the saliva of caries-susceptible rats. This does
not support the acid theory of dental caries. However, that
saliva has definite buffer action at all, and that caries
becomes more rampant when salivary glands are removed,
supports the concept that the buffering capacity of saliva
contributes to caries-resistance.

Although no difference was found between the pH of
salivas of resistant and susceptible rats, and the buffering
capacity was not correlated with caries age (even though the
saliva from susceptible rats showed a greater buffering
capacity) the data revealed an efficient buffer system in
whole and submaxillari-sublingual salivas of these animals.
That is, the pH (average 8.70) is decidedly higher than the
upper range of the buffer zone (approximately 7.5 to 6.0).
See figure 5. Therefore, saliva from these rats maintains

its maximum titratable alkalinity.

SUMMARY

Whole, parotid and submaxillari-sublingual saliva
from caries-resistant and caries-susceptible rats were
studied from various points of view to ascertain the sail-
vary factors that might contribute to resistance or suscep-
tibility to dental caries. These studies were the effect of
saliva on the growth of microorganisms, amylase activity,
viscosity, rate of flow, pH and buffering capacity.

Whole and submaxillari-sublingual saliva from the two
lines of rats did not materially affect the mean rates and
maximum limits of growth of five out of six strains of rat
oral lactobacilli when compared with a saline control.

Rat oral streptococci, when cultured in the presence
of whole and submaxillari-sublingual saliva from resistants
and susceptibles, showed an increase in the maximum amount
of growth, but not in the rate of growth as compared with a
saline control. Whole susceptible saliva supported a greater
maximum amount of growth than did whole resistant saliva.

No difference in the submaxillari-sublingual saliva from the
two lines of rats existed in this respect.

More than 99 percent of the anylase activity of saliva
originated in the parotid glands of these animals. Parotid
and whole salivas from susceptible rats showed greater amy-
lase activity than these salivas from resistant rats. However,
no significant correlation existed between amylase activity

and caries activity.
86

87

When the various salivas were collected at room temper-
ature, whole and submaxillari-sublingual salivas from re-
sistant rats were more viscous than'these salivas from sus-
ceptible rats (no real difference in the relative viscosity
was found in the parotid secretions between the two lines of
rats)., However, when whole saliva was collected in tubes
submerged in ice, the difference between resistants and
susceptibles no longer was observed. Comparison of salivas
within the lines of rats showed parotid saliva to be less
viscous than submaxillari-sublingual saliva: whereas, whole
saliva gave intenmediate values. A correlation analysis
between caries experience and relative viscosity revealed
no significant relation between these two traits.

The mean rate of flow and pH of the parotid, submaxil-‘
lari-sublingual, and whole secretions were not essentially
different in resistant and susceptible rats. Within each
line of rat, however, the rate of flow of each saliva
differed materially. Whole saliva from unoperated rats
showed the greatest flow rate, parotid saliva was slowest,
and submaxillari-sublingual saliva was intermediate.

Buffering capacity, determined as titratable alkalin-
ity, was significantly greater in susceptible whole and sub-
maxillari-sublingual salivas, but this salivary property

was not correlated with caries activity.

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, 1954. The relationship of titratable acidity,
titratable alkalinity, and pH to the incidence of
dental caries. J. dent. Res., 33:55.

 

Van Kesteren, M., Bibby, B. G., and Berry, G. P., 1942.
Studies on the antibacterial factors of human saliva.

J. Bact., 43:573.

Wah Leung, S., 1951. A demonstration of the importance of
bicarbonate as a salivary buffer. J. dent. Res., 30:

403.

Weigmann, F., and H6121, H., 1940. Untersuchungen Uber die
antagonistische Wirkung von Streptokokken gegenuber
Diphghgrie und anderen Bakterien. Z. Hyg. Infektkr.,
122: 7 .

Weigmann, F., and Koehn, A., 1936. Weitere Untersuchungen
uber die Wirkung des menschlichen Speichels auf Diph-
theriebacillen. Z. Hyg. Infektkr., 118:507.

