A Simple Colorimetric Method for the Diagnosis of Caries Activity

A SIMPLE COLORIMETRIC METHOD FOR THE DIAGNOSIS OF CARIES ACTIVITY B y M a r s h a l l L . S n y d e r , P h.D ., A n n A rb or, M ich .

H E use o f a sim ple colorim etric m ethod 1 fo r the diagnosis o f caries a ctiv ity is described fo r ordinary clin ical use b y the p ra cticin g dentist. Based on the rate o f color ch an ge in a selective aga r m edium (p H 4.7-5.0), it requires on ly a fe w pipets and an in cu b ator to give results in forty-eight hours w h ich com pare fa v o ra b ly w ith those obtained b y the q u an titative es­ tim ation o f lacto bacilli in the saliva.2’ 3 Since the latter technic requires at least fo u r days and is alm ost entirely lim ited to the bacteriologic laboratory, the ad vantages o f a colorim etric test in speed, econom y and accu racy are obvious. E vid en ce th at the colorim etric m ethod is satisfactory fo r the diagnosis o f caries a ctiv ity was secured b y cu ltu rin g, peri­ od ically over thirteen m onths, w ith both procedures, the saliva o f sixty-three yo u n g children and com parin g the re­ sults w ith clin ical exam inations a t the be­ gin n in g, m iddle and end o f this period. T h e present paper em bodies the d a ta o f study.

T

m a t e r ia l a n d m e t h o d s

M o st o f the sixty-three children tested w ere in the kindergarten and first, second, and th ird grades o f the U n iv ersity E le ­ m en tary School and o f an average o f 5-8 years. T h e y w ere selected fo r tw o rea so n s: 1. T h e incidence o f caries is less, w ith youn ger children. 2. M a n y rem ain six years in the school and are given rou­ tine dental and x -ra y exam ination. T h ese conditions perm it a longitudinal From the Hygienic Laboratory, University of M ichigan.

Jour. A.D.A., Vol. 28, January 1941

study w ith a sm all num ber o f subjects abou t w h o m detailed inform ation is available. Specim ens o f saliva w ere obtained b y h a vin g the ch ildren chew paraffin tablets fo r three m inutes, du rin g w hich tim e the saliva w as collected in sterile test tubes or bottles. C ollections w ere m ade from 8 :1 5 to 9 :30 a.m . a t approxim ately m onthly intervals, interru p ted on ce b y illness and once b y the sum m er vacation. A m ax­ im um o f nine specim ens w ere secured fro m a n y one child , the average being betw een six an d seven. A fe w o f the recent inclusions h ad on ly tw o or three tests recorded. T h e specim ens o f saliva w ere taken to the lab oratory, w h ere they w ere shaken vigorously b efore variable am ounts (0.10.2 cc.) w ere ad ded d irectly to tubes o f m elted m edium a t 450 C . T h e respective tubes w ere then solidified and incubated fo r fo u r days, w ith d a ily observations. A t first, b oth lactose and dextrose infusion a g a r (p H 5.0) contain ing brom cresol green as the ind icator w ere used. T h e re­ sults indicated th a t dextrose was m ore reliable th an lactose and th at 0.2 cc. o f saliva was th e optim um am ount fo r use in inoculation. L ater, a specially p re­ pared dextrose-agar m edium * was found better than the others tested and is now rou tinely em ployed w ith 0.2 cc. o f saliva as the standard inoculum . T h e color re­ actions induced b y bacterial action range from no alteration o f the bluish green *T h is medium can be obtained from the D ifco Laboratories, Detroit, M ich., in dehy­ drated form or already prepared in screw-top tubes under the name bromcresol-green dex­ trose agar.

44

S n y d e r — C o l o r im e t r ic M

D ia g n o s is

ethod fo r

color through stages to a distinct yellow . A lterations w ere arb itrarily estim ated and only those recorded as 3 or 4, in w hich green was no longer the dom inant color and w h ich w ere rea d ily distinguished from u n in oculated controls, w ere consid­ ered significant. F or the purpose o f estim ating the lactobacillus content o f the saliva, 0.1-0.2 cc. o f the specimens o f saliva w ere placed on tom ato ju ice-a g ar plates (p H 4.8-5.0) and sm eared b y m eans of sterile bent glass rods. A larger q u an tity (0.6-0.8 cc.) was inoculated into acid-glucose broth in a q u alitative test fo r the presence or ab ­ sence in forty-eigh t hours o f gram -posi­ tive rods considered to be lactobacilli. A fte r ninety-six hours, the plates w ere

