STUDIES ON GROWTH MEASUREMENT AND EVIDENCE FOR A POSSIBLE CARBON DIOXEE REQUékEMENT EN TETRAJ-{YMENA ?YRIFORMES W Thesis {or ”19 Degree of DH. D. MICHIGAN STATE UNIVERSITY Claude Alton Welch 1957 {twists This is to certify that the thesis entitled "Studies on growth measurement and evidence for a possible carbon dioxide requirement in Tetrahymena pyrif‘ormis W." presented by Claude A. Welch has been accepted towards fulfillment of the requirements for Ph. D. degree in 200103 Major professor ’- ' Date February 12, 1957 0-169 Michigan Sta tc University *— a ! I i. s Q Ti “44:.” - a" 1m STUDIES ON GROWTH MEASUREMENT AND EVIDENCE FOR.A POSSIBLE CARBON DIOXIDE REQUIREMENT IN TETRAHYMENA PYRIFORMIS W BY Claude Alton welch AN ABSTRACT Submitted to the School for.Advanced Graduate Studies of Michigan State University of Agriculture and Applied Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department of Zoology ‘Year 1957 Approved (flawed. Claude A. welch ABSTRACT This investigation was undertaken in order to reevaluate several of the growth measuring devices used in micrdbiology and to utilize the findings in an attempt to examine the problem of carbon dioxide fixation in the protoaoan, Tetrahymena. ‘Cultures of Tetrahymena‘pyriformis W'were grown in several types of bactone and chemically defined media. In some‘cases, growth of the populations was estimated by optical density (Klett-Summerson photom- eter), direct cell count (Sedgewick-Rai‘ter chamber) and dry-weight determinations. Direct cell counts were also made on clone cultures grown in Van Tieghem chambers. In some cases the Van Tieghem chambers contained isosmotic solutions of either KOH, KCl or Na acetate as moisteners. The KOH was used to absorb carbon dioxide from the sealed chamber; isosmotic solutions of KCl or Na acetate were used as controls. A modified Van Tieghem chamber was constructed by soldering two small brass tubes into holes drilled into the opposite sides of thelarass ring portion of the chamber. With suitable connections attached to the brass tubes, a constant flow of air can be drawn through the chamber; this permits one to control the gaseous environment of the culture. Optical density measurements were not well correlated to either the direct cell counts or the estimated total mean protoplasmic area. . No attempt was made to measure changes in the opacity of cells. Optical density measurements taken on the supernatant of the culture media showed a gradual increase in Optical density. The increase in Claude A. Welch Abstract, Page 2 Optical density, however, was not proportional to the number of cells. In fact, the supernatant of a culture grown in a bactone enriched (yeast extract) medium gave a lower optical density than a non-enriched bactone culture supernatant even though the maximum growth (cell count) in the enriched medium was three times as high as the non-enriched medium. Neither the red, green nor blue filter was affective in negating the Optical density change which occurred in the supernatant. The supernatant of the chemically defined medium, compared to the bactone media, showed a slower and smaller change in its Optical density. It is recommended that the supernatant Of the culture medium be used as the standardization medium for opticaldensity measurements. At least, it seems advisable to continually check the Optical density of the supernatant for possible variation. Optical density changes which may occur in the supernatant not only can produce inaccurate growth measure- ments, but may provide helpful information concerning metabolism. Good growth curves were obtained from clone cultures grown in the Van Tieghem chambers. This culture method gives accurate cell counts of live cells during the important early phases Of the growth curve. Accurate cell counts can be made at maximum growth by photo- micrography (Kodak XXX, 1/1000 seconds). Growth of Tetrabymena was not suppressed when COz-free air was bubbled through the culture tubes. This confirms earlier investigations. Howevezy..a reduced growth rate was produced in Van Tieghem chambers containing 0.3 molal KOH. More consistent results were Obtained when Coz-free air was passed through Van Tieghem chambers containing single organisms in chemically defined medium. Claude A. welch Abstract, Page 3 Attempts to substitute various carboxylic acids (malic, succinic, fumaric, aspartic, alpha ketoglutaric) for this possible carbon dioxide requirement were unsuccessful. Tests were not made to ascertain whether or not these acids were able to penetrate the cell membrane. Claude Alton welsh candidate for the degree of Doctor of Philosophy Final Examination: February 12, 1957 Dissertation: Studies on Growth Measurement and Evidence for a Possible Carbon Dioxide Requirement in Tetrahymena Pyriformis‘w Outline of Studies: Major subject: Zoology Minor subjects: Physiology, Biochemistry Biographical Items: Born: October 2h, 1921, Flint, Michigan Undergraduate Studies: Highland Park Junior College l9hO-l9h2, Colorado School of Mines l9h3, Michigan State University l9h6- l9h8 Graduate Studies: Michigan State University l9h8-l957 Experience: Member of the United States Army l9h3¢l9h6; Graduate Assistant, Department of Zoology, Michigan State University 1951- 1952; Instructor, Department of Natural Science, Michigan State University 1952-1957 Professional Memberships: Society of Sigma Xi, Society of Protozoologists ii ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. R. A. Fennell, Dr. E. P. Reineke and to Dr. C. A. Hoppert for their guidance and interest during the course of this investigation. iii LIST OF FIGURES LIST OF TABLES. LIST OF PLATES. INTRODUCTION. . MATERIALS AND NETHODS RESULTS . TAbLb OF CONTENTS o o o o O O o o O o o o O O O O o o o 0 0 A. Observations on growth in Tetrahymena . . . . . . . . l. 2. 3. The relationship between the direct cell count, Optical density and the total mean protOplasmic area. . . . . . . . . . . . . . . Klett scale readings of various portions of bactone and bactone enriched media . . . . . . Klett scale readings obtained on the cell culture supernatant using’blue, green and red filters. . . . . . . . . . . . . . . . . . . . DW’WeightSOooooooooooooooo Filters...............o.o The variation of the optical density of the supernatant with various types of media. . . . Bactone medium . . . . . . . . . . . . . . Bactone medium inoculated from a vitamin enriched culture . . . . . . . . . . . . Bactone medium "E“ . . . . . . . . . . . . Bactone medium supplemented with MgC12 . . Chemically defined medium. . . . . . . . . 8. Studies on carbon dioxide requirement in Tetrabymena. 1. 2. 3. h. 5. Aeration flask eXperiments . . . . . . . Desiccator-depression slide experiments. Petri dish-depression slide experiments. Moist-chamber experiments. . . . . . . . Van Tieghem chamber eXperiments. . . . . Isosmotic moisteners . . . . . . . . Experiments testing pH constancy and moistener molalities . . . . . . . . . . iv Page vi viii 3h 31: TABLE OF CONTENTS (Cont.) Page Dubnoff shaking incubator with chemically defined medium . . . . . . . . . . . . . lOl Dubnoff shaking indubator with bactone medium . . . . . . . . . . . . . . . . . 103 Chemically defined medium supplemented with certain dicarboxylic acids. . . . . 103 6. The use of a modified Van Tieghem chamber to remove carbon dioxide from the environment of the cell . . . . . . . . . . . . . . . . . . . 108 Dicarboxylic acids . . . . . . . . . . . . 112 DIWUSSI ON. C O O . O O 0 O O O O O O O O . O O O O O . O O O O O 113 A. Observations on growth in Tetrahymena . . . . . . . . 113 B. Studies on carbon dioxide requirement in Tetrahymena. 126 SUhJflAhY O O O O O O O O O C O O O O O O O O O O O O O O O \ O O O . 13S LITLRATURL CITLD. O O O O O O O O O O O O O I O O 0 O O O O O O O 138 Figure ‘0 IO 11 LIST OF FIGURES Relation between the molality and the product of the molality and the camotic coefficient for KCl, KOH and Na acetate. . . . . . . . . . . . . . . . . . . . . Graphs of the total mean protoplasmic area and the Klett scale reading plotted against time. . . . . . . . . . . Graphs of the number of cells per cubic millimeter and the Klett scale reading plotted against time. . . . . . Graphs of the Klett scale readings for cultures grown in a bactone medium and in an enriched bactone medium plotted againSt time. 0 O O O O O O O O O O O O O O O 0 Graphs of the Klett scale readings of the culture solution using red, green and blue filters plotted against time. Graphs of the corrected Klett scale readings of the culture solution using red, green and blue filters plotted against time. . . . . . . . . . . . . . . . . . Graphs of the Klett scale readings of the culture medium (bactone), the supernatant, and the numerical difference between these two measurements plotted against time . . Graphs of the Klett scale readings of the culture medium (bactone), the supernatant, and the numerical difference between these two measurements plotted against time . . Graphs of the Klett scale readings of the culture medium (bactone medium "E"), the supernatant, and the numerical difference between these two measurements plotted against time. . . . . . . . . . . . . . . . . . Graphs of the Klett scale readings of the culture medium (chemically defined medium), the supernatant, and the numerical difference between these two measurements plotted against time. . . . . . . . . . . . . . . . . . Graphs of the Klett scale readings of several types of culture supernatants, using fresh medium as the standard solution, plotted against time . . . . . . . . Vi Page 21 h3 h5 SO Sh 56 bl 65 69 7h 77 LIST OF FIGURES (Cont.) Figure 12 Graphs of the rate of change in the cell count using various concentrations of KOH as moisteners in the Van Tieghem chambers. . . . . . . . . . . . . . . . 13 Graphs of the rate of change in cell count using KOH, KCl and Na acetate as moisteners in the Van Tieghem chambers. 0 0 o o o o o o O o o o o o o o o o o O 0 1h Graphs of the rate of change in cell counts using Van Tieghem chambers and the Dubnoff Metabolic Shaking Incubator . O C O O O O O O O I O O O O O 0 O I O O 15 Graphs of the rate of change in cell counts using the modified Van Tieghem chamber pictured in Plates 3 and h 0 O O O O O 0 O O O O O O O 0 O O O O O O O O Page 88 98 105 110 Table II III IV VI VII VIII IX XI XII XIII XIV XV XVI XVII LIST OF TABLES Chemically defined media. . . . . . . . . . . . . . . . . Practical osmotic coefficients. . . . . . . . . . . . . . Values of the product of the practical osmotic coeffi- cients and molalities for K01, KOH and Na acetate . . . An example of the data sheet used in Part 1 . . . . . . . An example of the data sheet used in Part 1 . . . . . . . A comparison of data taken on 20 cells with the same data taken on 270 cells. . . . . . . . . . . . . . . . . . . A summary of the data collected in Part 1 ... . . . . . . An interpretation of sane Of the data from Table VII. . . Klett scale readings taken on various portions of two different culture media in Part 2 . . . . . . . . . . . Summary of data collected for Part 3. . . . . . . . . . . A summary of Klett scale readings taken in an experiment using a two per cent bactone medium . . . . . . . . . . A summary Of Klett scale readings taken in an experiment using a two per cent bactone medium . . . . . . . . . . A summary of Klett scale readings taken in an eXperiment using bactone medium "E". . . . . . . . . . . . . . . . A summary of Klett scale readings taken in an experiment using a two per cent bactone medium enriched with h1g0120000000000000000000...... A summary of Klett scale readings obtained in an experiment using a chemically defined medium (Elliott). An interpretation Of some of the data from Table XV . . . Symbols employed in Section B . . . . . . . . . . . . . . viii 20 3‘3 37 38 39 A? A9 60 6h 68 72 73 76 81 Table XVIII XIX XX XXI XXII XXIII XXIV LIST OF TABLES (Cont.) Page Results of experiments in Parts 1, 2 and 3 which involved various methods used in an attempt to remove carbon dioxide from the environment of the cells . . . . . . . 82 Number of organisms per culture using sealed depression Slides. 0 O O O O O O O O O O l O O C O O O O O O O O C 85 Cell counts in Van Tieghem chambers using various con- centrations of XOR as a moistener . . . . . . . . . . . 86 Cell counts using Van Tieghem chambers containing 0.3 molal Na acetate, 0.31 molal KCl and 0.3 molal KOR as mOiSteners O O O O O I O O O O O O O O O O O O O O O 97 Cell counts using Van Tieghem chambers which were placed in the Dubnoff Metabolic Shaking Incubator. Culture medim: 5y ' O O O O O O 0 I O O 0 O O O O O O O O O O O 1()2 Cell counts using Van Tieghem chambers which were placed in the Dubnoff Metabolic Shaking Incubator. Culture medium: 8' . . . . . . . . . . . . . . . . . . . . . . 10h Cell counts using the modified Van Tieghem chamber, as pictured in Plates 3 and h, with various types of culture media . C C O O O O . O O O O O O C O O C . C . C C C C 109 ix LIST or muss Plate Page 1 Apparatus used in the aeration-flask experiments. . . . . . 23 2 Modified Van Tieghem chamber apparatus. . . . . . . . . . . 28 3 Modified Van Tieghem chamber apparatus (detail) . . . . . . 30 h Van Tieghem chambers. . . . . . . . . . . . . . . . . . . . 32 S PhotomicrOgraphs of live cells as they appear in the drOp of a Van Tieghem chamber . . . . . . . . . . . . . . . 90 Fig. 1. Distilled water moistener; and Fig. 2. 0.2 molar KOH moistener. 6 PhotomicrOgraphs of live cells as they appear in the culture drop of a Van Tieghem chamber . . . . . . . . . . . 92 Fig. 1. 0.3 molar KOH moistener; and Fig. 2. 0.5 molar KOH (note tyrosine crystal). 7 Photomicrograph of live cells in the culture drop of a van Tieghem chamber. The organisms are cultured in a bactone medium supplemented with 0.35 per cent agar . . . . . . . . 