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Kassanis, B. and Kleczkowski A. 1944. The effect of formaldehyde and
mercuric chloride on tobacco mosaic virus. Biochemical Journal. 38 (1),
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© 1 January 1944, Portland Press Ltd.
The Effect of Formaldehyde
BY B. KASSANIS AiN A. KLECZKOWSKI,Rothameted Experimental Staton, Harpenden,Herts (Reeeived4December 1943)
The action of formaldehyde in killing bacteriaand
inactivatingviruses without affecting their sero-logicalreactions is wellknown and widely usedin
theproduction ofvaccines. Themechanism under-lying thistype ofinactivationisby'no means
under-stood, and there is considerable contradiction in published work as towhether the changes are
re-versibleor not. Schultz&Gebhardt(1935)reported that a Staphylococcus phage treated with formalde-hyde couldbe reactivatedmerely by dilutionand
storage. Ross & Stanley (1938) reported that tobaccomosaic virus treated withformaldehyde at pH7could be partially reactivated by dialyzingat
pH 3, that groups reacting with Folin's reagent
wereaffected by theformaldehyde treatment and thatdialysis restored these groups to their former
stateindegreeproportionalto the amount of reacti-vation.
Onthe other hand, Galli & Vieuchange (1939) and Levaditi&Reinie (1939)couldnotregain anyinfectivity in neurovacciniavirustreated with
Reactivation of viruses inactivated by mercuric chloridehas also beenclaimedby variousworkers.
Went (1937) reported that all the infectivity of tobaccomosaic viruspreparations couldbe
dilution,and Manil(1938) claimed
reactivation ofatobacco necrosis virusby
precipi-tating the mercuric ions withanmmonium carbonate.
Kreuger& Baldwin (1933) found thata
bacteriophageofS. aureus which hadbeen
inacti-vated by mercuric chlorideatpH 7could be
com-pletely reactivated by precipitating the mercury
The present paper describes
inactivating effects offormaldehyde and mercuric chloride on tobacco mosaic virus under different
conditionsand thefailureofattemptstoreactivate inactivatedpreparations.
Purified tobacco mosaic virus(T.M.V.)intheliquid
crystal-linestate was used in alltheexperiments. This was pre-pared by methods described by Bawden & Pirie (1943).
Some preparations had been incubated with trypsin and
others not;nodifferenceswerefound betweenpreparations
made in the twoways.
Unlessotherwisestated, virus solutions inphosphateor
with 2%formaldehyde or0.5
%mercuric chloride. Before being tested for infectivity or serological reactions the treated virus solutions were either dialyzed thoroughly
against distilled water, when the virus content war measuredbymioro-Kjeldahl, or diluted to below the limits at whichthe reagents have any effects on the plant or the virus. To avoid damage to the plants, theconoentration offormaldehyde must be less than
0.5%and of mercuric chloride less than 0-05%; at pH 7 solutions of 0.25% formaldehyde and 0.01% mercuric chloride have no de-tectable effect on infectivity after contact for 24 hr. Infectivity tests were made by the local lesion method, withNicotiana
glutinosa.Folin's colourtests were made
atpH8by the method described by Ross &Stanley (1938).
Preliminary experiments showed that vaiation in thehydrogen-ionconcen,trationoverthepHrange
3-7 had no effect on the
infectivityof the virus preparations. Sampleswereincubated for 24 hr. at
differentpH valuesand wereequally infectivewhen inoculatedafterneutralization or afterconsiderable dilutionin water.
Table 1. Theeffect of pH oninactvation ofT.M.V.
formaldehydeand by mercuric chloride
005% solutions of the virus in buffers atdifferent pH
values.were incubated for24 hr. with 2% formaldehyde
5%mercuricchloride,thendilutedand inoculated. Infectivity
(numbers of lesionsonsixleaves)
6*0 7 0 Controls 7-0
Formaldehyde Dilu- Dilu-tion tion 1/100 1/1000
Dilu-tion tion 1/100 1/1000
176 122 129 2
62 27 30 0
400 55 183 45
Virus solutions in buffers of different
pHvalues were incubated for 24 hr. with 2
and tested for
infectivity.The results in Table I show that
INACTIVATION OF TOBACCO
the whole range tested,although the rate of inacti-vation was minimal around the isoelectric point, pH 3-5. With longer exposure to theseconditions complete inactivation with formaldehyde. can be obtained at any of these pH values. Myrback (1926) found that the inactivation of invertase by HCHO and
HgCl2was similarly affected by pH.
