il | | Ill il IO oon in “ LIBRARY | Michigan State ‘eq University . ve Rstahd sites SENTOR THESIS. "PURITY OF YEASTS” +t L ‘ JENNETTE C. CARPENTER. Michigan Agricultural College,1898. e ‘ ) ( ’ ~ ve | THESIS Purity of Yeasts, Tne object of the following investigation has been to demon- strate the relative pukity of a few of the vrincipal commercial yeasts and to determine the growth and action of the yeast plant and the impurities upon the different media, In each case a portion of the yeast cake was suspended in a definite amovnt of sterilised water, Agar and gelatin plates were made from this suspended material; when the colonies were sufficiently developed they were carefully studied,and the micro- organisms examined by means of a hanging drop, Cultures from each kind of colonies were made in tubes of gelatin, bouillon,agar, potato, milk,2% cane sugar bouilldn,2% lactose bouillon,2% glucose bouillon and 1% flour stareh bouillon, The cultures were grown in the or- dinary temperture of the room and studied during their growth, Those of bouillon,milk,glucose bouillon,kactose bouillon,cane sugar bouillon and starch bouillon,were tested for lactic and acetic acids, The number of colonies was counted by means of the Wolfhii- gil's apparatus for the purpose of determining the ratio existing between the yeast plant and the contaminating varieties. Stain- ed specimens,from which drawings were made,were mounted from the agar tube cultures, Specimen No. I. Fleischman's Condensed Yeast, 24 hours old:- ,0l1 grams of the yeast cake was suspended in 1000 cc. of sterilized water, (2) Gelatin and agar plates of .lec,.4cc and .7cc were made from this suspended material, In twenty-four hours the plates had developed sufficiently to be studied, Two forms of colonies and one mould were found,which are classified as No 1,No 2 and No 3, No. 1,-Yeast. Golonies,- The border was regular and even,with a clear and dis- tinct outline. The form was round,being lighter in color at the center and darker at the edges, The color of the colony was that of cream,(a,Fig 1) Hanging-drop,.- Very small round of slightly oval cells. Many were single while others were connected in chains.(b,Fig 1) Motion.- None. Gelatin tube cultures,- The growth was along the entire line of inoculation in distinct white dots,which were close together, The surface growth was of thin tiny white rings,the edges of which were raised above the center. Radiating lines extended out- wards from the body of the colony, Inclined agar tube cultures,- A surface growth of moist white ap- vearance not covering the entire surface but only sprinkled over it, Potato tube cultures.- The surface was partly covered with a dry rough gmowth of white connecting colonies, A few single “eolonies were present, Bouillon tube cultures,- The liquid was slightly cloudy,with lit- tle colonies grouvned together at the bottom and floating in the liouid, (3) Starch bouillon cultures,.- The liquid was a little cloudy, A sed- iment formed later, Cane sugar bouillon cultures.- The liquid was more cloudy than the starch or bouillon, No sediment, Lactose bouillon cultures.- The liquid was scarcely clouded. Glucose bouillon cultures,.- The lincuid was heavily clouded. Milk tube.- Showed no change, Growth.- Ravid, Acids,- No acetic acid was produced in apyof the cultures, Lac- tic acid was produced in all of the cultures,varying in in- tensity with milk the least,through glucose,cane sugar, bouil- lion,lactose and starch. None of the reactions were strong, Behavior to gelatin,- No liquffaction, No 2. Colonies.- Small,with regular even borders,but a sharp outline. To the nakeceye they sppeared yellow,but the microscope re- vealed a brown colony composed of concentric rings which be- came darker toward 2 nearky black center.(a,Fig 2) Hanging-drop.- Small white round orranisms varying in size from . a tiny speck to a pin head, They were not connected.(b,Fig 2) Motion.- A slight brownean motion. Gelatin tube culture.- The growth extended along half the length of inoculation. The surface showed a growth of cream colored granular colonies from which a very slight white feathery Prowth extended toward the bottom (4) Inclined agar tube culture.- The surface showed tiny unconnected cream colored colonies, Potato tube culture.- A brownish granular growth along the surface, Bouillon tube cultures,.- The liquid was cloudy with a thick cream colored ring along the surface next the tube and a slight cream colored scum on the surface, Starch bouillon cultures,- The surface of the liquid was covered with a growth of white svecks. A ring of the same color was on the surface next the tube, Cane sugar bouillon cultures,.