EXPEJUMENTAL PARASITOLOGY 41, 329-334
(1977)
Schistosoma mansoni and S. japonicum: Methylene Blue Test for the Viability of Schistosomula in Vitro S. Y. LI HsB,
H. F. Hsii, P. ISACSON,
AND
H. F.
CHENG
*
Departments of Preventive Medicine and Environmental Health, and * Radiology, College of Medicine, University of Iowa, Iowa City, Iowa 52242, U.S.A. (Accepted for publication
22 June 1976)
Hsii, S. Y. Lr, Hsii, H. F., ISACSON, P., AND CHENG, H. F. 1977. Schistosomu munsoni and S. japonicum: Methylene blue test for the viability of schistosomula in vitro. Expetimental Parasitology 41, 329-334. Methylene blue staining has already been used to help in determining the viability of schistosomula, but precise criteria were lacking. Such criteria have now been established using the schistosomula of Schistosomu mansoni and S. japonicum. Living schistosomula, depending on the time of reading, are either ( 1) unstained and motile or (2) a deep blue, contracted, and motionless. Dead organisms are (1) a light green-blue and flattened or (2) colorless and degenerate. Optimum readings are made in uncovered preparations, 50-60 min after addition of the dye, when all initially motile schistosomula will have contracted and been stained. INDEX DESCRIPTORS: Schistosomula; Schistosoma mansoni; Schistosoma japonicum; Blood flukes; Trematodes; Methylene blue; In vitro viability; Snails; Biomphalaria gZ&ata; Oncome,!ania nosophora; Rhesus monkey serum; Rat skin penetration.
even slightly motile ones, were considered Methylene bIue is a basic metachromatic to be dead because of blue staining. In our dye. It has been widely used for cytological studies concerning the viability of schistoand histopathological staining. As a vital somula (Hsii and Hsii 1975) we were comstain, it is often used to test viability, be- pelled to use motility-morphology criteria cause living cells have been found to have because of difficulties, detailed below, in the ability to exclude the dye. Methylene using methylene blue. Nevertheless, the method had drawblue also has been used as an indicator of motility-morphology backs, being time-consuming and not the activity of dehydrogenases in enzydecisively accurate. matic analysis because it is an oxidizingAs in &TO testing of schistosomular vireducing agent that turns bright blue when ability is important for the determination oxidized and colorless when reduced. Basing their work upon the principle of of immune serum’s role in immunity to dye exclusion by living cells, Clegg and schistosomes, a simple but more accurate Smithers (1968) used methylene blue to test is needed. Therefore, a further study determine the viability of schistosomula of methylene blue staining and the viability isolated from the skin of mice; it was of schistosomula has been undertaken. claimed that the dye was an accurate indicator of viability. Living schistosomula MATERIALS AND METHODS were described as completely unstained and dead organisms were described as Cercariae of Schistosoma mansoni and bright blue. All contracted schistosomula, S. japonicum were used. The cercariae of 329 Copyright 0 1977 by Academic Press, Inc. All tights of reproduction in any form reserved.
ISSN 0014-4894
330
HSii ET AL.
S. munsoni emerged from the snail Biomphularia glabrata originating from Puerto Rico, and the cercariae of S. japonicum from the snail Oncomelania nosophora obtained from Kofu, Japan. The sera were obtained from two normal rhesus monkeys and four wellimmunized monkeys-two immunized with S. mansoni cercariae exposed to 48,000 R and two immunized with S. japonicum cercariae similarly exposed to X irradiation. The rat skin technique of Stirewalt and Uy (1969) was used for the collection of schistosomula which were harvested in Earle’s balanced saline containing 0.65% lactalbumin hydrolysate, 2% glucose, 100 units/ml of penicillin, and 100 pg/ml of streptomycin. The schistosomula were then removed to the culture medium which consisted of equal volumes of either normal or immune serum and the harvest fluid. About 30-60 schistosomula were cultivated in 16 x 125-mm capped plastic tissue-culture tubes containing 1 ml of the culture medium placed in a 37 C incubator in an atmosphere of air and 5% COz. Examination of the viability of the schistosomula with the methylene blue method was done upon specimens immediately after their collection in the harvest fluid and upon those kept in the culture medium for l-5 days. The schistosomula were pipetted in drops onto a slide and stained by addition of equivalent drops of 0.03% methylene blue (Fisher Scientific Co. EA-21) in Earle’s saline. The prepared slides were handled in the following three ways: (1) the slide was not covered with a coverslip during the examination period, (2) the slide was covered with a 25 x 25 coverslip, and (3) the slide was not covered with a coverslip but was kept in a moist chamber at room temperature. The appearance, motility, and coloration of schistosomula were examined microscopically at 40 and 100 x .
