Research

A METHOD OF COUNTING ORAL BACTERIA

JOHN B. Tl~~% AND AXS ILARTMAN, JERSEY CITY, N. ,J.

T HE counting of oral bacteria, particularly lactobacillus, in saliva has at- tracted a great deal of attention as a possible method of determining the

effectiveness of various methods of caries reduction or prevention. Correlation between the numbers of lactobacilli and caries has been reported by Becks’ and his co-workers. A recent paper by Boyd, Cheyne, and Wessels” contradicts this premise of correlation. Becks3 reported that, he used the Hadley4 method of counting Lactobacillus acidophilus, while Boyd does not give the method used.

While the problem of counting the number of bacteria in a cubic centimeter of saliva is not insurmountable, when this number is used as a measure of caries activity, certain other factors are involved which greatly influence the correla- tion of these observat,ions.

Of these factors, the one responsible for the greatest error is that in col- lecting the specimen. The number of bacteria in saliva collected by chewing a paraffin plug is not necessarily representative of the true oral population. The uncounted numbers of bacteria in cavities and crevices of the teeth and t,he immeasurable faetor of salivary flow contribute errata of such magnitude that even accurate counts may be meaningless.

Another unpredictable factor contributing to the difficulty of relating the numbers of L. acidophilus to the caries rate is the variation in numbers of bac- teria in the mouth from week to week” and even from hour to hour.

There are also difficulties attendant to methods of counting. We have found no medium which is completely specific for only one organism. With the Hadley modification of Kulp’s tomato juice agar, the recognition of lactobacillus colonies is not absolute, as other organisms show similar growth forms. We have adopted a medium which, while open to the same criticism, plainly indicates all acido- genie colonies, which are probably as significant as L. acidophilus. In over two hundred randomly chosen colonies which were stained and examined micro- scopically, not one showed organisms other than lactobacilli. However, we have since isolated several organisms on this medium which were not L. acidophilus. We estimate an error of 5 per cent to 10 per cent in counting other organisms as lactobacilli.

Many workers have reported methods of counting bacteria, which may be divided into two major classifications as follows :

1. The Direct Method-not distinguishing between living and dead or- ganisms.

2. The Culture Method-only counting organisms able to grow on the medium used.

FDXTI the Research Laboratory of Amm-i-dent. Inc.

1200

METHOD OF COUNTING ORAL BACTERIA 1201

The accuracy of all methods is subject to the cumulative errors of dilution, distribution of the organisms in the media, and the ability of the organism to reproduce when transferred. The difficulty in starting with a known number of bacteria per cubic centimeter is so great that evaluation of any method must be largely statistical whereby the errors are appraised, generalized, and rein- tegrated into the count.

As a first step in a project to determine whether correlation existed between L. acidophilus (or acidogens) and cavity formation rate, we examined the methods of counting used by various workers. Since Keselev 7 and Beck9 re- ported the use of the Hadley method, this technique was chosen as a starting point.

Upon detailed examination of the Hadley method it is apparent that the dilutions are inaccurate. The saliva samples were made up to 10 CC. from pa- tients giving 2 to 8 CC. This gives 20 per cent to 80 per cent of the bacterial count of the original saliva. As the saliva is not measured, it is impossible to determine within any reasonable range its dilution at this point. The only cal- culation given is for the dilutions of 15 and 1:20, which excludes completely the first dilution which may extend from 1:5 to 4 :5, introducing a gross error.

TABLE I. COMPARISON OF EFFECTIVE TO THEORETICAL DILUTIONS WITH THE HADLEY METHOD

SIZE OF SALNA

SAMPLE C.C.

