Document 358545

Yeasts Isolated from Tideland Mud, Wu-chi
Shih-Chin Sung體
Few investigations on marine yeasts have been reported. Waksman(7) described
the presence of Torula in sea water in 1931. Two more reports of marine yeasts
were presented by
Phaff的 in
1952 and Wood(8) in 1952. Recently, extensive studies
on various aspects of marine yeasts have been carried out by Suehiro (5)“:) at
Kyushu University. However, no work has been reported on marine yeasts in Taiwan.
The present investigation was to isolate marine yeasts from tideland mud and to
measure the adsorption of yeasts by the mud.
M·Y Broth.Malt extract (Difeo) 3gm., yeast extract (Difco) 3gm., peptone
(Wako) 5gm吋 dextrose (Cica) 15gm吋 and sodium chloride (Cica) 30gm. were dissolved in 1000 ml. distilled water in a 2000 ml. flask by heating gently and stir(1)
ring constantly with a glass rod. When it cooled, pH was adjusted to 4.0 土 0.2 with
5N HCI. Then an aliquot of 5ml. was distributed to each of 20ml. test tubes. Tubes
were sterilized at 120 。C for 15 minutes.
(2) M-Y agar. 2.5 percent M-Y agar medium was prepared by addition of 25g
!11· ag,ar to the M-Y broth formula. The ingredients were also dissolved by heating.
pH was about 5.6. An aliquot of 15ml. or 5ml. of the medium was distributed to
each of 20ml. or lOml. test tubes for pouring agar plates or preparing agar slant民
respectively. Tubes were autoclaved at 120°C for 15 minutes.
(3) Brath for Fermentation Tests. Yeast extract 4.5m忌, peptone 7. 5 gm., sodium
chloride 30gm. were dissolved in lOOOml. of distilled water by heating gently.
After cooled, lrnl. of bromthyrnol blue (1. 6 percent alcoholic solution) was introdticed to the flask, then shaken well. An aliquot of 4ml. of the broth was distributed
to each lOrnl. test tubes (12 × lOOmm.) and a Duhdam tube was introduced. Sterilized
at 120。C for 15 minutes. Then, 2ml.of 6 percent dextrose or an e quivalent of
galactose, m altose, sucrose or lactose were introduced respectively i nto one of a
five-tube set. Sugar solutions were filter sterilized.
* Instructor, Department of- Botany, Taiwan Provincial Chung Hsing University.
** This project was supported by a grant from the National Council on Science Development.
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n~ 學織第二期
的 Broth for Sugar Assimilation Tests. 6. 7gm. yeast nitrogen base (Difeo) and
5gm. of dextrose or an equivalent of galactose, maltose, sucrose or lactose were
dissolved in lOOml. of sterilized distilled water. This lOX strength solution was then
filter sterilized, kept in the refrigerator and used as needed. The final medium
was prepared by pipetting 0.5ml. of lOX strength solution into a lOml. test tube
containing 4.5ml. of 3.0 percent sterilized sodium chloride solution. Tubes were
shaken well and ready for inoculation.
(5) Broth for Nitrate Assimilation Tests. 11. 7gm. yeast carbon base and 0. 78
gm. potassium nitrate were dissolved in lOOml. distilled water. This lOX strength
solution was filter sterilized and kept in refrigerator. Final medium was prep·
ared by pipetting 0.5ml. of lOX strength solution into a lOml. test tube containing
4.5ml. 3.0 percent sterilized sodium chloride solution. Tubes were shaken well
and ready for inoculation.
Isolation and Identification of Yeasts. Collections of mud were made at Wu
-chi, a small fishery harbor located on the west coast of this island near Taichung City. Each sample was collected with a sterilized spoon, put into a
50ml. sterilized cotton-plugged flask, and brought back to the laboratory immedi·
ately. One gram of mud was suspended in 5ml. sterilized sea water in a 20ml. test
tube, and shaken well. In order to inhibit the growth of bacteria and molds which
are normally predominant in mud, mud suspension was firstly inoculated in M-Y
broth containing 3 percent sodium chloride with pH of 4.0 士 0.2. Four tubes were
inoculated for each sample ; two inoculated tubes of each four were poured with
enough sterilized melted vaseline on the top of broth to make a seal at least lcm.
thick. The low pH inhibits the growth of bacteria, and the anaerobic condition
is unfavorable to molds. Tubes were incubated at 25。c for 5 days, then were
streaked on M-Y agar plates, ~lso incubated at 25。C. Representative yeast colonies
were selected to prepare smears for microscopic study to test for purity. The remainder of each of the colonies was ’, fished’, to duplicate M-Y agar slants. which
were incubated at 25。C for 48 hours and kept in the refrigerator for further
taxonomic identification.
Taxonomic identification was made by using the procedures of Wickerham盼
and Lodder and kreger-Van Rij(2).
Morphological studies were made cf asexual reproduction, cell morphology,
colonial characteristics and sporulation. Asexual reproduction, cell morphology
and colonial c haracteristics were studied by fo llowing the dalmau plate technique
suggested by W ickerham.
Following sterilization, 15ml. of melted M-Y agar was poured into each
sterilized petri dish The dishes were allowed to stand at room temperature for
one or two days before using to assure a dry surface. A single very light inoculation
from a slant culture was made near one side of the plate (as from the relative
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A Study of the Marine Yeast• ln Taiwan.
Yeasts Isolated from Tideland Mud, Wu-chi Distri官Z
positions 10 o’clock to 2 o’clock) In addition to the single inoculation, two point
inoculations were made near the other side of the plate (as at positions 4 o' clock
and 8 o’ clock) A central section of the streak inoculation and one of the point
inoculations were covered with cover glasses. With forceps a cover glass was
removed from absolute alcohol, drained momentarily, and the excess alcohol was
burned off by passing over a low flame. When the cover glass had cooled, one
edge was placed on the agar and the cover glass was allowed to fall across the
central portion of the inoculated streak, and a second cover glass was placed over
one point inoculation. The plate was incubated at 25。 C for one week. The great
advantage of the dalmau plate technique is that the cover glass removed from the
plate, after staining, could be directly used for microscopic studies of the hyphae
for filamentous species. Basic fuchsin was used to stain vegetative cells for
general studies.
Old cultures grown on M-Y agar slant were desirable for studying sporulation.
This was conducted by keeping the well-grown culture at refrigerator tempera ”
ture for more than six weeks. Decrease in moisture to a certain degree is
favorable for spore formation. Spores were stained with malachite green and
were then counterstained with safranin for microscopic studies.
Physiological studies was conducted to include the formation of pellicle, fermentation, sugar assimilation and nitrate assimilation.
The cultures grown in M-Y broth for 4 days were observed for pellicle formation. The fermentation tests were carried out in the yeast-peptone broth as above mentioned. Yeasts used for this test were taken from 48 hours slant cultures.
Five ml. sterilized distilled water was introduced into the slant tube to make a
suspension. Inoculum was O. lml. These inoculated test tubes were studied at 1,
2, 4, 6 and 10 days. Fermentation was indicated by accumulation of gas in the
Durham tube and the color change of the indicator.
Difeo yeast nitrogen base broth containing 0.5 percent various sugars were
used of testing sugar assimilation. Cultures of 24 hours were used as inoculum.
The media were slightly inoculated and kept at 25。C. After 7 days and again
at 21 days, tubes were shaken to suspend growth and then were placed against
a white card bearing lines approximately 3/4 of a millimeter wide. The lines
were drawn with India ink. If the lines could not. seen through the culture, or if
the lines appeared as diffuse, broad bands, the test was regarded as positive. If
the lin~-t~e distinguishable as such, the test was negative. The.two ..observations
indicate which reactions occurred rapidly and which occurred latently (3).
Nitrate assimilations were carried out in the broth prepared with Difeo yeast
carbon base and potassium nitrate. Procedures were similar to that of sugar assi
milation tests. However, yeasts grown on a rich medium may carry a reserve of
nitrogen in t he form of protein. Possible errors due to this reserve were eliminated by making two serial transfers in the nitrate a臼imilation broth . When the
first transfer was 7 days old, the culture wa.s shaken one loopful was transfered
n~ ' 鼠,第二期弓
'to; '.1 second tub,e. If .a· positive test was obtained when. the secon<l ·culture ·was· 7
days old, the organism was consi<lered· able to assimilate nitrate;:
The adsorption of yeasts by mud.
Yeast suspension was filtered with No. 2 filter paper to eliminate clumps
of yeast and the filtrate was poured into two test tubes. One of them was poured
into dried mud. Both tubes were shaken for one minute and allowed to stand for 30
minutes. 1'he supernatant was used to inoculat the M-Y agar plate. At 25°C after
4-5 days, the number of yeasts was counted and the adsorption rate was determind
by the following formula:
Y': Number of yeasts in the test tube with mud.
Y: Number of yeasts in the test tube without mud.
Yeasts morphology and physiology.
Seven strains have been isolated from the tideland mud collected from Wuchi the morphiogical characteristics of each strain obtained in the present
work does not follow exactly the typical descriptions given by Lodder and
Kreger-Van Rij份). Following Kriss's suggestion(l),in this paper only the name of
the most closely related species is given to each strain isolated.
The influence of the sea water environment to which the marine yeasts are
subjected also may he one of the factors which affect the variation. Further
studies on both morphology and physiology are necessary for definite identificat-1on.
Strain# 1.. Cells almost round(3一5)µ, Round cells together with short oval ones
seldom found, usually single. Thin pellicle and sediment formed. in M-Y broth. On
da!mau pfate. streak culture white color, surface smooth and glistening; no
pseudomyce!ium formed at covered portion.
Usually one spore developed in ascus.
Fermentation; negative
Assimilation:一 Glucose
h卜 Maltose
+ Lactose +
+ Nitrate 一
Related to Debraryomyces hansenii (Zopf) (Fig. 1 ,勾心
2. Cells short-oval or oval, (2. 5一5)×(~的μ﹔ single or in pairs.
Sediment fo rmed in M ‘ Y broth. On dalmau plate, streak culture red color, strongly
glistening, surface smooth and :mucous, margin smooth. No pse udomycelitini
formed at covered portion.
Ferr.nentation: negative
Assimlation: Glucose + Maltose 十
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A Study of the Marine Yeasts in Taiwan
Yeasts Isolated from Tideland Mu溢, Wu-chi Disi'rict
Galactose - Lactose .• Sucrose
+ Nitrate
Related to Rhodotorula
(Jorg) (Fig. 3,4)
Fig. 1
Fig. 2 × 1500
Fig. 3
Fig. 4× 1500
Strain# 3. Cells long-oval, (2-4) X (4-1日 µ, single or in pairs. Sediment and
ring formed. On dalmau plate, streak culture orange, surface mucous and smooth,
slightly rugose in middle. Margin surrounded by primitive pseudomycelium.
Primitive pseudomycelium formed at co耳rered portion.
Fermentation: Glucose
+ Maltose +
Galactos~ - Lactose Sucrose + ,
Assimilation: Glucose + Ma』tose +
Galactose - -.:Lactose Sucrose
十 N,itrate
Related to Saccharomyees pastori"am的( Hansen) (Fig. 5,6)
Strain# 4. Cells lemon-shaped or long-oval, (2-5)X(4-10) µ, single or in pairs
Sediment and thin ring formed in M-Y broth. On dalmaq plate, stre ak culture
greyish-yellow, surface, smooth and glistening. Border surrounded by p seudomycelium. Pseudomycelium formed at covered portion.
F ermentation: Glucose
+ Maltoe 一
Galactose - Lactose Sucrose
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,,,.者 ll 第二期
Assimilation: Glucose
+ Maltose 一
Gala:'cfo-se 0 - Lactosi:! :.:..
Related to Kloeckera apiculata (Rees emend. Kloeker)
(Fig. 7 ,8)
Fig. 6 × 1500
Fig. 5
F!g. 7
Strain# 5. Gells short-oval to oval, sometimes rounded, (2. 5-9) X (3-12) µ.,
budding, usually single, occastionally paired or chained. Sediment ~nd ring formed
in M-Y broth. On dalmau plate, streak culture cream to yellowish, surface rough
folded. Pseudomy:;.elium and true mycelium extended out from margin. Pseudomycelium, true mycelium and blastospores formed at covered pcrtion.
Fermentation: Glucose ·+ Maltose +
Galactose + Lactose Sucrose
Assimilation: Glucose + Maltose 十
Galactose + Sucrose +
- Nitrate 一
Related to Candida troρicaUs (Cast.) (Fig. 8,9)
s-::rain# 6 . Cells round or short :. oval, (2-5) X(3 -6)µ .• many !Jng pseudomycelial
cells seen a mong ordinary cells. Sediment and ring formed in M-Y broth. Old
σlttture sh::>w n slight pellicle. On dalmau pla妞, streak culture milky color, surface
soft, smooth and glistening. Abundant vseudomycelium and blastospores formed at
covered portion.
Fermentation: Glucose + Maltose 一
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A Study of the Marine Yeasts in Taiwan
Yeasts Isolated from Tideland Mud, Wu-chi Distri'Ct
Galactose - Lactose
Sucrose +
Assimilation: Glucose
+ Maltose
Galac-Cose 十 Lactose
Related to Candt"da gut"ZUermondit" (Cast.) (Fig. 11,12)
Fig. 9
Fig. 10x1500
Fig. 11
Fig. 12 × 1500
Strain# 7. Cells round, oval er occasionally long-oval, (2-5.5)X (3-6, )f-1.,single
or in pairs. Sediment, ring and pellicle formed in M-Y broth. On dalmau plate,
streak culture p"'.ll~ yellow, surface smooth, mucous and glistening. No pseudomy•
celium fbrmed a:- covered portion.
Fermentation: negative
Assimilation: Glucose
+ Maltose +
Galactose + Lactose +
Rela:·ed to
Cry ρtococcus
十 Nitrate
laurentii (Kufferath) (Fig. 13,14)
Fig. 13
Fig. 14 × 1500
一 207 一
n1 學 n.
The adsorption of yeasts by tideland mud.
The particle constitution of the tested mud is given in the following table:
Table 1
\空空m. of 叫 icle.
No. 1, Surface mud near
high tide
No. 2, Depth of 30 cm.
near high tide
No. 3, Sandy mud near
low tide
The adsorption
~~~~~~ --『』-----『』』『
rates of yeasts by different mud samples are
shown in
following table:
Table 2
No. 1
No. 3
2。一 30%
Cαndidα tropicalis
Debraryomyces hαnsenii
Cryptococcu.& Jαu.rentii
Rhodotorula mucilagjnosa
No. 2
The weights of tested muds were lgm. per 5ml. of yeast suspension and the
Debraryomyces hanseniz", Cry ρtococcus
laurentit" and Rhodotorula mucilaginosa. Table 2, shows that Debraryomyces
hansenii Cryρtococcus laurentit' and Rhodotorula muet"laginosa are well adsorbed
by all mud wheres Candida troρicalis is poorly adsorbed by Sandy mud This
tested cultures were Candida
may be due to the nature of the different species of yeaets. The adsorption rate is
related to the particle constitution of different mud samples, the larger the
proportion of the smaller mud particles.the higher the adsorption rate.
Seven strains of marine yeasts have been isolated from the tideland mud collected from Wu-chi, a small fishery harbor near Taichung. Descriptions of cell
morphology. colonial characteristics, asexual reproduction, sporulation, along with
observations on pellicle formation, fermentation, sugar and nitrate assimilation
the p robable identification of these species ; Debraryomyces hansenii,
Rhodotorula mucz"laginosa, Saccharomyces pastorianus, Kloeckera apiculata,
Candida troPicaUs, Candida guiUiermondii and Cryρtococcus laurent t"i.
The adsorption rate of yeast by mud is correlated with the nature of different
species and t he parti-:;le constitution of the mud ; the brger the proportion of
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A Study of the M~rine Yeasts in Taiwan
Yeasts Isolated from Tideland Mud, Wu-chi District
smaller mud particles, the higher the adsorption rate.
Literature cited
1. Kriss, A.E. Marine Microbiology (Eng. Edi.,) pp . 184-236. 1963.
2. Ladder, J. and N.J.W. Kreger-van Rij. The Yeasts, a Taxonomic Study.
Interscience publishers, Inc. New york 1952.
3. Wickerham, Lynferd J. Taxonomy of Yeasts. U. S. Dspt. Agric. Tech. Bull., No.
1029, 1951.
4. Phaff H.J., E.M. ; Mrakk & O.B. Williams Mycologia 44: 443, 1952.
5. Suehiro, Sumio Sci. Bull. Agri. Kyushu Univ. 17; 443-449 1960 •
6. 一一一一一, ibid, 20; 223, 1963.
7. Waksman, S.A. Sci. Month., 38 :話, 1931.
8. Wood, E.J.F. Australian Jour. Marine & Fresh Water Res. 4: 160, 1953.
殖方式,袍子之有無及形蔥,培養湯中有無膜之形成,發酵情形,酷類及硝酸鹽之間化情形依 Loddet
民檢索表鑑定,但本文所得之各商林之形慧,生理特徵與標準說明略有出入,按 Kriss 氏之意見,
,自俟進一步比較觀察。相近種名 :R·
Debraryomyces hansenz'i, R hodotorula mucilag仰osa,
Saccharomyces Pastorianus, Kloeckera api'culata, Candida. troPicalis, Candida
guilliermondii 及 Cryρtococcus laurentii.
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