Rapid fermentation testing of anaerobic bacteria

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Rapid Fermentation

Testing

of

Anaerobic Bacteria

PAUL C. SCHRECKENBERGERI* AND DONNA J. BLAZEVIC

Department ofLaboratory Medicine and Pathology, University ofMinnesota, Minneapolis, Minnesota55455

Received for publication 29July 1975

Rapid tests for glucose, maltose, lactose, sucrose, and starch fermentation

wereperformedon 112 strains ofanaerobic bacteria. The testswereincubated

underaerobic conditions, and resultswereread within4h. An overall

correla-tionof89% wasachieved between the rapidtestsand the Virginia Polytechnic

Institutemethod.

In a previous paper (7), we described a series ofrapid biochemical testsfor use with anaero-bic bacteria. These tests were incubated in an aerobic environment, and the results could be read within 4 h. The tests utilized a large inocu-lum and relied upon preformed bacterial en-zymes that reacted with a small amount of substrate to produce the desired end product. The present paper expands this concept to in-clude the rapid detection ofcarbohydrate fer-mentationby anaerobicbacteria.

MATERIALS AND METHODS

Test organisms. All anaerobic bacteria used in the study were recent laboratory isolates saved in

chopped meat broth at room temperature in the dark. All organisms had been previously identified by the Virginia Polytechnic Institute (VPI) method-ology (3). There were 112 organisms used in the study, consisting of: 1Arachnia, 10 Bacteroides fra-gilis, 10Bacteroides species, 10 Bifidobacterium, 10 Clostridium perfringens, 10Clostridium species, 10 Eubacterium, 6Fusobacterium, 5 Lactobacillus, 10 Peptococcus, 10 Peptostreptococcus, 10 Propionibacte-rium, and 10 Veillonella.

Growth media. All prereduced media were pur-chased from Scott Laboratories(Fiskeville, R.I.) and contained hemin and vitamin K. The media were inoculated using the VPIAnaerobic Culture System (Bellco). Prior to testing, each strain was subcul-tured to a fresh blood agar plate prepared with Trypticase soy agar,5%sheepblood, and1% hemin-vitamin Ksolution (ScottLaboratories). The plates were incubated anaerobically in a vented GasPak jarevacuated and filled three times with90% C02

and10% H2. Ifthe culture waspure, asingle well-isolatedcolonywaspicked and transferredto prere-ducedpeptone-yeast-glucosebrothorchopped meat-glucose broth.All furthersubculturingwascarried outfrom this broth.Inthecaseof therapidtests,a fresh blood agarplatesupplementedwith hemin and vitamin K wasinoculated andincubated anaerobi-callyfor 24 to 48 handwasthesource of inoculum for all therapidtests.

IPresent address: Curriculum in Medical Laboratory Sciences, University of IllinoisMedical Center, Chicago,

Ill.60612.

Conventional tests. Prereduced peptone-yeast-carbohydrate broths for glucose, maltose, sucrose, lactose, and starch were used asthe conventional tests for determining carbohydrate fermentation. The tests were performed by the procedures outlined inthe VPI manual (3). Each broth wasinoculated with 4 drops of an actively growing culture from either peptone-yeast-glucose or chopped

meat-glu-cose broth. Peptone-yeast broth without

carbohy-drate was included with eachsetof peptone-yeast-carbohydrate broths thatwasinoculated.Acid pro-ductionfrom carbohydrate fermentationwas deter-mined by measuring the pH of the broth cultures after7days of incubationat 35C.The criteria used for differentiating between weak and strong acid production was that establishedinthe VPI manual; namely, pH 5.5 to 6.0equals weak acid production, and pH below 5.5 equals strong acid production. However, if the peptone-yeastbroth without carbo-hydrate had a final pH below 6.0, then 0.2 was subtracted from the finalreadingtodetermine the cutoff for a weak acid reaction, and 0.4 was sub-tractedto determine the cutoff for strong acid pro-duction.