Weigmann, F., and Noeske, H., 1937. Untersuchungen Uber den
antibakterielle wirksmen Faktor im menschlichen Spei-
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Weisberger, 0., Nelson, C. T., and Boyle, P. E., 1940. The
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White, B. J., and H111, T. J., 1949. Antagonistic relation-
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109

Whyte, R., 1943. A diatetic, dental, and bacteriological
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7

75:2 , 273. 301.

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Ph. D. Dissertation, Michigan State University.

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, 1959. Studies on the antibacterial action of
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Preprintea AEstracts, p. 39.

 

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1 :507.

APPENDIX

110

111

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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112

TABLE 2. Paired data analysis of the effect of caries-re-
sistant and caries-susceptible whole rat salivas
on the growth of rat oral streptococci

 

Maximum_§;owth in 0.0.

 

 

 

 

 

 

Streptococcus
Culture Difference
Number Susceptible Saliva Resistant Saliva g(d)
l 1.70 1.00 0.70
2 1.80 1.40 0.40
3 1.90 1.20 0.70
3 1.40 1.10 0.30
S 1.90 0.90 1.00
5 1.30 0 85 0.45
6 1 50 1.10 0.40
S- =1 n 3 0.0931 id = 3.95
d (M) (n)
a _ O 3 3 0.5643
t z 53 3 5.051 (5 c1)2 =15.6025

Degrees of freedon 3 6

(s d)2 = 2.2289
n

P =<o.o1 2
Ed = 2.5925

113

 

 

 

 

 

 

 

 

 

TABLE 3. Paired data analysis of the effect of caries-
resistant and caries-susceptible whole rat salivas
on the growth of rat oral streptococci

Slope of Growth Curve
Streptococcus

Culture Difference

Number Susceptible Saliva Resistant Saliva gig)
I 2.13 2.50 -0.37
2 3.77 3.63 0.14
3 2.74 2.42 0.32
3 2.01 2.00 0.01
5 4.11 4.12 -0.01
5 3.87 4.50 -0.63
6 2.46 1.76 0.70
7 4.59 4.13 0.46
8 4.22 3.94 0.28
dZ-gzcuz §d=0.90

S' 3 n 3 0.1368 -

d \ (nu-l) (nT d = 0.1000
3. 0 (2 d)2 = 0.8100

1: 2 T:— : .

d 0 73' (S. d)2 = 0.0900
n
Degrees of freedom - 8 Edz : 1.4360

P 3 7’0.S

114

TABLE 4. Paired data analysis of the effect of caries-
resistant and caries-susceptible submaxillari-
sublingual rat salivas on the growth of rat oral

streptococci

 

Maximum Growth in 0.0.

 

 

 

 

 

 

Streptococcus
Culture Difference
Number Susceptible Saliva Resistant Saliva (d)
1 1.20 1.00 0.20
2 1.20 0.92 0.28
3 0.98 0.97 0.01
3 0.90 0.88 0.02
5 0.75 0.79 -0.04
5 0.72 0.76 -0.04
6 1.10 0.95 0.15
202 - (2012 id = 0.58
33 :J fl = 0.05106 _
Th-l) (n) d 3 0.0829
_ (2.02 = 0.3361.
t 2 933—0- : 1.52 ‘Zdlz 3 0.01881
n
Degrees of freedom 3 6 zdz : 0.1446

P: >002

115

TABLE 5. Paired data analysis of the effect of caries-
resistant and caries-susceptible submaxillari-
sublingual rat salivas on the growth of rat oral
streptococci '

 

Slope of growth Curve

 

 

 

 

 

 

Streptococcus
Culture Difference
Number Susceptible Saliva Resistant Saliva (d)
l 2.19 3.08 0.89
2 2.80 4.18 1.38
3 2.50 2.36 -0.14
3 1.79 2.12 0.33
S ' 4.07 3.76 -0.31
5 4.49 3.79 -0.70
6 1.91 1.78 -0.13
7 3.77 4.12 0.35
8 3.71 3.60 -0.11
‘ 26 = 1.56
_ _ Zdz - 2 d 2 -
Sd - J i__fil_'= 0.2138 d 3 0.1733
("'11 (n) _
(zd)2 - 2.4336
3 - 0 (2612 = 0.2700
t = 53 : 0.8106 n
Degrees of freedon 3 8 Zdz 3 3.5626

P = >0Jl

116

 

