comparative quantitative studies fungitoxicity fungal spores … · comparative and quantitative...

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Comparative and Quantitative Studies of Fungitoxicity against Fungal Spores and Mycelia1 TOMOMICHI YANAGITA2 AND SABURO YAMAGISHI Institute of Food Microbiology and School of Pharmacy, Chiba University, Narashino, Chiba-ken, Japan Received for publication February 25, 1958 In studies on the mode of action of antifuingal agents, it may be valuable to reveal the difference in drug sensitivity between fungal spores and mycelia. Various methods employed in the evaluation of antifungal substances do not necessarily apply to the solution of this problem. For the estimation of "sporostatic" activity of an agent, the inhibitory effect on the course of germination of spores can be taken as a measure of (luantitative evaluation of the activity. On the other hand, the quantitation of growth inhibitory activity oni mycelia ("mycostatic" activity) of an agent is much more complicated. In the present experiment, mycelial fragments pre- pared from pellets formed in the submerged culture of a fungus were used for the quantitative evaluation of mycostatic as well as the "mycocidal" activities of a given agent. The mycelial fragments formed individual colonies on agar media. Therefore, techniques for bac- teriostatic and bactericidal tests routinely employed could be easily modified to mycostatic and mycocidal tests. MATERIALS AND METHOI)S The stock culture of Aspergillus niger strain 16173 was grown on an agar slant of medium of the following composition: peptone, 20 g; yeast extract, 0.5 g; KH2PO4, 1 g; MgSO4- 1OH20, 0.5 g; glucose, 10 g; agar, 25 g; and water to 1000 ml (pH adjusted to 6.0). The same medium with or without addition of agar was used for test media throughout these experiments. The stock culture was used after at least 1 week of incuba- tion at 30 C. Dry preparation of spores used for the present ex- periments was obtained by the following procedure, which is the modification of the method of Yanagita (1957). A sterilized processing tray (26 by 20 by 4 cm) covered with paper (used for Japanese paper-screen) was poured with 400 ml of the agar medium. After the solidification of the agar, 10 ml of sterile distilled water 1 Stupported in part by a grant-iin-aid for Fundamental Sci- entific Research donated by the MIinistry of Education of Japan. 2 Member of the Tokugawa Institute for Biological Re- search, Tokyo, Japan. 3Kindly suipplied by Dr. J. W. Foster of the University of Texas. was spread over the agar surface, and a sheet of sterilized filter paper (26 by 20 cm) was laid down on the agar. P'recaution was paid to prevent the inclusion of air bubbles between the paper and agar. Then spore suspension made from a stock culture was sprayed on the filter paper and incubated for 1 week in a wet box at 30 C. Abundant formation of spores could then be achieved. The spore bearing filter paper was separated aseptically from the agar medium, and the tray was emptied by discarding the wasted medium. The spore bearing paper was then replaced in the empty tray, anid incubated for a few days in a dry chamber at 30 C until the margin of the paper curled up by drying. For collecting the dry spores, an electric vacuum brush4 was found to be very convenient. In this case the paper bag instead of cloth bag was used for the final collection of spores. The spores thus collected were screened through fine silk cloth to separate the contaminating mycelia, conidiophores, and other forms of the cell. The yield of dry spores was 0.6 to 0.8 g per tray. These spores were usable for many months provided that they were stored at 4 C in a small tube with a tight rubber stopper. The substances tested were: five synthetic fungi- cides; sorbic acid, dehydroacetic acid (DHA), sodium pentachlorophenolate (PCP), 2-butylthio-1 ,4-naph- thoquinone (BTN), and phenylthiolbenzoate; and three antifungal antibiotics; actidione, mycelin (Aiso et al., 1952), and eurocidin (Nakazawa, 1955). Eurocidin is a pentaene and mycelin is an antibiotic closely re- lated to fradicin (Swart et al., 1950). A. Tests for Sporostatic and Sporocidal Activities During the course of spore germination, the usual marked increase in the size of spores occurs, followed by the emergence of germ tubes. Thus, in the test for the sporostatic activity of antifungal substances, there are two criteria of judgment, namely, the inhibition of conidial swelling and that of germ tube emergence. Previously, it was observed that the cytochemical and metabolic pattern of the germinating spores changes conspicuously at the time of germ tube emergence (Yanagita, 1957). In other words, at this stage of ger- mination, the cytochemical and metabolic patterns are 4 Matsushita Electric Works, Tokyo, Japan. 375 on April 20, 2020 by guest http://aem.asm.org/ Downloaded from

