VITAL STAINING OF FUNGI IN PURE CULTURES

AND IN SOIL WITH FLUORESCEIN DIACETATE

B. E. ‘%IDERSTR~~M
University of Lund, Department of Microbial Ecology. Ecology Building.
HelgonavLgen 5. S--223 62 Lund. Sweden

(Acceptrti 22 Marc/z
1976)

Summary-A method for vital staining of fungal mycelium with Ruorescein diacetate (FDA) is described.
in experiments with pure cultures, good correlation was obtained between relative staining efficiency.
growth rate. and,respiration. FDA thus appears to be a true vital stain. in that it stains only metaboli-
cally active my&a.
In fresh soil suspensions stained with FDA, brightly fluorescent hyphae and portions of hyphae
were observed. The applicability of the method for measurement of active mycelium in the soil is
discussed.

England) containing 2 mg.ml-’ acetone and kept at

-20°C. dilutions were made to 10 /~g.mi-’ in phos-
Fungi play an important role in the mineralization phate buffer (60 rnM* pH 7.5). The organisms to be
processes in the soil, and in many soils the fungal stained were suspended in the diluted solutions for
biomass greatly exceeds the biomass of other micro- 3 min and then collected by filtration through a non-
organisms. The method most generally used for esti- fluorescent membrane filter (Miiiipore MF Black
mating fungal biomass in the soil is the agar-film filter, 0.8 /cm) in a Pyrex microanalysis filter holder.
technique described by Jones and Moiiison (1948). The filter was gently sucked off and placed on a glass
They claimed that hyphae stained by phenolic aniline slide. A drop of nonfluorescent immersion oil (Car-
blue were to be considered as living hyphac. Later, gille Type B) was added to the topside of the filter,
they rejected this idea (Skinner, Jones and Mollison, which then was covered with a coverglass. The prep-
1952), and it is still an open question whether blue- aration thus obtained was immediately studied mic-
stained hyphae are living or not (Parkinson, Gray roscopically. The staining of the organisms was esti-
and Williams, 1971). mated as excellent, good, faint, or nil respectively. The
Since the living and metabolically active parts of microscope used was a Reichert Zetopan, with a
the hyphae in the soil are the biologically most inter- Binoiux light source (HBO 200 Hg lamp), equipped
esting components of the fungi, the fact that living for incident illumination. As primary filter a 3 mm
mycelia cannot be distinguished from dead. is a very BGl2 was used, and as secondary filter I mm
important limitation of the agar-film technique. A GG9 + 2 mm OG515. The objective (40 x) had a
number of methods have been worked out in order high numerical aperture (NA 0.9). The microphoto-
to allow a direct study of the living part of the soil graphs were taken with Agfapan 100. The film was
microflora. The most promising methods employ exposed for 4 s, and then developed in Agfa Rodinai
staining with fluorescent brighteners, e.g. Anderson (1 -t 25) for 10 min at 25 C.
and Westmoreland (1971). Anderson and Slinger
(1975). Weaver and Zibilske (1975) have shown that
Calcofluor White MR (a stilbene sulfonic acid) stains To stain mycelia in soil, a known amount of soil
cellular proteins of nonlysed bacteria. However, the was suspended in phosphate buffer (pH 7.5), FDA
method does not give any information about the was added (10 LLg.rnl-‘), and after 3 min the suspen-
metabolic activity of the organisms. sions were filtered and examined as described above.
The present paper describes a technique, which The dispersion of the suspensions of organogenic soils
enables the metabolically active hyphae to be was made with a MSE ATO-mixer at 6000 rev. min- ’
observed after staining with fluorescein diacetate for 2 min. For the study of mineral soils, the suspen-
(FDA). FDA is a fluorogenic substrate. which is non- sions were shaken energetically by hand for some
fluorescent until it is enzymatically hydrolysed. The minutes.
properties of this substance are discussed in more
detail by Rotman and Papermaster (1966) and tirowth md respiration erprritwnt.s with pure cultures
Ziegler, Ziegler and Witzenhausen (1975). Growth experiments were carried out with S&&r-
omycrs cerecisiae and ~~~rt~~r~~~~~rfi~~u~~zi~J~~
grown
in malt extract broth (Oxoid) in Erlenmayer flasks
MATERlALS AND METHODS
on a rotary shaker at 30-C. Inoculation of S. cercvi-
siae was made from a liquid over-night culture, while
Staining procedure md microscopy M. r~~u~~~~~u was inoculated with a spore suspen-
From a stock solution of fluorescein diacetate sion filtered through glass wool. The growth was
(FDA) (Koch-Light Laboratories Ltd. Colnbrook, measured as an increase in optical density (OD) in
59
60 B. E. SC~IERSTR~I