 

 

 

 

 

 

 

TABLE 6. Paired data analysis of the effect of caries-
resistant whole rat saliva and saline control on
the growth of rat oral streptococci

Maximum Growth in 0.0.
Streptococcus
Culture Difference
Number Resistant Saliva ngine Control 1d)
1 1.00 0.70 0.30
2 1.40 0.58 0.82
3 1.20 0.67 0.53
3 1.10 0.72 0.38
5 0.90 0.38 0.52
5 0.85 0.41 0.44
6 1.10 0.54 0.56
' “ Ed 3 3.55
_ 282 - (2812 _
S; - n 3 0.0627 d 3 0.5071
N 54) (n1 2 _
- (2d) - 12.6025
d - 0
t .-. T : 8.088 g d)2 3 1.8004
De rees of freedom 3 6
9 2612 = 1.9653

117

TABLE 7. Paired data analysis of the effect of caries-re-
sistant whole rat saliva and saline control on
the growth of rat oral streptococci

 

Slope of Growth Curve
Streptococcus

 

 

 

 

 

 

Culture Difference
Number Resistant Saliva Saline Control 1d)
1 2.50 3.20 0.70
2 3.63 1.99 -l.64
3 “ 2.42 3.13 0.71
3 2.00 2.47 0.47
5 4.12 3.08 -l.04
5 4.50 3.63 -0.87
6 1.76 1.82 0.06
7 4.13 5.19 1.06
8 3 94 5.29 1 35
262-12612 Zd=0.80
5; 3 n 3 0.3464 _
(n-IT (m d = 0.0889
- (2d12 = 0.6400
_ d - 0 2 -
t -T20.257 (2d) '0.0711
n
f = 8
Degrees of reedom 2dz = 8.6928

P 3 > 0.5

118

TABLE 8. Paired data analysis of the effect of caries-
resistant submaxillarl-sublingual rat saliva and
saline control on the growth of rat oral strepto-

 

 

 

 

 

 

 

 

 

cocci
Maximum Growth in 0.0.
Streptococcus
Culture Difference
Number Resistant Saliva Saline Control (d)
1 1.00 0.70 0.30
2 0.92 0.58 0.34
3 0.97 0.67 0.30
3 0.88 0.72 0.16
5 0.79 0.38 0.41
5 0.76 0.41 0.35
6 0.95 0.54 0.41
6 “2 d 2 id = 2.27
S3 3‘} n 3 0.0323 8 3 0.3243
d ("'T7 TnT 2 _
8 0 (Ed) - 5.1529
t = T5 = 10.040 (26112 = 0.7361
n
Degrees of freedom 3 6 Z.d2 3 0.7799

P 3 (‘0.01

119

TABLE 9. Paired data analysis of the effect of caries-
resistant submaxillari-sublingual rat saliva
and saline control on the growth of rat oral

streptococci

 

Slope of Growth Curve

 

 

 

 

 

Streptococcus
Culture Difference
Number Resistant Saliva Saline Control 01d)
1 3.08 3.20 0.12
2 4.18 1.99 -2.19
3 2.36 3.13 0.77
3 2.12 2.47 0.35
5 3.76 3.08 -0.68
5 3.79 3.63 -0.16
6 1.78 1.82 0.04
7 4.12 5.19 1.07
8 3.60 5.29 1.69
582-12612 26111.01
53 z n : 003709 -
(h-T} (n) d 3 0.1122
5- 0 (Ed)2 3
t 3 ‘33-_'3 0.302 2
d {Zidl 3 0.1133
Degrees of freedom 3 8 Zldz 310.0165

P = >0.5

120

 

 

 

 

 

 

 

TABLE 10. Paired data analysis of the effect of caries-
susceptible whole rat saliva and saline control
on the growth of rat oral streptococci

Maximum Growth in 0.0.