Failure of attempts to reactivate tobacco mosaic virus inactivated by
Using the method of Ross & Stanley (1938), we attemptedunsuccessfully to reactivate preparations
of T.M.V. partially inactivated by formaldehyde. The results of one such experiment are given in Table 2.
over opposite half-leaves. Formaldehyde-treated virus diluted
1/100andstored for 3daysat20 was
tested in the same way. The infectivity of boththe
diluted and the dialyzed sample was still most
nearly approached by the control solution
con-tainingbetween 0-15x10'6and 0-3x106 g./ml. Inaddition, the dialyzed and the diluted samples of formaldehyde-treated virus were compared directly at 2 x 10-6 g./ml. by inoculation to the opposite halves of 80leaves. Thediluted samplegave
atotal of 610 and thedialyzed sampleatotal of 590 lesions. Thus there is no evidence that dialysis at
pH 3 0 had in any way affected the infectivity of formaldehyde-treated virus. In similar experiments the loss of infectivity by incubation with 2% formaldehyde for24hr. at pH7-0 varied between
Table 2. The effect of
dialysisat pH 3-0 onT.M.V. treated with 2
%formaldehyde at pH 7-2
Testafterincubation for Testwithformaldehyde-treated Testwithformaldehyde-treated
24hr. withformaldehyde virusdiluted 1/100 and virusdialyzed for
atpH7-2. stored for3days. 3daysatpH 3-0.
Averageno.of lesions Average no.of lesions Averageno.of lesions per
leaf.formedby perleafformed by perleafformedby
Conoentration , -_-_ __,_- ____,_
_-of the oontrol Control at Treated virus Control at Treated virus Control at Treatedvirus
virusx10-5 varying at 2-0x10-4 varying at2-0x10-4 varying at 2-0x 10-4 (g./ml.) dilutions
38 24 14
64 52 49
70 76 8
108 81 40
%solution of T.M.V. wasincubated for24hr.
at pH 7-2 with 2
%formaldehyde. A sample was
then taken and a 1/100 dilution in water made,
toreduce the concentration of formaldehydewell
below the level at which it has any appreciable effect. The bulk of thesamplewasstoredat20, and the remainder was used todetermine the extent of inactivation. This was doneby7comparing on oppo-site half-leaves the infectivity of the sample with thatof the controlat variousdilutions,and deter-mining the dilution of the control which gave
approximatelythe samenumber of lesions. It will beseenthatinfectivityoftheformaldehyde-treated preparation containing 2-0 x 10-4 g./ml. was most nearly approached by the control solution con-taining between 0-15x10-5 and 0-3 x10-6g./ml., so that the residual infectivity amountedtobetween vs and
Tiwof the control.
Thebulk oftheformaldehyde-treated virus was dialyzed for 1 hr. against distilled waterandthen for 3 days at
20against frequently changed
citrate buffer of
pH3. Then itwas
first againstdistilled water andthen against m/15 phosphate buffer of pH 7-2.The dialyzed prepara-tion offormaldehyde-treatedvirus was thendiluted
to aconcentration of2-0 x10-4g./ml. and compared with the control at various dilutions by rubbing
lostmoreinfectivity. This additional losswas pre-vented
infectivity was regained by eithertreatment.
'Several attempts were also made to reactivate
virus whichhadbeenpartially inactivated
in infectivityduringincubationwasless than when treatmentwasmadeatpH about 7-0,
between90 and 98
Again,both dilu-tion anddialysis stopped further-inactivation, but
neitherprocedure ledtoany increasein
pH 8-0colour test
Ross'&Stanley (1938) foundthattreatingT.M.V.
withformaldehyde ledto a drop of about 50
the colour value given by Folin's pH 8-0 test.
They suggestedthat the extent of the
dropin the colour value was correlated with the
pH3 gave an
increase in the colour value
correspondingto the amountofreactivation.
