Evaluation of Enrichment, Storage, and Age of Blood Agar Medium in Relation to Its Ability to Support Growth of

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1976, p Copyright 1976 Americn Society for Microbiology Vol. 4, No. 5 Printed in U.S.A. Evlution of Enrichment, Storge, nd Age of Blood Agr Medium in Reltion to Its Ability to Support Growth of Anerobic Bcteri CHARLES W. HANSON* AND WILLIAM J. MARTIN Deprtment ofpthology, UCLA Center for the Helth Sciences, Los Angeles, Cliforni Received for publiction 30 April 1976 By mesuring the colony size of vriety of nerobic bcteri isolted from clinicl specimens, n evlution ws mde of the benefits derived from the ddition of severl enrichments to blood gr medium commonly used for the growth of nerobes. Similr methods were used to study the effects of vrious storge conditions nd ge ofthe medium. The results were compred with those obtined on freshly prepred nd enriched blood gr pltes s well s commercilly vilble blood gr pltes. Freshly prepred nd enriched blood gr ws found to give substntilly lrger colonies thn could be grown on commercilly obtined blood gr pltes when both were inoculted nd incubted under identicl conditions. Storge of plting medi under CO2 for periods of up to 72 h hd only minor effect on the growth of the nerobic bcteri studied, but longer periods of storge under CO2 resulted in less efficient plting medium. Nonenriched brin hert infusion (BHI) ws found to be better bsl medium thn Trypticse soy gr (TSA) medium. Colony size on fully enriched BHI blood gr pltes ws > nonenriched BHI > nonenriched TSA > commercilly prepred nonenriched TSA pltes. The dt suggest tht freshness of the pltes my be s importnt s using rich medi. In recent yers there hs been incresing interest in culturing nerobic bcteri from clinicl infections. Mrked improvements hve been mde in this direction through the development of simplified techniques for the isoltion of nerobes. For exmple, the introduction of the GsPk jr (1) ws mjor brekthrough for smll- to medium-sized clinicl lbortories. Other innovtions such s the use of the holding jr (4) nd simplified schemes (2, 5) for the presumptive identifiction of the most common nerobic isoltes hve mde it possible for dequte nerobic bcteriology to be prcticed even in smll clinicl lbortories. One of the most importnt decisions for lbortories tht use plting medi is the proper selection of such medi upon which these rther fstidious orgnisms cn be isolted nd grown. Most of the literture suggests tht freshly prepred, highly enriched, nd properly stored medi re essentil (3, 4, 7). However, the preprtion of these medi cn be imprcticl, prticulrly in smll lbortories. Indeed, mny my question its vlidity nd continue to use commercilly prepred blood gr pltes. The experimentl procedure ofthis study ws designed to follow closely the routine procedure tht would be used in the verge clinicl lbortory with respect to tretment of specimens. Therefore, by necessity, the scope of this investigtion ws limited to qulittive study of the growth chrcteristics of nerobic bcteri on both commercil nd fresh blood gr pltes. The results showed tht fresh rich medium ws superior to commercil blood gr pltes in tht it produced lrger colonies nd enhnced certin colonil chrcteristics tht permitted the colonies to be more redily recognizble s nerobes. MATERIALS AND METHODS Orgnisms. The orgnisms used were those routinely isolted from vriety of clinicl specimens. Smples of the isoltes were retined s stock cultures for further testing. The grm-negtive bcilli nd members of the genus Clostridium were identified s to species by use of the API system (Anlytb Products, Plinview, N.Y.). The gener of the nonsporeforming grm-positive bcilli nd cocci isolted were determined presumptively by the methods of Mrtin (5). Veillonell species were identified on the bsis of Grm stin nd morphology. In most instnces, the species or t lest the gener of the grm-positive 394 nd grm-negtive bcilli were confirmed by gs chromtogrphy on Dohrmnn model 15C-3 gs chromtogrph (Dohrmnn Division, Envirotech Corp., Mountin View, Clif.). Medi. AnO2BA medium consisted of brin hert infusion broth (Difco Lbortories, Detroit, Mich.1