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Page 1: Comparative Quantitative Studies Fungitoxicity Fungal Spores … · Comparative and Quantitative Studies of Fungitoxicity against Fungal Spores and Mycelia1 TOMOMICHI YANAGITA2 AND

Comparative and Quantitative Studies of Fungitoxicityagainst Fungal Spores and Mycelia1

TOMOMICHI YANAGITA2 AND SABURO YAMAGISHI

Institute of Food Microbiology and School of Pharmacy, Chiba University, Narashino, Chiba-ken, Japan

Received for publication February 25, 1958

In studies on the mode of action of antifuingal agents,it may be valuable to reveal the difference in drugsensitivity between fungal spores and mycelia. Variousmethods employed in the evaluation of antifungalsubstances do not necessarily apply to the solution ofthis problem. For the estimation of "sporostatic"activity of an agent, the inhibitory effect on the courseof germination of spores can be taken as a measure of(luantitative evaluation of the activity. On the otherhand, the quantitation of growth inhibitory activityoni mycelia ("mycostatic" activity) of an agent is muchmore complicated.

In the present experiment, mycelial fragments pre-pared from pellets formed in the submerged culture ofa fungus were used for the quantitative evaluation ofmycostatic as well as the "mycocidal" activities of agiven agent. The mycelial fragments formed individualcolonies on agar media. Therefore, techniques for bac-teriostatic and bactericidal tests routinely employedcould be easily modified to mycostatic and mycocidaltests.

MATERIALS AND METHOI)S

The stock culture of Aspergillus niger strain 16173was grown on an agar slant of medium of the followingcomposition: peptone, 20 g; yeast extract, 0.5 g;KH2PO4, 1 g; MgSO4- 1OH20, 0.5 g; glucose, 10 g; agar,25 g; and water to 1000 ml (pH adjusted to 6.0). Thesame medium with or without addition of agar wasused for test media throughout these experiments. Thestock culture was used after at least 1 week of incuba-tion at 30 C.Dry preparation of spores used for the present ex-

periments was obtained by the following procedure,which is the modification of the method of Yanagita(1957). A sterilized processing tray (26 by 20 by 4 cm)covered with paper (used for Japanese paper-screen)was poured with 400 ml of the agar medium. After thesolidification of the agar, 10 ml of sterile distilled water

1 Stupported in part by a grant-iin-aid for Fundamental Sci-entific Research donated by the MIinistry of Education ofJapan.

2 Member of the Tokugawa Institute for Biological Re-search, Tokyo, Japan.

3Kindly suipplied by Dr. J. W. Foster of the University ofTexas.

was spread over the agar surface, and a sheet ofsterilized filter paper (26 by 20 cm) was laid down onthe agar. P'recaution was paid to prevent the inclusionof air bubbles between the paper and agar. Then sporesuspension made from a stock culture was sprayed onthe filter paper and incubated for 1 week in a wet boxat 30 C. Abundant formation of spores could then beachieved. The spore bearing filter paper was separatedaseptically from the agar medium, and the tray wasemptied by discarding the wasted medium. The sporebearing paper was then replaced in the empty tray, anidincubated for a few days in a dry chamber at 30 Cuntil the margin of the paper curled up by drying. Forcollecting the dry spores, an electric vacuum brush4was found to be very convenient. In this case the paperbag instead of cloth bag was used for the final collectionof spores. The spores thus collected were screenedthrough fine silk cloth to separate the contaminatingmycelia, conidiophores, and other forms of the cell.The yield of dry spores was 0.6 to 0.8 g per tray. Thesespores were usable for many months provided thatthey were stored at 4 C in a small tube with a tightrubber stopper.The substances tested were: five synthetic fungi-

