A COMPARATIVE STUDY 0? THE SALIVA AND OF THE BLOOD OF THE CARIES SUSCEPTIBLE AND CARIES RESISTANT STRAINS OF HUNT-HOPPERT RATS MELVIN A. BENARDE A THESIS ubmitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied. Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology 1954 ProQuest Number: 10008261 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008261 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 r AG KIMCWLDDGEMENT Sincere appreciation is accorded to a friend and teacher, Dr. Fred W. Fabian, whose lessons ex­ tended beyond the lecture hall. To Dr. Sam Rosen for a friendship that I hope will grow with the years, my deep respect. To our Department of Bacteriology whose "open door policy'1’ left nothing to be desired, I express my appreciation for attempting a difficult job-my training. Especial thanks are extended to Dr. Hans Lillivik of the Department of Chemistry under whose guidance the study of serum, protein electrophoreeis was carried out. TABLE OF CONTENTS Page Introduction ................................... 1 Experimental Methods and Materials .......... Animals Employed ........................ Drug S c h e d u l e ............. 4 7 8 Outline of Procedures ............... I. II. . . . . . 10 Bacteriological Studies of Hunt-Hoppert Rat S a l i v a .............................. 11 Review of Literature ................... 11 P r o c e d u r e .................................. 13 R e s u l t s .................................... 14 Antibiotic Activity of Saliva ........... 18 Review of Literature ................... 18 P r o c e d u r e .................................. 22 R e s u l t s .................................... 24 III. A m y l a s e .................................... 85 ................... 25 Review of Literature P r o c e d u r e .................................. 27 R e s u l t s .................................... 28 IV. Electrophoresis ......................... Review of Literature ................... Procedure ............................... R e s u l t s ............... V. Determination of Agglutinins in Saliva and S e r u m ...................................... 47 Saliva Review of Literature ........... P r o c e d u r e ......................... R e s u l t s ........................... (ii) Serum R e s u l t s ........................... 32 33 33 36 (i) VI. 48 49 51 56 Salivary Leucocytes ...................... 61 ................... 61 Review of Literature P r o c e d u r e .................................. 63 R e s u l t s .................................... 65 -- continued -- TABLE OF GOH TEATS -Continuedpage VII* Physical Determinations on Pat Saliva . 71 (i) Surface T e n s i o n ................ P r o c e d u r e ................... R e s u l t s ...................... 71 71 72 (ii) Refractive Index ............. P r o c e d u r e ................... R e s u l t s ..................... 75 75 75 (lii) V i s c o s i t y ...................... Review of Literature . . . . P r o c e d u r e ................. . . R e s u l t s ..................... 78 78 78 79 SpecificGravity .............. P r o c e d u r e ................... R e s u l t s ..................... 81 82 83 Discussion..... ............................... 85 Summary 88 (iili) .................................... Conclusion Bibliography * .............. .......... 90 91 LIST OF TABLES Table No. Page 1. Bacterial counts per ml of Hunt-Hoppert rat s a l i v a ............................................16 2. Per cent occurrence of oral lactobacilli and streptococci isolated on selective media . . . . 17 3. Amylase determinations of pooled Hunt-Hoppert rat s a l i v a ....................................... 29 4. Mobilities and per cent total area of serum protein fractions of Hunt-Hoppert rat sera . . 38 Agglutinin titers of pooled Hunt-Hoppert rat s a l i v a .......... 54 6. Per cent of total occurrences per unit of titer 55 7. Per cent total occurrences showing antibody titer of 32 or greater ........................ 55 8. Titers of Hunt-Hoppert pooled rat sera 9. Per cent total occurrences per unit of titer 5. . . . . 58 . 59 10. Per cent total occurrences showing antibody titer of 1280 or g r e a t e r ........................ 59 11. Comparison of serum titers of Hunt-Hoppert rats with other rats and m a m m a l s ...................... 60 12. Leucocytes per ml of Hunt-Hoppert rat saliva 13. Differential count of salivary leucocytes . . . 68 14. Surface tension of pooled Hunt-Hoppert rat s a l i v a ............... 74 . 67 15. Refractive index of pooled Hunt-Hoppert rat s a l i v a ............................................77 16. Dropping time of pooled Hunt-Hoppert rat saliva 80 17. Relative viscosity of Hunt-Hoppert rat saliva • 80 18. Specific gravity of pooled Hunt-Hoppert rat s a l i v a ............. 84 LIST OF FIGURES Figure No. 1. 2. Page Photographs of multi-unit animal con­ tainer ................ Photograph of zones of hydrolysis on starch agar gels ........... 5 31 Electrophoretic patterns of caries resis­ tant and caries susceptible rat sera 3. Caries in R e s i s t a n t s ................ 39 4. Advanced caries stage .............. 40 5. Developmental level ................ 41 6. Pre-caries s t a g e ............. 7. 8. 9. 10. 11. 42 The relation of albumin area to caries e x p e r i e n c e ............................ 43 The relation of alpha globulin area to caries experience ..................... 44 The relation of beta globulin area to caries experience ............... . . . 45 The relation of gamma globulin area to caries experience ..................... 46 Photomicrograph of salivary leucocytes 70 INTRODUCTION Approximately ten years ago Hunt and Hoppert (31) published the results of many years of experimentation. They succeeded in evolving two distinct strains of albino rats; one was caries resistant, the other caries suscept­ ible, Both groups were maintained on the Hoppert caries producing diet. Their work proved that, for these animals, heredity is an Important factor in the development of dental caries. The fundamental nature of their work serves as a basis from which many problems may be drawn. In fact, the research problem to be described presently is a direct con­ sequence of their findings. Because of the complex nature of the overall problem, discretion necessitates partition into smaller but specific areas of Investigation. These investigations are, therefore, only several facets of this larger problem. Since the carious condition is, in part, one result of heredity, it is permissible to speculate that certain physiological and chemical qualities have been altered in one of the groups to a degree sufficient to initiate caries. The immediate problem can be succinctly stated. "’hat are the differences between these two groups of rats disposing one of them to dental carles? It is not often that two such Ideal groups of animals are available for observation, each serving as a control for s. the other. Specimens taken for study from both groups at the same time, under the exact conditions, receiving equivalent treatment, could be expected to yield signifi-*cant results whether differences or similarities were found between the two strains. It is understood, however, that only differences between the two strains might possibly explain the disposition of the susceptible group to caries. Added direction for this study was obtained from the early recognition of wide bacteriological differences in the oral micrcflora of these animals . Rosen &t al_ (5S) were able to recover lactobacilli more frequently and in greater numbers from susceptible animals than In the resis­ tant group. Utilizing the Hunt-Hoppert animals and Rosen’s find­ ings as a point of departure, it was deemed logical at this time to undertake a study of the saliva and blood of these animals. These two materials would be subjected to a var­ iety of tests in an attempt to discover any significant qualities that might explain the difference between the two strains of rats. The caries time (the number of days elapsing from the date on which the animal was placed on the cariogenic diet to the date on which the first carious cavity could defin­ itely be established in a lower molar) is approximately 70 days for the susceptible animals and 300-500 days for th e resistant animals. The procedure adopted was to collect the necessary specimens from four developmental levels: the pre-caries stage, the period of caries development, period of advanced caries, and the period when caries appear in the resistant strain. In this way a complete picture depicting the conditions prevailing at strategic stages of growth would be obtained0 EXPERIMENTAL METHOD, ir,\ m p t i J. _ j 11 For this study to proceed according to adopted pro­ tocol s adequate Quantities of saliva for the various pro­ cedures had to be obtained. The immediate problem was to devise a method for the collection of saliva. After several unsuccessful, attempts in which various types of suction devices were used, chemical inducement of salivation was employed with fair success. It was desir­ able not only to obtain a sufficient quantity of saliva but also to assure the survival of the animals since they would be required for more than one test. A necessary piece of equipment in which to hold the animals while anesthetized and salivating had to be desig­ ned. Figure 1 shows the multiple unit animal holder with and without animals. The holder has the following measurements Overall length overall width height with cover number of sections width of each section width of separation head opening width of bottle rack individual bottle holder height of bottle angle of incline of unit 33-J inches 14i " 8-1 ° 2 Tl 8 4 inches 3/16 inch ij inch diameter 2-5/16 inches If inch outside dia. 3 inches 8 degrees The sides and dividing partitions were made of Ms sonIte R Figure 1 ► Photographs of multi-unit animal container J. Multiple unit animal container (empty) K. Container full and cover in place L. Close-up view of animals salivating Before the anesthetized animal is placed in the unit, a piece of brown wrapping paper can be slipped into the compartment. This reduces the necessity of cleaning to a minimum. The Masonite cover prevents the animals leaving their units. The size and lightness of weight of the holder enables anyone to carry it about easily and to set up oper­ ations in small quarters. Animals Employed The animals used in this investigation were obtained from the colony maintained by Hunt and Hoppert. These albino rats have been selectively bred; one strain for caries susceptibility and the other for caries resistance. The data to be presented were obtained from 