RESULTS
Staining Reactions of Methylene Blue on Schistosomula of Schistosoma mansoni in Harvest Fluid Shortly after Skin Penetration a. Uncovered. Immediately after methylene blue addition, the dead schistosomula were stained a light green-blue; they had a flattened appearance and protrusion of the acetabulum was characteristic (Figs. 1-3). From 5 to 15 min, the live schistosomula, activeIy contracting and extending, were not stained (Fig. 1, Table I). At 20 min, a few fine purpleblue granules became visible and the body stained a light purple-blue (Fig. 2). At 30 min, a light blue tint appeared at the posterior end, and the worms became less active. At 40 min, while the purple-blue granules remained, the bodies of the schistosomula appeared a deeper blue. At 50 min, the schistosomula were contracted and practically nonmotile, except for an occasional sluggish movement at the anterior end. At 1 hr, the whole body was stained a deep blue and appeared nonmotile (Fig. 3). Dead schistosomula remained a light green-blue, differing distinctly from the deep blue, nonmotile, and contracted organisms. b. Uncovered and kept in moist chamber. As described above, the dead schistosomula were stained a light green-blue. As to the living schistosomula, the time of appearance of the purple-blue granules was about the same as that for the schistosomula not kept in a moist chamber, but the staining reaction took longer (Table I). The appearance of the blue color occurred later and the schistosomula were alive for 2 hr or more. c. Covered with coverslip. Dead schistosomula were stained as described above. The staining reaction of living schistosomula was variable (Table I). Oftentimes, the schistosomula were not stained for 1 hr. When they were stained, the purple-blue granules and blue coloration
Schistosoma munsoni AND S. japonicum:
METHYLENE
BLUE VIABILITY
FIGS. l-3. Sclzistosoma mansoni, methylene blue test, uncovered, in harvest fluid, x80. FIG. 1. One dead schistosomulum (D), stained light green-blue, showing a flattened appearance and a characteristic protruding acetabulum; one living schistosomulum, unstained and extending. Test was read immediately after addition of the dye. Fro. 2. One dead schistosomulum (D), stained light green-blue; four living schistosomula, with granules stained purple-blue, contracting and extending. Test was read 20 min after addition of the dye. FIG. 3. One dead schistosomulum (D), stained light green-blue; four moribund schistosomula, stained deep blue, contracted, and motionless. Test was read 1 hr after addition of the dye.
in the body also were found to a lesser degree and grew fainter with time. The original blue color was restored if the cover&p was removed and a drop of saline was added. Staining Reaction of Methylene Blue on Schistosomula of Schistosoma mansoni in Culture Medium There were no appreciable differences in the staining reactions with the addition
-It++ +++ -k-t+ ++I+ + zk -
hfd
-I-t+ ++ -t+ ++
PG’
Uncovered
f + ++ +++
Bj
Schi&osornula
+++ +++ +++ -I-++ ++ NE0 NE +
M
f + SE NE -I++
PG
Cnouvered, in moist chamber
NE NE +
B
+++ +++ ++ + f NE -
h1 i f +* + -!NE ++
PG
Stained*
+ + NE
-
B --NE NE --
PO
UnstainedC
+++ +++ +4 + * NE NE -
M
I
KE SE
-
B +++ +++ +++ ++ * NE NE
f + -l--t t-t +-INE NE
++ +++ NE KF,
of Sch,i.dosoma mansoni with
TA4BLE
Covered with cover&p
In harvest fluid, shortly after skin penetration
of Living
+++ ++i+-t+ +I++ ++ + zk
31
+ f-ifC -t++ +++ ++i-
PG
+
B
+
+++ +++ ++ ++ + -t-
hl
f
B
c++ * ++
+ + ++ ++
PC
* -
-t++ -t++ ++ + * *
b1
---
-----
PG
Dnalaine&
Covered with coverslip Stainerl~
In normal serum, 1 day’
Blue
Uncovered, in moist rhambrr
Methylene
B -
a Reactions of living whtitoaomula of S. ~lansoni in normal .wrum medium for days 2-5 and immune serum medium for days 1-6 are referred to in the text. b Living schistosomula showed positive staining reactions in 1 day. = Living schistosomule showed no staining reactions in 1 hr. d M, Motility (&, only occasional sluggish movement at the anterior end: 4, occasionally contrwliug and extending; $ +, moderately contracting and extending; ++-I-. actively contracting and extending). e PG. Purple-blue granules ( &. a few hardly recognized granules : + , D few fine psoules ; + +, %Imoderate numh~r of Ene granules ; + f-l-, umny c.o&rse granules). f B, Blue color (A, trace of hght blue color at the posterior end; +, light blue color in the nrholc body: f C, Italian blue in the whole borly: + ++, peacock blue in the whole body). s NE, Not examined.