cides; sorbic acid, dehydroacetic acid (DHA), sodiumpentachlorophenolate (PCP), 2-butylthio-1 ,4-naph-thoquinone (BTN), and phenylthiolbenzoate; andthree antifungal antibiotics; actidione, mycelin (Aisoet al., 1952), and eurocidin (Nakazawa, 1955). Eurocidinis a pentaene and mycelin is an antibiotic closely re-lated to fradicin (Swart et al., 1950).

A. Tests for Sporostatic and Sporocidal Activities

During the course of spore germination, the usualmarked increase in the size of spores occurs, followedby the emergence of germ tubes. Thus, in the test forthe sporostatic activity of antifungal substances, thereare two criteria of judgment, namely, the inhibitionof conidial swelling and that of germ tube emergence.Previously, it was observed that the cytochemicaland metabolic pattern of the germinating spores changesconspicuously at the time of germ tube emergence(Yanagita, 1957). In other words, at this stage of ger-mination, the cytochemical and metabolic patterns are

4 Matsushita Electric Works, Tokyo, Japan.

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T. YANAGITA AND S. YAMAGISHI

vigorously shifting from those of spores toward those ofmycelia. Therefore, as suggested by Horsfall (1956),it is probable that the concentration of an agent in-hibiting swelling may differ from that inhibiting sprout-ing. To clarify this point, a method for the simultaneousdetermination of swelling and sprouting inhibition wasdeveloped.

Sporostatic test method. Ten mg of dry spores weresuspended in 4 ml of 0.02 per cent aqueous solution ofTween 80.5 A 0.05 ml aliquot of the suspension wasspread with a bent glass rod over a duplicate series ofagar plates (10 ml of medium per plate) containingserially diluted test fungicide. Two plates without

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addition of test substance, but inoculated with thesame amount of spores, served as controls. Each platethus inoculated contained about 5.7 X 106 spores perplate (about 1.1 X 105 spores per cm2). All of theseplates were incubated at 30 C. After 4 hr incubationone series of plates was cooled in a refrigerator at 3 Cto stop the process of conidial swelling. The other serieswas cooled after 8 hr incubation to stop the process ofgerm tube emergence. The percentage of swollen andsprouted spores on 4- and 8-hr cultures, respectively,was determined by microscopical examination. Thediameter of the resting spores averaged 3 A, whereasthat of swollen spores cultured for 4 hr averaged be-tween 5 and 10 A (see figure 1). The number of swollenspores, whose diameters were arbitrarily set at more

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Figure 1. Photomicrographs of spores of Aspergillus niger treated with various concentrations of pentachlorophenol for 20 min.These were taken after a 4-hr incubation period at 30 C after treatment. (1) Untreated resting spores; (2) to (5) treated by 2 X10-3, X 10-3, 5 X 10-i, and 2.5 X 10-4 M pentachlorophenol; (6) spores grown through the period of swelling in the absence of pen-tachlorophenol.

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Page 3: Comparative Quantitative Studies Fungitoxicity Fungal Spores … · Comparative and Quantitative Studies of Fungitoxicity against Fungal Spores and Mycelia1 TOMOMICHI YANAGITA2 AND

FUNGITOXICITY AGAINST SPORES AND MYCELIA

than 5 ,u, was scored after the examination of about 200spores per plate. Sprouted spores, on the other hand,denoted those which were deformed or had alreadysprouted. These configurations could easily be differ-entiated from globular ungerminated ones. The degreeof sporostatic activity, I., was calculated for both casesas shown in formula 1.

1=(1S)X 100 (1)

where S and SO represent the percentages of swollenor sprouted spores on test and control plates, respec-tively. The value S. usually gave 95 and 90 per cent forthe process of swelling and sprouting, respectively,under the present experimental conditions.

