APPLIED MICROBIOLOGY, Oct. 1972, p. 638-644Copyright 0 1972 American Society for Microbiology

Vol. 24, No. 4Printed in U.S.A.

Survival of Human Dental Plaque Flora inVarious Transport Media

SALAM A. SYED AND WALTER J. LOESCHEDepartment of Oral Biology, School of Dentistry, The University of Michigan, Ann Arbor, Michigan 48104

Received for publication 5 June 1972

Dental plaque samples from (i) subjects with no apparent oral disease, (ii)mentally retarded subjects with periodontal disease, and (iii) subjects with ac-tive caries were collected in three transport media viz. a dithiothreitol poisedbalanced mineral salt solution designated as reduced transport fluid (RTF),VMG II, and modified Stuart medium (SBL). The samples were dispersed bysonic treatment, diluted in the respective medium in which they were col-lected, and cultured on MM10 sucrose agar. The efficiency of the transportmedia in the survival of dental plaque flora was determined by comparing thequantitative recovery (expressed as percentage of the initial viable count) fromthe specimens stored for various lengths of time. The data showed a great vari-ation in the recovery of the oral bacterial flora from the plaque samples. VMGII and SBL served better than RTF as storage media for non-disease-associateddental plaque cultured under strict anaerobic conditions. Recoveries of bacteriafrom periodontal plaque specimens stored in RTF were higher than SBL andVMG II under identical conditions. The organisms present in the cariousplaque samples appeared to survive much better in RTF and VMG II than inSBL as determined by conventional anaerobic culturing technique. However,VMG II showed a higher recovery of organisms from these specimens with anincrease in the storage period, suggesting multiplication of the plaque flora.RTF did not allow the growth of oral bacterial flora under all experimentalconditions. On the basis of the relative performance of these media it is sug-gested that RTF is a statisfactory medium for the transport of oral bacteriapresent in the samples.

Human oral flora contains a large proportionof anaerobic bacteria (2, 8, 10, 11, 22; W. J.Loesche, R. N. Hockett, and S. A. Syed, Arch.Oral Biol., in press) which differ markedly intheir sensitivity to oxygen (16). Although con-siderable efforts have been made in recentyears to improve and evaluate the varioustechniques (2, 7, 11, 12, 15, 18, 23), little hasbeen done to determine the efficacy of the ex-isting transport media in maintaining the via-bility of the oral bacteria in the sample. Conse-quently, limited information is available in theliterature as to the relative performance of thetransport media in the survival of these orga-nisms. Mtller (19) introduced a transport me-dium (VMG II) which he considered superiorfor demonstrating streptococci and anaerobicnonsporulating bacteria in the endodonticsample. Jordon et al. (13) used VMG II me-dium in their studies, in which caries-inducingstreptococci were isolated from human dentalplaque. These investigators showed that VMG

638

II medium retained the reproductive capacityof the organisms present in samples of dentalplaque and preserved the viability of mostplaque streptococci for several days at roomtemperature. GAstrin et al. (9) investigated theeffect of storage of various clinically importantpathogenic bacteria in VMG IV medium,Stuart medium as modified by Ringertz (21),and SBL medium. They found a marked de-crease, i.e., 1 to 2 log, in the recovery of bac-teria after their storage for 1 hr in the testmedia. VMG IV medium allowed multiplica-tion of most bacteria examined by these inves-tigators. On the basis of the comparison of thethree transport media, GAstrin et al. recom-mended SBL medium as a suitable transportmedium in different types of bacteriologicalexaminations.

In the present study, a balanced mineral saltsolution (4) poised with dithiothreitol (DTT),and containing sodium ethylenediaminetet-raacetate (EDTA) was evaluated for its use-

on July 25, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

SURVIVAL OF HUMAN PLAQUE FLORA

fulness as a transport medium for various oralbacterial specimens. The efficiency of thistransport medium, designated as reducedtransport fluid (RTF), was compared with thatof VMG II and SBL media in maintaining theviability of bacterial flora present in the sam-

ples. Results of the experiments with RTF andthe other two transport media are reported inthis paper.

