JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1975, p. 30-36Copyright ( 1975 American Society for Microbiology

Vol. 1, No. 1Printed in U.S.A.

Practical Aerobic Membrane Filtration Blood CultureTechnique: Development of Procedure

NADINE M. SULLIVAN, VERA L. SUTTER, AND SYDNEY M. FINEGOLD*

Research and Medical Services, Wadsworth Hospital Center, Veterans Administration, Los Angeles, California90073,* and the Department of Medicine, University of California at Los Angeles School of Medicine, Los

Angeles, California 90024

Received for publication 24 October 1974

The advantages of a membrane filter system for blood culturing have beenrealized for many years. Lysing of the blood prior to filtration is a convenient wayto proceed, but previously described lysing procedures result in loss of certainorganisms, particularly gram-negative bacilli. Four concentrations of TritonX-100 and sodium carbonate were studied in vitro, and their lysing andantibacterial properties were observed. A solution of 0.08'7c; Na2CO3 and 0.005%Triton X-100 was found to have the least antibacterial effect and gaveconsistently good lysis and filtration times (under 3 min). An 8.3-ml amount ofblood added to 190 ml of this concentration of lysing solution, filtered throughthree 47-mm membrane filters (0.45-,m pore size), led to recovery of 85% or moreof various aerobic and facultative organisms in studies of artificially seededblood.

Despite the availability of excellent antimi-crobial drugs, the mortality from bacteremiaremains high, especially when shock accom-panies sepsis. It is imperative that the identifi-cation and susceptibility of the infecting orga-nism be determined as early as possible in thecourse of bacteremia, since it has been observedthat early administration of the appropriateantimicrobial drug greatly improves thechances for survival of the patient.Our group has been studying different ap-

proaches for more efficient blood culturing andhas shown several advantages of the membranefilter technique over conventional blood cultureprocedures for the rapid diagnosis of bacteremia(4, 8, 11). The advantages of the membranefilter procedure include faster growth, ability toremove inhibitory agents (normal bactericidalfactors and drugs which have been adminis-tered), and growth in the form of discretecolonies on the filter, which facilitates fasteridentification of the infecting organism. Theearlier procedures (2, 10, 12) for membranefiltration have been too cumbersome and timeconsuming for practical clinical use. The hemol-ysis of blood cells was tried many years ago byBraun and Kelsh (2) and by Tidwell and Gee(10); however, it was only possible to filter verysmall quantities of blood with their procedures.An efficient lysing system utilizing 0.8% Na2CO3and 0.05% Triton X-100 was introduced morerecently (7). However, two important difficul-

ties with this system were an antibacterialeffect of the lysing solution on gram-negativebacilli and certain sensitive gram-positive orga-nisms (3) and significant filtration difficultiesrelated to plugged filters. Unpublished studiesin our laboratory of the lysing procedure of Roseand Bradley (7) revealed the same problemsdescribed by Farmer and Komorowski (3). Ourapproach to overcoming these problems in-volves use of a system employing a larger filterarea and the utilization of lower concentrationsof sodium carbonate and Triton X-100(Na2CO3-TX- 100).The procedure which we have developed has

the advantage of being accurate and yet simpleenough for practical use in the clinical labora-tory. It minimizes the problems of organism lossand filter plugging.

MATERIALS AND METHODSEffect of various lysing solutions. Four concen-

trations of Na2CO3-TX-100 were tested: (i) 0.4%Na2CO3-0.025% TX-100; (ii) 0.16% Na2CO3-0.01%TX-100; (iii) 0.12% Na2CO3-0.007% TX-100; and (iv)0.08% Na2CO3-0.005% TX-100. The pH of thesesolutions was 10.0 to 10.3. Blood (8.3 ml) was collecteddirectly into a Vacutainer tube containing sodiumpolyanethol sulfonate (SPS, 1.7 ml of 0.35% solution;this yields a final concentration of 0.06%) (BD Inc.)and mixed. The blood was then added via syringe to190 ml of the appropriate Na2CO3-TX-100 (Rohm andHaas) solution. The solution was mixed until goodlysis was observed (about 1 min) and then filtered,

30

on March 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

VOL. 1, 1975 AEROBIC MEMBRANE FILTRATION FOR BLOOD CULTURING

using a vacuum of 25 inches (ca. 64 cm) of Hg per min.A 100-ml wash with saline followed the initial filtra-tion in certain studies. The filter first used in thesystem was a 90-mm membrane filter (0.45-Mm poresize; Millipore Corp.), but later studies employed adisposable unit containing three 47-mm filters (0.45-Mm pore size; Gelman). This unit (kindly supplied byMcGaw Laboratories, Glendale, Calif.) is shown inFig. 1.

