biological assay of fungicides against yeasts in vitro using a coulter counter

mykosen 19 (10) 361-372 0 Grosse Verlag 1976

Eingegangen am 4. Marz 1976

Department of Chemotherapy, Schering A. G., Berlin

Biological Assay of Fungicides against Yeasts in vitro using a Coulter Counter

JANET BROTHERTON

Summary

1. Saccharomyces cerevisiae and Candida albicans were successfully counted and sized in a Coulter Counter during an 8 hour biological assay against a variety of fungicides.

2. The EDso and M. I. C . (minimum inhibitory concentration) values for many fungicides, but especially some new imidazoles, were determined from the dose-response curves and calculated in terms of fmoles/cell.

3. For many of the compounds tested, the greater the inhibition of cell division, the larger the mean size of the cells that grew, in a linear relationship that was characteristic for each species and compound.

4. This relationship was calculated as the "cytotoxic efficiency" and allowed separate assessments of the degree of inhibition of cell division and of protein synthesis.

Zusammenfassung

1. Bei Saccharomyces cerevisiae und Candida albicans wurden erfolgreich im Coulter Counter in einem 8 Stunden dauernden biologischen Versuch unter Zusatz von Fungiziden Zahlungen und Grofienbestimmungen durchgefuhrt.

2. Die EDGa und MHK-(minimale Hemmkonzentration)-Werte fur viele Fungizide, insbesondere fur neue Imidazole, wurden in der Dosis-Wirkungskurve bestimmt und in fnioles/Zelle angegeben.

3. Fur viele der getesteten Substanzen trifft zu, dai3 je groi3er die Hemmung der Zell- aufteilung ist, um so grofier ist die Durchschnittsgrolle der Zellen, die in der Kultur wuchsen. Dabei liefi sich eine lineare Beziehung nachweisen, die charakteristisch fur jede Spezies und Verbindung ist.

4. Diese Beziehung wurde als ,,cytotoxische Wirksamkeit" berechnet und gestattete unterschiedliche Begutachtungen des Hemmungsgrades der Zellaufteilung und der Pro- teinsynthese.

The many technical problems involved in the assessment of antifungal drugs in vitro have been described by DESAI (1968). The present investigation was undertaken to see if a Coulter Counter could be used for the final assessment of yeast cell growth, instead of the usual turbidimetric methods. This instrument gives a n exact cell count and assays of greater accuracy and sensitivity, as has been described previously for the action of anti- inflammatory corticosteroids against skin fibroblasts (BROTHERTON 1971).

Key words: Fungicides, Bioassay, Yeasts, Coulter Counter, Candida, Saccharornyces, Clotrima- zole, Miconazole, Diclonazole, Imidazoles.

362 JANET BROTHERTON

Table 1: Machine settings for the counting and sizin of yeasts in the Coulter Counter, Model ZB Indus t r iab

__ -. - _ _ . . Saccharornyces cerevisiae Nuclei’” Nuclci Cells’‘ Candida albicans Nuclei Nuclei’’ Cells’:. Cells Cells Cells Cells

Combination number 1 2 3 4 5 7 9

- -- -. . -

~~

Madiine settings I 114 0.177 114 0.354 114 112 1

(M = 2, G = 0) A (= B) 114 2 112 112 1 1 1

Machine value IBIM 0.03125 0.04425 0.0625 0.0885 0.125 0.25 0.5 Volume factor, pm3 F = VIT 2 2.828 4 5.656 8 16 32

’’ Usual optimum combination ’‘’‘ For an aperture of nominal diameter of 100 pm, the rnadiine constant K = M . V _= 64