,5 min 10 min 15 min 20 min 30 min 40 min 50 min 1 hr
Time
Reactions
/j
x 2:
Schktosoma
munsoni AND S. japonicum:
of normal or immune serum to the medium. Dead schistosomula were stained a light green-blue but deteriorated organisms were essentially unstained. The staining reactions of living schistosoma maintained in the culture medium for 1 day were, in general, similar to those occurring with the specimens maintained in the harvest fluid, except that in the uncovered organisms the purple-blue granules might be seen at 10 or 15 min, and the blue color might be quite deep at 30 min. The staining reactions of the uncovered schistosomula incubated in culture medium for 2-5 days differed from those cultured for only 1 day, or from those maintained in the harvest fluid. In 2-5 day cultures: (1) the purple-blue granules did not always appear at 15-20 min, and even when they appeared, were very indistinct; (2) the blue coloration appeared at lo-20 min instead of 30 min; and (3) the schistosomula became contracted and nonmotile at 2040 min. In the moist chamber, the schistosomula may appear alive, actively contracting and extending, for 17 hr or more. Staining Reactions of Methylene Blue on Schistosomula of Schistosoma japonicum Methylene blue staining of S. japonicum schistosomula, observed on limited occasions, did not differ from the staining of S. mansoni. DISCUSSION
Our results with the methylene blue stain show that the viability of schistosomula can be classified as follows: living and unstained (Fig. 1); living but with scattered granules stained purple-blue (Fig. moribund or nonmotile and stained 2); deep blue (Fig. 3); and dead and stained light green-blue or unstained (Figs. l-3). The living schistosomula were active, extending and contracting, whether they were unstained or had purple-blue granulcs. The moribund schistosomula were characteristically contracted, nonmotile or
METHYLFXE
BLUE VIABILITY
333
sluggish, and stained a deep blue. The dead schistosomula were flattened, often with a protruding ventral sucker, and stained a light green-blue or were unstained, according to the length of time since death. During the staining test, the dead schistosomula, if stainable, usually color immediately while the living ones take the stair1 gradually and metachromatically, i.e., from purple-blue to deep blue. For this reason, schistosomula eventually seen to be contracted and stained deep blue must be recognized as having been the living ones at the beginning of the test. The rationale for the color changes of schistosomula in the methylene blue test may be as follows: The oxidized form of the dye is bright blue, while the reduced product is a leuco compound. In the presence of oxygen, the leuco form may regain its color by nonenzymatic oxidation. When methylene blue is added to living schistosomula, the dye is rapidly reduced by enzymes to its leuco form and the organisms are not stained. The groups of enzymes known as dehydrogenases, which bring about the transfer of hydrogen to methylene blue, have been reported in schistosomes (von Brand 1973). However, near the surface of schistosomula, nonenzymatic oxidation keeps methylene blue in the oxidized form which appears as purple-blue granules, possibly representing the reactions in the subtegumental cells. When the schistosomula gradually become moribund, the enzymatic reaction of the dehydrogenases diminishes. Accordingly, the oxidized form of the dye takes its place, and the organisms appear deep blue. If the schistosomula have been dead for some time, the organisms may become acidic and the staining reaction will be light green-blue or even colorless. On the other hand, the occasional lack of staining of schistosomula in the methylene blue test when they were covered with a glass slip, evidently is due to the lack of oxygen. This
334
HSti ET AL.