- The liquid showed a heavy white scum on the surface, Lactose bouillon culture.- The liquid was cloudy, A thin ring of white on the surface around the tube, and a thin scum cov- ered the surface, Glucose bouillon cultures,- The liquid was slightly cloudy. A very slight ring was about the tube. Milk.- No change was visible. Growth.- Rapid, Acids.- Acetic acid was found in very small quantity in the starch, Lactic acid was fornd in small amount in glucose bouillon, in- creasing in the lactose bouillon,and milk, Behavior of gelatin.- Does not liquefy. No 3,- Mould, Colonies,- The colonies first appeared as delicate white stars which later s»read out as dry,flat whitish masses, The out- (5) line consisting got mycelial threads radiating from the cen- ter.(a,Fig 3) Microscopic examination.- The spores were separated. Rectangular in shape.(b,Fig 3) Gelatin tube cultures.- The growth occurred along the entire line of inoegulation. The surface growth was a grayish white dry growth with a branching net-work of threads extending down into the gelati®. Inclined agar tube culture.- A grayish white,thin,feathery growth, confined to the lines of inoculation and not spreading over the entire surface. Potato cultures.- Surface covered with a heavy white growth. Bouillon cultures.- The surface of the liquid was covered with a white feathery growth, Starch bouillon cultures.- The surface of the liquid was about half covered with a white fan-shaped growth. Cane sugar cultures.- The liquid showed a thick white 6lo.ud floating near the surface,but no surface growth. Lactose bouillon cultures.=- The liquid had a slight white growth throughout,with a heavy white surface growth. Glucose bouillon cultures.- The liquid had a thick white surface growth. Milk.- Showed no growth. Behavior to gelatin.- No liqué@faction. (6) In the gelatin plate made from .1 cc.,449035 colonies of Nol, 1863 colonies of No 2,and 12 colonies of No 3 were founds; making a ratio of 1 colony of No 2 to 8 of Nol. Speeimen No. 2. Sunlight Yeast(Dry,age unknown):- .1 gram of the yeast cake was } suspended in 100 cc. of sterilized water. Gelatin and agar Plates of .1 ¢0C0.,-5 CC.,-05 ce. and 03 cc. were made, Up- onexamination,when developed,two kinds of colonies and one. mould were found,which are classified as No 1,No0 2 and No 5, No 1,- Yeast, Colonies.- The colonies were star shaped with a distinct outline. The center was heavy and had lines not closely crowded to- gether radiating from it. The color was creamy white.(&,Fig 4 Hanging-drop.- Large white,round cells,closely massed together. (>, Fig 4) Motion.- None. Gelatin tube cultures,- The growth extended along the entire line of inoculation in yellowish white specks. The surface growth was white. Inclined agar tube cultures,.- The growth was of round or oval distinct colonies,varying slightly in size, Potato tube culture.- The growth was a dry whitish mass spreading outwards from a few central cells. Bouillon tube culture,- The liquid was very cloildy,. (7) Starch bouillon cultures,.- Somewhat cloudy. Cane sygar bouillon cultures,- This culture showed the least growth. Lactose bouillon cxltures.- The liquid was scarcely cloudy. Glucose bouillon cultupes.- The liquid wis very cloudy,but less so than the bouillon. Milk tube.- Showed no growth. Growth.- Not rapid. Acids.- No acetic acid produced in any of the eultures, All gave lactic acid;bouillon gave the least,then starch bouillon, milk,lactose bouillon,cane sugar bouillon,and glucose bouillon. Behavior to gelatin.- No liqu#Paction. No 2. Colonies,.- The colonies were round with a regular and even border, and a sharp and distinct outline. The center was a light brown with a darker brown edge,.(a,Fig 5) Hanging-drop.- Small white or colorless rods about as wide as long. (vo, Fig 5) Motion.- Ranid rotary motion. Gelatin tube cultures.- A moist yellowish white surface growth. A funnel-shaved growth extended along the entire line of in- oculation. Inclined agar tube cultures.- The surface was covered with a thin slimy yellowish white growth of connected dots, Potato.- A slight yellowish white granular growth. (8) Bouillon tube cultures.- The liquid was very slightly cloudy. A white sediment formed 2t the bottom of the liquid. Starch bouillon cultures.- The liquid was about as clo'dy as the bouillon, Cane sugar bouillon culture,.- The liquid was cloudy and the sed- iment formed at the bottom, Lactose bouillon culture.- The liquid was slightly cloudy, but not so much s0 as the cane sugar bouillon. A sediment form} ed at the bottom. Glucoss bouillon cultures.