IB Ti/%

Methods

Inocula in Sabouraud liquid medium (Difco) containing 20% w/v glucose were prepared from slopes of Saccharomyces cerevisiae, A T C C 9763, and from vials of Can- dida albicans (laboratory strain) which had been stored as suspensions of blastospores in ethanol/solid carbon dioxide. Assays were carried out a t 30’ C, using 10 ml medium in tubes for Sacch. cerevisiae and in SO ml conical flasks in a shaking water bath for C. albicans. Inoculum sizes were 1.3-5.4 x loo cells/ml medium for Sacch. cerevisiae and 0.1-0.5 x lo6 celldm1 medium for C. albicans. All assays were for 7.5-8.0 hours. Cells were counted and sized using a Coulter Counter, Model ZB Industrial, with a n aperture of nominal diameter 100 pm and the machine settings shown in Table 1 . A particular combination of machine settings was used so that those cells with mean size were located somewhere in the middle of the threshold scale (BROTHERTON 1971). Fungicides were first screened against Sacch. cerevisiae a t 10 pg/ml and then for the most interesting compounds the M. I . C. and ED5, values were determined accurately against both yeasts using serial dilutions.

Results

Growth curves For both yeasts there was a lag phase of 2-3 hours followed by exponential growth

for a further 4-5 hours. Table 2 shows the total number of cells achieved in the assays after about 8 hours together with the mean cell sizes. The generation time for Sacch. cerevisiae was about 2.5 hours and for C. albicans about 45 mins. At the start of the assays the cells of C. albicans were smaller and the size distributions slightly narrower than the cells of Sacch. cerevisiae. For all the assays the volume measured a t the mean in the size distribution curves was 146.0 pms for C. albicans compared with 185.8 pm3 for Sacch. cerevisiae. A t the end of the assays the cells in the control cultures were significantly larger in nearly every case. The average increase in mean cell size for Sacch. cerevisiae was 13.5 % whereas for C. albicans it was 57.1 %, which made it necessary to change the settings on the Coulter Counter to obtain valid measurements. A few pseudohyphae were

mykosen 19, Heft 10 (1976)

Biological Assay of Fungicides against Yeasts in vitro using a Coulter Counter 363

Table 2: Growth achieved in each assay with the mean sizes of the cells

Cells x Io-Vculture Cell size Cell size Change in volume during assay %

at start at end N, Yeast assay Nl No Full v, d V, number

Inoculum growth growth >tms !im ltm3 tim

Saccharomyces 1 4 7 8 9 10 11 12 16

cerevisiae 23.90 14.50 22.20 53.35 34.53 32.19 2 1.94 15.96 12.72

28.70 14.60 23.40 46.86 34.53 32.24 25.67 15.60 13.80

70.30 59.60 155.90 138.50 145.84 102.22 83.12 59.60 73.01

192.0 220.0 160.0 156.0 152.0 184.0 200.0 198.0 2 10.0

7.16 7.55 6.74 6.68 6.62 7.06 7.26 7.23 7.38

2 10.3 240.0 170.0 190.0 194.0 200.0 230.0 238.0 217.8

7.37 7.77 6.87 7.13 7.18 7.26 7.60 7.69 7.47

+ 9.5 + 9.1 + 6.3

+ 21.8 + 27.6 + s.7

+ 15.0 + 20.2 + 3.7

Mean 185.8 7.07 210.0 7.37 + 13.5

Nl Candida albicans

6 2.45 1.54 13 2.88 2.25 14 2.89 15 2.53 17 2.65 1s 1.03 19 5.44 20 3.09

N,

79.40 59.36 102.20 71.30 5 1.20 19.95 171.30 138.00

147.0 124.0 100.0 134.0 198.0 124.0 1 1 8.0 222.0

6.55 6.19 5.76 6.35 7.23 6.12 6.09 7.51

150.0 272.0 272.0 266.0 266.0 186.0 168.0 216.0

6.59 8.04 8.04 7.98 7.98 7.08 6.85 7.45

+ 2.0 + 118.5 + 172.0 + 98.5 + 34.3 + 50.0 + 42.4 - 2.7

Mean 146.0 6.47 225.0 7.50 + 57.1

V = volume, d = spherical diameter.

seen in all the cultures of C. albicans, even when 50 pg/ml cysteine hydrochloride was added. These were so small in number relative to the number of yeast cells that they had no effect on the total count or the mean size.

