Class Book ()qpyiig]it>]°_ Ci)K»:lGRT DEPOSm SOIL BIOLOGY LABORATORY MANUAL BJ ALBERT LEMUEL WHITING, Ph.D., Associate in Soil Biology in University of Illinois, College of Agriculture and Agricultural Experiment Station FIBST EDITION NEW YORK JOHN WILEY & SONS, Inc. London: CHAPMAN & HALL, Limited 1917 >/- Copyright, 1917, BY ALBERT LEMUEL WHITING MAR 20 1917- Stanbope jprcss F. H. GILSON COMPANY BOSTON, U.S.A. ICI.A455963 PREFACE. Soil biology treats of the microorganisms which inhabit soils in their relation to soil fertiUty, crop production, and permanent agriculture. It includes the rapidly develop- ing fields of soil bacteriology, soil protozoology, soil mycology, and others which may later merit study. The purpose of this manual is to present the important prin- ciples of soil biology, particularly as they point to the inteUigent control of the essential elements of plant food. The principles are incorporated in practices which acquaint the student with the various forms of life in the soil and their activity. Special attention is given to all biochemi- cal reactions influencing soil conditions. The sequence of arrangement of the practices is not fixed but that given has been found best in this laboratory. The choice of materials which are tested is based upon farm practice. Students are encouraged to undertake these studies on their own soils. ^The laboratory course is a part of a five-hour course consisting of two lectures, one quiz, and three laboratory periods per week. Soil fertility and bacteriology are prerequisites while organic chemistry and plant physiology are desirable for the course as outHned. The questions, problems, and references accompanying the practices have been found by experience to be valuable supplements in fixing the principles and applying the information obtained. Emphasis is laid upon quantitative results and the measure appHed consists of biochemical and chemical methods. The results thus obtained are interpreted as far as possible in terms of soil.fertility and crop yields. iv PREFACE In Part II are included bacteriological, chemical, me- chanical, and pot-culture methods as appHed or developed in this laboratory. In the last section will be found Suggestions for Instructors and Students Preparing to Teach. An attempt has been made to satisfy the demand to have this information ever at hand and in a classified form. This little work would not be complete without an acknowledgment to Professor C. G. Hopkins for sugges- tions and encouragement in its development and to Mr. Warren R. Schoonover, assistant in soil biology, whose able assistance and careful observations have proved invaluable; also to certain former graduate students for testing new methods. A. L. WHITING. Urbana, Illinois, February, 1917. TABLE OF CONTENTS. PART I pagb Examination of Microorganisms in Soils and Manures 3 Occurrence of Bacteria at Different Depths in Soils 6 Quantitative Bacteriological Examination of Soils 6 Occmrence of Non-vegetative Forms of Bacteria and Fungi in Soils 10 Occurrence of Fungi at Different Depths and in Different SoUs 12 Ammonification in Soils 14 Influence of Moisture Content on the Process 14 Ammonification of Urea and Isolation of Urea Organisms. . . 17 Nitritation 20 Oxidation of Ammonia to Nitrite by Nitrosomonas .... 20 Influence Exerted by Carbonates, Ignited Soil, Soluble Organic Matter and Aeration 20 Nitratation 23 Oxidation of Nitrite to Nitrate by Nitrobacter 23 Influence Exerted by Carbonates, Ignited Soil, Soluble Organic Matter and Aeration 23 Isolation and Study in Pure Culture of Nitrosomonas and Nitrobacter 26 Production of Inorganic Nitrogen in Soils 28 Comparative Ammonification and Nitration of Crop Residues, Green Manures, and Farm Manures ' in Typical Soils 28 Influence Exerted by Carbonates, Soil Type, Moisture and the Conditions of the Organic Substances 28 Carbon Dioxid Production 36 CeUiilose Decomposition ....'. 39 Aerobic Decomposition by Fungi and Bacteria 39 Cellulose Decomposition 41 Anaerobic Decomposition 41 Symbiotic Nitrogen Fixation 43 Inoculation of Legume Seeds 43 Growth of Nodules and Determination of Nitrogen Fixed 43 V Vi TABLE OF CONTENTS Page Isolation of B. Radicicola from Legume Nodules 46 Non-Symbiotic Nitrogen Fixation 48 Aerobic Nitrogen Fixation in Soils ....*..... 48 Influence of Carbonates on the Process 48 Nitrogen Fixation in Solution by Soil Bacteria 51 Isolation of Azotobacter from Soil 53 Non-symbiotic Anaerobic Nitrogen Fixation and Isolation of B. Clostridium Pasteurianum 55 Denitrification and Formation of Calcium Carbonate 57 Sulfofication and Desulfofication in Soils 60 Fungi in Soils 63 Relation to Soil Nitrogen 63 Factors Influencing Fungous Growths in Soils 63 Protozoa in Soils 65 Isolation and Study of Amoebse, Ciliates and Flagellates from Soil 65 Determination of Active Protozoa in Soils 65 Algse in Soils 68 Relation to Soil Nitrogen 68 Comparative Numbers in Different Soils 68 Factors Influencing the Growth of Algse in Soils 68 A Study of Enzymes 71 Growth and Study of Iron Bacteria of Soils 73 Denitrification in Solution by Soil Bacteria 75 Active Protozoa in Soils 76 Decomposition of Cyanamid 78 Protein Formation in Soils 81 Flagella Staining of B. Radicicola, B. Nitrosomonas, B. Denitrificans, B. SubtUis, B. Typhosus .... 83 Cross Inoculation of Legumes 84 Solvent Action of Soil Bacteria on Minerals 86 Soluble Phosphorus and Calcium Produced by Nitrosomonas 86 PART II Bacteriological Methods 91 Food of Soil Microorganisms 91 Preparation of Culture Media 93 Formulae of Solutions (Liquid Media) 95 Formulae of Solid Culture Media 97 Special Media 99 SHica Jelly 99 Magnesium Plaster of Paris Blocks 100 TABLE OF CONTENTS VU Page Cellulose Solution 100 Reaction of Culture Media 101 Staining and Preparation of Stains 103 Simple Staining 103 Special Staining 103 Flagella Stains (Loeffler's) 104 Spore Stain (Hansen's) 104 Capsule Stain (Hiss') 105 Nodule Tissue Stain (Flemming's) 105 Protozoa Fixative Stain and Mount (Martin and Lewin .• 105 JProtozoa Stain 106 Algse Fixative and Stain 107 Formulae of Stains 107 Formulae of Stock Solutions of Simple Stains 107 Formulae of Simple Stains 108 Formulae of Stock Solutions of Disinfectants 109 Formulae of Special Stains 109 Fixatives 110 Chemical Methods Ill Quantitative Determination of Nitrogen 108 Total Nitrogen in Soil Ill Total Organic Nitrogen in Soil 112 Total Nitrogen in Microorganisms, Plants and Other Organic Materials 112 Ammonia Nitrogen 112 Ammonia Nitrogen by Aeration 113 Nitrite Nitrogen 113 Nitrate Nitrogen 113 Nitrite and Nitrate Nitrogen 114 Inorganic Nitrogen 114 Qualitative Tests for Nitrogen 115 Organic Nitrogen . 115 Test for Organic Nitrogen and Sulfur when Present Together 116 Ammonia 116 Nitrites -. 116 Nitrates 117 Quantitative Determination of Sulfur 117 Total Sulfur in Soil and Organic Materials 117 Sulfates in Soil 118 Qualitative Test for Sulfur 119 Organic SuKur 119 VIU TABLE OF CONTENTS Page Inorganic Sulfate 119 Hydrogen Sulfid 119 Determination of Phosphorus, Carbon, Dry Matter, Acidity and Magnesium 119 Determination of Calcium 119 Determination of Iron (Total) 119 Determination of Carbon Dioxid 120 Mechanical Methods 121 Collecting Soil Samples for Biochemical Analysis . . 121 Collecting SoU Samples for Bacteriological Analysis 121 Preparation of Soil Samples 122 Sampling of Crops 123 Shaking 123 Preparing a Soil Infusion 123 Ignition of Soil 123 Centrifuge ". . 123 Filtration 124 Cleaning Glassware 124 Autoclave 125 Hot Air Oven 126 Sterilization of Glassware 126 Sterilization of Seeds 127 Sterilization of Nodules 128 Sterilization of Parts of Plants 128 Sterilization of Soil 128 Moist Heat 129 . Dry Heat 129 Volatile Antiseptics 131 Sterilization of Sand 131 Pot Culture Methods 132 Containers 132 Sand Medium 132 Soil Medium 132 Moisture 132 Plant Food 133 Inoculation of Legume Seeds 133 Planting 133 Crops 134 Growth of Plants Under Sterile Conditions 134 Records 134 Suggestions for Instructors and Students Preparing to Teach 136 Acid, Alkah and Other Standard Solutions 136 Indicators 137 TABLE OF CONTENTS IX Page Colorometric Reagents 138 Chemicals Used by Students in Soil Biology 139 Apparatus 141 Special Apparatus 142 PART I. LABORATORY PRACTICES. 1. Pkactices 1-25 2. Class Practices 1-2 3. Advanced Practices. . 1-6 SOIL BIOLOGY PRACTICE 1. EXAMINATION OF MICROORGANISMS IN SOILS AND MANURES. Many kinds of microorganisms inhabit soils and ma- nures. Their presence in soils is essential to agriculture in general since the soil is the basis of all agriculture. It is also true that life would cease on the earth were it not for the activity of these organisms. Study the typical forms as they occur in their natural media as outlined below : (a) Prepare an infusion of each of the following samples: 1. Fresh horse manure. 2. Sandy loam. 3. Rich loam. Place 50 grams in 100 cc. of sterile water contained in a 200 cc. sterile Erlenmeyer flask. Shake vigorously for 5 minutes and then allow it to stand until after the follow- ing procedure has been carefully carried out. (6) 1. Place the microscope on the table in a position which per- mits of comfortable use. 2. Bring the draw tube to standard length. 3. Remove the eyepiece and arrange the plane mirror, using Abbe condenser, so that the field of light is clear and free from obstructions such as window bars, trees, etc. - 4. The illumination should be central. (Transmitted axial light.) 5. Examine with medium power a specimen of algae, diatom, a sand or soil grain, cotton fiber or an air-bubble. 3 4 SOIL BIOLOGY 6. Change the illumination to oblique (transmitted oblique light) by placing the finger below and half over the light opening of the iris diaphragm of the condenser. Note the advantage of oblique light for surface and mor- phological studies. 7. Repeat numbers 5 and 6, varying the opening of the iris diaphragm as follows: Wide open, open |, and f the diameter of the rear lens opening of the objective. This is determined by removing eyepiece and looking into the tube. 8. Focus oil immersion objective and lower it with coarse adjustment until it comes into contact with the oil. Determine this by watching carefully with the head to one side, complete focusing with fine adjustment. Always focus upward when looking into the instrument. Examine each in the following manner: By means of a sterile glass rod remove two drops of the liquid. Place it upon a clean slide. Examine with low, medium, and oil immersion objectives, using a cover slip. In a similar manner, make stained preparations on the slides, using carbol-fuchsin, methylene blue, gentian violet, and iodine, and examine with oil immersion objective. Do not use cover slips for the stains on the slides. INote the different forms present and which one predominates. Examine for cells of higher plants, fungous growths (mycelial and sporal), algae, diatoms, and protozoa (amoebae, ciliates, flagellates). MICROORGANISMS Material Different forms found Predominant class of bacteria Bacillus Coccus Spirillum References. 