MATERIALS AND METHODSTransport media. SBL medium was prepared

according to the recommendation of Gastrin et al.except that 0.08% agar was added. VMG II mediumwas prepared as described by Jordan et al. (13). RTFwas prepared according to the formula shown inTable 1. This medium was filter-sterilized by using a

membrane filter (0.22-um pore size). Prior to its use,

each medium was tested for sterility under aerobicand anaerobic conditions. For the collection of sam-

ples, all transport media were dispensed in 10-mlsamples in 18 by 150 mm test tubes. These mediawere also used as diluents and therefore dispensed in9-ml amounts in 16 by 125 mm screw-cap culturetubes.

Plating medium. A modification of the M 10medium of Caldwell and Bryant (4) was used as a

nonselective medium for the samples. When 5% su-

crose is added to this medium, polysaccharide-forming colonies of bacteria such as Streptococcusmutans, Streptococcus sanguis, and Streptococcussalivarius are readily recognized. The constituents ofthis medium, referred to as MM10 sucrose agar, are

shown in Table 2.Anaerobic glove box. An anaerobic glove box

similar to the one described by Aranki et al. (2) was

used for culturing of the non-disease-associated (nor-mal) and periodontal plaque specimens under strictanaerobic conditions and for anaerobic incubation of

TABLE 1. Composition of reduced transport fluid(RTF)a

Components Amount (ml)

1. Stock mineral salt solution no. 1 . ..... 752. Stock mineral salt solution no. 2' ..... 753. 0.1M EDTA ........................ 104. 8% Na2CO3 ........................ 55. 1% dithiothreitol (freshly prepared) 20

6. Resazurin 0.1% (optional) ..... ....... 17. Distilled water ........ ............. 814

a Formula per liter of RTF. Prepared by membranefilter sterilization by using membrane filter (0.22-gmpore size) and dispensed into 16 by 125 mm screw-

cap tubes (dilution tubes) and 18 by 150 mm testtubes (sample collection tubes). The pH of thismedium was 8 + 0.2 without adjustment, and itdecreased to 7 in 48 hr in the anaerobic glove boxatmosphere (85% N., 10% H2, 5% C0).

b Contained 0.6% K2HPO4.c Contained 1.2% NaCl, 1.2% (NH4)2S04, 0.6%

KH2PO4, and 0.25% MgSO4.

TABLE 2. Composition of MM1O sucrose agara

Medium Amount

Trypticase (BBL) ............ ......... 2 gYeast extract (Difco) ......... ......... 0.5 gHemin ................. ............. 0.001 gSodium lactate (60%) ......... ........ 4 mlSodium formate ...................... 1.0 gPotassium nitrate ........... ......... 0.5 gMineral salt solution no. l ............. 38 mlMineral salt solution no. 2c ...... ...... 38 mlGlucose ............................. 1 gSucrose ............................. 50 gAgar (Difco) ......................... 15 gDistilled water ....................... 874 ml

Autoclave the above at 121 C for15 min and then add

8% Na2CO, solutiond.................. 5 ml0.05% menadione solution ...... ...... 1 ml1% dithiothreitol solution.d............. 20 mlDefibrinated sheep blood ....... ....... 20 ml

a Formula per liter. Medium prepared and pouredon bench top and introduced into the anaerobicglove box.

b Contained 0.6% K2HPO4.c Contained 1.2% NaCl, 1.2% (NHj2SO4, 0.6%

KH2PO4, 0.12% CaCl2.d Sterilized by using membrane filter (0.22-Mm pore

size) and added to the sterile medium, cooled to 50C. Final pH, 7.2 :i: 0.2.

inoculated plates. It contained a mixture of 85% ni-trogen, 10% hydrogen, and 5% carbon dioxide, whichwas circulated over a palladium catalyst to eliminatetrace oxygen. The temperature of the glove box wasmaintained at 37 C by using a heating element andthermoregulator. The plating medium and the trans-port media were prereduced by incubating them inthe anaerobic glove box for 48 hr prior to their usefor the collection and culturing of normal and perio-dontal plaque samples.Sample collection. Dental plaque samples were

collected from three different populations. Theseincluded (i) subjects with no active caries and noevidence of periodontal disease, (ii) patients withactive caries and known to harbor S. mutans in theirplaque, and (iii) institutionalized children with per-iodontal disease. The first group of individuals hadfour males and one female. The age of these individ-uals ranged from 24 to 37 years. The second groupconsisted of six male subjects with age between 10 to18 years. The last group consisted of four male andone female patients. All patients belonging to thisgroup were mentally retarded and handicapped indi-viduals. Their age varied between 11 to 22 years. Inthe first two populations, the dental plaque sampleswere removed by means of sterile, unwaxed dentalfloss which was passed interproximally between ei-ther the mandibular or maxillary second premolarand first molar. Three samples were collected fromeach subject. The floss (approximately 1 inch [2.5cm] in length), carrying the plaque sample, was in-troduced into each of the three test media. When

