口腔衛 生学雑誌 第22巻 第1号 昭和 47 年 3 月

Original

DIFFUSATE MEDIA FOR CULTIVATION OF ORAL

ANAEROBIC BACTERIA

Paul A. Mashimo* and Solon A. Ellison*

Abstract: Antigen-free media suitable for cultivating anaerobic bacteria from the human

gingival crevice were devised, based upon supplementing diffusates from standard media in-

gredients. Growth in these is at least as good as in customary media. Formulations suitablefor the major species encountered are described and compared.

概要:口 腔内嫌気性培養用に考案 した透析培地について,真 下 彰*,S・A・ エ リソン*.口 腔内諸

疾患 の免疫学的研究 を,と くに 口内細菌 の抗原性について しらべた いときに,そ の培養基 に由来す る抗

原物質 にいろいろ と妨害 され る.一 方多 くの口腔 細菌 は理想的 な合成培地 に,な かなか うま く発育 して

くれない。そこで,抗 原物質をふ くまないで,一 定 の培養後培地内容物質 を全部透折 で除 くことが出来

るい くつかの培地 を考案 し,他 に応用 して好成績 を得 てい るのでその処方 と代表的な結果 を報告す る.

The growth requirements for the several species of anaerobic bacteria resident in the

gingival crevice are largely unknown12). A few instances can be noted in which addition of

specific chemicals to standard media bases has shown the need for supplementation of these

media in order to obtain good growth, e.g. the apparent requirement for hemin and mena-

dione, by some types of Bacteroides3). The available media are correspondingly complex.

Simple defined media are a great convenience, almost a necessity for immunologic studies of

bacteria. An alternative which has often been satisfactory is to employ diffusates7) prepared

from standard media. Since our present work was particularly concerned with soluble antigens-

of oral bacteria, it was decided to attempt to develop a series of such media using which

we might obtain consistent growth of the organisms under study.

Materials and Methods

1. General

The bacterial strains tested are listed by genus in Table 1. All strains except those

of Corynebacteria were obligately anaerobic. The organisms were isolated from the

indicated sources by streaking into nutrient agar with 10% sheep blood. Bacteroides mela-

ninogenicus (BM) was obtained from Dr. Ronald Gibbons, Forsyth Dental Center , Boston.When growth was adequate the colonies were transfered into blood agar plates and restreaked

to check purity. Spirochetes were isolated into S-3 medium (see below). Inocula were placed

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* Department of Oral Biology School of Dentistry, State University of New York at Buffalo (Buffalo ,New York 14226)

* ニ ュ ー ヨー ク州 立 バ フ ァ ロー大 学歯 学部 口腔 生 物 学 教室

昭 和46年12月1日 受 付

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Table 1. Characteristics of strains of bacteria

into wells made by a glass tube with an attached rubber cap for aspiration. Cultures were

incubated anaerobically in Brewer jars, and daily observations were made for up to 7 days.

Spirochete-laden agar was then obtained by pipette for transfer.

Growth in the media tested was estimated in several ways. In selected instances

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turbidity measurements were made; in others simple observations of the amount of bacterial

sediment was adequate for our purpose. When turbidity measurements were used to assay

growth, a standard suspension (T. of 10% at 450mƒÊ) of each organism was prepared in

0.9% saline and 0.1ml of this used to inoculate the various media under comparison.

Periodically, the growing cultures were well suspended and their optical densities determined.

On the 5th, 10th and 14th days of incubation, cultures were examined in the darkfield

microscope. Comparison was thus made of bacterial morphology in standard and diffusate

media.

2. Media

The standard media employed for comparison were Bacto-Heart Infusion (Difco Lab.)

(BHI), and Thioglycollate medium without indicator (Difco Lab)(TG). The compositions of

diffusate media which proved most useful are listed below.

A similar procedure was followed in all cases. An initial mixture was prepared. This

was dialyzed against a volume of water such that the final concentrations of ingredients

would be appropriate. The diffusates were collected after 24 hours of dialysis, and after

adjusting pH, were sterilized by autoclaving at 15 lbs. pressure for 15 minutes. In a few

instances, sterile addition of other ingredients was done after autoclaving.