Screening Table 3 shows the growth achieved with all the compounds screened against Sacch. cere-

visiae, expressed as a percentage of the growth in the control cultures. Results were cor- rected t o 100 91 purity of each compound and for any solvent effect (very small). I t is considered that the percentage growth values are accurate t o within 5 %, for example the highest value was 104.8 5% and the highest "zero" value was 4.6 % (nystatin).

Accurate assays Figure 1 (a, b and c) shows a composite picture of the assays of the most potent com-

pounds against Sacch. cerevisiae and Table 4 shows their EDso and M. I. C. values. So that


364

150

100 8 0 - 6 0 -

40

30

20

10

JANET BROTHERTON

- - -

- - -

1 1 1 1 1 I

- 60 - 40 - 30 - 20 -

10 ' I I I I I I 1 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

200

100 80 60

40 30

log nmol/lOml Fig. 1 : Biological assays of fungicides against Saccbaromyces cerevisiae.



Table 3: Summary of the effects of all the compounds screened against S a c c h a r o m y c e s c e r e v i s i a e

Compound Percentage growth at various concentrations, pg/ml

100 10 1 0.1 0.01

I . N o effect a t 10 glml 6-Nitrobenzirni&zole Ethidium bromide, 97 % Fluocortolone Thiosemicarbazide Ethyl potassium xanthate Emetine hydrochloride

11. Some effect at 10 pglml Colchicine Puromycin Chlortetracycline 2,4-Dinitro hen01 Fusidic a c i l sodium salt Sodium fluoride 6-Mercaptopurine 2-amino-5-nitropyridine Acridinium &loride Potassium cyanide

Flunidazole 2-Amino-5-nitropyridine Clotrimazole Rotenone, 90 Sodium malonatc Actinomycin D N-Phenylthiourca Sodium iodoacetate Sodium diethyldithiocarbsmatc 4-n-Hexylresorcinol Diclonazole Sodium arsenite 8-Hydroxyquinoline

Miconazole Metronidazole Phenazine Proflavine hernisulphate Pentachlorphenol 8- Azaguanine Acriflavine hydrochloride

5-Fluorouracil

111. No effect at 10 pglml Tributyltin acetate

104.8 102.6

71,6 101.4 98.6 96.7 95.1

92.9 88.6 85.5 85.1 84.2 82.9 81.3 80.9 80.2

22.1 79.0 43.0 83.7

78.3 72.6 72.4 91.3 69.2 63.7 60.3 58.3 57.3 51.9 51.6 50.9 77.9 84.6 81.7

0 36.0 99.8 48.5 36.5 34.6 88.4 95.0

94.6 31.0 28.5 27.0 18.0 15.4 9.5 3.1 65.9 7.4 5.6 50.8 99.8

1.7 0 81.0

Haloprogin 0.2 0 51.4 98.7

Nystatin

1V. N o effect at 1 pglml Amphotericin B, 55 % Phenylmercuric acetate Cycloheximide

0.1 4.6 31.0 99.7

0 0 85.7 0 1.9 52.4 100.2 0 0 37.4 100.3



Table 4: Assay of fungicides against S a c c h a r o m y c e s c e r e v i s i a e

Yeast ED50 M. I . C. assay nrnolesl fmoles/ nmoles/ frnoled no. 10 rnl cell 10 rnl cell

Compound

Potassium cyanide 12 8800 551 58,900 3690

Clotrirnazole 4 840 57.5 1480 102

Sodium arsenite 12 720 45.1 1820 114

Diclonazole 7 350 15.0 1000 45.0

5-Fluorouracil 12 74.6 4.67 933 58.5

Acriflavine hydroddoride 16 62.0 4.47 407 32.0

Tributyltin acetate 1 1 95.0 3.70 282 12.8

Miconazole 8 167 3.56 813 15.2

Haloprogin 1 1 35.8 1.39 166 7.57

Cyclohexirnide 4 2.98 0.204 10.7 7.39

Nystatin 1 1 4.85 0.189 43.7 1.99

Phenylrnercuric acetate 1 3.64 0.127 19.5 0.816

Amphotericin B 8 1.36 0.0290 11.48 0.216

the potencies of all the compounds could be directly compared with each other, these values have been calculated in terms of nmoles (i. e. 10-9 moles) and fmoles (i. e. lO-!5 moles). In this way the action of the fungicides can be related to the number of receptors in each cell. I t is seen that some assay curves are straight while others are bent. The curves for miconazole, diclonazole (econazole) and clotrimazole were particularly bent with very long parts of the dose-response curve showing a slight at very low dilutions. I t was very difficult to determine the minimum concentrations effect at which an effect began, but there were sharply defined concentrations at which a complete kill was achieved (the M. I. C.).