9h INTRODUCTION The measurement of growth is the measurement of one of the fundamental attributes of microorganisms. As pointed out by Richards (l9hl, p. 51?), the analysis of population growth requires knowledge of the environment, the individuals, and the interactions of each on the other. Furthermore, rarely is a single type of measurement adee ouate to give a picture sufficiently complete for analytical studies. Populations Of Protozoa are usually estimated by using a Sedgewick-Rafter chamber with a Whipple disc in the ocular lens of the microscope (of. Hall gt;§l., 1935). Elliott (1939) attempted to ascertain growth by centrifugation using a hematocrit tube fused to a centrifuge tube.. - The population of pigmented or granular forms of microorganisms may be estimated from the optical density of the culture medium, by means of some device constructed to measure the transmission of light through the suspension. Various types of apparatuses have been devised to do this (of. Richards and Jahn, 1933; Stier gt_gl., l93h3 and Mestri, 1935), but since 1935 some very excellent photoelectric color- imeters have been commercially available. Although these commercial photometers are primarily designed for chemical analysis they can be used as turbidimeters. Monod (1919) has this comment concerning the current use of the photometer as a growth measuring device: The time—honored method of looking at a tube, shaking it, and looking again before writing down a + or O in the lab-book has led to many a great discovery. Its gradual replacement by determinations of "turbity at 16 hours" testifies to technical progress, primarily in the manufacturing and advertising of photoelectric instruments. ‘This technique however is not, prOperly speaking, quantitative, since the quantity measured is not defined. It might be a rate, or a yield, or a combination of both. Richards (19hl, p. 519) points out that optical density depends on the number of organisms present, the distribution of organisms of various sizes, and their metabolic condition (e.g., storage products). Another variable, apparently usually ignored, is that the optical density might also depend upon changes which might occur in the culture supernatant due to the metabolic activity of the cells or'simply due to an unstable type of culture medium. One of the objectives of this investigation is to critically examine several of the methods currentLy used to measure growth of microorganisms. It was hoped that any conclusions drawn concerning the validity of any of the methods chosen for analysis could be‘emplqyed in an attack upon the Specific problem of carbon dioxide fixation in Tetrahymena. Jahn (1935) and Pace and Ireland (19h5) reported that Tetrabymena grew best in Coz-free air when cultured in a 2 per cent bactone medium. 0n the other hand, Hahn (l9hl) has shown that the flagellated protozoans Astasia longs and Polytomella caeca showed a reduced growth rate if the carbon dioxide is constantly removed from the culture medium. Van Niel (l9h2) and Lynch and Calvin (1952) demonstrated a very high incorporation of radioactive carbon dioxide into the carboxyl group of succinic acid by Tetrabymena. In fact, buchanan and Hastings (19h6) state that Tetrahymena possesses one of the most active mechanisms for the assimilation of carbon dioxide of those systems so far studied. Non-photosynthetic (heterotrophic) carbon dioxide fixation by bacteria was clearly demonstrated in theclassical work of‘Wood and werkman (1936). Evans and Slotin (19b0) working with pigeon liver showed that this tissue could synthesize oxalacetate from pyruvate and carbon dioxide. These data were supported by the fact that the rate of synthesis of alpha ketoglutarate in pigeon liver depended on the con- centration of carbon dioxide. Wood, Vennesland and Evans (l9h5) reported that radioactive carbon, from bicarbonate, administered to fasting rats was found in the third and fourth carbons of glucose. Calculations showed about one in every eight carbon atoms of glycogen was derived from the bicarbonate. As pointed out by Buchanan (19b6): “The detailed investigations with isotopic carbon of the utilization of carbon dioxide by bacteria and mammalian cells has removed one of the oldest distinctions'between plant and animal life." In addition to the protozoa mentioned above, heterotrOphic carbon dioxide fixation has been found in the following organisms: (l) in bacteria by Slade (19h2), Gitterman and Knight (1952), Tomlinson and Baker (19514). Abelson 333;. (1952), Stoppani et_,__a__1_. (1955), Bolton m]: (1952); (2) in molds by Foster 933;. (19141), Rockwell and Highberger (1927). Heplar and Tatum (195k); (3) in frogs by Cohen (195h), and Flickinger (195k); (h) in fowl by Donaldson and Marshall (1956); (5) in rats by Delluva and Wilson (l9h6); and (6) in rabbits by Donaldson et a1. (195k). Wood (1951) remarks as follows: Much remains to be done before the full significance of CO fixation can be assessed in metabolism. One of the questions that remains unanswered is whether or not 002 fixation is an essential step in normal heterotrophic metabolism or whether the occurence of fixed CO is due largely to side reactions which are not required for'metabolism. Krebs (1951) has estimated that, for mammalian cells, the amount of carbon dioxide which reeneters metabolism by fixation is less than 10 per cent of that formed. As pointed out by Kidder (1951, p. 395) Tetrahymena (and probably ciliates in general) possess enzyme systems somewhat comparable to those of higher animals. One would suspect, therefore, that if a carbon dioxide requirement does exist for Tetra- hymgng it would not be of the order of magnitude as that found in bacteria and molds. The objectives of this investigation are twofold: (1) to reexamine several of the current methods used for growth measurement in order to evaluate them according to their ability to measure various aspects of growth, and (2) to utilize these growth measuring devices in an attempt to ascertain whether or not carbon dioxide is required for growth in Tetrabymena pyriformis W. ’ “' Mai-1.x" MATERIALS AND METHODS A. NON-CHEMICALLY DLFINLD MEDIA (BACTGNb thIA E) One of the two general culture media that was used in this study is a non-chemically defined medhmlwhose basic constituent is Bacto-Tryptone (Difco Laboratories, Detroit, MiChigan). Bacto-Tryptone, as stated by the Difco Laboratories,contains proteoses, peptones, cystine, tyrosine, tryptOphane, iron, magnesium, calcium, potassium, sodium, chlorine, phosphorous, and sulfur. Fourteen per<3ent of the powder is nitrogen. A one per cent solution of Bacto-Tryptone after fifteen minutes autoclaving has a pH of 7.2. Although a one per cent Bacto-Tryptone solution with no addi- tional metabolites will support a growth of about 180 cells per cubic millimeter for about two weeks, the addition of a buffer and thiamine greatby increases the total number of cells (350 cells per cubic millimeter) as well as the duration of the culture. The bactone medium (hereafter designated as bactone medium E) is essentially that used by Dr. James F. Hogg (personal communication) and consists of the following: Bacto-Tryptone 10.0 grams; yeast extract 0.1 grams; glucose 1.0 grams; KZHPOH 1.0 grams; KHZPOh 1.0 grams; thiamine H01 0.002 mg.; sodium acetate 1.0 grams; and H20 1000 m1. This medium is adjusted to pH 7.2 using 0.1N NaOH and autoclaved at 15 Pounds pressure for 15 minutes. B. CHbMICALLY DEFINED MEDIA The chemically defined media used in this study are those of Elliott gt_al. (195k) and Kidder and Dewey (1951, p. 392). The con- stituents of these media are given in Table I. The vitamins are kept in a stock solution of 25 per cent alcohol in such concentration that one milliliter is used per liter of medium. The stock solution of salts, excluding KHZPOh and K2HP0h, are concentrated so that 50 milli- 'liters of the stock solution will fulfill the salt requirement for one liter of medium. The KHZPOu and K HPOh are kept in separate stock 2 solutions and added separately because these two salts will form an insoluble precipitate if mixed with the other salt stock solution. The dextrose is autoclaved separately as recommended by Kidder (1951). After the pH is adjusted to 7.b using 0.1N NaOH, the chemically defined medium is autoclaved for 15 minutes at 15 pounds pressure. The bactone medium as well as the synthetic media are autoclaved in the vessels which are tolae used in the experiment. Stock cultures are grown in test tubes (20 x 2.5 cm.) held in a vertical position. All vessels containing autoclaved medium which is not to be used immediately, are sealed with Parafilm and stored in the refrigerator. 1. Indirect cell counts. Photoelectric colorimetgy. Growth curves were established using a Klett-Summerson Photoelectric Calorimeter. A blue filter (Klett #h25h00-500 millimicrons) was used as recommended by Elliott (19h9) and, in some cases, the red filter (Klett #6636h0-700 millimicrons) was used as recommended by Kidder (1951, p. 395); additional measurements CHEMICALLY DEFINED MEDIA TABLE I Elliott Medium (micrograms Kidder and Dewey Medium 2c (micrograms Compounds per milliliter) per milliliter) DL Alanine none 1100 L Arginine 150 860 L Aspartic Acid none 1220 Glycine none 100 L Glutamic Acid none 2330 L Histidine 110 h20 DL Isoleucine 100 1260 L Lenoine 70 l9h0 L Lysine 35 1520 DL Iethionine 35 680 L Phenylalanine 100 1000 L Praline none 175h DL Serine 180 lShO DL Threonine 180 - 1760 L Tryptophane 20 2h0 DL Valine 60 1320 Ca pantothenate 0.10 0.50 Nicotinamide 0.10 0.50 Byridoxine HCl 2.00 5.00 Ryridoxal HCl none 0.50 Ryridoxamine HCl none 0.50 Riboflavin 0.1 0. 0 Pterqylglutamic acid 0.01 0. 5 Thiamine H01 1.00 5.00 Biotin none 0.002 Choline chloride none 5.00 Thioctic acida 0.001 0.001 Adenylic acid 25.0 200.0 Cytidylic acid 25.0 250.0 Guaqylic acid 25.0 300.0 Uracil 25.0 100.0 Fe ‘7 0 0.5 none “5533-7330 10.0 11.0.0 ?e(th)2(30h)3-6H20 none 62.5 lnClz‘szO none 1.25 TABLE I - Continued _=- - ‘=- L A“ :- Kidder and Dewey Elliott Medium Medium 2c (micrograms (micrograms Compounds per milliliter) per milliliter) Zn012 none 0.125 Ca012'2H20 none 30.0 Cu012°2H20 0.5 3.0 FeCl '6H20 none 0.75 was? 100.0 500.0 KB POL none 500.0 Zn(N02)2'6H20 5.0 none Dextrose 1000 ' 2500 Sodium acetate 1000 none aProtogen was used in the original medium. bNa DL Thioctate was kindly provided by Dr. E. L. R. Stokstad, Lederle Laboratories, Pearl River, New Yorke 9 were made with a green filter (Klett #5hg500-570 millimicrons). Klett calibrated test tubes were used and were checked for accuracy. In some cases the cultures were grown directLy in the Klett tubes, but in most cases they were grown in 125 milliliter pyrex Erlenmyer flasks and then transferred to the Klett tubes for measurement. After the instrument is adjusted using distilled water, the optical density of the culture is compared to the "blank standard." The "blank standard" in these experiments was either: (a) fresh culture medium inwhich no organisms had ever been grown or (b) the supernatant of the medium in wiich the organisms had been grown. When the "blank standard" was as is stated in (b) above the organisms were first centrifuged at moderate speed for 15 minutes and the supernatant immediately drawn off using a 10 milli- liter syringe. To facilitate the removal of the supernatant a 2 inch length of plastic tubing was fastened to the end of the syringe to be used in place of a needle. The renoval of the supernatant must be carried out immediately upon completion of centrifugation because the organisms will quickly migrate back into the supernatant. In fact, the supernatant was drawn off as soon as the tubes came to a stand still, but before the tubes were removed from the centrifuge. This procedure produced a cell-free supernatant. In some cases the supernatant, after centrifugation, was fil- tered using a Seitz vacuum type filter with a low vacuum. Because of their very elastic cell membrane, Tetrahymena can not be filtered out using ordinary filter paper. All glassware used in this study was cleaned with potassium dichrcmate cleaning solution and well rinsed with large quantities of tap water followed by several final rinsings 10 of distilled water. As reported by Richards (1936), unless all glass- ware cleaned with acid solutions is thoroughly rinsed, serious errors may enter into the experiments in which the glassware is used. For this reason a rack was constructed which held the Erlenmyer flasks in an inverted position over a stream of water directed upward and thus onto the bottom of the flask. In this way, a continuous supply of fresh water is flushed through the inside of the flask. Drydweight determinations. A measured volume of the culture was centrifuged and the supernatant drawn off with a syringe as described in the previous section. The cells were washed with distilled water to remove any remaining medium and recentrifuged. This process was repeated three times to insure the complete removal of medium. The cells were then transferred to a weighing bottle and dried for twenty- four hours in a drying oven at 100°C. The weighing bottles were allowed to cool in a desiccator before final weighings were made. Heating was continued until a constant dry-weight was obtained. The amount of original culture used, of course, depended upon the age of the culture. A three day old culture, for example, required a much larger volume of culture in order to obtain a weighable dry-weight of organisms. Results are always reported as milligrams dry-weight per milliliter of culture. 2, Direct cell count. The Sedgewick-Rafter counting chamber. Direct cell counts can be made using the Sedgewick-Rafter counting chamber along with the Whipple Micrometer. The technique is similar to that used by Hall et a1. (1935) and by Whipple (1927). 