2 VOL. 4, 1976 contining 2.5% gr (Difco) nd 5% sheep blood (Clinicl Stndrds Lbortory, Torrnce, Clif.). Unless otherwise indicted, the AnO2BA medium ws enriched with 0.5 jig of vitmin K (Aqu-Mephyton, Merck, Shrp nd Dohme, West Point, P.) per ml, 5.0 ug of hemin (Sigm Chemicl Co., St. Louis, Mo.) per ml, 0.5% yest extrct (Difco), nd 0.3 mg of cysteine hydrochloride per ml. CMBA (commercil blood gr plte) consisted of Trypticse soy with 6% sheep blood nd 1.5% gr (Clinicl Stndrds Lbortory). TSA (Trypticse soy gr; BBL, Division of Becton, Dickinson nd Co., Cockeysville, Md.) with 5% sheep blood (Clinicl Stndrds Lbortory) nd 1.5% gr (Difco) nd BHI (brin hert infusion gr, nonenriched; Difco) with 5% sheep blood (Clinicl Stndrds Lbortory) nd 1.5% gr (Difco) were lso used. Specimen hndling. The specimens were received in prereduced nerobiclly sterilized trnsport tubes contining 0.5 ml of peptone-yest extrctglucose prepred ccording to the method of the Virgini Polytechnic Institute mnul (3). Immeditely upon receipt, the specimen ws plted on freshly prepred blood gr pltes (AnO2BA) nd portion ws inoculted into tube of thioglycolte 135C without indictor, enriched with 1.0 ml of sterile rbbit serum (Clinicl Stndrds Lbortory). For the purposes of this investigtion, CMBA plte ws lso streked. After inocultion, ll of the bove medi were (t rndom) put in holding jr nd mintined under constnt flow of 100% CO2 (Liquid Crbonics, Chicgo, Ill.) until full (1 to 4 h), t which time the jr ws chrged with GsPk (BioQuest, Cockeysville, Md.) nd incubted for 48 h t 35 C (4). In ddition, CMBA plte ws streked nd incubted under 5% CO2 t 35 C. This plte served to monitor erobic growth nd ny possible contmintion. For some experiments, fresh TSA nd nonenriched BHI, both contining 5% sheep blood nd 1.5% gr (Difco), were prepred. Colony size mesurement. After 48 h of incubtion, well-isolted colonies on the pltes were mesured with x5 oculr micrometer through 1.3 power objective lens in Busch & Lomb dissecting microscope. The colony size ws recorded in millimeters. For most experiments, the colony sizes of ll orgnisms in given group (i.e., ll Bcteroides frgilis, ll Bcteroides species, ll Peptococcus species, etc.) were verged. The dt re presented s the verge colony size on the medium in question for the entire group. In one experiment (Tble 2), orgnisms from the stock cultures were streked on CMBA nd AnO2BA s well s on AnO2BA pltes lcking one or more of the vrious enrichments normlly dded to the AnO2BA medium. After 48 h of incubtion, the colony sizes were mesured s described previously. The medium showing the lrgest colony ws given vlue of 1.00, nd the colonies mesured on the other medi were expressed s percentge vlue of the lrgest colony. The percentge vlues of ll orgnisms in the group were then verged nd the results were tbulted. ph mesurements. The ph of the blood gr medium ws determined by flooding the plte with ANAEROBIC PLATING MEDIA 395 distilled wter, letting it stnd for 20 min t room temperture, nd inserting the electrode of Corning model 7 (Corning Glss Works, Corning, N.Y.) ph meter into the blood gr-wter mixture. RESULTS The results of the colony size mesurements on the two medi cn be seen in Tble 1. The freshly prepred AnO2BA medium gve colony sizes 20 to 30% lrger thn the CMBA pltes when plted nd incubted under identicl conditions. The following exceptions were noted: two isoltes ech ofbcteroides melninogenicus, two Peptococcus species, nd one isolte of Eubcterium species would not grow on the CMBA pltes, further emphsizing the vlue of the freshly prepred AnO2BA medium. The results of five Fusobcterium species isolted during the course of this study re lso shown in Tble 1. Two of the isoltes grew only on AnO2BA pltes. Two dditionl Fusobcterium species hd colony sizes tht were 22% lrger on CMBA, wheres the fifth Fusobcterium species isolted ws 38% lrger on the AnO2BA plte. Six Clostridium