Vol. 58, No. 11APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1992, p. 3488-34930099-2240/92/113488-06$02.00/0Copyright X) 1992, American Society for Microbiology

Viability of Cryptosporidium parvum Oocysts: Correlation ofIn Vitro Excystation with Inclusion or Exclusion of

Fluorogenic Vital DyesA. T. CAMPBELL, L. J. ROBERTSON, AND H. V. SMITH*

Scottish Parasite Diagnostic Laboratory, Stobhill General Hospital,Springbum, Glasgow, Scotland G21 3UW

Received 9 April 1992/Accepted 3 August 1992

A viability assay for oocysts of Cryptosporidium parnum based on the inclusion or exclusion oftwo fluorogenicvital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide, was developed by using severaldifferent isolates of oocysts. Correlation of this assay with viability measured by in vitro excystation was highlystatistically significant, with a calculated correlation coefficient of 0.997. In this research, two similarexcystation protocols were utilized, and no significant difference between excystation protocols was detected.Percent excystation of oocyst suspensions could be increased or reduced by inclusion of a preincubationtreatment in either excystation protocol, and this alteration was also demonstrated in the viability assay.Oocysts which excluded both dyes would not excyst in vitro unless a further trigger was provided and weremore resistant to acid or alkali treatment. The results of this research provide a reproducible, user-friendlyassay which is applicable to individual oocysts and also provides a useful adjunct for identification of oocystsin water and environmental samples.

Transmission of protozoan parasites of the gastrointestinaltracts of humans, such as Giardia intestinalis and Crypto-sporidium parvum, via water is well documented (12, 26),and surveys of the occurrence of transmissive stages of theseparasites (cysts and oocysts, respectively) indicate thatthese stages occur commonly in the aquatic environment (9,25, 29). The public health significance of dead transmissive-stage organisms is minimal; however, when the organismsare infective, the risk to public health can be enormous.Hence, information about cyst and oocyst viability is ofconsiderable importance. To this end, methods for determin-ing the viability of cysts of Giardia spp. have receivedattention in recent years. Techniques used for assessing theviability and/or infectivity of Giardia cysts include in vitroexcystation (3, 19), exclusion or inclusion of vital dyes (2,11, 13, 14, 23, 24), and infectivity in animal models (17, 22,23). In general, cysts and oocysts occur in low numbers inthe aquatic environment, and the minimum infectious dose islikewise low (between 10 and 100 cysts or oocysts). It istherefore important to be able to assess whether individualorganisms isolated from water are viable. Assessment ofviability by in vitro excystation and infectivity of animalsrequires organisms in a relatively clean and concentratedsuspension, and these methods are therefore unsuitable forassessing the viability of individuals or of small numbers ofthe transmissive stage. Thus, exclusion or inclusion of vitaldyes and Nomarski differential interference contrast (DIC)microscopy are the techniques that have shown most prom-ise for assessing viability of individual Giardia cysts isolatedfrom water.Within the last 10 years, the importance of transmission of

C. parvum in water has been acknowledged worldwide. Sixoutbreaks of waterborne cryptosporidiosis have been docu-mented: four occurred in the United Kingdom, and twooccurred in the United States (1). In the United Kingdom,

* Corresponding author.

a group of experts have recommended that cryptosporidio-sis in humans become a reportable disease (1). Followingacceptance of the public health significance of both crypto-sporidiosis and the waterborne route of infection, attentionhas focused on development of methods which indicatewhether sporozoites contained within an individual oocystare viable or nonviable. The development of a reproducible,sensitive, user-friendly viability assay which could be usedto determine whether the sporozoites contained withinoocysts are capable of excysting and which would paralleland be compatible with in vitro excystation would be ofvalue. It should be applicable to individual or small numbersof oocysts, not only because oocysts may normally occur inlow numbers in potable water (26), but also because cur-rently recommended techniques for the isolation of oocystsfrom water are inefficient (9 to 59% recovery efficiency [26]).Such an assay would not only have an obvious applied use inlaboratories concerned with the detection of protozoa inwater but would also be a valuable tool in further research.A series of 27 fluorogenic dyes have been assessed (5) todetermine whether inclusion or exclusion of them correlatedwith viability as assessed by in vitro excystation. Of thesedyes, a simultaneous dual labeling with 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) showedsignificant promise.The aims of this communication are (i) to compare inclusion

or exclusion of the fluorogenic vital dyes DAPI and PI with invitro excystation, (ii) to assess the importance of knowntriggers of oocyst excystation such as incubation at 37°C withbile and acid induction on both efficiency of in vitro excysta-tion and fluorogenic dye inclusion or exclusion, and (iii) toassess the usefulness of these dyes as surrogate indicators ofviability for small numbers of C. parvum oocysts.