of

45

C a r ie s

Positive color reaction in 24-48 hours : definite activity. Positive reaction in 12-24 hours : m arked activity. In a sim ilar w a y, caries activity was estim ated b y absence o f or a rise and fall in num bers of lactobacilli in the saliva. B oth m ethods are lim ited by the fa ct that a single bacteriologic exam ination is not sufficient to in d icate w hether caries is active or not. A positive or negative diagnosis m ust depend on repeated tests a t intervals to rule ou t chance findings. O n this basis, sporadically positive or n egative results are elim inated. RESU LTS

T h e sixty-three children w ere divided

T a b l e 1 .— C o m p a r i s o n o f L a b o r a t o r y a n d C l i n i c a l D i a g n o s e s o f C a r i e s A c t i v i t y i n S i x t y T h r e e Y o u n g C h il d r e n Laboratory Diagnosis o f Caries Activity Clinical Diagnosis of Caries Activity Negative Questionable Active

Negative No. Children Color

23 17 23

22

13

0

Active

Questionable

Lacto­ bacilli 21 11 2

exam ined fo r characteristic colonies o f lactobacilli and oth er acid u ric types, the numbers o f w h ich w ere estim ated b y the aid o f a b in ocular dissecting m icroscope. T hus, a com parison w as m ade between the colorim etric test and quan titative lactobacillus counts as m ethods for the d iag­ nosis o f caries activity. F ro m previous experience, it was learned th a t positive color reactions in forty-eigh t hours are significant, and, in the present study, th e diagnosis o f caries activity was m ade in respect to the colori­ m etric test on the fo llo w in g b asis: N o color chan ge in 96 h o u r s : no ac­ tivity. Positive color reaction in 48-96 h o u r s : slight activity.

Color

Lacto­ bacilli

0 2

4

3

1

2

Color 1 2 20

Lacto­ bacilli 1 2

19

on the basis o f sum m ation o f clinical a c ­ tiv ity in the follow in g m a n n e r: a n ega ­ tive group o f tw enty-three children w ho showed neither cavities nor fillings, an active group o f tw enty-three children in w hom there was a definite increase in the num bers o f cavities and fillings and a grou p o f seventeen children whose clin ­ ical status was questionable. T h e clin ically questionable individuals included those subjects in w hom dou b tfu l areas w ere fou n d or in w h om there was little or no ch an ge in num bers o f previously re­ corded cavities or fillings. T h e diagnosis o f caries a ctiv ity b y colorim etric tests and q u an titative lactobacillus counts is com ­ pared w ith the clin ical conditions o f these three groups in T a b le 1.

46

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T a b le 1 shows th at o f the twenty-three cases fou n d to be clin ica lly negative, only one was estim ated to be caries active b y both lab oratory m ethods, w hile frequent cultures o f lactobacilli, althou gh in low num bers, m ad e the diagnosis questionable in another case b y the qu an titative pro­ cedure. In the clin ica lly questionable group, b oth cultural technics indicated the same tw o cases as caries-active, b u t only two questionable estim ates w ere m ade by the colorim etric tests instead o f fou r b y lactobacillus counts. O n the w hole, the colorim etric m ethod separated the chil­ dren into distinct groups m ore easily than lactobacillus counts, since it was easier to interpret eith er consistent or interm ittent positive or n egative co lo r reactions than va ryin g num bers o f lactobacilli.

m etric m ethod fo r diagnosing caries a c ­ tiv ity involves not on ly the practical con­ siderations o f sim plicity, accu racy and econom y, bu t also the idea that acidogen ic or acid uric organism s other than lactobacilli h a ve some share in the proc­ ess. T h u s, instead o f n arrow in g the eti­ olog y to one species or type, there w ould be th e broader con cep t o f acid prod u c­ tion irrespective o f num bers or sp ecies; but, in this study as in an earlier one,5 the on ly correlation betw een the presence and num bers o f acid u ric or acidogenic b acteria in the saliva and caries activity w as th at w h ich w as fou n d w ith the la c ­ tobacilli and yeasts. N eith er streptococci nor staph ylococci w ere isolated a n y more frequ ently in the caries-active th an the caries-negative cases.