perfringens were isolted, but due to the spreding nture of the colonies only three could be mesured ccurtely. In ny cse, ll strins of C. perfringens showed good growth on both AnO2BA nd CMBA. The three strins ofb. melninogenicus tht grew on both types of medi were found to hve more intense brick-red fluorescence under long-wvelength ultrviolet light fter 48 h of incubtion on AnO2BA thn ws observed on CMBA pltes. However, the blck pigmenttion of these orgnisms becme pprent sooner on CMBA thn on AnO2BA pltes. The gr concentrtion used in the AnO2BA medium ws 2.5%, s recommended in the Virgini Polytechnic Institute mnul (3). Since 1.5% gr ws used in ll other plting medi, it ws necessry to compre the effect of gr concentrtion on colony size. Duplicte sets of AnO2BA pltes were prepred with 1.5 nd 2.5% gr nd streked with series of stock cultures, nd colony sizes were mesured fter 48 h of incubtion. In generl, the more rpidly growing nerobes such sb. frgilis nd Clostridium species gve slightly smller colonies on the higher gr concentrtion, but the slower-growing nerobic bcteri like Propionibcterium species, Peptococcus species, nd B. melninogenicus showed essentilly the sme size colonies on medi prepred with either gr concentrtion. In nother experiment, representtive smples of the vrious groups of nerobic bcteri were tken from the working stock cultures

3 396 HANSON AND MARTIN nd streked on CMBA, AnO2BA, nd AnO2BA deficient in one or more of the vrious enrichments normlly dded to the AnO2BA medium (Tble 2). As expected, colony size ws lrger with incresed enrichment. The dt clerly indicte tht fully enriched AnO2BA is by fr the best medium for both growth nd enhncement of colony size when incubted under similr conditions. Furthermore, ll strins of B. frgilis gve their most chrcteristic ppernce on this medium when exmined under the dissecting microscope. Most of the literture on the subject emphsizes the importnce of using freshly prepred medi for the isoltion of nerobic bcteri (3, TABLE 1. 4, 7). To test this contention, two sets of AnO2BA pltes were prepred. One set ws stored t room temperture in the CO2 holding chmber (4) for 12 dys, nd nother set ws stored in plstic bg t 4 C for 12 dys (12 dys ws the pproximte ge of the CMBA pltes being used for routine erobic bcteriology). At the end of the storge period, both the 12-dy-old AnO2BA kept t 4 C nd the AnO2BA pltes stored under constnt flow of CO2 were streked. In ddition, 12-dy-old CMBA nd freshly prepred AnO2BA pltes were similrly streked. Agin the freshly prepred AnO2BA pltes were superior (Tble 3). It lso is worthy of note tht the 12-dy-old Comprison of colony size of nerobic bcteri isolted on freshly prepred AnO2BA nd CMBA pltes Avg colony size (mm) on: % Lrger on No. of isoltes Orgnism AnO2BA com- AnO2BA CMBA pred CMBA with 20 Bcteroides frgilis Bcteroides species B. melninogenicus B. melninogenicus 0.40 NG 2 Fusobcterium species 1.35 NG 2 Fusobcterium species b 1 Fusobcterium species Clostridium perfringens Clostridium species Peptococcus species Peptococcus species 0.97 NG 7 Peptostreptococcus species Veillonell species Bifidobcterium species Eubcterium species Eubcterium species 0.75 NG NG, No growth. b The verge colony size ws lrger on CMBA thn on AnO2BA. J. CLIN. MICROBIOL. TABLE 2. Comprison of colony size of nerobic bcteri isolted on freshly prepred nerobic blood gr pltes (AnO2BA), on pltes deficient in one or more of the enrichments normlly dded to AnO2BA, nd on commercil blood pltes (CMBA) BHI + yest BHI + No. of yest OrimBHI5 + extrct + extrct + AnO2BA CMBA isoltes blood blood cysteine + blood 7 Bcteroides frgilis B. melninogenicus 0.72c Fusobcterium species 0.61c 0.59c 0.81r e 3 Peptococcus species Peptostreptococcus species Veillonell species Bifidobcterium species ,51 3 Propionibcterium species The medium showing the lrgest colony ws given vlue of 1.00, nd the colonies mesured on the other medi were expressed s percentge vlue of the lrgest colony. RBHI, Brin hert infusion gr (the bsl medium of the AnO2BA pltes). c Some strins did not grow.