MATERLALS AND METHODS

Sources and purification of oocysts. C. parvum oocystswere obtained from the following sources. Purified cervine-

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ovine oocysts (c-o oocysts) were purchased from the More-dun Research Institute (MRI), Edinburgh, Scotland. Thisstrain, originally isolated from deer feces, has been passagedin sheep by MRI. Human oocysts were isolated from fecalsamples submitted from individuals with cryptosporidiosisfor routine examination to the Scottish Parasite DiagnosticLaboratory (SPDL), Stobhill Hospital, Glasgow, Scotland.Oocysts purified from each stool were kept as separateisolates. Bovine oocysts were isolated and purified frombovine fecal samples obtained either from a study farm byGlasgow Veterinary School or from MRI. Purified bovineoocysts were purchased from MRI. This isolate has beenpassaged in calves by MRI. Oocysts purchased from MRIhad been purified by a semiautomated method which in-volved incubation of the oocysts in 1% sodium dodecylsulfate and both acid sedimentation and sucrose flotation(30). Oocysts were obtained suspended in phosphate-buff-ered saline (PBS; pH 7.2) containing 100 U of penicillin and100 pg of streptomycin per ml.Oocysts from bovine and human fecal samples were

purified at SPDL as follows. Fecal samples (ca. 5 g) weremade up to 50 ml with reverse osmosis (RO) water, emulsi-fied by vortexing, and then centrifuged at 900 x g for 5 min,and the supernatant was removed by aspiration and dis-carded. This washing procedure was repeated three to fivetimes until the supernatant was clear. The pellet was resus-pended in 10 ml of RO water and overlaid with an equalvolume of diethyl ether. After being thoroughly mixed byshaking, the samples were centrifuged at 900 x g for 5 min,and fat and supematant layers were discarded. The pelletwas washed twice in RO water as described above to removetraces of diethyl ether. Further purification of oocysts wasperformed by resuspending the pellet in 10 ml of RO waterwhich was underlayered with a sucrose solution (1.18 spe-cific gravity when cold) and centrifuging it at 900 x g for 15min. The interface was recovered, diluted with RO water,and washed. Oocyst purification by cold sucrose flotationwas repeated until the oocyst suspension was free of excesscontaminating matter. All oocyst suspensions were stored at4°C in RO water, and aliquots were sampled for bacterial andfungal contaminants by routine culture on blood agar andSabouraud agar plates.

Incubation of oocysts with DAPI and PI. Working solutionsof DAPI (2 mg/ml in absolute methanol) and PI (1 mg/ml in0.1 M PBS, pH 7.2) were prepared and stored at 4°C in thedark. Purified oocysts were suspended in Hanks balancedsalt solution (HBSS) (2 x 104 oocysts per ,ul of HBSS), and100 ,ul of suspension was incubated simultaneously with 10,ul of DAPI working solution and 10 ,ul of PI working solutionat 37°C. Initially, suspensions were sampled regularly toensure that oocysts in subsequent experiments were incu-bated with DAPI and PI for the length of time optimal forproducing maximal dye uptake. Oocysts were washed twicein HBSS before being viewed by epifluorescence micros-copy.

Microscopy. Ten-microliter aliquots of oocyst suspensionwere viewed under both DIC (Nomarski) optics and epifluo-rescence with an Olympus BH2 microscope equipped with aUV filter block (350-nm excitation, 450-nm emission) forDAPI and a green filter block (500-nm excitation, 630-nmemission) for PI. Proportions of ruptured (ghost), PI-positive(PI+), DAPI-positive PI-negative (DAPI+ PI-), DAPI-negative PI-negative (DAPI- PI-) oocysts (Table 1) were

quantified by enumerating more than 100 oocysts in eachsample. Ghost oocysts were easily identified under Nomar-ski optics, being nonrefractile apart from the residual body.

