40 AMERICAN JOURNAL OF DIGESTIVE DISEASES AND NUTRITION

R E F E R E N C E S 1. Meleney, H. E. and Frye, W. W. : Studies of Endamoeba histo-

lyrics in Tennessee: V. A comparison of five strains of E. histolytiva with reference to their pathogenicity for kittens. Amer. Jour. Hyg., XVII, 637-655, May, 1933.

2. Meleney, H. E. and Frye, W. W.: The pathogenicity of En- damoeba histolytica. Trans. Roy. Soc. Trop. Med. and Hyg., XXIX, 369-379, Jan. , 1936-

8. Walker, E. L., and Sellards, A. W.: Experimental entamoebic dysentery. Philip. Jour. Sei.. VIII , Sec. B., 253-381, Aug., 1913.

4. Bundesen, H. N., ConnolIy, J . L., Rawllngs, L D., Gorman, A. E.,

McCoy, G. W., and Hardy, A, V. : Epidemic amebic dysentery : the Chicago outbreak of 1933. Nt. Inst. Health Bull., 166, March, 1936.

5. Spector, B. K., Baylis, J . R., and Gullins, O.: Effectiveness of filtration in removing from water , and of chlorine in killing, the causative organism of amoebic dysentery. U. S. Pub, Health Rep., XLIX, 786-800, July 6, 1934.

6. Baylis, J . R., Gullins, O., and Spector, B. K.: The efficiency of rapid sand filters in removing the cysts of the amoebic dysentery organisms from water. U . S . Pub. Health Rep., LI, 1567-1576, Nov. 13, 1936.

The Relationship Between Oral and Gastric Bacterial Flora By

MARION HOOD, Ph.D.

and

LLOYD ARNOLD, M.D. CHICAGO, ILLINOIS

T HE re l a t ionsh ip between the bacter ia l flora of the oral and gas t r ic regions has received l i t t le a t ten-

t ion, a l though considerable work has been done upon the flora in each locality. The correla t ion or in t e r - dependence of the two types of microbic populat ion seems to have beeH given bu t scant considerat ion.

Var ious observers have a t tempted to show tha t cer- t a i n re t rogress ive or degenera t ive oral lesions, such as pyorrhea, seeded the s tomach and upper levels of

the in t e s t ina l t r ac t wi th pyogenic bacter ia , derived f rom the mouth. Hurs t , (1924) (1927) (1933) has called a t t en t ion for m a n y years to the impor tance of pyogenic foci in the mouth, t h r o a t and nose upon

gas t r ic and duodenal bac ter ia l flora. The p resen t work classifies the o rgan isms cul tured

f r o m the mout l l and s tomach and de te rmines the re- la t ion which these bea r to each other. Effor t has been made to discover the effect of cont inuous en t rance of the exogenous flora and to s tudy the protect ive mech- an i sm by which the body defends i tself aga i n s t th is

entrance. G E N E R A L T E C H N I C

All experiments were performed on patients in the medical wards of the Research and Educational Hospital of the University of Illinois, College of Medicine. Although none of these persons could be considered entirely normal from a health standpoint, those exhibiting any particular dysfunction of the gastro-intestinal tract or suffering from primary anemias were not used. Cultures were made on plain agar from material aspirated from the stomach, if only the possibility of growth was to be determined. For classification, blood agar and Endo plates, as well as sugars for biochemical reactions were also used. No anaerobic cultures were made. For culturing the stomach contents one standard plat inum loop of material was placed on the surface of solid media and distributed with a sterile glass spreader which was then carried to a second

*From the Research Laboratories of the Illinois Depar tment of Public Health and the Depar tment of Bacteriology and Public Health, and the Depar tment of Medicine, Univers i ty of Illinois, College of Medicine.

Submitted October 7. 1986.

similar plate. To obtain cultures from the mouth, sterile swabs were rubbed across the back of the tongue, then passed twice across a sterile agar plate. With a second sterile applicator the material was spread over the sur- face. This swab was then carried to the second plate. By this procedure sufficient dilution was usually obtained to procure well isolated colonies.