Figure 2 shows a composite diagram of similar assays against C. albicans and Table 5 shows their EDso and M. I. C. values. It is seen that phenylmercuric acetate, amphotericin B and haloprogin are still among the most potent compounds while nystatin and cycloheximide are less so and miconazole, diclonazole and clotrimazole have joined the potent compounds. These three imidazoles showed totally differently shaped dose-response curves from all those found previously, being concave. I t was now possible to determine the minimum concentration at which they exerted an effect. This was a t 0.01-0.06 fmoles/ cell while only phenylmercuric acetate began to be effective a t a lower concentration, that is at about 0.0002 fmoles/cell. However it was now impossible to determine the M. I. C. of these imidazoles as the amount of growth did not decrease to zero before the limits of solubility of the compounds in the medium were reached.

Effect o f fungicides on cell size Of the compounds tested against Sacch. cerevisiae, phenazine, 5-fluorouracil, 8-hydroxy-

quinoline, proflavine hemisulphate, sodium arsenite, potassium cyanide, acriflavine hydrochloride and cycloheximide were found to bring about an increase in the mean cell size that was significantly greater than that observed during the normal progress of the assay. This effect was also observed for C. albicans with miconazole, diclonazole, clotrimazole,

mvkosen 19, Heft S O (1976)


Fig. 2: Composite diagram of the action of fungicides against Candida albicans.

Assay 12 Assav 16

Mean volume of cells, pm3

Fig. 3 : Relationship between inhibition of cell division and increase in mean cell size for three experiments with Sac&aromyces cerevisiae.



Table 5: Assay of fungicides against C a n d i d a a 1 b i c a n s

Yeast EDSO M. I. C. Compound assay nmolesl fmolesl nmoles/ fmoles/

10 ml cell 10 ml cell no.

5-Fluorouraci~ Cycloheximide Sodium arscnite Potassium cyanide Nystatin Amphotericin B Phenylmercuric acetate Haloprogin Clotrimazole Diclonazole Miconazole

18

15

19

20

19

14

6

17

17

18 15

4870

1720

2410

153

128

0.985

0.459

0.447

0.263

0.0752 0.0846

4730

680

443

49.5

23.5

0.341

0.298

0. I 69

0.0992

0.0730 0.0334

630,000

23,400

26,900

5620

I480 2.88

2.95

41.7

? 1 ?

613,000

9270

4950

1820

272

0.998

1.92

93.3

? ? ?

cycloheximide, 5-fluorouracil, sodium arsenite and nystatin. The effect was only observed a t concentrations of compound at which there was a partial inhibition of growth. When there was sufficient data for a particular compound, a plot of the mean cell volume against the number of cells per tube, both on logarithmic scales, showed a straight-line relationship going through the point Nn,Vr (as defined in Table 2 ) - see Figures 3 and 4. The greater the inhibition of growth, the larger the size of the cells that grew. Each compound produced a different degree of increase in cell size.

