J Clin Pathol 1986;39:672-676

Technique for measuring 50% end points incytotoxicity assays for Clostridium difficile toxinsSARA W ROTHMAN

From the Department ofBiological Chemistry, Walter Reed Army Institute ofResearch, Washington, DCUnited States

SUMMARY Serial dilutions of Clostridium difficile culture filtrates were incubated overnight withHeLa cell monolayers. Cells were fixed in formalin, stained with crystal violet, rinsed, and drained.Cell rounding could be observed microscopically in the stained monolayers. Absorbance of theretained dye on monolayers in the drained wells was measured at 595 nm-405 nm. End points couldalso be estimated visually. The dilution at which dye absorbance was reduced by 50% agreed withthat determined by microscopic observations. Five replicate dilution series showed high re-producibility. Specificity was verified by neutralisation with crude rabbit antibody to C difficiletoxins. Cytotoxicity in faecal specimens was assayed in the same way, allowing reporting of titres,comparison with standard toxin preparations, and determination of the extent of neutralisation tobe made. This novel assay technique has proved effective and reliable in a clinical setting and shouldallow the gathering of more information on the epidemiology of antibiotic associated colitis.

Determination of endpoints in the tissue culture assaycurrently used in clinical laboratories for recognisingClostridium difficile toxins in faecal specimens is cum-bersome and subjective, entailing the laboriouscounting of cells with cytopathogenic effects.15 Un-til an objective and accurate quantitative assay be-comes available, the predictive value of the toxin titrein the clinical course of the disease will not be accu-rately assessed. Some laboratories have tried tosubstitute other assays, including counter-immunoelectrophoresis. The usefulness of thismethod for C difficile toxin assays is questionable.6-8Recently, an elegant enzyme linked immunosorbentassay (ELISA) for toxins A and B has beendeveloped9 and will certainly be the preferred methodin the future. At present, its widespread use is re-stricted because monospecific antibodies for toxins Aand B are available in only very few laboratories. Thelack of specific antibodies results in a high percentageof false negative readings in the latex agglutinationassay.'0 The tissue culture assay is still the mostwidely useful for detecting C diflicile toxins as it canbe carried out without the use of either purified toxinsor monospecific antibody.

In purifying C difficile toxins A and B, I developeda technique for easily and accurately measuring end-points in the tissue culture assay by reading absorb-

Accepted for publication 23 January 1986

ance of stained monolayers in a microtitre platereader." I have now explored further the quan-titation of this assay, the possibility of reproduciblemeasurement of a 50% end point, and the usefulnessof the technique for identifying and assaying Cdifficile toxins in clinical specimens.

Material and methods

TOXIN AND ANTITOXINCrude C difficile culture filtrates were used as toxinpreparations. Preparation of filtrates and rabbit anti-toxin were used as previously described." 12

CELLS AND CULTURE CONDITIONSHeLa cells and maintenance conditions have been de-scribed previously.'3 To prepare assay plates HeLacells were suspended from monolayers by treatmentwith trypsin and edetic acid, counted, and adjusted toa concentration of 160 000 cells/ml in growth mediumconsisting of Eagle's minimum essential medium withEarle's salts (MEM) (HEM Research, Rockville,Maryland) supplemented with 10% heat inactivatedfetal bovine serum, 2 mM glutamine, 300 U of peni-cillin, and 300 Mg streptomycin/ml. One tenth mlsamples were pipetted into 96 well microtitre plates(Costar, Cambridge, Massachusetts). Monolayerswere established by 24 hours' incubation at 35°C in a5% carbon dioxide atmosphere.

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Fig. 1 Microscopic appearances ofstained intoxicated HeLa cell monolayers. HeLa cell monolayers were incubatedovernight after addition of0 1 ml ofstool extract (b, d). One tenth ml ofphysiological saline was added to controlmonolayers (a, c). Cells werefixed and stained with crystal violet (c, d). Unfixed cells are shown in (a) and (b).Phase-contrast, original magnification x 160, Kodak No II filter (yellow-green).