In vitro studies were done with each of the foursolutions. Test organisms from human clinical mate-rial (Escherichia coli, Streptococcus pneumoniae, andStaphylococcus aureus) were diluted so as to yieldapproximately 100 to 300 organisms/ml (counts veri-fied in triplicate by pour plates in all experiments).One milliliter of test organism was added to theSPS-containing tube, which already contained 8.3 mlof normal human blood. The contents of this tubewere then added to 190 ml of the test lysing solution,mixed for 1 min, and then filtered. The filters weresubsequently placed on blood agar plates and incu-

FIG. 1. Filtration unit (McGaw) with three mem-brane filter holders. The lysed blood specimen isdivided evenly between the three filters.

bated aerobically at 35 C. Colonies were counted after24 h.

Effect of prolonged exposure of organisms toleast harmful lysing solution. To establish themaximal length of time organisms could remainviable in the lysing solution, a killing curve experi-ment was performed. E. coli, Klebsiella pneumoniae,Pseudomonas aeruginosa, and S. aureus were tested.Relatively small numbers of each test organism wereadded directly to 190 ml of the lysing solution. Three1-ml pour plates were made from each test setup atthe following time intervals: 0 (control), 5, 10, 15, 30,and 60 min. One milliliter of the solution was added to19 ml of melted cooled Brucella agar for the pourplates. These were incubated aerobically at 35 C andcounted after 24 h.Need for wash after flltration. In our earlier

filtration procedures (utilizing smaller volumes offluid during filtration), it was found necessary to washthe filter because residual antibiotics and normalserum inhibitory factors remained on the filter (4, 12).

Figure 2A from earlier studies with our previoustechnique shows a filter, improperly washed, withresidual antibiotic. There is inhibition of the indicatororganism by antibiotic in the filter strip in the centerof the petri dish. Figure 2B shows a similar setup withproper washing. The necessity of a wash for thecurrently described procedure (using 190 ml of the0.08% Na2CO3-0.005% TX-100 lysing solution) wastherefore tested. Four antibiotics were used in concen-trations higher than or equal to those normallyattained in serum during antimicrobial therapy. Thetest organisms and antibiotics were: E. coli and 20 Mgof chloramphenicol per ml, group A Streptococcusand 2 Mg of penicillin G per ml, Enterobacteraerogenes and 20 Mg of chloramphenicol per ml, E.aerogenes and 30 Mg of tetracycline per ml, and P.aeruginosa and 100 Mig of polymyxin B per ml. Thebacterium (to be used as an indicator of residual anti-microbial activity) was diluted to 101 organisms/mlin broth and then spread evenly over a blood agarplate with a cotton swab. The corresponding anti-biotic, in the appropriate concentration, was addedto two Vacutainer-SPS tubes containing 8.3 ml ofnormal human blood. The tubes were mixed, addedto the lysing solution, mixed well for 1 min, and thenfiltered. One filtration in each pair was followed by

_ i ~~~~B

FIG. 2. Need for wash after filtration with old membrane filter technique. (A) Unwashed filter has adsorbedantibiotic from blood specimen and inhibits test organism on plate; (B) washed filter; antibiotic has beenwashed away so there is no longer inhibition of test organism.

31

on March 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

SULLIVAN, SUTTER, AND FINEGOLD

filtration of 100 ml of normal saline; the other was not.The filters were cut in half and placed on the bloodagar plates which had been seeded with sensitiveindicator strains and incubated aerobically at 35 C.Control broths containing 105 organisms/ml and thecorresponding antibiotics in the same concentrationswere incubated at the same time as the filters.

RESULTS

Effect of various lysing solutions. Ten to 20filtrations were run with each of the four con-

centrations of Na2CO3-TX-100, and the filtra-tion time was noted. The average filtration timewith the 0.4% Na2CO3-0.025% TX-100 was 41 s;

with the 0.16% Na2CO3-0.01% TX-100, 61 s;

with the 0.12% Na2CO3-0.007% TX-100, 42 s;

and with the 0.08% Na2CO3-0.005% TX-100, 42s. The slowest filtration time was always under3 min in each group.

The results of the in vitro experiments withthe four concentrations of Na2CO3-TX-100 are

given in Table 1. The gram-positive cocci (S.pneumoniae and S. aureus) were affected little,if at all, by even the strongest lysing solutiontested. E. coli, however, sustained significantkilling in the higher concentrations of lysingsolution and showed only a small loss, if any,

with the lowest concentration. One study em-

ploying E. aerogenes was done and, althoughonly a minor bacterial loss was observed even atthe highest concentration, colonial morphologywas somewhat distorted at this concentration.