assertion is further supported by the development of coloration in the organisms when oxygen is made available by removing the coverslip and adding a drop of fresh saline. According to Clegg and Smithers ( 1968), there were three kinds of staining reactions for schistosomula in the methlyene blue test: (1) nonviable schistosomula which were stained bright blue, (2) living schistosomula which were completely unstained, and (3) contracted and moribund schistosomula which were stained intensely with methylene blue, They classified only the unstained schistosomula as “living” and all the stained as “dead.” According to our results, the dead schistosomula were stained a light greenblue or unstained, and the living ones were unstained at first but later stained a purple-blue and then a deep blue. The differentiation of dead schistosomula from living ones should be made by the characteristic changes of the staining reactions, considering the time factor involved in these changes. Since contracted schistosomula seen during later times in the experiment were typical of organisms that had been alive initially, we do not agree with Clegg and Smithers (1968) in counting these schistosomula as “dead.” Examination for the viability of schistosomula usually involves 30 to 40 organisms per slide. Hence, it is difficult to make a reasonable estimate. The diagnostic help provided by the methylene blue staining
is necessary. Since all the living schistosomula in the uncovered method will assume a contracted form and stain a deep blue in 50 to 60 min, and since the resulting deep blue color contrasts distinctly with the light green-blue or colorless dead schistosomula, it should be easy to make a differential count of the living and dead organisms by coloration (Fig. 3). The coverslip method is unsuitable for this test because of the uncertainty of the color reaction appearing in the test. The moist chamber method also has its drawbacks because a comparatively long time to required to stain the living organisms a deep blue. ACKNOWLEDGMENTS This investigation was supported in part by U.S. Naval Research Contract N00014-68-01960007, and by a grant from the Edna McConnell Clark Foundation. REFERENCES CLEGG, J. A., AND SMITHERS, S. R. 1968. Death
of schistosome cercariae during penetration of the skin. II. Penetration of mammalian skin by Schistosoma mansoni. Parasitology 58, 111-128. Hsti, S. Y. LI, AND Hsii, H. F. 1975. Recovery of schistosomula in the skin of rhesus monkeys immunized with cercariae of Schistosoma japonicum exposed to high doses of X-irradiation. Journal of Parasitology 61, 1108-1109. STIREWALT, M. A., AND UY, A. 1969. Schistosoma mansoni: Cercarial penetration and schistosomula collection in an in vitro system. Experimental Parasitology 26, 17-28. BRAND, T. 1973. “Biochemistry of Parasites,”
VON
2nd ed., pp. l-499. Academic Press, New York.
(1977)
Schistosoma mansoni and S. japonicum: Methylene Blue Test for the Viability of Schistosomula in Vitro S. Y. LI HsB,
H. F. Hsii, P. ISACSON,
AND
H. F.
CHENG
*
Departments of Preventive Medicine and Environmental Health, and * Radiology, College of Medicine, University of Iowa, Iowa City, Iowa 52242, U.S.A. (Accepted for publication
22 June 1976)
Hsii, S. Y. Lr, Hsii, H. F., ISACSON, P., AND CHENG, H. F. 1977. Schistosomu munsoni and S. japonicum: Methylene blue test for the viability of schistosomula in vitro. Expetimental Parasitology 41, 329-334. Methylene blue staining has already been used to help in determining the viability of schistosomula, but precise criteria were lacking. Such criteria have now been established using the schistosomula of Schistosomu mansoni and S. japonicum. Living schistosomula, depending on the time of reading, are either ( 1) unstained and motile or (2) a deep blue, contracted, and motionless. Dead organisms are (1) a light green-blue and flattened or (2) colorless and degenerate. Optimum readings are made in uncovered preparations, 50-60 min after addition of the dye, when all initially motile schistosomula will have contracted and been stained. INDEX DESCRIPTORS: Schistosomula; Schistosoma mansoni; Schistosoma japonicum; Blood flukes; Trematodes; Methylene blue; In vitro viability; Snails; Biomphalaria gZ&ata; Oncome,!ania nosophora; Rhesus monkey serum; Rat skin penetration.