11 One milliliter is withdrawn from the growing culture. One to several milliliters, depending upon the age of the culture, of 10 per cent formalin is added to the original one milliliter. The formalin solution thus dilutes the culture as well as kills the cells. One milliliter of the diluted, dead cells is then transferred to the Sedgewick-Rafter counting chamber and the cells allowed to settle to to the bottom. Using a 10x objective lens and a 10x eyepiece, the largest dimensional area circumscribed by a Whipple Micrometer is one square millimeter. Since the Sedgewick-Rafter counting chamber has a depth of one millimeter, the cells present on one square millimeter of the bottom actually represent the cells that have settled out of one cubic millimeter. The Dilution Factor is equal to the sum of the original one milliliter of cells plus the number of milliliters of formalin which were added as a "fixing-dilution" agent. Therefore, the total number of cells per cubic millimeter is equal to the actual count of the cells per cubic millimeter multiplied by the Dilution Factor. The counting chamber holds 1,000 cubic millimeters and 10 individual areas occupying one cubic millimeter each are chosen at random and an average of these 10 areas is used as the cell count prior to multiplication by the Dilution Factor. The Van Tieghem chamber. The Van Tieghem chamber as described by Duggar (1909) is a type of moist-chamber preparation most often used for cultures of fungi. These chambers are also very adaptable to the growth of protozoa and can be used very nicely to obtain direct cell counts for establishing accurate growth curves. Since a thorough account of the procedure for making the Van Tieghem chamber could not 12 be found in the literature, the method had to be worked out by the author. Some of the important steps are given in detail. The main constituents of the Van Tieghem chamber are a micro- scope slide, a cover glass and a metal or glass ring about 10 milli- meters high and 10 millimeters inside diameter. The ring and microscope slide can be sterilized in a Petri dish using dry heat. The ends of the glass ring are then touched to the surface of melted vaseline. This technique produces a neat, even layer of vaseline on both ends of the ring. The ring can be placed on the center of the slide while the vaseline is still liquid thus a good seal is obtained between the ring and the slide. The ring, sealed to the slide, is then returned to the Petri dish and stored ready for use. The cover glasses are very important since they are the objects which actually hold the culture and thus must be perfectly clean and sterile. The cover glasses are allowed to soak in absolute alcohol for several weeks after being cleaned in acid cleaning solution and thoroughly rinsed. They are then individually rinsed, using a small forceps, in separate beakers of absolute alcohol or metasilicate as suggested by White (19511) and allowed to air dry. A very satisfactory rack for drying large quantities of cover glasses can be constructed by tacldng a small coil spring (10 centimeters long and 0.75 centimeters in diameter) which has been stretched beyond its elastic limit (to about 20 centimeters in length) to a 10 inch board. A piece of absorbent paper may be placed between the spring and the board; the cover glasses are then placed between the 100ps of the extended spring. In this way the cover glasses dry quite free of lint and water marks 13 because they are adequately separated from each other. After they are dry, all of the cover glasses are placed in one clean Petri dish or a weighing bottle and dry-heat sterilized. After sterilization it is important that the cover glasses be cooled very slowly. In fact, allowing them to remain in the oven as the oven cools is very satis- factory. If the cover glasses are cooled rapidly, much dust enters the Petri dish, as the volume of air contracts, and deposits on the cover glasses. This dust not only destroys the sterility but inhibits the proper drop formation when the culture is made. If the cover glass contains anything on its surface which gives it a wettable surface it is impossible to form a satisfactory hanging drop culture for the dr0p will tend to spread and to coalesce with the small droplets deposited from the moistening material in the bottom of the chamber. After it has cooled to room temperature, the Petri dish with the enclosed cover enlasses should be kept in a vaseline-sealed desiccator to insure cleanliness and prolong sterility. The cover glasses are resterilized every few days. It is not satisfactory to flame the cover glass just prior to the placing of the culture drop because a wettable surface, presumably from deposited carbon, results. Most of the Van Tieghem chambers used in this study contained hanging drops which possessed only one organism at the beginning of the experiment. The following procedure was found to be the most Satisfactory for isolating single organisms and at the same time main- taining the best conditions for sterility. Five milliliter aliquots of media are autoclaved'in 10 milliliter Erlenmyer flasks. Just prior to setting up an experiment, the 5 milliliter aliquot is inoculated using 1b a flame-sterilized transfer needle in which the wire had been bent to form a loop having a diameter of about 2 millimeters. The following equipment and materials have been found necessary for the setting up of Van Tieghem cultures in quantity: ’ a microscope equipped with a h8 millimeter objective lens and a 10x ocular lens or a 32 millimeter objective lens used with a 6x ocular lens; a sufficient quantity of sterilized units, as mentioned previously, consisting of a Petri dish containing a slide with the glass ring sealed to it by vaseline. The free end of the ring has also been previously coated with vaseline; a supply of clean, sterile cover glasses; a transfer needle containing a loop having a diameter of about 2 millimeters; and a glass ring which has been permanently cemented to a microsc0pe slide. The procedure for setting up the Van Tieghem.culture is as follows. The unit consisting of a glass ring cemented to a slide is grassed through a flame and allowed to cool. A sterile cover glass is then placed on top of the ring and a dr0p of culture medium from the previously inoculated 5 milliliter aliquot is placed on the cover glass Ilsing the small sterile transfer loop. The cover glass is quickly ilrwerted so that the drop extends down into the hollow of the glass ring. Here again, if the cover glasses are not perfectly clean the drOp will spred as the cover glass is inverted. The entire drop can now be examined under the microscope if the proper objective and ocular 1ens combination is used. In this way one can select the drop or reject it before sealing the cover glass to a permanent Van Tieghem chamber. The dr0p, upon this trial inspection, may contain none to several \ 15 organisms depending upon the size of the original inoculum. In general, it was found that if the 5 milliliter aliquot is inoculated by the transfer of a single 2 millimeter loop from a stock culture of maximum growth, the drops withdrawn from the aliquot would stand the best chance of containing only one organisn. This procedure, of course, is best adjusted to meet the needs of the experiment. Usually 5 or 6 dr0ps had to be examined for every one that was selected. This is somewhat time consuming but is necessary if all of the drops are to contain an equal number of organisms. Once the dr0p is selected, the cover glass is then transferred to the Van Tieghem chamber which had been previously prepared. If desirable, a few drops ofa suitable moistener may be added to the bottom of the chamber to insure adequate moisture. Cultures of Tetrahymena can be grown without any additional moistener, but for some purposes the moistener may be desirable. The choice of the moistener is of utmost importance because of the thermal distillation of water which will occur between the moistener and the culture drop. A few drops of the same medium of which the culture drop consists is the most osmotically suitable. However, it is liable to become con- taminated so that the choice of a suitable moistener is best made by examining various salt solutions which contain no nutrients. If it is possible to calculate the ionic strength of the culture media, a 801ution of equal osmotic activity of some non-volatile salt will serve as a good moistener. For non-chemically defined media, such as a bactone medium, the pr0per concentration for the moistener can best be found empirically. This is done most easily be setting up a series of hanging draps of pure medium, containing no organisms, over moisteners 16 of varying concentrations. The diameter of the drop can be measured using an ocular micrometer. The drOp will increase in size if the ionic strength of the moistener is less than that of the culture medium. The drop. will decrease in size if the ionic strength of the moistener is greater than that of culture medium. A moistener which has an ionic strength which is greater than that of the medium.is to be avoided for the withdrawal of water from the culture medium due to distillation has a strong inhibitory effect on the growth of the culture. Moisteners 1which have an ionic strength less than that of the medium lose water to ‘the culture medium thereby diluting it. However, this effect is not inhibitory for excellent growth can be obtained even if distilled water i.s used as a moistener. The problem of carbon dioxide fixation as approached in some ziarts of this particular study, involved the use of ionized moisteners. ITt was necessary that the moisteners chosen, although they might have (zertain chemical properties which were greatly different, must possess identical osmotic properties. Thus in the choice of moisteners one unust carefully consider their Practical Osmotic Coefficients. _The I’ractical Osmotic Coefficient (O) can be obtained by isopiestic measure- nuants which are made in an apparatus, which.in.princip1e, is very similar t4) the Van Tieghem chamber. Robinson and Stokes (1955) describe an iSapiestic measurement as follows: Let X andfi! be two solutions initially at the same temperature, the vapor pressure of X being initially greater than that of‘f and let them be connected by a path through which vapor'can ‘pass. The solvent will distil from solution X to Solution 1, resulting in a cooling of X and a heating of Y from the heat of vaporization generated during the process. Because of these temperature changes the vapor pressure of X decreases and that 17 of Y increases and if perfect thermal insulation could be main— tained between the two solutions, a steady state would be set up with a temperature difference between the two solutions sufficient to equalize the vapor pressures. If thermal conductance is allowed however, for example starting with two solutions each containing one gram of water and sufficient Na and KCl to make each solution O.h molal, the distillation of 61 mg. of water will concentrate the K01 solution to b.260 molal and dilute the NaCl to 3.770 molal at which concentrations the vapor pressures are equal. The Practical Osmotic Coefficients taken from Conway (1952) for KOH, KCl and Na02H302 are given in Table II. The following formula (see Harned and Owen, 1950) expresses the relationship of the molality (m) of the salt to the activity of the water (aw): RT ln aw = ngS (¢m) )0 where P. is the gas constant, T the absolute temperature and C the Practical Osmotic Coefficient. Since it is the activity of the water which must be taken into consideration in the Van Tieghem chamber, the following relationship can be shown for the comparison of two salts: l f” 53“ #1’ mm a" = 2.303 x 55.5 W“) for salt #2, log a' = 1 (O'm') 10 W 20303 X 5505 If aw must equal a'w, which is the requirement for a prOper osmotic control, then: 1 ( > 1 2.303 X 55.5 2-303 X 55.5 and Om O'm' (43W) TABLE II PRACTICAL OSMOTIC COEFFICIENTS (t) (AFTER CONWAY, 1952) A KOH K01 Holality NaCziiJO2 ‘_ ___ 0.1 0.9h0 0.9hh 0.927 0.2 0.939 0.936 0.913 0.3 0.9h5 0.938 0.906 0.h 0.951 0.9hh 0.902 0.5 0.959 0.953 0.899 0.6 0.967 0.962 0.898 0.7 0.977 0.972 0.897 0.8 0.986 0.983 0.897 0.9 0.99h 0.993 0.897 1.0 1.002 1.003 '0.897 19 Therefore, in order to find two solutions which have an equal osmotic effect upon the distillation of water, one must find the molalities at which the value of 01: is the same. Table 111 gives the values of 4), m and Gin for KOH, K01 and 151302I‘1302 used in these experiments. The best way to find the proper molalities at which two salts have equal water activity is to plot a graph of the molality versus the product of the molality and the practical osmotic coefficient. This relationship is given in Figure 1. One can see from this graph, for example, that a 0.3 molal solution of KOH would have the same osmotic effect as a 0.31 molal solution of KCl. D. STUDIES ON CARBON DIOXIDE YBQUIREMENT 1_L Aeration flask emeriments. The apparatus as pictured in Plate 1 was used in several ex[Deriments in an attempt to remove carbon dioxide from the culture me(iii-mu. Atmospheric air was bubbled, using a vacuum system, first through a 20 per cent row solution and then through a tube of 8-20 mesh Ascarite before entering the medium itself. Fritted gas dispersion chinders were used in the culture medium as well as in the KGB solu- tions in order to decrease the bubble size and thereby increase the absoli‘ption area. The inlet and outlet for air entering the 20 x 2.5 c‘i'v‘n‘tilneter test tubes used as culture tubes contained sterile cotton. The Culture tubes with cotton filled drying tubes attached were auto- claVed as a unit to insure sterile conditions. Air for the control 1mhes was bubbled through water instead of XOR. TABLE III VALUES OF THh PRODUCT or THE PRACTICAL OSHOTIC COEFFICIENT (0) AND MOLALITIES FOR KCl, KOH AND Na02H302 1010211302 x014 x01 lolality (m) i in «b n g ch in 0.1 0.9t0 0.09h0 0.9th 0.09uh3 0.927 0.0927 0.2 0.939 0.1878 0.936 0.1872 0.913 0.1826 0.3 1 0.9hs 0.2835 0.938 1 0.281h 0.906 0.2718 0.h ! 