Streptococcus

Culture Difference
Number Susceptible Saliva Saline Control (d)
l 1.70 0.70 1.00
2 1.80 0.58 1.22
3 1.90 0.67 1.23
3 1.40 0.72 0.68
5 1.90 0.38 1.52
5 1.30 0.41 0.89
6 1.50 0.54 0.96
Ed 3 7.50
282 -12 612 -
5:1: T 1) (3L: 3 0.1039 d = 1.0714
n- n
(26112 = 56.2500
3 - 0 _ (2 .112 = 8.0357
t 3 Sa ' 10.312 n
282 " 8.4878

Degrees of freedon 3 6

P 3 < 0.01

121

 

 

 

 

 

 

TABLE 11. Paired data analysis of the effect of caries-
susceptible whole rat saliva and saline control
on the growth of rat oral streptococci

Slope of Growth Curve
Streptococcus
Culture Difference
Number Susceptible Saliva Saline Control (d)
I 2013 3020 -3007
2 3.77 1.99 1.78
3 2071+ 3013 -0039
3 2.01 2.47 -0.46
5 4.11 3.08 1.03
5 3.87 3.63 0.24
6 2.46 1.82 0.64
7 4.59 5.19 -0.60
8 4.22 5.29 -l.07
TE d2 - g 2 6112 _ - _
SE 3 n - 0.3271 d - 0.0111
\1 W!) (n) 2 -
- (2d) - 0.0100
d - 0 2 -
t : 15'— : 0.031, (2d) - 0.0011
n
Degrees of freedom 3 8 idz 3 7.7100

P 3 > 0.5

122

TABLE 12. Paired data analysis of the effect of caries-
susceptible submaxillari-sublingual rat saliva
and saline control on the growth of rat oral
streptococci

 

Haxlmum GLrowth in 0.9;

 

 

 

 

 

 

Streptococcus
Culture Difference
Number Susceptible Saliva Saline Control 1d)
1 1.20 0.70 0.50
2 1.20 0.58 0.62
3 0.98 0.67 0.31
3 0.90 0.72 0.18
5 0.75 0.38 0.37
5 0.72 0.41 0.31
6 1.10 0.54 0.56
Ed 1' 2.85
282 - {$012 -
58 3 n 3 0.0188 d 3 0.4071
TH‘ITTH) 2 _
(Ed) - 8.1225
5-0 (2.1123 1.1604
t 3 83 3 21.654 n
202 = 1.3095

Degrees of freedom 3 6

P 3 (0.01

123

TABLE 13. Paired data analysis of the effect of caries-
. susceptible submaxillari-sublingual rat saliva
and saline control on the growth of rat oral

streptococci

 

Slope of Growth Curve

 

 

 

 

Streptococcus
Culture Difference
Number Susceptible Saliva Saline Control (d)
1 2.19 3.20 1.01
2 2.80 '099 ”008'
3 2.50 3.13 0.63
3 1.79 2.47 0.68
S 4.07 3.08 -0099
5 4.49 3.63 -0.86
6 1.91 1.82 -0.09
7 3.77 5.19 1.42
8 3.71 5.29 1.58
12.12 Ed 2 Ed 3 2.57
' = I ( 1%? = 0.3342 5 = 0.2856
11" n
\ (2-d)2 = 6.6049
21 - 0 (2612 = 0.7339
t 3 ga 3 0.855 n
282 2 8.7761

Degrees of freedom 3 8

P = >0.5

124

TABLE 14. Data used in the correlation analysis between amy-
. lase activity of whole saliva from caries-suscep- .

_ tible rats gfirst experiment) and caries age
Rat Number y ase Activ ty* aries g

 

 

(x0 (v)
14890 1140 109
14978 2470 109
15127 1270 93
15130 1260 79
15131 1470 79
15122 876 93
15123 3420 79
15124 2040 93
15128 1900 93
15125 1260 67
15129 1560 107
15579 2340 78
15582 1180 78
15800 1340 71
15803 792 71
15804 1970 71
15878 843 78
15879 1320 78
15885 2260 64
15886 1140 64

 

*3 Amylase activity : mg glucose per m1 saliva.
** Caries age : days of age when a carious lesion first

appeared.
ny - L2 x) (12.1.).
1'1

“xv ‘ 1 [2x2 - 2.9.2 x 21 [82 - 2.42.3371

: ‘1 0.082

 

 

125

TABLE 15. Data used in the correlation analysis between amy-
lase activity of whole saliva from caries-suscep-
tible rats (second experiment) and caries age

 

 

Rat Number Amylase—Activity* *_Cér1es Age**'
(30_ (v)
16036 2450 76
16037 1680 76
16034 3080 90
161335 1860 76
16038 2000 76
16111 1970 55
16112 1610 55
16113 2530 55
16114 1960 55
16115 3520 55
16116 3300 55
16117 2180 55
16118 3040 55
16144 1180 58
16147 2540 58
16148 2190 53
16141 648 58
16142 838 58
16143 570 58

 

* Amylase activity : mg glucose per ml saliva.
** Caries age : days of age when a carious lesion first

appeared.