even slightly motile ones, were considered Methylene bIue is a basic metachromatic to be dead because of blue staining. In our dye. It has been widely used for cytological studies concerning the viability of schistoand histopathological staining. As a vital somula (Hsii and Hsii 1975) we were comstain, it is often used to test viability, be- pelled to use motility-morphology criteria cause living cells have been found to have because of difficulties, detailed below, in the ability to exclude the dye. Methylene using methylene blue. Nevertheless, the method had drawblue also has been used as an indicator of motility-morphology backs, being time-consuming and not the activity of dehydrogenases in enzydecisively accurate. matic analysis because it is an oxidizingAs in &TO testing of schistosomular vireducing agent that turns bright blue when ability is important for the determination oxidized and colorless when reduced. Basing their work upon the principle of of immune serum’s role in immunity to dye exclusion by living cells, Clegg and schistosomes, a simple but more accurate Smithers (1968) used methylene blue to test is needed. Therefore, a further study determine the viability of schistosomula of methylene blue staining and the viability isolated from the skin of mice; it was of schistosomula has been undertaken. claimed that the dye was an accurate indicator of viability. Living schistosomula MATERIALS AND METHODS were described as completely unstained and dead organisms were described as Cercariae of Schistosoma mansoni and bright blue. All contracted schistosomula, S. japonicum were used. The cercariae of 329 Copyright 0 1977 by Academic Press, Inc. All tights of reproduction in any form reserved.
ISSN 0014-4894
330
HSii ET AL.
S. munsoni emerged from the snail Biomphularia glabrata originating from Puerto Rico, and the cercariae of S. japonicum from the snail Oncomelania nosophora obtained from Kofu, Japan. The sera were obtained from two normal rhesus monkeys and four wellimmunized monkeys-two immunized with S. mansoni cercariae exposed to 48,000 R and two immunized with S. japonicum cercariae similarly exposed to X irradiation. The rat skin technique of Stirewalt and Uy (1969) was used for the collection of schistosomula which were harvested in Earle’s balanced saline containing 0.65% lactalbumin hydrolysate, 2% glucose, 100 units/ml of penicillin, and 100 pg/ml of streptomycin. The schistosomula were then removed to the culture medium which consisted of equal volumes of either normal or immune serum and the harvest fluid. About 30-60 schistosomula were cultivated in 16 x 125-mm capped plastic tissue-culture tubes containing 1 ml of the culture medium placed in a 37 C incubator in an atmosphere of air and 5% COz. Examination of the viability of the schistosomula with the methylene blue method was done upon specimens immediately after their collection in the harvest fluid and upon those kept in the culture medium for l-5 days. The schistosomula were pipetted in drops onto a slide and stained by addition of equivalent drops of 0.03% methylene blue (Fisher Scientific Co. EA-21) in Earle’s saline. The prepared slides were handled in the following three ways: (1) the slide was not covered with a coverslip during the examination period, (2) the slide was covered with a 25 x 25 coverslip, and (3) the slide was not covered with a coverslip but was kept in a moist chamber at room temperature. The appearance, motility, and coloration of schistosomula were examined microscopically at 40 and 100 x .
RESULTS
Staining Reactions of Methylene Blue on Schistosomula of Schistosoma mansoni in Harvest Fluid Shortly after Skin Penetration a. Uncovered. Immediately after methylene blue addition, the dead schistosomula were stained a light green-blue; they had a flattened appearance and protrusion of the acetabulum was characteristic (Figs. 1-3). From 5 to 15 min, the live schistosomula, activeIy contracting and extending, were not stained (Fig. 1, Table I). At 20 min, a few fine purpleblue granules became visible and the body stained a light purple-blue (Fig. 2). At 30 min, a light blue tint appeared at the posterior end, and the worms became less active. At 40 min, while the purple-blue granules remained, the bodies of the schistosomula appeared a deeper blue. At 50 min, the schistosomula were contracted and practically nonmotile, except for an occasional sluggish movement at the anterior end. At 1 hr, the whole body was stained a deep blue and appeared nonmotile (Fig. 3). Dead schistosomula remained a light green-blue, differing distinctly from the deep blue, nonmotile, and contracted organisms. b. Uncovered and kept in moist chamber. As described above, the dead schistosomula were stained a light green-blue. As to the living schistosomula, the time of appearance of the purple-blue granules was about the same as that for the schistosomula not kept in a moist chamber, but the staining reaction took longer (Table I). The appearance of the blue color occurred later and the schistosomula were alive for 2 hr or more. c. Covered with coverslip. Dead schistosomula were stained as described above. The staining reaction of living schistosomula was variable (Table I). Oftentimes, the schistosomula were not stained for 1 hr. When they were stained, the purple-blue granules and blue coloration
Schistosoma munsoni AND S. japonicum:
METHYLENE
BLUE VIABILITY
FIGS. l-3. Sclzistosoma mansoni, methylene blue test, uncovered, in harvest fluid, x80. FIG. 1. One dead schistosomulum (D), stained light green-blue, showing a flattened appearance and a characteristic protruding acetabulum; one living schistosomulum, unstained and extending. Test was read immediately after addition of the dye. Fro. 2. One dead schistosomulum (D), stained light green-blue; four living schistosomula, with granules stained purple-blue, contracting and extending. Test was read 20 min after addition of the dye. FIG. 3. One dead schistosomulum (D), stained light green-blue; four moribund schistosomula, stained deep blue, contracted, and motionless. Test was read 1 hr after addition of the dye.