0.951 0.380h 0.9uh E 0.3776‘ 0.902 0.3608 0-5 ‘ 0.959 0-h795 0-953 ’ 0oh765 0.899 0ohh95 0.6 l! 0.96? 0.5902 ‘ 0.898 0.5388 0.962-i0.5772 L..- —. 20 FI GURE 1 The relation between the molality and the product of the molality and the osmotic coefficient for KCl, KOH and NaC2H302. 21 9‘0 0¢.0 and 0nd $34.62 x e 0N0 0N0 9.0 0.0 q 1 4 d ..0 «.0 t0 0.0 ALIWV‘IOW 23 PLATE 1 The apparatus used in the aeration-flask eXperiments. Legend: 1. Tubing leading to the vacuum pump; 2. culture tubes (control); 3. gas washing bottles; h. Ascarite tube; and 5. culture tubes (experimental). - u «r- C513? ~vI.‘ Rj‘p, 4 ‘14 .o v_ a .... I. "A A u A.‘ ‘ n v.. . \‘V- I a ‘K ‘ H| t ' . II‘ . t‘L 2S 2. Desiccator-depression slide experiments. Another method used to remove carbon dioxide involved a desic- cator (l6 centimeter diameter) in which a depression slide was placed. Several small vessels containing 20 per cent KOH were placed in a sealed desiccator and a depression slide containing a drop of inoculated medium was placed between the vessels containing the XOR. At the end of four days the depression slide was removed from theldesiccator and the organisms were counted under the microSCOpe. 3. Petri dish-depression slide experiments. Six or seven microscope slides are stacked horizontally in a Petri dish, the t0p slide being a depression slide. A 20 per cent solution of KOH is pipetted into the space around the slides and then the Petriciish cover is sealed with vaseline on the inside,or a layer of Parafilm is wrapped around the outside. The culturecirop located within the depression of the uppermost slide can be viewed through the Petri dish cover with a h8 millimeter objective lens and a count of the organisms taken. L. Van Tieghem chamber experiments. The Van Tieghem chambers as described in the previous section were used in the carbon dioxide experiments. An attempt was made to remove all carbon dioxide by placing the proper molalities of KOH in the bottom of the chamber. Slight modifications were made by increas- ing the volume of XOR until it nearly filled the chamber in which case the hanging drOp under the cover glass occupied the air space formed by the meniscus of the KOH solution. In other cases, when onxy a few drape 26 of moistener were used, 8 x 12 millimeter pieces of filter paper were inserted in a vertical position within the chamber to facilitate the absorption of the carbon dioxide by the KGB. Several experiments were conducted in which the Van Tieghem chambers with added filter paper were placed in a Dubnoff Metabolic Shaking Incubator. The incubator portion of the apparatus was not utilized because room temperature was sufficient. The cultures, however, were kept covered and the apparatus set at a rate producing one complete oscillation per second. A basic alteration of the Van Tieghem chamber is pictured in Plate b. A brass ring with a 10 millimeter inside diameter and a height of 10 millimeters is used for the main chamber. Two small holes were drilled on Opposing sides of the ring and a one centimeter piece of brass tubing having a diameter of 2 millimeters was soldered into each hole. -This chamber, sealed to a microscOpe slide with vaseline, can be clamped to a microscOpe using two Double Clamps #20h (Harvard Apparatus Company). Using a small vacuum and the necessary hose connections the air can be made to pass through the chamber after the cover slip is sealed in place with vaseline. To inhibit desiccation of thetirop due to the continual passage of air, the air is drawn through distilled water before it enters the chamber and a few drops of moistener are placed in the bottom of the chamber at the beginning of the experiment. To remove the carbon dioxide from the incoming air, the air is bubbled through two Erlenmyer flasks containing 100 cubic centimeters of 20 per cent KOH and then through a 6 inch tube of 8-20 mesh Ascarite. A tube containing sterile cotton is placed at the incoming end of the gas chain. The air, after passing through the Ascarite, is bubbled through 27 distilled water before it enters the chamber. This apparatus is pic- tured in Plates 2 and 3. The passage of air can be controlled by a hose clamp placed between the chamber and the vacuum pump. This procedure enables one to carefully control the rate of air flow so that the experiments can be well controlled. The rate of air flow for these experiments was usually held at about 160 bubbles per minute. This rate is equivalent to about 20 cubic centimeters per minute. If the volume of the chamber, with a few draps of water in the bottom, is estimated at about 0.5 cubic centimeters, this rate of air flow would change the air in the chamber once every 1.5 seconds. Plate b shows the usual type of Van Tieghem chamber on the left and the modified chamber containing the adaptation for continual air passage on the right. 28 PLATE 2 Modified Van Tieghem chamber apparatus. Legend: 1. 2. 3. h. Tubing which leads to vacuum pump; clamp used to regulate air flow; Van Tieghem chamber; and Ascarite tube. PLATE 3 Mod‘ lfied Van Tie ghe m chamber appar atus ( deta' ll). 30 32 PLATE h The Van Tieghem chambers. Legend: 1. 2. Ordinary Van Tieghem chamber consisting of a glass ring sealed to a microscope slide with vaseline; and modified Van Tieghem chamber containing small tubes soldered to a brass ring. a! g . 2‘4 hhSULTS A. QBSndVATIONS ON GROWTH IN TETRAHYMENA 1. The relationship between the direct cell count, optical density and the total mean protgplasmic area. Organisms used in this experiment were cultured in a one per cent bactone medium, and in thiamine enriched (0.2 gamma per milliliter) bactone medium. Estimates of growth were made with a Sedgewick—Rafter counting chamber, and a Klett—Summerson photoelectric colorimeter fitted with a blue filter. An ocular micrometer was used for ascertaining length and width measurements. Forcietails concerning procedures for making measurements and recording data, consult Tables IV and V. Table VI suggests that the error in the various measurements (mean length, mean width, etc.) varied from about 1J5 to 6.h per cent when the number of cells measured was decreased from 270 to 20. Table VII summarizes the data obtained in this series of experiments. It is evident from Table VII that a maximum of 192 cells per cubic millimeter was obtained in the bactone medium (11111 hour culture) which was equivalent to a Klett scale reading of 2h.5. 0n the other Zhand, the maximum cell count using the bactone enriched medium (168 hours) increased to 273 cells per cubic millimeter which was equivalent ‘to a Klett scale reading of 55.5. Thus, the ratio between maximum 2122333 of cells in the bactone enriched medium (273 cells per cubic 35 TABLE IV AN EXAMPLE OF THE DATA SHEET USED IN PART 1 .: —_‘ -; _Experiment. . . #1 Culture Media . 1% bactone Date. . . . . . 8/15 Hour. . . . . . 10 a.m. Total Hours . . 73 A. Direct Cell Count C. Cell Dimensions Trial Sample Sample Miss“ ...ii. #2 2211 Lease.“ 1mm“ 1 62 85 1 hS 21 2 66 7h 2 3h 18 3 70 07 3 ht 20 h 68 88 b h6 21 5 79 75 5 83 17 6 6h 105 6 50 23 7 52 67 7 uh 20 8 69 85 8 h? 16 9 76 89 . 9 53 19 10 7h 78 10 51 22 11 SS 23 Mean 68 81 12 h2 17 General mean 714 13 1:6 20 Dilution factorb 2 1h hh 21 Cells/mm 1&8 15 h2 21 16 hS 20 17 56 25 18 56 23 19 h2 21 B. Photometric Measurements 20 53 20 Sample Sample lean h6.9 20.b #1 #2 Calibration Scale Reading 21 2h factore 0.9h 0.9h ' Meanf(microns) hh.0 19.1 Mean scale reading 22.5 Area per cell 2,60h Optical densityc 0.0h5 Areag per mm3 385,392 aTrial number: Ten microscopic fields were chosen from each of the‘two samples. bDilution factor: This term was described under the discussion °f the Sedgewick-Rafter chamber in the- previous section. Specifically, t is the number which when multiplied by the number of cells counted '11]- Sive the corrected total of cells per cubic millimeter. 36 TABLE IV - Continued chtical densigx: The Optical density equals 0.002 multiplied by the scale reading. dLength and width measurements: These values are expressed in units of the ocular micrometer. The calibration factor is discussed in footnote "e" below. eCalibration factor: One micrometer unit equals 0.0009h millimeters or 0.9B microns. (Area of cell: On the assumption that the area of the cell can best be calculated as the area of an ellipse, the "protoplasmic area" of the cell was calculated as follows: Area of the cell :11ab, where "a" and "b" are the semiaxes of the elipse, or in this case, the length and width of the cell. ghrea per mm3: Mean area per cell multiplied by the total cells per cubic millimeter will give the total protoplasmic area in square microns per cubic millimeter. TABLE V AN EXAMPLE OF THE DATA SHEET USED IN PART 18 37 EXperiment. . #1 Culture Media Date. . . . . . 8/20 Hours a o e o o 10 acme Total Hours . . 193 1% bactone plus thiamine (0.2 gamma per milliliter) A. Direct Cell Count Trial Sample Sample #2 Number #1 1 97 9h 2 89 81 3 96 66 h 91 60 S 107 102 6 78 81 7 8h 89 8 113 77 9 85 130 10 7h 101 Mean 91.L 88.1 General mean 89 . 7 Dilution factor 3 Cells/mm3 269 B. Photanetric Measurements Sample Sample #1 #2 Scale reading 50 53 Mean scale reading 51-5 Optical density 0.103 K C. Cell Dimensions 0811 Length l 52 2 h2 3 h6 b L6 5 51 6 36 7 56 8 h9 9 h8 ; 10 83 I 11 L3 ' 12 38 13 h2 1h Sb 15 h? 16 51 17 hS ; 18 h? t 19 to E 20 h3 i Mean h5.9 : Calibration ‘ factor 0.9h Mean (microns) h3-1 Area per cell Area per mm m 2h 18 22 2h 25 17 25 2b 2b 20 16 19 20 2h 22 26 21 23 2o 23 21.8 0.9b 20.5 29739 7369791 8Explanations for the terms used in this table can be found in the footnotes to Table IV. 38 TABLE VI A COMPARISON OF DATA TAKEN ON 20 CELLS WITH THE SAME DATA TAKEN ON 270 CELLS Per cent 20 Cells 270 Cells Difference A.— lean length h7.8 hh.9 6.h lean width 22.2 21.2 h.7 lean area 1067.7 963.5 h.5 Mean EZL‘EEE 2.16 2.13 1.5 Width 39 .>H 64969 on :we epocpoom memo .46664444443 you 6356M m.ov omaamana £643.66364hco :6465466 ocouomn RH <9 .Aumaws mo madam mm 6946 scopahupuoaomm amaw H 1.6.4V :oapsaom onopahuuuopomm R4 46 664.6 6.4m m.64 466.6 6.~m «A 664 W 666.6 p.64 6.6m 666.6 6.6m 6m4 66m m mm6.64 646.6 6.66 66 6mm 664.6 4.46 6.44 666.6 6.64 644 64m 6m6.6 6.64 6.64 4m6.6 6.64 64 64m mam.6 6.64 6.6m 664.6 6.46 6mm 466 646.6 6.m4 m.m~ 446.6 6.64 44 466 ~6~.6 6.64 4.m4 664.6 6.46 m6~ 646 644.6 .e.m4 6.4m 446.6 6.64 66 646 ~6e.6 6.64 6.64 464.6 6.66 mam 64m 4m4.64 4.64 4.6m NM6.6 6.64 mm 646 6me.6 m.6~ 4.m4 464.6 6.4m 666 «64 6m4.6 n.64 6.64 6m6.6 6.64 464 664 666.6 6.66 6.44 444.6 6.6m new 664 666.6 n.64 6.mm 646.6 6.46 464 664 mma.6 m.- 6.64 664.6 6.4m 666 444 6mm.6 4.64 4.64 646.6 6.46 604 444 6ma.6 6.66 4.64 666.6 6.44 mmm 66 m44.6 4.64 6.m4 466.6 m.e~ em4 66 666.6 n.6m n.64 466.6 m.mm 646 me mmm.6 4.64 6.44 446.6 6.66 _ 644 me as wca as .66: :4 mas mew mas .mum :4 mom £9645 newcoq muwmcoa venom men caspaso you :66“: summon wimcoa lemma mom caspaso 66666 4466 4466 46646m6 64666 64466 66 666 66664 4466 4466 4664666 64666 64466 66 66¢ Docwfidg 4.5%... 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S w on 1 3 8 3 m 96 N 9 00 cm t7 the standard, C(FS); and (e) the scale reading of the filtered superna- tant using fresh media as the standard, F30“. All Klett scale readings were obtained using the blue filter. The results are summarized in Table IX. Figure )4 shows the rate of change in the optical density Of the fractions described in (c) and (d) above. Data presented in the preceding section indicated that the Optical density was affected by factors other than cell count and total protoplasmic area. It was decided that an examination should be made of the Optical density changes which might occur other than that due to the presence Of cells. An examination of Table IX clearly indicates that the Optical density of the filtered supernatant (F3), from all ages of bactone and enriched bactone cultures, does indeed change with time. For example, light absorption by materials in the supernatant accounted for approximately one half of the optical density of 72 hour bactone enriched cultures. Furthermore, this "light-absorbing factor" in the supernatant was brought about by the presence of the cells because as can be seen by a comparison of fresh media (l) with the filtered fresh media (FM), practically no change occurred in fresh , media even though it had been eXposed to the air for the same number of hours. At 168 hours after inoculation, the Klett scale reading for the Don-enriched bactone culture medium, usingfresh medium as the St'am-iar'd, C“), was 58 and the scale reading of the supernatant, F5“), was 22. Thus, the scale reading due to the cells alone, C(FS), was 36. LikeWise, the maximal growth of the culture, C(M), appeared to occur at 168 hOurs in non-enriched bactone medium, but if the supernatant was used as the standard, 0””, maximal growth occurred at 1% hours. ha The previous discussion seems to indicate that in addition to the variables found in Part 1, namely, the cell count and total protoplasmic area, other factors can affect the Optical density of the culture medium. An additional "light absorbing factor," which is present in the supernatant, must be taken into consideration if one wishes to use Optical density measurements for estimating growth of protozoa. It appears that the best way to minimize the effect of the supernatant is to measure the optical density of the culture solution using the supernatant as the standard solution. 