107 animals. This does not include the animals used in prelim­ inary trials required to perfect techniques. Of the 107 animals used, 78 were males and 29 were females. Of these, 90 animals were used for the saliva study and Included 65 males and 25 females. Only three of these animals were re­ used for saliva studies. Forty-five animals were used for the serum studies of which 17 were not used in the saliva experiments. Included among these 45 were 55 male and 10 female animals. For the bulk of the studies reported, pools of saliva and serum from four animals were used. young animals, In the case of very individual samples or pools from two animal’s 8. ■saliva were- subjected to the various tests because of the scarcity of animals in that category during the time of this investigation. Drug Schedule When mechanical devices failed to yield sufficient saliva, a chemical stimulant was sought. Hoick (23), writing in Griffith and Farris, lists many drugs used in rats. From this list pilocarpine nitrate was chosen as a salivary stimulant. Nembutal (a barbiturate) was selected to produce light anesthesia as it was discovered early in this work that a struggling animal would not yield saliva. Hoick1s list is a compilation culled from the litera­ ture and can only be used as a point of departure. His list suggests that 40-160 mg per kilo of body weight would yield the typical action of pilocarpine. One hundred milli­ grams was administered along with nembutal at 40 mg per kilo of body weight. Nembutal was given first by intraperi- toneal injection to bring on the anesthesia. This was fol­ lowed by subcutaneous injection of the pilocarpine after the anesthetic had taken effect. in many deaths. only This dosage resulted The pilocarpine was reduced to 50 mg per kilo and the nembutal to 20 mg per kilo. This schedule also caused enough deaths to warrant further study. It was ob­ served that after injection of the pilocarpine the animal becomes more flaccid than from nembutal alone. appears to enhance the effect of nembutal. Pilocarpine This observation led to a reduction of pilocarpine to 5 me; per kilo. was sufficient to induce adequate salivation. was equal to that obtained with 100 mg perkilo. pine was equally effective at the This In fact, it Pilocar­ 5mg per kilo level for all ages and weights and for both sexes, A.n initial injection of 20 mg per kilo of body weight was administered. In cases wherethis dose did the desired anesthesia additional not achieve 5mg per kilo increments were given until the anesthesia was obtained. ation in response was found among the animals. k large vari­ Some were anesthetized by the initial 20 mg per kilo, others required as much as 50 mg per kilo. This variation did not appear to be determined by sex, weight or age. to nembutal was unpredictable. The response of animals This variation may be charged to irconstitutional differences” among individual animals. When the dose was standardized the animals responded well with approximately one death in eight each time a group was processed. After the drug was injected the animals were placed In the holder and allowed to salivate until sufficient quanti­ ties were obtained. This procedure was employed for all saliva used in this Investigation. Collections were made in the animal house and shortly thereafter the samples were brought to the laboratory for analysis. The saliva and serum were subjected to various tests. These are listed in outline form.. a unit and Is described separately. ha eh procedure constitutes 10. OUTLINE CP PRGCEDUR Bacteriological I.- (a) Total counts of oral microflcra (b) Lactobacilli and streptococci counts II. Antibacterial properties of saliva Biochemical I. Amylase determinations of saliva II. Moving boundary electrophoresis cf sera Serology and Immuno 1 or,y I. Agglutination tests Saliva and sera II. Determination of leucocytes in saliva Physical . »■. —^ -—Measurements - - -- I. Refractive index saliva II. furface tension saliva and sera III. Viscosity IV. Specific Gravity saliva 11* I. BACTERIOLOGICAL STUDIES OF HUNT-HOPPERT RAT SALIVA The raicroflora of the oral cavity has long been associated with dental caries. It was not until 1882 that substantial experimentation removed these observations from the realm of pure speculation. W. D. Miller (44, 45) promulgated a theory of caries production that is popularly held today. He noted that the source of acids which decalcify teeth is carbohydrates degraded by oral bacteria. Bunting (10) was so convinced that Lactobacillus acidophilus was the prime agent of dental caries that he wrote, nThe presence or absence of Sac illus ac idophilus in the mouth constitutes a definite criterion of the activity of dental caries that is more accurate than any clinical estimation can be . . . . And there was a spontaneous cessation of caries coincident ’with the disappearance of B. acidophilus from the mouth.FT Arnold and McClure (3) identified lactobacill! in 90 per cent of cases of dental caries. They presented a posi­ tive correlation between the number of organisms in saliva and caries activity. Becks (5) noted that a high caries Incidence was related directly to a high aciduric micro-organism count. Rosen and his group (59) showed that lactic acid bacteria are a permanent microflora cf caries susceptible rats whereas the same organisms do not persist in caries 12. resistant rats. Rosebury (57), on the other hand, stated that of 40 healthy albino rats all had L. acidophilus as a part of the normal oral flora. He believed that these sciduric bacteria may be present in the mouth without giving rise to dental caries. Streptococci have also been incriminated as etiologic agents in caries. Belding and Belding (S) made the obser­ vation that Streptococcus odontolyticus is the principal agent in dental caries. Canby and Bernier (12) suggested that, since strep­ tococcus is rarely isolated from deep carious dentin, great emphasis must not be given them as an agent of caries, Bibby, Volker and Von Kesteren (8) using three dif­ ferent media estimated the organisms present in saliva. They found that in carious mouths lactobacilli made up about 1/2000 of the total numbers and less than 1/1300 of the acidogenic aerobic organisms. Streptococci wer° present in 20 times greater numbers than the lactobacilli but appeared to be numerically unimportant. They believed that the acid production in the mouth is due to acidogenic types (mainly streptococci) which were not identified in their study. They found that the streptococci produced acid more rapidly than the lactobacilli. 13. From these few reports, which are typical of the bulk of the literature, it is plain that although acid pro­ duction in the mouth is generally held to be a direct causal factor in caries production, the organism or organisms in­ volved are uncertain. The present study was undertaken with the intention of describing the condition of the saliva with respect to both the total numbers of micro-organisms and the numbers of lactobacilli and streptococci present at each stage of caries activity. Procedure Saliva was obtained in a manner previously described. Ten-fold serial dilutions of the saliva were made in sterile distilled water. Platings of 0.1 ml portions were then made on four different media. For an approximation of the types and numbers of aerobic bacteria to be found in saliva, tryptone glucose extract agar (Difco), a general purpose medium was selected. To enumerate the lactobacilli present the specific lactobacillus medium of Rogosa (B3L) and tomato juice agar spe­ cial (Difco) with 0.2 per cent sodium azide added to in­ hibit the growth of gram-negative forms were employed. Azide dextrose broth (Difco) with 1.5 per cent agar added was employed for the selective cultivation of strepto­ cocci. 14. One-tenth milliliter portions of the various dilutions of saliva were dropped onto the agar surfaces and spread by means of a sterile glass rod bent at approximately a 4-5° angle. Incubation was carried out at 37° C. for from 24 to 72 hours. The colonies on tryptone glucose extract agar (TGR) were counted after 24 hours; the colonies on lactobacillus selection medium (LBS), tomato juice agar special (TJAS) and azide dextrose agar (ADA) were counted after 72 hours, Re suits Table 1 shows that the resistant animals at each level of caries activity exhibit a lower total count than do the susceptible strain. Lactobacilli and streptococci are pre­ sent in both strains with the streptococci far outnumbering the lactobacilli. This observation is in accord with the general observations noted in the literature. The total numbers increase successively from group to group in both the resistant and susceptible animals. At the pre-caries stage the susceptible strain shows a three­ fold higher incidence of oral bacteria. In the developmen­ tal period this same strain exhibits -a 2.5 times greater incidence in total numbers. A six-fold greater occurrence of oral bacteria in the advanced caries stage and a nine­ fold increase in the oldest group marks the susceptible? as consistently higher with respect to total numbers. 15, Lactobacilli were present in greater numbers in the susceptible animals especially during the period of caries development. There was a ten-fold increase when lactobac- illus selection medium was used and a 17-fold increase when tomato.juice agar was employed. The zero counts obtained in both strains of animals on lactobacillus selection medium during the pre-caries, stage is probably not a true reflec­ tion of the prevailing condition. The nzide dextrose agar discloses.fairly large numbers of streptococci present in all stages. The susceptible strain during the developments! period exhibits a nine-fold increase over the resistants. It may be significant that between the pre-caries and developmental stages of caries activity, ”the sensitive stage,” large increases in acidogenic bacteria become appar­ ent. The possibility of sustained acid formation during this ,Tsensitive stage” may initiate the carious process. It was also noted that the increase in total numbers of bacteria from the pre-caries level to the period when caries appeared in the resistant animals was 60-fold. Should total numbers be more significant than any one organ­ ism this observation may be of considerable importance. Table 2 shows the per cent relative incidence of certain bacteria found in the saliva.. The predominance of streptococci over lactobacilli is evident. 