4 VOL. 4, 1976 ANAEROBIC PLATING MEDIA 397 AnO2BA pltes were superior to CMBA pltes. Furthermore, storge of AnO2BA pltes t 4 C hd less of n effect on colony size thn storge of pltes under constnt flow of CO2. The reson for the gretly incresed verge colony size for the Fusobcterium species grown on the 4 C AnO2BA plte over those grown on fresh AnO2BA is not cler t this time, but ws possibly due to the presence of unstble inhibitory substnces for this bcterium in the fresher medium. The effect of holding pltes under CO2 for periods up to 72 h ws studied. One set of AnO2BA pltes ws stored in the CO2 holding chmber under constnt flow of CO2 for 72 h, nd nother set ws stored t 4 C in plstic bg for the sme period of time. The pltes were then streked nd fter 48 h of incubtion red s described erlier. Storge ofthe pltes under CO2 hd little effect on the colony size of the bcteri except for Peptostreptococcus nd possibly Bifidobcterium nd Propionibcterium species (Tble 4). The ph of CMBA nd AnO2BA ws determined fter 0, 2, 3, nd 12 dys of storge in ir nd under constnt flow of CO2. Storge under CO2 for up to 72 h or up to 12 dys in ir t room temperture resulted in minor ph chnges, but 12 dys of storge under CO2 cused more serious decreses in the ph of the medi (Tble 5). Since the bsic medium of the CMBA plte is Trypticse soy nd the bsic medium of the AnO2BA plte is BHI, it ws desirble to compre colony size on freshly prepred nonenriched TSA nd BHI blood gr pltes nd relte these results to colony sizes obtined on CMBA nd AnO2BA pltes run simultneously. For most species tested, the lrgest colonies were found on the AnO2BA pltes (Tble 6). The colonies on fresh nonenriched BHI pltes were slightly lrger thn those obtined on fresh nonenriched TSA pltes, but considerbly lrger thn the colonies on CMBA pltes. This ltter result suggests tht freshly prepred pltes re t lest s importnt s the enrichments dded to the AnO2BA medium. The one Peptostreptococcus species tested in TABLE 3. Comprison ofcolony size ofnerobic bcteri isolted on freshlyprepredano2ba pltes, CMBA pltes, AnO2BA pltes stored for 12 dys under constnt flow of CO2, nd AnOBA pltes stored t 4'C for 12 dys No. of Avg colony size (mm) on: OrgnisCBAmnOBA t CAnO2BA t 24'0 isoltes Orgni8m CMBA AnO2BA t 4 C for 12 dys under CO2 for 12 dys AnO2BA fresh 6 Bcteroides frgilis Fusobcterium species Clostridium perfringens Peptococcus species Peptostreptococcus species Veillonell species Bifidobcterium species Propionibcterium species Some strins did not grow. TABLE 4. Comprison of colony size of nerobic bcteri isolted on freshly prepred AnO2BA pltes stored for 72 h t 4 C nd AnOBA pltes stored for 72 h t 24 C under constnt flow of CO2 % Increse in Pltes stored colony size on No. of isoltes Orgnism Pltes t 40C for under CO2 for pltes t 72 h 4on 72 h over pltes stored under C02 6 Bcteroides frgilis Fusobcterium species Peptococcus species Peptostreptococcus species Bifidobcterium species l 3 Propionibcterium species The verge colony size for Bifidobcterium species ws 11% lrger on the pltes stored under C02 thn on the pltes stored t 40C.