EXY " (2x11 2 11
1'1

rxy 3' \IIT‘XZ _ 1.2.113] [Y2 _ ‘21:)2

 

 

 

] 3 o 0.105

1'1 1’1

126

TABLE 16. Data used in the correlation analysis between amy-
lase activity of whole saliva from caries-resis-
tant rats (second experiment) and caries age

 

 

Rat NumEer Amylase Activity? _:CEr1es Age**’
(x) 1y)
16043 1460 488
16044 1470 366
16046 1250 289
16039 1170 366
16041 1270 355
16056 829 346
16057 792 304
16058 1660 318
16049 956 165
16066 964 328
16068 2080 343
16069 810 277
16072 621 207
16065 474 154

 

* Amylase activity 3 mg glucose per ml saliva.
** Caries age 2 days of age‘when a carious lesion first

ap eared.

ZXY ' LLX 1217)
r1

rxy = \J‘sz - M] [2Y2 .. .L-Z—nflf]

 

 

= 1 0.651

1'1

127

Correlation analysis between amylase activity and caries
age using combined data of susceptible and resistant rats
used in the second experiment

 

 

 

SP 9 SP
xY1 Xyz
r (average 1 and 2) 3 Jr __
XY
1555X1 1 55x2) (ssy' 1 ssyz
where
xI 3 Amylase activity of susceptible rats
x2 3 Amylase activity of resistant rats
y‘ 3 Caries age of susceptible rats
y2 3 Caries age of resistant rats
SSX 3 2(x' - 5:112 3 2x12 - (Z xl)2
1 ""fi"'
55x2: 206233-5212: szz' (2X212
'__'TT_-'
ssy = 20, - 1,12 = 2,12 - (z y,12
' _"'""11
g _ - 2 = 2 - 2
55,2 5172 Y2) Zyz (E yzl
N
Sny' :Zb‘] ' 3]) (Y‘ " 7]) = Z XlY' " (2X1) (2Y1)
N

Snyz :EO‘Z ' ’22) (Y2 " 72) : 2x272 '(XX2)(EY21
fl

 

128

 

 

 

TABLE 17. Data used in the correlation analysis between
relative viscosity of whole saliva from caries-
susceptible rats (first experiment) and caries age

Rat Number' Relative Viscosity Caries Age

(x) (y)

14890 2.51 109

14978 2.02 109

15131 1.69 79

15123 1.70 79

15124 1.44 93

15128 1.31 93

15125 1.50 67

15127 1.61 93

15129 1.39 107

15122 1.60 93

15130 1.59 79.

15579 1.80 78

15582 2.02 78

15800 1.71 71

15803 2.00 71

15804 1.72 71

15878 1.36 78

15879 1.50 78

15885 1.49 64

15886 1.69 64

15890 1.50 63

15895 1.94 63

15977 1.88 95

15972 1.45 53

15973 1.30 81

'ixy - 1 2 x) ( ;_JQ_
n
”“' e + 0.251

 

 

 

1'1

rxy : [[2322 - ('2: Q1317 “72 -

_”m_2- 22

n

129

 

 

TABLE 18. Data used in the correlation analysis between
relative viscosity of whole saliva from caries-
:giceptible rats (second experiment) and caries

'fiét Number ifielative Viscosity ‘Céries Age

(x) 11y)

16036 1.96 76

16037 2.12 76

16034 1.88 90

16035 1.95 76

16038 2.24 76

16111 1.77 55

16112 1.68 55

16113 1.78 55

16114 1.86 55

16115 1.89 55

16116 1.87 55

16117 1.86 55

16118 1.76 55

16141 2.00 58

16142 2.02 58

16143 1.91 58

16147 1.67 58

16148 2.13 58

 

rxy : A 2 - 2
x 1 Z x)

280'“ 1.2.x0 (22911
1'1

 

 

n

1 128-

n

2 * ij': + 0.452

130

TABLE 19. Data used in the correlation analysis between
relative viscosity of whole saliva from caries-
resistant rats (second‘experiment) and caries age.