Sporocidal test method. In this experiment a knownnumber of spores were subjected to contact with certainconcentrations of fungicide in a liquid medium for adefinite time interval, taken out of the environment,and then plated on an agar medium without addedfungicide. The spores swelling on the medium werethen counted for survivals.A 0.2 ml aliquot of the spore suspension in water

(2.5 mg dry spores per ml) was inoculated in 4.8 mlmedium, with or without the addition of various con-

centrations of test fungicide, the final concentration ofspores being 100 ,ug per ml. After 13 min incubation withthe fungicide at 30 C, the spores were harvested bycentrifugation. The time of contact with the initialconcentration of fungicide was exactly 20 min, includingthe time of centrifugation. Spores thus treated werewashed once or twice with 5 ml of medium (free fromthe fungicide) and resuspended in 0.3 ml of medium.One-tenth ml of the suspension was spread over anagar plate free from the fungicide, incubated for 4 hrat 30 C, and the percentage of swollen spores scored.The percentage of surviving fraction was calculatedfrom formula 1.

B. Tests for Mycostatic and Mycocidal Activities

Preparation of mycelial suspension. Klarmann et al.(1934) used mycelial fragments, prepared by breakingup a 10- to 15-day-old mycelial mat of a fungus, forfungicidal tests. However, the method is not fitted fortesting of mycostatic activity per se, since the mycelialmat of the fungi might bear a variety of spores andother forms of the cell. In the present experiments,mycelial fragments were prepared by breaking up 2-day-old pellets grown in the submerged culture of a fungus,In this case the fungus culture was free from spores.

Figure 2. Phase contrast photomicrographs showing various types of mycelial fragments. For the method of preparation of thesefragments see text.

1958] 377

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T. YANAGITA AND S. YAMAGISHI

One loopful of spores taken from a stock culture oftest fungus was inoculated into 60 ml liquid mediumcontained in a shaking flask. It was cultured at 30 Cfor 2 days on a reciprocal shaker (130 rpm; stroke:7 cm). If it was cultured more than 2 days, conidio-spores formed around the lower neck of the flask.Since this situation may cause the unfavorable con-tamination of the mycelial preparation with spores, theduration of the culture should be less than 2 days.The fungal culture with abundant formation of pelletswas then stirred vigorously for 2 min in a sterile WaringBlendor (10,000 rpm). The homogenate thus preparedwas filtered through three sheets of sterile gauze. Thefiltrate was a homogeneous turbid suspension of my-celial fragments, as shown in figure 2. When 0.1 ml ofthe suspension was spread over the agar plate, about100 recognizable colonies formed after 1 day of cultureat 30 C. Thus, 1 ml of the homogeneous mycelial sus-pension contained about 1000 colony-forming centers.The viability of the mycelial fragments in the suspen-sion thus prepared fell off rather rapidly. After theywere kept for 20 hr at 3 C in the culture fluid, about40 per cent of the colony-forming centers lost viability.When the freshly prepared fragments were resuspendedin M/15 phosphate buffer at pH 6.0 and stored at 3 Cfor 20 hr, almost the same degree of death occurred.Consequently a freshly prepared mycelial suspensionwas always used.

MIycostatic test method. Agar plates were preparedwith or without additioni of various concentrations ofantifunigal substance. One-tenth ml of the mycelialsuspension was spread over the agar surface by the useof a bent glass rod. After 1 day of incubation at 30 C,the colonies formed on each plate were counted. Thedegree of inhibition, I, is given as shown by formula 2

1=( - X 100 (2)

in which M and Mo represent the number of colonies onthe plates with and without added test substance,respectively.

MIycocidal test method. One ml of mycelial suspensionwas harvested by centrifugation and was resuspendedin 5 ml of medium with or without the addition ofvNarious concentrations of test substance. The sus-pensions were incubated at 30 C for 13 min, followedby centrifugation. The time of contact with the initialconcentration of the fungicide was 20 miii, including thetime of centrifugation, as in the case of the sporocidaltest. The spores thus treated were then washed once,and resuspended in 1 ml of medium. One-tenth ml ofthe suspension was inoculated on an agar plate con-taining no fungicide, incubated for 1 day at 30 C, andthe resultant surviving colonies counted. The colonycount of untreated mycelial suspension was taken as acontrol and the survival ratio was calculated at eachconcentration of the test substance.

