JOURNAL OF SCIENCE & ENGINEERING, MAY.1964. VOL Il PP. 201 ~ 209 A STUDY OF THE MARINE YEASTS IN TAIWAN Yeasts Isolated from Tideland Mud, Wu-chi District輔 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. MATER』AιS AND METHODS Med.ia 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. -201 一 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. Methods 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 -202 一 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 -203-· 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: A.dsorp Y': Number of yeasts in the test tube with mud. Y: Number of yeasts in the test tube without mud. RESULTS AND DISCUSSION 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 Galactose Sucrose 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 S訂ain# 一 204 一 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 -1 n珊az'lagi·nosa (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 一 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 -gos 『 ,,,.者 ll 第二期 Assimilation: Glucose + Maltose 一 Gala:'cfo-se 0 - Lactosi:! :.:.. Sucrose - Mitrate 一 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 + Lactcse - 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 一 206 一 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 Sucrose + Nitrate 一 + 一 Related to Candt"da gut"ZUermondit" (Cast.) (Fig. 11,12) Fig. 9 Fig. 10x1500 哥華 發 這當 唱齡 議會 嗜血態 青海 犧/纜臨睡鵝騙 棚," 2海-·--- 事 因 國 機能 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 + Sucrose Rela:·ed to Cry ρtococcus 十 Nitrate laurentii (Kufferath) (Fig. 13,14) Fig. 13 Fig. 14 × 1500 一 207 一 n1 學 n. 第二期 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. Mud ~~ i (mm.) 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 1.5-0.3 0.5-0.3 0.3-0.2 1.7% 5% 6.5% 86.8頁6 H當 3.5% 5.5% 90% 7% 25% 18% 50% <0.2 ~~~~~~ --『』-----『』』『 rates of yeasts by different mud samples are shown in following table: Table 2 Yeasts No. 1 ~~~~︱ No. 3 99% 99% 2。一 30% 99.5% 99.5% 80-90% 99.5% 99.5% 85-903石 99.1% 99.65石 99% 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 troρicalis, 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. SUMMARY 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. permit 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 一 2仿一 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 • I 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. 臺灣海洋音字母商之研究 給1晏海洋潮汐區淤泥中分離之酵母茵 宋世謹* 由梧棲海岸潮汐i區淤泥中分離獲得主海洋酵母菌,各茵掛己經整整理後共得七菌緣。因酵母盟主’,其 綠甚多,海洋酵母菌之璟坡特殊及過去之研究資料有限,本文觀察各菌隊之粗抱形態,茵蒂特鐵,生 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, 遠予確認為某種,或遲予命名均難免有玲:哥拉立嘆,放僅敘述其特徵並暫智、其與某穫相近,而不于確霞、 ,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. 酵母菌之吸著率與菌除特性及淤泥粒子組成有關,一般為淤泥粒子中小粒子較多者,其咬著率亦 較高。 *畫畫灣省立中興犬學植物學系講師 一 209 一
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