1. U. S. Dept. Agr., O. E. S., Bui. 194, 6, 7, and 13. 2. Read the booklet accompanying your microscope. 3. Elementary Chemical Microscopy, Chamot (1915), 1-53, 102-158. 4. Bacteriological methods, pages 91-93, this manual. Questions. -1. Which of the microorganisms found are vegetable and which animal? 2. Discuss the distribution of bacteria in soils. 3. How do the microorganisms obtain their food in a soU? 4. Name the sources of the twelve essential elements for soil microorganisms. 5. Which class of the microorganisms is the most important and why? PRACTICE 2. OCCURRENCE OF BACTERIA AT DIFFERENT DEPTHS IN SOILS. QUANTITATIVE BACTERIOLOGICAL EXAMINATION OF SOILS. Fresh samples of the surface soil (6f inches) and the subsoil (at 35^0 inches) are collected in the manner pre- scribed on page 121. Place 100 grams of the soil in a sterile 400 cc. shaker bottle and add 200 cc. of sterile water. Submit the mix- ture to five minutes shaking in the mechanical shaker or by hand. This soil infusion is used for inoculating pur- poses. Allow it to settle 15 minutes to facilitate measur- ing. By means of sterile pipettes make the following dilutions : 2 cc. of the infusion in 98 cc. of sterile water (A) 1-100. 10 cc. of (A) into 90 cc. of sterile water (B) 1-1000. 10 cc. of (B) into 90 cc. of sterile water (C) 1-10,000. 10 cc. of (C) into 90 cc. of sterile water (D) 1-100,000. 10 cc. of (D) into 90 cc. of sterUe water (E) 1-1,000,000. 10 cc. of (E) into 90 cc. of sterile water (F)* 1-10,000,000. 20 cc. of (F) into 80 cc. of sterile water ((?)* 1-50,000,000. Boiling flasks containing the correct amounts of sterile water will be found on the supply sheK. Letter the flasks as above. Place 1 cc. of dilutions (D), (E), (F), and (G), with sodium asparaginate or synthetic agar. Place the 1 cc. in the sterile Petri dish and pour the agar quickly, tilting the dish to effect uniform seeding. Reserve dilu- tions (C) and (D) for further use in practices 3 and 4. When cool, place in the Petri dish containers and invert * (F) and ((?) not necessary if a poor soil. 6 BACTERIA 7 the container. Place in room-temperature incubator. Count plates after 3-^ days. After counting, replace plates in incubator and allow the colonies to develop for a week or 10 days. Make further observations on the growth, using the Society of American Bacteriologists' Chart for descriptive notes. Consult laboratory charts for identifying colony characteristics. Each student should enter his results together with the results of another student using the same soil on the data sheet below. SOIL BIOLOGY Date Soil surface Me- dium Incubation Dilution Num- ber per 1 CO. of dilution Av. per gram of soil No. per acre Notes Temp. Time Average Sub-soil Average BACTERIA 9 References. 1. Agricultural Bacteriology, Percival (1910), 118-124. 2. Iowa Exp. Sta., Research Bui. (1912), 8. 3. U. S. Dept. Agr., O. E. S., Bid. 194, 8-13. Problems. 1. Calculate in pounds per acre the dry weight of the bacteria found in this soil. 500 miUion dry bacteria weigh 0.2 milligram, living 1 milligram. 2. Calculate in cubic feet the volume occupied by the living bacteria in this soil. 100 million occupy 0.2 cubic millimeter. 3. How many pounds of nitrogen and phosphorus are contained in the bacterial bodies of ah acre, as based upon the dry weight figures obtained mider number one ? Analysis of Bacteria. (Dry Basis.) Nitrogen 2.3 per cent. Phosphorus 1.2 per cent. Questions. 1. Do numbers of bacteria in a soil indicate efficiency as to biochemical reactions? 2. Explain the differences fovind between the surface and subsoil? 3. How does the number of bacteria compare with the number of soil particles in a gram of a silt loam? Explain the reason for this difference. PRACTICE 3. OCCURRENCE OF NON-VEGETATIVE FORMS OF BACTERIA AND FUNGI IN SOILS. The number of non-vegetative forms is determined in samples of the surface soil of the same type as that used in the previous practices. Add to each tube of melted agar (synthetic or sodium asparaginate) and melted fungi gelatin 1 cc. of dilution (d). Heat duplicate tubes of each medium at the following temperatures, 70°, 85°, and 100° C. for ten minutes. Pour plates at 40-42° C. and, when cool, place in Petri dish container, invert and place them in the room-temperature incubator. Examine at the end of two days. Count in the manner already described after 3-4 days. Return the plates to the incubator and allow the colonies to further develop for 2 weeks or longer. Note pigment formation and colony characteristics. Observe which form disappears at the various tempera- tures. Calculate the percentage of the total number that are in the non vegetative stage. 10 BACTERIA And fungi 11 Date Soil Me- dium Incubation Dilu- tion Heated to temp. Num- ber per 1 cc. of dilu- tion Av. per gram of soil No. per acre Notes Temp. Time Question. 1. How do you explain the presence of the non-vegetative forms in normal soils? PRACTICE 4. OCCURRENCE OF FUNGI AT DIFFERENT DEPTHS AND IN DIFFERENT SOILS. Prepare 1-10,000 dilutions of the surface soils and 1-1000 dilutions of the subsoils used in practice 2. Plate in duplicate 1 cc. of each dilution employing the fungi gelatin. Incubate at room temperature for 3-4 days or until the colonies are easily recognized as fungi. Make the counts at this time, using the binocular or hand lens. Study the microscopic appearance of the colony. Draw the typical colonies under the binocular. Allow the fruiting bodies to mature. Examine the mycelia and fruiting bodies carefully. Which are septate or non- septate? Are the fruiting bodies perfect or imperfect? The I objective is used for the fruiting bodies. Date Soil Mer dium Incubation Dilu- tion Depth No. per cc. Av. per gram No. per acre Notes Temp. Time References. 1. Household Bacteriology, Buchanan (1913), 50-84, 487-523. 2. Cornell Agr, Expt. Sta., Bui. 315 (1912), 415-419, 437-501. 12 OCCURRENCE OF FUNGI 13 Questions. 1. What kinds of fungi inhabit soils? 2. Which of these kinds predominate? 3. Of the factors necessary for growth which are most important for soil fungi? 4. In the struggle for food how are soil fungi at an advantage? PRACTICE 5. AMMONIFICATION IN SOILS.* INFLUENCE OF MOISTURE CONTENT ON THE PROCESS. Ammonification, which is the production of ammonia from organic compounds by microorganisms, is greatly influenced by the moisture content of a soil. There is an optimum moisture content for all bacterial activities in soils and it is determined as outlined in this exercise with the exception that periodic determinations are usually made while here one suffices to show the influence the moisture factor exerts on this process. On the bulletin board will be posted the soil type, the organic matter (kind and amount) and the amount of sterile water to add to each treatment in addition to that indicated below. Weigh ten 100 gram portions of the air-dry sieved soil into the jelly glasses. Add the organic matter with a sterile spatula and thoroughly mix. Add the sterile water as indicated below with a sterile pipette slowly and evenly throughout the entire mass. Leave the surface level. 1 and 2 6 cc. 3 and 4 12 cc. 5 and 6 18 cc. 7 and 8 24 cc. 9 and 10 30 cc. * Organisms concerned in the liberation of ammonia from or- ganic compounds are not isolated in this course as they have been studied in the general course in bacteriology. They are furnished to those students who wish to further study them from pure cultures. 14 AMMONIFICATION IN SOILS 15 Tlace the tin covers on the glasses and set them in the room-temperature incubator. After 14 days remove and transfer contents to a Kjeldahl flask. Determine am- monia by direct distillation with magnesium oxide as outlined on page 112. Group Blank on Method mgs. Soil Type 1 cc. NH4OH mgs. N. Date Sample num- ber Period of incuba- tion, days Treat- ment HCl < = NH40H Titrated back, NH4OH Equiv- alent in sam- ple, NH4OH Mgs. N Notes - References. 1. Consult the data sheets on the reference shelf. Pfoblems. 1. Prepare a graph on cross-section paper, the abscissas repre- senting percentages of moisture, the ordinates percentages of nitro- gen recovered of the initial applied. Nitrogen content of the materials used wiU be foimd on the bulletin board. 16 SOIL BIOLOGY 2. Show by chemical equations the reactions which yield am- monia from proteia, protein derivatives, amides, and amino acids. 3. ' Tabulate in your laboratory manual the names of ten typical ammomfiers. Questions. 1. Ammonification is the result of cell activity; what are the active agents which enable the cell to assimilate and digest organic material? 2. How does moisture influence these agents? 3. What farm crops are able to utilize ammonia directly? 4. How does ground limestone and rock phosphate influence ammonia production? 5. What is the normal ammonia content of the corn-belt soil? 6. What influence does the mechanical composition of a soil exert on ammonification? 7. How does chemical composition influence ammonification? 8. What effects do the biological factors have on ammonification? PRACTICE 6. AMMONIFICATION OF UREA AND ISOLATION OF UREA ORGANISMS. Place 20 cc. of urea solution in each of six 200 cc. Erlen- meyer flasks, plug and sterilize in the autoclave at 10 pounds pressure for 10 minutes. Inoculate as follows : 1 and 2, nothing (sterile). 