 

 

Rat Number Relative Viscosity Caries Age
(X) (Y)
16043 2.04 488
16044 2.02 366
16046 2.23 340
16039 2.15 366
16040 2.06 355
16041 1.92 355
16049 2.44 148
16056 1.92 246
16057 2.08 304
16058 1.92 318
16065 2.03 159
16066 2.00 328
16068 2.16 343
16069 1.76 277
16070 1.71 249
16072 1.85 207

 

XY

Exy-sz) (2.1)
n

 

- \J [x2 - inx 315172-:“531‘31 a: -0.004

131

Correlation analysis between relative viscosity and caries
age using combined data of susceptible and resistant rats
used in the second experiment

SP + SP

rxy (average 1 and 2) 3 XY' xyz

 

{ SS“) 71553557755" 7) =
+ 0.038

(55

1"

where
x] 3 Relative viscosity of susceptible rats

Relative viscosity of resistant rats

X
11

Caries age of susceptible rats
Caries age of resistant rats
' 3 (X1 - 3112 = Ex.2 -(2x.12
“'TT"'
' 2 (x2 - 53212 E x22 - (5x212
2 '—"_11
1 N
- 2 2 2
Y2 Z (72 ' 1'2) Zyz - (Zyzl
___Tr___
spxy‘ =21x. - $2,) (y.-9.1= zxm - 12x.) 12y.)
N

(0%
MN
II
I

U) U)
m m
1 1

01
U3
ll

 

SPXY2 :20‘2 " 322) (Y2 " 72) : Xzyz " (2X2) (2Y2)
N

 

N 3 Number of rats used

132

TABLE 20. Bod ‘wei hts in rams of rats with (operated) and
wit out control their parotid ducts removed

 

 

 

 

 

 

 

 

Male Femgle
Control Operated Control Operated
425 402 252 278
451 456 234 279
472 444 244 250
423 440 225 280
492 523 244 261
421 441 257 230
407 388 250 216
530 413 224 259
395 415 240 240
382 467 236 205
436 449 272 ' 184
412 339 232 197
452 424 236 252
468 420 236 248
422 420 260 204
380 334 236 250
376 396 240 232
372 378 312 244
404 420 232 240
408 448 204 236
432' 372 276 230
430 424 260 220
500 380 200 242
332 462 336 264
384 344 372 280
400 364 208 196
348 416 220 232
364 380 252 224
328 324 224 204
356 388 212 230
388 392 220 232
348 332 270 252
380 370 208 200
356 352 232 240
3 0 328 220 240

3 400 226 240

 

133

TABLE 21. Absolute weight in milligrams of the submaxillari-
sublingual g ands from rats with (operated) and
without (control) their parotid ducts removed

 

 

 

 

 

 

 

Jie‘le Fm
Control Operated Contrpl Operated
654 699 540 541
709 697 493 531
616 709 535 493
650 740 528 578
756 801 494 498
757 704 504 593
631 661 506 513
768 672 440 614
652 707 559 539
647 545 500 560
707 678 556 522
696 509 494 553
670 718 527 611
740 790 446 500
745 686 547 412
641 523 496 500
663 731 524 539
680 685 595 502
719 661 583 544
660 740 486 556
690 648 643 493
868 577 571 520
788 588 505 567
649 802 678 670
771 636 697 608
735 650 510 485
553 980 493 549
549 774 600 597
609 593 534 569
651 670 483 566
638 741 611 633
638 592 616 586
711 525 461 466
619 613 524 546
653 535 486 593

 

134

TABLE 22. Relative weights* in milligrams of the submaxillari-
sublingual glands from rats wIth (operated).and
without (control) their parotid ducts removed

 

Male .Eemals;

 

 

 

 

 