TABLE 1. Correlation of oocyst viability, visualization of oocystcontents by Nomarski (DIC) microscopy, and exclusion or

inclusion of DAPI and PI

Contents seen InclusionType of by Nomarski of: Viabilitymicroscopy PI DAPI

Ghost No No No DeadPI+ Yes Yes Yes DeadDAPI+ PI- Yes No Yes Viable at assayDAPI- PI- Yes No No Viable after further trigger

a DAPI- PI- oocysts can be converted to DAPI+ Pl- oocysts and viceversa.

PI+ oocysts fluoresced bright red under the green filterblock, and this red fluorescence varied from distinct pointsof intense fluorescence corresponding to the locations ofsporozoite nuclei to a more-diffuse fluorescence within theoocyst. Oocysts were considered DAPI+ PI- only if theydid not include PI and if the nuclei of the sporozoitesfluoresced a distinctive sky blue under the UV filter block.Those oocysts which were neither PI+ nor ghosts and whichshowed either a rim fluorescence or an absence of fluores-cence under the UV filter block were considered DAPI-PI- (Table 1).

Excystation. Two similar excystation protocols, S and M,were used on oocyst suspensions following either preincuba-tion treatment or incubation with vital dyes. For excystationprotocol S, 200 p.l of bile solution (1% bovine bile in Hanksminimal essential medium) and 50 .1l of sodium hydrogencarbonate solution (0.44% sodium hydrogen carbonate in ROwater) were added to 100 p,l of purified oocyst suspension.The reactants were mixed thoroughly prior to incubation at37°C. For excystation protocol M, 10 ,ul of sodium deoxy-cholate solution (1% sodium deoxycholate in Hanks minimalessential medium) and 10 ,ul of sodium hydrogen carbonate(2.2% sodium hydrogen carbonate in HBSS) were added to100 p.l of purified oocyst suspension. The reactants weremixed thoroughly prior to incubation at 37°C.For both excystation protocols, reagents were made up

freshly (<30 min before use). Initially, suspensions weresampled at regular time intervals to determine excystationdynamics and to ensure that oocysts in subsequent experi-ments were incubated in an excystation protocol for thelength of time optimal for maximizing excystation.Enumeration of excystation percentages and sporozoite ra-

tios. Ten-microliter aliquots of excystation suspensions wereviewed under both fluorescence and Nomarski optics, andthe proportions of ghosts, partially excysted oocysts, andnonexcysted oocysts were determined. In selected excysta-tions, free sporozoites were also enumerated. In excystationsuspensions previously incubated with vital dyes, exclusionor inclusion of DAPI and PI was also recorded. For eachenumeration, at least 100 oocysts were counted. The percentexcystation was calculated as follows: [(number of ghosts +number of partially excysted oocysts)/total number ofoocysts counted] x 100, where the number of ghosts equaledthe number preexcystation subtracted from the numberpostexcystation. This correction was also used when calcu-lating the sporozoite ratio according to the following for-mula: number of free sporozoites/(number of ghosts +number of partially excysted oocysts).

Preexcystation treatments. Before being subjected either toincubation with vital dyes or to in vitro excystation, suspen-

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c

18.2

00 -

80 -

---- different human Isolates

* bovine Isolate---- carvine/ovine Isolate (mean t sd, n-iS0)

60 -a

40 -

20 --- ----a.... ... 3u~~~~~~~~~~~~~~------..........o0 .-

0 5 10 15Time (hours)

.218n.U)

1C9

8

20 25 predicted

FIG. 1. Excystation of different isolates of C. parvum oocystsover time.

sions of purified oocysts were preincubated in solutionsknown to affect excystation dynamics (21). Following thepreincubation treatment, oocysts were washed three times inHBSS before being subjected either to incubation with DAPIand PI or to an excystation protocol. All of the followingpreincubation treatments were for 1 h: (i) 100 ,ul of oocystsuspension and 1 ml of 0.1 M sodium hydroxide (NaOH) atroom temperature; (ii) 100 ,ul of oocyst suspension and 1 mlof 0.1 M hydrochloric acid (HCI) at room temperature; (iii)100 pl of oocyst suspension and 1 ml of RO water at roomtemperature (control for i and ii); (iv) 100 >il of oocystsuspension and 1 ml of HBSS acidified to pH 2.75 with HCI(20 ml HBSS:200 ,ul of 1 M HCI) at 37°C; (v) 100 ,ul of oocystsuspension and 1 ml of HBSS at 37°C (control for iv).

Statistics. Calculation of correlation coefficients, linearregression analysis, analysis of covariance, and Mann-Whit-ney U tests were performed with a MINITAB statisticalpackage.

RESULTS

Uptake of DAPI and PI. Uptake of PI by oocysts wasmaximal after a 5-min incubation at 37°C; however, uptakeof DAPI did not maximize until oocysts had been incubatedfor 2 h or longer at 37°C. Therefore, in all subsequentexperiments described here, both DAPI and PI were incu-bated simultaneously at 37°C for 2 h. The proportion ofoocysts which took up the dyes varied between oocystisolates; c-o isolates contained a considerably greater pro-portion of DAPI+ PI- oocysts than the other isolatestested.

Excystation dynamics and correlation of excystation proto-cols. For human, bovine, and c-o isolates of C. parvumoocysts excysted by excystation protocol S or M, theexcystation efficiency was maximal after 4 h (Fig. 1). Excys-tation efficiencies varied between oocyst isolates, with thec-o isolates having a considerably higher maximal excysta-tion efficiency than the other isolates tested. Correlation ofexcystation protocol S with excystation protocol M gave acorrelation coefficient of 0.996. This correlation also held foroocyst suspensions that had been preincubated with acidi-fied HBSS before being subjected to the excystation proto-cols (Fig. 2).

Prediction ofmaximal (4-h) excystation efficiency with DAPIand PI. It was hypothesized that inclusion or exclusion ofDAPI and PI following a 2-h incubation at 37°C could beused to predict the viability of C. parvum oocysts, theproportion of DAPI+ PI- oocysts being the predictedviability and the observed or actual viability being calculatedfrom maximal in vitro excystation. Oocysts were considered

l0 ____/

10 /

30

O0 .20.10

0 10 20 30 40 50 60 70 80 90 100

% excystation using excystation protocol S

FIG. 2. Correlation of excystation protocols S and M.

viable if at least one sporozoite was ejected (partially ortotally excysted) during the in vitro excystation.For human, bovine, and c-o oocysts, excystation effi-

ciency was maximal after 4 h. The predicted viabilities (after2 h of incubation with DAPI and PI at 37°C) were compara-ble to the maximum observed excystation efficiencies (Fig.1). Correlation of this excystation efficiency (observed via-bility) with predicted viability (inclusion or exclusion ofDAPI and PI) gave a correlation coefficient of 0.997. Linearregression analysis provided the following mathematicalrelationship between predicted viability (x) and observedviability (y): y = 1.85 + 0.936x (d2 adjusted for degrees offreedom = 99.3%). Analysis of covariance demonstrated nosignificant difference between this relationship (y = 1.85 +0.936x) and the simpler mathematical relationshipy = x (Fig.3).

Since a simple correlation between predicted viability(percentage of DAPI+ PI- oocysts after a 2-h incubationwith vital dyes) and observed viability (maximal excystationefficiency) had been derived, it was suggested that thoseoocysts which excysted during a 4-h excystation protocolwould be those which were DAPI+ PI-. Excystation timetrials showed that for all oocyst isolates, there was a markeddecrease in the proportion of DAPI+ PI- oocysts with time.The proportion of DAPI- PI- oocysts fluctuated but did notdecrease significantly with time (Fig. 4). The sporozoitesseen in the excystation medium all contained fluorescent

100-

O cernate/ovine isolate

* bovine isolates80 /

o3 human isolates <

y-a

2 60

4040

X .2

O D20-O~Oz

0

0 20 40 60 80 100

Predicted viability (Percentage DAPI+)

FIG. 3. Correlation of predicted and observed viabilities fordifferent isolates of C. parvum oocysts.

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'I*-J0

40

TABLE 2. Inclusion or exclusion of DAPI and PI in c-o oocystsfollowing preincubation treatments

Preincubation % Inclusion (mean ± SD)' % Deadtreatment DAPI+ PI- DAPI- PI-

RO water 76.5 + 0.6 4.9 ± 1.3 18.6 ± 1.90.1 M HCI 63.4 + 2.9 3.3 ± 0.6 33.3 ± 2.30.1 M NaOH 5.2 + 3.0 0.0 ± 0.0 94.8 ± 3.0

an = 5.

q9u-o-o---------------------------------------- < 0.05). A similar pattern was observed with preincubation201t \ of oocysts with 0.1 M HCI at room temperature (Tables 2-tOx-and 3). A significant increase in oocyst death was observed+S~~~~~~~~~~~~~forboth isolates (c-o isolate, P c 0.05; human isolate, P <

t 8 for both isolates(c-oisolate, PT0.05;human isolate,120.01). However, whereas the viability of the c-o isolate was0 4 8 12 116 20 24 significantly reduced (Table 2; P c 0.05), the proportion of

viable (DAPI+ PI-) oocysts in the human isolate wasExcystaton tine (hours) significantly increased (Table 3; P c 0.01).

J. 4. Alterations in the proportion of DAPI+ PI- and DAPI- (ii) Preincubation in acidified HBSS. Preincubation in acid-solates over time during excystation. Symbols: -0-, DAPI+ ified HBSS was not associated with any significant differenceolate (mean; n = 10); --0 --, DAPI- c-o isolate (mean; n = in the percentage of dead oocysts for either oocyst isolate-@- DAPI+ human isolate; --0--, DAPI- human isolate; (Table 4; P > 0.3). In the suspension of c-o oocysts, the-, DAPI+ bovine isolate; -- O --, DAPI- bovine isolate. proportion of viable (DAPI+ PI-) oocysts was not signifi-

cantly different after preincubation treatment (P > 0.3), andthe mean proportion of viable oocysts remained higher than

?I+ PI-) nuclei. Free sporozoites with nonfluorescent 70% (Table 4). For bovine oocysts, the suspension whichi were never observed. had not been acidified had relatively low proportions ofeincubation treatments. Preincubation treatments per- DAPI+ PI- oocysts, but this proportion was significantlyed on oocysts (see below) had a significant impact on increased (Table 4; P c 0.009) by incubation in acidifiedlity, both observed (maximal excystation) and predicted HBSS.bation with DAPI and PI). For all preincubation treat- In both the c-o isolate and the bovine isolate, the propor-;s, a good correlation between observed and predicted tion of DAPI- PI- oocysts was reduced significantly bylities was observed (Fig. 5), with a correlation coeffi- preincubation with acidified HBSS (Table 4; c-o isolate, P <of 0.989. 0.006; bovine isolate, P < 0.009).Preincubation with 0.1 M NaOH and 0.1 M HCI. In both Sporozoite ratios. Sporozoite ratios were enumerated dur-nd human isolates, preincubation with 0.1 M NaOH at ing excystation of c-o and bovine (purified at SPDL) oocysttemperature was associated with a significant increase isolates (Table 5). High variability was observed, particu-cyst death (Tables 2 and 3; c-o isolate, P < 0.01; human larly in the c-o isolate, as demonstrated by the relatively highte, P c 0.05). In the c-o isolate after incubation with standard deviations (Table 5). Sporozoite ratios were alsoH, a significant reduction in the proportion of viable enumerated during excystations of oocyst suspensions thatPI+ PI-) oocysts was observed (Table 2; P c 0.001). had been preincubated in acidified HBSS. For both isolates,ever, in the human isolate, a dynamic situation was sporozoite ratios tended to fall over the duration of therved, with a significant decrease in DAPI- PI- oocysts excystation, presumably because of lysis of the sporozoites.l significant increase in DAPI+ PI- oocysts (Table 3; P After 4 h, the sporozoite ratio never exceeded 1 (Table 5).

For both isolates, preincubation in acidified HBSS tended tobe associated with an enhanced sporozoite ratio during the

100] initial stages of excystation, and this association was signif-/Z icant (P < 0.009) in the bovine isolate (Table 5).

:5

00

80

60

40

201

0 20 40 60 80 100

Prdccted viabty

FIG. 5. Correlation of observed and predicted viabilities afterpreincubation treatments. Symbols: 0, RO water at room temper-ature; A, 0.1 M NaOH at room temperature; 0l, 0.1 M HCO at roomtemperature; 0, HBSS at 37C; A, acidified HBSS at 37°C; M, 1%

trypsin-HBSS at 37'C; x, 1% trypsin-acidified HBSS at 37°C;x =y.