We have confirmed the decrease in the colour
atpH 7-0,but wereunable to reverse thisdecrease by prolonged
diilysisat pH 3-0. Neither was there
any real correlation between infectivity and the colourvalue. If viruspreparationsaretreatedwith
formaldehydefordifferentlengthsof time, thereis
anapparent correlation, forboth colour value and
infectivity decrease with increasing length of
ex-posure. But wehave found that the colour value also decreases after treatment of the virus with
formaldehydeat pH 3-0. By treatment of the virus with formaldehyde at different pH values for
different lengths of time, preparations can be
ob-tained with higher colour values than others with higherinfectivity. Thedecreasein the colour value
after a prolonged formaldehyde treatment of the
virusatpH 3 0 is about 20
%,whereas at pH 7 it is
%.Boththedecreasein colour value and
formalde-hyde treatment is carried out at pH 7-0 than at
pH 3-0, s0 that ifthe virus is incubatedfor equal
lengths oftime withformaldehyde at the two pH
values there is again an apparent correlation be-tweenthecolourvalue and infectivity. But if the
virus is incubated with formaldehyde for longer
periods atpH 3-0 than at pH 7-0, the infectivity may be lower though the colour value may be
higher. Forexample,in one test when 1
of the viruswere incubated with 2
atthetwopHvalues for24hr.,at pH 7-0 the colour
value was 55
%and thenumber of lesions 4
%of the original, and at pH 3-0 the colour value was 90
%andthe number oflesions .35
pH 7-0for 16 hr. had a colour value of 65% and
the numberoflesions 7
%of the original,whereas thepreparation treated for 72 hr. at pH 3-0 had a
colour value of80
%althoughthenumber of lesions
was only 1-5
%of the original.
Ross& Stanley(1938)attributed the decrease in
Folin'spH8-0colourvalueafter formaldehyde treat-mentofT.M.v.at pH 7-0 to changes intryptophan.
They deduced thisfrom thefact that the colour value
givenbytryptophan is decreased after incubation at pH 7-0 with formaldehyde, whereas the colour
valueoftyrosine, the only otherconstituent of the
virusknowntogive the reaction, remainsunaltered. We have tested the effect of formaldehyde on tryptophan and tyrosine at pH 8-0 and 3-0. 0-2
solutions of the amino-acids in buffers at pH 3-0
and8-0 were incubated for 48 hr. at 370 with 2
%formaldehyde. The crystalline precipitate that
ap-pearedin the tryptophan treated with formaldehyde at pH 8-0 was dissolved by heating before the
value:Thecolour value of
tryptophanwas reduced to about 60
%after treat-ment atboth pH values, whereas that of tyrosine wasunaffected. The fact that both
tryptophanand T.M.v.have their colour value reduced when treated
with formaldehyde in acid andalkaline conditions supports the view thatchanges in tryptophan occur
during formaldehydetreatment of the virus. Never-theless,the interpretation is by no means
straight-forward. The colour value of isolated tryptophan falls by approximately the same amount after treatment with formaldehyde at pH 3-0 or 8-0, whereas that of the virusfallsmuch more atpH7-0 than atpH 3-0. Ifthetyrosine :tryptophan ratio in T.M.V.is about 0-8(Knight&Stanley, 1941) and the ratio ofFolin's pH 8-0 colourvalue given by tyrosine to that given by an equal weight of
tryptophanis about 1-3,thecolour value ofT.M.V.
treatedwithformaldehydeatpH3-0fallstoavalue that would be expected from the behaviour of
isolated tryptophan; however, thefall after treat-mentwithformaldehyde atpH about 7-0 is much greater thanexpected.There seem to be twoequally possible explanations: the virusmay contain certain
other constituentswhich react with Folin'sreagent
and are affected bytreatment with formaldehyde
at pH 7-0 but not at pH 3-0; or tryptophan as
combinedinthe vwus is affectedmoreby
formalde-hyde treatment at pH 7-0 than when it is free. There is evidence that the mner in which
con-stituents arecombined inaproteindoes affect the colour value, for heat denaturation increases by about40
%thecolourvaluegiven by T.M.v.,and a
similar effect of denaturation has been described
with pepsinogen (Herriott, 1938). Therefore, the
simply a summation of the values of individual
constituents, but also depends on the manner in
which thesearearranged. Becauseofthisit is im-possibletointerpretthechangesinthe virusonthe
Wadsworth & Pangborn (1936) found that the
crystalline product formed
treatment oftryptophan at
pH8-0was 3, 4, 5,
obtained by Jacobs & Craig
tryptophanwithformaldehydeinacidmedium. We have obtained a crystalline product from T.M.V.
formedduring incubationoftryptophan with form-aldehyde at pH 8-0. The
virus was dialyzed, denatured by heat, incubated
%commercial trypsin for 3 days at 370 and then left for 6 days at 20. No attempt was
made to identify the crystals, but they were not
formed in control virus solutions treated similarly
Slyke teat for amino-nitrogen
10mg. of T.M.V. was found by the Van Slyke method for estimation of free
INACTIVATION OF TOBACCO MOSAIC VIRUS
(at 200) increase ofpressurecomparedwithablank.