T a b l e 2 .— C o m p a r i s o n o f I n c i d e n c e o f L a c t o b a c i l l i a n d Y e a s t i n S p e c i m e n s o f S a l i v a w i t h A c i d P r o d u c t i o n i n B r o m c r e s o l G r e e n - D e x t r o s e - A g a r (pH 4 .7 5 ) I n o c u l a t e d w i t h 0 .2 Cc. Sa l iv a

Summation of Clinical ActivityNegative Questionable Active

No. Chil­ dren

23 17 23

No. Speci­ mens

89 78 106

No. Lacto­ bacilli

No. Yeast Organ­ isms

9 16 96

3 4 12

A lth o u gh a past history o f caries ac­ tivity p laced m a n y ch ild ren in the clin­ ically questionable grou p , there w ere tw enty-three subjects in w hom definite increases in num bers o f cavities and fill­ ings could be determ ined. In this group, the color test indicated tw en ty cases to be caries-active and three questionable, w hile lactobacillus counts indicated nine­ teen as active, tw o questionable and two caries-negative. T h ese results w ou ld sug­ gest th at the colorim etric m ethod was m ore accurate than lactobacillus counts in diagnosing caries a ctivity, b u t m ore recent findings (unpublished) establish the tw o m ethods as o f about equal efficacy. H ow ever, acceptance o f the colori,-

Positive Color Reactions

24

48

72

96

0 2 34

3 8 88

10 14 10 1

14 16 102

Because variab le am ounts o f saliva w ere inocu lated into differen t carbo h y­ drate m edia in the early part o f the in­ vestigation, the incidence o f lactobacilli and yeasts in the specimens o f saliva is com pared w ith respective color reactions in T a b le 2 fo r on ly that portion where a single m edium , brom cresol green-dextrose-agar was used w ith a constant in ­ oculum o f 0.2 cc. o f saliva. D espite the apparen t selection o f data, there are in­ cluded a m a jo rity o f the specimens of saliva and the tim e intervals over w hich final diagnoses w ere m ade. T a b le 2 has several interesting features. T h e 10 per cen t incidence o f lactobacilli in the specim ens o f saliva from the n ega­ tive group and the 10 per cent absence

S n y d e r — C o l o r im e t r ic M

ethod fo r

from th e n egative group, instead o f being m arked exceptions to etiologic relation­ ship, o n ly em phasize m ore clearly the trend o f clin ical a c tiv ity ; th at is, in any group o f cases, some are becom ing either caries-active or caries-negative, and the clin ical chan ge is reflected in o r pre­ d ictab le b y the cultu ral findings. Y easts w ere seldom isolated from the clin ica lly negative group, bu t their in­ creased presence in the saliva o f caries-

D ia g n o sis

of

47

C a r ie s

action in v iv o , there is, in respect to acid p rod u ction in the test tube, a critical tim e interval betw een forty-eigh t and seventytw o hours before w h ich positive color re­ actions are of m uch significance, bu t a fter w hich th ey ad d little to the d ia g ­ nosis o f caries activity. T o em phasize fu rth er the predom inant role o f lacto bacilli in dental caries, it is interesting to com pare in T a b le 3 their num bers in the same specimens o f saliva

T a b l e 3 . — C o m p a r i s o n o f F r e q u e n c y a n d T i m e o f C o l o r C h a n g e i n B.r o m c r e s o l G r e e n - D e x t r o s e - A g a r w i t h L a c t o b a c i l l u s C o u n t s i n R e s p e c t i v e S p e c i m e n s o f S a l iv a

No. Children

23

Summation o f Caries Activity Negative

Lactobacillus Counts

No. Speci­ mens

0 0-100 100-1,000

80 4 4

1 ,000-10,000

Total

17

Questionable

0 0-100 100-1,000 1 ,000-10,000 10,000-100,000

Total

23

Active

0 0-100 100-1,000 1 ,000-10,000 10,000-100,000 100,000-1,000,000

Total

active cases is such th at they can not be statistically disregarded. N u m erically, it is a p p aren t th a t their rôle is a secondary one, b u t their influence on acid prod u c­ tion is im portant in view o f the w ork of Fosdick and H ansen,6 w ho have shown th at the yield of lactic acid in cultures o f lacto bacilli is grea tly enhanced b y the ad dition o f yeast. T h e y explain this synergy on the basis that lactobacilli are rich in reductase, bu t deficient in phos­ phatase, w hich is essential fo r the b e­ gin n in g degradation o f carbohydrate and is furnished b y the yeast.7 W h atever the