CYTOTOXICITY ASSAYFour fold serial dilutions of toxin were prepared;MEM was used as diluent. One tenth ml sample of adilution was added per well, and the plates were incu-bated overnight. Negative controls consisted of 0O I mlMEM. For neutralisation serial twofold dilutions ofrabbit antiserum in MEM were mixed with an equalvolume of C difficile toxin at a 1/16 dilution. Thisdilution was chosen because in my experience positive

patient specimens have always been cytotoxic at thisdilution. Negative antibody controls were preparedby mixing antibody dilutions with equal volumes ofMEM. The toxin dilution used in the neutralisationassay was mixed with an equal volume ofMEM as apositive toxin control. One tenth ml samples of toxin-antitoxin mixtures and controls were incubated withcell monolayers.

After incubation medium and toxin were emptied

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from the plate by vigorous shaking. Cells were fixedwith a 2% solution of formalin in 0-067M phosphatebuffered saline (pH 7.2) for one minute; the fixativewas removed and the plates were stained with 0 13%crystal violet in 5% ethanol and 2% formalin andphosphate buffered saline for 15 minutes. Excess stainwas removed by careful water rinsing, and the plateswere air dried.

For quantitation of the endpoint the absorbance ofthe dye in the dried plates was measured at 595 nm ina microtitre plate reader (Multiskan MC; Flow Labo-ratories, McLean, Virginia). Absorbance at 405 nmwas subtracted so that anomalies in the plastic wouldnot affect accuracy. The toxin dilution, resulting in a50% decrease in absorbance, was defined as the 50%cytotoxic dose (CD50) and was chosen as the endpoint for the assay. To show that cells did not detachfrom the monolayer 12 control and 12 intoxicatedmonolayers were fixed and stained as describedabove. The dye was dissolved in 50% ethanol, 1%sodium dodecyl sulfate, and the absorbance of thedye solution was measured in the microtitre platereader. Previous studies have shown that dye concen-tration, as determined by absorbance measurements,is proportional to the number of cells remainingattached to the surface of the well after intoxi-cation.1415 There was no decrease in dye concen-tration in intoxicated monolayers, proving that cellshad not detached.

Results

APPEARANCE OF STAINED INTOXICATED CELLSStaining of cell monolayers in the cytotoxicity assayfor C difficile toxin was used to avoid the laboriouscounting of cytopathic effects. After overnight incu-bation the toxin cells were fixed and then stainedwith crystal violet. Fig. I shows the microscopic ap-pearance of stained intoxicated cells. These cells wereheavily stained and, like the unstained cells, showedthe rounding typical of cytotoxic effects of C difficiletoxins. They had not detached, but they occupied arelatively small amount of the surface of the well. Thecontrol cells, on the other hand, were spread overmost of the plastic surface and also became heavilystained. The assay prepared in this way could bestored indefinitely at room temperature if protectedfrom light to prevent fading of the crystal violet.

QUANTITATION OF THE CYTOTOXICITY ASSAYReproducible 50% end points were quickly obtainedafter fixing and staining cells. Direct visual exam-ination of fixed and stained plates permitted rapidscreening for positive specimens and visual estimationof end points (Fig. 2 inset). The intoxicated cells al-lowed more light through than the controls because

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Fig. 2 Determination of50% cytotoxic dose. Five replicatefourfold dilutions ofcrude toxin were added to HeLa cellmonolayers, 0 1 mlper well (inset: Wells B-F 2, 4 -1; B-F1 1,41'°). Remaining wells were MEM controls. Cells werefixed and stained after overnight incubation and absorbanceofmonolayers was measured. Means and standard deviationsofreplicate dilutions are represented graphically. Meanabsorbance ofcontrol wells was 0 54 ((SD) 0 04), n = 10.

of the small amount of surface they covered, and soappeared lighter to the eye. Because of the differencein light absorbance between stained intoxicated andstained control cells, as measured in the microtitreplate reader, we were able to construct an absorbancecurve (Fig. 2). From the absorbance curve we calcu-lated the 50% absorbance value as follows: (max-imum absorbance minus minimum absorbance) /2 = 50% absorbance. The toxin dilution at the 50%absorbance point was obtained by extrapolating fromthis point on the curve to the log scale of the toxindilution on the X axis. The toxin dilution so deter-mined (4-6.5) contains 50% of the amount of toxinneeded to cause maximum rounding of all cells in themonolayer and is known as the 50% cytotoxic dose(CD50). The reproducibility of this assay is shown bythe small standard deviations apparent in the curvefor the four fold serial dilution. Five separate five foldserial dilutions of the same toxin preparation wereassayed on a different plate, and the mean 50% endpoint was exactly the same as that obtained with fourfold dilutions.

