Scand. J. Dent. Res. 1975: 83: 274-278 (Key words: dentat ptaque; microbiology, orat)

Comparison of five growth media and two anaerobic techniques for isolating bacteria from dental plaque JORGEN SLOTS Departments of Periodontology and Microbiology, Royal Dental College, Copenhagen, Denmark ABSTRACT — Five agar media in combination with the Hungate roll tube technique and conventional anaerobic jar technique (Baird & Tatlock® jars) were evaluated to determine the most suitable medium for non-selective isolation of the viable microorganisms in dental plaque. The highest colony count was obtained by using "Modified Medium 10" with the roll tube technique. About twice as many plaque colonies were recovered by roll tube technique as by the conventional anaerobic technique. With the MM 10 roll tube technique, 7 d of incubation revealed only 93 % of the colonies that could be detected after 14 d of incubation. (Received for publication 14 April, accepted 30 May 1975)

The oral microflora has only been partially identified because the previously used microbiologic culture methods revealed only a small fraction of the total microflora (SocRANSKY, GIBBONS, DALE, BORTNiCK, RosENTHAL & MACDONALD 1963, GIBBONS, SOCRANSKY, ARAUJO & HOUTE 1964). Recently, new techniques for culturing anaerobic bacteria have been employed, namely the Hungate roll tube technique (HUNGATE 1950) and the anaerobic glove

how various growth media affect the total colony counts is limited, The present study was conducted to compare five growth media commonly used for non-selective isolation of oral bacteHa, to determine which one permits the highest recovery of viable dental plaque microorganisms. Anaerobic colony growth was tested by use of the Hungate roll tube technique and by the conventional anaerobic jar technique.

box technique (ARANKI, SYED, KENNEY & FRETER 1969), and much information is

Material and methods

now available concerning their efficiency (ARANKI et al.

1969,

GORDON, STUTMAN

; imi T TT P & LOESGHE 1971, LOESCHE, HoGKETT & SYED 1972). However, information on

GROWTH MEDIA ^,

The agar media tested were: i."piaque" medium (JENSEN, THEILADE 1968).

LOE, SCHIOTT

&

BACTERIA FROM DENTAL PLAQUE 2. N2C (A^utrient-Cystein medium) (CILMOUR & PooLE 1970). 3. HIA-10% B (Heart /nfusion ^gar - 10% Blood) (GORDON et al. 1971). 4. BHIA-Suppl. (jBrain Heart /nfusion .i4garSupplemented) (HOLDEMAN & MOORE 1973). 5. MM 10 (Modified iWedium 10) (LOESCHE & SYED 1973).

The media for the roll tube and the conventional anerobic jar technique were identical except that resazurin was added to the roll tube media as an indicator of the anaerobic condition. The media were prepared according to the original recommendations. Furthermore, the roll tube media were handled according to the procedures outlined by HOLDEMAN & MOORE (1973). The media were used within 4 d after preparation. SAMPLE GOLLEGTION

Supra- and subgingival dental plaque samples were removed from 10 males and females aged 20 to 69 years by the use of periodontal curettes. No attempts were made to relate the samples to the age, dental health, and oral hygiene of the subjects, nor to the age of the dental plaque. Plaque in connection with caries lesions and filling materials was not collected. The individual samples consisted of material from one or two molar tooth surfaces in the maxilla and mandible. During the collection of the samples the area was constantly flushed with a gas mixture comprising 85 % Na, 10 % H2, and 5 % CO2. The mixture was made oxygen-free by passing a quartz tube containing hot reduced copper powder. After removal, the plaque was immediately transferred to a test tube (1.5x9.5 mm) containing 4.5 ml Reduced Transport Fluid (RTF) (SYED & LOESGHE 1972).

275

SAMPLE PLATING

One-tenth ml of each of the dilutions 10-^, and 10-^ were plated on the various growth media. Duplicate tubes and plates were prepared from each dilution and medium. The samples were cultured within 4 h after collection. Between platings, the suspensions were stored in ice. The plating procedure was as follows: Roll tube technique — The aliquots were inoculated while maintaining the melted medium at 45-50°C. After inoculation the tubes were stoppered, and the roll tubes were prepared by rapid rotation under cold tap water. The roll tubes were incubated at 37 °C for 14 d. Conventional anaerobic jar technique (Baird & Tatlock® jars equipped with catalyst and indicator) - The aliquots were spread over the surface of the agar media with a bent glass rod. Prior to plating, the agar plates had been placed for 1 d in anaerobic jars filled with the oxygen-free gas to reduce the oxygen tension in the media. The platings were performed at room atmosphere. Plating and reincubation were carried out within 30 min. For each incubation procedure the jars were evacuated and filled twice. The agar plates were incubated at 37°C for 7 d.