Effect of prolonged exposure of the orga-nism to the least harmful lysing solution.Figures 3 and 4 show the results from the killingcurve experiments. The mean number of colo-

nies per 1-ml pour plate is plotted against thetime the organisms were exposed to the lysingsolution. It was found that a small loss some-times occurred between 10 and 15 min, but thatnot until 30 to 60 min was there an appreciableloss of organisms (and then only with two of thefour test organisms, E. coli and K.pneumoniae).Need for wash after filtration. A zone of

inhibition around the filter and no growth underthe filter would be observed if residual antibi-otic remained on the filter. Failure of growth inthe broth control would verify susceptibility ofthe indicator strain to the corresponding drug.Table 2 shows the results of the experiment. Inall cases there was no growth in the brothcontrol containing the test organism plus theantibiotic, and full growth was observed underand around all filters. Therefore, with thepresent procedure, washing is unnecessary.

DISCUSSIONThe need for better techniques for the diagno-

sis of bacteremia has been appreciated for manyyears. The conventional broths used today groworganisms relatively slowly, and additional timeis required for subculture before even prelimi-nary identification can be determined.The potential of the membrane filter system

in preventing the rapid killing of bacteria by thenormal bactericidal factors in serum (5) and inavoiding inhibition or killing by residual an-

timicrobial compounds which cannot be inacti-vated has been recognized for some time. Thereare also certain other desirable features of the

TABLE 1. Effect of four concentrations of Na2CO,-TX-100 on certain organismsAvg recovery (%)

(range)Concn of lysing solution E. coli S. S. aureus Gross colonial morphology

(6 strains) pneumonae (2 strains)(3 straihis)

0.4% Na2CO3-0.025% TX-100 20.6 100 93 Gram-positive organisms: mor-(40-9) (100) (100-85) phology typical; E. coli and

E. aerogenes: morphologydistorted (colonies spreadout)

0.16% Na2CO3-0.01% TX-100 50.6 -a 85 Typical in all cases(75-33) (100-75)

0.12% Na2CO3-0.007% TX-100 59 98 -a Typical in all cases(75-50) (100-95)

0.08% Na2CO3-0.005% TX-100 86.4 100 100 Typical in all cases(100-60) (100) (100)doneatthisconcentra

a No studies done at this concentration.

32 J. CLIN. MICROBIOL.

on March 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

VOL 1, 1975 AEROBIC MEMBRANE FILTRATION FOR BLOOD CULTURING

membrane filter technique, such as rapidgrowth of organisms as colonies with typicalmorphology. Earlier filter systems did demon-

60

50_/

40-

E 30-

,26-z

0

erginOS{I

221

14/i

45N T S.a10-~~~~

10 20 30 40 50 60

TIME ( MINUTES)

FIG. 3. Effect of least harmful lysing solution(Of$0% Na2CO3-0.005% TX-100) versus P. aeruginosa,E. coli, and S. aureus as a function of time. There isno antibacterial effect for at least 15 min.

S.

z

C-

z0

J

z0

strate the ability of the technique to provideearly detection and identification of organismscausing bacteremia (12); however, the problemsof a cumbersome system and inability to filteran adequate volume of blood limited the practi-cal application of this technique. A later system(7) permitted use of somewhat larger volumes ofblood, but filter plugging was still a problemand there was significant loss of certain orga-nisms caused by the solution used to lyse theblood in this system.

10 20 30 40 50 60TIME ( MINUTES )

FIG. 4. Effect of least harmful lysing solution ver-

suts Klebsiella pneumoniae as a function of time.There is no antibacterial effect for the first 15 min.

TABLE 2. Studies concerning need for wash after filtration procedure

Normal saline wash(100 ml) No wash

Test organism AAntibiotic Zone of Growth Zoneof Growth Control(MUg/ml) inhibition r inhibition udr biotic

around under around under antbotcfilter filter filter filter

E. coli Chloramphenicol (20) None + None + No growthGroup A Streptococcus Penicillin G (2) None + None + No growthE. aerogenes Chloramphenicol (20) None + None + No growthE. aerogenes Tetracycline (30) None + None + No growthP. aeruginosa Polymyxin B (100) None + None + No growth