Control . Operated Control fl _§§533flg31
154 174 214 195
157 153 211 . 190
130 160 219 197.
154 168 235 206
154 153 202 181
180 160 196 258
155 170 202 238
145 163 196 237'
165 170 233 225
169 117 212 273
162 151 204 284
169 150 213 281
148 169 223 242
158 188 189 . .202
176 163' 210 172
169 157 210 200
176 185 218 232
183 181 191 ~ 206
178 157 251 227
162 165 238 236
160 174 233 214
202 136 220 236
158 155 252 234
195 174 202 254
201 185 187 217
184 179 . 245 247
159 236 224 237
151 204 238 266
186 183 238 279
183 . 173 228 246
164 189 278 . 273
183 178 228 - 232
187 . 142 222 233
174 174 226 228 ~
187 163 221 249
198 1

90 215 ' .255

 

* Relative weight of glands 3 milligrams of gland weight
per 100 grams of body weight.

135

Formulae used in testing the significance of the differences
between the bod weights, and absolute and relative weights
of the submaxil ari-sublingual glands in rats with (operated)
and without (control) their parotid ducts removed

S tandard error 0”

 

 

 

-Jixz - _(__Z_.x 2
0X "

 

 

IN
N31
, 272-£2.21}.
07‘7" N
\ N-l

 

where x 3 sanples from control rat

samples from operated rat

Y

N nuaber of rats used

Standard error of the means - S.E. 3 0’

W

 

t : Differnce between means :

Stanaara error of die difference between means
52 -5,
«5.13.1; . (s.1-:.)y

 

336

TABLE 23. Data used in the correlation analysis between
titratable alkalinity of whole saliva from caries-

susceptible rats and caries age

 

 

Rat Number Titratable Alkalinity* (ml) Caries Age**

(x3 (y)
1&890 2.30 109
lh978 2.h0 l09
l5l27 2.38 93
l5l30 2.l0 79
l5l3l 2.25 79
l5l22 2.#5 93
l5l2h 2.30 93
l5129 2.60 107
l5579 2.55 78
l5582 2.40 78
l5800 l.60 7l
l580h 2.05 7l
l5879 2.l5 78
l5885 2.50 6h
l5890 2.50 63
l5977 2.05 95
l5973 2.05 8i
l6036 2.85 76
l6037 2.h5 76
l603h 2.l0 90
l6035 2.00 76
l6038 2.30 76
l6lll 2.l0 55
l6ll2 2.l5 55
l6ll3 2.20 55
l6llh 2.00 55
l6ll5 2.35 55
l6ll6 2.40 55
l6ll7 2.25 55
l6ll8 2.30 55
l6lh7 2.h0 58
l6lhl 2.l5 58
l6lh2 2.l3 58

 

I Titratable alkalinity : milliliters of0.0235N “5' re-
quired to adjust l ml saliva, diluted l to 5, to pH ll.5

t 0.2.
** Caries age : days of age when a carious lesion first ap-

peared.

ny- (2)1121)
n 3 + 0.192

‘
I

 

XY

 

[x2 -: g fnxlj] [2Y2 ' i i {if}

\

'37

TABLE 2h. Characteristics of the rat oral lactobacilli used
to study the effect of rat saliva on the growth

oflmicroorganismS'

 

Characteristic Lactobacilli
(Laboratory Culture Number)

I h 9 19 ll 14

Dextrose t
Dextrln +3’5 - - - +' -
Starch - - - - 4,2 _
Arabinose .+' +' +' +1 +3 +1
Lactose +3'5 +2 . ,2 ,l ,l *2
Litmus Milk - A'“ - A'“ 3'5 A21“
Gas f ,3-5 ,3-5 ti _ ‘2
Micro-aerophilic -+ + -+ + + +
Catalase - - - - - -
Nitrate reduction - - - - - -

Colony size
Rogosa SL (Surface) l.5 l.5 1.5 0.5 NG 0.75

Rogosa SL (Subsurface) 3 2.5 3 3 l 3
Growth in micro str. str. str. str. gr. str.
inoculum broth cl. cl. cl. cl. . cl.

 

Superscripts signify day at which positive reaction was
first noted.

A : 863d: C = curd; N6 = no growth; 3 3 weak or variable
reaction; str. : strong; cl. : cloudy; gr. : granular.

138

 

 

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