RESULTS

Comparison of sporostatic and mycostatic activities ofantifungal substances. The data obtained are graphicallyexpressed in terms of the relationship between thedegree of inhibition and the concentration (logarithmicscale) of test substances. Sporostatic activities, asjudged by inhibition of swelling and sprouting, seemto differ in some cases. Figure 3 shows the typicaldata of the dosage-response curves for the inhibitionof conidial swelling, germ tube emergence, and mycelialgrowth. In the case of PCIP, the curve for sproutinginhibition fell between those for mycelial growth aiidconidial swelling inhibitions. The reason why the slopeof the sprouting inhibition curve is less than that ofthe others is uncertain. Another typical example wasfound in the case of phenylthiolbenzoate. This fungicideshowed a quite different pattern of dosage-response

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Figutre 3. Comparison of dosage-response curves for anti-fungal agents against mycelia and spores of Aspergillus niger.PCP and DHA represent, respectively, pentachlorophenol ariddehydroacetic acid.

TABLE 1Comparison of sporostatic and mycostatic activities of

antifungal substances on Aspergiltuis niger

Antifungal Substance

Pentachlorophenol ......Dehydroacetic acid......Sorbic acid..............Phenylthiolbenzoate ....

Butylthio-1, 4-naphtho-quinone ...............

Mycelin .................Eurocidin ...............Actidione ...............

50 Per cent InhibitionConcentration

Sporostasis Mycostasis

Af

7.4 X1.4 x3.3 X1.4 X

10-510-310-310-4

3.4 X 10-4pAg/ltl!>100>5056

A

1.4 X 10-63.8 X 10-i1.2 X 10-35.9 X 10-5

2.5 X 10-4Ag/lMl7.85.5

56

Ratio,Sporostatic:Myostatic

52.83.82.82.4

1.4

>13>9

1

Note: Sporostatic activity was judged by the swellinginhibitioin.

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FUNGITOXICITY AGAINST SPORES AND MI\YCELIA

curves. The activity for sprouting inhibition was thehighest and those for mycelial growth and conidialswelling inhibitions were successively less. In the com-parative experiments on the sporostatic and mycostaticactivities of various substances performed below, theconidial swelling inbibition was taken as the measure ofsporostasis.As shown in figure 3, the sporostatic and mycostatic

activities of actidione differed from those of PCP.Table 1 shows the concentrations of various antifungalsubstances which caused 50 per cent sporostasis andmycostasis. The ratios of the two activities are alsoindicated in the table. These results show that thereare three types of mode of action among various agents:(1) the activities of sporostasis and mycostasis showthe same order of magnitude, (2) the mycostatic activ-ity is far stronger than the sporostatic activity, and(3) difference between the two activities is not so wideas in type (2). No substance has been found whichexhibits stronger sporostatic than mycostatic activity.Actidione and BTN are of the first type; PCP, mycelinand eurocidin of the second; and sorbic acid, DHAand phenylthiolbenzoate of the third.

Comparison of sporocidal and mycocidal activities ofantifungal substances. A 20-min exposure of spores andmycelial fragments to D)HA, even in high concentra-tions, did not have any influence on the survival ratioof these cells. In a parallel experiment with PC1P, bothsporocidal and mycocidal activities were clearly demon-strated. In this case, dosage-response curves for sporo-cidal and mycocidal activities almost paralleled thoseof sporostatic and mycostatic activities. The 50 percent inhibition concentrations for sporocidal and myco-

TABLE 2

Comparison of growth inhibitory and killing activities ofpentachlorophenol on A.spergillus niger

Antifungal Activity 50 Per cent Inhibition Conc Ratio, Cidal: Static

Mycostatic 1.4 X 10-6 21Mycocidal 3.0 X 10-5

Sporostatic......... 7.4 X 10-5 10Sporocidal .......... 7.5 X 10-i

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cidal activities of PCP are listed in table 2, as comparedwith those for sporostatic and mycostatic acti-vities.The ratios, cidal: static, indicate wide differenees be-tween the concentrations causing growth inhibition anddeath of spores and mycelia.