in the body also were found to a lesser degree and grew fainter with time. The original blue color was restored if the cover&p was removed and a drop of saline was added. Staining Reaction of Methylene Blue on Schistosomula of Schistosoma mansoni in Culture Medium There were no appreciable differences in the staining reactions with the addition
-It++ +++ -k-t+ ++I+ + zk -
hfd
-I-t+ ++ -t+ ++
PG’
Uncovered
f + ++ +++
Bj
Schi&osornula
+++ +++ +++ -I-++ ++ NE0 NE +
M
f + SE NE -I++
PG
Cnouvered, in moist chamber
NE NE +
B
+++ +++ ++ + f NE -
h1 i f +* + -!NE ++
PG
Stained*
+ + NE
-
B --NE NE --
PO
UnstainedC
+++ +++ +4 + * NE NE -
M
I
KE SE
-
B +++ +++ +++ ++ * NE NE
f + -l--t t-t +-INE NE
++ +++ NE KF,
of Sch,i.dosoma mansoni with
TA4BLE
Covered with cover&p
In harvest fluid, shortly after skin penetration
of Living
+++ ++i+-t+ +I++ ++ + zk
31
+ f-ifC -t++ +++ ++i-
PG
+
B
+
+++ +++ ++ ++ + -t-
hl
f
B
c++ * ++
+ + ++ ++
PC
* -
-t++ -t++ ++ + * *
b1
---
-----
PG
Dnalaine&
Covered with coverslip Stainerl~
In normal serum, 1 day’
Blue
Uncovered, in moist rhambrr
Methylene
B -
a Reactions of living whtitoaomula of S. ~lansoni in normal .wrum medium for days 2-5 and immune serum medium for days 1-6 are referred to in the text. b Living schistosomula showed positive staining reactions in 1 day. = Living schistosomule showed no staining reactions in 1 hr. d M, Motility (&, only occasional sluggish movement at the anterior end: 4, occasionally contrwliug and extending; $ +, moderately contracting and extending; ++-I-. actively contracting and extending). e PG. Purple-blue granules ( &. a few hardly recognized granules : + , D few fine psoules ; + +, %Imoderate numh~r of Ene granules ; + f-l-, umny c.o&rse granules). f B, Blue color (A, trace of hght blue color at the posterior end; +, light blue color in the nrholc body: f C, Italian blue in the whole borly: + ++, peacock blue in the whole body). s NE, Not examined.
,5 min 10 min 15 min 20 min 30 min 40 min 50 min 1 hr
Time
Reactions
/j
x 2:
Schktosoma
munsoni AND S. japonicum:
of normal or immune serum to the medium. Dead schistosomula were stained a light green-blue but deteriorated organisms were essentially unstained. The staining reactions of living schistosoma maintained in the culture medium for 1 day were, in general, similar to those occurring with the specimens maintained in the harvest fluid, except that in the uncovered organisms the purple-blue granules might be seen at 10 or 15 min, and the blue color might be quite deep at 30 min. The staining reactions of the uncovered schistosomula incubated in culture medium for 2-5 days differed from those cultured for only 1 day, or from those maintained in the harvest fluid. In 2-5 day cultures: (1) the purple-blue granules did not always appear at 15-20 min, and even when they appeared, were very indistinct; (2) the blue coloration appeared at lo-20 min instead of 30 min; and (3) the schistosomula became contracted and nonmotile at 2040 min. In the moist chamber, the schistosomula may appear alive, actively contracting and extending, for 17 hr or more. Staining Reactions of Methylene Blue on Schistosomula of Schistosoma japonicum Methylene blue staining of S. japonicum schistosomula, observed on limited occasions, did not differ from the staining of S. mansoni. DISCUSSION
Our results with the methylene blue stain show that the viability of schistosomula can be classified as follows: living and unstained (Fig. 1); living but with scattered granules stained purple-blue (Fig. moribund or nonmotile and stained 2); deep blue (Fig. 3); and dead and stained light green-blue or unstained (Figs. l-3). The living schistosomula were active, extending and contracting, whether they were unstained or had purple-blue granulcs. The moribund schistosomula were characteristically contracted, nonmotile or
METHYLFXE
BLUE VIABILITY
333