639VOL. 24, 1972

on July 25, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

SYED AND LOESCHE

normal subjects were used, the dispersion and cul-turing process was accomplished within a few min-utes, so that there was a minimal delay betweencollection and culturing. The samples from thedental caries patients were processed 12 to 15 hrafter collection.The samples from the institutionalized children

were removed by periodontal scalers, placed in apreweighed aluminum boat, weighed, and then intro-duced into the sample collection tubes of variousprereduced transport media. Three samples for eachmedium for a total of nine samples were collectedfrom each individual. One set of samples, repre-senting all the transport media, was cultured at 4 hr;other sets were stored at refrigeration temperaturefor either 1 or 2 weeks before culturing.Sample dispersion. The dispersion of periodontal

plaque and normal plaque samples was accom-plished by sonically treating each of the samples for10 sec at the highest intensity of the microtip (soni-fier model W185 D, Ultrasonic Inc.). Exposure of thesamples to air during dispersion was minimized byequilibrating the gas space in the tube with an ox-ygen-free gas mixture of 95% nitrogen and 5% carbondioxide. A set-up similar to the one recommendedby the Anaerobic Laboratory, Virginia PolytechnicInstitute, (1) was used to gas the sample tubes duringthe entire process. The dispersed samples wereclosed by rubber stoppers and transferred immedi-ately to the anaerobic glove box for dilution andplating process.

Carious plaque samples were dispersed in thesame manner as other specimens except that no gasmixture was used during the process.Sample dilution and plating procedure. The

plaque samples from normal subjects were collectedin prereduced transport media and handled understrict anaerobic conditions throughout the experi-ment. They were diluted anaerobically in the sametype of medium in which they were collected andstored. Serial 10-fold dilutions were prepared, and thelast three dilutions were used for plating on a pre-reduced medium. After the initial plating was com-pleted, the diluted samples were kept inside theanaerobic glove box until the second plating wasdone 6 hr later. Then the tubes were closed by rubberstoppers, removed from the glove box, and stored at10 C except when used for the experiment.

Periodontal plaque samples were diluted andplated in the anaerobic glove box. The procedureused was similar to the one used for normal plaquesamples except that there was an initial 4-hr delaybetween the collection of the specimens and cul-turing procedure.The samples obtained from the caries-active pa-

tients were collected in freshly prepared transportmedia, diluted and plated on the bench top in theroom atmosphere, and incubated in the same anaer-obic glove box used for other samples. Each samplewas diluted in the same type of medium in which itwas collected and stored.

RESULTSThe recovery of the bacteria from normal

plaque specimens was followed for 14 days.

The viable counts obtained from the first cul-turing were considered as a reference value(100%). The results are shown in Table 3.There was approximately a 19% reduction inthe viable counts after 6 hr of storage of thespecimens in RTF. The VMG II and SBLshowed a slight increase in the recovery of theorganisms, indicating possible multiplication.Approximately 70% of the organisms were de-stroyed after 1 day of storage in RTF as com-pared to 28 to 30% loss in viability in VMG IIand SBL medium under identical conditions.The survival of bacteria appeared to be thehighest in VMG II medium. After an initialdecrease in viability, the number of bacteriaremained stable in this medium during theentire storage period.At 7 and 14 days, samples were plated from

either dilutions prepared on day 0 or from newdilutions of the initial collection tubes. Therewas no apparent difference between the freshor old dilutions on the recovery of organismsfrom the samples.Our next experiments were concerned with

field studies involving collection of plaquefrom periodontally involved, mentally retardedindividuals. These subjects had considerableplaque accumulations on their tooth surfacesso that it was possible to collect and weigh theplaque samples. Therefore, in this manner, thedata could be referred to a milligram wetweight reference. Nine samples were removedfrom each of five subjects and were returned tothe laboratory in the respective transportmedia. One set of samples was dispersed andplated at 4 hr. The other sets were stored at 10C until dispersed and plated at either 7 or 14days. The results are given in Table 4. At alltime periods, the recoveries per milligram ofplaque wet weight were superior for the RTF.After 14 days, there was about a 40% decreasefrom the 4-hr value with RTF. However, withVMG II there was a 65% decrease and withSBL a 35% decrease.