Comparison of heat sensitivity of spores and mycelia.It is generally accepted that the difference in heatsensitivity between spores and vegetative cells is muchless in fungi than in bacteria. In the latter organisms,the difference can easily be demonstrated (luanti-tatively; on the other hand, such qluantitative com-parison has been difficult to demonstrate in fungi. Thepresent device of counting viable cells of fungal myceliaprovides a way of obtaining new information on thecomparative representation of the heat sensitivitiesof fungal mycelia and spores.The spores of the test organism used in this experi-

ment do not respond to so-called heat shock. A shortexposure of the spores to a high temperature results ininstant death. Both the spores and mycelia of thisfungus were exposed to various temperatures for 3 min,and the viability of these cells examined by the tech-niques used in the sporocidal and mycocidal tests.From this experiment it has been shown (figure 4)

that the temperatures which caused the death of 50per cent of these cells differed by only 5 C between thespores and mycelia. The fact that the slopes of thesetwo curves were almost parallel suggests that the varia-tion of heat sensitivity of spores may be of the sameorder of magnitude as that of mycelia.Change in heat and germicidal sensitivities of spores

during the course of germination. The results obtainedin the experiments described above show that the heatand germicidal sensitivities in some cases differ mark-edly between spores and mycelia. In this connectionthe question may be raised as to when, during thecourse of germination, the resistance to heat and drugswill be lost. The following experiments were designed toanswer this (uestion.

Five mg of dry spores were inoculated in 50 ml ofgermination medium containing L-alanine, glucose, andphosphate (Yanagita, 1957), and cultured with shakingat 30 C. A 5 ml aliquot of the culture was taken at

0 51

TEMPERATURE, C

Figure 4. Comparison of heat resistance of mycelial frag-ments and resting spores. These cells were heated for 3 min atdesiredi temper.attures in peptone-yeast extract-gluicose media.

2

TIME IN HOURS

Figuire 5. Loss of heat and germicidal resistance in germinat-ing spores of Aspergillus niger. Heat treatment: 49 C for 3 min;germicidal treatment: 2 X 10-4 M pentachlorophenol (PCP) fot

20 min at 30 C.

5 \

I PCPio I

HEAT \5 .4

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1958] C-7 J

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T. YANAGITA AND S. YAMAGISHI

various time intervals and subjected to treatment,withheat or 1PCP. The heat treatment of the germincatingspores comprised 3 min heating at 49 C in the germina-tion medium. The spores thus treated were tested forv-iability by scoring of swvollen spores after 6 hr cultiva-tioin at :30 C. The treatment with the fungicide wasperformed by the addition of l'Cl' to the ali(uot ofculture to make the finial concentration of 2 X 10-4 M,followed bv the iincubation for 20 min alt 30 C. Afterthe exposure of spores to the fungicide, the culture wasinstantly filtered through at nmembrane filter,6 washedwith saline, and the residual spores resuspended insaline. The spore suspension was subjected to the testsfor viability (colony forming ability oni the peptone-yeast extracet agar after I day of culture) and for totalcell countinlg. Thus the percentage of survivals wascalculated.The heat senisitivity of the spores was founid to

change abruptly after only 30 min inicubation in thegerminationi medium (figure 5). Under the presentexperimenttal coniditions, the spores begain to emergegerm tubes after 6) hr. It should be noted that sporesincubated in distilled water at the same temperaturedid not show aniy sign of loss of heat resistance. Asmentioned previously, the spores used in this experi-ment did not swell or emerge germ tubes in the distilledwater (Yaniagita, 1957). F11rom these facts, it seems thatthe loss of heat resistance in the initial phase of germi-nation is directly related to the metabolic changes as-sociated with germination.The loss of PCP-resistance of germinatiing spores

occurred later thani the loss of heat resistance. Figure5 also shows that a conspicuous change in PCl' sensi-tivity occurs between 1 and 2 hr of culture. It cannotbe concluded that the loss of PCP-resistance at thismoment is caused by a change in the permeability of theagent through the cell wall or by a change in the meta-bolic pattern of the spores.