2 5 8

HADLEY DILUTIONS TRUE COUNT HADLEY COUNT 1:50 1:200 OF BACTERIA/ OF BACTERIA/ PER CENT EFFECTNE DILUTIONS C.C. C.C. ERROR

1:250 l:l,OOO 1,000 5,000 500 1:lOO 1:500 1,000 2,000 100 1:62.5 I:250 1,000 1,250 25

Also, the technique of shaking thirty seconds by hand does not permit re- producible samples to be taken on the same saliva, as the bacteria are clumped and distribution is poor.5

A method using indicator media was devised by Snyder,* but unfortunately there are many acidogens in saliva and their rate of acid production is im- mensely variable. Because of this, the Snyder technique has not been found useful as it more accurately portrays the speed of acid production by the or- ganism rather than the number of organisms.

Media

An investigation of various media was made to find a starting point for the development of a method. Purple trypsin digest agar was most promising. However, the numbers of organisms which grew upon it and the luxurious growth of the acidogens, which changed the color of the indicator before the colonies fully appeared, made its use too difficult for routine techniques.

The media in Table II were tried. From these, the Hadley medium and the Snyder bromcresol green medium

were chosen as being best suited to our purposes. Further investigation showed the Snyder medium to be superior for the recognition of colonies and for speed in counting.

1202 JOHN B. LEWIS AND ANN HABTMAN

Due to our difficulties in obtaining reproducible counts from the same sa- liva, we were forced to the conclusion that the distribution of bacteria in the saliva was faulty. To remedy this we obtained a Boerner shaker and, by shaking the collection tubes with glass beads for various times, we found that two min- utes’ shaking produced maximum counts. It was impossible, however, to break all the chains of lactob.acillns into single organisms; therefore all counts showed from one-half to one-sixth the number of single bacteria.

TABLE II

MEDIUM

Peptonized milk

Tomato juice agar

FORMUI,A

Difco

Kulp Difco

P* L. a.

6.5 Best broth

6.1 Very good

OTHER ORGANISMS

Poor

Poor

Trypsin digest agar Cheplin Difco

6.1 Excellent Excellent

Purple lactose agar Difco 6.8 Fair Poor

Lactose broth Difco 6.7 Poor Poor

Purple trypsin digest Difco + 0.0025% 6.8 Excellent Excellent agar Bromcresol

purple

Thioglycollate fluid Brewer 7.2 Poor Excellent Difco

Purple beef glucose agar Lewis 7.6 Poor Not tested

Tomato juice agar (Hadley)

Difeo + 2% agar

5.0 Very good Very poor

Beef dextrose broth Lewis 7.2 Fair Poor

Bromcresol green dex- trose agar

Snyder 4.8 Best agar Very poor

Apparatus

Boerner shaker and special bar. Glass beads, 0.33 mm. in diameter. Shell vials, O.D., 20 mm., I.D., 18 mm., H., 69 mm., with No. 2 rubber

stoppers. Test tubes, 16 by 150 mm. Test tubes, 11 by 100 mm., with shell vials, O.D., 14.5 mm., I.D., 11.7 mm.,

H., 25 mm. Petri dishes, 15 by 100 mm. Pipettes, 1 cc., graduated in 0.01 cc. Pipettes, 1 cc., graduated in 0.1 cc. Thermoregulated water bath with booster, 45’ C. Quebec colony counter, preferably “dark field.”

Materials

Paraffin plugs-made by heating paraffin until sterile (about one hour at 50° C.) and pouring into suppository molds. Plugs are bullet-shaped, maxi- mum diameter, 11 mm., height, 31 mm.

METHOD OF COUNTING ORAL BACTERIA 1203

Medium used is the so-called “Snyder Test Agar ” made by Difco Labora- tories, Detroit, Mich., of the following formula :

Bacto Tryptase 20 Gm. Bacto Dextrose 20 Gm. Sodium chloride 5 Cm. Agar 20 Cm. Bromcresol green,

indicator grade 0.02 Gm. Water, q.s. l,ooo.o C.C.

Diluent : Sterile 0.85 per cent sodium chloride solution.

Method

1. Collection of Specimens-A sterile rubber stoppered shell vial with three glass beads is used to collect the saliva. A paraffin plug is chewed for three minutes alternating from side to side. The saliva is expectorated from time to time as it is excreted.