ENUMERATION AND STATISTIGAL TEST

The total colony counts were obtained by use of a dissecting microscope at X 10—40 magnification. Colonies were counted on the agar plates after 7 d of incubation, and in the roll tubes after 4, 7, and 14 d. The colony counts after 7 d of incubation were analyzed statistically by means of the Wilcoxon matched pairs signed rank test.

SAMPLE DISPERSION

Results

The plaque samples were dispersed by sonication under a continuous flow of the oxygenfree gas using an ultrasonic disintegrator 100 W MSE (MSE Incorporated, Westlake, Ohio), used with a micro-probe of 3 mm in end diameter. The sonication was performed for 45 sec with an amplitude of 8 jxm. After sonication, the plaque suspension was serially diluted stepwise 10 times in RTF under anaerobic conditions. The resazurin indicator remained colorless during this procedure.

The increase in the colony counts during 14 d of incubation was determined in seven experiments with five roll tube media (Table 1). After an incubation period of 4—7 d, which is common, a significant part of the colonies present after 14 d was not detected. By use of MM 10, about 7 % of the colonies present after 14 d could not be found earlier.

SLOTS

276

Table 1

;

The increase in colony counts through 14 d of incubation of five roll tube media Incubation time Media

7 d

4d «

"Plaque" medium N2C H I A - 10% B BHIA Suppl. MM 10

»

Range*

X*

Range*

75.0-92.5 81.4-96.3 68.2-99.4 69.0-98.8 80.5-96.8

93.6 95.8 93.9 97.6 92.9

87.1- -100.0 93.6-- 98.8 80.0-- 98.4 89.5--102.1 81.5-- 99.2

•

85.2 92.8 85.4 91.0 88.8

14 d

100.0** 100.0 100.0 100.0 100.0

* Data based on seven experiments. ** The figures indicate the number of colonies detectable, expressed in percentages of the 14 d recoveries for each medium.

Comparison of the culturing techniques — The mean recovery of organisms for the various media was 51-109% higher using the roll tube technique than using the anaerobic jar technique (Table 2). Comparison of the media - Of the media used with the roll tube technique (Table 3), MM 10 produced the highest colony counts in 6 of the 10 samples and it yielded the highest mean culture counts. MM 10 grew statistically significantly more colonies (at the 2 % level of significance) than did "Plaque" medium, N^C, H I A 10% B, and BHIA-Suppl. Within the latter media no statistically significant dif-

ferences were observed. The large ranges in recovery rates found for the roll tube media established with MM 10 as baseline indicate a large person-to-person variation. The media used with the anaerobic jar technique showed less difference in the recovery of organisms (Table 4 ) . With HIA—10 % used as baseline, the anaerobic plates also showed large ranges in recovery rates. The following differences were statistically significant ( 2 % level): MM 10/

Table 3 Effect of roll tube media composition on the recovery of microorganisms from 10 dental plaque samples

Table 2 Effect of roll tube versus anaerobic jar method on the recovery of microorganisms from 10 dental plaque samples Media

No. of colonies Roll tube/ anaerobic jar Range

"Plaque" medium N2C

1.89 1.60

0.60-3.60 0.92-2.75

HIA-10%B

1.51

0.92-2.60

BHIA Suppl. MM 10

1.75 2.09

0.71-2.57 1.38-2.92

Media

No. of samples with highest colony counts

No. of colonies Media/MM 10 Range*

"Plaque" medium N2C HIA-10%B BHIA Suppl. MM 10

2 1

0.75 0.34-1.16 0.65 0.38-1.17

1 0 6

0.73 0.46-1.06 0.69 0.50-0.88 1.00

* Related to MM 10 having highest colony counts in most of the samples.

BACTERIA FROM DENTAL PLAQUE Table 4 Effect of anaerobic jar media composition on the recovery of microorganisms from 10 dental plaque samples Media

No. of samples with highest colony counts

"Plaque" medium NgC

1 1

HIA-10%B BHIA Suppl. MM 10

5 1 2

No. of colonies Media/HIA10 % B X*

Range*

0.80 0.85 1.00 0.79 1.01

0.46-1.13 0.51-1.53 —

0.61-1.22 0.63-1.44

* Related to HIA - 10 % B having highest colony counts in most of the samples.

"Plaque" medium, H I A - 1 0 % B/"Plaque" medium and H I A - 1 0 % B/BHIA Suppl. Discussion

The results of this study emphasize the importance of strictly anaerobic conditions, such as in the Hungate roll tube technique, for optimally non-selectively isolating microorganisms from dental plaque. The conventional anaerobic jar technique was in this respect only about half as effective as the roll tube technique. This was the case even though specific efforts were made to minimize oxygen exposure to the anaerobic plates. The importance of these procedures was stressed by LOESGHE (1969). Such efforts comprised sample handling under anaerobic conditions, anaerobic storing of plates until plating, and performing plating and reincubation procedures as quickly as possible. The result obtained agrees well with previous studies comparing the anaerobic jar technique with the roll tube technique (MGMINN

&

CRAWFORD 1970,

GORDON

et al. 1971) and the glove box technique

277

(ARANKI et al. 1969). The roll tube technique and the glove box technique are considered to be similar in permitting cultivation of anaerobes from clinical specimens (KiLLGORE, STARR, DELBENE, WHA-

LEY & DOWELL 1973).