These results have been expressed quantitatively as in Table 6. Values shown for the cell count (N) and the mean cell volume (V) were taken either from Figures I and 6 or from Table 3. By multiplication of the cell count and the mean cell volume, a value (NV) was produced representative of the total volume of cell substance in a culture. This was used to calculate the value "p" as shown and this was taken as being proportional to the percentage inhibition of protein synthesis. This proportionality will only be exactly true if the ratio of total cell volume to nuclear volume remains constant even when the cells are much larger. The "cytotoxic efficiency" of the fungicides was calculated as the ratio P/M and gives a value for the inhibition of "protein synthesis" relative to the inhibition of cell division. I t is seen from Table 6 that where there are a series of 4 or 5 points for a particular compound, the ratio P/M increased slightly as the inhibition of both parameters increased, that is as the cells got larger. This is believed to be due to the nuclear size not increasing at the same rate as the total cell size. Nevertheless this effect was small and the results are sufficiently close together to allow a mean to be calculated. Results for the estimation of P/M in two experiments for both 5-fluorouracil and acriflavine hydrochloride against Sacch. cerevisiae were very similar, indicating the validity and repro- ducibility of this method of calculation.

The mean values of P/M are all less than unity (or less than 100 %) indicating that for these compounds protein synthesis was not inhibited as much as cell division over the concentration range where there was partial inhibition of the latter. The degree of this effect varied considerably. I t was least for sodium arsenite against C. albicans, with a cytotoxic efficiency of 94.7 %, but the same compound showed a cytotoxic efficiency of


CioloSical Assay of Fungicidcs against Yeasts in vitro using a Coulter Counter 369

I I 1 1 I 1 1 I I I ' N1

Assay 18

Fi . 4 . Relationship between inhibition of cell division and incrcase in mean ccll size for Candida a h c a n s .

only 30.7 % against Sacch. cerevisiae. The cytotoxic efficiencies of cycloheximide and 5-fluorouracil were also considerably less against Sacch. cerevisiae and the latter was the least efficient compound tested against Sacch. cerevisiae (21.8 W). Diclonazole, miconazole and clotrimazole were all very inefficient against C. albicans (1 8-32 W ) and some specta- cular increases in mean cell volume were observed. This in itself made it somewhat difficult to measure the mean cell size as the size distribution curves were wide, f la t and low.

The cytotoxic efficiency of these three imidazoles againsa Sac&. cerevisiae appeared to be 100 %, in that no increases in cell size were observed. The cytotoxic efficiency of haloprogin against both yeasts was also 100 %.

No decreases in mean cell size were ever observed over the concentration range for partial inhibition of cell growth, that is n o compound appeared to inhibit protein synthesis to a greater degree than it inhibited cell division.

Discussion Cell size

Although the yeasts appeared to be growing in clusters, the mean cell volumes found are consistant with the measurement of each cell singly as it passed through the aperture of the Coulter Counter, although the volume of any bud would be included. Using linear measurements and calculating the volume of cells of Sacch. cerevisiae as a n ellipse, it has


3 70 JANET BROTHERTON

Table 6: Cytotoxic efficiency of fungicides

Yeast assay Compound no.

Cells Mean x 10-6 volume Cytotoxic

per of cells efficiency

N V M P 100 P/M mean 10ml (pY)