Hydrogen ion concentration was the method used for de- termining the acidity of the various substances. The colori- metric method was employed using standard buffers made according to the specifications of Clark (1928). All buf- fers were standardized by a Leeds Northrup potentio- meter. The hydrogen ion concentration gives the effective acid concentration which is the most important factor l imiting bacterial growth. When control tests showed that the hydrogen ion concentration of the stomach contents was too acid to make probable the growth of organisms, varying amounts of buffered alkali made according to Clark (1928) were added. I t was difficult to alkalinize the stomach. There are several possible explanations for this fact. The alkali may have passed directly through the stomach into the duodenum or it may have stimulated increased acid secretion according to Boldyreff's (1914) principle of the self-regulation of the acidity of gastric contents. The alkali was sometimes given in milk in an effort to stabilize the acid-base balance as described by Arnold and Brody (1926).

E X P E R I M E N T A L P R O C E D U R E S

Experiment I: The Relation Between the Oral and Gastric Bacterial Flora. Twenty-five fast ing stomachs were aspirated by means of Rehfuss tubes. Cultures were made from this material and at the same time from the mouths to determine whether or not the micro-organisms cultured from the oral cavities were similar to those found in the stomach contents as reported by Bartle and Harkins (1925) and Kopeloff (1922).

In a series of sixteen cases a preliminary procedure was carried out to determine whether there was variation in the flora in different parts of the mouth. Cultures were taken from the lips, the tip of the tongue, the back Of the tongue, the gums, and from the saliva. As was also found by Arnold and Stuart (1935) there was some variation in

HOOD AND ARNOLD---THE RELATIONSHIP BETWEEN ORAL AND GASTRIC BACTERIAL FLORA 41

organisms from the different areas and at different times from the same area. As the back of the tongue showed the heaviest, as well as the most diverse flora, the mouth swabs for subsequent work were taken from this area.

The following list summarizes the findings, showing re- I i ! I

Organisms Location ~~<I Tongue Stomach }

/

Staphylococcus albus 21 19 ,,/" Staphylococcus aureus 16 15 Staphylococcus citreus 2 0 Bacillus coli 0 3 " ," Bacillus acidophil aerogenes 6 9

. /

Bacillus subtilis 3 2 :~i Gram positive diplococci 5 4 ~ 4 \ :'J( Gram negative cocco-bacilli 3 3 ; Gram positive cocci 2 2 ~ :~ Micrococcus tetragenous 4 3 ~ i ::! Sarcina lutea 3 3 ~ - Streptococcus salivarius 9 6 ~ 7 Streptococcus ignavius 5 2 Streptococcus pyogenes 2 1 2 ~ Streptococcus mitis 2 2 7 Streptococcus equinus 1 0 Streptococcus anginosus 0 1 Leptothrix 0 1 l : 2/' Yeast 0 2

/ ,

markable s imilar i ty between the type of organisms which £ont~o] constitute the oral and gastr ic bacter ial flora. The numbers indicate the times the given organisms were cul- tured from the total series of twenty-five cases. The streptococci were classified according to the method of Holman (1916).

Experiment II: The Rate of the Disappearance of Test Organisms from the Mouth and Stomach.

When an exogenous f lora is i n t roduced in to the mouth the bulk of the m a t e r i a l passes into the s tomach. T h e r e is, however , a con t amina t i on of the oral cav i ty wi th cont inuous seed ing of t he s tomach fo r some t ime. I f the in t roduced m i c r o - o r g a n i s m is pa tho- genic th i s may be an i m p o r t a n t f a c t o r in the produc- t ion of disease.

A s tudy of th i s p rob lem was u n d e r t a k e n by us ing the eas i ly recognizab le t e s t o rgan i sm, B. p rod ig iosus . Control cu l tu res showed t h a t m a t e r i a l ob ta ined by means of Re h fuss tubes f r o m the f a s t i n g s tomachs of f i f teen p a t i e n t s was f r ee f r o m the exogenous flora to be used and hyd rogen ion d e t e r m i n a t i o n s proved i t was sufficiently a lka l ine to s u p p o r t g rowth . The mouths were • then washed wi th 100 e.e. of normal sa l ine in which was suspended the su r f ace g rowth of one a g a r p la te of B. p rod ig iosus . The. s tomachs were a s p i r a t e d and swabs t aken f r o m the back of the tongue a t t h i r t y minu t e in te rva l s . H y d r o g e n ion concent ra - t ions were de t e rmined on all g a s t r i c f r ac t i ons and cu l tu res were made on all spec imens taken. These cu l tu res showed t h a t t he ave r age d i s appea rance r a t e fo r t e s t o rgan i sms f rom the mou th was two hours and t h i r t y - s i x minu tes , w i th a m i n i m u m of no g r o w t h and a m a x i m u m of five hours . The ave rage d i s a p p e a r a n c e r a t e in t he s tomach was one hour and e igh teen minu t e s w i th m i n i m u m of no g r o w t h and a m a x i m u m of t h r e e hours . This is i l l u s t r a t ed in G r a p h 1.