Preparation of rapid tests. Rapid fermentation tests for glucose, maltose, sucrose, lactose, and starchwereprepared by adding 1.0gof the appro-priatecarbohydratetoabuffer-salt solution consist-ing of dipotassium phosphate (0.04 g), potassium

dihydrogen phosphate (0.01 g), potassium chloride (0.8 g), bromthymol blue (0.2 ml of 1% aqueous

solution), and distilledwater(100ml). A fermenta-tionbase mediumwasalsoprepared and consisted of buffer-saltsolution without the addition of any car-bohydrate. All the fermentation media were ad-justedto apH 7.2,dispensedin0.5-mlaliquots,and

frozen at-20 Cuntil used.

Inoculation and incubation of therapidtests.All rapidtests wereperformed by usingasterileloop (3 mmindiameter) and scraping upaloopfulofgrowth

from the surface of theagarmediumandinoculating directlyintothe substrate thathad beenpreheated

to 35 to 37 C. All tests wereincubatedaerobicallyat 35 to 37 C and read after4h of incubation.

Interpretation of rapid tests. Carbohydrate fer-mentation was demonstrated by a change in the color of the indicator from blue(at thestartingpHof 7.2)togreen(thatwasinterpretedasweakly acid)to yellow (thatwasconsideredacid, indicating fermen-313

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tation). A blue coloratthe end of4 hwasinterpreted asnegative. Questionablereactionswerecompared with the fermentation basemedium, whichwas in-oculated with the sameorganismbut containedno

carbohydrate. Freshbromthymolblue solution(0.5 ml, adjusted with 0.1 N NaOHto giveadeep-blue

color)wasaddedtosometubesat the end of incuba-tiontoaid in theinterpretationofquestionable

reac-tions.

All media and reagents werechecked with posi-tive andnegativecontrols toensuretheiraccuracy.

RESULTS

Table 1 shows the correlation between the

rapid and conventional tests for carbohydrate fermentation. A total of560 rapid tests were

run on 112 organisms utilizing five different substrates. A total of498, or 89%, of the tests were incomplete agreement with the conven-tionaltests. Thirty-eightof therapidtests(7%) appeared to be either less sensitive or falsely negative when compared to the conventional

tests, whereas theremaining24 tests (4%)

ap-pearedto be more sensitiveorfalsely positive by comparison. Lactose gavethehighest corre-lation (94%) of the substrates tested, with su-crose (90%), starch (88%), and maltose (87%) showing decreasing agreement.

Seventy-eight of the 112 organisms tested

(70%)showedcompleteagreement betweenthe

rapidand conventionaltests.Table2lists those

organismswhich accounted for the 62 discrep-ancies. The bifidobacteriawere responsible for the highest number of discrepancies between

the conventional and rapid methods,

account-ingfornearly27%of thedisagreements noted.

In each case the discrepancy was due to a weakly acidic rapid test, whereas strong acid wasproduced in the conventional method. In

fact, 17of the22 teststhatresulted in thistype ofdiscrepancy were due tofive strains of

Bifi-dobacterium. Therewere sevenstrains of Clos-tridium species that were responsible for 12

discrepancies between the conventional and rapid methods. Four of the discrepancies were duetoonestrain ofClostridium septicumthat strongly fermented glucose, maltose, and

lac-toseandweaklyfermentedsucrosebutfailedto

produce any acid in the rapid test system. Of the eight discrepancies produced by strains of Peptostreptococcus, three were attributed to one strain thatproduced weak acid from

mal-tose inthe conventionaltestbutwas negative inthe rapidtest. This strain alsowasnegative for acidproductionfrom sucroseand starch in the conventional test but was weakly positive in the rapidtest. Two strains of Eubacterium resulted in seven discrepancies. Both strains produced acid from glucose and maltose with the rapidtestbutwerenegative withthe

con-ventional test. One strain produced acid from

sucrose, and the other strain produced weak

acid, but both failedtoferment sucrosein the

conventional test. One strain produced acid from starchinthe rapidtestbutwas negative inthe conventional method.Insummary,ofthe 112 organismstested forcarbohydrate fermen-tation, 9 organisms, including 5 Bifidobacter-ium, 1Clostridium, 1Peptostreptococcus, and 2 Eubacterium,accounted for 31 (50%) of the total numberof discrepancies noted.