3 and 4, 1 gram of fresh soil. 5 and 6, 1 gram of fresh horse-manure. Place in the incubator at room temperature and after 48 hours remove 5 cc. of each treatment with a sterile pipette, filter and titrate against standard acids. Use weak acid for No. 1 and 2, strong acid for the others. Make a second titration in a similar manner 24 hours later. Calculate the per cent of urea changed at each period. Isolation of Urea Organisms. ■ — Pour plates of each of the soil and manure treatments by transferring a loopf ul to 10 cc. of sterile water. From these dilutions, inoculate tubes of sterile, liquefied, and cooled (40° C.) urea agar. Plate as usual. Incubate 2-4 days at 20° C. and transfer after 4 days to other plates and further transfer until pure cultures are obtained. Stain the organisms and examine in a hanging drop. Describe them carefully as to size, shape, motility, and rateof ammonia production. Inoculate tubes containing 10 cc. of sterile urea solution with a loopful from typical colonies. Note turbidity. Titrate to obtain ammonia production at end of 2 days. Determine whether the 17 18 SOIL BIOLOGY animonia is produced under aerobic or anaerobic conditions by heating 10 cc. of urea solution to expel dissolved gases; cool and inoculate, immediately covering the surface with 1 inch of heavy oil and plugging the test tube tightly with cotton. Group Soil Type Blank on Method mgs. N. 1 cc. NH4OH mgs. N Date Sample num- ber Period of incu- bation, days Treat- ment HCl s = NH40H Titrated back, NH4OH Equiva- lent in sample, NH4OH "ff- Notes 85. References. 1. Handbuch der technischen Mykologie, Lafar (1904-6), 3, 71- 2. Centbl. f. Bakt. 2 Abt. (1913-14), 39, 209-358, plates after 358. Problem. 1. Calculate how many pounds of nitrogen, as nitrate, are possible from the lu-ea occurring in the urine of one cow, for a year, if 20 pounds of urine are voided per day. UREA 19 Questions. 1. Write the chemical reactions showing the ammomfication of urea and the calories yielded. 2. Are the common ammonifiers able to decompose urea? 3. Of what special imporiiance are the urea organisms? PRACTICE 7. NITRITATION. OXIDATION OF AMMONIA TO NITRITE BY NITROSOMONAS. Influence Exerted by Carbonates, Ignited Soil, Soluble Organic Matter, and Aeration. (This experiment also illustrates denitrification.) Place 25 cc. of the salt solution for nitrite formation (page 95) in each of 12 one-liter Erlenmeyer flasks (ratio of depth to diameter 1 : 20-22). Make the following additions: 1 and 2, nothing. 3 and 4, 1 gram ground limestone or dolomite. 5 and 6, 1 gram magnesium carbonate. 7 and 8, 50 grams ignited soil. 9 and 10, 50 grams ignited soil + 1 gram limestone or dolomite. 11 and 12, 0.5 gram dextrose + 1 gram limestone or dolomite. Some students will perform this practice using 100 cc. flasks (ratio, depth to diameter, 1 : 3-4), plugging tightly with cotton. Plug the flasks loosely and sterilize in the autoclave at 12 pounds pressure for 15 minutes. When cool, add with a sterile graduated pipette the required amount of a standard solution of ammonium sulfate, carbonate, or nitrate, to give 10 milligrams of nitrogen per flask. Inoculate each with 1 gram of fresh soil obtained pref- erably from some continuous soil or crop experiment. Place flasks in the 30° or the room-temperature incubator 20 NITRITATION 21 as indicated by instructor. At the end of 2, 3, and 4 weeks test all for nitrite by the method outlined on page 116. Show each test to the instructor. The quantitative determination will be made upon advice of the instructor. Determine the nitrite in 1-10 inclusive by the per- manganate method (see page 113). Group Soil Type Blank on Method. . .mgs. N. 1 cc. N/10 KMn04 mgs. N. No. Treat- ment cc. N/10 KMn04 taken cc. N/10 NaS203 titrated Equiv- alent N/10 KMn04 Mgs. N Per cent of initial nitrogen changed Qualitative tests - - - - - - - „ - - - - - - - References. 1. U. S. Dept. Agr., O. E. S., Bui. 194, 57-67. 2. Agricultural Bacteriology, Percival (1910), 134-168. 3. N. J. Agr. Exp. Sta. Ann. Kept. (1908), 29, 117-119. Questions. 1. Write the reaction for this oxidation, vising ammonium sul- fate, carbonate, and nitrate in the presence of calcium carbonate. 2. Do nitrite exist in normal soUs? 3. Are nitrites assimilable by plants? 22 SOIL BIOLOGY 4. What three factors are most detrimental to the growth of this organism mider field conditions? 5. Give the notable exception exhibited by this organism in its nutrition. 6. What is the chief som-ce of this element? 7. Name 10 elements which wiU suflSce to neutralize the acid formed. 8. What substances retard and check nitritation and in what concentrations? 9. What substances accelerate nitritation? 10. Discuss the importance of nitrite production from the stand- point of the liberation of insoluble minerals. PRACTICE 8. NITRATATION. OXIDATION OF NITRITE TO NITRATE BY NITROBACTER. Influence Exerted by Carbonates, Ignited Soil, Soluble Organic Matter, and Aeration. {This experiment further illustrates denitrification.) Place 25 cc. of the salt solution for nitrate formation (page 95) in each of 12 one-liter flasks. Make the following additions: 1 and 2, nothing. 3 and 4, 1 gram ground limestone or dolomite. 5 and 6, 1 gram magnesium carbonate. 7 and 8, 0.025 gram sodium carbonate. 9 and 10, 50 grams ignited soil. 11 and 12, 0.5 gram dextrose + 1 gram ground lime- stone or dolomite. As in the previous practice, this practice will also be conducted with 100 cc. flasks plugged tightly with cotton. Plug flasks loosely and sterilize in the autoclave at 12 pounds pressure for 15 minutes. When cool add to each the required amount of standard sodium nitrite solution which gives 10 milligrams of nitrogen per flask. Inoculate each flask with 1 gram of soil as in the previous practice. Place flasks in the 30° or room-temperature incubator as indicated by instructor. At the end of one week test quahtatively for nitrate by the method given on page 117. At the end of 2 weeks test for nitrites by the method used in the previous practice and then deter- mine nitrate nitrogen in all but 11 and 12 by the method given on page 113. 23 24 SOIL BIOLOGY Group Soil Type . . . . Blank on Method Mes. 1 cc. NH4OH. .Mgs. No. Treat- ment HCI < NH4OH Titrated back, NH4OH Equiva- lent in sample, NH4OH Mgs. N Per cent of ini- tial N changed Qualita- tive tests - - - - References. 1. Centbl. f. Bakt. 2 Abt. (1899), 5, 329, 377, 429. 2. Centbl. f. Bakt. 2 Abt. (1910), 27, 169. 3. Science (1912), 35, 996. 4. Expt. Sta. Record (1908-9), 20, 518, 519-520. 5. Ohio Expt. Sta. Bui. Tech. Series (1915), 7. 6. Ann. Inst. Pasteur (1904), 18, 181-196. Problem. 1. Calculate the pounds of carbon dioxid reduced when 35 pounds of nitrogen are oxidized. Questions 1. What is the function of the base in this reaction? 2. Explain the effect of sodium carbonate. 3. How is the effect represented by the action of sodium carbon- ate obviated under field conditions? NITRATATION 25 4. What is the cause of the action produced by the ignited soil? 5. Name ten nitrites which are changed to nitrates. 6. What substances retard or check nitratation, and in what con- centrations ? 7. What substances accelerate nitratation? 8. Write the reaction for this transformation. 9. Discuss the value of nitrates compared with nitrites, ammonia, and soluble organic nitrogen in soils. PRACTICE 9. ISOLATION AND STUDY IN PURE CULTURE OF NITROSOMONAS AND NITROBACTER. In the isolation of the nitrite and nitrate organisms advantage is taken of the already vigorous growths exist- ing in treatments 9 and 10 of practices 7 and 8. When the qualitative tests have given a strong reaction (consult instructor for advice at this time), follow the procedure outhne below. Isolation of NiTROSOMONAS. — Procedure: 1. Place 1 cc. of the solution (numbers 7 and 8 in prac- tice 7) in a sterile Petri dish. 2. Add 10 cc. of sterile silica jelly (Solution I) in the Petri dish and thoroughly mix. 3. Add 1 cc. of sterile solution of sodium carbonate and ammonium sulfate (Solution II), and immediately tilt to mix the contents of the dish as the jelly solidifies rapidly. Caution should be exercised in the manipulation de- scribed under 3 as rough plates resulting from uneven mixing of the carbonate solution with the jelly make it ■difficult to see the colonies. The plates should be left level until solid. An excess of moisture is undesirable and will not occur if the above procedure is followed. After the medium has become solid, label the plates and place them in Petri dish containers and incubate at 30° C. AHow the colonies to develop until the centers become yellow or orange, when the microscopical study should begin. Test colonies with nitrite and nitrate reagents before transferring. Transfer from typical colonies to magnesium ammonium phosphate agar. When typical colonies are well developed (4-6 days), transfer to the 26 NITROSOMONAS ' 27 sterile magnesium plaster of Paris blocks, which are half submerged in a solution for nitrite formation, in Petri dishes. Inoculate sihca jelly and agar slants. Study the colony characteristic on the various media. Stain the or- ganism with carbol-fuschin, gentian violet, and methylene blue. Study the size and shape, and compare with nitrate organism. Prepare a permanent slide. Inoculate a small sterile flask containing ignited soil and solution for nitrite formation and incubate at 30° C. Test for nitrite pro- duction at the end of 5 days. This method together with the microscopic study will determine if your culture is pure. Isolation of Nitrobacter. — Proceed as in the isola- tion of Nitrosomonas, using instead inoculating solution from 9 or 10, practice 8, and 1 cc. of a solution of sodium carbonate and sodium nitrite (Solution III). Omit the use of magnesium ammonium phosphate agar, otherwise make similar studies with this organism and inoculate a small flask after studying the organism in pure culture. References. 1. Exp. Sta. Rec. (1890), 2, 751-757 (Winogradsky). - 2. Jour. Chem. Soc. (London) (1878), 33, 44 (1898), 59, 484 (Warington) . 3. Jour. Chem. Soc. (London) (1891), 60, 352 (Franklands). Questions. 1. From the qualitative tests which organism is the more rapid grower and what are the comparative rates of oxidation? 2. Give the group number for both these organisms (Soc. Am. Bact. Chart). PRACTICE 10. PRODUCTION OF INORGANIC NITROGEN IN SOILS. COMPARATIVE AMMONIFICATION AND NITRATATION OF CROP RESroUES, GREEN MANURES, AND FARM MANURES IN TYPICAL SOILS. Influence Exerted by Carbonates, Soil Type, Mois- ture, AND THE Condition of the Organic Sub- stances. This experiment is designed to show the weekly am- monia and nitrate production from organic materials, such as are used in agricultural practice and under as nearty similar conditions as possible. Such materials as clover, sweet clover, soybeans, cowpea, and alfalfa hays, corn stalks, wheat and oat straw, and farm manures are applied in both the green and dry condition according to common usage. It would be advisable to use fresh soil for this practice if convenient. The materials applied in the dry condition should all pass a 10-mesh sieve. Green materials may be applied much coarser. This practice is conducted in groups. Students are permitted to use soils from their own farms or ones in which they are particularly interested. Each group of students conducts four sets of treatment on a given soil. Weigh out fifty-six 100 gram portions of the soil to be studied. Place in the jelly glasses and make the following applications : 1-14, nothing. 