Experiment HI: The Effect of Rinsing the Mouth on the Disappearance Rate of the Microorganisms.

To d e t e r m i n e w h e t h e r th i s pe r iod of con t inuous seed ing could be e l imina ted or shor t ened the fo l lowing

G r a p h I A v e r a Q ~ o ( [ 1 6 t e e n ~ c a ~ e s , ~ h o ~ n ~ t h e r a t e o f ' S ~ 5 a p ~ a r a rice oN t£ s t o v e a r l S ms

( y o r e E~Le m o u t h a n d s t o m a c h ' [ [ ' r N I [ 2 . h 2 . . . . . . I ! . /

!~ i 7 / ~ i ± 1 i i i i IpI _ . l i ~ ~,oL--°~' 3 T 1 7 5 E o r ~ a r l l D m 5 tForr i ~ ~ Z;- ': ' -I ~to~ach-~-q 7 " ~ / [ -~ Test. i , ° r Q a n ' s m 5 ' o f r o m . . . . ' ~ 0

t ~ . . . . . ~ _ . r n u t h . . . . . - - ~ - j , ~ T - - ~. - ~ . - : . . . . . .

' 4 _ ~ i : - / + ~ ' #-~ . . . . . . . '. : - 5 - -~; 9 i ~ o , ;' i , , _ 0 0 ' _ c : - ~ , " I? - - x - : _ : - T - -

_ ~ _ :; _i 17~ ;iZ-L -7 7 - - 4 o o

2 - [ ' " .... ~ - I ~ ~ !-l~ ~;-. ; ' . . : . = o o . . . . . , - ] : • ; i - : ' i i

i . , / ~ t , ,~ : , : l

l h r Z h r 5 5hF.I

U T £

[ --

I

3hrt~ 4 h r ~

p rocedure was ca r r i ed out. Reh fus s tubes were in- s e r t e d into the f a s t i n g s tomachs of f ou r pa t i en t s . A s p i r a t i o n s were made and th i s m a t e r i a l as well as swabs f rom the back of the tongue were cul tured . As B. p rod ig iosus was to be in t roduced l a t e r as t e s t o rgan i sms , these f i rs t cu l tu res se rved as cont ro l s to e l imina te the pos s ib i l i t y of p rev ious seed ing w i t h the exogenous flora. The Rehfuss tubes were l e f t in place and the mouths were washed wi th t w e n t y - f o u r h o u r b ro th cu l tu res of B. p rod ig iosus and then r i n sed a t v a r y i n g in t e rva l s w i th s t e r i l e no rma l sal ine or hexyl - resorc ina l , 1:4000. Cul tures were made a t ha l f hour in t e rva l s fo r two hours . The d a t a a r e shown in G r a p h II , A and B. L i t t l e u n i f o r m i t y in resu l t s was found in th i s ser ies of exper imen t s . I t does ind ica te however , t h a t any d i m i n u t i o n in number s of b a c t e r i a is due more to the mechanica l ac t ion of r i n s i n g the mou th than to the an t i s ep t i c ac t ion of the fluid used. N o r m a l sa l ine proved equal ly as efficient as t h e hexy l recorc i - nol, 1:4000.

C o n t r a r y evidence is offered by F e i r e r and Leona rd (1931) who r e p o r t e d t h a t t h e r e is a d i u r n a l t i de of m i c r o - o r g a n i s m s in the mouth equal to +297 pe r cent even w i thou t the i n t a k e of food. U s i n g t h r e e d i f fe ren t so lu t ions as mou th washes they found t h a t t h i s in- c rease was only +139 p e r cent and +179 p e r cent re - spec t ive ly w i th p roduc t A and B and was - - 7 4 per cent u s ing hexyl resorc ino l , 1:3000.

Experiment IV: The Effect of the Removal of Saliva on the Hydrogen Ion Concentration and the Bacterial Flora of the Gastric Contents.

Rehfuss tubes were inse r t ed in to t he f a s t i n g s tomachs of t h r e e pa t i en t s . By means of den ta l suc- t ion tubes the sa l iva was removed as comple te ly as poss ible f r o m the mou ths of these pa t i en t s . Cu l tu res were m a d e f r o m the mouths , t he sal iva, and the a sp i r - a ted g a s t r i c contents and the p H de t e rmined on the

42 A M E R I C A N J O U R N A L O F D I G E S T I V E D I S E A S E S A N D N U Y R : T I O N

la t te r both before and af te r the deflection of the saliva from the stomach. This was done in the hope tha t the comparison of these results might determine whether or not the swallowed saliva exerted a notice-

c.c. and the est imated number of culturable bacter ia for this twenty-four hour amount varied from 2,592¢ 000 to 79,120,800. The following table gives the sum- mary of the results.

The number of micro-organisms in the saliva secreted during twenty-four hours

Case

P a t i e n t

Amoun t of sa l iva in two hour s

Amoun t of sa l iva in t w e n t y - f o u r hours

N u m b e r of colonies p e r c.c.

N u m b e r eu l t u r ab l e b a c t e r i a p e r twenty-four hours

I

No. 44976

70 c.c.

840 c.c.

6240

5,241,600

II

No. 45351

163 c.c.

1956 c.c.

19,000

35,208.000

I I I

No. 45600

22 c.c.

264 c.c.

29%700

79,120,800

I V

No 45677

40 c.c.

480 c.c.

14,409

2,592,000

V

No, 45103

190 c.e.

[20 c.c.

48,000

41,040,000

*Calcula ted on bas is of two hour observed per iod .

able influence on the hydrogen ion concentration and the bacterial population of the gas t r ic contents. Al- though the tests were made a t ten minute intervals over a two hour period no significant change could be noted in any' case, ei ther in the hydrogen ion concen- t r a t ion of the gas t r ic contents or in the number of colonies in the cultures from the three sources. The results are shown in Graph II I . Bell and MacAdam (1924) reported no change in the acidi ty of the stomach when the saliva was withheld. Kopeloff (1922) concluded f rom his work tha t the continued swallowing of saliva is the chief fac tor which deter- mines the bacterial content of the stomach.

Experiment V: The Number of Organisms in the Saliva During Twenty-Four Hours.

Although the preceding series of cases showed no diminution in the number of colonies cultured from the gastr ic contents when the saliva was removed, it seemed advisable to culture the collected saliva and est imate the approximate number of organisms per cubic centimeter. The total amount removed dur ing the two hour period was well mixed and dilutions were made f rom 1:2 up to 1:64. F rom a pipette which de- livered fifteen drops per cubic centimeter, one drop of each of the dilutions was plated out on plain agar plates. The colonies from the cultur e showing best dilution were counted and the number of organisms per cubic cent imeter computed. The amount of saliva per twenty- four hours calculated on the basis of the two hour observed period varied from 264 c.c. to 2280

Average of gout cases sLo~'lnQ th~ ¢:-!~:c on th~ dlsa~p=~:".~:c of test o~anisrri5 when t}~c mouth 15 pln~¢~ "MIt}l tlOFh]~ ~ali[ie

Llnterval between woshlrlp mouth with test orVanisrr~ and rln$1nD with norlne~ ~ahne J

i leo g

1- T3 ~

All tes ts were made between eight and ten a.m. be- fore the pat ients had eaten breakfas t . This may ac- count for the fact t ha t in general both the amount of saliva and the number of bacter ia per cubic centi- meter were less in this group of experiments than had been reported by other workers.

Crowley and Richet (1935), using different technic found a much higher bacter ial count per cubic centi- meter of saliva. Thei r experiment was directed toward producing growth of the maximum number of organ- isms, while in the present case the in teres t lax in com- par ing the, growth findings from the saliva with those obtained from the gas t r ic contents, therefore s imilar technic was used for both.

Hawk and Bergeim (1931) s tated tha t the amount of saliva secreted by an adult is from 1000 to 1500 c.c. per twenty-four hours. Zaus and Fosdick (1934) reported tha t the basal flow of saliva averaged 0.5 c.c. per minute or 720 c.c. per twenty- four hours. During st imulat ion the amount increased to 2.5 c.c. per minute or 3600 c.c. per twenty-four hours. According to the counts made by Fe i r e r and Leonard (1931) approxi- mately 2,788,000 organisms could be grown on plain agar from 1 c.c. of fluid used as a mouth wash. They noted tha t there is a marked increase in organisms dur ing the day even when no food is taken.