16. -p © © P CD pH Cm •M TD -P © CO > •rH rH © CD. ad © to o © p T-H cd p > Cd TO o © Q PL ad rH cd p a id © £3 *h Cl P o © l—1 CL 03 > © Q rH C m © o •rH Oh £Q © © -P bD o © cd -p ao to © i—i cd Eh o at* C— o o LO m O O LO o o o o o o cd O O O O LO LO O DO •N 1---1 O O LO *\ CO EH O o CO «v 1 —1 rH o o o O o o O o o LO C— i—i •% rH o oo to •% 1—1 o LO c•> CO ah O O of O o o o O LO o CO CD *\ CO CV) 1 © © r-H P Ph •* lO rH Oi o to to ** (30 rH LO l—1 ah CQ CO CD -p © a •<-« 03 P o © C.3 O o CV3 C- CO to CO •H 1 —1 1 —1 rH J i -p c 3 • K p a Cm o o o p co © -P o PQ -P PI O © r—1 — *rH 1 —1 rH ■rH CD O Cd rQ O 1-3 P O Eh Cd rH *H © O o o <3 o p n p. © -A t , p CQ CD © Pd, © iH P •rH 3-D !m a; o CO © GO II II P Ci © P (fj •rH P3 > o p © o N Eh P I Eh P a it II II 11 c0 W 03 ”"*1 co PO t-D Q Eh —1 Eh amylase bacterial or salivary C lgH 8S0 g i p H 2R°11 2C6H 12°6 (Siucose) Id dd ll + ___________^ ________ '2* maltase (bacter ial) -r CfiH15> C L . ______________________ n Fermentative enzymes' (bacter ial) (maltose) 2C~H.0~ (lactic acid) Should food particles be retained by teeth, starch would be present as a potential source of acid. The amount of amylase present in the mouth might be an important factor in determining the amount of acid produced. 27 . Procedure A preliminary study was made to determine the pre­ sence or absence of amylase in the saliva of the animals. Saliva was harvested and used in the achromic end point test as described in Hawk, Oser and Summerson (25). It was found that the end point (disappearance of blue color) was reached approximately three times faster with the saliva from susceptible animals than with that of the resistant animals, indicating that the susceptible animals produced more amylase. However, due to the limitations of the accuracy in determining end points, the method of Stark et_ aJL (62) was adopted for use. This is a simple yet strikingly demon­ strative test. Filter paper discs placed on starch agar gels are impregnated with a calculated amount of test saliva. This is allowed to incubate for ten hours after which the plate is flooded with a dilute iodine solution and the excess poured off. Diameters of the colorless circular areas (against a deep blue background) are easily measured with a millimeter rule. Two discs were used on each starch agar plate; one containing the saliva of resistants, the other of the sus­ ceptible strain. This was done in order to obtain measure­ ments under the same conditions. Tests were carried out in duplicate and an average of the zones obtained. 28. Results Tho measurements of zones of hydrolyses, measured in millimeters, appear in Table 3. It is evident that group for group the resistant strain exhibited smaller zones of hydrolysis, which indicates that less amylase is present in the saliva of this strain. If these data portray the condition prevailing in the mouths of most of the animals, it may bear directly on the caries problem. If the amylase present can split suffici­ ent starch molecules to maltose rapidly enough for the oral microflora to degrade the maltose and produce a sustained acid action, one of the conditions for caries production would be satisfied. As pointed out in another section, the saliva of sus­ ceptible animals contained far greater numbers of bacteria than did that of resistant animals. The combination of amylase and large numbers of acldogcnic bacteria being pre­ sent at the same time in the susceptible animals is doubt­ less a condition favorable to caries activity. If the results of this study are borne out in further studies on a larger number of animals, this evaluation would take on greater significance. concept is only suggested. For the present such a Table 3. Amylase determination of pooled caries resis­ tant and caries susceptible Hunt-Hoppert rat saliva at different stages of caries develop­ ment . Diameter of zones in mm Resistant Susceptible Pre-caries 29.5 31.5 Developmental period 27 .7 33.2 Advanced caries 24.5 27.0 Caries appear in resistant animals 24.5 28.1 30. Figure 2. Photographs showing zones of hydrolysis produced by amylase present in the saliva of resistant and susceptible rats. M. Pre-caries level Upper clear zone represents resis­ tant saliva.; lower zone, susceptible saliva. N. Period of caries development Upper zone represents resistant sal­ iva; lower zone, susceptible saliva. 0. Caries appear in resistant level Upper zone represents resistant saliva; lower zone, susceptible saliva. F. Advanced caries level Upper zone represents susceptible sal­ iva; lower zone, resistant saliva. IV. ELECTROPHORESIS The moving boundary method of electrophoretic analy­ sis reveals a detailed and reliable representation of the serum, proteins. It may be considered to be of greater value than any other chemical or physical technique for the determination of serum proteins available at this time. The procedure yields quantitative information on a whole series of well-defined serum and plasma protein components which cannot be differentiated as readily by other methods. Two excellent features of this technique are its reo^uirement of small amounts of material and that the conditions of analysis are relatively non-drastic to the protein. The principle of electrophoresis Is that a sharp boundary between protein solution and its solvent (usu­ ally buffer) is formed and observed as the charged protein migrates in an electric field. Should the solution contain only one species of protein, the boundary remains symmet­ rical and single. If there are different species of charged proteins in the solution they move at different speeds, so that the initial sharp boundary separates into several boundaries, each representing a different protein fraction of different charge. It was earlier noted that a diagnostic tool to distin­ guish between resistant and susceptible animals might evolve from one or a combination of the methods employed. The 33. delicate procedures and extensive time required for elect­ rophoretic analysis might be reduced and adapted for diag­ nostic purposes, if a simple distinguishing feature of the pattern results could be detected. The usefulness of this procedure is the fundamental information regarding serum protein differences it can yield. It was for this purpose that It was used in the invest igatI on• Among the many papers available on the general sub­ ject of serum electrophoretic analysis, those of Stern and Reiner (64), Henley and Stern (26), Leutscher (38), Longsworth (40), and Moore and White (49) are pertinent. Literature on electrophoresis related to the subject of dental caries could not be found. Procedure Blood was obtained from the anesthetized animals by cardiac puncture. The whole blood was pooled (usually from two or three animals of each group) and serum obtained by standard techniques. The serum was separated from the clot by means of a suction pipette and diluted with two volumes of buffer. The buffer used in this study was a barbiturate (veronal and sodium veronal) solution of pH 8.6, 0.1 M ionic strength made up at 24° 0. It is stated by Longsworth that this buffer produces better resolution of the delta and epsilon from the gamma globulin boundaries. 34. The buffer-serum sample was placed in a Visking^ casing and allowed to dialyze against ICO ml of buffer for two hours at which time 100 ml of fresh buffer was substi­ tuted. Three hours later this buffer was again changed for 300 ml of fresh buffer for overnight equilibration against the protein solution. Constant agitation of the with membrane-filled^solut1on was maintained at all times to in­ sure rapid equilibrium. The final dialysis time was carried out in a cold room at 4° 0. clarified, The equilibrated serum was if necessary, by centrifugation for 15 minutes at 2000 rpm in the cold room. The buffer-serum sample was then placed in the electg rophoresis aparatus and allowed to operate for 7200 seconds at 7.5 milliampsres which appears to give the best resolution of the. serum proteins. Resistance of buffer solution and protein solution 3 was measured with a conductivity bridge (Model RC-la) and conductivity cell with cell constant 0.4892 to obtain specific conductivity data. The resolution of the serum proteins produced the electrophoretic patterns. Perkin-Blmor Model 38 Tiselius Electrophoresis apparatus. 2 Industrial Instruments, Inc. ^Membrane of seamless, regenerated viscose process cellulose. 35 Each serum protein component mobility was determined, as recommended by Longsworth and Maclnnes (39), from the measured distance in centimeters between the initial boun­ dary and the ordinate dividing the area of the component in half. This migration distance (d) was substituted into the following formula of Longsworth along with other pertinent data for the determination of mobility. 4 = d q K SP i t The potential gradients were evaluated, by supplying data into the following formula: Potential gradient z i___ where: d q t i L n = distance migrated in centimeters = cross-sectional area of the cell r time of migration in seconds = current in amperes = specific conductance of protein = conductivity cell constant resistance of protein in ohms The area under each protein component was computed in the manner suggested by Greenberg (41). The negative con­ taining the descending'boundaries was placed on centimeter ruled graph paper. A strong light source overlaid with a glass plate was backed with graph paper. The boxes in each area were counted and a per cent of total concentration for each component was obtained.* 36. Results The patterns are listed in Figs. 3,. 4, 5, and 6. The information obtained from the boundary patterns are listed in tabular form (Table 4). This table is, in part, reproduced in the form of line graphs. These line graphs, Figs. 7, 8, 9, and 10, indicate little, if any, differences in proteins composition exists in the sera of the animals studied• A minimum difference of 10 per cent between any of the component fractions was arbitrarily established as a basis for considering the results significant. With this criterion it became apparent that the albumin fraction offered no point of difference. Close scrutiny of the pattern from susceptible ani­ mals of the caries developmental period reveals a discon­ tinuity in the boundary of the alpha-globulins and a needle-like projection rising cut of it. be due to imperfections in the apparatus. This effect may Ice not made of distilled water may carry entrapped material that might interfere with the light going to the camera lens. This explanation is suspected to be a more correct Interpreta­ tion of this pottern. The remaining beta globulins and gamma globulins clearly do not indicate the 10 per cent difference as there is a factor of experimental error to be considered. It is 37 . believed that 10 per cent is not an excessive figure to establish as a standard. This procedure apparently does not reveal any unusual or unique patterns that might shed light on the factor of differ ence between the two animal strains. 4. Mobilities and per cent total area Hunt-Hoppert rat sera at different of serum protein stages of caries fractions of activity. 38. \ d CH 3 X & d X cd P O fn i— I i— I o to • (H o 02 • rH 02 sf ♦ o o o X X X • O 03 « 1 ---1 X 02 ♦ 0 2 0 2 rH 1 —i * X O « X 0 0 0 • 0 • CO • 02 X sf C h • 1 —I 03 sf • 02 CD 1 —1 IH X O 02 • X • 0 2 0 <3 to o cd 03 d Cd I >s X X X O X ^ |H Cd 0 Pi 03 05 rH -P 3 CD X) SS rH d I X CJ rH • EH Od C3 OX 0 o Pi sf sf CO • rH Sf IH • 1 —1 IH iH CO i—1 X 1 —1 X rH X rH 03 03 « rH i—1 o « X EH 03 • CO 03 X • CD O to • 02 X 00 m X EH 1 —1 « 02 CO i—1 CD 02 X (X! CD 03 03 X CO rH O 02 X 02 CO CD * CO sf X • X X C h • sf 03 X • X 03 1 —1 • (H O X • LO 02 • X IH X • X X X sf IH X CO sf X X sf sf EH X X ’sf .V, jri X X • St' X X 4 » X IH 03 • X st4 OJ * IH X CO « St' O X « X O O • X X 03 » X I i —1 o ■H I>5 5 x i —I X x O X £3X CD P i >5 X Cm X O > CD X &0 O X ai 1 0 I Sf * X 02 1 —1 O X X 1 —1 1 —1 0 i—1 cd X d 03 •iH P| cd o » 0 Pi P-, a 0H O i—t 03 > 03 to C C S CD EH iH • 02 X cd 1 —1 0 > 0 i—1 •i— I sf « CO cd x! d in • I PQ o X I>s rH X X to O *H cd C C S Table O • H i— I X in ID 0 X p 0 o X 0 C D d 0 > X •d4 , to O 03 03 d X C DX rj d 0 Cu I f j X Pi 0 U'j 0 X «M 03 P, ID to 0 0 to C D Pi s q .u oH).S f 3 G V 0 > 0 0 to X 0 0 d cd > X <3 to O 02 X d C Dx Cd X d Cd m in P. 0 -iH (d X UJ 03 Pi 0 Ph cd Pi D to C 0 d s a x q iq .d e o s n g AP Figure S. Electrophoretic patterns of e e M and car lee. susceptible rat sera • res istant ‘re-caries Stage Ascend ing Liesc end in' Res 1stant pH 8,6; Veronal Buffer M - 0,10; 7000 Serum diluted with two volumes of Buffer Ascending tis cectible pH 3*5; i'c r one.1. Ouf f e Serum dll uted p 1th two v s ; 6,64 volts 43. THE RELATION OF ALBUMIN AREA TO CARIES EXPERIENCE too □ RESISTANT • SUSCEPTIBLE TOTAL AREA 75 SO r^ PERCENT I d (/) 25 UJ QC UJ > UJ cn UJ cr < o > UJ UJ tr 2 Q. O _l UJ O < 0 1 LU OC CL O z in SO < UJ Q. CL o < UJ o z in UJ < > o < UJ > UJ Q too tr /SO cr < o 200 RAT AGE ( DAYS) FIGURE 10 2SC 300 + THE RELATION OF ALPHA GLOBULIN AREA TO CARIES EXPERIENCE RESISTANT 100 SUSCEPTIBLE 73 PER CO UJ UJ UJ UJ UJ -J CO UJ UJ CL UJ Q. O UJ O SO UJ UJ CENT TOTAL AREA Q UJ O CL 25 o 50 100 iso RAT AGE 200 250 (DAYS) FIGURE 7 500 + THE RELATION OF BETA GLOBULIN AREA TO CARIES EXPERIENCE 0 RESISTANT • SUSCEPTIBLE AREA /oo co co UJ PERCENT TOTAL UJ UJ UJ SO UJ UJ UJ Ul o Ul o Q. Ul UJ o Ul o 25 o CO UJ so o /oo /50 zoo RAT AGE (DAYS) FIGURE 8 THE RELATION OF GAMMA GLOBULIN AREA TO CARIES EXPERIENCE RESISTANT SUSCEPTIBLE TOTAL AREA 75 htn LU 50 cn UJ UJ PERCENT UJ CL UJ UJ o UJ o Q_ O _J UJ UJ UJ o UJ — 0 50 /oo - □ £ > /SO 200 250 RAT AGE ( DAYS ) FIGURE 9 47. V. DETERMINATION OF AGC-LUTIKINS IN SALIVA AND SERUM To elucidate further differences that might influ­ ence the rate of caries development, an investigation into the presence of antibodies (humoral agents that may offer some measure of protection) in the serum and saliva of these rats was undertaken. It might be well to note here that among the reasons for the performance of each of the procedures undertaken was the desire to obtain a tool for the ready diagnosis of incipient caries. The immunity implied may be defined as a state of re­ sistance to the development of a disease. Should the dis­ ease be initiated by an infectious agent then the immunity may be due to substances in the blood or in the tissues which assist in eliminating the organisms. Dental caries cannot be strictly termed an infectious disease with the concomitant symptom complex as seen in connective or epithelial tissue infections and therefore one would not expect to find immune bodies protecting dir­ ectly against carles. However, caries resistant indivi­ duals exhibit an immunity which is unexplainable but is suggestive of an immunological !ip r i n c i p l e w h i c h may be active through either the blood or the saliva. In an attempt to procure information relative to the two objectives men­ tioned, the tube agglutination test was employed* 48. Extensive as the literature is on dental caries, re­ latively few reports document the findings of agglutinins in serum or saliva against the lactobacilli. McIntosh, James and Lazarus-Barlow (43) reported finding agglutinins against L. acidophilus odontolyticus in the sera of seven out of ten persons to a titer of 1:160. In 1943, Dietz, et_ al^ (18), in a study of agglutinins for lactobacilli in 15 caries susceptible and 15 caries free men, concluded that the highest titers of oral lactobacillus agglutinins are accompanied by a low incidence of lactobacilli, but not necessarily a low caries experience. He obtained titers of 1:13-1:88 in non-caries subjects and 0-1:72 in subjects with caries. Further observations with serum, agglutinins are con­ cerned with attempts to induce titers by vaccines of lactobacilli and correlate them with caries experience. In 1927, Ross, Krssnow and Samet (60) reported agglu­ tinins to a titer of 1:8 with 14 or 18 rabbits inoculated with vaccines prepared from two strains of L. acidophilus. The following year Jay and his group (33) were unable to produce agglutinins in rats by vaccines of mixed types \ of organisms. His attempts to use autogenus: vaccines in children resulted in abcesses ascribed to the rough colony type of L. acidophilus. Canby and Bernier (12) vaccinated twenty cariessusceptible men with vaccines prepared from strains of 49. Ll«._ 9.cidophi 1u s isolated from serious dentin. The average count of lactobacilli in the saliva was reduced in 19 sub­ jects and increased in one. The agglutinin titsr of serum was increased in ten, unchanged in three and undetermined in seven. Williams (71) reported in 1944 on an investigation employing 23 volunteers who were inoculated subcutaneously four times at weekly intervals. They each received equal mixtures of two widely cross-agglutinating organisms with both live and heat-killed vaccines. He observed (72) that salivary counts for lactobacilli are not appreciably affected by increased blood agglutinin titers after vaccin­ ation. Procedures The following organisms were used to prepare the antigen for all the agglutination tests to be described. for both serum and saliva: TS T£ LS LS 109 116 110 149 These organisms are described by Rosen et el (57) as satisfying the genus Lactobacillus. All four organisms were isolated from the mouths of susceptible rats. TL 109 and TS116 were isolated on tomato juice agar special and LS 110 and 149 were isolated on L3S medium. These organisms were maintained on micro assay culture agar (Lifco). 50o When an agglutinate.on test was to be performed, 24hour micro inoculum broth (Difcc) cultures of each of the above isolates was used as antigen. The broth cultures were aggregated by centrifugation and the broth removed. The cells were resuspended in 0.85 per cent saline diluted 1:1000. This dilution was used in order to mini­ mize auto-agglutination since it has been shown that lacto­ bacilli generally agglutinate spontaneously in 0.85 per cent salt (70, 69, 51). Despite this procedure, one of the isolates, TO 109, agglutinated spontaneously and was not used. Therefore, only three of the four isolates previously described were used to prepare antigens. Five-tenth per cent phenol was added to the bacterial suspension as s. preservative.. These suspensions were di­ luted to a turbidity, equal to that of tube ^5.0 of McFar­ land* s nephalometer. Seven Wasserman tubes were set up containing 1.0 ml of distilled water in which the saliva or serum dilutions were made. It is apparent that the final saline concentra­ tion is quite small, but sufficient to manifest the second stage of antigen-antibody combination (69). Subsequent procedure is standard and need not be described here. 51. Results The results shown in Tables 5 , 6 , and 7 indicate that agglutinating antibodies were present in the saliva of these animals. These agglutinins were present at all levels of caries activity in both resistant and susceptible animal strains to some degree. In the pre-caries stage greater agglutination was exhibited, by the resistant animals. This was of a variable nature differing between the throe antigens which was not unexpected. These isolates were not the same in respect to biochemical definition and their antigenic characteristics may differ also. The variable titers obtained from trial to trial with a particular antigen was not anticipated. This will be discussed presently. ho difference in antibody content was apparent between the groups during the period of caries development. The same occasional variability was evidenced here. At the advanced caries level all three antigens of the resistant group exhibit marked increases over the sus­ ceptible group with less variation within any single antigen over four trials. In the final group, where caries were s^en in the re­ sistant animala, isolate TT 116, generally the strongest agglutinating strain, appeared tc hevs combined with four times as much agglutinin in the resistant saliva as compared. 52. with the susceptible. r' -hen s.11 antigens were taken toget­ her, the difference still existed but was less obvious. In this group the variation within the antigens v/a.s also found but to a lesser degree. It should be noted that the saliva of the resistant group exhibited titers of 1:32 or greater, 53.-3 per cent of the time as compared with 15.1 per cent for the susceptibles. However, the variability within a single antigen suppressed an attempt to regard this as significant. for the most part pooled saliva was examined. least three animals were used for a pool. At The same animals of any specific group were not used repeatedly in gathering data. This use of different animals may be one factor re­ sponsible for the occasional variation of titer with a single antigen. Three resistant animals were used twice. These animals were those in the period of caries develop­ ment. They ’ were tested 34 days apart (trials 1 and 3, Table 5) and this interval might explain the increase in titer exhibited between these two trials. The antibody present in the saliva may represent leakage from the blood serum. may oe reflected in the saliva. fluctuations in the serum Another factor that may well be responsible for the trial to trial titer variations are the bacterial isolates themselves. It has been reported that lactobacilli can undergo changes while being trans­ ferred through ordinary media. fs these isolates were 53. transferred with regularity in order to prevent their dying out, antigenic changes may have occurred. (16, 24). Orland (51) writes that nthe apparent suppression or loss, at least temporarily, of a major somatic antigen among lactobacilli constitutes a rather interesting immunologi­ cal phenomenon, though the mechanism involved is highly uncertain at this time.n There is a further possibility that the agglutina­ tion obtained is of a non-specific nature. It remains for further experiments to prove that the lactobacilli are either agglutinated by a substance with the characteristics of an immune body and produced in response to the presence of antigen in the tissues or that the clumping is only the non-specific manifestation of the presence of salts or proteins capable of educing bacterial aggregation. w O CO CO (D CO i—i CO CD o L T D i —l w 00 O CD i —1 CD 1 o i —1 CD r l m P CO 1—1 CO -p o o o •fH Eh to -p Pi cd -p cn *i—t to a? 04 -4 oo CO •v W CD CD CO i —l CD i —1 to rH cd ■H CV2 P. Eh i—1 Pl CD nO •H -P W i—l 1—1 1 OO w W w CO CO 1 —1 CD 00 " C F • a * CD CD CD CD * 1 w CO I o 1 CD » W O CO o o CO o o co o a> H H ^ i—1 i—1 i—t CD O CD CD O CD CO o CD rH iH i —1 1 S P —1 ' i —t r H —1 i —1 —1 i —i rH |H i —1 i —) i —I 1 cfl Eh Eh co PI y] PI a.) CO CO PI PI CO 00 CO Eh r4 . 1 Ul Eh C O -P Pi C D P> UJ • r H C O xi o CD P i •rH P i 1—I CD > CD i—1 Oj (D •r-l P, od a 1 1 CD u Ph CD Q* —1 CO -p Pi 0 s pi o —t CD > 0 ) Q CO CD •r-t Pi •H p C O o P <0 C D f i r TP Cd CD O P cd > r d C O 0 • r H Pi c d o Ul PI OJ i 4 of data* o 00 W O CO lack o 1 indicates oo 1 (-) W CO P. E-h serum. O « rH Cu undiluted -P P. (D O o (1.0 ml) of the & Kf OO CD oo O CD i —I volume O rH O per unit CD CD O CD W units •rH W •rH -P o CO CO CD •rH p CO o CO CD rH P •rH -P Ph o: o C/J p CQ CQ i—1Cvi CO •i— l Si Eh rH C m O in CO (D P CO P CO p P CD Eh cd C m C m +r~1 TO -P p P a (0 P a •M 00 05 Ph m rH CO CD iH Cd P P o o 02 CO i—i CQ 02 co P CD qO •tH P P P O o CO rH CO O o O CO H1 LO CO 02 o o o O o 02 02 Oi CO CO CO o CO CO o o NT* CO o o o CO 00 1 —1 o O o CO 02 i—1 o o o o o CO 02 i—1 o CO oo rH o CO 02 rH O o CO LO CO 02 CO rH i—1 m Eh 05 o rH i—1 rH U l cn m2 P CO i—1 i o o Of CO CO Cl p o Eh Eh Eh CD C O ' o o o oo Nt< 02 oo CO CO 1—1 o CD ID OO O CO LO 02 O oo 02 rH o CO LD 02 o O 00 P 03 CO i—1 o CO 02 i—1 o o CO 02 CO i—! O O o CO CO ID CO 02 rH 02 O CO LD 02 o OO 02 r-l ® a, o W t p P p m i—1 CD > CD rH O o CO CD 02 07 rH l—1 o o o CO CO 00 CO 02 rH i—1 o CO LO 02 o 03 02 rH o CO 02 i—I o CO LD 02 O O 03 CO 02 02 rH ,— i O CO 02 i—1 O CO 02 i—I O CO 02 i—1 O CO 02 i—1 O CO LD 02 O CO 02 rH O CO LD 02 CO i—i rH a) P o 1 —i rH Ul 05 'L* rH m l»'»1 CO i—l i—1 DO Eh o 05 1 —1 1—1 rH U l CQ Ml P 05 CO O rH i— 1 < —1 rH U l fQ U l P E~h P rH TO o *1--I P O'* Ph i— 1 <3 P P a Ul 02 •rH p cd o able 8. CD •rH P CD 0 1 CD P Ph O CO ID 02 O OO 02 i—1 P Eh m Eh O CO 02 i—) el P-r o rH CD > O P Tf CD O P Cd > Td P •rH P 0j C'j P H Qd P P Ul P p it c Oh P lit OJ cn •rH Ul •rH C P P cd o 59. Table 9. Per cent total occurrences per unit of titer. Rat Sera A Resistant Number of occurrences Per cent of total occurrences o• o 5.7 2 160 0.0 o• o 7 320 22.8 8 640 52.8 22.8 8 1280 1—i 20.0 22.2 160 0 320 0 640 4 11.1 1280 19 CVl 0 8 • 00 to 5 2560 35 36 Table 10. Titer 3 0 2560 . Number of occurrences CD • CD Titer Susceptible Per cent of total occurrences of antibody titer of 1280 or greater. Resistant InTumber "~of titers Susceptible Titers "of 1280 or greater Number of titers 24 36 35 Titers of 1280 or greater 15 Per cent Per cent 6 6 .6 42 .8 60. Table 11. Ant igen Comparison of serum titers of Funt-^oppert rats with other rats and mammals. Rabb it Human Rats I Rats II 1 1 2 1 2 1 2 T3116 8 0 16 8 LE110 0 0 8 0 266 256 256 256 LS149 0 0 0 0 2-56 256 128 256 = Bes istant S * Susceptible 2 2048 4096 1024 1024 HuntHoppert rats RC- £ 1792 1120 1024 720 700 1120 61. VI. SALIVARY LEUCOCYTES The presence of leucocytes in the blood and their role as phagocytic agents have been recognized since the work of Metchnikoff, The literature is replete with studies of these cellular elements. But this is not so in the case of leucocytes present in saliva. Despite the fact that cer­ tain leucocytes are known to exhibit a phagocytic action toward many bacteria, virtually no work has been done to establish the function of these salivary corpuscles. Appleton (2) stated that polymorphonuclear leucocytes were constantly passing through the epithelium of the oral mucosa to the free surface. He suggested that these cells were potentially phagocytic and may contribute to the diminution of bacteria present in the mouth. Bibby, Hine and Clough (7) shared the opinion of Appleton that leucocytes were phagocytic and reduced the population of oral bacteria. From their work, Isaacs and Danielian (32) concluded that saliva, as it appeared at the opening of the duct of the salivary glands, was free of leucocytes. They believed that leucocytes present in the saliva had wandered through the oral mucous membrane. Orban and Reinmann (50) reported finding fewer leu­ cocytes in stained preparations of caries free individuals than in caries susceptible persons. 62 o The leucocytes from the mouths of susceptible individuals were in better condition than those from r e s i s t a n t s T mouths. Wright and Jenkins (73) found no difference in the total number of leucocytes in caries free and caries sus­ ceptible individuals. They found that the number of intact cells were four times greater in the caries free subjects. Many cells were found to be in various stages of disintegration. The sparse literature dealing specifically with numbers of leucocytes present in the saliva of caries free and caries susceptible individuals is not conclusive. Agreement is also lacking as to the meaning of leucocytes in saliva. The possibility presented itself that salivary leu­ cocytes might be instrumental in reducing the oral lacto­ bacilli population. Rosen (59) observed that these bacteria in the saliva of the Hunt-Hoppert rats were a permanent flora of the susceptible rats and were transient in the re­ sistant strain. In keeping with the idea of a large screening program that would point the way for future investigations, a study of the saliva with respect to leucocytes was incorporated into the program. Two objectives were sought: to demon­ strate the presence of leucocytes in the saliva of tnese animals and to quantitatively describe the difference in numbers between them; and secondly, if possible, obtain a diagnostic tool whereby a leucocyte count would indicate the advent of the caries condition. Procedure Glass slides were scrupulously cleaned in soap solu­ tion, followed by immersion in a bichromate bath. ^ith the aid of a tungsten-carbide pencil, a 7/8 inch (22 x 22 sq. mm) square was etched into the slides. Saliva, obtained in the manner previously described, was pooled and 0 . 0 1 ml ’ was placed in the center of the etched square from a 0.2 ml pipette. The glass jar con­ taining the pooled saliva was vigorously rotated immedi­ ately prior to removing the 0.01 ml sample tc distribute the leucocytes evenly throughout the saliva. This portion was evenly spread to the edges of the square and quickly fixed in a current of warm air which was obtained by placing a bunsen flame behind a fan rotating at moderate speed. Both a susceptible and a resistant smear were made in rapid succession. These were then ready for staining. stain proved roust satisfactory for this purpose. Wright’s Cover slip preparations did not prove as effective as the slide preparat ions. Staining Procedure Wright’s powder Crl yc er in Methyl alcohol 0.3 g m . 3.0 c c . 97.0 c c . 64. The powder and glycerin were ground in a mortar and the methyl alcohol added. The stain was then placed in a dark brown bottle and allowed to incubate at 37° C for at least two weeks. Buffer Monopotassium phosphate Disodium-phosphate anhydrous Distilled water - 6.63 gm. - 2.56 gm. - one liter (1) Ten drops of dye were placed on the prepared slide for two minutes. (2) Ten drops of buffer were added and allowed to stand for seven more minutes after which a bright metallic sheen appeared. (3) The preparation was washed with distilled water in a manner calculated to lift off the dye. (4) The stain was then dried between blotting paper. The cytoplasm of the leucocytes appeared blue-purple and the nuclei stained mauve. purple. Bacteria stained deep blue- This preparation was not examined under the oil immersion lens of the microscope. Haemaeytometer preparations, including those stained with Randolph’s stain, in an Ac Spencer Bright-Line Haemacytometer did not yield the fine results obtained with dry mounts, and, of course, the dry mounts are a permanent record. The high magnification that can be used with the dry mounts allow differential counts to be made easily* All counts were translated to numbers present in 1*0 ml of saliva. The calculation is made according to the following formula: No. of cells counted 484 " 484___ area covered (sq. mm) x jqq s ” No. of cell per ml - the total area of the etched square Area covered = Area of one field under oil immersion multiplied by the number of fields observed. The formula may be simplified further after the area of one field is calculated. In this case the area of an 2 oil immersion field was 0.08 mm . The formula then becomes No. of cells counted x 6 ,050_______ No, of fields x counted Only intact dells were counted. ..nn = No. of cells -per ml Fragments of cells that could be identified were not Included in the total count. This procedure did not impose difficulty as only a very small number of the total cells appeared to be in some degree of disintegration. Results A glance at Table 12 reveals that leucocytes are pre­ sent in the mouths of Hunt-Hoppert rats. The data show that there is a distinct difference between the numbers of leucocytes present in the resistant and susceptible strains 66. It is to be noted that in. only the pre-caries stage are leucocytes present in larger numbers in the resistant animals. There is an approximately three-fold higher leu­ cocyte count among the resistant animals. Eut the figures represent averages of several trials and in this instance may not indicate the true condition prevailing. Three separate trials on susceptible saliva yielded consistent counts averaging 12,165 cells per ml. On the other hand, the trials with the resistant animals did not reveal this type of consistency. One of the trials showed a zero count and another trial a very high count. The average in this case, although a higher figure than the susceptible, may lead to false interpretation. Each successive stage thereafter exhibits a marked difference In leucocyte numbers with the susceptible animals showing far higher counts. A three-fold increment is noted in the developmental period and four-fold increases are seen in both the advanced caries and caries in resis­ tant groups. The type of leucocytes found is noteworthy. Poly­ morphonuclear leucocytes predominate in the susceptible strain with monocytes second. Lymphocytes, on the other hand, predominate in the resistant strain with monocytes again second and polymorphonuclear leucocytes being rela­ tively few in number. Table 13 presents a tabulation of this differential count. 