Reproducibility of the fermentation reactions wasattimesaproblem. One strain of Clostrid-ium species produced acid from starch in the conventional test but was negative for starch fermentationwhen the testwas repeated. An-otherstrain of Clostridium thatfirst produced acidfrom lactose in the conventional testwas

negative when repeated. Three strains of Pro-pionibacterium were negative for glucose fer-mentation with the conventional tests. When the tests were repeated, one of the negative

strains became positive, another produced weak acid, and the thirdstrain still remained negative. Two other strains of

Propionibacte-TABLE 1. Resultsof conventional and rapid fermentation testsa performed on 112 strains of anaerobic bacteria

No. of testsb

Test or substrate No. of tests in % Tests in

agree-a w - w - - a a w agreement ment

a w - a a w w -

-Lactose 46 1 58 5 1 0 1 0 0 105 94

Sucrose 48 1 52 5 1 2 0 1 2 101 90

Starch 29 1 69 3 0 5 0 2 4 98 88

Glucose 62 10 25 4 1 1 5 4 0 97 87

Maltose 44 4 49 5 1 4 2 3 0 97 87

Total 228 17 253 22 4 12 8 10 6 498 89

a Rapidtestswere incubated aerobically.

ba,Strong acid; w, weak acid, -, negative reaction. Top row of symbols is for the rapid tests; bottom row ofsymbolsis forthe conventional tests.

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FERMENTATION TESTING OF ANAEROBIC BACTERIA

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riumproduced weak acid from glucose the first timetheVPI test was run but produced strong

acid when the test was repeated. All five strains of Propionibacterium mentioned were positivefor glucose fermentation with the rapid test, both initially and upon repetition. Three strainsofPeptostreptococcus were negative for glucose fermentationwith theconventionaltest but were weakly positive when the test was repeated. The same three strains initially pro-duced weak acid from starch but were negative when the conventional test was repeated. One strainof Eubacteriumwasnegativeforglucose fermentation with the conventional test but was weakly positive when the test was

re-peated. One strain of Fusobacterium that was negativefor all the carbohydrates tested with the conventionaltestswaspositive for glucose, maltose, and sucrose fermentation when the testswererepeated. Two strains of Lactobacil-lus that failed to ferment one or more sugars initially produced acid from the same sugars when the conventional tests were repeated. Re-producibility with the rapid fermentation tests wasalsosomewhat ofaproblem, but no more so than that experienced with the conventional method. When a discrepancy occurred between the first and second runs of a test, the results obtained in the second run were used in the comparison.

The addition of fresh indicator to the rapid fermentation tubes after incubationand theuse of a carbohydrate-free fermentation base for comparisongreatly aidedinthe interpretation of the rapid fermentation reactions. Turbidity of the cell suspensions didnotaffect the color of the indicator solution.

DISCUSSION

The substrate used for the rapid fermenta-tion tests wasbasedonthe formula ofKellogg and Turner (4), except thatasmaller concentra-tionofcarbohydratewasused andbromthymol

bluewassubstitutedforphenolredasthe indi-cator. The pK ofphenol red is 7.9, with apH

range of6.8 to 8.4. Inthe presence ofaheavy

inoculumthe changeincolor from redtoorange wasindistinct. Bromthymolblue, withapKof 7.0andapHrangeof6.0 to7.6,providedbetter discrimination between negative, weak acid,

and strong acid reactions. Bromthymolblueis also the indicator recommendedby the

anaer-obe laboratory at the CenterforDisease Con-trol foruseintheir fermentationbasemedium

(2).