5 398 HANSON AND MARTIN this experiment gve lrger colonies on TSA nd BHI thn on AnO2BA. It is lso importnt to note tht the strin offusobcterium necrophorum tested filed to grow on CMBA nd TSA, both of which contin the sme bsl medium. DISCUSSION Except for few specilized techniques, nd equipment uniquely required for the cultivtion of nerobic bcteri, the methods described here differ little from those used in routine dignostic bcteriology (6). One of the importnt considertions for good nerobic bcteriology is the use of fresh rich medium. The results of this study clerly indicte tht more luxurint growth (s reflected by colony size) cn be obtined on freshly prepred rich medi. Recently Wilkins et l. (9) suggested tht the presence of blood in gr medi exerts n inhibitory effect on the growth of some strins of B. frgilis. The inhibition ws reversed by the ddition of hemin. This observtion my prtilly explin the smller colony size obtined on CMBA to which no hemin ws dded, but cnnot explin the results from the TABLE 5. ph of CMBA nd AnO2BA pltes fter 0, 2, 3, nd 12 dys of storge in ir nd under constnt flow of CO2 ph t: Medium 0 dys 2 dys 3 dys 12 dys CMBA In ir t 24 C Under CO, t 24 C AnO2BA In ir t 24'C Under C02 t 24 C TABLE 6. non-hemin-contining BHI nd TSA blood gr pltes. Only with some of the Fusobcterium species were the colonies lrger on CMBA thn on fresh AnO2BA pltes. The presence of the lrger Fusobcterium colonies on the poorer medium is difficult to explin, but it my indicte tht the dded enrichments lso contin inhibitors to the growth of these bcteri. On the other hnd, severl of the Fusobcterium species isolted filed to grow on the CMBA pltes. Neither plte ws found to be better thn the other in its bility to grow reltively lrger numbers of given bcterium. Ellner et l. (2) were ble to cultivte greter numbers nd lrger colonies ofpeptococcus nd Fusobcterium species when blood gr pltes were exposed to n nerobic tmosphere before inocultion. These investigtors dded plldium chloride to keep the pltes reduced during severl weeks of erobic storge. In this study, cysteine hydrochloride ws dded s reducing gent but, more importntly, fresh pltes were used. The fct tht blck pigmenttion of B. melninogenicus colonies occurred erlier on CMBA thn on AnO2BA is surprising since blood gr pltes enriched with hemin enhnce this chrcteristic (7). However, the slower pigmenttion ofb. melninogenicus on AnO2BA ws not considered mjor drwbck, since the presence of these orgnisms cn usully be detected before pigmenttion occurs by scnning the pltes with long-wvelength ultrviolet light nd looking for brick-red fluorescence. As noted in Results, this fluorescence ws found to be more intense on the AnO2BA thn on the CMBA pltes. Since the AnO2BA pltes were either inocu- Comprison ofcolony size ofnerobic bcteri isolted on freshly prepred AnO?BA pltes, CMBA pltes, freshly prepred BHI pltes, nd freshly prepred TSA pltes % gravg col- % gravg col- %Lre Avg col- Lrger ony % Lrger ony size % Lrger Avg col- No. of on oysz (mmnnn (mm)aoa onoba ony size isoltes Orgnism (mym) on An%BA nonen- AnO,BA nonen- AnO,BA (mm)o CMBA thmba riched thn riched tbhi AnO2BA CMA TSA TSA BHI J. CLIN. MICROBIOL. 2 Bcteroides frgilis B. melninogenicus Fusobcterium necropho- NG NG rum 1 F. mortiferum Clostridium species Peptococcus species b Peptostreptococcus species c c 1.50 NG, No growth. b The Peptococcus species were lrger on BHI thn on AnO2BA. c The Peptostreptococcus species ws lrger on TSA nd BHI thn on AnO2BA. BH