67. G CD P> -P 03 G P-r Pi EUP 05 CD •H CD CD CD © •rH G _io G CD Eh CD Cm Cm •rH Xl G1 to G» to •N CO LD rH 00 1 G ’ to *\ o CO CO 1—1 03 LD O •x 03 co G ed P 03 LD rH *\ LD to i—I CO CO o 03 CO 1—1 G 03 c•1 03 to 03 CO rH CO rH to to G o •rH X> CD o G CO > X> 65 CO •rH G C5 CO 05 05 > •H i—1 tti CD to to ao o**> LD G< CO co CO rH rH LD CD O CO O rH rH LD •\ O LO CO to O 03 LO LD •N to LO CO D CO G+ LD CO •% «% LD to tr 03 ■—l GO Gl rH •% G1 O i—1 03 CO 03 CO CO O O 03 tO 03 CO rH G1 03 LD t—i 1—1 •\ 03 CD CO 1—1 rH •N CO rH O CO LD •\ O rH LD CO i—1 -> CO rH • * to to UQ > CO •x to to G1 G-> 1 to IH i—1 cv -P cd G i—1 -P P G 03 a P. cd G CD CG G, O CO 1 -P G G o 0 X) O •rH G © Oa 1—1 CD o o•\ CO oo LD CO to i—I O o 03 «\ to D/i CO GH rH i—1 CO LD CO CO to to 1—1 > -p •r~l K f' •i—1 «M -P O o CO i—i C /3 s CD G •rH Cl' G P . cd O 65 © n CD © •rH G i—t 05 © O > 1 CIO © 1—1 G 00 CO CO o 1 c co Ph CD cp, -P O O o o C/j © to Cd G CD P PI CD to CD i—1 a 1—1 to CO tH G1 vt£ <1? 1—1 CO rD © G G en •rH & CD G -P o o G G it o n i 01 cd Pi - P CO !>s CD O £j H >> o d >> i>> O XI > Cvi H co LO LO CD LO CO CO a rH O CO 1 O d 1 LO CVi CO to CD Cd ^ ' ID to CVI 5 O o o P — i i o o i— 1 ffl rH CD Xj CD P* -P E> CVi cvi CO O CO CO t- r— 1 i— 1 O i— 1 i— 1 CO LO i— i cvi CO o to C~~ CO d* LD CO I— 1 CO I— ! CO 1— 1 CO H CVI CO i— I cvi to d 1 1— 1 Cvi o X 1 O CD CD d o -p >> S as seen P O cCV2 o s 1 i— 1 ci •rH l o count Xi rx P-'i Ol CO O' 1 O 01 05 CD d 1 P CD rH u Eh 03 -P TJ O •rH . CD p P a rH 05 -P CD CD •rH P, P C5 1 O ’* d Ch d CD $ b V| o rH CD > 05 01 CD •1— 1 Oo -P m iH CQ CD p . u CD C5 ra G5 o d Cd > 1 j -d d H t 01 05 -I— ( d CO o the actual Pi O d CD -p CO -rH -H cvi represent 1 O S *P (— i in the stained P. -P j d 1 CVi 01 CD (D a U d 1 1 O XI p- ID O numbers rQ •H D P O 3 CD i-1 rH O pH O o Those Table 13, Differential count of salivary leucocytes as found in stained preparations of saliva of Hunt-Hoppert rats at different stages of caries activity. O x; Or Pi G preparation. 68. Figure 11. Photomicrograph of salivary leucocytes (Each space represents 10 micron.) A. lion-filamentous polymorphonuclear leucocyte and small lymphocyte. B. Monocyte, upper right; lymphocyte, lower left. C. Group of small lymphocytes; monocyte off to right. D* Clump of small lymphocytes; monocyte, lower right. E. Nuclei of epithelial cell, upper left; poly­ morphonuclear leucocyte, center right; monocyte, center left. F. Monocytes G. Monocytes H. Won-filamentous polymorphonuclear leucocyte and small lymphocyte. I. Non-filamentous polymorphonuclear leucocyte. % Hi*. » • V c # 71. VII. PHYSICAL DETERMINATIONS ON RAT SALIVA Very few physical determinations have been performed on saliva in pursuit of the riddle of dental caries. To describe as completely as possible the conditions prevailing in the mouths of the animals studied, the phy­ sical nature of saliva required definition. measurements.;— The four surface tension, specific gravity, refrac­ tive index and viscosity-- would amply describe the physical character of the saliva under investigation. Surface Tension A search of the literature failed to uncover observa­ tions concerning the surfacd tension of rat saliva or observations concerning surface tension of saliva from carious mouths. Procedure Surface tension was measured by a torsion balance method in which a Cenco Du Nouy Interfacial Tensiometer* was employed. The interfacial tensiometer is a direct reading instrument and was calibrated against boiled dis­ tilled water (72.0 dynes/cm) and C.P. Benzene (28.2 dynes/ cm) at 23°±0.5°C. One milliliter samples of saliva were pipetted onto acid cleaned watch glasses for measurement. ^Serial No. 890, No'.""70542 PietinunTRing - Mean circum­ ference 5.995 cm. R/r 5 3 .6 . 720 Results Table 14- presents the figures obtained. It is obvious that little difference exists between the two groups of animals. Four-tenths of a dyne per cm differ­ ence between the animals of the pre-caries level and 0.9 dynes per cm difference in the period of caries develop­ ment are well within the limits of experimental error. The slightly higher differences obtained with the remaining two groups, 1.8 dynes per cm and 2.7 dynes per cm in that order, likewise were within the limits of ex­ perimental error. Preliminary tests had shown that vari­ ations from one to four dynes per sample could be antici­ pated. Although these figures do not indicate differences between the two strains, the very nature of the figures themselves are interesting. The low surface tension, (average of 42.9 for the resistants and 43.7 for the susceptibles), content. is quite provoking considering its high water When the values for saliva are compared with a surface tension depressant such as 0,1 per cent sodium oleate solution which has a surface tension of 42.2 dynes per cm or to water with a surface tension of 72.0 dynes per cm, one cannot escape the conclusion that saliva has great wetting characteristics. This may account for the effici­ ency of saliva in moistening feed materials. 73. It was observed as early as 1925, by Albus (1), that b* acidophilus could grow well at a surf'soe tension of* 36 dynes per cm. This low surface tension vitiates any anti- lactobac illus effects due to surface tension. Since both strains exhibit the same surface tension, it is doubtful that the difference in bacterial population can be attribu­ ted to this factor of saliva. It is of more than passing interest to note that LBS medium selective for lactobacilli contains 0.1 per cent Tween 80, a surfactant, as an integral part of its formula. The object of this medium is to grow lactobacilli selective­ ly to the exclusion of all other microbial forms and a low surface tension is a contributing factor. The incorpora­ tion of a surface tension reducing reagent obtains a sur­ face tension of approximately 45 dynes per centimeter for this medium. It may be reasonable to speculate that an equally low order of surface tension present in the saliva of the rats limits the types of microflora pros ont. Table 14, Surface tension* of pooled Hunt-Hoppert rat saliva at different stages of caries activity. Trial Resistant Pre-caries level Develop­ mental level Oar ies appear in resistant 1 4-3 .1 43.6 43.1 43.7 2 42.8 42.8 41 .9 42.0 42.1 44 .3 3 Sus ceptible Advanced car* ies Avg. 42.9 43.2 42.3 43.3 1 42.6 42.5 45.2 46. 3 2 43.0 42.2 45.0 43.2 3 42.2 44.8 46.0 Avg. 42.5 45 •0 45.1 42.3 uLleasured in dynes per centimeter 75 Refractive Index Careful, searching of the literature on dental caries failed to reveal papers concerned with the refractive index of saliva. Since differences in the intrinsic con­ centration of saliva could be revealed, this procedure might prove of value in characterizing the saliva. Procedure Por the determinations to be made an AC Spencer Refractometer (series 424) was employed. The scales of this instrument are calibrated directly in refractive index as measured with the nD !f line of the sodium spectrum. The scale may be read directly to the third decimal place and the fourth may be estimated with an accuracy of *t 0,0002. A drop of saliva was spread evenly on the ground surface of the auxiliary prism. The lever arm was moved until a dividing line was observed through the telescope. O The refractive index L Q »D -7 was read directly from the Alidade scale. Results The information obtained from this procedure is pre­ sented in Table 15. The magnitude of difference among any of the groups, or between resistants and susceptibles of 76 any one group is negligible. The index of refraction obtained•tends to indicate that the saliva contains a low concentration of dissolved solids. But it is sufficiently high to affect the saliva such that detection of differ­ ences can be accomplished with other procedures. Although this study presents .new information that can be applied to Hunt-Hoppert rat saliva, it does not appear to yield data, the nature of which can shed light on the caries problem. 77. Table 15. Refractive index of pooled Hunt-Hoppert ret saliva at different stages of caries activity. Tr ial Pre-caries level Devpi opmental Advanced car ies Caries appear in res istant 1 1.3341 1.3339 1.3350 1.3358 2 1.3340 1.3340 1.3340 1.3557 3 1.3339 1.3340 1.3340 1.5357 1 .3340 1.3340 1.5 343 1.3554 1 1.3340 1.3343 1.3351 1.3360 2 1.3339 1.3343 1.3347 1.3361 3 1.3340 1.3341 1.a347 1.3359 1.3340 1•3342 1.3343 1.3360 Rea istant Avg. Susceptible Avg. 78 Viscosity Viscosity measurements of saliva, both animal and human are conspicuous by their absence from the dental literature • Hewat (28), in one of the very few reports avail­ able , observed that of the children he studied those ex­ hibiting caries had salivas with a relative viscosity of 1*23. Saliva from caries free mouths had a relative vis­ cosity of 1.02. He believes that the more viscous saliva tends to bind particles of food (especially sugar) to the teeth. Procedure Viscosity measurements were carried out on the saliva of the Hunt-Hoppert rats in an attempt to ascertain the con­ dition of difference between the two groups as a function of the internal triction of the liquid. The viscosity then, would reflect the resistance experienced by the molecules in moving around in the interior of the lioxuid due to inter-molecular forces. One milliliter volumetric pipettes were softened in a Bunsen flame and drawn out to a length, such that dis­ tilled water, falling between two points, yielded dropping times of twenty seconds. Standard viscometers were not available for the small volumes required by this study. After filtration through a Swinney apparatus, the 79. saliva was drawn into a pipette by suction bulb. Dropping time was measured with the aid of a sweep hand stop watch. All measurements are relative to distilled water with a dropping time of twenty seconds. Results The figures in Table 16 are most interesting. In every instance the susceptible groups exhibit a lower viscosity than the resistant group. Table 17 indicates that the rela* tive ‘viscosities are on the average 9.0 per cent higher for the resistant animals as compared with the susceptible ani­ mals. Considering