S. mutans has been implicated as the etio-logic agent of dental caries in certain animalmodel systems (14). Considerable efforts arecurrently being made to determine the pres-ence and level of this organism in human pop-ulations. In the next series of experiments, in-terproximal plaque was removed from sixteen-age subjects known to harbor S. mutansand S. sanguis in their plaque. As these orga-nisms are microaerophilic, the anaerobic pre-cautions taken earlier with other samples wereomitted and the samples were dispersed, di-luted, and plated on the bench top prior toincubation in the anaerobic glove box. Thismethod of sample manipulation would more

640 APPL. MICROBIOL.

on July 25, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

SURVIVAL OF HUMAN PLAQUE FLORA

TABLE 3. Recovery of dental plaque flora of normal subjects from three transport media under strictanaerobic conditionsa

Culturing Percent survival of bacteria Statistical significancehin daysafter 1 2 3 vs 2 1 vs 3 2 vs 3

collection RTF VMG II SBL 1V s3 2v

0 100 100 100(14.5i 7.7 x 106)c (18.6 ± 5.9 x 10') (18.6 4 12.6 x 106)

1/4 81.2 102.8 103.1 NSd <0.05 NS1 30.6 72.4 70.3 <0.01 < 0.005 NS2 23 73.7 62.8 <0.001 < 0.005 NS4 5.5 78 28.9 <0.001 <0.01 <0.0257 4.0 78.3 21.7 <0.001 <0.001 <0.00114 3.8 78.4 16.4 <0.001 <0.001 <0.001

a Floss samples were cultured in the anaerobic glove box and stored anaerobically at 10 C.& Based on t test (3).c Numbers in parentheses: mean counts of five samples on pre-reduced MM10 sucrose agar, plus or minus

standard deviation. These counts are given as a reference value.d Not significant.

TABLE 4. Recovery of dental plaque flora of periodontal patients from three transport media under differentexperimental conditionsa

Sample Viable counts x 108/mg wet weight Statistical significancebstorage 1 2 3indays RTF VMG II SBL | vs 2 | vs 3 2 vs 3

16 3.9 ± 0.9c 2.08 ± 0.9' 2.3 ± 0.2' <0.025 <0.01 NSd7 2.3 ± 2.8 0.9 ±0.7 1.0± 1.0 NS NS NS14 2.4 ± 2.8 0.95 ± 0.9 1.5 ± 1.9 NS NS NS

a Samples were stored undispersed in the transport media at 10 C and sonically treated on the day theywere used for culturing.

" Based on t test (3).c Represents mean counts of five plaque samples for each transport medium, plus or minus standard devia-

tion.d Not significant.

closely approximate the conditions for han-dling similar samples in epidemiologic sur-veys. There was a 12- to 15-hr delay betweencollection and the first culturing of the sam-ples. The samples after dispersion were storedat 10 C and cultured over a 14-day period todetermine the relative performance of thetransport media in the survival of the plaqueflora. The results (Table 5) indicate that therecovery of plaque bacteria from SBL trans-port medium was very poor. There was al-most 100% loss of the viability of the organismsin this medium. The recovery of the plaquebacteria from RTF and VMG II within the firstweek dropped to 23% and 33%, respectively. Inthe second week of storage, the recovery oforganisms from VMG II medium was in-creased tremendously, suggesting multiplica-tion of bacteria or yeast, or both.