15-28, 1 gram of carbonate (limestone or dolomite). 29-42, organic matter. 43-56, 1 gram carbonate + organic matter. 28 INORGANIC ' NITROGEN 29 The students are permitted to choose the kind of organic matter they desire to test and the amomits to be added are posted. The sterile water to be added will be found on the bulletin board and is the optimum for the various treat- ments. Mix thoroughly and place glasses in the room-tempera- ture incubator. Each week 1-3 cc. of sterile water 'is added to each glass to compensate for loss due to evapora- tion. Ammonia and nitrate determinations are made on du- plicates of all treatments at the beginning of the experiment, and every 7 days, for 7-10 weeks.* The sample is divided into two equal parts by weight. On one-half determine the ammonia by direct distillation or by aeration. Dry the other half at 108° C. for 6-8 hours in the electric oven, then add 300 cc. of dilute hydro- chloric acid (5 cc. per liter). Shake vigorously several times. Allow the solution to settle a few minutes when 200 cc. is removed by suction. Proceed as indicated for the determination of nitrites and nitrates, page 113. If the total nitrogen content is not known it will be -necessary to determine it on the soil used. The total nitrogen content of the typical soil types and the organic materials will be posted. * It is sometimes convenient to make the intervals 9 and 1 1 days to conform to the laboratory periods. Other materials such as raw rock phosphate are used in this experiment. The number of jelly glasses may be increased to extend through the growing period of a crop, 30 SOIL BIOLOGY AMMONIA DETERMINATIONS. Date Sample No. Treat- ment HCl ^ SNH4OH Titrated back, NH4OH Equiva- lent in sample, NH4OH Mgs. P.P.M. Notes INORGANIC NITROGEN 31 AMMONIA DETERMINATIONS. Date Sample No. Treat- ment HCl =: = NH40H Titrated back, NH4OH Equiva- lent in sample, NH4OH Mgs. N P.P.M. Notes - 32 SOIL BIOLOGY NITRATE DETERMINATIONS. 1 p fl 02 a 1 S £ 1 1 5 , IS 1 PM 1 1 INORGANIC NITROGEN 33 NITRATE DETERMINATIONS, p sa a ^ ■ 34 SOIL BIOLOGY SUMMARY OF THE NITROGEN DETERMINATIONS. Group . Group . Soil Type . Soil Type . Treat- ment Nitrogen as Parts per million of nitrogen in water-free soil Pounds Date of determinations per acre Begin- ning Av. Nitrate Ammonia Nitrate Ammonia Nitrate Ammonia Nitrate Ammonia Nitrate Ammonia Nitrate Ammonia Nitrate Ammonia Nitrate Ammonia References. 1. Soil Fertility and Permanent Agriculture, Hopkins, 194-198. 2. Soil Conditions and Plant Growth, Russell (1913), 78-89. 3. Centbl. f. Bakt. 2 Abt. (1908), 25, 64. 4. Centbl. f. Bakt. 2 Abt. (1910), 27, 169-186. Exp. Sta. Reed. (1910), 23, 721. 5. Hawaii Agr. Exp. Sta. Bui. 39 (1915), 24-25. 6. Jour. Indus. & Eng. Chem. (1915), 7, 521. 7. N. J. Rept. of Soil Chemist and Bacteriologist (1914), 217, 220. Problems. 1. Plot the ammonia and nitrate data for all treatments on cross- section paper, the abscissas representing the time and the ordinates the milligrams of nitrogen as ammonia and nitrate. Use red for nitrate, blue for ammonia and solid, broken, single, and double dotted lines for the treatments. INORGANIC NITROGEN 35 2. Explain the fluctuations of both the ammonia and nitrate curves. 3. Calculate the yield of oats, wheat, and corn possible from the nitrate and ammonia found. 4. Correlate the ammonia and nitrate production on the soil used with crop yields. Questions. 1. Under field conditions is there need of an application of nitrate nitrogen? 2. Does nitratation go on in acid soils? 3. Do non-nitrifiable soils exist? 4. How should a non-nitrifiable soil be treated? 5. How does the crop influence nitrate and ammonia formation? 6. What effect does cultivation have on nitratation? 7. Are nitrifying organisms active in the fall and winter months? 8. What means may be used to check the loss of ammonia and nitrate from soils? 9. Is the loss of ammonia from the brown silt loam appreciable? 10. Why does organic matter not inhibit the growth and ac- tivity of these organisms in soils? 11. What factors are most important in nitrate production in field soils? 12. What factors are most important in ammonia production in field soils? 13. What is the average annual loss of nitrogen per acre? _ 14. Is there any nitrification below the surface soil? 15. How does the nitrate content of a soil vary from the surface soil to a depth of 5 feet? PRACTICE 11. CARBON DIOXID PRODUCTION. Carbon dioxid is produced by respiration of the micro- organisms in soils. It is evolved from soil into the air in large amounts. A large amount of carbon dioxid bathes the soil and liberates insoluble elements by the production of acid and salts. The importance of the carbon cycle is understood. The rate and amount of carbon dioxid evolved depends upon many factors, chiefly the kind, amount, and stage of decomposition of organic matter present in the soil. A determination of nitrogen as ammonia and nitrate makes possible a calculation of the carbon nitrogen ratio of de- composition. Place six 100-gram portions of the soil to be tested in a beaker and add the organic materials. Thoroughly mix and place in 500 cc. Erlenmeyer flasks equipped with glass tubes on the end of which are rubber tubings which may be opened for the admission of air and a pair of Wort- mann valves (Greiner and Friedricks, Cat. No. 3459), one above the other. Into both valves place 10 cc. of stand- ard potassium hydroxid. The carbon dioxid from the soil is collected in the lower valve, while the upper valve serves as a trap collecting carbon dioxid from the air. Arrange as follows : 1 and 2, soil alone. 3 and 4, soil + 2 grams dextrose. 5 and 6, soil + 2 grams organic matter. Place the stopper containing the glass tube and valves in the flask and allow the flasks to remain at room tempera- ture in a reasonably shaded place (dark not necessary), 36 CARBON DIOXID .PRODUCTION 37 Every two days remove the valves and titrate the contents of the lower one as indicated on page 12. At the end determine the ammonia on 50 grams and the nitrate on 50 grams of treatment numbers 5 and 6. Group Soil Type lOcc. KOH cc. . .Acid 1 cc. KOH mgs. CO2 Num- ber Treat- ment Milligrams carbon dioxid P.P.M. total CO2 P.P.M. NH3 P.P.M. NO3 CO2/NO3 ratio Days - - - - ~ - - - - - - f-' - - - ' References. 1. Soil Fertility and Permanent Agriculture, Hopkins, 33. 2. Soil Conditions and Plant Growth, Russell (1913), 78. 3. lU. Agr. Exp. Sta. Bui. 145, 105-111, 121. 4. U. S. Dept. Agr., O. E. S. Bui. 194, 55-56. 5. Iowa Exp. Sta. Research Bui. 3, 135-154. 6. Jour. Agr.'Sci. (1915), 7, 44. 7. Centbl. f. Bakt. 2 Abt. (1910), 28, 45. 38 SOIL BIOLOGY Problems. 1. Calculate how long the carbon supply of the atmosphere over an acre would be sufficient for 100 bushel, crops of corn if micro- organisms failed to maintain the supply. 2. From the figures obtained in this practice, what can be deduced as to the stage of decomposition of the soil and the organic matter used? Indicate this by carbon, nitrogen ratios. Questions. 1. How does carbon dioxid originate from soil organic matter? 2. WMch classes of organisms are most active in carbon dioxid production and under what conditions? 3. What per cent of carbon dioxid is found in a normal soil , atmosphere? 4. What factors are important in the fluctuations occurring in the carbon dioxid content of the soil atmosphere? 5. In what way does carbon dioxid prove injurious? 6. What beneficial action does it produce? 7. How would you overcome the injury arising from planting immediately after plowing under a green crop? PRACTICE 12. CELLULOSE DECOMPOSITION. AEROBIC DECOMPOSITION BY FUNGI AND BACTERIA. Contrary to earlier conceptions, the decomposition of cellulose and fibroiis residues takes place very actively under aerobic conditions. Fungi play a very important role in this decomposition. Place 2 grams of filter paper, cut into squares of about I of an inch, and 2 grams of corn stover or straw in jelly glasses with 100 grams of soil. Add sterile water to make the optimum moisture content. Place all in the 30° C. incubator for 10-14 days, observing the fungous growth at frequent intervals. 1 and 2, paper 2 grams. 3 and 4, straw or stover 2 grams. 5 and 6, soil, only. Plate from 1, 3, and 5 on cellulose and starch agar. Incubate at 30° C. for 3 weeks, then transfer to cellulose and starch agar. At this time expose plates of cellulose and agar to the laboratory air for 5 minutes. Study the dissolving of the cellulose in the zone around the colony. Stain the organisms with carbol-fuchsin. Describe the organism and draw colonies and individuals. Allow 2, 4, and 6 to remain 8-10 weeks, when an examination of the residual cellulose or stover should be made under the microscope. References. 1. Centbl. f. Bakt. 2 Abt. (1904), 11, 689-695; (1908), 21, 700; (1909), 23, 300-304; (1910), 26, 222-227; (1910), 27, 1-7, 449-451, 633. 39 40 SOIL BIOLOGY 2. Centbl. f. Bakt. 2 Abt. (1912), 34, 63, 485-494. 3. U. S. Dept. Agr. Bur. PL I. Bui. 266 (1913). 4. SoU Science, 1916, 1, 437-487. Questions. 1. What kinds of fungi decompose cellulose? 2. What kinds of bacteria decompose cellulose? 3. Is there a selective action exhibited by these organisms? 4. What products are formed in the aerobic decomposition of cellulose? 5. What is the enzyme concerned? 6. Wni the cellulose of a green crop be more easily attacked than that of a dry drop? 7. Name the important processes for which cellulose serves as a source of energy. PRACTICE 13. ANAEROBIC CELLULOSE DECOMPOSITION. In each of five 200 cc. Erlenmeyer flasks, place 150 cc. of solution for anaerobic cellulose decomposition. Weigh accurately 3 sets, of 3 each of 5| cc. diameter filter paper and place a set in each of three flasks, making sure that the papers are entirely submerged. In the other two flasks, place 3 grams of wheat or oat straw. Plug and sterilize at 12 pounds pressure for 10 minutes. Inoculate as follows: 1, 2, and 3 with 5 cc. of a filtered infusion of well- rotted horse-manure. 4 and 5 with 5 cc. of a filtered infusion from surface soil. Place in the room-temperature incubator. After de- composition has started, as judged by the frayed appear- ance of the edge of the paper, transfer, with a platinum needle, a small portion to a tube to be used in isolating the organism. Allow these flasks to remain several weeks or until such a time when the papers are apparently decomposed. Note the odor of hydrogen sulfide. Test with lead acetate paper. When decomposition has progressed suffi- ciently, filter the contents of the flasks on a weighed filter paper, wash with water, dry, and weigh. Report the loss of carbon. Isolate the organisms under anaerobic condi- tions, and test their ability to reduce sulfur compounds and to produce hydrogen sulfide from inorganic and organic sources. 41 42 SOIL BIOLOGY Paper Straw 1 2 3 4 5 Initial weight Final weight , . . . Loss Weight after drying Weight of filter paper used as filter Final weight References. L Microbiology, Marshall (1912), 246-249. 2. Vorlesungen tiber landwirtschaftliche Bakteriologie, Lohnis (1913), 171-177. 3. Centbl. f. Bakt. 2 Abt. (1902), 8, 192-201, 225-231, 257-263, 289-294, 321-326, 385-391 (Omelianski) ; (1904), 11, 369-377; (1904), 12, 33-43 (1906), 15, 673-687. 4. Centbl. f. Bakt. 2 Abt. (1908), 20, 682; (1912), 34, 485-494. Problems. 1. Calculate how many pounds of straw per acre will be de- composed under anaerobic conditions in 6 months at the rate found in this experiment. Questions. 1. Which decomposes more rapidly the straw or pure cellulose? 2. What are the chief products formed? 3. What beneficial purposes may cellulose serve imder anaerobic conditions? 4. Why is an excess of straw or coarse manure often very in- jurious to soil? 5. For what 3 processes does cellulose serve as a source of energy under anaerobic conditions? PRACTICE 14. SYMBIOTIC NITROGEN FIXATION. INOCULATION OF LEGUME SEEDS. Growth of Nodules and Determination of Nitrogen Fixed. {B. radicicola is to he isolated from nodules obtained in this practice.) This practice may be omitted if the students have already had the work given in soil fertility or its equiva- lent. Place 12 pounds of washed sand in each of 6 one-gallon earthen jars and in two others place 10 pounds of brown silt loam. The jars may be rinsed with 1-300 mercuric chloride solution and then with sterile water if recently used for similar work. Select 5 of the larger legume seeds (cowpeas) and 15 of the smaller (clover) and steriUze by the method outlined on page 127. Arrange the jars as below, and place them in the greenhouse. 1 and 2, inoculate with soil. 3 and 4, inoculated by the glue method. 5 and 6, uninoculated. 7 and 8, inoculated (plant in soil). Plant the seeds in the usual manner and water with sterile water. Apply sterile plant food solution every ten days, omitting the nitrogen. After about 10-15 days, depending upon the legume, wash out the plants from numbers 1, 3, and 5, and examine the roots carefully. Study the nodules. Make a drawing showing the manner of attachment, shape, and size, and carefully describe the 43 44 SOIL BIOLOGY appearance as to color and surface. Make cross and longitudinal sections and describe the internal appearance. Compare your description with that of other groups and report the differences found between the various legumes grown. After 20-30 days analyze 3 plants, either green or dry, of each of the inoculated and uninoculated, for total nitrogen. Remove small nodules from some of the plants washed out in 1 or 3 and proceed with practice 15. Sample No. Treat- ment HCl ^ -- NH4OH Titrated back, NH4OH Equiva- lent in sample, NH4OH Mgs. Gain from nodules References. 1. 111. Agr. Exp. Sta. Cir. 86, Bui. 94. 2. lU. Agr. Exp. Sta. Bui. 179, 471-482, 493-499, 503-522. 3. Consult article dealing with Legume Inoculation (Reference Shelf). 4. Vorlesungen iiber landwirtschaftliche Bakteriologie, Lohnia (1913), 361-378. Questions. 1. Explain why the soil and glue methods are superior to the chemical cultures at the present time. 2. Why is the glue method an ideal method? SYMBIOTIC NITROGEN FIXATION 45 3. Explain the method of collecting, storing, and inoculating with soil. 4. Name the legume groups as arranged for cross inoculating under field conditions. 5. What percentage of the total nitrogen is derived from the air by inoculated legumes, on normal soils? 6. Discuss the nitrogen enrichment of a sandy soU, a sUt soil, and a heavy clay soU by this symbiosis. 7. Calculate the gain in nitrogen due to inoculation on the basis of standard crops of cowpeas and soy beans. 8. Through what organs do legumes obtain the atmospheric nitrogen? 9. What American scientists first noted and carefully studied nitrogen fixation? PRACTICE 15. ISOLATION OF B. RADICICOLA FROM LEGUME NODULES. If the previous practice was not conducted, proceed as follows: Plant the various legume seeds in both sand and soil in the one-gallon earthen jars, in the ordinary way, inoculating either by the glue method or by an infusion from the nodule. When the plants in the sand are well started, remove a small nodule with sterile forceps and place in a 1-500 mer- curic chloride solution for 3 minutes, remove and rinse in sterile water, pass into three test tubes of sterile water and finally crush in 1 cc. sterile water with a sterile glass rod or sterile forceps. Pour 5 loopfuls in a test tube of sac- charose-ash-agar, agitate thoroughly and remove 5 loop- fuls to a second tube of the agar. Pour these plates in the usual way and incubate at 30° C. It is essential to transfer several times on the saccharose-ash-agar before making the detailed study. After 10-12 days examine with a hand lens and describe the colony characteristics. Stain the organisms with aniline gentian violet and carbol- fuchsin. Examine in hanging drop. Describe the or- ganisms as to size, shape, motility, and reaction to stain. Make a permanent slide for your own collection. Ex- amine slides of other groups, and make notes on the organisms from the different legumes. Remove an old nodule from a legume plant growing in the soil and examine the organisms in the hanging drop. Draw the bacteroids as you see them. Stain the bac- teroids with carbol-f uchsin, heating to steaming twice with an interval of two minutes between. Examine some of 46 ISOLATION FROM B. RADICICOLA 47 the bacteroids from the liquid-culture media which will be furnished. References. 1. 111. Agr. Exp. Sta. Bui. 179, 482-488. 2. Centbl. f. Bakt. 2 Abt. (1909), 23, 59-91. 3. Vir. Agr. Exp. Sta. Ann. Rpt. (1909-10), 123-142, 145-174. 4. Centbl. f. Bakt. 2 Abt. (1907), 19, 264-272, 426-441. Questions. 1. Are these bacteroids an involution form? 2. How do they differ from bacilli? 3. In what form does the organism exist in the soU? 4. How long are the organisms viable under field conditions? 5. Does freezing of the soil kill the organisms? 6. What effect does drying a soil have on these organisms? 7. WUl these bacteria live in an acid soil? 8. Are these organisms able to fix nitrogen without the legume plant? 9. Discuss the importance of this symbiosis as related to a per- manent agriculture. PRACTICE 16. NON-SYMBIOTIC NITROGEN FIXATION. AEROBIC NITROGEN FIXATION IN SOILS. Influence of Carbonates on the Process. Fifty grams of soil (see posted sheet for soil type as- signed to your group) are placed in each of ten jelly glasses. Treat as follows: 1 and 2, nothing. 3 and 4, 0.3 gm. mannite. 5 and 6, 0.3 gm. mannite and 1 gm. CaCOs. 7 and 8, 15 gms. fresh clover or rye tops. 9 and 10, rich algae slime, place in the direct light. The amount of water to be added will be found on the posted sheet. Place in the room-temperature incubator for four weeks, adding 3 cc. sterile water each week and stirring the soil in all biit 9 and 10 after adding it. At the end of this time dry the soil and grind it to pass 100-mesh sieve and determine the total nitrogen on 10-gram samples of each treatment. The total nitrogen content of the original soil before incubation together with the nitrogen content of the clover, algse, carbonate, etc., wiU be posted. 48 NON-SYMBIOTIC NITROGEN FIXATION 49 Group Soil Type Blank on Method mgs. N. 1 cc. NH4OH mgs. N. -2 .Q ■11 il cog 1 1 2; ^ S S 1 References. 1. Centbl. f. Bakt. 2 Abt. (1901), 7, 561-582. Centbl. f. Bakt. 2 Abt. (1902), 9, 3-43. 2. N. J. Exp. Sta. Ann. Rpt. (1903), 24, 217-286; (1904), 25, 237-268; (1906), 27, 178-187; (1907), 28, 141-169; (1908), 29, 137-147. 3. Wis. Agr. Exp. Sta. Research Bui. 12 (1910). 4. Col. Agr. Exp. Sta. Bui. 155. 5. Soil Conditions and Plant Growth, Russell (1913), 93-95. 6. lU. Agr. Exp. Sta. Bui". 179, 494. Problems. 1. How many pounds of wheat straw, corn stover, and fresh clover tops must be oxidized to yield a fixation of nitrogen sufficient to supply a 50 bushel wheat crop and 100 bushel corn crop? 50 SOIL BIOLOGY 2. Calculate the fixation of nitrogen per acre, subtracting the results obtained in numbers 1 and 2 and the nitrogen content of the added materials from the various treatments. 3. Explain the practical difficulties arising in determining the activity of Azotobader in normal soil, such as a brown silt loam. 4. Explain the difference between Azotobader and B. radicicola in relation to organic carbon and the carbon cycle. Questions. 1. Name the common Azotobader organisms. 2. Why is isolation from a soil easier in the fall or winter? 3. What influence does the reaction of the soil have on the growth of Azotobader? 4. Name the chief sources of organic carbon for nitrogen fixation by Azotobader. 5. What part may algae play in assisting Azotobader fix nitrogen? 6. In what kind of soils does the greatest fixation occur? 7. What organisms usually are found associated with Azoto- bader? PRACTICE 17. NITROGEN FIXATION IN SOLUTION BY SOIL BACTERIA. Place 25 cc. of solution for aerobic non-symbiotic nitro- gen fixation in each of ten • 450 cc. Erlenmeyer flasks. Add 20 grams of ignited soil, plug with cotton and sterilize at 12 pounds pressure for 15 minutes. Inoculate as below with one gram of soil. 1 and 2, brown silt loam. , 3 and 4, gray silt loam. 5 and 6, brown sandy loam. 7 and 8, yellow silt loam. 9 and 10, nothing. Incubate at 30° C. for 25 days and do not disturb the scum any more than possible when transferring. Note any pecuHar odors during incubation. Analyze the con- tents of each flask for total nitrogen. Calculate the fixation. 