the technique employed, the data presen­ ted are well beyond the limits of experimental error* This nine per* cent difference in relative viscosity may be indicative of smaller, more mobile molecules in the interior of the susceptible saliva as compared with larger molecules in the resistant saliva. These smaller molecules may be the result of enzyme action on carbohydrates. If this is true, then it might be reasoned that the susceptible strain of animals would be the recipient of greater acid production in their mouths with subsequent caries activity. The mechanism of carbohydrate degradation with resultant acid formation is presented in the section on Amylase determinations. The viscosity data suggested that amylase determina­ tions be incorporated into the body of the investigation. Tie enzymatic destruction of large molecules to smaller fragments might reasonably yield the viscosity data presented in the tables• Table 16* Dropping times# of pooled Hunt-Hoppert rat saliva at different stages of carles activity* Rasistants Trial Pre-carles level 1 2 3 24*02 24*16 4 Develop­ mental level Advanced Caries carles appear in resistant 24 *12 23.SO 83.43 22.60 23.84 23*72 28.50 23.06 23,52 23*38 24.02 Avg* 24.14 23.16 23.66 1 22*36 @2*64 21.32 23*33 2 22.24 22.72 21.30 21*40 21.26 21*16 22*29 21.40 21*60 22.09 21.29 21.96 Susceptible® 4 AVg* ♦Measured in seconds* Table 17* Relative Viscosity of Hunt-Hoppert rat saliva. Resistant Susceptible Pre-earlas level 1*20 1*11 Developmental period Advanced carles 1.18 1*17 1*10 1*06 Caries appear in resistant® 1*20 1.10 81. Specific brevity Specific ere.vity measurements are a. convenient aid in characterizing liquids. They have been proven to be value.ole in differentiating between similar compounds. Specific gravity, t , which is defined as the mans, m, of a substance at t° C. relative to the mass, m , of an o? equal volume of water at t° C, is therefore a dimensionless number. The most common method of specific gravity determina­ tion consists in finding the weight of liquid, occupying a known volume defined by the shape of a given vessel. The vessel is calibrated in terms of the weight of pure water which it holds. Fycnometers are the usual vessels employed. There is a paucity of information on determinations of specific gravity performed on salivas of persons, or animals, with and without caries. Hewat (28), who obtained saliva from children with and without dental caries, re­ ported. that specific gravity of saliva from the non-carious children was 0.9937. The figure presented, for the saliva from carious mouths was 0.9918. The incorporation of specific gravity measurements into this study was to further characterize the saliva obtained from the Hunt-Hoppert rats. As no specific gra­ vity determinations have ever been performed on the saliva 83. of* these animals, such a determination was needed and might indicate a factor of difference between the animals. Procedure Since only small volumes of saliva were available, a 1.0 ml Lipkin-type* pycnometer was employed. This is a bi-capillary vessel with one of the capillary arms bent at an angle of 140° so as to allow self-filling. The liquid is first drawn into the pycnometer by capillary action and the pycnometer then fills by siphoning. This hook is also convenient for hanging the pycnometer on the Ainsworth precision balance for the weighing operation. Four place accuracy can be obtained with this procedure. After filtration through a Swinney apparatus, the filtered saliva was placed in a glass tube (l/2-inch tall by l/2-inch external diameter) and the hook end of the pycnometer immersed below the surface of the saliva. The saliva was allowed to rise to any arbitrary level on the pycnometer scale. The filled vessel was then hung on a precision balance and weighed. After flushing and rinsing with distilled water the same procedure-was followed using distilled water, except that the water was brought to the same level as the saliva. From the weights of equal volumes of saliva and water, relative specific gravity measurements were obtained. ^Available commercially from the H. Indiana. l.. Mastin Co., c.vanston, 83. Results Table 18 lists the data obtained. It is generally seen that little or no difference in specific gravity was the rule. The salivas of both animal groups exhibited no differences at the pre-caries level. at the developmental level. This was also true An average difference of 0.0053 units was obtained between the two strains at the advanced caries level. This represented a reduction in density from the developmental period. But this was-not reflected in the older animals as would be anticipated since caries activity was present in both strains. As the specific gravity in this group was similar to that in the developmental period, the reauction obtained at the advan­ ced caries level In the susceptible saliva appeared not to be ai-oonsequence of the carious condition. Although the data collected In this study had never been obtained previously, it does not appear to delineate the problem of differences between the two strains of animals• Table 18. Specific gravity of pooled Hunt-Kcppert ret saliva at different stages of caries activity. Trial Pre-caries level Develop­ mental period 1 1.Q00S 1.0003 1.0010 1.0003 2 1.0005 1.0004 1.0001 1.0004 1.0003 1.0006 1.0006 1.0006 1.0005 1.0008 1.0006 1 1.0008 1.0003 D .9958 1.0004 2 1.0006 1.0003 0.9948 1.0003 1.0002 0.9950 1.0004 1.0002 0.9952 1.0003 Advanced Caries caries appear in resistant Res istant 3 Avg. Susceptibles 3 Avg. 1.0007 DISCUSSION Of the various tests employed in an attempt to reveal differences between the t™ro strains of rats, several showed promise of being effective. In the area of physical measurements only viscosity determinations offered significant differences. The resis­ tant strain had a nine per cent higher viscosity which may be correlated with the higher amylase content present in the saliva of the susceptible animals. Contemporary with these findings was the further observation that larger numbers of bacteria were recovered from the saliva of susceptible animals. Not only was the total bacterial population larger, but the acidogenic bac­ teria represented by the lactobacilli and streptococci, were also present in larger numbers. The existence of three potentially interrelated con^ ditions may offer an avenue for speculation regarding the initiation of caries in the susceptible strain of rats. Particles of the carbohydrate containing diets may probably lodge in the crevices and fissures of molar teeth. Con­ tained in the saliva bathing these teeth are starch-hydro­ lyzing enzymes (amylases) that may aid in the degradation of the larger polysaccharide molecules to smaller disac­ charide and monosaccharide sugars. This reduction of large molecules to smaller ones may be mirrored in the lower 86. viscosity of the saliva* The amylases may be either seli- vary or bacterial in origin, or 0 combination of the two. The large numbers of oral bacteria present could con­ ceivably produce quantities of this enzyme such that the resulting hydrolysates, disaccharide and monosaccharide sugars, would be available for bacterial fermentation with subsequent production of a sustained acid action on the enamel of the teeth. This synthesis, though in agreement with the current­ ly popular theory of caries initiation, may in no way con­ tribute to actual caries formation, but may bo the result of the genetic differences between the two strains. Never­ theless, the observations suggest the direction future investigations should take. If the r e s u l t s of this study are further verified with larger numbers of animals the mechanism of carles development presented ho^e will assume greater meaning. It was mentioned earlier that dental caries is a unique disease. pecause of the nature of the tissue in­ volved, dental lesions do not lend themselves to strict definition of infection with its symptom complex. Particu­ larly lacking is the condition of tissue repair and replace­ ment. This study establishes the presence of leucocytes in the saliva of these rats. These cellular elements are present in 0 manner suggestive of a positive attraction. The numbers of leucocytes, particularly pelymorphoruelears 87. and. monocytes present in higher numbers at each stage of* caries activity in the caries susceptible animals indicates some type of stimulative mechanism in operation. The large numbers present may be in response to the dental infection. The possibility that a chemical fftriggertT liberated in the diseased area sets the mechanism in motion, is in accord with the vitalistic theory promulgated recent­ ly. On the other hand, the presence of these leucocytes may be in response to a chemotactic attraction toward the large numbers of bacteria present. The higher polymorph and monocyte count obtained with susceptible saliva as compared with the high lymphocytes of the resistant saliva suggests the possibility that phagocytosis is being attempted by the macrophage system. The aspects of chemotaxis and phagocytosis could not be studied at this time. 88. SUMMARY A screening study was made of the saliva and blood of the Hunt-Hoppert caries susceptible and resistant rats. Of the eight tests applied to these body fluids, only four showed promise of helping to differentiate between the two strains. Viscosity measurements appear promising as a means of elucidating the caries problem* A nine per cent differ­ ence between the two strains was obtained. animals had the lower viscosity. The susceptible The other physical tests employed, viz., specific gravity, refractive index, and surface tension, showed no significant difference between the two strains of rats. The numbers of lactic acid bacteria and ntotal,? numbers of bacteria recovered from susceptible saliva was substantially greater at all levels of caries activity as compared with the numbers recovered from the resistant saliva• The amylase content of caries susceptible rat saliva was consistently higher at all levels of caries activity than is the saliva of non-carious rats. Salivary leucocytes were present in the saliva of both strains of Hunt-Hoppert rats. However, there was a marked difference in numbers and types of leucocytes pre­ sent in the two strains of rats. The susceptible animals 89. had the greater numbers in their saliva. Agglutination tests run on the saliva and blood sera on both strains of the Hunt-Hoppert rats showed no significant differences between the two strains* The electrophoretic pattern of the serum proteins of these animals did not yield significant differences b e ­ tween the two strains. Saliva collected from both strains of rats and tested for its antibacterial action against organisms normally present in the mouth of these rats showed no acti­ vity against these organisms. 