CLINICAL ASSAY FOR C DIFFICILE TOXINSFig. 3 shows the format of the microtitre plate assayfor the identification and quantitation of C didiMletoxins in clinical specimens. Four fold dilutions ofcrude toxin or stool filtrates permitted a visual esti-mation of the CD50's. In each case this was the 4-5

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1 2 3 4 5 6 7 8 9 10 11 12Fig. 3 Cytotoxicity assayfor detection and measurement ofC difficile toxins in humanfaecal specimens. Columns 1, 11,

and 12 are MEM controls; columns 2 and 3faecal specimentitration; columns 4 and 5 C difficile standard toxin titration;both arefourfold dilution series: row G, lowest dilution(4 '); row B, highest dilution (4 6). Columns 6 and 7 are Cdifficile antitoxin neutralisation assays; columns 8 and 9antitoxin controls. Twofold dilutions ofantitoxin were addedto equal volumes of 1/16 dilution offaecal specimen (6, 7) or

MEM (8, 9); row G lowest dilution, row B, highest dilution.Column 10 (E, F, G) isfaecal specimen control; 1/16 dilutionwas added to equal volume ofMEM. In all cases 0 1 mlvolume was addedper well.

(1/1024) dilution (Fig. 3). This result agreed with theend point read from the absorbance curve (notshown). The medium control could easily be seen asnegative and also had expected high absorbance. Theeffect of neutralisation by crude C difficile antitoxinwas also obvious by rapid visual inspection,confirmed by absorbance readings. The antibodycontrol was done to ensure that cytotoxicity by non-specific factors in the serum could not mask theneutralisation of toxin. For the specimen control incolumn 10 the dilution of stool prepared for the neu-tralisation assay was tested to ensure its toxicity andthe validity of the assay. Using this assay method,stool extracts can be tested for toxicity, identified ashaving C difficile toxins, and a titre can be estimatedimmediately on inspection of the plate.

Discussion

In this report I have described a rapid, accurate, andreproducible technique for measuring 50% end pointsin C difficile toxin assays. The technique is useful inassaying clinical specimens, allowing measurement oftoxin titres, and showing specificity through the use ofneutralising antibody, without any need to count cellsby light microscopy. Multiple determinations usingtwo different dilution series showed that the end pointwas reproducible. As noted by Reed and Muench,"6 a

50% end point is more reproducible than the 100%

675

end point previously used." 1 7 Accuracy is increasedby bypassing the need for judgment in assessing cyto-pathic effects. The assays can be quickly observed bylight microscopy to check that typical cell changes arepresent, or compared later with other specimens fromthe same patient. The assay can be used with crudeantibody, thus making it available to any laboratorythat can obtain tissue culture monolayers.The neutral red vital stain method of Finter'8 has

been used by Giugliano in assaying C difficile culturefiltrates.'9 Damaged cells take up less neutral redthan healthy ones, and determination of dye concen-tration allows an estimate of the extent of cell damageto be made. Unfortunately, this vital stain method iseight to 16 times less sensitive than microscopic exam-ination, although it is more precise. The assay re-ported here is not based on a vital stain, nor is it ameasure of dye concentration. The decrease in ab-sorbance in intoxicated monolayers is due to the mor-phological change caused by the cytotoxin. Theintoxicated cells round up and occupy a smaller sur-face area than the normal cells: this assay, therefore,directly measures the change in cell shape and is inagreement with microscopic observations.The technique reported here for assaying C difficile

toxins can be easily used by any laboratory with amicrotitre plate reader. The time needed to measuretoxin titres can be considerably reduced and accuracyenhanced. The data from the plate reader can easilybe transferred into a computer for analysis. This as-say method should permit an accurate assessment ofwhether higher stool titres are associated with moresevere disease and certain strains of C difficile in anti-biotic associated colitis.