sluggish, and stained a deep blue. The dead schistosomula were flattened, often with a protruding ventral sucker, and stained a light green-blue or were unstained, according to the length of time since death. During the staining test, the dead schistosomula, if stainable, usually color immediately while the living ones take the stair1 gradually and metachromatically, i.e., from purple-blue to deep blue. For this reason, schistosomula eventually seen to be contracted and stained deep blue must be recognized as having been the living ones at the beginning of the test. The rationale for the color changes of schistosomula in the methylene blue test may be as follows: The oxidized form of the dye is bright blue, while the reduced product is a leuco compound. In the presence of oxygen, the leuco form may regain its color by nonenzymatic oxidation. When methylene blue is added to living schistosomula, the dye is rapidly reduced by enzymes to its leuco form and the organisms are not stained. The groups of enzymes known as dehydrogenases, which bring about the transfer of hydrogen to methylene blue, have been reported in schistosomes (von Brand 1973). However, near the surface of schistosomula, nonenzymatic oxidation keeps methylene blue in the oxidized form which appears as purple-blue granules, possibly representing the reactions in the subtegumental cells. When the schistosomula gradually become moribund, the enzymatic reaction of the dehydrogenases diminishes. Accordingly, the oxidized form of the dye takes its place, and the organisms appear deep blue. If the schistosomula have been dead for some time, the organisms may become acidic and the staining reaction will be light green-blue or even colorless. On the other hand, the occasional lack of staining of schistosomula in the methylene blue test when they were covered with a glass slip, evidently is due to the lack of oxygen. This
334
HSti ET AL.
assertion is further supported by the development of coloration in the organisms when oxygen is made available by removing the coverslip and adding a drop of fresh saline. According to Clegg and Smithers ( 1968), there were three kinds of staining reactions for schistosomula in the methlyene blue test: (1) nonviable schistosomula which were stained bright blue, (2) living schistosomula which were completely unstained, and (3) contracted and moribund schistosomula which were stained intensely with methylene blue, They classified only the unstained schistosomula as “living” and all the stained as “dead.” According to our results, the dead schistosomula were stained a light greenblue or unstained, and the living ones were unstained at first but later stained a purple-blue and then a deep blue. The differentiation of dead schistosomula from living ones should be made by the characteristic changes of the staining reactions, considering the time factor involved in these changes. Since contracted schistosomula seen during later times in the experiment were typical of organisms that had been alive initially, we do not agree with Clegg and Smithers (1968) in counting these schistosomula as “dead.” Examination for the viability of schistosomula usually involves 30 to 40 organisms per slide. Hence, it is difficult to make a reasonable estimate. The diagnostic help provided by the methylene blue staining
is necessary. Since all the living schistosomula in the uncovered method will assume a contracted form and stain a deep blue in 50 to 60 min, and since the resulting deep blue color contrasts distinctly with the light green-blue or colorless dead schistosomula, it should be easy to make a differential count of the living and dead organisms by coloration (Fig. 3). The coverslip method is unsuitable for this test because of the uncertainty of the color reaction appearing in the test. The moist chamber method also has its drawbacks because a comparatively long time to required to stain the living organisms a deep blue. ACKNOWLEDGMENTS This investigation was supported in part by U.S. Naval Research Contract N00014-68-01960007, and by a grant from the Edna McConnell Clark Foundation. REFERENCES CLEGG, J. A., AND SMITHERS, S. R. 1968. Death
of schistosome cercariae during penetration of the skin. II. Penetration of mammalian skin by Schistosoma mansoni. Parasitology 58, 111-128. Hsti, S. Y. LI, AND Hsii, H. F. 1975. Recovery of schistosomula in the skin of rhesus monkeys immunized with cercariae of Schistosoma japonicum exposed to high doses of X-irradiation. Journal of Parasitology 61, 1108-1109. STIREWALT, M. A., AND UY, A. 1969. Schistosoma mansoni: Cercarial penetration and schistosomula collection in an in vitro system. Experimental Parasitology 26, 17-28. BRAND, T. 1973. “Biochemistry of Parasites,”
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2nd ed., pp. l-499. Academic Press, New York.
