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Mercurochrome (di-brom oxy mercuri fluorescin) as a fungicidal agent in the growth of amphibian embryos.

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MERC*UROCHROSIE (DI-BROM OXY MERCURI
FLUORESCIN) A S A FUNGICIDAL AGENT I N
T H E GROWTH O F AMPHIBIAN EMBRYOS
8, R. DETWILElZ A N D G, E. McKENNON
Department of Anatomy, College of Physicians and Surgeons, Columbia Umiuersity
I n the present communication we wish to report some
observations which we have made upon the application of
Mercurochron=e as a fungicidal agent in the growth of
Amlnlystoma embryos. During the operating season of 1928
(March t o May) the conditions under which we worked
favored the growth of molds, and the larvae which had been
employed in various types of tissue-grafting experiments
early became infected.
The chief mold which we encountered was Saprolegnia,
although there seemed to be various other strains which we,
unfortunately, did not identify. The first infections occurred
in March. Several series of larvae which had been operated
upon early in March and which were in healthy condition
several weeks after the operation suddenly began t o show
the presence of mold. The growth occurred usually in the
form of small tufts which grew out from the gills, the legs,
and from the sides of the body, particularly along the lateralline sense organs. I n a number of cases the infection was
so severe that death ensued before treatment could be applied.
Attempts were made in the beginning to reduce the infection by changing the animals daily into fresh spring water
which had previously been aerated. Tliis did not reduce the
infection. At the suggestion of Doctor Noble, of the American JIuscum of Natural History, we then tried to reduce the
growth by placing copper coins in the water-a method which
he employed with partial success in treating older animals.
205
'l'his also seemed to 'have no effect.. We then learned from
M r . Otto Gneiding, a. breeder of fancy fish in Ridgefield Park,
Sew Jersey, that he wa.s able to prevent. the grov7t.h of molds
which frequently develop in t.he wounds of fish by adding
Mclrcnrochrome to their aquaria. With this informat.ion a t
!raid, we set out t o investigate the possibilities of using this
aiitisept~icin t.he ca.se of infected embryos. As a preliminary
test, several aiii.mals with well-developed t,ufts of mold on the
fore limbs and gills were placed in a solution of 1.: 500,000
Mercuroclirome made up in spring water.' 'In twenty-f oiir
honrs after treat,ment, .t,hehitherto vigorous hyphae had colI q s e d , and t.he whole growth was easily removed with fine
forceps. No effects upon the larvae were noticed. After tlic
mold had been removed, t,he larvae were placed back in spring
water. In several other c.ases, however, in which the mold
h a l already covered the whole body, Mercurochrome t reatment. had no effect, and the animals died.
Tnvestigations were then made i n an attempt to find a concimtra.tion which would destroy the mold and in which larvae
c?onld hc reared wi.thout injury and retardation of growth.
This was of ut.most importance, since temporary treat.ment,
altliough effective, would be of little value if the animals hail
to be placed back in spring water where reinfection took place
so readily. At. this juncture we were encountering tl general
growt.h of mold upon many of the larvae in a number of
experimental series. .A concentra.tion of 1: 750,000 u7as made
lip, and all infected animals of the various series were placed
in this solution. !Phe larvae at this period were ent.ering the
f c d i i i g stage (two to tliree weeks after tail-bud sta.ge). The
resu1t.s of the treatment arc shown in table 1. Tn this table
four series are listed. There were siut.y-nine animals, of
which fifty-six showed t.hc growth of mold to varying degrees.
Tap-witer coiilil not be used in thc experiments, hecause -it was found to
Ernlbryos placed. in tap-waLtCr whcn ill st:tge 29 (tail-bud stag<,) showed
no effeets a.s regards growth and renet,ions up until the time when the circulation
1wg:in. Thc 1:trvao t,lieii ceasrtd t,o grow, t h c gill filaments riecrosed, cpithelkil
sloughing occurred, the circulation failed, and death ensued. Control cmhrpox
in a erat ed spring wat cr un (1CI'w PILt. 11orni:il drrelopment .
tie toxic.