Differential counts of S. mutans and S. san-guis were made on MM10 sucrose agar. Theseorganisms form characteristic colonies due to

polysaccharide formation which can be easilyidentified. In addition, certain key tests wereperformed on the representative and doubtfulisolates to confirm their identification. Theseincluded mannitol fermentation, ability toform ethanol-insoluble dextran, and heavyplaque formation on wire (17). The recovery ofthese organisms from the transport media wasfollowed for 14 days. The results of this studyare presented in Table 6. There was a rapiddecrease in the viability of S. mutans and S.sanguis in SBL medium as compared to theother transport media. There was a 75 to 90%decrease in the viability of the organisms inRTF medium over a 10-day period. S. mutansappeared to survive best in VMG II. However,this medium promoted growth of S. sanguis asshown in Table 6.

DISCUSSIONThe problem of delay between the collection

of the specimens and subsequent culturing is

VOL. 24, 1972 641

on July 25, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

TABLE 5. Survival of dental plaque flora of dental caries patients in three transport media cultured atdifferent time periodsa

Percent survival of bacteria Statistical significance'Culturingindays 1 2 3 | 2 vs 3RTF VMGII1 SBL ls v3 2s

½/2 100 100 100(16.2i 8.0 x 106) (19.0 i 13.0 x 106) (10.1 + 5.0 x 106)

4 32.2 47.3 13.2 NSd <0.01 <0.0057 23.0 32.9 1.7 NS < 0.001 < 0.00110 16.7 98.8 0.1 < 0.005 < 0.005 < 0.00514 2.4 96.7 0 <0.005

a Samples were handled aerobically and stored at 10 C. The inoculated MM10 sucrose agar plates wereincubated in the anaerobic glove box.

'Based on t test (3).cNumbers in parentheses: mean counts of six floss samples, plus or minus standard deviation. These

counts are given as a reference value.d Not significant.

TABLE 6. Effect of storage on the recovery of Streptococcus mutans and S. sanguis from three transportmediaa

Percent survival of S. mutans and S. sanguis.Culturing RTF VMG II SBLin days

S. mutans S. sanguis S. mutans S. sanguis S. mutans S. sanguis

1/, 100 100 100 100 100 100(3.7 i 4.7)b (20.3 + 16.6) (28.5 i 63) (9.5 ± 10.6) (7.7 ± 2.9) (6.75 + 5.1)

4 31.4 64 80.7 74.3 5.4 9.127 12.0 31 77.2 88.4 0.65 0.1410 10.3 24.3 15.7 205.0 0 0.3214 0.8 0.9 2.3 185.0 0 0

a Results are based on the mean of six carious plaque samples collected in the transport media from pa-tients known to harbor S. mutans. Viable counts were made on MM 10 sucrose agar.

,'Numbers in parentheses: mean viable counts x 0l per dental floss, plus or minus standard deviation.These counts are given as a reference value.

not uncommon. Ideally, our aim was to selecta reasonably -good medium which could main-tain the viability of the overall population ofthe organisms present in the plaque withoutappreciably altering their relative proportionover a period of time. VMG II and SBL mediawere selected as they are routinely used for thetransport of various specimens for bacterialanalyses. RTF is currently being used as atransport medium in our laboratory. Therefore,the performance of this medium was comparedwith other two media under various condi-tions.

Culturing of the normal plaque samplesunder continuous anaerobiosis throughout theentire experimental period showed that SBLand VMG II medium served better as storagemedia than RTF. The viability of the plaquebacteria was well maintained in these mediafor several days under these conditions. How-

ever, these ideal conditions would not be du-plicated in the usual circumstances underwhich plaque is collected and cultured.When the samples were collected from

periodontal patients under field conditions andmanipulated and plated anaerobically, theRTF yielded the highest recoveries on a milli-gram (wet weight) basis at all time intervals.This would suggest that for plaque associatedwith periodontal disease the RTF might be thetransport medium of choice. This performancemay be attributed to the low En of the RTFwhich could maintain the viability of the an-aerobes which tend to be present in high pro-portions in periodontal plaque (Loesche et al.,Arch. Oral Biol., in press).The results obtained from the cultural study

of carious plaque samples showed that SBLmedium did not serve well as a storage me-dium. The RTF and VMG II were comparable

642 SYED AND LOESCHE APPL. MICROBIOL.

on July 25, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

SURVIVAL OF HUMAN PLAQUE FLORA

for the first 7 days, but, with further storage,VMG II gave rise to higher recoveries. Acareful analysis of the composition of theVMG II provides the basis of its apparentlyexcellent performance. VMG II medium con-tains 1.1% peptones (gelatin, thiotone, andtryptose) and 1% glycerophosphate, which mayserve as growth-promoting substances. Mbller(19) used small amounts of phenyl mercuricacetate (.003%) as a bacteriostatic agent in thismedium apparently to keep growth in check.However, the presence of SH-containing com-pounds, such as thioglycollic acid (.05%) andcysteine (.05%), could neutralize the bacterio-static effect of the mercurials in the medium(21), thereby allowing growth to occur.