Reed and Orr(6)reportedthat certain

anaer-obes have the ability to reduce

bromthymol

blue.Thesefindingswereconfirmedduringthe

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course of our study, and it was therefore de-cided toincludeafermentation blank with each set ofcarbohydrates that were inoculated. In cases where the indicator wasreduced,as evi-denced by a green color in the

carbohydrate-free fermentationsubstrate, the final reactions werereadafter the addition of fresh indicatorto each tube. In some casesthe blue color of the original indicator could not be restored inthe fermentationblank, due probablytotheacidity of some inocula. However, interpretations could be made by comparing the color in the fermentation blank with the color achieved in the tubes containing carbohydrate. Inspite of theproblemsencountered with organisms that werecapable ofreducing the bromthymol blue dye, it was decided to continue including the indicator with the substrate so that early detec-tion of fermentadetec-tion reacdetec-tions could be made with those organisms not capable of reducing the dye. Indeed, many of the strong acid reac-tions, such asthose that occurred with Bacte-roides fragilis and Clostridium perfringens, took place within 2 h, with some reactions occurringasearlyas 30minafter inoculation. Thecarbohydratesused inthisstudy (glucose,

maltose, sucrose, lactose, and starch) were selectedasbeing representative,and since each showed the sameapproximate degreeof

accu-racy in our evaluation, it is reasonableto sus-pect that the equivalent results might be achieved with othercarbohydrates aswell.

The1%concentration of carbohydrate used in the rapid tests was selected as a compromise between the2%solutions used by Kelloggand Turner (4) and the0.6% concentration used by the Center for Disease Control (2). The final concentration ofstarch was lowered to 0.5%, since this amount was easier to solubilize and didnotadversely affect the outcome of the reac-tions. Substrate concentration, however, was not considered to be nearly as critical as inocu-lum size. Brown (1) modified the rapid fermen-tationprocedure of Kellogg and Turner (4) and found results to be much improved when the inoculum size was tripled. Indeed, we found this to be true with our tests, especially with the slower growing anaerobes thatproduced a smalleramount of inoculum. Whentesting the morefastidious organisms it was necessary to use half the growth from one plate toobtain a loopful of inoculum sufficient to inoculate one tube of medium. In the case of the hardier organisms, however, this was not necessary, sinceone plate supplied enough growth to inoc-ulateall five tubes of fermentation media plus the noncarbohydrate-containing fermentation control.

Each of the carbohydrates, with the excep-tionof starch, wasdissolved in the buffer-indi-catorsolution withoutheating. The starch sus-pensionwas autoclaved for 15minto obtain a soluble suspension. Heating the starch didnot seem toproduce any adverse effectonthe sub-strate.

Although each test in the conventional method wasinoculated with an actively grow-ingculture, goodgrowth didnotalways occur, thus producing results whichwereinconsistent withthedefined characteristics ofagiven orga-nism. Forexample, weobserved five strains of Propionibacterium that failed to produce strong acid from glucose even though glucose fermentation is one of the main identifying characteristics of this organism (3). These strains werewellcharacterized andwere iden-tified onthe basis of Gram smearmorphology and gaschromatography. However, evenafter repeated testing, onestrainremainednegative and another was only weakly positive, al-though allfive strainsproduced strongacidin the rapid procedure.

The inconsistencies reported with the con-ventional fermentation tests may be partially

explained bythe fact that theprereduced carbo-hydrate broths contain, inadditiontothe par-ticularcarbohydrate,acomplexof peptones and yeast extracts which may support avariety of reactions. Thus, the end product often becomes a balance between acid production from the breakdown ofcarbohydrate and alkali produc-tion from the breakdown of protein constitu-ents. In the rapid test system multiple reac-tions were notlikely to occur, since the system wasdevoidof anynutrientsexceptforthe sin-gle carbohydrate being tested. To aid in the interpretation of the VPI fermentation tests, peptone-yeast brothwithoutcarbohydrate was inoculated routinely with each set of carbohy-drates. Inspite ofthis precaution, interpreta-tion of the reactions was often difficult. With some organisms the pH of the peptone-yeast brothwoulddrop to as low as 5.5 and was often around 5.8, whereas a nonfermented carbohy-drate inthe same series mighthave had apH above6.0.Thus the boundarybetween calling a reaction negative, weak acid, or strong acid often became arbitrary.