6 VOL. 4, 1976 lted immeditely fter preprtion or held under flow of CO2 for no longer thn 48 to 72 h, it ws desirble to test the effect ofthis procedure on the growth of the bcteri. Apprently only the Peptostreptococcus were ffected to ny extent (Tble 4). On the other hnd, AnO2BA pltes tht were stored under CO2 for 12 dys were mrkedly less effective for growing bcteri when compred with the fresh AnO2BA pltes, lthough still better thn the CMBA pltes. Storge of AnO2BA pltes t 40C for 12 dys hd miniml effect on the growth of the nerobes used in this study, notwithstnding the frequent references to the possible formtion of orgnic peroxides (7) nd incresed 02 bsorption t colder tempertures. The AnO2BA held for 12 dys t room temperture under CO2 showed the poorest growth of ny of the AnO2BA pltes. The most likely explntion for this observtion is probbly twofold: the drying out of pltes over the 12-dy period, nd the formtion of crbonic cid due to the continuous exposure to CO2. Regrdless of buffering effects in the medium, stedy decrese in ph ws found to occur with incresed storge periods of the pltes under C02 (Tble 5). Freshly prepred nonenriched TSA nd BHI pltes supported firly good growth compred with tht obtined with the AnO2BA pltes nd very good growth compred with tht on the CMBA pltes. The results of this experiment emphsize the importnce of using freshly prepred s well s enriched medi for the isoltion nd growth of nerobic bcteri. Another commonly used medium bse is brucell broth (8); however, its bility to grow nerobic bcteri reltive to BHI nd TSA ws not investigted in this study. ANAEROBIC PLATING MEDIA 399 Since the results of this investigtion clerly show tht fresh rich medium mrkedly enhnces the growth of nerobic bcteril isoltes, we re now in position to expnd the scope of these studies in terms of ssessing the quntittive recovery of nerobic bcteri from routine clinicl specimens. It is obvious tht such informtion would be invluble to clinicl microbiologists nd clinicins in the mngement of nerobic infections. LITERATURE CITED 1. Brewer, J. H., nd D. L. Allgeier Sfe self-contined crbon dioxide-hydrogen nerobic system. Appl. Microbiol. 14: Ellner, P. D., P. A. Grntto, nd C. B. My Recovery nd identifiction of nerobes: system suitble for the routine clinicl lbortory. Appl. Microbiol. 26: Holdemn, L. V., nd W. E. C. Moore Anerobic bcteriology mnul. Virgini Polytechnic Institute nd Stte University, Blcksburg. 4. Mrtin, W. J Prcticl method for isoltion of nerobic bcteri in the clinicl lbortory. Appl. Microbiol. 22: Mrtin, W. J Isoltion nd identifiction of nerobic bcteri in the clinicl lbortory. Myo Clin. Proc. 49: Smith, L. DS Introduction to nerobic bcteri, p In E. H. Lennette, E. H. Spulding, nd J. P. Trunt (ed.), Mnul of clinicl microbiology, 2nd ed. Americn Society for Microbiology, Wshington, D.C. 7. Smith, L. DS The pthogenic nerobic bcteri. Chrles C Thoms Co., Springfield, ill. 8. Sutter, V. L., H. R. Attebery, J. E. Rosenbltt, K. Bricknell, nd S. M. Finegold Anerobic bcteriology mnul. Extension Division, University of Cliforni, Los Angeles. 9. Wilkins, T. D., S. L. Chlgren, F. Jimenez-Ulte, C. R. Drke, nd J. L. Johnson Inhibition ofbcteroides frgilis on blood gr pltes nd reversl of inhibition by dded hemin. J. Clin. Microbiol. 3: