0095-1137/78/0007-0524$02.OO/O
Copyright © 1978 American Society forMicrobiology Printed in U.S.A.
Comparative Evaluation
of
aModified
Zinc Sulfate
Flotation Technique
MARILYN S. BARTLETT,`* KATHLEEN HARPER,2 NANCY SMITH,'PATRICIAVERBANAC,'
ANDJAMES W. SMITH1
Departmentof Clinical Pathology, UniversityHospital, Indiana University Schoolof Medicine,
Indianapolis, Indiana46202,1 and MicrobiologyDivision, Bureau ofLaboratories,Indiana State Board of
Health,Indianapolis,Indiana 462062
Receivedforpublication12December1977
Amodified zinc sulfateflotation technique using Formalinized fecal specimens
(F-ZnSO4) was comparedtothe Formalin-ether (FE) concentration method for
thelaboratorydiagnosisofintestinal parasites. Manylaboratorieshavedifficulty
storing,using, and disposing of ether and needaprocedure forconcentratingfecal
specimens which doesnotrequire ether.Comparativedatawereobtained for the
recoveryofprotozoan cystsandhelmintheggsand larvae from fecespreservedin
Formalinless thanand longer than1month. Whereas the FE methodwasfound
generally to be more efficient, F-ZnSO4 was apparently more effective for the recoveryofsomespecies of parasites. F-ZnSO4wasnotsatisfactory forrecovery
ofschistosome eggs. Weconclude that, exceptfor schistosomes, F-ZnSO4
com-paresfavorablytothe FEmethod fordetectinginfections of clinical significance.
The originalzinc sulfatecentrifugal flotation
(ZnSO4)technique (2) since its developmenthas
been variously modified for use as a routine
concentrationprocedure for detecting intestinal
helminthsand protozoa (4).Instudies
compar-ing the recovery of parasites by the original
ZnSO4 method, and some modifications, with
recovery by the Formalin-ether sedimentation
(FE) technique, the latter generally has
ap-peared more efficient (4-7). However, the FE
method has the disadvantage of employing
ether, which is combustible andrequires special
safetyprecautionsforstorage, use,anddisposal.
One modification of theZnSO4method is the
Formalin-zinc sulfate flotation (F-ZnSO4)
tech-nique (3, 4), which incorporates some of the
advantages of the FE technique and has been
employed routinely in the Indiana University
laboratory for6yearsandinthe Indiana State
BoardofHealthfor15years. This method
elim-inateswashingsteps andutilizesFormalin
fixa-tion to clear internal structures of protozoan
cysts and prevent distortion commonly
associ-ated with salt solutionsofhighspecific gravity.
Furthermore, the methodisadaptableto
varia-tions inindividual laboratory schedulesinthat
the preliminaryfixation offecalspecimenscan
beperformedseparatelyfromtheactual
concen-trationformicroscopic examination.
Ourexperiencewiththisparticular procedure
hassuggestedthat itis anacceptablesubstitute
for the FE method. The present investigation
was undertaken to compare the F-ZnSO4 and
FEtechniques, since no such study has
previ-ously been published and because many
labo-ratories desire a concentration technique that
doesnotpresentthestorageanddisposal
prob-lems found with ether. The study also compares
the recovery ofparasites from fecal specimens
fixed inFormalin less than1month and longer
than 1month.
MATERIALS AND METHODS
Specimens.Fecalspecimens knowntocontainone
or more species of protozoa andhelminthswere ob-tained fromlaboratories in the UnitedStates, Puerto Rico,Japan, and Egypt. Specimens were submitted either in screw-cap mixing bottles (33 by 70 mm) containing15ml of 10%Formalin and fiveglassbeads (5 mm),orin vials ofFormalinonly andsubsequently
transferredtothe mixing bottles forprocessing.
Ap-proximately 1 part offeces to 3 to 5 parts of 10%
Formalin wasemployed.The dateofpreservationin
Formalinwasrequestedfor eachsample.
Specimenswereseparatedintotwogroups accord-ingtothelength oftime they had beenpreservedin
Formalin: less than1month, andlongerthan1month. Samplesweregiver%identification numbersand there-afterprocessed in arandom manner asblind
speci-mens. Priortoconcentration, theyweremixed for10
to15 s on aVortex mixersetatmaximumspeed.Equal
portions of eachspecimenwereconcentratedby both methods, and thesesamples offinelysuspended feces were alwaysobtainedimmediatelyafter mixing. Each specimenwasconcentratedby both methods within4
days.
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F-ZnSO4 method.Forthe F-ZnSO4flotation
mod-ification (3, 4), each sample offecal-Formalin
suspen-sion wasstrained through onelayer ofgauzeinto a
conical paper cup and immediately poured into a
round-bottomtube(100by 16mm) towithin3/4inch (ca. 19.05mm) of the rim. The suspensionwasthen
centrifuged for 3.5min at 1,800rpm (750 xg). (All
centrifugations were effected without mechanical
braking.) Thesupernatantwasdecanted, thelastdrop wasdrainedontoacleansection ofpapertowel, and aqueousZnSO4solution (1.195to 1.200specific
grav-ity)wasaddedtowithin1 inch(ca. 25.4 mm) of the
rimof the tube. Thepacked sedimentwasthen
resus-pended, usingtwo applicator sticks, until no coarse
particles remained.Thissuspensionwasimmediately centrifugedat1,500rpm(500xg) for 1.5 min,
trans-ferredwithoutagitationtoarack which held itupright,
andallowedtostand for1minto compensateforany
disruptivemovement occurring during transfer from
the centrifuge. With awire loop 7 mmin diameter,
bentat aright angle to thestem, two loops of the
surfacefilmweretransferredtoadrop of 0.85%saline
andtoadropof Dobelland O'Connor iodine(1, 4)on aglass slide (3by2inches; ca.76.2by 50.8 mm) for wet-mount examination. The specific gravity of the zincsulfate solution was checked frequently with a
calibratedhydrometerforheavyliquidswithaspecific
gravityrangeof 1.00to 1.22throughoutthestudy.
FE method. The FE methodasdescribedforuse
with Formalin-fixed feces (4) was employed in this
study. Briefly, 10 mlofstrained fecal-Formalin
sus-pension, as preparedfor the F-ZnSO4 modification, wastransferredtoaconical centrifugetube,etherwas
added, and the tubewasstoppered, shaken vigorously,
andcentrifuged. Thetopthree layersweredecanted,
andasaline andaniodinewetmountwereprepared
fromthesediment.
Examinationofwetmounts.Onesaline andone
iodinepreparationweresystematically examined from
each concentrated FE sediment orF-ZnSO4 surface
film. Microscopywasperformed byoneof three
indi-viduals competent in identifying intestinal protozoa and helminths. The two concentration procedures
were not performed concurrently, and if the same
parasitologistexaminedmountsfromboth the FE and F-ZnSO4 concentrations ofagiven specimen,itwasby
chance.
Findings were recorded as to species, parasitic
stages, and semiquantitative numbers, with five or
fewer organisms per coverslip mount designated as
rare;6to20, few;20to40,moderate;andmorethan
40,many.
Determinationofrelative efficiencies of
recov-ery.Since theobjective of this studywastocompare
onlythe relative efficiencyof the two concentration
techniques in recovering the individualspeciesof
par-asites, alldataanalyseswerebasedonacceptance of 100%recovery ofa species being equal to the total
specimens in which it was found, regardless of the
methodorcombination ofmethodsyielding the
recov-ery.
RESULTS
In the262 Formalin-fixed fecal samples
con-centrated by both the F-ZnSO4 and the FE
methods for Formalinized specimens, a total of
505intestinalparasites werefound (263as
pro-tozoan cysts, 224 as helminth eggs, 18 as
hel-minthlarvae).
Table 1 shows the relative efficiency of the
two techniquesfor recoveringstages of the
in-dividual species. Of the 505 findings, 379 were
detected by both methods. The FE technique
yielded 91% (462) of the total parasites found
and F-ZnSO4 yielded 84% (422). The greatest
difference between the two methods was
ob-served in the recovery of helminth eggs, with
89%of the totalfindings byeithermethodbeing
recovered by the FE method and 77% by
F-ZnSO4. When data pertaining to protozoa only
were considered, this difference was less (FE,
94%;F-ZnSO4, 89%).
Theefficiency of each method forthe recovery
of individualspeciesis shown inTable 1.When
the null hypothesis was applied to determine
significant differences (Table 1) in recovery by
thetwo methods of the 19species studied,
sig-nificantdifferences wereobserved for four
spe-cies:Entamoeba coli, Endolimax nana,
hook-worm, andSchistosoma mansoni.
Therecovery ofparasitesfromfecespreserved
inFormalin less than 1 month andlonger than
1 month is shown in Table 2. Storage time
appeared to have some effect on certain
para-sites.UsingthebasisdescribedinMaterials and
Methods for determining relative efficiency,
100% recovery ofEntamoeba histolytica cysts
wasobtained bybothmethodsfromspecimens
preserved in Formalin less than 1 month,
whereasonly 50%recoverywasobtainedbythe
FEconcentration of fecespreservedinFormalin
longer than1month. TheF-ZnSO4method
ap-parentlywasmoreefficient fordetecting
Enta-moebahartmanni cysts and Clonorchis sinensis
eggs inthe olderFormalin-preserved specimens,
whereasthe relative efficiency ofthe FE
tech-niqueincreased from50 to 100% forEnterobius
vermicularis eggs when only long-term
pre-servedspecimenswere concerned. Variances of
lesserdegrees were observed for other species.
DISCUSSION
Although,ingeneral,theFEmethodwasthe
more sensitive of thetwo, variances related to
the species of parasite and length of fixation
timeinFormalinwereobserved.
Incomparingtherecovery oforganismsfrom
fecespreservedinFormalin less thanandlonger
than1month,differences observed suggest that
fecal specimens preserved in Formalin for
ex-tended periodsof time may not alwaysbe
suit-able forevaluatingconcentrationtechniquesfor
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TABLE 1. Recovery ofparasites by F-ZnSO4 and FE concentration methods from 262 fecal specimens
Parasites Total identifi- F-ZnSO4 FEtotal/oy' Both meth- Probability
Parasites
~cations
towa/only'
F ods PoabtyProtozoa(cysts) 263 234 247 218
Entamoeba histolytica 22 22/5 17/0 17 <0.1
Entamoeba hartmanni 34 29/3 31/5 26 >0.5
Entamoebacoli 71 61/2 69/10 59 <0.05+
Endolimaxnana 68 58/2 66/10 56 <0.05+
Iodamoeba butschlii 17 14/2 15/3 12 >0.5
Giardia lamblia 46 45/2 44/1 43 >0.5
Chilomastix mesnili 5 5/0 5/0 5
Helminths(eggs) 224 172 200 148
Enterobius vermicularis 13 12/5 8/1 7 <0.5
Trichuris trichiura 56 39/8 48/17 31 <0.5
Ascaris lumbricoides 47 39/2 45/8 37 <0.5
Hookwormspp. 51 36/4 47/15 32 <0.02+
Taeniaspp. 3 2/0 3/1 2 >0.5
Hymenolepis nana 13 13/2 11/0 11 <0.5
Hymenolepis diminuta 2 1/0 2/1 1 >0.5
Diphyllobothrium latum 11 11/0 11/0 11
Schistosoma mansoni 10 2/0 10/8 2 <0.01+
Fasciolahepatica 1 1/0 1/0 1
Clonorchissinensis 17 16/3 14/1 13 >0.5
Helminths (larvae) 18 16 15 13
Strongyloidesstercoralis 13 13/2 11/0 11 >0.5
Hookworm spp. 5 3/1 4/2 2 <0.5
aTotalrecovered/recovered only by this method.
bProbabilityofsignificant difference of F-ZnSO4and FEresults. +,Significant difference of<0.05.
use in laboratory diagnoses. In this study, the
useof1monthasthedividingtime inattempting
to obtain some indicaton of the effect of
pro-longed storage offeces in Formalinresulted in
greaterdifferencesthan had beenexpected.
One month had been selected asthe
separa-tion time for the purposes of this study, since
previous experience with the F-ZnSO4 method
byoneof the authors hadindicatedthat
concen-tration results were comparable for specimens
preservedin Formalinatintervals upto 1month,
but thatat sometime thereafter itappearedthat
reproducible results were not always possible.
Theeffect oflong-timestorage isimportant
be-cause publichealth laboratories receiving
For-malinizedspecimensthroughthemailmay
per-form concentrations on specimens preserved
longer than1month. Inaddition,theproficiency
testspecimensused forevaluation oflaboratory
performancearespecimens held in Formalin for
longperiods of time.
Of the four speciesfor which significant
dif-ference in total efficiency of the two methods
was determined (Table 1), two, E. coli and E.
nana, are considered harmless commensals and notclinicallyimportant.Thegreatest difference
inpercentage of recovery occurred with E.nana
in the long-term preserved samples. The two
speciesof protozoacapableofproducing clinical
disease, E. histolytica and Giardia lamblia,
weredetected in all but one instance by the
F-ZnSO4 technique, whereas five E. histolytica
and two G. lamblia were missed by the FE
method.
The other two species for which significant
differences were found were hookworm andS.
mansoni. TheF-ZnSO4method in thissudywas
found ineffective for the recovery of schistosome eggs.
Mostspecimensin thisstudywereprocessed
without priorknowledgeof thespecies of
para-sites present. However, with specimensfor
re-coveringS. mansonieggs, the situationwas
dif-ferent in thatwehadtoseekmaterialcontaining
specificallythese eggs. Of the21specialsamples
of fecal material with S. mansoni eggs, in only
9instances wereeggs foundby either
concentra-tionmethod. These results suggest that neither
method isfully adequatefordetectingtheseeggs
and that if schistosomiasis issuspected multiple
specimens should be examined by one of the
more sensitive methods recommended for the
laboratory diagnosis of schistosomiasis.
Hookworm eggs, for which therewas a
signif-526 ET AL. J. CLIN. MICROBIOL.
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TABLE 2. Relative recoveriesofparasites from fecal specimens according to preservation time in Formalin
<Month inFormalin >Month inFormalin
Parasites Total iden- FZnSOn(%) FE (%)
Ttialidn-
F-ZnSO4(%) FE (%) tifications F-nO E tifications FZS4M FProtozoa(cysts)
Entamoebahistolytica 12 100 100 10 100 50
Entamoebahartmanni 15 67 93 19 100 89
Entamoeba coli 45 84 96 26 88 100
Endolimaxnana 34 91 100 34 79 94
Iodamoebabutschlii 13 85 92 4 75 75
Giardia lamblia 26 96 96 20 100 95
Chilomastix mesnili 4 100 100 1 100 100
Helminths(eggs)
Enterobiusvermicularis 10 90 50 3 100 100
Trichuris trichiura 26 73 92 30 67 80
Ascarislumbricoides 29 86 93 18 78 100
Hookworm spp. 19 74 95 32 69 91
Taeniaspp. 2 50 100 1 100 100
Hymenolepisnana 8 100 88 5 100 80
Hymenolepis diminuta 1 0 100 1 100 100
Diphyllobothrium latum 11 100 100
Schistosoma mansoni 7 0 100 3 67 100
Fasciolahepatica 1 100 100
Clonorchis sinensis 7 86 100 10 100 70
Helninths (larvae)
Strongyloides stercoralis 8 100 88 5 100 80
Hookworm spp. 5 60 80
icant difference in recoveries, were found 51
times.Only63% were detected by both methods.
The F-ZnSO4 method yielded 71%ofthe total
51andFE, 92%. On further analysis of the data,
in 24of the51recoveries hookwormeggs were
found in rare numbers (five or fewer eggs per
cover slip). When these 24 specimens were
ex-cluded, thereappearedtobe no significant
dif-ference.Sincehookworm egg countsof lessthan
2,600 perml of fecesaregenerallynotconsidered
indicativeofclinically significant infections (4),
itwouldseemthat thefindingornotfinding of
rare eggs by the F-ZnSO4 concentration would
alsohavelittle clinicalsignificance. Theeffect of
employing ZnS04 solutionin a range ofspecific
gravitymeasurementsfrom1.195 to 1.200 rather
thanprecisely 1.200mayhave had some small
influence, and preservation of specimensin
For-malinlonger than1weekmayalso have affected
theflotation of hookwormeggs. Further
inves-tigation of the influence of these variables is
proposed.
Although the FE concentration method was
found to detect rare helminth eggs more
fre-quentlythanF-ZnSO4,in mostinstancesof
clin-icallysignificantinfectiontherewillbesufficient
numbers of eggs to be detected with either
method. Inthisstudy, 33% ofthe total helminth
eggfindings yieldedegg counts of five or less.
TheZnSO4method ofconcentrating feces has
generallybeenconsideredunsatisfactory for the
recovery ofoperculatedeggs. Inthis study,
op-erculated eggs were found 29 times, 28 by
F-ZnSO4and26by FE. ExperiencewithF-ZnSO4
modificationin thisstudy andinpreviously
un-published comparisons of the two methods at
the IndianaUniversity Medical Center and
In-dianaStateBoardof Health indicate thatthe
F-ZnSO4technique isasefficientastheFE inthe
recovery ofoperculatedeggs.
The
F-ZnSO4
modificationhas certainadvan-tages. Wetmountspreparedfromsurfacefilms
have lessbackground fecaldetritusthando FE
wetmounts; this ishelpful forindividuals with
limited experience in microscopic screening.
Protozoan cysts areclearedsothatidentification
doesnotrequire the routineuseofiodine, which
can obscure important identifying structures.
Neithercysts nor eggs aredistortedby thehigh
salt concentration when specimens have been
initially fixed in Forinalin. Schistosome eggs,
whenfound, show no distortion and are easily
identified. Zinc sulfate flotation techniques do
not impose the hazards involving the use of
ether. When the F-ZnSO4 modification is
em-ployed, parasitological examinations can be
readily coordinated with clinical laboratory
schedules without reducing the quality of
ser-vice.
The results of this comparison of theF-ZnSO4
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and Feconcentration techniques suggest that F-ZnSO4 is an adequate method for the detection
ofmostintestinal parasite species and is
essen-tially comparable totheFE methodwhenonly
clinically significant infections are considered.
ACKNOWLEDGMENTS
We wish to express our appreciation to the individuals and agencies contributing to this study. Specimens were provided by theparasitology laboratories of Florida State Department of Health andRehabilitative Services, Kansas Department of Health and Environment, South Carolina Department of Healthand EnvironmentalControl, Mississippi State Board ofHealth, Alabama Department of Public Health, Tennessee Department of Public Health, Minnesota Department of Health,California Department of Health,Alaska Department ofHealth and Social Services, Hawaii Department of Health, and Commonwealth of Puerto Rico Department of Health and by Microbiology Section, Proficiency Testing Branch, Center for DiseaseControl,Atlanta, Ga.; Hyland, Division of Travenol Laboratories Inc., CostaMesa,Calif.; Naval Medical Research Unit 3,Cairo, Egypt; Russell M.McQuay, Mt. Sinai Hospital,Chicago; Wilda B. Knight, San Juan Laboratories, Centerfor Disease Control, San Juan, Puerto Rico; and Yoichi Ishii, Kyushu University, Fukuoka, Japan. We thank James A.Norton, Biostatistics,Department of Psychiatry, Indiana University School of Medicine, for assistance in analyzing data.
LITERATURE CITED
1. Dobell, C., and F. W. O'Connor. 1921. Intestinal pro-tozoaof man.William Wood, New York.
2. Faust, E. C., J. S.D'Antoni, V. Odom, M. J.Miller, C. Peres, W. Sawitz, L. F. Thomen, J. E. Tobie, and J. H.Walker. 1938. A criticalstudy of clinical labora-torytechniques for the diagnosis of protozoan cysts and helmintheggs in feces. Am. J.Trop. Med. 18:169-183. 3. Harper, K. 1964. Routine laboratory procedures for in-testinalparasitology. Procedure manual.Microbiology Division, Bureau of Laboratories, Indiana State Board of Health.
4. Melvin, D. M., and M. M. Brooke. 1974. Laboratory procedures for the diagnosis of intestinal parasites. U.S. Department of Health, Education and Welfare publi-cation no.(CDC) 75-8282, Atlanta, Ga.
5. Ritchie, L. S., C.Pan, and G. W. Hunter m. 1952. A comparison of the zinc sulfate and the MGL (formalin-ether) technics. J. Parasitol.38(Suppl):16.
6. Ritchie, L.S., C. Pan, and G. W. Hunter m. 1953. A comparison of the zinc sulfate and the formalin-ether (406thMGL) technic. Med. Bull. U.S. Army Far East 1:111-113.
7.Vinayak, V. K., B. N.Tandon, and0. Prakash. 1967. Acomparativeevaluation forformol-ether, zinc sulfate and magnesium sulfate concentration techniques for diagnosis of helminthicovaandprotozoal cysts. Ind. J. Med. Res.55:134-138.
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