51 52 SOIL BIOLOGY Group Blank on Method . .mgs. N. 1 cc. NH4OH mgs. N. p E S a c • e b oo c3+i ^ 3 ® ^;^ cort -g (3 o o 3 -Sag -SgM-g 03 — o 2 - ^ p § o "d-g-S ■^ -' 3 g^ ?^s — ' o'ffl °"^ a c3 o. s <" s a j30fl00pc!M3.; "ii a a. a g fcDG=;j 3 S a o §•£3 SS5-3 3.15 g rt3 ^ 3 o S '- ■" j"H;a^;a h'd-o^ 96 SOIL BIOLOGY W g a g o o t— I ~-^ H P o CO O o OW 03 ■ - bO C S ■2 g.2-S ^■^ o tio.2 OJ3 2: 9'3« o o o - o = o.2Sc >}-S -O to o ? "5 o p '^S 12; "^ C« bO C3 g >1 1 d lO - d S T3 o o o fcH H t- cccoco o :3 bC (£ d S d g S S c S £ ., mo ■?g S ° " m:^ Oj? 9 S i S «J--'3 ■2|S-g'igs§ • ^ +^ I o ra 2 ^ o o 5 fl ho rs.ri oj.i o E ca n! nla g S o g S fl t-l F^ .. Ih l-l i-l 3.3.3 3.3 3 o<^<» o<^ o s 3 • s-^ g'^-2 g S OS S-3 3 ,^ O 03 (iHOWPt, SOLID MEDIA (AGAR) 97 p4 < o g o 03 o Pi o Starch. 500 cc. '.o '■ \ ■ :i ! : i : : i° : : ! ; uj ::::'.: ITS j :-(d : : : : : -^ : - ; a _o 1 o m '.•SB i ; : i : i : : i : i : : : : d o m • ■ 6 ■ ■ « : :§ ■ -o Fungi gelatin. 1000 cc. : Iff, ; : ■ : :=^ ; lo !(M j • ; ;d 1 1 1 1 8 : : : 1" : : : : : : ! '. • : • .(M • • J 1 •1 ^ CO ■in ■ ■ ■ "5 • : : : : : i"^ : : » -i .2'3 s M g. l\s\\ : : : : : : : : : -u^c H . -o • ■ ■ -o g o ■- ., ; : ; ; ; : T3 : 3 : m : : - .1 1 o O 1 Bouillon d 2 o o o 3- "S" Peptone. . ._ Starch solution Dextrose Maltose Mannite solution Mono-potassium phosphate Mono-ammonium pnospnate Magnesium ammonium phosphate Ammonium sulfate Magnesium sulfate Potassium sulfate Sodium nitrate Ammonium nitrate Sodium nitrite Ferric chloride Sodium chloride Calcium carbonate Sodium asparaginate 98 SOIL BIOLOGY » So m S O lis .2.2 1 . o o ;co CO o o S a ho O 0) ? ©, a :2S-: ^.2 qacoocoflc ca S3 ca.,i< o.,i o o3 B iggSPSQSg«J 3.2c"|oa°i sgsSog.gsa gpH SPECIAL MEDIA 99 SPECIAL MEDIA. Silica Jelly. — This solid medium possesses the ad- vantage of being exceptionally selective for the growth of the nitrite and nitrate organisms. Its successful prepara- tion and use require careful observance of the following details. Procedure. — Standardize a sufficient amount of a solu- tion of sodium silicate to last several years. This is easily accomplished by adding hydrochloric acid, filter, wash, dry, and weigh as silicic anhydride (Si02). Stock solu- tions containing 4-5 per cent of silicic anhydride are pre- pared from this solution as desired. Prepare a 4-5 per cent solution of silicic anhydride in water and a standard solution of hydrochloric acid of equivalent strength, using methyl orange as the indicator. This (4-5 per cent silicic anhydride) stock solution of sodium silicate will keep indefinitely. When it is desired to prepare the jelly for plating proceed as follows: Solution I. 106 cc. standard hydrochloric acid. Add the following salts to the acid. Poxu" the sodium silicate solution in the acid. 100 cc. sodium silicate solution (4-5 per cent silicic anhydride). 200 milligrams di-potassium phosphate. 100 milligrams calcium chloride. 40 milligrams magnesium sulfate. 1 drop ferric chloride. Add methyl orange as indicator and sterilize in the auto- clave at 12 pounds pressure for 15 minutes. Dilute two 10 cc. portions to 100 cc. and titrate against solution II, III, or IV (sodium carbonate solution 7.5 grams per liter), to determine the amount necessary to solidify the jelly. One cc. only should be added and if more is required, add more carbonate to the solution. 100 SOIL BIOLOGY Solution I should not stand more than 5-6 days before using. It is best used immediately. Solution II For nitrite organisms Sodium carbonate 7.5 grams per liter Ammonium sulfate. ... 10 grams per liter Solution III For nitrate organisms Sodium carbonate 7.5 grams per liter Sodium nitrite 10 grams per liter Solution IV For nitrite and nitrate or- ganisms Sodium carbonate 7.5 grams per liter Ammonium sulfate .... 5 grams per liter Sodium nitrite 5 grams per liter Solutions II, III, IV, when it is necessary to sterilize them, should not be autoclaved, but the dry salts should be placed in sterile water in sterile flasks. Pour plates as below: I. Place 1 cc. of the solution to be plated in the sterile Petri dish. II. Add 10 cc. of Solution I and thoroughly mix. III. Add 1 cc. of Solution II, III, or IV as desired and tilt to mix. The jelly solidifies rapidly, changing in color from red to pale yellow, and care must be exercised to keep the plates level. Place in the tin boxes and incubate at 28° and 22° C. Transferring the organisms from the silica jelly to ammonium, magnesium, phosphate agar, and nitrite agar has been found very advantageous, as they grow more rapidly on these media than on the silica jelly, but for first plating the silica jelly is unexcelled. Magnesium Plaster of Paris Blocks. — These blocks are sometimes used for the growth of nitrite, ni- trate, and nitrogen fixing organisms. SPECIAL, MEDIA 101 Procedure. — Thoroughly mix 1 gram of magnesium carbonate with 200 grams of plaster of Paris and slowly add water until the mass becomes pasty. By means of a spatula spread the mixture on a piece of glass and mark into the desired shape (squares are very convenient for use in Petri dishes). When dry they may be pried loose and stored indefinitely. When desired, place in Petri dishes or other receptacles and pour in enough of the nitrite, nitrate, or nitrogen fixation solution to half submerge the block. Sterilize in the autoclave at 10 pounds pressure for 10 minutes. Inoculate with 1 cc. of infusion or 3 loopfuls of medium. Cellulose Solution (Scales Method) . — Dilute 100 cc. of concentrated sulfuric acid with 60 cc. of distilled water in a 2 liter Erlenmeyer flask. Keep the acid at 60° C. Moisten 5 grains of white ribbon S. & S. filter paper with water and add to the acid. Agitate until the paper is dissolved and quickly fill the flask with cold tap water. Do not take over 1 minute for this operation. Filter the precipitate and wash free of acid and then make up to 500 cc. with water. To prepare a solution for anaerobic studies or the agar consult the table of formulae for solid and liquid media. Reaction of Cultuhe Media. — When it is desired to bring the medium to a definite degree of acidity or alkalinity, proceed as follows: Place 5 cc. of the solution in an evaporating dish or casserole. Add 45 cc. of water and boil 1 minute. Add 1 cc. of phenolphthalein, and then run in from a burette A7'/20 NaOH or HCl until a faint but stable pink or no color with HCl remains after 1 minute of boiling. Make several titrations and average the results. The formula, cc. iV/20 NaOH or HCl used , 20 • ^■•^- 1000 cc, makes the calculation of the number of cc. of normal 102 SOIL BIOLOGY HCl or NaOH necessary to be added to neutralize one liter a simple matter. X is the number of cc. of normal alkali or acid required to exactly neutralize one liter of medium. The amount of alkali or acid actually added to any medium to give a definite degree of alkalinity or acidity is easily calculated as below. One cc, of normal alkali or acid per liter is designated as +1° for the acid and —1° for the alkali on Fuller's scale. Neutral solutions are indicated as on this scale. Example. — Prepare a medium +8° on Fuller's scale and one —8°. When the initial titration was made with NaOH then X — 8 = number cc. normal alkali to add to give +8°. X -\- 8 = number cc. normal alkali to add to give —8°. When the initial titration was made with HCl, then X -\- 8 = number cc. normal acid to add to give +8°. X — 8 = number cc. normal acid to add to give —8°. The erroneous use of per cent of acidity or alkalinity in this connection is omitted here. If the student meets this method in the literature, he should consider only degrees + or — as they are cc. of normal acids or alkalies per liter and from them correct calculations may be made. Per cent cannot be relied upon as indicative of normality or hydrogen ion concentration. The student and instructors as well should bear in mind that true acidity means hydrogen ion concentration. In adjusting the reactions of solutions where accuracy is desired, they had best be titrated cold or after sterilizing as some of the constituents of certain solutions react and hydrogen is lost. Many of the culture media outlined in this manual require no adjustment of the reaction. In most experi- ments for soil bacteria when it becomes necessary to adjust the medium, +8° is commonly used. STAINING AND PREPARATION OF STAINS 103 STAINING AND PREPARATION OF STAINS. Simple Staining. In general, the simple stains are most satisfactory in soil biological studies. For simple staining the procedure below should be followed: 1. By means of clean cornet forceps pass the clean slide through the flame three times to remove the alcohol. 2. By means of a sterile platinum loop place a small drop of sterile water in the center of the slide; this serves to give a better spreading of organisms and to indicate to the student if his slide is clean. With a sterile loop place the bacterial substance in the water and spread. 3. Air dry and fix by passing through the flame 3 times, film side up. With the forefinger at the tip of the forceps and describing a circle about 1 foot in diameter one round per second there is no danger of overheating. 4. Flood with the stain desired. Heating is permissible if carefully done and sometimes is a great advantage. Allow Loeffler's methylene blue and gentian violet to act 3 minutes, carbol-fuchsin 30 seconds, iodine 5 minutes, Lugol's iodine 3 minutes, picro-sulfuric acid aigrosin 24 hours, Bismarck brown 3 minutes. 5. Wash with distilled water, taking care not to injure the film. 6. Dry and examine with the oil immersion lens. 7. For permanent mounts the stain should be made on the cover ,slip and mounted in Canada balsam properly labeled, with date, name of student, and organism. Special Staining. Without the aid of special stains the presence and study of flagella, capsules, spores, and other characteristics would be unknown. Many of these methods require extreme care and patience to obtain successful results. 