u

z0

0u

33

on March 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

SULLIVAN, SUTTER, AND FINEGOLD

Using a smaller concentration of Na2CO3 andTX-100 and a larger filter area, we have usuallybeen able to recover 85 to 100% of the organismsused in artificial bacteremias. Since there maybe very few organisms per milliliter of blood inclinical bacteremia, particularly when patientsare on antimicrobial therapy (4), the more bloodwhich is cultured the better the chance ofdetecting a low level bacteremia. In our presentsystem, using the commercially available Vacu-tainer tube with SPS (BD, Inc.), 8.3 ml of bloodcan be filtered rapidly. The entire procedure forprocessing the specimen from the time of blooddrawing ordinarily takes only 2 to 5 min. TheSPS in the tube is an anticoagulant; it promptlyneutralizes the normal bactericidal power ofblood, interferes with phagocytosis, and inacti-vates certain antibiotics (1, 4, 11). Sodiumamylosulfate shows the same types of activitiesas SPS and may offer some advantages (6).With our system, filtrations taking longer

than 10 to 15 min might lead to loss of orga-nisms. However, such filtration problems areextremely rare. It should be pointed out that thelate loss of gram-negative bacteria in lysingsolutions may be related simply to the pH itselfof the lysing solution, rather than to the specificingredients.With the large volume of lysing solution used

it proved unnecessary to wash the filter toremove residual inhibitory substances.Two clinical blood culture studies have been

done using this system. In one study, 300patients undergoing genitourinary tract manip-ulation were studied for transient bacteremias(8). Various blood culture systems were com-pared. The membrane filter system did not doas well as a prereduced osmotically stabilizedbroth in terms of total number of recoveries, butpositive cultures were detected fastest in ninecultures by this procedure. The other study,reported in the accompanying paper (9), dem-onstrated the advantages of the improved mem-brane filter system; it far surpassed the othersystems, both in total recovery of organisms andin speed of detection of the bacteremia.We were working primarily with uninfected

people in the first clinical blood culture studyand had no problems with the filtration process.However, in the second study we noted filtersplugging on occasion. The blood of patients withhigh leukocyte counts gelled in the lysing solu-tion, thereby causing the filter to plug before allof the solution could be filtered. A commercialstreptokinase-streptodornase (SK-SD) com-pound (Varidase, Lederle Laboratories) addedto the lysing solution-blood mixture resulted in

breaking up of the gelled mass and permittedfiltration without problem. Further details onthis problem and its solution are presented inthe accompanying paper (9).A membrane filter setup comprised of three

47-mm filters (0.45-Mm pore size) and a lysingsolution of 0.08% Na2CO3-0.005% TX-100 plusSK-SD constitutes an excellent system for thediagnosis of bacteremia due to aerobic andfacultative organisms. Since this system has notyet been tested with anaerobes, it is suggestedthat an appropriate broth for anaerobes beincluded in the system. There are a few impor-tant points that must not be overlooked if thesystem is to function properly.

(i) The blood which has been anticoagulatedwith SPS must not be refrigerated and must befiltered before 6 h to avoid coagulation. One ofthe major advantages of the system is to greatlyexpedite detection of bacteremia. Since speedin diagnosis and therapy is a major factor inimproving the patient's prognosis, specimensshould always be processed immediately when-ever possible.

(ii) The lysing solution must be at roomtemperature before use; below 16 C it does notfunction properly.

(iii) Only 3 ml of SK-SD (of a 1:20 dilution inwater) should be added to the 190 ml of lysingsolution (add immediately after the blood); 9 mlor more of SK-SD is bactericidal.

APPENDIX

The complete procedure for the membrane filtersystem is as follows.An 8.3-ml amount of blood is collected directly into

a Vacutainer tube (BD, Inc.) containing 1.7 ml of a0.35% SPS solution and mixed immediately. TheVacutainer tube stopper is decontaminated by vigor-ous scrubbing and a 2-min exposure to 70% alcohol.The blood is then injected into 190 ml of 0.08%Na2CO3-0.005% TX-100 (Rohm and Haas) solution(Fig. 5) after first removing the protective metal cap(similar to that on a standard bottle of solution forintravenous administration). If the patient is knownto have an elevated leukocyte count, addition of 3 mlof a 1:20 dilution of SK-SD in water to the lysingsolution is necessary. This solution is mixed for 1 min.The rubber diaphragm over the stopper of the lysingsolution bottle is removed. The protective cap isremoved from the spike at the bottom of the filterunit. The spike is then pushed through the centraldepression in the stopper of the bottle of lysingsolution. A vacuum hose is attached to the port on topof the filter unit and a vacuum of 25 inches (ca. 64 cm)of Hg is applied. The unit contains three 47-mmfilters (0.45-pm pore size) (Fig. 6). A 90-mm filter(0.45-gm pore size) may also be used. When the

34 J C LIN . MICROBIOL .

on March 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

VOL. 1, 1975 AEROBIC MEMBRANE FILTRATION FOR BLOOD CULTURING

FIG. 5. The blood is being transferred from theVacutainer tube to a bottle of lysing solution.