Saccbaromyces cerevisiae 7 Cycloheximide

9 Phenazine 8-Hydrox yquinolinc Acriflavine HCI

10 Proflavine hemisulphatc

12 Potassium cyanide

Sodium arsenite

5-Fluorouracil

10 5-Fluorouracil

16 Acriflavin HCI

Candida albicans 15 Miconazole

Clotrimazole

Cycloheximidc

18 5-Fluorouracil

Diclonazole

19 Sodium arsenite

Nystatin

120 100 90 80

66.3 75.1 45.0

51.9

50 40 30 20

50 40 30 20

50 40 30 20

38.5

60 50 40 301

15.3 70.9

21.2

22.2 46.6

15 10

5

10 8 5 2

100 60

29.7 87.5

n

198 229 255 287

290 230 260

280

253 271 296 335

272 320 395 530

278 334 424 592

427

228 238 250 266

904 226

744

374 288

212 252 278 344

388 417 640

1485

176 182

220 196

34.1 20.4 60.0 47.7 23.9 50.0 51.6 54.5 25.4 46.5 61.4 31.6 51.5

71.5 39.3 54.9 63.5 47.8 75.2 90.5 72.0 79.5

73.0 40.7 55.8

22.7 14.8 65.2 45.5 31.1 68.5 63.9 68.2 48.9 71.6 90.9 68.6 75.0

22.7 6.2 27.4 45.5 13.4 29.6 30.7 68.2 21.0 30.7 90.9 33.3 36.6

22.7 3.5 15.5 45.5 8.4 18.5 68.2 14.2 20.8 21.8 90.9 22.1 24.3

93.6 27.6 30.1

22.0 17.2 78.4 38.9 30.8 79.4 80.7 55.8 45.5 81.3 72.7 61.0 84.0

81.4 27.6 33.9 31.9 0.5 15.8 29.8

72.8 17.2 23.6

71.4 57.2 80.2 81.7 35.9 29.8 83.1

26.1 17.6 67.5 52.6 33.3 63.3 66.7 63.2 41.5 65.7 79.0 55.6 70.8

52.6 0 63.2 10.5 16.7 79.0 14.3 18.1 18.6 94.9 20.7 21.8

41.7 39.7 95.4 67.1 63.1 94.1 94.7

85.5 79.1 92.6 41.7 41.3 81.8 87.2

rnvkosen 19, Heft 10 (1976)


been shown that the cell volume varies between 230 and 350 pm3 during the budding cycle (JOHNSON 1965). These volumes are slightly larger than those reported here. This may be due to the calculation of volumes from linear dimensions, which is a procedure subject to large errors, o r to slightly different growth conditions or to strain differences. Mean cell size has been shown to vary considerable with growth conditions for skin fibroblasts, being very large in monolayer culture and very small in stirred deep culture with con- tinuous medium adjustment (BROTHERTON 1971). This has been interpreted as a decreasing amount of cytoplasmic (protein) synthesis with increasing speed of cell division. Under the growth conditions described here, the size increases for Sacch. cerevisiae were quite small while those for C. albicans were large and approached the changes previously observed for skin fibroblasts. At no time were germ tubes observed during the growth of C. albicans which would indicate the beginning of the phase change from the yeast t o the niycelial form (CASSONE et al. 1973; BERNANDER and EDEBO 1969). The size increases observed for C. albicans are therefore seen as real increases in the volume of the yeast forms.

The effect of fungicides in further increasing yeast cell size has not been reported before, although corticosteroids have been shown to have this effect on skin fibroblasts (BROTHERTON, 1971). The detection of such an effect requires the use of a Coulter Counter for the accurate measurement of cell volumes. Using this technique i t is now possible to measure cell "growth" as two parameters, that is as cell division and as cytoplasmic synthesis, and by multiplying the number of cells by the mean volume, to obtain a value for the total tissue synthesis.

Fungicidal action Although a great deal of study has been devoted to the mode of action of most of the

fungicides examined here, it is difficult to find data on their precise cytotoxic potency against the species studied here. Quoted M. I. C. values often refer to diffusion methods on solid media. The M. I. C. and EDao values given here have been related directly to the number of cells present and the number of receptors in each cell for that particular fungi- cide. They are not directly comparable with M. I. C. values given in terms of pg/ml under a variety of growth conditions and inoculum sizes, but they give much more information about the effects of each compound.

Comparative chemotherapy The similarities and differences between the effects of the fungicides against the two

species of yeasts are interesting. Phenylmercuric acetate, which is one of the most potent cell poisons known with a general effect on SH groups (THRASHER 1973), had an ED,, of

Key to table 6 : Calculation of cytotoxic efficiency

P

CE

Initial number of cells Final number of cells Initial mean volume of cells, pmJ Final mean volume of cells, pmS Percentage inhibition of cell division (mitosis) 100 - 100 IN -N. I ~~

iNprlrl1 Percentage inhibition of protein synthesis

(No allowance made for mean volume of the Percentage cytotoxic efficiency = 100 P/M

nucleus)



0.1 27 fmoles/cell against Sacch. cerevisiae and 0.298 fmoles/cell against C. albicans. These values are very similar and probably reflect the number of SH groups, mostly in the cell wall, which must be oxidised to give this degree of inhibition.