A. FM-3 Medium

a) Initial solution

Trypticase (BBL) 15 grams

Bacto-yeast extract (Difco Lab) 5 •V

Dextrose (Fisher Scientific Co.) 5 •V

NaCl (Fisher Scientific Co.) 5 •V

DL-tryptophan (Mann Res. Lab) 0.2 •V

Calcium pantothenate (Mann Res Lab) 0.5 •V

H2O 150ml.

b) Water volume for dialysis: 850ml.

c) Final pH: 7.2

B. EX-1 medium Formula 1 Formula 2

a) Initial solution

Bacto-Brain Heart Infusion (Difco Lab) 6.0 grams 20.0 grams

Biosate (BBL) 4.5 •V 10.0 •V

Bacto-yeast extract (Difco Lab) 1.8 •V 10.0 •V

Trypticase (BBL) 6.0 •V 7.0 •V

Polypeptone (BBL) 6.0 •V 20.0 •V

Sodium thioglycollate 0.75 •V 0.75 •V

Cysteine 0.2 •V 0.2 •V

Glucose 0.05 •V

Proteose peptone (Difco) 1.0 •V

Sodium chloride 1.0 •V

Water 150ml. 150ml.

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b) Water volume for dialysis: 850ml. 850ml.

c) Final pH: 7.0

C. S-3 and S-4 Medium

a) Initial solution: S-3 S-4

Biosate (BBL) 4.5 grams 3 grams

Bacto-Yeast extract (Difco Lab) 1.5 •V 3 •V

Casitone (Difco Lab) 6 .0 •V 6 •V

Bacto-proteose peptone (Difco) 1.0 •V 1 •V

Bacto-neopeptone (Difco) 1.0 •V

Bacto-peptone (Difco) 1.0 •V

Bacto-brain heart infusion (Difco) 4.5 •V

NaCl 1.0 •V

Arginine 0.03 •V

H2O 150ml. 30ml.

b) Water volume for dialysis: 850ml. 270ml.

c) Final pH: 7.2 7.0

D. V-2 Medium

This was made in a slightly different fashion.

a) Basic solution

Na2HPO4 6.95 grams

KH2PO4 2.46 •V

NA Amine, type A 20 •V

Biotone 20 •V

Glucose 10 •V

Sodium thioglycollate 1 •V

CaCl2•E2H2O 10 mgms.

MgSO4•E7H2O 150 •V

Adenine sulfate 10 •V

Uracil 10 •V

Water 494ml.

b) The following were added into the basic solution:

MnCl2•E4H2O 0.04% sol. 0.1ml.

CuSO4•E5H2O 0.1% sol. •V

ZnSO4•E7H2O 0.1% sol. •V

Fe2(SO4) 3 0.1% sol. 1.0ml.

Nicotinic acid 0.1% sol. 1.0ml.

Thiamine HCl 0.1% sol. 1.0 ml.

Pyridoxamine HCI •V •V

Ca-pantothenate •V •V

Riboflavin •V 0.5ml.

Biotin 0.005% sol. 0.1ml.

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Folic acid 0.005 % sol. 0.1 ml.

c) The following solution was prepared and then dialyzed against

425 ml. water.

Tween 80 (Difco) 5 ml.

Trypticase (BBL) 5 grams

Biosate (BBL) 2.5 grams

Water 75 ml.

The diffusate was added to an equal amount of basic solution and autoclaved.

Hemin (equine) 2x cryst. (Mann Res. Lab) was always added into all media when

Bacteroides melaninogenicus was examined following the recommendation of Gibbons and

MacDonald3)4).

Results

The results are summarrized in Tables 2 and 3, and Figs. 1 and 2. EX-1 yielded good growth

of the most of the strains tested. Veillonella grew better in this medium than in BHI. FM-3

proved particularly useful for fusobacteria. Bacteroides strains grew well in FM-1 supplemented

Table 2. b Growth of selected strains of bacteria, as determined turbidometrically

a. Measurements were made after five days incubation, at the following wave-lengths:

S-3 and V-11-420mƒÊ; FM-3-430mƒÊ; EX-1, BHI, and TG-450mƒÊ.

0=O.D. less than 0.09

1+=O.D. 0.1-0.19

2+=O.D. 0.2-0.29

3+=O.D. 0.3-0.49

4+=O.D. more than 0.5

b. See Table 1 for abbreviations.

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FUSOBACTERIUM F-18

Figure 1. Growth of Fusobacterium F-18in various media, estimatedturbidometrically. Measurem-ents were made daily. (See textfor abbreviations. See Table 2for wave lengths.)

CORYNEBACTERIUM C-1

Figure 2. Growth of Corynebacterium C-1 in various media, estimatedturbidometrically. Measurem-ents were made daily. (See textfor abbreviations. See Table 2for wave lengths.)

as described earlier. The "S" group of media, although useful for spirochetes, and also for

mixed cultures of samples taken from the gingival crevice, were not otherwise very satis-

factory. Oral spirochetes could be maintained well in S-3 with 0.6% agarose added. Without

this addition growth was not obtained. The appearance of initial cultures is shown in Fig. 3.