2. The sample is shaken for two minutes on a Boerner shaker to diffuse the organisms thoroughly, and then 1 C.C. is transferred to 9 cc. of sterile saline solution in a 16 by 150 mm. test tube with three glass beads and shaken for two minutes. This gives a final dilution of 1 :lOO as 0.1 C.C. is used per plate. If necessary, as in some cases with very high counts, 1 C.C. of this dilution is added to 9 C.C. of sterile saline and shaken two minutes, as above. This likewise pro- duces a 1 :l,OOO dilution.

(Note : The violence of shaking is adjusted by raising or lowering the suspending bar and/or the counterbalance flask. The agitation used is just less than that required to throw the rubber stoppers out of the tubes.)

3. Four cc. of liquefied bromcresol green agar is placed in an 11 by 100 mm. test tube capped with a shell vial and maintained at a temperature of +45O C. in the water bath. One-tenth cc. of the dilution is then added and the tube shaken gently by hand for one minute (foaming results if the Boerner shaker is used). This is then poured on Petri dishes previously poured with 15 C.C. of the medium. The tubes are drained for a slow count of five. The dishes are then rotated to cover the surface evenly and allowed to cool on a level surface. They are then incubated at 37O C. for seventy-two hours.

4. Counts are made using the Quebec dark-field colony counter. If less than 100 colonies occur, the whole plate is counted. If more than 100 colonies occur, two rows of 8 squares each at right angles to one another are counted and the result multiplied by 3.975 and the dilution, to give the number of colonies per cubic centimeter of saliva. Colonies which become jade green from the medium are counted. Colonies which do not absorb the dye are arbitrarily dis- carded even though they may change the color of the surrounding medium by acid production.

Experimental Verification of Accuracy

Ten separate dilutions were made of four salivas representing four ranges of numbers of lactobacilli. Two experienced workers counted each plate twice,

1204 JOHN B. LEWIS AND ANN HARTMAN

TABLE: 111

---z WORKER A -~__---

SALIVA NO. -i T SALIVA X0. i? SALIVA NO. 3 SALIVA NO. 1”

PLATE 1 COUNT COUNT

NO. --

NO. 1

290 180 210 290 200 310 250 220 180

10 220

----235 Avg.

NO. 2

280 210 200 280 230 340 250 220 180 230

242

- ___-~ COUNT

NO. 2

209,000 209,000 163,000 187,000 158,000 202,000 180,000 211,000 226,000 170,000

191,500 ___-

~- COUNT

NO. 2 ____ 83,900 81,900 98,600 83,500 70,800 98,600 87,400 86,700 83,500 80,300

___- 85,520

COUNT

NO. 1

227,000 218,000 165,000 179,000 159,000 200,000 171,000 218,000 227,000 171,000

193,500

COUNT

NO. 1

16,300 21,900 21,500 21,500 19,100 16,300 21,900 21,900 17,900 19,500 19,780

COUNT COUNT

NO. 2 NO. 1

16,700 80,300 21,900 80,300 22,700 100,600 22,300 86,300 17,900 81,900 S6,700 98,600 21,900 83,900 21,900 80,800 19,100 83,900 21,500 82,700

20.260 85,930

WORKER “I

-- I

0.3 / SALIVA

COUNT 1 COUNT

SALIVA NO. 1”

PLATE

NO.

1

2 3 4 5 6 7 8 9

IO

---TIP- Avg. 1 250

COUNT COUNT

NO. 1 NO. ?