I n both yeasts nystatin is about 100 times more potent than amphotericin B a t the ED,, values. As both antibiotics act by the same mechanism in that they bind to ergosterol in the cell wall so making it more permeable, this difference represents the greater structural fit of nystatin. Both compounds are considerably more potent against Sacch. cerevisiae re- flecting the higher amount of ergosterol in the cell wall of this yeast.

Clotrimazole (IWATA et al. 1973) and miconazole (VAN DEN BOSSCHE 1974) have also been postulated to act by damaging the permeability barrier but by reacting with the cell membrane. These compounds were mu& more potent against C. albicans than against Sacch. cerevisiae.

Potassium cyanide is a cytotoxic agent that acts by the formation of a complex with metalloenzymes but is preferentially active against cytochrome oxidase. This specificity is reflected in the ED50 values of 49.5 fmoles/cell for C. albicans, whose mitochondria1 elec- tron chain is similar to that in mammals and different from that of other fungi examined in being especially active in the step from N A D H to cytochrome b (YAMAGUCHI et al. 1971), and 551 fmoles/cell for Sacch. cerevisiae, which is without the phosphorylation step between N A D H and cytochrome b (SCHATZ and RACKER, 1966).

Differences between the metabolic pathways in the linkage of cytoplasmic protein synthesis to nuclear synthesis in the two yeasts are also shown by the fact that nystatin and the three imidazoles were not efficient in inhibiting protein synthesis in C. albicans whereas they were 100 % efficient in Sacch. cerevisiae. Conversely cycloheximide, potassium cyanide, sodium arsenite and 5-fluorouracil were more efficient a t inhibiting protein synthesis in C. albicans. However sodium arsenite, cycloheximide and 5-fluorouracil were considerably less cytotoxic in the inhibition of cell division in C. albicans compared with Sacch. cerevisiae.

Acknowledgements: I wish to thank Miss G. BOD’IN for excellent tedinical assistence.

References

BERNANDER, S. & L. EDEBO, 1969: Growth and phase conversion of Candida albicans in Du- bos medium. Sabouraudia 7, 146-155.

BROTHERTON, J., 1971 : Parallel-line biological assay of some anti-inflammatory steroids using skin fibroblasts. Cytobios 3, 225-238.

CASSONE, A., N. SIMONETTI & V. STRIPPOLI, 1973: Ultrastructural changes in the wall during germ-tube formation from blastos ores of Candida albicans. J. gen. Microbiof 77,

DESAI, S. C., 1968: Problems of assessment of antifungal drugs. In: Systemic Mycoses, G.E. W. Wolstenhome, R. Porter, eds., pp. 253-262, London: Churchill.

IWATA, K., H. YAMAGUCHI & T. HIRATANI, 1973: Mode of action of clotrimazole. Sabou- raudia 11, 158-166.

JOHNSON, B. F., 1965: Morphologic analysis of yeast cells. Adult cell volume of Saccharomy-

4 17-426.

ces cerevisiae. Exp. Cell. Res. 39, 577-583.

SCHATZ, G. & E. RACKER, 1966: Stable phos- phorylysing submitochondrial particles from Baker’s yeast. Biochem. Biophys. Res. Com- mun. 22, 579-584.

THRASHER, J. D., 1973: The effects of mercuric compounds on dividing cells. In: Drugs and the cell cycle. A. M. Zimmerman, G. M. Padilla, I. L. Cameron, eds., pp. 25-48. London: Academic Press.

VAN DEN BOSSCHE, H., 1974: Biochemical effects of miconazole on fungi. I. Effects on uptake and/or utilisation of purines pyrimi- dines, nucleosides, amino acids and giucose by Candida albicans. Biochem. Pharmacol. 23,

YAMAGLJCHI, H., Y. KANDA & K. IWATA, 1971 : Biochemical properties of mitochondria from Candida albicans. Sabouraudia 9, 221-130.

887-899.

Author’s address: Dr. JANET BROTHERTON, Abteilung fur Gynakologische Endokrinologie, Klini- kum Steglitz der Freien Univcrsitat Berlin, Hindenburgdamm 30, D-1000 Berlin 45.


biological assay of fungicides against yeasts in vitro using a coulter counter

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