3. Klett scale readings Obtained on the cell culture supernatant using blue, green and red filters. This eXperiment was planned to see if perhaps a suitable filter, other than blue, might possibly negate the Optical effect of the super- natant. A 2 per cent bactone medium enriched with thiamine (0.2 gamma per milliliter) was used for the culture medium. The data from this experiment is presented in Table X and Figures 5 and 6. Dry weights. The determination of dry-weights was unsatisfactory. Table X shows a gradual increase in dry-weight which could be accounted for by the accumulation of dead cells. The dry-weight determination Shows little correlation with either the direct cell count or the Klett scale reading. For example, at 72 hours there were 2140 cells per cubic millimeter with a total dry weight of 0.56 milligrams per milliliter; at 1111; hours there were 282 cells per cubic millimeter, and a dry- ‘eight of 1.75 milligrams per milliliter. Thus, in 72 hours the number °f cells per cubic millimeter increased by about 17 per cent and the drY'Weight by over 200 per cent. h? .TABLE IX KLETT SCALE READING TAKEN ON VARIOUS PORTIONS OF Two DIFFERENT CULTURE MEDIA IN PART 2a Total C(u) C(FI) M(FM) C(FS) FS(M) 3°“rs B EB B EB B EB B EB B EB 72 o 23 0 2h 0 -1 0 ll 0 12 9o u2 so u2 50 o o ’ 32 39 11 9 1th h8 78 h9 79 -1 +1 h3 63 6 1h 168 58 97 S9 98 -1 -1 36 81 22 18 216 h2 82 bl 81 +1 -1 3h 7h 7 13 26b 22 88 21 86 +1 -2 7 68 1h 18 ~— 8Definitions of symbols used in Table IX: B: One per cent bactone medium; EB: one per cent bactone medium enriched with thiamine HCl (0.2 gamma/ml.); C: culture medium containing cells; M: fresh culture medium in which cells have never been grown; FM: filtered fresh culture medium in which cells have never been grown; FS: filtered supernatant in which the cells have been growing; and C(n): this symbol means that a Klett scale reading is taken on C (the culture medium containing cells) using I (fresh culzur m di ) 3 he standa solution. C F“ If“?! (RPS and F5 I were comparisons made n a similar manner. E‘I GU85 h Graphs of the Klett scale readings for cultures grown in a bactone medium and in an enriched bactone medium plotted against time. See footnotes to Table IX for definitions of symbols. SO mmao: onN CON On. 00. . \ N /oy /\ in \\ .. o .6336 0:203 £9306 1 00 II. 1 l 23 2.562: 2.203 “3&0; 2.562: 2.292. 35.20 ”3.3010 85:3... 2.292. 35:5 "550.. 2.33 l 0. ON on o¢ SNIOVBH TWOS 1131)! OnN CON 9.50: on. ? .533... 2.203 "fin—vole E... .6390... 2.203 .350... 2.3.3... 2.203 250:5 .8...an .633... 2.203 cop-0.2m $2.306 2.000.. on o¢ SNIOVBH BWVOS 1.1.31)! 52 Filters. The Klett scale readings suggest that: (a) the fresh culture medium was quite stable and if a change did occur it was one which produced a decrease in light absorption instead of an increase; (b) none of the three filters was selective for cells alone because all filters showed an increase in the optical density of the supernatant with time; and (c) at the beginning of the culture growth it appeared as though the supernatant had actually lost some Optically absorbent material which was sensitive to the blue and green filter. The maximum cell count of 282 cells per cubic millimeter was reached at lhh hours. At this time the scale reading (using the blue filter) of the culture, when fresh media was used as the standard, C(M), had increased to 66, while the scale reading of the supernatant, 5(M), ind increased to 18. Thus about 30 per cent of the reading, C(M), was due to the optical density of the supernatant. At the same time (lhh hours) using the green filter it can be calculated that 5 per cent of the Optical density of the cultre medium, C(m), was due to the optical density of the supernatant. Likewise, using the red filter, 20 per cent of the Optical density of the culture, C(M), was due to the Optical density of the supernatant. It would seem from the observations cited above that a green filter provided the most accurate Klett scale reading for the measure- ment of growth. §;_ The variation of the optical density of the supernatant with various likes of media. The following experiment was carried out as part of a plan for ascertaining whether or not the "light absorbing factor" in the TABLE X SUMMARY or DATA COLLLCTED FOR PART 38 53 Direct Total Cell Photometric Measurements DtY gaunt L__ A_ AAA W?ight Hours Cells V mgms. per mm3) Filter “(3) C(u) 5(l) C(u) - 5(M) per ml.) Blue 183 0 -11 11 2h 2 Green 53 0 - 9 , 9 0 _, A Red 7.5 h 2.5 1.5 AA__ . Blue 183 50 0 50 72 2u0 Green 50 3s 0 35 0.56 Red 7 23 2-5 25.5 Blue 183 66 18 h8 lhh 282 Green us So 10 to 1.75 Red h 37 8 29 Blue :181 68 18 so 168 276 Green h6 h} 10 33 1.95 Red 6 30 8 22 Blue 183 h8 19 29 192 225 Green hS 28 8 20 1.83 Red 6 22 8 1h Blue 180 hh 27 17 216 85 Green hh 25 10 15 2.3? Red 6 g 15 5 10 Blue 179 hh 27 17 2h0 72 Green hS 25 9.5 15.5 2.61 Red 5 1h 6 8 afleaning of symbols used: Fresh media in which cells have not been grown; distilled water; culture media containing cells; supernatant, after centrifugation, of culture l: B: C: S: M(B): media in which cells have been grown; and Klett scale reading of fresh media using distilled water as a standard. Sh FIGURE 5 Graphs of the Klett scale readings of the culture solution using red,’ green and blue filters plotted against time. See footnotes to Table X for definitions of symbols. § 2 8 CELLS PER CUBIC MILLIMETER O D N mmaoz OnN nNN CON at on. nu. CO. on on nu ’l.’ /. .. \ l 0 /\v ole e/ I b . .20 .2... ......a O . 2... .350 :25 50.0.6 .260 :2... 2.3... 22082:... 0.3..0 3 $00.0 2.3... O O 8 n N 9NIOV38 BWVOS 1131)! O I) FI GUIE 6 Graphs of the corrected Klett scale readings Of the culture solution using red, green and blue filters plotted against time. See footnotes to Table X for definitions of symbols. CELLS PER CUBIC MILLIMETER 00. CON manor. com oNN 8w 3.. on. an. 00. oh . a . . j 4 . o/o ..zmAxB .2... e26 o / 2.0 .Gsmusen. :02: c029.» .l. .3230 .2... 2....-. ..0.0E_...E 0.2.0 .2. 2.00.6 a 2.000.. O . O r $333948§8 N I) SNIOVBU 31VOS .1131)! 58 supernatant was independent of or related to the constituents of the medium, e.g., proteoses, amino acids, etc. If this factor were found to be a constant and consistent quantity, then the scale readings could always be adjusted to give a more correct growth measurement. The data collected in this series of eXperiments were the following: (1) the Klett scale reading of the fresh media using distilled water as the standard, “(8); (2) the scale reading of the cell culture using fresh medium as the standard, C(M); (3) the scale reading of the supernatant using fresh media as the standard, 5(M); and (h) the arithmetic differ- ence between the scale reading for (2) minus the scale reading for (1), i.e., C(M) - 5(M). All Klett scale readings were taken using a blue filter. In this series of experiments the cell culture supernatant was not filtered because it was found that a clear supernatant could be obtained by centrifugation and a rapid drawing-off of the supernatant by way of a syringe (see "Materials and Methods"). Bactone medium. A 2 per cent bactone medium was used for the Culture medium and the flasks were inoculated with cells which had been maintained in 2 per cent bactone medium for at least one year. The data for this experiment are summarized in Table XI and Figure 7. It is interesting to note that 1-h6 hours subsequent to inocula- tion of the cultures, the Optical density was due to changes in the Supernatant. Microscopic examination of the supernatant revealed neither cells nor particulate matter. It was also evident that the scale readings of the culture media, C(m), did not portray the usual tYPe Of growth curve. On the other hand, if the scale reading of the supernatant was subtracted from the scale reading of the culture medium, 59 one Obtained a growth curve, as shown in Figure 7, which resembled the growth curve obtained by making direct cell counts. Figure 7 also shows that the graph of the Optical densities of the supernatant is nearly a straight line, and possibly indicative Of a direct prOpor- tionality of some kind. This is unusual in that one would expect the "light absorbing factor" in the supernatant to be related to the cell culture growth curve and thus should portray a somewhat logarithmic nature. Bactone medium inoculated from a vitamin enriched culture. This experiment was essentially identical to the previous experiment. The same medium (2 per cent bactone) was used and the scale readings were obtained using the same filter. The cells used for inoculation had been maintained by Dr. R. A. Fennell of the Michigan State Univer- Sity Zoology Department, on a vitamin enriched bactone medium for nEarly 12 months prior to this experiment. The question arose as to Whether or not the transfer of organisms from a vitamin enriched bactone medium to a bactone medium would modify the metabolism of the protozoa and as a consequence alter the Optical density of the supernatant. The data obtained in this experiment are given in Table XII and Figure 8. Table XII shows essentially the same results found in the PPeVious experiment except for a slightly enhanced growth curve which was probably attributable to the culturing of the organisms in the v11i'iitnin enriched medium prior to the transfer to the experimental Solutions. It is evident in the table that for the first 21. hours, the opt1981 density of the culture was essentially the same as the optical denSity of the supernatant. Thus the photometric readings appear to TABLE XI A SUMMARY OF KLETT SCALE READINGS TAKEN IN AN EXPERIMENT USING A TWO PER CENT BACTONE MEDIUMa A 333:. ..(8) cm sun can _ Sm) (Hours) 21 183 5 5 o 27 183 6 6 0 b6 183 9 9 O 56 182 23 o 17 71 182 hh 10 3h 81 182 60 12 hS 93 182 68 15 53 99 182 72 17 55 116 182 . 63 17 h6 122 182 61 20 b1 lhO 182 S6 26 30 1h6 182 SS 27 28 16h 182 h9 32 17 196 182{ 57 h3 it 262 g 182 67 60 7 aSee footnotes t Table X for t e definitions of symbols I B): ‘00” and S M). 60 61 FI SURE. 7 Graphs of the Klett scale readings of the culture medium (bactone), the supernatant, and the numerical difference between these two measure- ments plotted against time. See footnotes to Table X for definitions of symbols. mmDOI OmN O¢N ONN OON Om. OO. O! ON. 00. ON Om O¢ ON O . _ a a _ a _ a _ a _ in a 4 Q/ \O\G\d.d\l__ O- QIV V\ / \l‘ \ 1 ON ...» 1 on / v I O¢ ... \o I On \ \o/o /qs\ \ \0 0/0 o .. ow /O asvafivovud 1 Oh one m - .33 . .. 8 .2506 wcoooq : om 9N|OV38 BWVOS 11.31)! 63 be a measure of materials in the supernatant and not a measurement of growth of organisms. The second column in Table XII shows a relation- ship between optical density and age of the fresh bactone solution, i.e., as the age of the fresh medium increased there was a tendency for the optical density to decrease. It is evident in column four that the optical density of the supernatant increased from about 9 in 12 hour cultures to about 73 in 328 hour cultures. The graph of the scale readings of the supernatant, 50‘), again appears to be a straight line function of some unknown factor. Bactone medium "E". The highly nutritional medium designated as bactone medium "E" (see "Materials and Methods," Part A) was used in this eXperiment. This medium is well buffered in addition to the added growth factors present in yeast extract. In this eXperiment, as well as in all previous experiments, the flasks were inoculated from a stock culture with a bacteriological transfer loop. In allecperiments, the final concentration after inoculation came to about one cell. per cubic millimeter. The data are presented in Table XIII and the graphs of the various scale readings are given in Figure 9. One of the most striking differences between this experiment afid those cited in earlier sections was the lack of an increase in the optiCal density of the supernatant prior to cellular accumulation. Although the maximum growth was much higher than for a non-enriched 2 per cent bactone medium, the lag phase in the growth curve was very Similar as can be seen in Figure 9. The increase in the scale reading of the supernatant was obvious but, surprisingly, was a somewhat Slower- increase even though the total growth was over three times as TABLE XII a summaax or KLETT state BLADINGS TAKEN IN AN exetniuhNT USING A Two Pea CENT BACTONb Msoiuaa ___— 328 253,3; 77(8) cm 500 Cm , 56:) (Hours) 12 176 9 9 o 25 176 11 11 o h8 176 9 6 3 60 176 ‘ h? 13 36 73 176 1 8b 15 69 91 176 7 100 18 82 100 176 98 18 1 80 116 172 98 26 72 125 7 170 98 g 33 65 1&3 i 170 95 E 37 i 58 161 E 170 85 g ho ‘ 55 185 l 170 72 i 39 33 209 l 169 . 67 i h? ‘* 20 282 l 169 3 77 I 61. 1! 16 j 7 169 j 81 g 73 i bun—wk aSee footnotes to Table X. f r the definitions of the symbols n B , c(“ and 5(M). FIGURE 8 Graphs of the Klett scale readings of the culture medium (bactone), the supernatant and the numerical difference between these two measurements plotted against time. See footnote to Table X for definitions of symbol 5. ago: OtnONm OOnOON OON O¢N ONNOON 8. OO. O! ON. OO. 00 OO 0.? q . d J - + d _ ~o~ ~v 9 8 8 9 0 ID 8 omovaa arms 1131» O 8 7» 8 § ‘ 67 great in bactone medium "E". For example, from Table XIII it can be seen that at maximal growth the Klett scale reading for the culture, C(M), and for the supernatant, 8(M), was 3L8 and 17, respectively. Table XII shows a Klett scale reading for maximal growth of 100 for the culture, CW), and 18 for the supernatant, SW). It is interesting to note that although the maximal growth of the enriched bactone culture was over three times that in the non-enriched culture, the Optical density of the supernatant in each case was about the same. Bactone medium supplemented with ”3012' This experiment was undertaken to see if MgC12 would affect the nutritional balance in any way which could be detected by a photometric analysis. The medium used was a 2 per cent bactone medium enriched with MgC12 (1.0 milligram per milliliter). The same type of measurements were made as were obtained in the previous three experiments. The results are summarized in Table XIV. , The magnesium seemed to produce no particular effect. The scale readings were essentially similar to results obtained in 2 per cent, bactone medium. In both cases, comparing Table XIV to Table XII, the maXimal scale reading of the culture, Cm), was reached at about 90 hours subsequent to inoculation and at this time the scale readings of the Supernatant, 50‘), were nearly equal. Ehemicaliy defined medium. This eXperiment utilized a chemi- “nY defined medium (see "Materials and Methods," Part B, Elliott's Hedi“) ° The data are summarized in Table XV and Figure 10 shows the graphs or the scale readings taken on the culture medium and the su ' perm‘lltarit. as well as a graph of the difference between these two TABLE XIII A SUMMARY OF THE KLETT SCALE READINGS TAKEN IN AN EXPERIMbNT USING BACTONL MEDIUM "En“ :23: “(3) col) Sm Coo .. 