207
MIERCUROCHROXE AS A FUXGICIDAL AGEKT
In twenty-four to forty-eight hours after treatment, the mold
was either completely sloughed off o r was in the proccss of
sloughing.
As a control to these experiments we had a number of cases
in which molded animals were placed in fresh spring water.
The mold did not disappear until after these animals were
given treatment.
The treated animals were kept in the Mercurochrome solntion up until the time they were fixed, which ranged from
fifteen to fifty days. It was found that after several days the
solution began to lose color. A fresh solution of 1:750,000
TABLE 1'
Showing e f e c t s of Xercurochrome treatment in various series of experimental
embryos wliich developed molds
........
.
..............
~~~
. . . . .
.........
...
. .
........
..
...
lAiiimals badly infected were placed ill a coilwiltration of 1:800,000 f o r twentyf o u r t o forty-eight hours, a f t e r which the solution was diluted t o 1:750,000 or
1: 1,000,000.
concentration possesscs an iridescence and has the color of
weak eosin. After several days the solution begins to deteriorate. This deterioration appears to be hastened by the
presence of dead organic matter in the aquaria which accumulates from various forms of unconsumed food. For this
reason, the solution was entirely changed from time to time,
or enough of the new solution was added to the aquaria to
bring the color approximately up to that of a 1: 1,000,000 concentration. In several series of experiments (series CCRU),
in which the mold had developed to only a slight degree, a
concentration of 1: 1,000,000 was employed and appeared to
inhibit further growth.
208
S. R. DETWILER A N D G. 3:. MCKENNON
Many animals which were reared for several weeks in the
Mercurochrome and which showed no evidence of infection
were allowed to remain in the solution without further change.
Alt.hough deberioration took place in the course of several
weeks, most of these cases remained free of mold. In cases
o f reinfec.t.ioii,a new solution was employed.
S o delet.erious effect.s of the Mercurochrome upon t.he experimental larvae were observed. The feeding react.ions were
normal and the growth rate was in no way impaired. Vary.ing the concentrations within the limits of 1 : 500,000 t.o
1 : 1,000,000 appeared to have no eflect. I n fact, several cases
a i * c recorded in which older unoperatcd larvae, which became
infected about forty-five days after t.ail-bud st.age and which
were heated wit.h a. concentration of 1:210,000, suffered no
deleterious effects of t.his strong solution.
The application of Mercurochrome treatment, to experi.mciitnl.animals usually did not begin until from two t.o three
weeks aft.er the tail-bud stage. At this time the larvae are
entering the feeding stage and condit.ions enhanc.ing mold
grot\-t.h are then introduced, because food is placed in the
aquaria, thus exposing the dish more frequently t.o spores.
Mnc+h of t.liis food is not consumed, and with t,he accumulation
of dead organic matter tlie mold growth usually increases
ra.pid1j-.
During the ea.rly part of the experimental season we were
very successful in w r i n g infect.ed animals a.nd iiihibit.ing
further growt,h in the aquaria. Later in the season, however,
we eiiconntered a more vigorous or resistant mold which
g i ~ licavily
x
upon the dead daphnia in the aquaria, even in
the presence of stronger concentrat.ioiis of Mercurochromc
solutions (1 : 250,000) &an. previously employed. The fact
that. under these circumst.ances only a few a.nimaJs became
infected was regarded by us as indicating that the Mercuro(.bhromewas active in inhibiting growth upon tlie living larvae.
'L'his was suggest.ed further by the fact that larvae which
tlied from other unknown causes developed mold very rapidly,
as did the dead daplinia, but mold w1iic.h had gained a foot-
209
MERCUROCHROME AS A F U N G I C I D A L AGENT
hold on healthy living larvae nearly always sloughed off in
twenty-f our hours in the presence of the hfercurochrome solutions employed.