Gastrin et al. (9) found that most of the purecultures of pathogenic organisms included intheir study showed growth and multiplicationin VMG IV medium. Jordan et al. (13), work-ing with dental plaque samples, also observedthe same phenomenon and stated that thereproductive capacity of the bacterial cellswas well maintained in VMG II medium.Rundell et al. (B. B. Rundell, L. A. Thomson,W. J. Loesche, and H. M. Stiles, Abstr. In-ternational Association for Dental Research,no. 576, 1972) studied the survival of S. mu-tans, S. sanguis, S. mitis, and S. salivarius inVMG II, SBL, and RTF. They reported thatgrowth sometimes occurred in VMG II me-dium as determined by total viable countsof the organisms. SBL medium was found tobe the most toxic of the three transport mediatested by these investigators. RTF, on theother hand, gave rise to the most constantpopulation of oral streptococci under theseconditions.

In our storage studies, we found the growth-promoting ability of VMG II medium (Table 5)when the samples were dispersed underaerobic conditions. Higher recovery of S. san-guis over an increase storage period of the sam-ples (Table 6) would mean its multiplication inVMG II medium. These findings suggest thatthe facultative or microaerophilic bacteria, orboth, could multiply in this medium. Furtherstudies would be required to determine thequalitative nature of specific bacterial compo-nents of the plaque flora which could grow andmetabolize the nutrients present in this me-dium. Due to these limitations, VMG II shouldbe used with caution as a transport or storagemedium, especially when one is interested indetermining the relative proportion of the orga-nisms in the original samples.The present findings indicate that the ideal

medium for the storage and transport of all the

different types of bacterial specimens is notyet available. However, of the three mediatested, RTF would appear to be more satisfac-tory for dental samples. It is a nonselectivetransport and storage medium. With the ex-ception of 0.02% DTT which was added to themedium as a reducing agent, it does not haveany compound which could support the growthof bacteria. DTT was selected as a reducingagent because of its low oxidation-reductionpotential and minimal susceptibility to oxida-tion under aerobic conditions (6). The additionof very low concentrations of EDTA (0.001 M)to RTF appears to promote a more uniformdispersion of bacterial cells (Loesche et al.,Arch. Oral Biol., in press). Since EDTA acts asa chelating agent, its incorporation into RTFwas useful for binding divalent cations, such asCa2+, which are present in high concentrationin plaque and contribute to the aggregation ofplaque bacteria.RTF does not contain any bacteriostatic

agent that would affect the multiplication ofbacteria in the sample. Consequently, growthmight occur in this medium if the sample it-self contains enough nutrients to allow bac-terial multiplication for a limited period.However, our studies did not show any growthof the plaque organisms in this medium. Al-though its efficiency as a storage mediumvaried with the nature of the samples, the per-formance of RTF appears to be better than theother two media tested. Poising the mediumwith DTT makes it more resistant to oxidationunder aerobic conditions. This characteristic ofRTF may reflect its better performance in therecovery of the plaque flora from carious andperiodontal plaque samples. These findingssuggest that RTF could be used as a desirablemedium for transport and storage of oral spec-imens. The viability of the organisms can bebest maintained in this medium at refrigera-tion temperature (Rundell et al., Abstr. Inter-national Association for Dental Research, no.576, 1972).

ACKNOWLEDGMENTSThe technical assistance of Samuel Gregorio is gratefully

acknowledged. This investigation was supported by PublicHealth Service grants from the National Institute of DentalResearch, no. DE-03011-03 and DE-02731-05.