DISCUSSION

The results of this study demonstrate that oocysts whosewalls are permeable to DAPI but not to PI (DAPI+ PI-)

TABLE 3. Inclusion or exclusion of DAPI and PI in humanoocysts following preincubation treatment

Preicubation % Inclusion (mean + SD)' % Deadtreatment DAPI+ PI- DAPI- PI-

RO water 18.6 ± 1.9 76.8 ± 1.8 4.6 ± 0.60.1 M HCI 53.6 ± 3.1 23.9 ± 4.6 22.5 ± 2.00.1 M NaOH 58.0 ± 1.1 20.5 ± 1.7 21.5 ± 2.9

an =5.

100-

80

60-

40-1

0.aea

a.a

00

a.

FICPI- isc-o iS10); --0-

(DATnuclePt

form(viabi](incumentviabilcient

(i):c-o a:roomin ooisolatNaO](DATHowobsexand a

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TABLE 4. Inclusion or exclusion of DAPI and PI in c-o andbovinea isolates of oocysts following preincubation

in HBSS or acidified HBSS

Isolate and % Inclusion (mean + SD)btreatment ~~~~~~~%Deadtreatment DAPI+ Pl- DAPI- PI-

C-oHBSS 73.2 ± 0.8 3.4 ± 2.3 23.4 ± 2.4Acidified HBSS 74.1 + 4.6 0.3 ± 0.5 25.6 ± 5.0

BovineHBSS 11.1 ± 1.2 60.3 ± 4.2 28.6 ± 3.1Acidified HBSS 64.0 ± 2.2 5.3 ± 1.5 30.7 ± 1.2

a Purified at SPDL.bn = 3.

after a 2-h incubation at 37°C will excyst in an excystationprotocol during a 4-h incubation. Such oocysts are describedas viable at assay (Table 1). DAPI is an AT-selective DNAstain, and when binding to DNA occurs, there is an approx-imately 20-fold enhancement in fluorescence (16).The correlation of inclusion of DAPI and exclusion of PI

with excystation during a 4-h excystation protocol wasdemonstrated to hold for oocysts obtained from varioussources. This correlation held if excystation dynamics werealtered by a variety of preincubation treatments. The highviability observed in suspensions of c-o oocysts (low num-bers of DAPI- PI- oocysts) could be due to the oocystpurification method used at MRI, which involves contactbetween the oocysts and dilute acid; preincubation of SPDL-purified oocysts with acid was found to increase theirexcystation efficiencies to similar levels.PI+ oocysts have sporozoites with disrupted or broken

membranes and are considered dead (Table 1). It has beenreported (10) that only cells with disrupted or broken mem-branes can be stained with PI. In our experience, motilesporozoites never include PI. Oocysts are also considereddead if they have ruptured and lost their contents (ghostoocysts) before being subjected to an excystation protocol(Table 1).

Oocysts which exclude both DAPI and PI but appear byNomarski microscopy to be morphologically intact andundamaged (Table 1) require a further trigger before theywill excyst in one of the protocols described above. Inclu-sion of a selected preincubation treatment (e.g., incubationwith acidified HBSS for 1 h at 37°C) converts the majority ofthese DAPI- Pl- oocysts to DAPI+ Pl- oocysts, regard-less of source of oocysts or purification technique. Followingsuch a preincubation treatment, the oocyst wall becomes

TABLE 5. Sporozoite ratios during excystation of c-o isolate andbovine isolatea of oocysts following preincubation

in HBSS or acidified HBSS

Isolate and Sporozoite ratio (mean + SD) at':treatment 30 min 1 h 4 h

C-oHBSS 1.8 + 2.2 2.2 ± 1.6 0.5 + 0.2Acidified HBSS 4.0 ± 1.0 2.3 ± 1.2 0.4 + 0.3

BovineHBSS 0.2 ± 0.3 1.2 ± 0.5 0.6 ± 0.2Acidified HBSS 2.8 + 0.3 2.2 + 0.8 0.6 + 0.2a Purified at SPDL.bn = 3.

permeable to DAPI. The DAPI can then be incorporated intosporozoite DNA through intact sporozoite membranes. Theopposite conversion, of oocyst walls permeable to DAPIbecoming impermeable to DAPI, has also been demon-strated to occur (21), and such a conversion has been shownto be reversible (21). The requirement of an acid trigger,which would normally occur in vivo, to convert a proportionof the oocyst population from being DAPI impermeable tobeing DAPI permeable is important for two reasons.