With regard to the effect of the media, MM 10 proved to be a valuable non-selective medium with either the roll tube technique or the anaerobic jar technique. SYED & LOESGHE (1973), working with normal and disease-associated dental plaques, also reported a superior performance of MM 10 in comparison with NjC medium, Schaedler agar, and Mitis-Salivarius agar. As non-selective medium, MM 10 has demonstrated its usefulness also with isolation from biological materials outside the oral cavity (CALDWELL & BRYANT 1966,

ELLER, GRABILL & BRYANT

1971). The present study further emphasized the importance of extending the incubation time beyond 4 to 7 d in order to increase the number of colonies. This was especially the case with MM 10. In evaluating the present results, the large ranges in recovery efficiency for all the media tested were noteworthy. This may be explained by differences in growth requirements due to the large variety of plaque microorganisms. It seems that more investigations of the growth requirements of the plaque bacteria are needed in order to develop more efficient media for nonselective isolation of bacteria present in dental plaque.

References ARANKI, A.,

SYED, S. A., KENNEY, E . B . &

FRETER, R . : Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 1969: 17: 568-576. CALDWELL, D . R . & BRYANT, M . P.: Medium without rumen fluid for non-selective enumer-

278

SLOTS

ation and isolation of rumen bacteria. Appl. Microbiol. 1966: 14: 794-801. ELLER, C , CRABILL, M . R . & BRYANT, M . P.:

Anaerobic roll tube media for non-selective enumeration and isolation of bacteria in human feces. Appl. Microbiol. 1971: 22: 522529. GIBBONS, R . J., SOCRANSKY, S. S., DE ARAUJO,

W. C. & HouTE, J. V.: Studies of the predominant cultivable microbiota of dental plaque. Arch. Oral Biol. 1964: 9: 365-370. CILMOUR, M . N . & POOLE, A. E.: Crowth stimulation of the mixed microbial flora of human dental plaques by heamin. Arch. Oral Biol. 1970: 15: 1345-1353. GORDON, D . F., STUTMAN, M . & LOESCHE, W .

J.: Improved isolation of anaerobic bacteria from the gingival crevice area of man. Appl. Microbiol. 1971: 21: 1046-1050. HOLDEMAN, L . V. & MOORE, W . E . C : Anae-

mens. Am. J. Clin. Pathol. 1973: 59: 552559. LOESCHE, W . J.: Oxygen sensitivity of various anaerobic bacteria. Appl. Microbiol. 1969: 18: 723-727. LOESCHE, W . J., HOCKETT, R . N . & SYED, S.

A.: The predominant cultivable flora of tooth surface plaque removed from institutionalized subjects. Arch. Oral Biol. 1972: 17: 13111325. LOESCHE, W . J. & SYED, S. A.: The predomi-

nant cultivable flora of carious plaque and carious dentine. Caries Res. 1973: 7: 201216. McMiNN, M. T. & CRAWFORD, J. J.: Recovery of anaerobic microorganisms from clinical specimens in prereduced media versus recovery by routine clinical laboratory methods. Appl. Microbiol. 1970: 19: 207-213. SOCRANSKY, S. S., GIBBONS, R . J., DALE, A. C ,

BORTNICK, L., ROSENTHAL, E . & M A C D O N A L D , robe laboratory manual. Virginia Polytechnic J. B.: The microbiota of the gingival crevice Institute and State University, Blacksburg, area of man. I. Total microscopic and viable Virginia 1973. counts and counts of specific organisms. Arch. HUNGATE, R . E . : The anaerobic mesophilic celOral Biol. 1963: 8: 275-280. lulolytic bacteria. Bacteriol. Rev. 1950: 14: SYED, S. A. & LOESCHE, W . J.: Survival of 1-63. human dental plaque flora in various transJENSEN, S. B., LOE, H . , SCHIOTT, C . R . & THEIport media. Appl. Microbiol. 1972: 24: 638LADE, E.: Experimental gingivitis in man. / . 644. Periodontal Res. 1968: 3: 284-293. KiLLGORE, G. E., STARR, S. E., DELBENE, V. E., SYED, S. A. & LOESCHE, W . J.: Efficiency of WHALEY, D . N . & DowELL, V. R.: Comparivarious growth media in recovering oral bacson of three anaerobic systems for the isolaterial flora from human dental plaque. Appl. tion of anaerobic bacteria from clinical speciMicrobiol. 1973: 26: 459-465.

Address: Royal Dental College 160, Jagtvej DK-2100 Copenhagen 0 Denmark