Although concentrations of Mercurochrome ranging in
strength from 1:1,000,000 to 1: 250,000 showed no deleterious
effects upon larvae which were several weeks old when first
treated, a series of observations upon early embryos did
show marked effects of the Mercurochrome. For these observations, a series of normal embryos between stages 29
and 37 were placed in concentrations of Mercurochrome varying from 1 : 100,000 to 1: 1,000,000. These solutions were
TABLE 2
Showing survival (in days) of normal Amblystonta embryos (stages 29-37)
grown in different concentrations of Xercuroclrrumc
-_
-~
CONCENTKATION
-.
~
I
I
I-
1:100,000
1: 150,000
1: 250,000
1: 500,000
1: 750,000
1: 1,000,000
..___~
I
'
1
I
..-
changed every second day. The results are given in tables 2
and 3. From table 2 it is seen that embryos survived progressively longer and longer periods 8s weaker conceiitrations
were employed. Embryos in a 1: 100,000 concentration sixvived only one day, whereas those in a 1:1,000,000 survived
as long as thirty days. During the periods of treatment
gromth was greatly retarded and the larvae early developed
abnormal postures aiicl reactions. These developed earlier
and were most marked in the stronger concentrations (table
3). The abnormalities consisted, in all cases, of kppliosis
which was accompanied by a failure of progressive swimming.
In many cases the animals, when stimulated, underwent
vigorous tremors. The specific lethal effects of the Mcrcnro-
210
S. K. DETTVILER ARlJ G . E. MCBEKNON
clirome upoii these early larvae have not been det.ermi.iied.
Whatevclr they are, they have no effect upon larvae sevei-al
u-eeks of age, for these older larvae c.an be grown continuously in solutions a-s strong as 1: 500,000 wit.liout any noticea l ) k interferelice with normal growt.1~arid react-ions.
TABLE 3
Slr o rr iir g t'ivrr ( i?r d a y s ) uiliiju
A 111 711 oiiiu mibrgos (.utngr 3 7 ) ti,c?citcxl
w i t h ,71ercurochsosw first cd&iBiletl abnorniul reuc!tionrr
.....
.. .
.
'ii or.naal
.
..
(!ONCEATItAT10A'
-.
...
1 : 150,ooo
1 : ''50,000
I : 300,000
3 : 7.50,000
1 : 1,000,000
. -.
.
~~
. -.
.
..
~
.
DAYS
.. .
.
.
~
2
I
(i
x
x
8
SITMMARI'
1. Mcrcui-ochrome in conceiitratioiis rarigiiig from 1: 300,000 t o I : 1,000,000 has been employed successfully N S H
Puiigicitlal agent in tlie growth of Amblrstoma embryos.
2. Larvae several weeks of age caii be sncccssfnlly treated
n-ithoiit any dclctcrions influence upon tlic animal. Contiiiiious treatment over a period of two months has 110 noticeable
effect upon growth. Aiiimals feed and react iiormally in the
pi'cseiicc of tlic fuiigicidal solution.
3. Noltls growing on larvae usuallj- sloiigli off witliin two
days when in the presence of hlercurochrome (concentration
1: 750,000).
1. Molds of resistaiit strains grow upoii dead orgaiiiv
nintl(1r (unconsnmed food) in tlic preseiicc of JIcrcurochromc
(coiicentratioii 1: 500,000) , but this concentration usually inliihits growth upon healthy larvae.
+j.Fresh coiiceiitrations of 1: ,500,000 a i d 1 : i50,000 ~ O S S ~ S
mi iridcscence and have tlic color of weak eosin. This color
fades witliiii a week aiitl tlie solution loses its effectiveness.
Its cleterioraiioii appears to be liastoiiecl by the preseiice of
(lead orgaiiic matt cr.
S
MBRCUBOCHILOXl3 AS A FUNGICIDAL AGENT
211
6. Early embryos (stages 29 to 3’7) subjected to continuous
t reatmeiit of Mercurochrome in coiiceii1ra tioiis ranging from
1: 1,000,000 to 1 : 100,000 develop abnormal reactions, aiid
eventually die. Embryos survive progressively longer aiid
longer as weaker conceiitratioiis are employed. Oiily yomg
embryos are lethally affected.
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