-z%- 290 200 210 240 220 270 270 250 230 370 340 270 260 260 240 190 200 220 240

0. 4

COUNT

NO. 2

227000 24O:OOO 180,000 188,000 176,000 227,000 173,000 220,000 217,000 165,000

201,300

SALIVA

COUNT

NO. 1

80,300 81,500

101,800 80,700 71,200 93,800 89,000 94,600 81,100 81,500 -- 85,550

.N -

N -

COUNT COllNT

NO. 1 NO. 2

16,700 16,300 22,300 21,900 21,500 22,300 23,100 21,900 18,700 17,500 16,700 18,300 19,900 20,700 23,100 23,500 19,900 20,700 21,900 20,300

20.380 20,340

NO. 2 NO. 1

81,100 225OOU 87,100 237:OOO 85,000 174,000 79,500 198,000 77,900 178,000 93,000 235,000 94,600 172,000 92,600 230,000 80,300 224,000 83,900 148,000

85,500 202,100

*Shows 1 :lO dilution; all other dilutions are 1 :lOO.

independently, without knowledge of each other’s results. The counts were rounded and evaluated statistically, the rounding possibly contributing to the error.

The results of the separate counts are shown in Table III; the averages and errors are shown in Table IV.

TABLE IV

-___-- WORKER A

SALIVA OF SUB- COUNT STANDARD STANDARD COUNT STANDARD STANDARD

JECT A DEVIATION ERROR B DEVIATION ERROR

NO. # % # % # % # %

1 235 47 20.00 15 6.38 242 47 19.40 15 6.20 2 19780 2304 11.64 727 3.67 20260 2403 11.86 759 3.74 3 85900 7454 8.67 2360 2.74 85500 8265 9.66 2614 3.05 4 194000 27315 14.07 8638 4.45 192000 2 3 202 12.08 7337 3.82

WORKER B

SALIVA

OFSUB- COUiXT STANDARD STANDARD COUNT STANDARD STANDARD

JECT A DEVIATION ERROR 1~ DEVIATION ERROR

NO. # % # % # 7% # % ___-- 1 259 55 21.2 17 6.56 260

2294;: 16.40 13 5.20

2 20380 2418 11.8 765 3.75 20340 11.30 726 3.56 3 85500 8400 9.8 2834 3.30 85500 5790 6.77 1833 2.14 4 202000 32150 15.9 10167 5.03 201000 26946 13.30 8461 4.18

METHOD OF COUNTING ORAL BACTERIA 1205

Grand Averages

Standard deviation 13.36 per cent Standard error 4.23 per cent

The standard error was 4.23 per cent, which, for a bacteriological method, is excellent. The standard deviation of 13.36 per cent is of equal excellence. More variance was noted in counts of 250 or lower and 190,000 and higher, which detracts from the greater accuracy at 20,000 and 90,000 bacteria per cubic centimeter.

Summary

The inherent errors in methods of counting bacteria in saliva are discussed. A method of counting oral bacteria is described which, when statistically

appraised, shows an average standard error of 4.23 per cent and average stand- ard deviation of 13.36 per cent on counts of salivas ranging from 250 to 200,000 bacteria per cubic centimeter.

The greatest accuracy was obtained on counts of 20,000 and 90,000 bacteria per cubic centimeter.

The variation between two workers is shown to be negligible.

References

1. Becks, H., Jensen, A. L., and Millarr, C. B.: J. Am. Dent. A. 31: 1189, 1944. 2. Boyd, J. D., Cheyne, J. D., and WesseL, K. E.: Proc. Sot. Exper. Biol. & Med. 71:

535-537, 1949. 3. Becks, H.: Ref. 1, p. 1191. 4. Hadley, F. P.: J. D. Res. 13: 415-428, 1933. 5. Permar, D., Kitchin, P. C., and Robinson, H. B. G.: J. D. Res. 25: 475-486, 1946. 6. Kesel, R. G., O’Donnell, J. F., Kirch, E. R., and Wach, E. C.: J. Am. Dent. A. 33: 695-

714, 1946. 7. Kesel, R. G., O’Donnell, J. F., Kirch, E. R., and Wach, E. C.: Am. J. Orthodontics and

Oral Surg. (Oral Surg. Sect.) 33: 2, 1947. 8. Snyder, M. L.: J. D. Res. 19: 349-355, 1940.