500' 16 } 1ho 19 5 1b 2b 1&1 2b 7 17 bl 1ho 27 11 16 ha 139 30 9 21 67 139 159 11 lh8 112 139 ' 325 l 15 310 121 139 3&2 16 326 136 139 E 3u7 } 17 330 1th 139 E 3&8 17 331 170 139 g 3&6 18 328 193 139 3h5 21 32h 2&9 139 lhh 38 106 301 139 * 1&1 ' 72 69 397 139 7 131 76 65 aSee footnotes t definitions of symbols I {’8 Table X for the ), 0(8) and 5(M). 68 69 F1 (Mini 9 Graphs of the Klett scale readings of the culture medium (bactone medium "E"), the supernatant and the numerical difference between these two measurements plotted against time. See footnotes to Table X for the definitions of symbols. fl .fcmép.-. see user. 1.366 n:...4 [On 3 8 ONIOVBH TWOS 11.31)! 1 OON é 71 readings. Table XVI shows an interpretation Of some Of the data in Table XV. A direct cell count was made using the Sedgewick-Rafter counting chamber. Table XV, column two, shows that the chemically defined medium was stable. The optical density was very low compared to the bactone medium because only the essential metabolites were included in the Chemically defined medium. Also, the large molecules of the proteoses and peptones present in the bactone medium are not present, which would - probably account for the lower Optical density. In this experiment we do not find the supernatant accumulating optical density as rapidly as in the bactone medium. For example, Table XV shows that at 210 hours the scale reading of the supernatant, 5““, was only about 17, whereas the scale reading of the supernatant, 30“, of a 2 per cent bactone culture (Table XII) at 209 hours was 67. In fact, the chemically defined medium was the onlj,r culture medium used so far in which the cu“mire medium showed a scale reading before one was found for the Supernatant. The graph of the rate Of change for the scale reading of the supernatant found in Figure 10 again reveals a possible straight line function. When this graph is compared to the other supernatant graphs, as is done in Figure 11, one can see that the Optical density of the Slipernatant of the chemically defined medium is considerably less than when the bactone media were used. Figure 11 8150 shows that although bactone medium "E" produced the greatest cell growth, the supernatant of bactone medium "E" had the smallest Optical density “unber Of all the bactone media used. Table XVI shows the calculations made ‘ in Order to see if a proportionality constant could be found TABLE. XI V A SUHMARI OF THE KLETT SCALE HLADINGS TAKEN IN AN LXPEILIMLNT USING A TWO PER CENT BACTONL MEDIUM ENRICHED WITH MgCIZ (1.0 mg. per ml.)8 éfiitfiie M(B) C(“) 5(“) C(“) - 5(H) (Hours) 12 182 h h o 25 182 7 7 o hB 181 16 8 8 60 181 29 5 2h 73 181 62 10 52 91 181 90 16 7h 100 181 8h 17 67 116 181 92 25 67 125 179 8h 25 59 1L3 178 80 3o 50 161 178 66 27 - 39 185 177 68 36 32 209 t 176 62 35 27 281 l 176 61 ‘ hS 16 Lh9 i 176 ‘ 78 69 9 I aSee footnotes to Table I for the definitions of the symbols H B , C(“) and 3(l). TABLE XV A SUMMARY or KLhTT SCALE RLADINGS OBTAINED IN AN EXPsRImLNT USING A CHEMICALL! DEFINED MEDIUM (ELLIOTT)a 253.3; ”(8) cm 300 (00‘) .509) Cfiigs (Hours) mm 15 2 o o o 2 27 2 3 0 3 6 h8 2 1h 2 12 i 82 67 2 6h 7 S7 191 93 2 97 1o 87 ‘ 212 118 2 107 12 95 7 228 138 2 96 15 81 f 188 152 2 93 1h 79 E 180 176 2 81 13 68 g 178 193 2 69 15 7 St E 152 210 2 66 17 h9 i 108 262 2 Sb 18 36 ! 92 287 2 51 28 ’ 27 g 73 31a 2 E 1.1 32 ‘3 9 ’ 36 8See footnotes to Table X for the definitions of the symbols M(B) M) and S H“. 7h FIGURE 10 Graphs of the Klett scale readings of the culture medium (chemically defined medium), the supernatant and the numerical difference between these two measurements plotted against time. See footnotes to Table X for the definitions of symbols. Own mmaoz OON om. ON. _ q — q \. /o /\n\ 5. -3. - as mgem; ”2.0-6 venue; 9N|OV38 BWVOS 1131)! TABLE XVI AN INTERPRETATICN OF SOME OF THE DATA FROM TABLL xva A. 03:? CW) (C(M) _ 50.1)) Cells Per 111113 09113 Per mm3 mm C(M) (C(11) _ 8““) 82 1h 12 5.86 6.8 191 614 57 2.98 3.36 212 97 87 2.18 2.h2 228 107 95 2.13 2.1.0 188 96 81 1.96 2.32 180 93 79 1.9h 2.28 178 81 68 2.20 2.62 152 69 Sh 2.20 2.82 108 66 h9 1.6b 2.20 92 Sh 36 1.70 2.56 73 51 27 1.83 2.70 36 I h1 9 0.88 u.00 synmbo1s :ffi :33t2(§)f to Table 11 for the definitions of the 77 FIGURE 11 Graphs of the Klett scale readings of several types of culture super- natants, using fresh medium as the standard solution, plotted against time. See footnotes to Table X for the definition of the symbol 3(k). mmao: 00¢ own own 8N o¢~ OON 00. ON. on O? o a _ _ _ _ _ _ _ u \o x \ ._ .I.\..\\..tX~KV/. - e 0\ l O o v o\u\ \O\O.O\ Ilmnv\ \ o o 1 ¢~ \ o\o \ 0 O\ Q \ - .. C \VID\ v 1 we v o\ .EOEE £336 wanton 2.3.82.0 223-0 1 cm as 2.3. 2.225 :39. \ "3.8 232:. £2.90 \V 0 «SM: Osaoon :2VMI. l NF ‘58.. H Tn SNIOVBE BTVOS 1131M 7‘) between any Of the data in Table XV. It is fairly obvious that not only does the chemically defined medium produce the least Optically active supernatant, but that also the proportionality between the direct cell countand the corrected scale reading of the cell culture (by subtracting the scale reading Of the supernatant) is quite good. B. STUDIES ON CARBON DIOXIDE REQUIREMENT IN TETRAIKMENA In order to facilitate the presentation of the data for the experiments in this section, the symbols listed and defined in Table XVII will be employed. l; Aeration flask weriments. The apparatus pictured in Plate 1 was used for this series Of experiments. Chemically defined medium (see Materials and Methods," Part, B,’ Elliott's Medium minus glucose and sodium acetate) and bactone medium E minus glucose‘and sodium acetate were used as the culture media. The culture tubes containing 30 milliliters of medium were inoculated with one loop Of a five day old culture of the same type of medium. This inoculation amounts to about 500 cells per milliliter in the culture tube at the commencement Of the experiment. The Objective of this experiment was to see if the removal of carbon dioxide from the air which entered the culture tube would inhibit the growth rate. The results are tabulated in Table XVIII. A direct cell count was made “Sing the Sedgewick-Rafter chamber. The data in Table XVIII did not reveal any consistent results w . hlch c3<>l1ld be interpreted as indicating a carbon dioxide requirement 80 for growth in Tetrahymena. In fact the results show that the growth in COz-free air was at least as good and in some cases better than in the control tubes. The growth Of protozoa using the bactone medium did show a reduced rate when COz-free air was used in three out of the four trials but this decrease was not a significant amount. For reasons which will be stated in the Discussion this experimental set up was not considered a very satisfactory approach to the problem Of the carbon dioxide requirement for Tetrahymena. 2; Desiccator-dgiression slide egperiments. The apparatus described in "Materials and Methods,“ consisted of a sealed desiccator in which vessels of KOH had been placed in order to remove the carbon dioxide. Organisms we re cultured in depression slides in both bactone (B'), and a chemically defined medium (Sy'). The control for this experiment was a desiccator containing vessels of 20 per cent ROI in place Of the vessels Of 20 per cent KOH. The volume 01' the culture was about 0.5 millileters, and the inoculation was such that the initial concentration of organisms was about 5 cells per Culture drop. The control for this experiment was a desiccator contain- ing vessels of 20 per cent KCl in place of the vessels Of 20 per cent KOH. The data for this experiment are summarized in Table XVIII. The number Of cells present in both the control and the experi- mental cultures was essentially the me. There is no indication, at a _ "V rate, that the removal or carbon dioxide by absorption with KOH i Whit-Ed the growth of the culture. 81 TABLE XVII SYMBOLS EMPLOYED FOR SECTION B Symbol Definition I; Acetate radicle (C2H30é‘); B bactone media E (see "Materials and Methods,“ Part A); B' bactone media E minus glucose and sodium acetate; m molality, i.e., number of moles dissolved in 1000 grams of solvent; M molarity, i.e., number of moles dissolved in 1000 milliliters Of solvent; 3y chemically defined medium (see "Materials and lethods," Part B, Elliott's medium); 537' chemically defined medium (Elliott) minus glucose and sodium acetate; and T Trial or sample number of the culture used in a given experiment. TABLE XVIII 82 RESULTS OF EXPERIMENTS IN PARIS l, 2 AND 3 WHICH INVOLVED VARIOUS METHODS USED IN AN ATTEMPT TO RENOVE CARBON DIOXIDE FROM THE ENVIRONMLNT OF THE CELLS Description Trial Hours Control Experimental of Method Number Growth ‘“‘ 3' 5y I . Bl By" 1 92 633 1388 778 1268 Aeration 2 75 81 122 h3 186 Flasks 3 80 90 133 87 1L2 h 82 128 118 98 113 b b b b Desiccator- 1 72 850 630 700 ‘ 7140 ‘Depression Slide 2 80 600 630 510 h70 ‘Apparatus 3 76 580 700 630 7 730 b b b b Petri dish- 1 68 31:0 g 1150 1 1180 1420 Depression 7 1 Slide 2 72 550 300 1 720 680 1 Apparatus 3 72 800 t 600 bzo 75o ‘ l 308115 per cubic millimeter. bCells per culture. 83 3. Petri dish-depression slide experiments. This experiment was set up using the apparatus described in "Materials and Methods" in which a depression slide was placed on tOp of a stack Of six slides in a Petri dish. The Petri dish was nearly filled with a 20 per cent KO?! solution and a drOp (about 0.5 milli- liters) Of inoculated medium was placed in the depression Of the tOp slide. Both types Of media, as used in the previous two experiments, were employed. The initial concentration Of the drOp culture was about 5 cells per culture. The control for this experiment involved the use Of Petri dishes filled with 20 per cent KCl instead of KOH. The results are tabulated in Table XVIII. This type Of experiment did not produce any difference in growth rate, in eXperimental, and control solutions. The number of organisms in the culture were highly variable. Several additional attempts at this type of experiment resulted in bacterial and fungal contaminations. The fact that contamination occurred seems to indicate that the carbon dioxide was not adequately removed from the environment because mam baCteria and molds are very sensitive to low carbon dioxide tensions. The Very nature of the eXperiments in Parts 2 and 3 were such that it was difficult to maintain sterile conditions. Wat chamber eigeriments. This experiment is summarized in Table XIX. A depression slide was used in this experiment and a small drop of inoculated culture In 6dium Containing only one cell was placed within the depression. Ne ar the culture drop but not touching it was placed a small drop 0f St 0.3 molal KOH (eXperimental) or 0.31 molal KCl (control). A ring of vaseline was placed around the depression and a cover glass sealed to the vaseline, thus producing a sealed volume around the depreSsion containing the two drops. The same media as used in Part 3, namely 8' and Sy', were used. One can see from the results in Table XIX that the experimental cultures grew much better than the controls for at maximum growth there were over fourtimes as many cells in the depression slides which con- t'ained the XOR than in the control slides. It is also interesting to note. that the lag phase of the growth curve was somewhat longer for the experimental culture than for the control. 1- Van Tieghem chamber exPeriments. Experiments which utilized Van Tieghem chambers are summarized in Table XX. A bactone medium (B) was used for the culture medium. Three drOps ofmoistener were used for each chamber. Varying concentra 1”ions of XOR were used for the experimental cultures and three drops of distilled water were used as a moistener for the control cultures. Each culture drop contained four cells at the inception of the experi- ment. Table XX shows that the cultures were discontinued at various t'imfi‘s. This was done in order to take the pH of the cultures. In all cases, using pHydrion papers (MicroEssential Laboratory, Brooklyn, New York), the pH was between 7.0 and 7.b. Figure 12 shows the graphs of the rate of' change in cell count for the various concentrations of KOH used. Plate 5 shows several photomicrographs of live cells as they appear in Van Tieghem cultures. TABLE XIX NUMBER OF ORGANISHS PEH.CULTURE USING SEALED DEPRESSION SLIDES L Age of 0.31m K01 Moistener 0.3m KOH Moistener Culture (H°“rs) T1 T2 T3 T1 T2 T3 0 1 1 1 1 1 1 2h b 2 3 2 2 1 72 27 31 29 10 7 12 96 3h h? hl 32 13 20 18h 81 76 83 121 22 76 168 86 92 6h 3&1 108 202 192 72 83 37 h30 2h1 372 2L0 3h 53 27 521 h67 h22 288 13 17 11 h11 370 360 hoe 6 h 3 b2 312 260 86 .acoaameXo on» ma peace can» no poems ooaaavmap Spa: ooomaaou mm: coaasaom mom used cam maa cum a»: com com com 93 coo cme omo cmm cmma cm: coma coma «ma amm cmm cm: coo ome cmo omm ccoa om; coma coma ccea omoa ccoa am cam cam com omo com cmo coma cmoa oom coma cooa coma ccea oa coma ma a «mm «mm com cmm cmo com cmo coma coma coma «mm coma coma omm cooa oa ccea ma m am com com cam cam com cmm coma coma coma mmm coma coma m coma oa coea ma. m om Nma com cmo com coma com coma coma coma cma coma ccoa o mo oa coo ma m mm cma mas coo com coma com coma coma coma moa coma coma so am oa caa ma m a: mma cum coo com coma com coca coco ccc~ mma coo coma o ma oa ca ma m as moa com cmm coca coma com coca coma cccm oma mm cm: o co ma an ma ma am we mm cma cam cam cum cema cmea mama om an co . o m ea o oa ma mm mm «o ama cam omo cop coma coma coma a» m co m ma mm m mm ea as on cm mm cam mam cum coca coma cum om ma ma ma ma on m am ma am am mm on am No ao «ma mam «ca on ma ma oa cm an m am ma «N am am am m: mm on mm «ma mo mm ma ma oa ma ma m ea Na ma ma ma aa oa Na ma ea am ma aa ca aa ca m ca s ca m m m m o m o a ca ma a o s a a a a a a a a s a m m a a m m m o as we ac me we as me we ac me we ae me we aa me we as Ampsomv am.c am.c am.c :~.o :a.c cocoooaoa cocoacc mo mm< hocoemao: mom om: mmzwemnofi 43m< mox mo monemxeszzoo mocamé ozamm mmmmzéc gong 55 2a 2238 3mm 5“ .593. 