Ina recent study comparing three procedures for biochemical testing of anaerobic bacteria (5), an 84.6%correlationwasobtainedwhen 20 different biochemical tests wereperformed us-ing the thioglycollate-based media used by the CenterforDisease Control and the prereduced anaerobically sterilized media recommended by the Virginia Polytechnic Institute. This

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FERMENTATION TESTING OF ANAEROBIC BACTERIA

dence suggeststhatacertain amount of incon-sistency is inherent among the conventional methods available for biochemical testing of anaerobic bacteria.

Recently two micromethod multitest system for identification of anaerobes (Minitek [Bec-ton,Dickinson andCo.]andthe APIAnaerobe System [Analatab Products, Inc.]) have become commercially available. Comparisons of the latter system with conventional identification procedureshave foundit tobeareliablemeans ofidentifyingmostclinically significant anaer-obic isolates (5, 8). The major differences be-tween these systems and the procedure de-scribed here is anaerobic incubation for 48 h with the API and Minitek Systems asopposed to aerobic incubation for 4 h with our tests. Thus,although theAPIandMinitek are micro-tests and offer the convenience ofa multitest system, they do not offer any significant im-provement in incubation time, since many of the conventional test results can also be read after48hof incubation.

Itshould be stressed that the distribution of organisms selected for this study was some-what skewed so that eachgenus ofanaerobic

bacteria isolated in our laboratory would be significantlyrepresentedinthestudy. Many of the routinely isolated anaerobes showed good

correlation between therapidand conventional tests. However, many of the infrequently iso-lated anaerobes showed considerable disagree-ment. As stated earlier, 8% of the organisms tested, representing five strains of

Bifidobac-terium, one Clostridium septicum, one Pepto-streptococcus species, and two Eubacterium species, wereresponsiblefor 50%of the

discrep-anciesnoted. Yet, these organisms are notthe most common isolates in the clinical

micro-biologylaboratory. Indeed, ifacomparison be-tweenrapid and conventional tests were to be made on agroup oforganismsselectedon the

basis of their frequency of isolationinthe clin-ical laboratory, a much higher correlation might be expected.

Althoughfermentationisconsideredtobe an anaerobic process, the results presented here indicate that fermentation of carbohydrates may bedetected by using rapid tests that utilize a large inoculum and a small amount of sub-strate and which are incubated in an aerobic environment. Even though the anaerobic orga-nismsfailto grow underaerobic conditions,the enzymes responsible for their fermentation pathwaysapparentlycontinue tofunctioneven in the presence of oxygen. Further studies should be guided by the fact that the conven-tionalmethods presentlyinusefor biochemical testing of anaerobic bacteria have certain in-herent limitations and that ultimately the cor-rectidentification of the bacterium itselfisthe best standard from which to make a compari-son.

LITERATURE CITED

1. Brown, W.J. 1974. Modification of the rapid fermenta-tion testfor Neisseria gonorrhoeae. Appl.Microbiol. 27:1027-1030.

2. Dowell, V.R., Jr., and T. M. Hawkins. 1973. Laboratory methods in anaerobic bacteriology. In Center for Dis-ease Control laboratory manual. Center for Disease Control, Atlanta, Ga.

3. Holdeman, L. V., and W. E. C. Moore (ed.). 1973. An-aerobelaboratory manual. VirginiaPolytechnic In-stituteand StateUniversity,Blacksburg,Va. 4. Kellogg, D.S., and E. M. Turner. 1973. Rapid

fermenta-tion confirmafermenta-tion of Neisseriagonorrhoeae. Appl. Mi-crobiol. 25:550-552.

5. Moore, H.B., V. L. Sutter, and S. M. Finegold. 1975. Comparison of threeproceduresfor biochemical test-ingof anaerobic bacteria. J. Clin. Microbiol. 1:15-24. 6. Reed,G.B.,and J. H.Orr.1941.Rapididentification of

gasgangrene anaerobes. War Med. 1:493-510. 7. Schreckenberger,P.C., andD. J.Blazevic.1974.Rapid

methods for biochemical testing of anaerobic bacte-ria. Appl.Microbiol. 28:759-762.

8. Starr,S.E., F. S.Thompson,V. R.Dowell, Jr.,and A. Balows. 1973.Micromethodsystem for identification of anaerobic bacteria.Appl.Microbiol. 25:713-717.

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