DISCUSSION Cultures from the mouth and stomach show a s t r ik-

ing s imi lar i ty in bacter ial flora. This must mean tha t the oral flora is present in the stomach either as a

pH 7 A_

5

4

5-

2~

) -

o ~/~

Graph H B A v e r a g e o f f o u c c a s e s s h o w l n ~ t h e e f £ e c t o n ~ h e d i s a ea t ' an ce Of te3r oman,srn~ when th~ mouth z5 rmaed wi th Hexy~Psorcinol 1-4000

1 ~/2 2 o ½ l Ik2 z Time m hour3

t75

~ 1 3 0

50 a2

23

H O O D A N D A R N O L D - - - T H E R E L A T I O N S H I P B E T W E E N O R A L A N D G A S T R I C B A C T E R I A L F L O R A 43

G~-<w i , R . - - " \ ~ "c ~i" vh~c~ "~c5 s h o w i n g , c ~ - c t ~,~- kh¢ r c m o " . 3 l o f * s a b r a o n I:H,- pH of

' ~ : i : ~ ]3aeterml Rrowth ~rom s t o m a c h __ , p H ~ . ~ . J n H S / _ ~ , , ~ I , : I , I I I ~ . , V O , I "- - 1 . . . . . 8 0 0 , : . ~ 1 ]3aetenal ~ r o v ~ h t o m aa[ - - - m _ ~ . . . . . . .

: ~ : : :~X:VL- , ' , , , , , , , , . . . . . . I . . . . i - • .... L M , ' B ..... al~rg, cth~r' . . . . . ~-.- . . . . . . . . . . . .

7. t H'~,~.4L~i = L d q ~ t _ , ; ' t ! , ! ~, I ' - - : . . . . . . . : 7 0 0 , 1 . V . t L = i t ~ ~ ~ [ ~ . . . . . . . . . _ _~ A - I . _ L . . . . .

I 111 fll 1.I ~ " " : " " ~ • , ~ 2

O r t : , : n a l tO 2 0 5 0 4 0 ?,C 7C' 8 0 q o l O 0 110 I?C"

t rans ien t or as an established inhabi tant . Although these micro-organisms which are continually washed into the stomach are non-pathogenic for the most part , there is always the possibi l i ty of in t roducing a viru- lent type.

Many references have been made in the l i t e ra ture (Brink, 1933, Cotton, 1927, Deak, 1933 and others) r ega rd ing the presence of the Coli-aerogenes flora in the gas t r ic lumen of pat ients with hypochlorhydria. B. colt was found in only three of twenty-five gas t r ic aspira t ions made from this type of person during the present investigation. Even in these cases the sa l ivary flora was present in g rea te r concentration than were the colt types.

By means of a test organism it was found tha t bacter ia may remain active in the oral cavity for three and one-half hours a f te r contact with the mucosa and s t ructures within the cavity. This gives a long period during which organisms are continuously passed into the stomach. Thus if pathogenic bacter ia have been the contaminants there will be not only the original infect ing mater ia l for disinfection by the gastr ic juice but also this smaller but continuous inoculation over a long period during which time there may be fluctuations in the gas t r ic acidity. Simul- taneous cultures made from the mouth and stomach

contents showed tha t the average disappearance ra te of test organisms from the mouth was two and one- half hours and from the stomach one and one-quarter hours.

In an effort to shorten this continuous period of contaminat ion of the stomach the mouths of four pat ients were r insed at va ry ing intervals with e i ther normal saline or hexylresorcinol. This procedure did not yield very conclusive results, but i t did indicate that any diminution in the number of bacter ia was brought about as readi ly by normal saline as by the disinfectant . Evidently the diminution in the numbers of bacter ia is due to the mechanical washing away of organisms, ra ther than "to the dis infect ing powder of the fluid itself.

The first two of the preceding studies gave the im- pression tha t the swallowed saliva might exert a marked influence on the bacterial flora of the stomach. In checking this by culture when the saliva was re- moved as completely as possible, i t appeared tha t there was no significant change in the number of colonies cultured, or in the type of organisms grown. As the pat ients became quite uncomfortable, hawing the gastr ic tube as well as the saliva ejector in place, the test was continued fo r only two hours. Possibly the seeding f rom the mouth was too well established to be affected by this change over such a relat ively short period of time.

CONCLUSIONS

1. Marked s imi la r i ty in bacter ial flora was shown by cultures made f rom the oral cavit ies and gas t r ic contents of twenty-five patients.