a spectrophotometer (Bausch & Lomb Spectronic 20) Table I. Fungi tested for staining with FDA in pure culture
at 440 nm, using round cuvettes of 11.5 mm path
length. In the growth curves, time was plotted against
log OD. This is not a commonly used method for
growth measurements of hlamentous fungi. However, Moriicwlla isahvllinu
Trinci (1972) used this method for growth measure- M. nana
M. par~~isportr
ments of filamentous fungi, and in the present investi-
M. 1“1,,1(1,1,1,(1,1a
gation it was found that with a dilute spore suspen-
MlUW hicvwli.s
sion of M. ramunrtiuw. the turbidity increased logar-
ithmically during the first 12 h, i.e. as long as the
hyphae did not become entangled together
To test if ability to become stained was correlated
with a decrease in growth rate and respiration, a
metabolic inhibitor, HgCl,, was added during the
logarithmic growth phase to five different cultures of
each organism to final concentrations of 5, 10, 50,
100 and 200 pM. Another culture of each organism
was kept as a control. Samples from the cultures were
collected during different phases of growth and
stained with FDA.
Respiration determinations were carried out with
S. cerrvisiae in 50 ml samples withdrawn from cul-
tures 2 h after the addition of HgC12. These samples
were incubated at 30 C in closed bottles with vials
containing 5.0 ml 200 mM NaOH and stirred magneti-
cally. The incubation time varied between 20 and 60
min depending on the metabolic status of the cultures.
The carbonate was precipitated with an excess of
BaCl, and the remaining hydroxide titrated against
HCI. The CO2 evolved.min~ ’ at time r, was calcu-
lated according to the equation

where fI is the starting time and rz is the end of

the respiration experiment. I, is the slope of the
growth curve during this time and R,,,,, is the COz
evolved between rI and r2. The OD value at time
t, was converted to dry weight of cell mass by means
of a previously constructed standard curve. thus
making it possible to express the evolved CO, as
mmole g- ’ (dry weight). min I.

RESC'LTS

Pure cultures of about 50 fungal species were tested

for their staining ability (Table I). All the fungi tested
gave good or excellent fluorescence when stained with
FDA. Most fast growing species. like Mucor sp.. had
a tendency to brighter fluorescence. than slow grow-
ing ones like Oidiodendron sp.. but this was not always
the case.
The results of the growth and respiration exper-
iment with S. ceretiisiur are shown in Fig. 1 and Table
2. During the lag phase (1 h after the inoculation)
only faint fluorescence was observed. but cells from
cultures in the logarithmic growth phase (3 h) were
excellently stained. This was the case in all the paral-
lel cultures. The deterioration of the staining ability
in the presence of HgC12 (Table 2) was well correlated
to the inhibition of both the growth (Fig. 1) and the
respiration rate (Table 2). After 15 h of growth, when
the control culture was in the stationary phase, most
cells in this culture did not fluoresce at all. although
some of them showed good or faint fluorescence.
The growth and staining experiment with M.
rcmunnianu gave similar results to the one with S.
cerucisiae. The spores of M. ramanniana gave no fluor-
escence during the first hour after the inoculation.
After 2 h, when the spores were swollen, which is
the first sign of germination, they were excellently
stained, and the control culture kept the excellent
staining ability during the whole experimental period
(12 h). In the cultures supplied with 5 and 10 pM
HgCl, respectively. neither growth nor staining was
affected. In the culture with 50 PM HgCI,, M. raman-
niclm~ grew slowly (the slope of the growth curve
k = 0.08 hm I, control culture k = 0.20 hh’), and the
relative staining ability was good, but not excellent.
In the cultures with 100 and 200 pM HgClz no growth
could be measured, and the hyphae did not fluoresce.
Mycelia in natural soils were readily stained with
FDA (Figs 2 5). In Fig. 2 the conidiogenous struc-
tures of a monoverticillate Penicillium is shown. The
 Vital staining of fungi 61