DIscussIoN

The method described for counting colonies ofmycelial fragments may be useful in the investigation ofthe biological activities of fungal mycelia per se, sinceit precludes the presence of spores. There is, however,an importaint problem as to how the degree of mycelialfragmentation influences the colony-forming ability.As shown in figure 2, cytoplasm leaked out from the cutends of some of the mycelial fragments, whereas thoseof branching mycelia seemed to be intact. There is atpresent no evidence as to which type of cell fragmenthas colony-forming ability. In conclusion, it can besaid that only a small fraction of the fragments wereable to form colonies.

Comparison of the mycostatic anid sporostatic ac-tivities of several agents disclosed that these two activ-

ities were very close in some cases and quite differentin others. No evidence, however, was shown thatsporostatic activity is stronger than mycostatic activity.This was also found true in the experiments of Yama-gishi (1958). He tested new antifungal substances,phenylthiolacylate derivatives, by this method. Hefound that the introduction of some acyl group intothe substance caused a marked enhancement of themycostatic activity, while it left the sporostatic ac-tivity unchanged. He showed also that this (uantitativemethod is convenient for the demonstration of en-hancement, by an agen-t, of growth inhibitory action ofa funigicide.

In general, the fungistatic activity of various sub-stances seemed to have more effect on mycelial growththani oni germ tube emergence. It is worth noticing,howev%eser, that, contraiviwise, some of the fungicidesexhibited more effective actioni on germ tube growththan on mycelial growth. This fact suggests that durinigthe course of fungal growth, beginining from the timeof spore germination, the fungus does not n=ecessarilyshow the highest sensitivity to an agent at the myceliumstage. It may generally be c(oncluded that the fungusshows the highest resistance during the course of cellu-lar growth at the stage of conidial swelling. Mandelsand Darby (1953) presented a rapid method for as-sayiing fungitoxicants, by measuring inhibition ofconidial swelling. In the case of screening antifungalagents, however, it is probable that their method maybe less selective thani the conventional one based onthe inhibition of germ tube emergence or of mycelialgrowth.The difference in heat resistance between mycelium

and spore was found to be much less in this organismthan in bacteria. In a separate experiment using Asper-gillus clavatus as the test organism, similar results wereobtained (Yanagita, unpublished data). The tempera-tures showing 50 per cenit survival for the mycocidaland sporocidal experiments were 50.5 and 60.0 C,respectively, under the similar experimental conditions.The finding that a rather abrupt loss of heat and

germicidal resistance was observed in the initial step ofspore germination is of great interest. At this stage ofgermination, no distinct swelling begins and no appre-ciable synthesis of cellular high molecular weightsubstances occurs (Yanagita, 1957). Further biochem-ical studies on the initial phase of spore germinationmight give a clue to the elucidation of these importantphenomena.

ACKNOWLEDGMENTSThe heat resistance experiment on Aspergilluts clavatus

was carried out by one of the authors (T. Y.) at thelaboratory of Dr. J. W. Foster, University of Texas, towhom the author expresses his gratitude for his valuablesuggestions and encouragement. Thanks are also due toDr. H. Tamiya of the Tokugawa Institute of Biological

[VOL. 6380

11 Nr. 1, .'\Iembranefilter-Gesellsehaft. GUtingen, Germany.

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DISINFECTION OF CLINICAL THERMOMETERS

Research for his helpful criticism in the preparation ofthis paper.

SUMMARYMycelial fragments of Aspergillus niger strain 1617,

prepared by blending mycelial pellets, were found to beusable as a test organism in the quantitative evaluationof mycostatic (or mycocidal) activity of antifungalagents. Using these fragmented mycelia and spores, acomparative and quantitative method for determininigfungitoxicity against fungal mycelia and spores waspresented.The sensitivity to various agents and heating of

mycelia was compared with that of spores, and thegeneral concept of fungitoxicity was discussed. The lossof germicidal and heat resistance of the spores in theearly phase of germination was demonstrated.