87 One can see from Figure 12 that the number of cells per culture is inversely related to the molal concentration of the EON. When distilled water was substituted for KOH (columns six and seven, Table XX) the number of organisms in individual cultures rapidly increased. This experiment was notewortmr in that it showed the great inhibition of growth induced by hypertonic solutions of KOH when they are used as moisteners in Van Tieghem chambers. In subsequent experiments, controls were established by the substitution of either an isosmotic solution of KCl or sodium acetate for KOH as a moistener in the Van Tieghem chamber. The pH measurements showed a variation of about one- half of a pH unit during the experiment. Thus the absorption of carbon dioxide by the KOH does not raise the pH to any extent. Isosmotic moisteners. A series of experiments were performed in order to compare the growth of cells in Van Tieghem chambers in which isosmotic solutions of KCl, sodium acetate, and KOH were used as moisteners. Six drops of moistener were used in all culture chambers. The experimental chambers contained 0.3 molal KOH for the removal of carbon dioxide; the control chambers contained 0.3 molal sodium acetate or 0.31 molal KCl. The culture medium for this experiment was bactone medium "E" minus glucose and sodium acetate. The data are summarized in Tablx XXI and the graphs of the average cell counts for the three types of cultures are found in Figure 13. some of the observations that should be noted from a study of Table XXI and Figure ’13 are: (a) the rate of growth when KOH is used as a m01531:.ener was much slower than in the controls, however the maxi- m um growth in the “KOH cultures" ultimately exceeded the maximum growth FIGUE 12 Graphs of the rate of change in the cell count using various concen- trations of KOH as moisteners in the Van Tieghem chambers. 88 manor c0532: .22.. 3530-0 2.2.. oo. om on 2. om on oe IW . . _ _ a! _ [114 q I I I \V\\ v .o v 1 ON. 0 1 00. .5552... :9. End..- 95 232:2... 10x Ecdd . 1 com .3552: :0x 600.. 222:2... 10x Ewd-» “8:232: :9. 5.6... .. ova OQN 83:! 81133 3301100 9O PLATE 5 Photomicrographs of live cells as they appear in the culture drop of a Van Tieghem chamber. Fig. l. Distilled water moistener; and Fig. 2. 0.2 molar KOH moistener. 92 PLATE 6 PhotomicrOgraphs of live cells as they appear in the culture drop of a Van Tieghem chamber.r Fig. l. 0.3 molar KOH moistener; and Fig. 2. 0.5 molar KOH (note tyrosine crystal). PLATE 7 Photomicrograph of live cells in the culture drOp of a Van Tieghem chamber. The organisms are cultured in a bactone medium supplemented with 0.35 per cent agar. 9L C '0 A L. 96 of the controls (Figure 13); and (b) when sodium acetate was used as a moistener, the number of organisms decreased from a maximum in 200 hour CUltures to zero in 3110 hour cultures. It should be pointed out that the cytoplasm in the cells grown in "sodium acetate chambers“ became extremely granular soon after the maximum cell count was reached. The pH of'the culture solutions in the “sodium acetate chambers" decreased to about 5.5 when the number of organisms decreased to almost zero. The 13H of the other cultures utilizing KOH or KCl remained fairly con- stant at about pH7. Experiments testingng constancy and moistener molalities. (a) A series of Van Tieghem chambers were set up using both 8' and Eiy' as culture media and utilizing varying concentrations of KOH from 0.1 molal to 0.6 molal as moisteners. Six drops of the KGB solutions were used. The dr0ps of uninoculated medium were left in the Van Ti. eghem chambers for about 200 hours. The pH, using pHydrion Papers, of the medium was taken at the end of 200 hours. In all cases the pH of the media remained constant. There was no EEVixience that the XOR "creeped" along the walls of the chamber to reac‘hthe culture solution. It was felt, therefore,'that the pH was “0t a~‘Variable in these eXperiments. (b) The following combinations of medium and moisteners for the Van Tieghem chambers were made: (1) A 2 per cent bactone medium minus glucose and sodium acetate using O.h molal KOH as a moistener; (2) a h per cent bactone medium minus glucose and sodium acetate using a 0.5 molal KOH as a moistener; 97 TABLE. XXI CELL COUNTS USING VAN ‘I‘IEGHEM 011111113133 CONTAINING 0.3m Na ACETATE, 0.31m KC]. AND 0.3m KOH AS LIOISTENERS ...-- - M ...—......“ U Type of hoiste—ner Used in Van Tieghem Chamber Age of “Z “ ‘— Culture 0.31m KCl 0.3mjaltc 0.3m TECH (”W“) T1 T2 T3 Th T1 T2 '1‘; Th T1 T2 T3 Th 0 l l l 1 1 . 1 1 1 1 1 l 1 20 3 h h 2 b 2‘ h 3 h 2 3 h uh 7 8 9 9 6 8 8 9 S 6 h 7 73 17 12 19 15 13 15‘ 17 16 11 9 10 8 96 2h 28 31 21 29 33 27 32 15 16 1b 18 117 35 36 38 29 50 1:9 142 h? 17 21 15 23 1141 119 52 h? 116 57 53 W 55 19 211 17 31 189 51 50 b3 b1 65 60 62 51 22 31 21 116 237 37 148 140 33 23 1:3 145 37 no 38 28 51 280 36 bl 31 30 10 13 17 12 53 157 113 60 312 22 36 17 21 3 7 6 h 62 67 58 69 31.0 j 17 12 11; 8 o o o o 52 61 55 57 M .. h ___ ' _ 1 FIGURE 13 Graphs of the rate of change in cell count using KUH, K01 or Na acetate in the Van Ti eghem chambers. 9.501 Om» ONm OON O¢N OON Ow. ON. on ow O ‘fi/a a a a a a a a \ .\ \o - c. \. \e 1 ON 1. o \O I On 0 o /O .. 0e. 11 \ 1 on e c \ c.3330... IOv. End; vco 20:220.: 40v. 636.0 1 06 23.332... one: End... venue; on 3801'"an 83d $1130 100 (3) a 5 per cent bactone medium minus glucose and sodium acetate using 0.h molal KOH as a moistener; and (h) a chemically defined medium (Elliott's minus glucose and sodium acetate) in which all amino acid concentrations were doubled and using O.h molal ROH as the moistener. The purpose of the series listed in (1) through (b) above was to see if it were possible to increase the carbon dioxide absorbing capacity of the XOR sdlutions by increasing the molality of the XOR. Since the increased molality of the KGB requires an increased osmotic equivalent in the culture medium, an attempt was made to increase the osmotic activity of the medium by increasing its concentration of metabolites. The results of these experiments were completely negative. No concentration in the bactone or chemically defined media could compete osmotically with 0.h molal KOH. In all cases only a few cell divisions took place and after the second day the cells became inactive and resembled spherical "cysts." (c) A series of Van Tieghem chambers were set up, as described in "Haterials and Methods," in which 16 drops of moistener were used instead of just six drops. After placing 16 draps of moistener in the Chamber, the cover glass (containing the inoculated culture drap) was placed on tOp of the glass ring. Under these conditions the culture drOp was situated within the air space formed by the meniscus of the moistener. Both types of media, namely 3' and Sy', were used for this experiment. The concentrations of KOH and KCl were 0.3 molal and 0.31 molal, respectively. There wasxio significant difference between the results obtained using this method and those obtained when only six drops of moistener 101 were used (Table XXI), i.e., the growth of the cultures using KOH as the moistener was slower at the early stages but ultimately reached a higher maximum. In fact, in many cases the "KGB cultures" not only reached a higher maximum cell count, but the high cell count remained constant over a longer period of time. It seemed possible from these eXperiments that perhaps not enough of the carbon dioxide was removed from the environment and that alternative methods must be devised for a more efficient removal of carbon dioxide. Dubnoff shaking incubator with chemically defined, medium. The objective of these experiments was to find a more efficient method for removing carbon dioxide from the environment of the cell. Six draps of moistener were placed within the Van Tieghem chamber. A small piece of filter paper (8 by 12 millimeters) was inserted in a Vertical position within the Van Tieghem chamber. These chambers were Placed on a rack to which masking tape had been attached in such a way that the sticky side was facing upward. The rack was then placed in the Dubnoff Metabolic Shaking Incubator and the apparatus was set so as to PPOduce about one complete oscillation ,per second. The sticky portion 0f the tape held the chambers firmly in place when the shaking apparatus was in Operation. The chambers were positioned on the rack so that the plane of the filter paper within the Van Tieghem chamber was perpendicu- lar to the direction of oscillation. Chemically defined medium (8)“) was used. The moisteners were 0.31 molal KCl (control) and 0.3 molal KOH (experimental). The data are presented in Table XXII and the growth curves are depicted in Figure 1h. TABLE XXII CELL COUNTS USING VAN TIEGHEM CHAMBERS WHICH WERE PLACED IN THE DUBNOFF METABOLIC SHAKING INCUBATOR. CULTURE MEDIUM: A‘A‘.‘ Type of Moistener Used in the Van Tieghem Chamber QY'o Age of Culture 0.31m x01 0.3m KOH (Hours) T1 T2 T3 it Av. T1 T2 T3 Th Av. o 2 2 2 2 2 2 2 - 2 2 2 19 7 13 8 13 10 8“ 10 u u 6 27 1h 25 15 2a 19 9 16 u 6 9 no 26 t2 38 no 36 15 23 9 13 16 h? in u9 ho u1 hl 16 26 10 12 16 63 39 51 u1 u? uh 19 29 12 1h 18 7b to 53 hl hS b5 23 31 12 16 20 88 h2 52 h3 ht us 25 33 12 15 21 11b 37 51 h7 h8 b6 30 u3 13 17 26 136 3h 57 SS h9 h? 27 5h 1h 18 28 190 37 uh ho ho b2 23 39 11 17 22 218 32 38 37 38 36 21 27 12 16 19 102 103 Figure 1h shows that a hCl moistener produced better growth than a KOH moistener under the conditions described above. Likewise, it is evident that, in addition to a longer lag phase, the XOR culture also possessed a lower maximum cell count than the control. The results of this eXperiment will be compared to subsequent experiments which involved the use of a bactcne medium (8') instead of the chemically defined medium (Sy'). Dubnoff shaking incubator with bactone medium. These experi- ments are quite similar in plan to the previous experiment. The Dubnoff Metabolic Shaking Incubator was again used and the pieces of filter Paper were inserted into the Van Tieghem chamber. The primary differ- ence between the two experiments was that the bactone medium (13') was used for this experiment and sodium acetate (0.3 molal) was used as a Control in addition to KCl (0.31 molal). The data are presented in Table XXIII and Figure 11;. This experiment shows that when a shaking apparatus was used, the cell count in the bactone medium was about the same whether or not KOH 1was used as a moistener. The "KOH cultures" did not exhibit the long lag phase which was shown in previous encperiments. The signifi- cance Of the difference in growth between the "KOH cultures" for the two different media, 3' and S)", will be discussed later. Chemicallyr defined medium supplemented with certain dicarboxgflic %- These experiments were carried out in order to see whether or not any of the dicarboxylic acids, which are intermediates in the "Krebs cycle," are capable of enhancing the growth rate if the carbon di oxide is removed at the same time. The Dubnoff shaking apparatus "as 1011 TABLE XX I I I CELL COUNTS USING VAN TIEGHEM CHAMBERS WHICH WERE PLACED IN THE DUBNOFF METABOLIC SHAKING INCUBATOR. MEDIUM USED: 8'. -‘ ‘_-‘_‘ ‘AL Lu :‘—‘ Type of Moistener Used in the Van Tieghem Chamber Age of A Culture 0.31m K01 0.3m NaKE 0. 3m xon (Hours) * T1 T2 T3 Th Av. T1, T2 T3 Th Av. T1 T2 T3 Th Av. o 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 2 ' 2 2 3 2 2 2 2 1 2 3 2 2 2 2 26 3 h 2 h 3 3 2 2 3 3 3 2 3 3 3 1.6 6 7 3 6 6 6 h 8 7 6 8 6 7 5 6 69 912h888615121011912910 90 1b 1b 8 8 11 10 7 16 15 12 15 13 11; 12 13 115 16 17 1o 12 111 17 12 17 21 15 20 1h 16 17 17 139 17 19 11 16 16 18 13 21 26 19 21 1h 15 20 17 163 20 13 11 17 15 18 15 22 27 2o 15 12 18 2h 16 211 17 10 10 16 13 16 16 1h 19 16 18 16 15 32 20 259 12 7 3 1h 9 11 1h 12 17 13 15 8 13 30 16 105 FI GURE 1h Graphs of the rate of change in cell counts using Van Tieghem chambers and the Dubnoff Metabolic Shaking Incubator. The data were obtained from Tables XXII and XXIII. OON 02 OO. OS ON. OO. 00 OO O? ON .5552: 49. 66.0 .6338 .014 ago 22532: one: End .5325 .m.o 2.2.3.... :9. and .e=_3e.m.. 22.3.2. :9. and .5285 am.- 55336 1.02 5nd .6332.»qu 8:33 0. ON On O¢ On 380.1100 83d STUD 107 again used and filter paper inserted within the Van Tieghem chamber. The moisteners were 0.31 molal KCl (control) and 0.3 molal KOH (experimental). The following carboxylic acids were added to the chemically defined medium (Sy') so that they were present in 0.02 molar quantities: succinic acid, oxalacetic acid, malic acid, fumaric acid, aspartic acid and alpha-ketoglutaric acid. The pH was varied from 7.1.: to 6.0. The results were almost exactly the same as the growth curves obtained when the dicarboxylic acids were not present. The conclusion is, therefore, that if the lack of carbon dioxide is inhibiting growth, the addition of the dicarboxylic acids mentioned above will not relieve the inhibition. A further modification of this type of experiment was carried out by adding trans-1,2-cyc10pentanedicarboxylic acid (Krishell Laboratories, Portland, Oregon), 8 succinic acid analog, to the dicar- boxylic acid solutions described above so that the cyclopentane dicarbomdic acid has a final concentration of 0.0).; molar. Seaman and Honlihan (1950) showed evidence that the cell membrane of Tetrahymena '33 not, permeable to the dicarboxylic acids involved in the "Krebs cycle. " According to his observations, the addition of trans-1,2- °y°1°Pentanedicarboxylic acid to a medium containing succinate showed that Some of the succinate was removed from the culture medium. In the experiments conducted using Van Tieghem chambers this succinic acid analog had no effect on the growth rate of the "KOH cultures.