vacuoles. Also dark melanized hyphae were well

stained, and sometimes hyphae appearing almost
black when examined in the transmitted light micro-
scope, showed very good fluorescence.
When a specific number of pregrown spores of M.
ramanniana were added to sterile organic soil, a total
recovery of the spores added to the soil was obtained
1.6. after staining with FDA. This indicates that the
method can be used for quantification of fungi in the
1.7 -
soil.
1.6- In soil suspensions homogenized for a longer
O-Z-0 period at a higher speed of the mixer, the measured
1.5 -
amount of fluorescent hyphae dropped slightly. Ex-
1.4 -
tremely violent treatment of the suspensions resulted
in a heavy fall in numbers of fluorescent hyphae.
In all soil samples fluorescent bacteria were also
seen,
1.3
1.2- - /
-.- 0 and 0.005 mM HgC12 Table 2. Staining with FDA and respiration of S. cerevisiar
1.1 - . . . . . c* . . . . 0.0, I.
1,1..I, qmm,.,, 0.05 ,. in samples from cultures with different concentrations of
1.0. /*
-Mm 0.1 and 0.2 .. HgC12. (Growth curves of the identical cultures are given
in Fig. I). Staining was estimated as excellent (f + +),
0.9’ I I I I I I I I I I
good (+ +). faint (+). or nil (-). nd. = not determined
0 1 2 3 4 5 6 7 6 9 10
TIME(h) Staining with FDA before and
Fig. 1. Growth (as optical density) of S. cerrtisitrr in differ- Concentration after addition of HgCI,
ent concentrations of HgCI,. 5 /LM HgClz did not affect of H&l2 (/Ml
growth. while 10 PM rendered a slight decline in growth added after 4 h I I1 3h 6h Xh 10.5 h
rate. 50 PM decreased growth rate considerably. while 100
0 + +++ +++ +++ +++
and 200 pM HgC), completely inhibited growth.
5 + +++ +++ +++ +++
IO + +++ ++ ++ ++
50 + +++ + + _
nonfluorescent sections of the conidiophores are most 100 + +++ _ _ _
likely vacuoles. On one phialide a spore chain with 200 + +++ _ _ _
three conidia can be seen, where the oldest conidium Respiration
is only faintly fluorescent. Entirely ripe conidia of mmole COz.gm’ dry wt. mini’
pure cultures do not fluoresce. Figure 3 shows an 6h
unidentified sporulating fungus. In Figs 4 and 5 0 5. I
hyphae of basidiomycetes with clamp connections can 5 n.d.
be seen. In Fig. 4 most of the hyphae seem to be 10 4.9
50 0.8
empty and only a small section is brightly fluorescent.
loo 0
The cell walls show a faint autofluorescence. The 200 0
darker sections of the hyphae in Fig. 5 are possibly