This corresponds to afreeamino-nitrogen content
equivalentto0-8%ofthetotal nitrogen. Treatment with formaldehyde at pH 7-4 for 24 hr. decreased the value given by the Van Slyke test to about
60%of the value for untreated virus, and this remained unaffected by 3 days' dialysis at pH 3-0. This agrees with the results of Ross & Stanley
(1938). Non-infective virusproducedby formalde-hydetreatmentatpH3-0for3daysgavethesame
value in theVan Slyketest as controlvirus, which
suggeststhatchanges ingroups responsible for the test are not necessarily associated with loss of in-fectivity caused by formaldehyde. It must be
emphasized, however, that in view of the small number ofgroupsgiving thetest, small differences
would not be detectable, and that the test shows only changesnotreversible by thetreatmentwhich it involves.
Inactimation ofT.U.V. bymercuricchloride
Table 1 shows thatmercuric chloride inactivates
the virus only over a narrow range ofpH values,
having little effect in acid conditions. Loss of in-fectivity as aresult offormaldehyde treatment is
notaccompaniedbyanylossofserological activity, andnon-infectivepreparations of the virus give the
same titres in the precipitin test asfully infective
controls. Treatment withmercuricchlorideatpH's above 6-0 leads to a reduction of infectivity and
serologicalactivity;achange alsooccursinphysical properties,for treatedsolutionsbecomeincreasingly
viscous and losetheir birefringence.
Table3. The effect of mercuric chlorideatpH 7-0
ontheinfectivity and8erologialactivity ofT.M.V.
Time of incubation
-Infectivity; lesions as
number formedby thecontrol(538)
Serological test; precipitation
1/1600 1/1600 1/1600
Table 3 shows the results ofone experiment in
which 0-4%solutions ofT.M.V.wereincubated with
0-5%mercuricchlorideatpH 7-0 and then diluted
1/100 and inoculated into plants. Six leaveswere
inoculated with eachpreparation. It will be seen
that inthe early stages ofincubation there was a
significant drop in serological titre, while infectivity
wasactuallygreaterthan that of the control. After
prolongedincubation the ratios between infectivity and serological activity were reversed, for infec-tivity fell steadily without any corresponding
re-duction ofprecipitintitre. Fromsuch preparations material could be removed by centrifuigation at 10,000r.p.m., leaving serologically active material in thesupernatant fluid,which still showed
A somewhat similar result was obtained by
Bawden & Pirie (1940) with T.M.V. treated with alkali, when gentle treatment often led to an in-crease in infectivity,whilemorevigoroustreatment led to a decrease. They regardedthe increase as a result of thedisaggregation of large virus aggregates
into smaller infectiveunits.
It was shown in Table 1 that inactivation by mercuiric chloride occurs only at pH valuehigher
than 6-0, and then, asshowninTable 3,itproceeds
gradually. It can be stopped at any stage by
acidification,bydilutionsufficientto reducethe con-centration of mercuric chloridebelow0-01%,orby adding sufficient quantities (2-6%)of certainsalts.
Some of the salts, e.g. (NH4)2C00,
(NH4)2S,act by precipitating
Hgl+,and others, e.g. BaCI2 or
thesolutions. There are, however, some salts,such as NaCl or KCI, which neither precipitate
nor acidify the solutions, but which
preventinactivation. We havebeen unableto find
any indication that these treatments reversed in-activation. Bydilution,acidificationorby addition
of certain salts, inactivation could be stopped at any stage,
btutvirusalready inactivated couldnot bereactivated. Samples ofthevirus incubatedwith 0-5
infectivity when tested immediately after any of these treatments as when tested 24 hr. later. In somecases, especiallyduring the stagewhen inacti-vation proceeded most rapidly, acidification or addition ofsome saltsresultedin higherinfectivity
than that given by dilution. This differencecould beinterpreted by the assumption that such treat-ments stop the progre#s of inactivation more
mainipoints that call for discussion are the
discrepanciesbetweenour resultsand thoseof other
workers, whoclaimed that T.M.V. inactivated with
formaldehyde or with mercuric chloride could be
possible explanationis that we
workedlwith astrain of virus whichbehaves differently, but this is unlikely, as few differences
have been found in thebehaviour ofdifferentstrains ofT.m.v. Thediscrepancies probablyarise fromthe
unreliability ofinfectivitytests asmeasurements of the amount of active virus present in different inocula, because the number of lesions produced by an inoculum can be affected by many other factors.