I thank JE Brown for critical advice during the prepa-ration of this manuscript and BB Libys and LauraWatson for typing the manuscript.

References

'Aswell JE, Ehrich M, Van Tassell RL, Tsai C-C, Holdeman LV,Wilkins TD. Characterization and comparison of Clostridiumdifficile and other clostridial toxins. In: Schlessinger D, ed.Microbiology. Washington, DC: American Society for Micro-biology, 1979:272-5.

2Borriello SP. An evaluated micromethod for the detection of Clos-tridium difficile enterotoxin. Microbios 1978;7:25-8.

3Chang T-W, Lauermann M, Bartlett JG. Cytotoxicity assay inantibiotic-associated colitis. J Infect Dis 1979;140:765-70.

4George RH. A micro method for detecting toxins in pseudo-membranous colitis. J Clin Pathol 1979;32:303-4.

5 Rifkin GD, Fekety FR, Silva J Jr, Sack RB. Antibiotic-inducedcolitis-implication of a toxin neutralized by Clostridium sordelliiantitoxin. Lancet 1977;2:1103-6.

'Sands M, Yungbluth M, Sommers HM. The non-value of counter-immunoelectrophoresis for the direct rapid detection of Clostri-dium difficile toxin in stool filtrates. Am J Clin Pathol1983;79:375-7.

7Welch DF, Menge SK, Matsen JM. Identification of toxigenic

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Clostridium difficile by counterimmunoelectrophoresis. J ClinMicrobiol 1980;11:470-3.

8West SEH, Wilkins TD. Problems associated with counter-immunoelectrophoresis assays for detecting Clostridium difficiletoxin. J Clin Microbiol 1982;15:347-9.

9Laughon BE, Viscidi RP, Gdovin SL, Yolken RH, Bartlett JG.Enzyme immunoassays for detection of Clostridium difficile tox-ins A and B in fecal specimens. J Infect Dis 1984;149:781-8.

'° Shahrabadi MS, Bryan LE, Gaffney D, Coderre SE, Gordon R,Pai CH. Latex agglutination test for detection of Clostridiumdifficile toxin in stool samples. J Clin Microbiol 1984;20:339-41.

Rothman SW, Brown JE, Diecidue A, Foret DA. Differentialcytotoxic effects of toxins A and B isolated from Clostridiumdifficile. Infect Immun 1984;46:324-31.

12Rothman SW, Brown JE. Inhibition of membrane functions inintact HeLa cells by Clostridium difficile cytotoxic culturefiltrates. Current Microbiology 1981;6:221-4.

13Brown JE, Rothman SW, Doctor BP. Inhibition of protein syn-thesis in intact HeLa cells by Shigella dysenteriae 1 toxin. InfectImmun 1980;29:98-107.

"Gentry MK, Dalrymple JM. Quantitative microtiter cytotoxicity

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biological characterization of Shiga toxin from Shigella dys-enteriae 1. Infect Immun 1982;36:996-1005.

16 Reed LJ, Muench H. A simple method of estimating fifty percentendpoints. Am J Hyg 1938;27:493-7.

Sullivan NM, Pellett S, Wilkins TD. Purification and character-ization of toxins A and B of Clostridium difficile. Infect Immun1982;35:1032-40.

18Finter NB. Dye uptake methods for assessing viral cyto-pathogenicity and their application to interferon assays. J GenVirol 1969;5:419-27.

9Giugliano LG, Barer M, Mann GF, Drasar BS. Tissue culturesystems for the examination of bacterial virulence. In: Models ofanaerobic infection. Boston: Martinus Nijhoff, 1984:189-200.

Requests for reprints to: Dr Sara Rothman, Department ofBiological Chemistry, Walter Reed Army Institute of Re-search, Washington DC, 20307-5100, USA.

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