90. CONCLUSION The results of this work would indicate that the larger bacterial population, the lower viscosity measure­ ments, the higher amyla.se content and the greater numbers of salivary leucocytes appear to offer a means of differ­ entiating between the two strains of the Hunt-Hoppert rats. These differences may lead to greater knowledge of the mechanism of caries formation. 91. BIBLIOGRAPHY (1) Albus, V/. R., and Holm, G. F. Surface tension as a factor in the growth of Lactobacillus acido­ philus and Lactobacillus bulgaricus. Pact. Abstracts, 9:142, 1925. (2) Appleton, J. L. T. Bacterial Infection. Febiger, Phila., 1933. (3) Arnold, F. H., Jr., and McClure, F. J. A study of' the relationship of oral Lactobacillus acido­ philus and saliva chemistry to dental caries. Pub, Health Rep. 56: Ho. 30, 1941, (4) Barany, F. Resistance to caries in relation to cer­ tain properties of saliva. Acta. Med. Scand. 127:370, 1947. (5) Becks, H. Salivary and bacteriological considera­ tions in the control of dental caries. Jour. Am* Coll. of Dent., 9:104, 1942. (6) Belding, P. H. , and. Belding, L. J. Diet, bacteria and disease. Dent. Items of Interest, 62: 303, 1940. Lea and (7) Bibby, B. G. , Hine, M. K. , and Clough, 0. F. The anti-bacterial action of human saliva. J. A. D. A . , 25:1290, 1938. (8) Bibby, B. G., Volker, J. F., and Van Kesteren, M. Acid production and tooth decalcification by oral bacteria. J. Dent. Res., 21:61, 1942. (9) Black, G. 0. Operative Dentistry. Medico-Dental Publishing C o ., Chicago. Vol. I, 61. 1908. (10) Bunting, R. W. Studies of the relation of Bsc illus "Acidophilus to dental caries. J. Dent. Res., 8:222, 1928. (11) Bbnicke, R., Rief, F . , Arndt, J. The antibacterial activity of human saliva with special refer­ ence for daytime fluctuations. Z. Hyg. In­ fections, 136:348, 1953. (12) Canby, C. P ., and Bernier, J. L. Bacteriologic studies of carious dentin. J. An. Dent. Assoc., 23:2083, 1936o 92. (13) Clauberg, K. L. The bactericidal set;ion. of' human saliva with special reference to the diph­ theria bacillus. Zentr. Bakt. Fsrasiten, I, Abt. 134, p. 96-99, 1935. (14) 01 ough, 0. V/. Inhibition of bacterial growth by human saliva. J . Dent. Research, 14:164, 1934. (15 ) ____________ i Bibby, B. G., and Berry, G. P. The effect of saliva on Lactobacillus Acido­ philus. J . Dent. Res., 17:'493, 1938. (16) Cool idge, T. B. , Williams, N. B., Ebish, a . E. I ., and Hodges, E. A. Metabolic changes in oral lactobacilli. J . Infect. Pis., 85: 126, 1949. (17) Day, C. D. M. The amylalytic enzyme of the saliva in relation to dental curias. Dent. Cosmos. 76:683, 1934. (18) Dietz, V. H., Williams, N. B., and Lawton, W. E. R e ­ lationships between blood agglutinins for oral lactobacilli, salivary counts and caries. J. Am. Dent. Assoc., 30:838, 1943. (19) Dold, H., Lachele, P., and Du Dscheng Hsing. perties, scope and method of action antibacterial inhibitive substances bines) of human saliva. Zeit, Hyg. ionskrankh, 118:369-96, 1936. (20) Flora in, L., Essai sur la salive humsine et sur les propietes physiologiques du sulfocyanate de potassium. Gaz . Med,, de Paris, 60:317, 18 89 . (21) Florestano, H. J., Faber, J . E., and Janos, L. H. Studies on the relationship between diastatic activity of saliva and incidence of dental caries. J. Am. Dent. A^soc., 28: 1799, 1941. The pro­ of the (InhiInfekt- (22) Granados, H . , Glavincl, J., and Dam, H. Experimental dental caries, XIII. The effect of adding to the drinking water saliva from two sub­ jects with different caries susceptibility. Acta. Path. Microbiol. Lcand. 27:65, 1950, (23) Griffith, J. Q. , and Farris, E . J. The Pat in Labor­ atory Investigations. J. S. Lippincott Co., Hew York., 1942. 93. .124) Harrison, R. W. Studies on lactobacilli. III. Re­ lationship of immunological specificity and fermentative capacity. J. Infect. Dis., 70:69, 1942. (25) Hawk, P. B., Oser, B. L . , Summerson, A. H. Practical Physiological Chemistry, 18th ed. Blakiston Company, Phila., Pa. 1947. (26) Henley, A., and Stern, K. C-. Instrumentation and pro­ cedures in physical studies of proteins and other macromolecules, Proc. of the Inst. Soc. of Amer., Sept., 1952. (27) Hess, W. C., and Smith, B. T.. The salivary amylase activity of carious and ncn-cerious indivi­ duals. J. Dent. Res. 27:593, 194-8. (28) Hewat, R. E. Dental caries: An investigation in search of determining factors in its manifes­ tation. The Hew Z. Dent. J., 28:45, 1932. (29) Hubbell, R. G-. The chemical composition of saliva and blood serum of children in relation to dental caries. Am. J. Physiol., 105:436, 1933. (30) Hugenschmidt, 1. C. Experimental study of the diff er­ ent modes of protection of the oral cs.vity against pathogenic bacteria. Dent, Cosmos, 38:797, 1896. (31) Hunt, H. R., Hoppert, C. A., and Erwin, D. G. Inheri­ tance of susceptibility to caries in albino rets (Mus Norvegicus). J. Dent. Res., Vol. 23:385, 1944. (32) Isaacs, R., and Danielian, 4. C. Maintenance of Leu­ cocyte level and changes during irradiation. Am. J. Med. Be., 174:70, 1927. (23) Jay, P., Crowley, M ., Hadley, F. ?., and Bunting, R. T/V. Bacteriologic and immunologic studies of dental caries. J. Am. Dent. As see., 20:2130, 1933. (34) Kawanoto, T. The bactericidal action of human saliva on various bacteria. Japan. Zeit. hikrobial Path., 20:114-21, 1926. a/ w M (35) (36) O K e s e l , "R. G, Dental caries: Mechanism and DPfiP^nt control techniques. U. of Mich, Work­ shop Fvaluntion, K. A. Be slick, 9 d-, p. 90-92, 1948. ., O ’Donnell, J. F., Kirch, Tach, Ammonia production in the oral cavity and the use of ammoniuir salt for the control of dental caries. Am. J. of Orthodon. and Oral Burg., Vol. 3?:80, 1947. (37) Poland, S. A., Jr. An Electrophoretic and Chemical Fractionation Study of Sera from Fats Immunized Against the Nematode, Hippostronglus Muris. Doctoral thesis, Mich. State College, 1953. (38) Leutacher, J. A., Jr. Biological and medical appli­ cations of electrophoresis. Phys. Fev. 27:621, 1947. (39) Longsworth, L. G . , and Maclnnes, D. A. The interpre­ tations of simple electrophoretic patterns. J. Am. Chem. Roc., 62:705, 1940. (40) _________________ . Recent advances in the study of proteins by electrophoresis. Chem. Rev. 30:323, 1942. (41) Lundgrcn, H. P., and hard, h . H. Amino Acids and Pro­ teins. Greenberg, D. M. (ed.) C. C. Thomas, Springfield, 111. 1951. (42) McClure, F. J. The effect of fluorides on salivary amylase. U . £. Public Health Fep. 54: 2165, 1939. (43) McIntosh, J . , James, A. F ., and Lazarus-Barlow, P. An investigation into the etiology of dental caries. IV. (1) Reaction of the saliva, (2) acid resistance of teeth, (3) bacterio-tropic action of saliva. Brit. J, Fxptl. Path., 6:26C, 1925. (44) Miller, W. D. Der Einfluss der Micro-organismen auf die Carles dor Monschlichen Zahne. Arch. Fxpth. Path., 16:291, 12 82. (45) Agency of micro-organisms — — — 3 - human teeth. in decay of Dent. Cosmos, 25:1, 1883. — 95. (46) (47) (48) — ----- — Introduction to the study of iiomunity in its relation to the diseases of the mouth and teeth. Dent. Cosmos, 45:1, 1903. --------- . Experiments relating to the question of immunity. Dent. Cosmos. 45:689. 1903. ________ __ Further experiments relating to the question of immunity. J . Brit. Dent. Assoc., 25:12, 1904. (49) Moore, D. H., and White, J. U. A new compact tiselius electrophoresis apparatus. The Rev. of Sc. Instr., 19:700, 1948. (50) Orban, B., and Weinmsnn, J. P. The cellular elements of the saliva and their possible role in caries. Am. Dent. Assoc. J., 26: 2008, 1939. (51) Orland, F. J. (52)r> Pesch, K. L., and Damm, R. The bacterial action of saliva on pneumococci. Zeit. Hyg. In­ fect ionskrankh, 118:1-16, 1936. (53) Pickerill, H. P. The Prevention of Dental Caries and Oral Sepsis. Bailliere, Tindall and Cox Publishing Co., London, 1912. (54) Rigolet, D. (55) Robertson, W. (56) Rogosa, M., Mitchell, and Wiseman, R. F. A selecti ve medium for the isolation and enumer­ ation of oral lactobacil1i . J. Dent. Res., 30:682, 1951. (57) Rosebury, T. A correlation of antigenic character­ istics among certain bacteria of the lactobacillus group. J. Infect. Dis., 86:63, 1950. Asialorrhie et carie dontsire. tologie, 25:387, 1901. L !Crdo n- A Practical Treatise on the Diseases of Teeth. Longmans. Pub. Co., London, 1835. Studies on the aciduric bacteria of the mouth in relation to susceptibility and immunity to dental caries. J. Dent. Res., 10:403, 1930. 96. (58) Rosen, S. (59) Rosen, S., Hunt, R. W., and Benards, M. A. Micro­ biological differences in the oral cavity of Hunt-Hoppert caries-resistant and caries-susceptible rats. J. Dent. Res., 1954. (In print). (60) Ross, V., Krasnow, F., and Samet, J . Agglutinins in serum and saliva of rabbits inoculated with lacidophilus. J. Dent. Res., 7:537, 1927. (61) Sanarelli, D. Der Menschilhe Speichel and die Pathogenen, Mikroorganismine der Mundhohle. Centralbl. f. Bact. U. Ferasitenkunde, Bd. x, p. 818, 18 89. (62) Stark, R ., We H e r son, R., Jr., Tetrault, P. A., and Kossacks, 0. F. Pacterisl alpha amylase paper disc tests on starch agar. Appl. Micro., 1:236, 1953. (63) Stephens, D. J., and .Jones, P. Leucocytes in the saliva in normal and abnormal sublets. Proc. Soc. Rxoer. Biol, and Med., 31:879, 1934. (64) Stern, K. G., and Reiner, M. Electrophoresis in medi­ cine. Yale J. of 3iol. and Med., 19:67, 1947. (65) Thompson, R., Shibuya, M. The inhibitory action of saliva on the diphtheria bacillus: the antibiotic effect of salivary strepto­ cocci. J. of Pact., 51:671, 1946. (6 6 ) ___________ (67) Turner, N. 0., and Crane, P. Ivi. A relationship be­ tween dental caries and saliva. Science, 99:262, 1944. (66 ) Von Kesteren, M., Eibby, B. G., and Berry, G. P. Studies on the antibactericidal factors of human saliva. J. Fact., 43:573, 1942. (69) Weiss, J . F ., and Rettger, L. F. Lactobacillus bifidus, J. of Bact., 28:501, 1934. Personal Communication, 1954. Lysozyme and its relation to the anti­ bacterial properties of various tissues and secretions. Arch. Path. 30:1096, 1940. 97. £70) Williams, N. B. Antigenic components of lactobacilli of human oral origin. J . Infect. Dis ., 82:31, 1948. (71) ________________ Immunization of human beings with oral lactobacilli. J. Dent. Res., 23:403, 1944. (72) ________________ Salivary counts in humans following vaccination with oral lactobacilli. •J. Dent. Res., 23:196, 1944. (73) Wright, D. E., and Jenkins, N . G. Leucocytes in the saliva of caries-free and cariesactive subjects. J. Dent. Res., 32:511, 1953.