, ' '........

FIG. 7. After filtration of the lysed blood, the filtersare removed aseptically.

FIG. 8. The filter is rolled onto the surface of agarmedia in petri dishes.

FIG. 6. The filtration unit is attached to the bottleof lysing solution (via a plastic spike), and vacuum isbeing used to draw the lysing solution-blood mixturethrough the filters.

solution is completely filtered, the vacuum is releasedslowly to prevent the filters from puffing. The filtersare aseptically taken from the unit (Fig. 7) and rolledonto the solid media to' insure proper contact withoutair pockets (Fig. 8). If a 90-mm filter is used, the filtercan be cut, aseptically, into thirds. The media werecommend are: a chocolate blood agar plate under10% CO,, a blood agar plate under 10% CO., and aneosin-methylene blue or MacConkey plate aerobi-cally. Incubation is at 35 to 37 C.The filters are observed periodically; positive cul-

tures can be recognized as early as 9 to 12 h in certainbacteremias. Most positive cultures will be evident onthe filters within 1 to 4 days, but filters are notdiscarded until at least 14 days of incubation. Colo-nies on filters are observed carefully for uniquemorphological features. Hemolysis beneath the filteris observed from the under surface of the petri dish orby gently lifting the filter to observe the agar under-neath. Colonies are then used for various rapidbiochemical and other identification procedures andare subcultured for conventional identification andsusceptibility studies.

ACKNOWLEDGMENTS

This work was supported by McGaw Laboratories, Glen-dale, Calif., Division of American Hospital Supply Corpora-tion, who also supplied the special filter units as well as cer-tain other materials.We would like to express our appreciation to Don Bobo and

Gary Stroy for many helpful suggestions and to SidneyRosenblatt for technical assistance.

35

on March 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

36 SULLIVAN, SUTTER, AND FINEGOLD

LITERATURE CITED

1. Belding, M. E., and S. J. Klebanoff. 1972. Effect ofsodium polyanethol sulfonate on antimicrobial systemsin blood. Appl. Microbiol. 24:691-698.

2. Braun, W., and J. Kelsh. 1954. Improved method forcultivation of Brucella from the blood. Proc. Soc. Exp.Biol. Med. 85:154-155.

3. Farmer, S., and R. A. Komorowski. 1972. Evaluation ofthe Sterifil lysis-filtration blood culture system. Appl.Microbiol. 23:500-504.

4. Finegold, S. M., M. L. White, I. Ziment, and W. R. Winn.1969. Rapid diagnosis of bacteremia. Appl. Microbiol.18:458-463.

5. Khairat, 0. 1946. The bactericidal power of the blood forthe infecting organism in bacteremia. J. Pathol. Bacte-riol. 58:359-365.

6. Kocka, F. E., E. Arthur, R. L. Searcy, M. Smith, and B.Grodner. 1973. Clinical evaluation of sodium amylosul-fate in human blood cultures. Appl. Microbiol.26:421-422.

7. Rose, R. E., and W. J. Bradley. 1969. Using the mem-

J. CLIN. MICROBIOL.

brane filter in clinical microbiology. Med. Lab.3:22-23, 29, 43.

8. Sullivan, N. M., V. L. Sutter, W. T. Carter, H. R.Attebery, and S. M. Finegold. 1972. Bacteremia aftergenitourinary tract manipulation: bacteriological as-pects and evaluation of various blood culture systems.Appl. Microbiol. 23:1101-1106.

9. Sullivan, N. M., V. L. Sutter, and S. M. Finegold. 1975.Practical aerobic membrane filtration blood culturetechnique: clinical blood culture trial. J. Clin. Micro-biol. 1:37-43.

10. Tidwell, W., and L. L. Gee. 1955. Use of membrane filterin blood cultures. Proc. Soc. Exp. Biol. Med.88:561-563.

11. Traub, W. H., and B. L. Lowrance. 1970. Anticomple-mentary, anticoagulatory, and serum protein precipi-tating activity of sodium polyanethol sulfonate. Appl.Microbiol. 20:465-468.

12. Winn, W. R., M. L. White, W. T. Carter, A. B. Miller,and S. M. Finegold. 1966. Rapid diagnosis of bactere-mia with quantitative differential-membrane filtrationculture. J. Amer. Med. Ass. 197:539-548.

on March 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from