104 SOIL BIOLOGY ' Flagella Stain. The staining of flagella is perhaps the most difficult method encountered by the soil biological student. At times it is easy to stain flagella and at other times difficult. The cultures should be repeated transfers of young and moist colonies from agar, not over 48 hours old and should be examined in the hanging drop first to see if they are motile. Proceed as in simple staining until after spread- ing the bacteria, when they should be allowed to diffuse one-half hour; then spread with platinum needle and fix by gentle heating. Apply the mordant by filtering it upon the cover glass, warm the cover glass, allow mordant . to act 4-6 minutes, wash with water and apply stain im^ mediately, allow warm stain to act for 3 minutes. Wash with water, dry, and examine. Loeffler's Flagella Stain. 1. Mordant (warm, allow to act 4-6 minutes). Solution of tannin (20 per cent in water), 10 cc. Saturated cold aqueous solution of ferrous sulfate. Remove iron oxide by filtering. Use pure crystals, 5 cc. 2. Stain carbol-fuchsin (warm, allow to act 3 minutes). Spore Stain. (Hansen's Spore Stain.) It is sometimes desirable to stain for spores (endospores) and for this purpose, make a simple stain first which will partially demonstrate the presence of spores. Fix as usual and wash with chloroform to remove fat. Wash with water and dry. Fix again and then drop on carbol- fuchsin and heat 5 minutes over a flame, renewing the stain as it boils away. Nearly decolorize in dilute acetic acid (5 per cent). Wash and counterstain with dilute aqueous methylene blue or Loeffler's methylene blue. Wash, air dry, and examine. STAINING AND PREPARATION OF STAINS 105 Capsule Stain. In selecting bacteria for capsule staining a viscid growth on agar or a slimy appearance on the surface of a solution suggest the presence of capsulated forms. The capsules do not take up the simple stains sufficiently strong owing to their mucilaginous nature. The capsules surround the cell wall and when several capsules are united the mass is the so-called zooglea. Hiss Capsxjle Stain. ' Spread film, dry, and fix; then stain with a 5 per cent solution of gentian violet and steam slightly, wash at once with 20 per cent copper sulfate solution, dry between filters, and examine. Nodule Tissue Stain. (Flemming's Triple Stain.) This method of stain is especially adapted to differen- tiating tissues in the nodule (Microtome sections). The section is first placed in: (1) Safranin (sat. al. solution) 50 cc. Distilled water 50 cc. Aniline water 5 cc. After washing in water, it then goes into (2) : (2) Saturated aqueous solution of gentian violet. It is then washed in water and passed into (3) : (3) Aqueous solution orange G. or other suitable stain, strong or weak (about one-half saturated). Protozoa Fixative, Stain, and Mount. (Martin and Lewin Method.) Place soil in some receptacle which will give a large ratio of depth to diameter and add through a funnel to the bottom' of the soil layer enough of picric fixative to cover the soil and shake disk immediately. 106 SOIL BIOLOGY Picric Fixative. Picric acid, 1 per cent 100 cc. Alcohol, 70 per cent 100 cc. Float cover slips marked to indicate film side on the surface to obtain film. Stain and mount by the method below: Films may be obtained by the corrosive fixative. Mercuric chloride (sat. solution) 100 cc. Alcohol, 70 per cent 100 cc. Treat picric films as indicated below and corrosive films likewise, omitting No. 1 : 1. Corrosive solution 2 minutes 2. Alcohol, 70 per cent + iodine in potas- sium iodide 5 minutes 3. Water, distilled. [ hsematoxylin 1 gram water, distilled 1000 cc. sodium iodate 0.2 gram alum 50 grams, 5 minutes alum 1-2 grams, 3 times water 100 grams 6. Tap water (till blue) . 7. Alcohol, 70 per cent 5 minutes 8. Eosin in absolute alcohol 3-5 minutes 9. Absolute alcohol I 1 minute 10. Absolute alcohol II 1 minute 11. Xylol I 2 minutes 12. Xylol II 1 minute Mount in balsam and preserve to be used to demon- strate the presence of active protozoa in normal field soils. Protozoa Stain. The use of certain stains serves to emphasize some of the functions within the cell and to differentiate the structures. Neutral red (1-800 physiological salt solution or tap water) distinguishes nucleus, nutrition vacuoles (alkaline 4. Hsemalum a 5. Wash with b STAINING AND PREPARATION OF STAINS 107 yellowish red), acid fermentation granules, and fatty granules. Neutral violet colors the metachromosomes. Bismarck brown (1-20,000-30,000) colors the nutrition vacuoles. Congo red proves acid in the nutrition vacuoles. Litmus and ahzarin suKate also prove this. Vesuvin stains bac- teria in the nutrition vacuoles of protozoa a very brown color. Methylene blue and gentian violet in equal parts in aqueous solution are useful for distinguishing protozoa. Alg^ Fixative and Stain. (Picro-nigrosin . ) This stain has the advantage of fixing the organism and staining at the same time. It is useful in collecting specimens on that account. It should be allowed to act 24 hours. Combine in equal parts the aqueous solutions of picric acid and nigrosin. FORMUL.^ OF STAINS. It is of advantage to have certain stock solutions of the stains and of other materials which do not deteriorate and which are used in large amounts. FoKMULuE OF Stock Solutions of Simple Stains. Methylene blue 7 grams Alcohol, 95 per cent 100 cc. Gentian violet 8 grams Alcohol, 95 per cent 100 cc. Fuchsin 4 grams Alcohol, 95 per cent 100 cc. Aniline 10 cc. DistUled water ; 500 cc. The stock solutions are best made by the application, of heat. The aniline should be shaken for 10 minutes and filtered through filter paper (this solution will not keep over a week). 108 SOIL BIOLOGY Simple Stains. The simple stains are prepared as follows: It is always best to filter the stock solution. 1. Methylene blue: Saturated alcoholic solution methylene blue 30 cc. Potassium hydroxide, 0.01 per cent solution 100 cc. 2. Carbol-f uchsin : Satm-ated alcoholic solution fuchsin 5 cc. Carbolic acid, 5 per cent solution 45 cc. 3. Aniline gentian violet: Aniline solution 50 cc. Saturated solution gentian violet 8 cc. 4. Lugol's iodine solution: Iodine , 1 gram Potassium iodide 2 grams Water, distilled 300 cc. 5. Iodine solution: Iodine 3 grams Alcohol, 70 per cent 100 cc. 6. Picro-sulfuric acid: Picric acid 2.5 grams Sulfuric acid, cone 5 cc. Water, distilled 1000 cc. 7. Picric acid, alcoholic: Picric acid 10 . grams Water 1000 cc. Alcohol, 70 per cent 1000 cc. 8. Nigrosin, alcoholic: Nigrosin 1 gram Alcohol 196 cc. Water 4 cc. 9. Nigrosin, aqueous: Nigrosin 2 grams Water, distilled 1000 cc, 10. Bismarck brown: Bismarck brown 2 grams Alcohol, 70 per cent 100 cc. 11. Fuchsin, aqueous: Fuchsin 1 gram Water 100 cc. STAINING AND PREPARATION OF STAINS 109 FoEMUKE OP Stock Solutions op Disinfectants. 1. Carbolic acid 50 grama Water, distilled 1000 cc. 2. Carbolic acid 20 grams Alcohol 100 cc. 3. KOH (sticks) 1 . 25 grama Water, distilled 1000 cc. 4. Corrosive sublimate, HgCl2 sat. in water.. . . 1000 cc. Alcohol, 70 per cent : 1000 cc. 5. Corrosive subUmate, HgCU 1 gram Water 300 cc. 6. Corrosive sublimate, HgCl2 1 gram Water 500 cc. 7. Corrosive subUmate, HgCU 1 gram Water 1000 cc. 8. FormaUn alcohol: Commercial formalin (40 per cent formal- dehyde) 2 cc. Alcohol, 70 per cent 100 cc. 9. Formalin aqueous: Formalin 2 cc. Water, distilled 98 cc. 10. Formalin strong for preserving specimens: Formalin 4 cc. Water, distilled 100 cc. Special Stains. 1. Orange G.: Orange G. 1 gram Water, distilled 100 cc. 2. Safranin : Safranin 1 gram Alcohol, 95 per cent 50 cc. Water, distilled 50 cc. 3. HsematoxyUn: Saturated solution arnmonia alum 100 cc. Add drop by drop solution of hsematoxylin 6 cc. alcohol 1 gram Expose to air one week. FUter and add 25 cc. 'glycerin and 25 cc. methyl alcohol. Let age 2 months before using. 110 SOIL BIOLOGY 4. Carbol-methylene blue: Methylene blue 1.5 grams Absolute alcohol 10 cc. Titrate in an evaporating dish and add gradually carbolic acid, 5 per cent aque- ous solution 100 cc. 5. Eosin aqueous: Excellent for cell contents and cellulose walls. Eosin 1 gram Water 100 cc. 6. Hsemalum : Hsematoxylin 1 gram Alcohol, 95 per cent, hot 50 cc. Then add to: Alum 50 grams Water, distilled 1000 cc. Cool, let settle, filter, and preserve from mold by vise of a crystal of thymol. Fixatives. 1. Chromic acid, 1 per cent: Chromic acid 1 gram Water 100 cc. 2. Osmic acid, 2 per cent : Osmic acid 2 cc. Water, distilled 100 cc. 3. Picric acid: Picric acid 1 gi;am Water. 100 cc. Alcohol, 70 per cent 100 cc. 4. Corrosive fixative: Corrosive sublimate, sat. solution 100 cc. Alcohol, 70 per cent 100 cc. For other information on stains, fixatives, and special methods consult texts on bacteriology, Plant Anatomy, by Stevens, Methods in Plant Histology, by Chamberlain, Behrens Tabellen, bei W. Behrens, and botanical literature. CHEMICAL METHODS. The chemical methods described in this manual have -been selected from the various possibilities in the different fields of chemistry, after thorough and painstaking research conducted under the conditions required in soil biological studies. QUANTITATIVE DETERMINATION OF NITROGEN. In all these methods determinations should be made in duplicate and blank determinations run on all reagents. All analyses are reported in milligrams per 100 grams of water-free soil, or air-dry soil, and as pounds per acre. Total Nitrogen in Soil. — The Kjeldahl method modified to include nitrateS is the most reliable method for the determination of total nitrogen in soils. Procedure. — Place 10 grams of soil (5 grams if an alkali or marine soil, 2 grams if a peat) in a 500 cc. Kjel- dahl flask, add 20 or 30 cc. sulfuric acid (according to organic matter content) containing 1 gram of salicylic acid, mix thoroughly and add 5 grams of sodium thiosul- fate, heat slowly at first; after 10 minutes boiling add 1 drop of metalhc mercury (0.6 gram), continue digestion until the contents are grayish in color (about 2 hours), add potassium permanganate to a permanent pink color, transfer ,to a liter Kjeldahl flask (glass), using 250 cc. nitrogen-free distilled water. Place the receiving flask containing the standard acid in position and turn on the steam and air of the pipe distillation apparatus. Add the required alkali (60 cc.) containing the potassium sulfide to the Kjeldahl flask, connect with apparatus and distill at least 40 minutes, obtaining about 200 cc. of distillate. Titrate the distillate against ammonium hydroxid with sodium alizarin sulfonate or cochineal, as indicator. Ill 112 SOIL BIOLOGY Total Organic Nitrogen in Soil. — Employ the method previously described, omitting the salicylic acid and sodium thiosulfate. This method is very reliable where nitrates are