- The liquid was very cloudy. A heavy white sediment formed at the bottom. Milk tube .- Showed rio change, Growth.- Rapid. Acids.- None of the cultures gave lactie acid reactions. All the cult res except lactose bouillon gave the acetic acid reaction which was very slight in starch and glucose,but increased with the bouillon,milk and cane sugar, Behavior to gelatin.- No liquefaction. No 3.- Mould, Colonies.- A rounded,irregular,felt-like mass at first white and then greenish or bluish gray. This was composed of many hor- izontally arranged straight of slightly wavy jointed threads. (Fie 6) Microscopic examination.- The fo organs were forked and on tke ends were the intermediate spore bearers, Each of these in t turn bore a row of oval svores. The whole appeared like a (9) bush.(b, Fig 6) Gelatin tube cultures.- The surface was covered with a felt-like growth,at first white but later green. Inclined agar tube culture.- The surface was entirely covered with a felt-like growth,green at the center but white at the edges, Potato tube cultures.- The potato was completely covered with a green felt-like growth. Bouillon tube cultures.- The liquid had only a little of the whit- ish felt-like growth on the surface. Later it became green. Starch bouillon cultures.- The surface of the liquid was only dotted with little specks of a felt-like growth. These were white at the edges and green at the center. Cane sugar bouillon cultures.- The surface of the liquid was cover- ed with a large folded felt-like mass,which was green at the center and white at the edge. Lactose bouillon culture.- The surface of the liquid was half cov- ered with a felt-like mass,green at the center and white at the edge, Milk tube.- The milk showed a deep green felt-like growth. Growth.- Ranid,. Behavior to gelatin.- Liquefies gelatin. Agar plate .03 cc. of Specimen 2,contained 12022 colonies of No 1, 2769 colonies of No 2,and 1 colony of No 35; making a ratio of 3 colonies of No 2 to 14 of No l»p (10) Specimen 5, Magic Yeast.(Dry,age nét known):- .0l1 grams of the yeast cake was suspended in 100 Cee of sterilized water. Gelatin and agar plates of .1 cc., .o2 cc.,and .03 cc, were made. When develonved three kinds of coionies and one mould vere found,which are class- ified as No 1,No 2,No 5 and No 4, No 1.- Yeast, Colonies,- Star-shaned with a distinct outline, The center was yellowish white and had lines not closely crowded together radiating from it.(a,Fig 7) Hanging dron.- Round or oval cells. Very small and showing call divisions, Nearly all were massed together.(o,Fig 7) Gelatin tube cultures.- The growth ofcurred along.the entire ‘ line of inoculation in small distinctly separate colonies, The s :rface growth was heavy and white. Inclined agar tube cultures.- A whitish feathery surface growth, thicker at the center than at the edge, Potato tube.- A yellowish white growth, Bouillon tuhe cultures.- The liquid was very cloudy. Starch bouillon culture.- The liquid was more cloudy than the -“pouflion. Cane sugar bouillon culture.- The licuid was very cloudy. The most growth of any of the cultures, Lactose bouillon culture.- The liquid was about as Sloudy as the bouillon. e - ° . » = a oe ° . - . : - : ‘ . . oe . ' e e . e « 1 , my 2 . Cd « _ f° e e . o . e ' . e ° . te - e es e . . ‘ Cd . , ‘ °e e é : ’ ° ’ : , ‘ ’ . - se , e e - ° eo . - e - es » . ¢ - . 4 * * e ~ - . e , . . . . ° . - , ‘ e . . e oe ' . ~ « . e @ _. - . ' e ? ‘ . « ° ° . . » ° 1 * - ’ , 4 . . e e e . . ° ‘ e | (11) Glucose bouillon culture.- The liquid was slightly more cloudy than the lactose bouillon or bouillon cultures, Milk tube.* Showed no change, Growth.- Not ranid. Slower than yeast of Specimen 1; but about the same as that of Specimen No 2, Acids,- None of the cultures gave the acetic acid reaction, All excent bouillon gave lactic acid,gtarch bouillon gave the least,increasing with lactose bouillon,glucose bouilloh,cane sugar bouillon and milk. The reaction was not strong in any case, Behavior to gelatin.- No liquefaction, No 2. Colonies.- Border irregular,with a distinct outline. The colonies ‘were brown and much darker in color at the edge that at the center.(a,Fig 8) Hanging drop.- Small,white or colorless,round organisms,(b,Fig 8) Motion.- None, Gelatin tube cultures.- The growth extended along the line of in- oculation in white specks, On the surface it was a dry white fluffy errowth. Inclined agar tube culture.- A white granular slimy growth Spreadim outward from a center. Potato tube.- A yellowish white granular growth on the surface, Bouillon tube culture.- The liquid was scarcely cloudy, Starch bouillon cultures.- The liquid was about as cloudy as the bouillon, (12) Cane sugar bouillon culture.- The liquid was slightly cloudy and showed white particles floating in it. Lactose bouillon culture.- The liquid cloudy,with a white feathery floating mass, Glucose bouillon culture.- The liquid was not very much clouded, about the same as the bouillon, Milk tube.- Showed no change. Growth.- Rapid, Acids.- Neither acetic nor lactic acid was found, Behavior to gelatin.- No liquefaction, No 5, Colonies.- The surface colonies had an irregular border,but a clear and distinct outline. The deep colonies were round with an even resular border. They were dark brown at the edge and light brown at the center.(a,Fig 9) Hanging drop.- Tiny bacilli,somewhat longer than wide, Closely connected in a mass,.(bv,Fig 9) Motion.- A quick darting movement. Gelavin tube cultures.- The growth occurred along the entire line of inoculation in white dots, The surface growth was a white soreading one. In clined agar tube culture.- A yellowish white growth spread- ing over the surface, Later the agar turned green, Potato tube cultures,- A brown unewWen growth on the surface, (13) Bouillon tube cultures.- The linuid became green with a white scum on the surface and a white sediment at the bottom. Starch pouillon culture.- The liquid showed very little white scum on the surface,and was less cloudy than the other 6ul- tures, Cane sugar bouillon culture.- The liquid was cloudy with aheavy white swum on the surface,and a white sediment at the bottom. Lactose bouillon culture.- The liquid was cloudy with a heavy white scum on the surface,and a white sediment at the bottom, Glucose bouillon culture.- The liquid was not so cloudy as the lac- tose bouillon and cane sugar bouillon cult’res, It showed a thin white scum on the surface and a small amount of a white sediment at the bottom, Mil¥ tube.- No change visible. Growth.- Very slow. Acids.- Acetic acid was found in very small quantity in the lactose bouillon culture, ‘Lactic acid was found in all except the pouillon,in the following order: starch bouillon, lactose bouillon, glucose bouilion,cane sugar bouillon and milk. The amount found in starch bouillon and lactose bouillon was very small. Behavior to gelatin.- BEiquefies and turns green. No 4.- Mould, Tnis was the same as that in the Sunlight Yeasts:- Gelatin Plate .02 ce. contained 2106 colonies of No 1, 224 colonies of No 2, 78 colonies of No 5 and 1 colony of No 43 making a ratio of 1 colony of No 2 to 9 of No 1; and 1 colony of No 3 to 27 of No l. (14) Specimen 4, Yeast Foam(Dry,aze not known):- .01 grams of the yeast-cmke was suspended in 10° cc, of sterilized water. Gelatin and agar plates of 02 €C.,003 CC.,-04 CC.,.05 cc.,were made. Where sufficiently grown to be studied the plates contained two kinds of colonies and one mould,which were classified as No 1,No 2,MNo 5, Wo 1.- Yeast. Colonies.- Star@shaped with a clear and distinct outline. Cream color,the center being darker than the radiating lines.(a,PFig)) Hanging drop.- Oval,white or coloriess cells,often with one end slightly pointed. They were very large.(b,Fig 16) Gelatin tube culture.- The growth was along the entire line of in- oculation in small white dots. The surface growth was of a heavy white mass, Inclined agar tube culture.- A white feathery growth,thick at the center with thinner lines radiating outwards, Potato tube cultures,- Surface covered with a yellowish white growth. Bouillon tube cultures,.- The liquid was very cloudy, Stareh bouillon culture.- The liquid was very clbudy but not as cloudy as the bouillon, Cane sugar bouillon culture.- The liquid was very cloudy, This gave the most growth of any of the cultures, Lactose bouillon culture.- The liquid was more cloudy than the bouillon, (15) Glucose bouillon culture.- Theliquid was more cloudy than the lactose bouillon but not so much as the cane sugar bouillon, Milk tube.- Showed no change, Growth.- Quite rapid but less so than Specimen No l. Acids.- None of the cultures gave acetic acid reactions,but all exceovt bouillon and stagch gave lactic acid, Lactose bouillon gave the least,then glucose bouillon,cane sugar bouillon and milk, Behavior to gelatin.- No liquefaction. No 2. This colony was the same as colony No 2 of Specimen 3,and gave the same growth. No 3, This mould was the same as in Specimens No 2 and No 3. Gelatin plate .02 cc. contained 2773 colonies of No 1,376 colonies of No 2,and 2 colonies of No 33; making a ratio of 59 colonies of No 1 to 8 of No 2, 0°96 0% 09 oO ' /0