Fig. 2. A sporulating Peuicillium sp. stained with FDA in natural soil. 750 x
62 B. E. S~~DERSTR~~M
Fig. 3. Unidentified sporulating fungus in soil. 1000 x
DFKUSSIOY
Ziegler CT al. (1975) have described the membrane
properties in active, passive. and dead micro-
organisms with respect to FDA uptake and hydroly-
sis. They concluded that FDA can be regarded as
a true vital stain, which stains only metabolically
active organisms. The results obtained in the present
study support this conclusion.
In the experiments with pure cultures, the
organisms from the active growth phases were excel-
lently stained, while cells in resting stages, as spores
or aged yeast cultures. were not stained. When ac-
tively growing cultures were inhibited by HgC12, the
decrease in relative staining was very well correlated
to the decline in growth rate and respiration rate.
Thus it seems as If FDA stains only metabolically
active cells. Since acetates of phenolic bases are hyd-
Fig. 4. Basidiomyccte hyphae in the soil. A small section
is metabolically active. The cell walls of the empty hyphae
show a faint autofluorescence. 750 x
Fig. 5. An active basidiomycete hyphae in the soil. 600 x
rolyzed nonspecifically by many enzymes such as pro-
teinases, and esterases (Rotman and Papermaster,
1966), one can assume that all fungi can hydrolyze
FDA. This assumption is also supported by the good
staining of all the fungal species tested in the present
study. In a pure culture of a fungus it may also be
possible to make a distinction between different
degrees of activity, but in a mixed population this
is probably not possible. as different species may
show varying response to FDA.
Staining of fungal mycelium in soil with FDA was
very successful (Figs 2---5). It is also possible to
measure the biomass of metabolically active myce-
lium in the soil. This can be done either by photo-
graphing the hyphae after which the length can be
measured in a photographic enlarger. or by using the
intersection method for measurement of filamentous
organisms (Olson, 1950; Swift, 1973). However, all
the measurements must be performed very quickly,
as the fluorescence may fade after about only 30 s
illumination in light of short wavelength.
The quantification of mycelia in the soil is beset
by a major problem. To get a reasonable distribution
of the hyphae on the filter, the soil must be homo-
genized. When the agar-film technique (Jones and
Mollison, 1948) is used, the amount of hyphae
recorded is strongly dependent on the amount of dis-
persion. If the soil is poorly homogenized, hyphae
within soil aggregates or organic particles may remain
invisible. When these particles are broken, the embed-
ded hyphae are released and can be measured, but
prolonged mixing of the suspensions results in des-
truction df recognizable mycelium. It is, however,
possible to find an optimum of dispersion for the re-
corded amount of hyphae (Swift, 1973; E. BZth and
B. SaderstrGm, unpublished). It is likely that the same
is also applicable for metabolically active hyphae, but
it seems as if these hyphae are much more sensitive
to dispersion destruction. For that reason, the hom-
ogenizing treatment must be kept to a minimum
when active mycelia are studied. As the active hyphae
fluoresce when stained with FDA. it is sometimes
possible to observe and measure those within soil par-
ticles.
 Vital staining of fungi
In the microphotographs of myceliurn in soil some
interesting features can be noted. In Fig. 2 the phia-
lides of the P~~~i~i~li~ show the brightest fluores-
cence. This was always the case; the conidiogeno~
structures always seemed most active. This is not sur-
prising, since metabolic activity is high during the
production of conidia. It is, however, noteworthy that
the conidiophores are so heavily vacuolated. In pure
cultures on rich media, the upper parts of conidio-
phores generally possess rather small vacuoles.
The formation shown in Fig. 4, where a short active
section occurs along the length of an apparently
empty hypha, was often noted in the soil. This was