LITERATURE CITED1. Anaerobe Laboratory. 1970. Outline of clinical methods

in anaerobic bacteriology. Virginia Polytechnic Insti-tute, Blacksburg, Va.

2. Aranki, A., S. A. Syed, E. B. Kenney, and R. Freter.1969. Isolation of anaerobic bacteria from human gin-giva and mouse cecum by means of a simplified glovebox procedure. Appl. Microbiol. 17:568-576.

3. Brown, K. A. 1965. Statistical theory and methodology

643VOL. 24, 1972

on July 25, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

SYED AND LOESCHE

in science and engineering. John Wiley & Sons,Inc., New York.

4. Caldwell, D. R., and M. P. *Bryant. 1966. Mediumwithout rumen fluid for nonselective enumeration andisolation of rumen bacteria. Appl. Microbiol. 14:794-801.

5. Cary, G. S., and R. E. Blair. 1964. New transport me-dium for shipment of clinical specimens. I. Fecalspecimens. J. Bacteriol. 88:96-98.

6. Cleland, W. W. 1964. Dithiothreitol, a new protectivereagent for SH group. Biochemistry 3:137-160.

7. Drasar, B. S. 1967. Cultivation of anaerobic intestinalbacteria. J. Pathol. Bacteriol. 94:417-427.

8. Dwyer, D. M., and S. S. Socransky. 1968. Predominantcultivable microorganisms inhabiting periodontalpockets. Brit. Dent. J. 124:560-564.

9. Gastrin, B., L. 0. Kallings, and A. Marcetic. 1968. Thesurvival time for different bacteria in various trans-port media. Acta Pathol. Microbiol. Scand. 74:371-380.

10. Gibbons, R. J., S. S. Socransky, S. Sawyer, B. Kapsi-malis, and J. B. MacDonald. 1963. The microbiota ofthe gingival crevice area of man. I. The predominantcultivable organisms. Arch. Oral Biol. 8:281-289.

11. Gordon, D. F., M. Stutman, and W. J. Loesche. 1971.Improved isolation of anaerobic bacteria from gingivalcrevice area of man. Appl. Microbiol. 21:1046-1050.

12. Gordon, J. H., and R. Dubois. 1970. The anaerobic bac-terial flora of mouse cecum. J. Exp. Med. 132:251-260.

13. Jordan, H. V., B. Krasse, and A. Miller. 1968. A method

APPL. MICROBIOL.

of sampling human dental plaque for certain cariesinducing streptococci. Arch. Oral Biol. 13:919-927.

14. Keyes, P. H. 1968. Research in dental caries. J. Amer.Dent. Ass. 76:1357-1373.

15. Leach, P. A., J. J. Bullen, and I. D. Grant. 1971. Anaer-obic CO, cabinet for the cultivation of strict anaer-obes. Appl. Microbiol. 22:824-827.

16. Loesche, W. J. 1969. Oxygen sensitivity of various an-aerobic bacteria. Appl. Microbiol. 18:723-727.

17. McCabe, R. M., P. H. Keyes, and A. Howell, Jr. An invitro method for assessing the plaque forming abilityof oral bacteria. Arch. Oral Biol. 12:1653-1656.

18. McMinn, M. T., and J. J. Crawford. 1970. Recovery ofanaerobic organisms from clinical specimens in pre-reduced media versus recovery by routine clinical lab-oratory methods. Appl. Microbiol. 19:207-213.

19. Mdller, A. J. R. 1966. Microbiological examination ofroot canals and periapical tissues of human teeth.Odont. Tdskr. 74: no. 5 and 6. Special article.

20. Nungester, W. J., M. A. Hood, and M. K. Warren. 1943.Use of thioglycollate for testing disinfectants. Proc.Soc. Exp. Biol. Med. 52:287-289.

21. Ringertz, 0. 1960. A modified Stuart medium for thetransport of gonococcal specimens. Acta Pathol.Microbiol. Scand. 48:105-112.

22. Rosebury, T. 1962. Microorganisms indigenous to man.McGraw-Hill Book Co., N. Y.

23. Spears, R. W., and R. Freter. 1967. Improved isolationof anaerobic bacteria from the mouse cecum by main-taining continuous strict anaerobiosis. Proc. Soc. Exp.Biol. Med. 124:903-909.

644

on July 25, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from