Usually two concentric zones were obtained. The outer area contained Borrelia buccalis and

the inner contained Treponema macrodentium. After 5 days, the zones diffuse widely, and

the center area shows a condensed hazy zone in which Treponema microdentium is contained.

Anaerobic vibrios grew excellently in V-2 medium.

Some rough quantitative comparisons are shown in Table 3. These indicate that in

Figure 3. The appearance of cultures made of gingival crevice contents in

S-3 medium. Samples were inoculated into the central wells in

each instance. The concentric zones contained spirochetes.

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many instances the diffusate media compared well with standard media.

Fig. 1 shows the growth of Fusobacterium strain 18 in different media. This instance

is typical of the group. At the beginning, EX-1 and FM-3 showed somewhat better results

than the others. TG yielded good growth, gradually increasing until the 14th day. FM-3

was at least as good as the latter.

Fig. 2 indicates the typical growth of a strain of Corynebacterium. EX-1 medium

yielded excellent growth of this strain until the 7th day, after which the optical density

decreased. This was not due to autolysis, but to clumping, which rendered the optical density

measurements impossible. EX-1 medium was thus better than BHI or TG. FM-3, V-II and

S-3 media did also give good results with this organism.

Discussion

Our approach to devising the media described was of necessity pragmatic. Initially,

we had tried as others have done, the simple maneuver of cultivation in diffusates prepared

from customary laboratory media. Although growth was obtained, the yield was poor.

Hence, putative "growth-factors" were added based upon the relatively meager literature

concerning this aspect of oral microbiology.

The use for cultivation of fusiform bacteria of trypticase and yeast extract, supple-

mented with calcium pantothenate and tryptophan was based on the observations of Omata8)

and Omata and Disraely9). Alterations in the relative amounts of the ingredients in their

medium (FM)10) were necessary to produce an appropriate pH.

EX-1 medium was devised empirically. A rich medium, suitable for growing a large

number of the bacteria present in debris from the gingival crevice or in the exudates from

experimentally infected animals, was desired. Formula 2 is a simplified version of Formula 1.

Some strains of Bacteroides which did not multiply in TG or BHI grew well in EX-1.

The cultivation of spirochetes, although regularly achievable, still requires complex

media. The mixture used by Socransky et al. (1969)13), although greatly simplified as compared

to the older media, contains antigenic materials. Studies on the Reiter treponeme11)14)16) had

indicated that a medium consisting of an amino-acid mixture plus serum albumin supplemented

with several vitamins, purines, pyrimidines, and carbohydrates gave reasonable growth. The

S-3 medium described here yields good results when used both for isolation and maintenance

of strains. Solidification with 0.6-0.8% agar or 0.6% agarose is necessary for passage of

some strains. Although S-4 is useful for primary isolation of spirochetes, it is not good for

passage of pure cultures.

The V-II medium was designed for use with anaerobic vibrios. In its present formu-

lation, it is quite probably more complex than is needed to satisfy the minimal growth

requirements of these organisms.

These media, when carefully prepared, do not contain antigens as determined by im-

munization of rabbits and subsequent assay for antibody, using gel-diffusion techniques.

Through their use, the production of soluble antigens during growth of oral bacteria has

been shown5)6). Some of these react with serums from human patients and from experi-

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mentally-infected animals, and the significance of these reactions is now under study2).

Obviously, it would still be desirable to define growth requirements more precisely and

to develop completely defined media. Efforts along these lines are in progress.

Acknowledgements

This investigation was supported by Public Health Service Grant DE-01932 from the National

Institute of Dental Research.

本 論 文 の要 旨 は第46回 お よび 第47回IADR(1968年,1969年)の 総 会 に お い て一 部 報 告 した もの で ある 。

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promoting factors for the Reiter treponeme.J. Bacteriol, 56, 163-176, 1948.

2) Ellison, S.A. and Mashimo, P.A. Anti-body response in Fusospirochetal infections.Programs and Abstracts of Papers, I.A.D.R.46th Gen. Mtg., San Francisco. p.104,1968.

3) Gibbons, R.J. and MacDonald, J.B.: Heminand Vitamin K compounds as required factorsfor the cultivation of certain strains ofBacteroides melaninogeicus. J. Bacteriol. 80,164-170, 1960.

4) Gibbons, R.J. and MacDonald, J.B.: Degra-dation of collageneous substrate by Bacteroidesmelaninogenicus. J. Bacteriol., 81, 614-621,1961.

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6) Mashimo, P.A. and Ellison, S.A.: Antig-enic structures of Fusiform bacteria. Programsand Abstracts of papers, I.A.D.R. 47th Gen.Mtg., San Francisco, p.110, 1969.

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principle present in yeast extract. J. Bacteriol.65, 326-329, 1953.

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