1

89 62 5

2

5 4 78

10 2 11

36 36

11

106

Color Change (Hours)

24

48'

4 4

I

96 5 4 4

3

1 10

14

0 0 1

4

0 0 0 0 0

0 0

0 0 0

3

0

• 0

5

2 2

2 8

0 0 0

72

0 2

4

5 3 14 7

1 6

2 10

1 1

5

2

5 3 16

8 2 10

7 16

33 34

36 35

36 35

11

11 88

11 101

11 102

34

as listed in T a b le 2 w ith the respective color reactions. I t is very obvious in T a b le 3 th at not on ly do lacto bacilli appear m ore fre ­ q u en tly in the active than in the n egative and questionable groups, b u t also the average n um ber increases. C o lo r reactions also appeared m u ch m ore qu ickly in the active than in .th e negative group. T h e tim e differentials served to pick ou t the m ost active cases. As alread y noted, these changes can not be attributed solely to num bers o f lactobacilli, b u t must also be recognized as due to the presence or

48

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A m e r ic a n D e n t a l A s s o c ia t io n

absence o f acid form ation regardless o f numbers o f lactobacillus or their m orphologic types or other aciduric organisms. COM M ENT

O f tw o laboratory m ethods used fo r the diagnosis o f caries a ctiv ity as m eas­ ured b y the clin ical history o f the indi­ vid u al cases, the first, or quan titative m ethod, was th at w h ich estim ated n u m ­ bers of lactobacilli in the saliva as an in ­ dex o f caries activity. W ith this pro­ cedure, it is possible to dem onstrate a consistent relationship betw een th e pres­ ence or absence o f lactobacilli, as e x ­ pressed b y high, m edium , low and n egative counts and th e state o f caries activity. H ow ever, there are m a n y ob jec­ tions to the m ethod, both bacteriologic and interpretative. I t is alm ost needless to say that a n y attem pt to evalu ate a ccu ­ rate bacterial counts from a m aterial so variab le in chem ical and b io logic m akeup as saliva, fo r w hich there are as y et no accu rately defined norm al lim its, and cultured on a m edium d ifficult to stand­ ardize, is open to all the errors inherent in pla tin g m ethods. Also, p la te counts, w hile distinguishing the m orphologic types o f lactobacilli, d o not take into con­ sideration either the acid production o f these respective types8 or the influence by other organism s on this acid prod u c­ tion.9- 10-11 T h e colorim etric test was developed from an attem pt to sim plify the q u an tita ­ tive technic fo r ord inary clin ical use. It reflects the acid production o f all the organism s o f the saliva inocu lated in the selective carbohydrate m edium (p H 4.75.0 ). Since the M illerian concep t12 o f dental caries is based on the action o f acid d erived fro m b acterial ferm entation o f carbohydrate, it was suggested th at b y this color test reflecting acid form ation as a w hole, irrespective o f num bers or species o f bacteria, it should be possible to detect caries activity easily, q u ick ly and accurately. T h is possibility w as d em ­

onstrated after a thirteen months’ study of sixty-three young children. This simple method for the diagnosis of caries activity requires a relatively small amount of equipment. A few pipets and an incubator furnish the means for any dentist to determine easily, accurately and economically whether caries is ac­ tive or not and, to some extent, the de­ gree of activity. Its application would seem to be especially useful in the fields of orthodontia, in preventive dentistry for children and in public health surveys. At present, the test is apparently too in­ sensitive for use in following effects of diet therapy on control of caries activity, but this possibility is being further in­ vestigated. sum m ary

A simple colorimetric test based on acid production in a selective carbohydrate medium (pH 5.0) can be used as a means of diagnosing caries activity. In a group of sixty-three young children, it was pos­ sible to select very accurately the cariesactive and caries-negative cases as estab­ lished by clinical examination. b ib l io g r a p h y

1. S n y d e r , M . L .: August, 1940.

J.

D.