Many substances are known, such' as trypsin, serumproteins andsomeplantconstituents,which act as inhibitors of infectivity. When present in
inocula these greatly reduce infectivity, and by
adding andremoving themfrom virus preparations
apparently complete inactivation andreactivation can be produced (Bawden, 1943). Whether such
inhibitions are caused by affecting the host or by forming non-infective complexes with the virus is
still unsettled, but there is no evidence that the
inhibitors produce any changes within the virus
particles. In concentrationsover 0-01
chloride can act assuch an inhibitor at pH below
6-0,and thisprobably explains Went's (1937) claim
of complete reactivation by dilution in water of
solutions of T.M.V. containing mercuric chloride.
Went didnotcontrol the pH of her virus-mercuric
chloride mixtures, which were probably acid, a
condition inwhich true inactivationdoes not
pro-ceed, although inhibition might be complete and
there would probably be obvious damage to the
inoculated leaves.When dilution wassuchthat the concentration of mercuric chloride was reduced below the inhibiting level, the original infectivity
would be regained, and it would look as thoughthe
virushad been reactivated.
Similarly,the presence ofsmall amounts of
inhi-bitorsin the preparations used by Ross & Stanley
(1938) could explain the apparent smallamountof reactivation gained by dialyzing formaldehyde-treated virus preparations. It is possible that
dialysis at pH 3-0 either removed or destroyed
someimpuritywhich was reducinginfectivity.This could also explain the change in the Folin colour value after dialysis, which we wereunable to
con-firmin our tests.
Evenin the absence of inhibitors, however, infec-tivity doesnot depend solely on theweightofactive virus, but also on the number of virus particles.
It is known that T.M.V. particles can aggregate
linearlytoform large rods (Bawden & Pirie, 1937), and this affects the infectivity. That this effect is
partially reversible issuggested by the increase of
infectivity frequently givenby short exposuresto alkali (Bawden&Pirie, 1940) or mercuric chloride
(cf. Table 3). The increase caused by mercuric chloride was often greater than that shown in
Table 3, forin thistesttheconcentration was such that inactivation was also proceedingrapidly. The presenceofmoredilutemerciric chloride has often increased the infectivity to more than twice that of
control virus solutions. Thus the discrepancies be-tweenourresults and those of Ross & Stanley (1938)
could alsohaveresulted fromthe useofvirus
pre-parations indifferent degrees of aggregation, with
their degrees of aggregation being differently
af-fected by the processes which led to an apparent reactivation of infectivity.
1. Tobaccomosaic viruswasinactivated by 2 %/ formaldehyde at all pH valuesbetween 3 and 7-5;
the rate of inactivation was minimal at pH 3 5.
Inactivation could be stopped at any stage by dilution or dialysis,but there was noevidencethat inactivated viru.s regained infectivity by these
2. Loss of infectivity caused by formaldehyde does not depend on changes in groups giving the
Van Slyketestforamino-nitrogen, forpreparations
inactivated byformaldehyde treatment atpH 3-0
givethe satne value as control virus. Formaldehyde
treatment at pH near7-0 leadsto agreater fall in
the Folin pH 8-0 colour value than treatment at
pH 3-0. There is no real correlation between the
decrease ofinfectivityand ofthe colourvalue. 3. Mercuric chloride insufficient concentrations
acts as an inhibitor ofinfectivity. At pH values greater than 6-0 it causes loss of infectivity and serologicalactivity. Dilution, acidificationor addi-tion ofcertain salts prevent inactivation or inter-rupt its progress at any stage, but there is no
evidence that any of these treatments can reverse it.
One of us (A. K.) has been in receipt ofa grant from theAgricultural ResearchCouncil.
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