“ In other words, the presence of trans-1,2-cyc10pentane- d icar‘b‘ntylic acid did not relieve the inhibition of growth; the 108 inhibition of growth may or may not be due to a lack of carbon diox- ide o 6. The use of a modified Van Tieghem chamber to remove carbon dioxide from the environment of the cell. The apparatus used in these experiments is pictured in Plates 2 and 3 and was described in detail in "Materials and Methods," Part D,h. The culture media are the same as used in the previous experiments. The results of these experiments are summarized in Table HIV and Figure 15. Table XXIV (Sy' medium) shows that at 90 hours subsequent to inoculation, the number of cells in the control cultures was over four times as great as in the experimental cultures. In fact, even if the 9x98 r'imental culture was inoculated with two organisms while the control culture contained only one organism (337' medium, T3), the latter cul- tures possessed about three times as marv cells as the experimental cultures after 90 hours. If the Sy medium was used, the maximum number of organisms in the experimental cultures was about one sixth as great as in the control cult"Jlf‘es. The additional glucose and sodium acetate, which is present in the 3y medium, apparently enhanced the growth of the control cult“res, but had little effect on the experimental cultures. In contrast to the above results, the experimental cultures grew as well as the control cultures if bactone medium (3) '83 used- Figure 15 shows that the growth curves for the experimental cultures and the control cultures were vel'.v different if 33" medium was used, TAdLE XXIV CELL COUNTS USING THE HODIFIED VAN TIEGHEM CHAMBER, AS PICTURED IN PLATES 3 AND h, WITH VARIOUS TYPES OF CULTURE MEDIA . Age of Experimental Control Medium Culture (COZ-free air) (Atmospheric air) (Hours) T1- T2 T3 Av. T1 T2 _ T3 Av. 0 1 1 2 1 l l 1 1 15 2 1 3 2 2 h l 2 30 3 2 o b 7 9 h 7 Sy' hS 6 2 12 8 23 2h 25 2h 60 9 u 17 10 f 37 33 h6 39 75 19 7 39 21 93 76 102 90 9o 33 21 us 33 152 127 1b3 110 o 1 1' 1 1 y 1 1 1 1 15 b 3 - 3 5 h - h 30 - - 7 7 - - 9 9 fly b5 8 11 13 ll 19 16 21 19 60 - - 21 21 - - 52 52 75 11 26 36 2h 112 1&2 170 lhl 90 3h h3 58 hS 3h0 276 320 312 O l l l 1 1 l 15 2 - 2 2 - 2 3o 5 u s ' 6 15 9 8' us 18 1b 16 31 h9 32 1 60 _ - _ - - - 7S 58 h3 SO }1h2 176 122 90 - 93 93 - 220 220 O l l 1 t 1 1 1 15 h 2 3 ' 3 3 3 30 12 - 12 10 - 10 B us 3h 23 28 29 38 3h 60 63 67 65 S7 92 75 75 - 1H3 1&3 - 171 171 90 - 320 320 - 360 3&0 109 110 FIGURE 15 Graphs of the rate of change in cell counts using the modified Van Tie ghem chamber pictured in Plates 3 and 1;. Data were obtained from Table XXIV. cm 00 on on cc 0. o m . 4 a J . ||HJ 011 \\ 1 ON 1 O? 0 3 1 .I .. 8 S d 3 8 m .1 on n n m ...e 9:22.85... .5336 .0...- .. 00. use £0 3.7.00 .533... b-» 2... 2.21368 .523... .36 :3 3.7.00 .533... .xmo 1 Om. 2.3 112 whereas bactone medium (B) produced growth curves that were essentially similar. The significance of the different results obtained when different media (B and Sy') were used, will be discussed later. Dicarboxylic acids. Experiments utilizing the dicarboxylic acids of the "Krebs cycle" were set up in a manner similar to those in Part. 1;. Two-hundredths molar concentrations of succinate, oxalocetate, malate, fumarate, aspartate and alpha ketoglutarate ware added to the 5V ' medium. Additional cultures were set up in which 0.0).: molar trans-l,2-cyclopentanedicarboxylic acid was added in addition to the dElfiarboxylic acid solutions described above. The results of these experiments revealed that the experimental cultures containing the dicarboxylic acids did not grow any better than the experimental cultures in the previous experiment. In other words the reduced growth rate, using Sy' medium, produced by COZ-free air Could not be relieved by the addition of the dicarboxylic acids listed above, nor did the addition of trans-1,2-cyclopentanedicarboxylic acid Produce any further modification of the growth curve. The results of the last series of experiments, in which the - motiilfied Van Tieghem chamber was used, seemed to indicate that the growth rate of Tetrahymena pyrifogmis W was greatly reduced if the carbon dioxide was removed from the environment of the cell. DI SC USSI ON A. OBSERVATIONS ON GROWTH IN TETRAHYMENA Hall M. (1935) reports on error of about 5 per cent when the Sedgewick-Rafter chamber is used to measure pOpulation densities of protozoa. Richards (19111, p. 518) states that the chief source of error of this method depends on how closely the sample represents the Population. This, of course, is the perennial problem in any sampling Procedure. Allen (1921), Berkson e_t__§_1_. (1935), and Serfling (19149) haVe discussed the statistical errors inherent in sampling techniques as they apply to b1010gical problems. Experiments in this study in which the Sedgewick—Rafter chamber Was utilized were carried out in order to correlate direct cell counts v"31th other methods of growth measurement. No specific attempt was made to evaluate the use of the Sedgewick-Rafter chamber by itself because its limitations have been thoroughly discussed by Whipple (1927). R~1'Lchards (19111, p. 518) points out that the Sedgewick-Rafter chamber has long been the standard type of growth measuring device for Protozoa. Monod (191:9) discusses the fact, that although there is wide- Spread use of Optical techniques, not enough efforts have been made to Check them against direct estimation of cell concentrations or dry- "eights. The results of this study seemed to indicate that a direct cell count and the Optical density of the culture medium are not closely correlated. In fact, for cultures grown in bactone medium, the 11b. ratio of the cell count to the optical density measurements varied from a minimum of 2.58 to a maximum of 8.00 during the first 26h hours. On the other hand, the addition of a small amount of thiamine (0.2 gamma per milliliter) to the bactone medium, produced a variation of the same ratio from a minimum of h.90 to a maximum of 6.00 during the same number of hours. That is, the variation of the culture medium by the addition of just one vitamin has markedly affected the ratio between the cell count and the Optical density. The significance of this is that although the correlation of the cell count and the Optical density may be fairly good for one type of medium (e.g., thiamine enriched bactone), the correlation may be very poor if the medium is altered by a slight degree. In all of the types of media studied, however, the ratio between the direct cell count and the optical density of'the culture medium never approached anything like a proportionality constant. If these two variables, i.e., cell count and optical density, are directly proportional to each other, then the expression "cell count/ optical density : K" (where K is a prOportionality constant) should hold. Elliott (l9h9) maintains that the photometer measurements are indicative of cell volume rather than cell number. He also states that the absorption of light is a function of both the size and the contents of cells. The problem, of course, is: Just what does the Optical density of the culture indicate? The results of this study would agree that the Optical density is probably not a good measure of the cell count. In addition, one must ask: If the Optical density is a measure ~of cell volume and cell content, as maintained by Elliott, is there any way that one could decide which of the variables is being measured? 115 Experiments in this study showed that the optical density of a bactone medium was not correlated to the total mean protoplasmic area of the culture. The ratio of these two quantities increased from 57.111 at 73 hours to h0h.76 at 26h hours. This is about a seven-fold increase. The same ratio in a thiamine enriched bactone medium varied from 57.23 at 73 hours to 90.75 at 261, hours--the increase being by a factor of Here again the expression "optical density/total mean protoplasmic 1.6. area : ‘K" does not hold. It must be admitted that there are inherent errors in the method used to determine the total mean protoplasmic area. It would be difficult to believe, however, that the errors would be of the magnitude to produce a variation in the ratio equivalent to that Probably an important error in the estimation of Pop off which was found. protoplasm was the use of cell area instead of cell volume. (Cited by Richards, 19111, p. 521) calculated cell volumes of P. caudatum as equal'to hflLBT/Zh, where L, B and T are equal to length, breadth and thickness, respectively. This formula assumes the cell tote a spheroid. Since it was difficult to see how accurate measurements could be made on the cell thickness, the assumptions made by Popoff were not followed. Since neither the protoplasmic area nor the cell count seem to be correlated to the optical density, one should consider the "cell content" as a possible source of Optical density. Stier _e_t__a_l_. (1939) I""390r‘l’os that in yeasts a change from 0.170 to 0.1117 occurred in the optical density within six hours after the addition of 5 per cent glucose to the suspension. He maintained that there was no significant ch ange in the total cell count during the six hours but that an increase in the Opacity of the cells was evident. The above reference suggests 116 that cell content may be a very important variable that should be con- sidered in optical density measurements. In our study, the only obvious variation in the Opacity of the protoplasm occurred when sodium acetate was used as a moistener in the Van Tieghem chambers. In con- trasts to the experiments of Stier gt_gl., given above, are the results of Gauss (193h). In separate cultures of P. aurelia and P. candatum, with the same culture medium, he reported that the same total volume of protoplasm was produced by each species although a direct cell count revealed that there were over twice as many P. aurelia as P. candatum. It may be argued that a change in cell content constitutes a change in growth and consequently if the optical density can depict a change in cell content then this should be acceptable as a measurement of growth. The fact must be considered, however, that a change in the Opacity of the cell may be due to only a small quantity of a substance which is very Opaque whereas a different substance, even though present in a greater amount, could conceivably transmit a high percentage of the incident light. Thus one should carefully qualify any statement which attempts to correlate an increase in cell Opacity with an increase in cell growth. Stier ejLal. (1939) in his studies on the opacity of yeast cells made the observation that there was no change in the Optical density of the suspending medium during the duration of the experiment. In our studies it was realized that in addition to the variables already Stated, namely, cell count, cell volume and cell content, one should always consider the possibility that the optical density of the culture SuPernatant might change significantly during the course of the ll? experiment. This seems to be an easily overlooked possibility although Elliott (19h9) mentions that a gradual change in the color of the medium often occurs during incubation. He mentions as possible causes that this may be due to the consumption of certain foods, accumulation of wastes, or a shift in pH. Our measurements of the Optical density of the supernatant strongly indicate that there are significant changes in the Optical density of the supernatant as the culture ages. A study of Figure 11 reveals that this optical density change varies with the type of medium used. In addition to the fact that the Optical density of the supernatant appears to be a variable, and thus should be con- sidered in any optical measurements of the cultures, there are at least three additional observations that should be pointed out in connection with this fact. (1) In several cases (Tables XI, XII, XIV) it appears as though Optical changes occurred in the supernatant prior to any signifi- cant increase in the number of cells in the culture. When a 2 per cent bactone medium was used, for example, the optical density of the culture medium was actually due to optical changes in the supernatant. This is evident from the fact that the Optical density of the supernatant is equal to the Optical density of the culture medium. It is quite obvious from this Observation that one could erroneously interpret Optical density changes as an increase in protoplasm or cell content if one ignores the possibility of optical changes in the supernatant. This would be particularly important if one were comparing the stationary or lag phases produced by different types of media because, as described 118 above, important changes occur in the supernatant during these phases of the growth curve. (2) As was pointed out from Figure ll, the optical density of the supernatant was related to the presence of cells in the culture medium. That is, fresh culture medium did not increase in Optical density, but usually showed a slight decrease (Tables XI through XIV). However, the Optical density was not directly proportional to the increase in the number of cells because only a slight increase was shown in the Optical density of the supernatant during the logarithmic phase of growth. One would suspect, therefore, that the initiation of Optical changes in the supernatant was dependent on the presence of cells, but that the subsequent changes in the supernatant seemed to occur at a rate independent Of the increase in cell count. In many cases (Figures 7 through 10), the graph of the Optical