2. The average rate at which test organisms dis- appeared f rom the mouth was two hours and th i r ty - six minutes a f t e r seeding. The average disappearance rate from the stomach was one hour and eighteen minutes.

3. A mouth wash of normal saline was as effective as one of hexylresorcinol, 1:4000 in reducing the exogenous bacter ial flora of the mouth and the stomach. The actual reduction in numbers was prob- ably due to the mechanical action of r ins ing the mouth.

4. Although saliva contains many micro-organ- isms, the bacterial flora of the stomach was not notice- ably influenced by diver t ing the flow of saliva f rom the stomach for a period of two hours.

R E F E R E N C E S 1. Arnold , L.. and Brody, L . : The Gas t ro -Duodena l Bac te r ic ida l

Mechan i sm. Am. Jour, of Hyg., 6:672-683, 1926. 2. Arnold , L., and S tua r t , C . W . : Recen t D e v e l o p m e n t in t he S tudy

of Ora l Bac te r i a l F lora . Am. Jour. Dig. Dis. and Nutrit. , 2:275- 280, 1935.

3. Bar t le , H. J . , and H a r k i n s , M. J . : T h e Gas t r i c S e c r e t i o n : I t s Bac te r ic ida l Va lue to Man. Am. Jour. Med. Sci., 169:373-388, 1925.

4. Bell, J . H. , and Mac A d a m , W . : T h e V a r i a t i o n s in Gas t r i c Secre- t ion of t he N o r m a l Ind iv idua l . Am. Jour. Med. Sci., 167:520- 528, 1924.

5. Boldyreff , W. : T h e Se l f -Regu la t ion of the Ac id i ty of t h e Gas t r i c Con ten t s and the Real Ac id i ty of the Gas t r i c Ju ice . Quart. Jour. Exp. Physiol., 8:1-12, 1914.

6. Br inck , J . : Das V o r k o m m e n yon B a k t e r i e n der Col i -Aerogenes- Gruppe , i m M a g e n i n h a l t bet e rha l t ene r S a u r e p r o d u k t i o n . Ztschr. ]. klin. Med., 123:380-389, 1933.

7. Clark , W. M . : The D e t e r m i n a t i o n of H y d r o g e n Ions . The Wil - l i ams and Wi lk in s Co., Ba l t imore , 1928.

8. Colton. H. A . : The Rela t ion of Focal I n f e c t i o n to Men ta l Dis- eases. Ne.w York Med. Jour., 111:672-677, 1920.

9. Crowley, M.. and Rieher t , U. G. : A Method fo r E s t i m a t i n g the Bac te r ia l Conten t of the Mouth by Di rec t Count . Jour. Bact., 30:395-400, 1935.

10, Deak , E . : Colt und Pa raco l i be funde i m Magen und S tuhl bet d a r m g e s u n d e n und d a r m k r a n k e n Sa t ing l ingen . Ztschr. f. Kin- derk., 55:196-211, 1933.

11. Fe l r e r , W. A., and Leonard , V . : Ora l H y g i e n e (1) T h e D i u r n a l T ide of t he B a c t e r i a of the Mouth and I t s U s e in D e t e r m i n i n g the Ac tua l Va lue of P r e p a r a t i o n s Commonly E m p l o y e d as Ora l An t i sep t i c s . The Dental Cosznos, 73:338-342, 1931.

12. H a w k , P. B., and B e r g e i m , O. : P r a c t i c a l Phys io log ica l C h e m i s t r y . P. B lak i s ton ' s Son and Co., Inc. , 1931.

13. H o l m a n , W. L. : The Class i f ica t ion of St reptococci . Jour. Med. Re8.. 34:377-443, 1916.

14. H u r s t , A r t h u r F . : Clinical I m p o r t a n c e of Ach lo rhyd r i a . Brit. Med. Jour.. 11:665. 1934.

15. H u r s t , A r t h u r F. : Cons t i tu t iona l F a c t o r in Diseases . Brit. Med. Jour., 1:823, 1927.

16. H u r s t , A r t h u r F . : The U n i t y of Gas t r i c Disorders . Lancet, 11:89. 1933.

17. Kope]o.ff. N. : Bac te r io log ic Studies of Gas t r i c F r a c t i o n s Obta ined by Reh fus s Method. Jour. Inf. Dis., 30:613-621, 1922.

18. Zaus . E. A., and Fosdick , L. S . : Ef fec t s of Sa l iva U p o n Gas t r i c Diges t ion . Jmtr. Dent. Rcs., 14:3-13, 1934.