Res.,

19:349,

2. R o d r i g u e z , F. E .: Quantitative Inci­ dence of Lactobacillus Acidophilus in Oral C avity as Presumptive Index of Susceptibility to Dental Caries. J.A .D .A ., 18 :2 118 , Septem­ ber 1931. 3. H a d l e y , F. P.: J. D . Res., 13:415, O c ­ tober 1933. 4. G o r d o n , S. M . : Dental Science and Dental Art. (J a y , P h i l i p : Bacteriology and Immunology of Dental Caries.) Philadelphia: Lea and Febiger, 1938. 5. S n y d e r , M . L .: cember 1939.

] . D . Res., 18:497, D e­

6 . F o s d ic k , L. S., and H a n s e n , H . L . : Theoretical Considerations of Carbohydrate Degradation in Relation to Dental . Caries. J.A .D .A ., 23:401, M arch 1936. 7. F o s d ic k , L. S .; H a n s e n , H . W e s s in g e r , G . D .: Reductase A ctivity of V a ­ rious M outh Organisms. J.A .D .A ., 24:1445, September 1937.

L.,

and

49

S e l d in — N i t r o u s O x id e - O x y g e n A n e s t h e s i a 8. T r a c y , R . L . : /. Bacterid., 3 3 : 4 6 7 , April 1939. 9. A n d e r s o n , T . G ., and R e t t g e r , L . F . : /. D . Res., 16:489, December J937. 10. H a m m o n d , C a r o l y n , and T u n n i c l i f f , R u t h : / . D. Res., :1, February 1940.

19

11. B e l d i n g , P.

H., and B e l d in g , L. J. :

D. Items Int., 62:303, April 1940. 12. M der

il l e r ,

Mundhole.

W. D .:

Die Mikroorganisms

Leipsic:

George

Thieme,

1889.

THE RÔLE OF NITROUS OXIDE-OXYGEN ANES­ THESIA IN THE OFFICE OF THE GENERAL PRACTITIONER By

H ar r y

M . S e l d i n * D .D .S ., N ew Y o rk , N . Y .

E N E R A L as w ell as local anesthesia has its definite place in m outh sur­ gery. T h e trained anesthetist can induce and m aintain a smooth, even and pleasant narcosis w ith nitrous oxideoxygen fo r as long a period as m a y be required fo r the com pletion o f a n y com ­ p licated oral surgical procedure. G en eral anesthesia is especially desirable : 1. I n the presence o f acute infection. 2. W h ere the site o f the needle p u n c­ ture is infected : i.e., pericoronal infection of m and ibular third m olar. 3. W h en pericem ental inflam m ation prevents the action o f local anesthesia. 4. In operations on very yo u n g ch il­ dren. 5. F o r apprehensive and uncooperative adults. 6. F o r m ultiple extractions. I t m ust be borne in m ind, how ever, th a t the in d ivid ual subm itting him self fo r surgical procedures under general anesthesia depends upon the skill and ju d gm en t o f the anesthetist to gu id e him

G

•Consulting pital.

Oral

Surgeon,

Harlem

Hos­

Read before the Section on Oral Surgery, Exodontia and Anesthesia at the Eighty-First Annual Session of the American Dental Asso­ ciation, Milwaukee, Wis.

Jour. A.D.A., Vol. 28, January 1941

safely b ack to consciousness from the n ar­ cosis. It is in the hands o f the anesthetist th at the balance betw een life and death is often placed . I t becom es obvious, then, th at the gas m achin e should not b e con ­ sidered as a toy, nor a hum an life as a playth in g, and th at not on ly should the dentist be th o rou gh ly trained in the art o f anesthesia, b u t he should possess in his office the p rop er physical set-up and personnel b efore he attem pts surgical procedures u n der a general anesthetic. N o one, n o m atter h o w w ell trained, can act as anesthetist, surgeon and assistant at the sam e tim e, and still do justice to his patien t. I t is possible to m ake the average routine extractions under gas-oxygen n ar­ cosis w ith just one assistant w ho is an experienced anesthetist. H ow ever, it takes a team o f three— anesthetist, o p ­ erator and assistant— to handle m ajo r surgery, and the com plications that m ight fo llo w in rou tin e exodontia. Self-confidence is a v irtu e ; overconfi­ dence, how ever, ceases to be a virtu e and m ight prove even to be a serious d etri­ m ent. T h e dentist should select fo r gen ­ eral anesthesia on ly those patients w ho h e is certain can b e successfully m anaged b y him self an d his staff. T h e patien t w ill not think too w ell o f the dentist w h o a t­