REFERENCES

Aiso, K., ARAI, T., WASHIDA, K., AND TANAMI, T. 1952"Mycelin" a new antifungal substance extracted from the

mycelium of a Streptomyces. J. Antibiotics (Japan), 5,217-219.

HORSFALL, J. G. 1956 Principles offungicidal action. Chiro-nica Botanica Co., Waltham, Massachusetts.

KLARMANN, E., SHTERNOV, V. A., AND GATES, L. W. 1934The bactericidal and fungicidal action of homologoushalogen phenol derivatives and its "'quasi-specific'"character. J. Lab. Clin. Med., 19, 835-851.

MANDELS, G. R. AND DARBY, R. T. 1953 A rapid cell volumeassay for fungitoxicity using fungus spores. J. Bacteriol.,65, 16-26.

NAKAZAWA, K. 1955 Studies on Streptomycetes. Eurocidin,an antibiotic produced by Streptomyces albireticutli (III).J. Agr. Chem. Soc. Japan, 29, 650-652.

SWART, E. A., ROMANO, A. H., AND WAKSMAN, S. A. 1950Fradicin, an antifungal agent produced by Streptomycesfradiae. Proc. Soc. Exptl. Biol. Med., 73, 376-378.

YAMAGISHI, S. 1958 Studies on thiol esters (IV). Mycostaticand sporostatic effects on Aspergillus niger. J. Pharm.Soc. Japan, 78, 263-267.

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Studies on Disinfection of Clinical Thermometers

I. Oral Thermometers from a General Hospital

ELEANORE S. WRIGHT AND R. A. MUNDY

Research and Development Laboratories, Lehn & Fink Products Corporation, Bloomfield, New Jersey

Received for publication March 21, 1958

The literature contains many different test proceduresfor the determination of the efficacy of disinfectants for

the clinical thermometer. None of these methods is

representative of the actual conditions encountered in

the field. The purpose of our investigation was to de-

velop a test method that reflects actual field conditions

and to evaluate the efficacy of some common disinfect-

ants by this method.In the past, the investigators of thermometer disini-

fectants have used artificial conditions in their test

procedures. For example, Gershenfeld et al. (1951)contaminated thermometers with a broth culture of

microorganisms containing citrated plasma. Ritter

(1956) contaminated glass rods with mucin inoculatedwith microorganisms. Ecker and Smith (1937) smeared

thermometers with sputum taken from patients with

Type 1 lobar pneumonia, and Frobisher et al. (1953)also used sputum, but from patients with active pul-monary tuberculosis. It is obvious that broth cultures

of microorganisms with organic matter do not duplicatepractical field conditions. Sputum serves as a source of

microorganisms in a natural environment; however, it is

unlikely that sputum would be found on oral thermom-

eters after use. In our study we decided to use ther-mometers from mouths of patients suffering with avariety of infections in order to obtain on the thermom-eter a representative spectrum of microorganisms intheir natural environment.

MATERIALS AND METHODS

T'hermometers. Becton, Dickinson' oral thermometers,from which the mercury was removed by breaking theupper end and centrifuging, were used for the test. Thebroken end was sealed and fire polished before using.

Disinfectantts. All disinfectant solutions were preparedon a volume per volume basis in sterile distilled water.The following disinfectants were tested:

Ethyl alcoholSynthetic phenolic (p.c. 10)2 orthohydroxydiphenyl

and para-tertiary-amyl phenol solubilized by potassiumricinoleate.

Synthetic phenolic (p.c. 5) orthohydroxydiphenylsolubilized by potassium ricinoleate.Commercial iodophor, iodine solubilized by nonionic

detergent; 1.6 per cent available iodine.1 Becton, DickinQon and Company, Rutherford, New Jersey.2 p. c.= Phenol coefficient.

3811958]

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