First, for optimized in vitro excystation, the followingseries of stimuli should be included: (i) temperature eleva-tion to 37°C, as little excystation is detected at lowertemperatures (8, 28), and above 37°C, sporozoites do notsurvive (17a); (ii) incubation in dilute acid for 1 h; and (iii)incubation with bile salt or sodium deoxycholate in sodiumhydrogen carbonate for 4 h. Incubation with chemicals otherthan acid might have the same trigger effect, althoughwhether this is achieved by the same mechanism is un-known. For example, one recommended excystation proto-col (7) involves a 10-min preincubation of oocysts in 1.05%sodium hypochlorite on ice. We suggest that dilute acid ismore appropriate, as it more closely resembles the environ-ment expected to be encountered by ingested oocysts in vivo(21).Second, permeability of oocyst walls varies within an

isolate. The degree of permeability may affect the abilities ofindividual oocysts to withstand environmental pressures; theproportion killed by incubation in either 0.1 M NaOH or 0.1M HCl was significantly lower in the isolate whose oocystswere originally impermeable to DAPI. We suggest thatoocyst permeability should be considered when results fromenvironmental and disinfection studies are interpreted (4-6,15, 18, 20, 27).DAPI staining of sporozoite nuclei gives a characteristic

sky blue fluorescence under a UV filter block. Besidesindicating oocyst viability, visualization of the sporozoitenuclei provides a useful adjunct for more-definitive tech-niques of recognition and identification of Cryptosporidiumoocysts. Visualization of sporozoite nuclei can be used inconjunction with the more-conventional techniques involv-ing fluorescence-labeled monoclonal antibodies. It has beenrecommended that recognition of cysts of Giardia spp. inwater rely on the identification of three morphological fea-tures (morphometry and the presence and distribution of atleast two internal organelles) (12). The use of DAPI inconjunction with a fluorescence-labeled monoclonal anti-body appears to fulfill such criteria for viable oocysts (5).To maximize the proportion of oocysts which include

DAPI and minimize the proportion of DAPI- PI- oocysts,a 1-h preincubation in acidified HBSS at 37°C is recom-mended. The other preincubation treatments investigatedhere increased the proportion of dead oocysts in either all(0.1 M NaOH) or some (0.1 M HCI) of the isolates of C.parvum oocysts tested. It should be borne in mind thatoocysts detected in environmental or water samples mayhave already been exposed to alkaline or acid pHs.Enumeration of sporozoite ratios at maximized excysta-

tion (4 h) provides information of little value, as most of thesporozoites appear to have lysed. The fragility of sporozo-ites necessitates that any interpretation of sporozoite ratiodata be treated with caution.The correlation of viability and in vivo infectivity is an

important issue that the research described here did notaddress. A correlation between in vitro excystation andinfection of mice with large inocula (105 oocysts per mouse)

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VIABILITY OF C. PARVUM OOCYSTS 3493

of Cryptosponidium oocysts has, however, been reported(4).

In conclusion, simultaneous staining with DAPI and PI isan excellent, reproducible, user-friendly indicator of viabil-ity of C. parvum oocysts as defined by in vitro excystationand correlates well when excystation is increased by an

acidic preincubation step or when excystation is reduced byharsher preincubation treatments that are lethal to some

oocysts. A study of the permeability of oocyst walls to DAPImight provide interesting data on the degree of viability andthe hardiness of oocysts. The use of DAPI in conjunctionwith fluorescence-labeled monoclonal antibodies could alsobe useful in the identification of oocysts in environmentaland water samples (5).

ACKNOWLEDGMENTS

This work was supported by the U.K. Department of the Envi-ronment, with administration of funds through the Water ResearchCentre.

REFERENCES1. Anonymous. 1990. Cryptosporidium in water supplies. Report of

the group of experts. Department of the Environment, Depart-ment of Health. Her Majesty's Stationery Office, London.

2. Bingham, A. K., E. L. Jarroll, E. A. Meyer, and S. Radelescu.1979. Giardia sp.: physical factors of excystation in vitro andexcystation v.s. eosin exclusion as determinants of viability.Exp. Parasitol. 47:281-291.

3. Bingham, A. K., and E. A. Meyer. 1979. Giardia excystation can

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