probably not an effect of the homogenizing treatment,
since septa also could be seen in the empty hyphae
at some distance from the active part.
It was an interesting fact that fluorescent bacteria
were regularly seen in the soil preparations. This may
indicate that FDA is also suitable for vital staining
of bacteria. Babiuk and Paul (1970) recorded unsatis-
factory staining efficiency with bacteria stained with
FDA, while Ziegler et ul. (1975) obtained good results
with a number of different bacteria. The attempts of
the present author to stain Laboratory cultures of dif-
ferent bacteria gave very poor results. However, when
freshly isolated bacteria from soil were stained, most
of them, but not all, became fluorescent with FDA.
Notably, some of them lost their staining ability after
some transfers on laboratory media, while others, e.g.
Buei~~~,~rereus, stained excellently after 30 transfers.
The method for measurement of active mycelium
in the soil described here has for some time been
in use for regular studies of fungal mycelium in a
pine forest soil in Sweden. The preliminary results
indicate that during the periods of high fungal acti-
vity, only about i-5% of the total amount of myce-
hum measured with the Jones and Mollison technique
are metabolically active. Some attempts to pick up
fluorescent hyphae under the microscope and culti-
vate them (Warcup, 19.55) have also been made with
promising results. The method can also be used for
the study of fungi on solid particles like leaf litter,
or to study the growth of fungi in pure culture.
Acknowledgemrllts-This work was carried out within the
Swedish Coniferous Forest Project supported by the
Swedish Natural Science Research Council, the Swedish
63
Environm~tal Protection Board, the Swedish Council of
Forestry and Agricultural Research, and the Wallenberg
Foundation. The author is much indebted to Professor B.
Nor&n for his help and encouragement, and also to Mr.
E. BHBth.
REFERENCES
ANDERSONJ. R. and SLINGERJ. M. (1975) Europium
chelate and fluorescent brightener staining of soil propa-
gules and their photomicrographic counting-I.
Methods. Soil. Biol. Biochem. 7, 205209.
ANDERSONJ. R. and WESTMOREXAND D. (1971) Direct
counts of soil organisms using a fluorescent brightener
and a europium chelate. Soil Bin/. Biochem. 3, 85-87.
BA~IUKL. A, and PAUL (1970) The use of Ruorescein iso-
thiocyanate in the determination of the bacterial bio-
mass of grassland soil. Con. J. Microbial. 16, 57-62.
JONES P. C. T. and MOLLISONJ. E. (1948) A technique
for the quantitative estimation of soil micro-organisms.
J. gen. Microbial. 2, 54-69.
OLSONF. C. W. (1950) Quantitative estimates of fdamen-
tous algae. Truns. Am.-microsc. Sot. 69, 272..279.
PARKINSOND.. GRAY T. R. G. and Wrr.~~khisS. T. (1971)
Methods fo; Studying the Ecology qf Soil Micro:
organisms. Handbook 19, Blackwell-IBP, Oxford.
ROTMANB. and PAPERMASTER B. W. (1966) Membrane
properties of living mammalian cells as studied by enzy-
matic hydrolysis of fluorogenic esters. Proc. nutn. Acnd.
Sci. U.S.A. 55. 134-141.
SKINNERF. A., JONESP. C. T. and MOLLIX~NJ. E. (1952)
A comparison of a direct- and a plate-countin tech-
nique for the quantitative estimation of soil micro-
organisms. J. g&t. ~~~rffb~ot. 6, 261-271.
SWIFT M. J. (1973) Estimation of mvcelial growth during
decomposition of plant litter. In Modern kethods in thz
Study ofMicrobial Ecology. (T. Rosswall, Ed.); Buli. ecal.
Res. Comm. 17, 3233328.
TRINCI A. P. J. (1972) Culture turbidity as a measure of
mould growth. Trans. Br. mycol. Sue. 58, 467-473.
WARCUPJ. H. (1955) Isolation of fungi from hyphae pres-
ent in soil. Nature, Lond. 175. 953-954.
WEAVERR. W. and ZIBILSKEL. (1975) Affinity of cellular
constituents of two bacteria for fluorescent brighteners.
Appl. Microbial. 29, 287-292.
ZIEGLERG. B., ZIEGLERE. and WITZENHAUSEN R. (1975)
Nachweis der Stoffwechselaktivitlt von Microor-
aanismen durch Vital-Fluorochromieruna mit 3’,6’-Dia-
cetylfluorescein. Zmtbl. Bakt. Hyg. Aht. 7. Orig. A 230,
252-264.