not a factor in soil studies. Total Nitrogen in Microorganisms, Plants, and Other Organic Materials. — The following method developed in this laboratory is a modification of the Kjeldahl-Gunning Arnold method and has given excellent success. It is rapid, accurate, and convenient. This method has been used constantly for determining nitrogen in green and dry plants and under these conditions" it is unexcelled. Procedure. — Place the sample (0.2-1 gram) in the 500 cc. Kjeldahl, add 20 cc. sulfuric acid, about 6-8 grams of potassium bisulfate (fused) and mercury as in Kjeldahl method. Digest 1^-2 hours. Proceed to distill as in the Kjeldahl method, using eitlier pipe or tank distillation apparatus. The acid and alkali should be about A'^/20 for accuracy. Sulfuric acid and sodium hydroxid are used when the amounts of nitrogen are small. If nitrates are a factor, apply the salicylic acid and sodium thio- sulfate modification and delay the addition of the mer- cury until reduction has proceeded 10-15 minutes. Titrate as usual. Ammonia Nitrogen. — The ammonia in soils, espe- cially in ammonification studies where large amounts are present, is most easily and satisfactorily determined by direct distillation with magnesium oxide. A slight hydroly- sis occurs, but aside from this the method is very reliable in the hands of students with the pipe distillation appara- tus used in this laboratory which has entirely eliminated the usual troubles. Procedure. — Place 100 grams (more may be used) of soil, either dry or moist, in a liter Kjeldahl flask, add 250 cc. water and 6-8 grams magnesium oxide. Place receiving flasks in position, turn on steam and air and connect CHEMICAL METHODS 113 Kjeldahl flasks with apparatus, light burners, and distill 45 minutes. Titrate as in total nitrogen method. Ammonia Nitrogen by Aeration. — The aeration method for the determination of ammonia is accurate and applicable to small amounts. The method herein described is a modification of the Folin method applied to soil and was developed in this laboratory. Procedure. — Place 50 grams of fresh soil (dry may be used) in a 500 cc. Kjeldahl flask, add 100 cc. water and 5 grams of heated magnesium oxide, connect with a 400 cc. shaker bottle containing standard sulfuric acid (iV/20) . The apparatus is set up in battery of as many as 20 or more, and connected with a vacuum pump run by a motor. The air is washed with sodium hydroxid and sulfuric acid and drawn through the apparatus for 17-19 hours, or con- veniently over night. The titration is made against weak alkali (A7'/30), using rosolic acid indicator where small amounts of ammonia are present. Nitrite Nitrogen. — The determination of nitrite nitrogen in solutions is easily accomplished by the follow- ing method in which ammonia and nitrate do not interfere. Procedure. — ■ Filter the soil or the medium containing the nitrite into a 250 cc. Jena beaker and wash residue 2-3 times with distilled water, add 50 cc. of dilute sulfuric acid (4 CC.-4000 water) very slowly, keep cool, and then add an excess of iV/10 potassium permanganate, let stand 5 minutes, and then add 5 cc. of 10 per cent potassium iodide solution and titrate the free iodine with iV/10 sodium thiosulfate. (Hot starch solution may be used but is not necessary.) 1 cc. iV/10 permanganate = 0.7005 milli- grams of nitrogen. Nitrate Nitrogen. — The determination of nitrates alone is made by the Devarda method or the aluminum re- duction method. Ammonia and nitrites must be expelled. Procedure. — (1) The filtered solution or acid .extract of a soil is first made alkaline with nitrogen-free potassium 114 SOIL BIOLOGY hydroxide and boiled until the ammonia is expelled, which requires about 20-30 minutes, depending upon the solution and the amount of ammonia present. Acidify with strong acetic acid, adding it frequently during evaporation. Evaporate to dryness on steam bath and take up with 5 cc. acid and again run to dryness. The procedure in both methods is identical to this point. (2) In the Devarda method transfer to a 500 cc. Kjeldahl flask using 200 cc. water and add 0.5 gram De- varda metal and 4 cc. potassium hydroxid (300 grams per liter), distill one hour using heat, collecting in standard hydrochloric acid. Titrate as usual. (3) In the aluminum reduction method after running to dryness, the salts are transferred to a reduction tubs, 50 cc. of water added, and 1-4 cc. nitrogen-free potassium hydroxid, a strip of aluminum, and reduction allowed to proceed over night. With solutions high in organic matter reduction is slow, and, the solutions should be tested for absence of nitrites or nitrates with diphenylamine sulfuric acid or a little Devarda metal should be added when dis- tillation is carried out. Distill 40 minutes and titrate as usual. Both these methods are accurate and convenient. The Devarda method is more rapid. The aluminum method is convenient where large numbers of analyses are to be made, and especially where only a limited amount of apparatus is available during a laboratory period. Nitrite and Nitrate Nitrogen. — The determina- tion of both nitrites and nitrates is made by either the Devarda or aluminum reduction methods. Procedure. — The solution should be made alkaline and the ammonia expelled; and then follows the procedure as outlined in the method for the determination of nitrate nitrogen under 2 or 3. Inorganic Nitrogen. — The determination of total mineral nitrogen is made by direct reduction and dis- tillation with Devarda metal or by reducing in the cold CHEMICAL METHODS 115 and then distilling with the aluminum, taking care in the latter method to make the water in the trap acid, as ammonia may be given off if present in large amounts. Qualitative Tests for Nitrogen. . Organic Nitrogen. — Ignite a small portion of the substance on platinum foil and test the residue for inor- ganic salts. Test some of the original substance for nitrate. Prepare a stock solution by placing a piece of clean metallic sodium (sodium is kept in kerosene; remove and clean so no vapors of the oil will be mistaken for the sodium vapors) the size of a pea in a small 2-inch test tube. Place test tube in an iron clamp attached to an iron stand, add a little material and heat until the vapors of sodium form a layer | inch high. Place 3 drops of the liquid, or if a solid an equivalent amount, letting it fall at intervals of one to two seconds upon the sodium vapors, taking care not to let the substance touch the side walls. Quickly add a second piece of sodium and ignite strongly. By means of a pair of forceps carefully lower the hot tube into 10 cc. of distilled water in a beaker. Warm, filter, and use this stock solution for the tests of nitrogen and sulfur. It may also be used to test for chlorine, bro- mine, and iodine, if desired. If ammonia is a factor, add strong alkali and note the odor of ammonia. Organic am- monium salts give the nitrogen test and odor with alkali. Procedure. — Boil a few cc. of the alkaline stock solu- tion for two minutes with five drops FeS04 solution and one drop of FeCls solution. Cool, acidify carefully with HCl. If the precipitate does not disappear, leaving a blue or bluish green precipitate or a clear yellow solution, warm gently. Cool, and filter through a clean white filter paper and wash. A blue precipitate of Prussian blue shows the presence of nitrogen. Often, if iodine is present, a blue coloration may appear at this point. To distinguish from 116 SOIL BIOLOGY the nitrogen test, wash the filter with alcohol to dissolve out the iodine. Test for Organic Nitrogen and Sulfur when Present Together. — Acidify one cc. of the stock solu- tion with HNO3 and add a drop of ferric chloride. A deep red coloration is due to the formation of ferric sulfo- cyanide. Always carry on tests for organic nitrogen and organic sulfur together with this test. Ammonia. — Place a few drops of the solution to be tested in a test tube or a Nessler tube, add 10 cc. or 50 cc. of water to mark on tube, and then add 0.1 cc. Nessler reagent. Yellowish coloration indicates ammonia. Test is accurate to 0.00025 of a part per million. Large amounts of ammonia are best shown by the ammonium chloride test. Whenever possible distill a portion of the solution to be tested. Nitrites. — The value of this qualitative test depends upon its use in the presence of nitrates and ammonia. The Greiss method is well suited to demonstrate the presence of nitrite. It is a delicate test and cannot be relied upon to indicate quantity unless performed as below. Place 0.2 cc. of the solution to be tested in 10 cc. of water, another 0.2 cc. in 20 cc. of water in test tubes, add 0.5 cc. sulfanilic acid, and then 0.5 cc. of alpha naphthyl- amine acetate freshly made up. Note the number of minutes required at room temperature (22°-24° C.) for the color to appear as faint, medium, and strong. Compare with the table found below : Time, min. Color 1 mg. of N 1 1 3 as nitrite from 50 cc solution in 10 cc 10 cc 20 cc 10 cc 11-2 5 5 1 3 3 5 12 i-i faint medium faint 3 " 10 cc strong 3 " 20 cc. ... 3 3 20 cc " 20 cc medium strong strong strong 5 " " 10 CO. . . 10 10 cc CHEMICAL METHODS 117 A standard solution will be available for comparison. Always run blanks on water and reagents. Nitrates. — The most useful test for nitrates where nitrites are present is the paratoluidine sulfate reagent. Nitrites do not interfere permanently and 100 parts per million of nitrogen as nitrite may be present and not cause error on the nitrate determination. There m.ust be at least 80 parts per million of nitrogen as nitrate to give a reliable test. This test serves to show nitrates in sufficient amounts to be relied upon for making transfers and to determine when quantitative analysis should be attempted. Place 1 cc. of solution to be tested in a test tube, add an equal volume of concentrated sulfuric acid without mixing the liquids; then add 4 drops of the reagent paratoluidine sulfate solution. The test should stand 3-5 minutes. A red ring at the point of contact indicates the presence of nitrates. Nitrites give a yellowish brown coloration.. There is present at least 4 milligrams in 50 cc. of solution when a good red ring develops after 3 minutes. Brucine sulfuric acid is a delicate and excellent reagent for nitrates, giving a red coloration which later becomes yellowish red. Diphenylamine sulfuric acid gives an excellent blue colora- tion with the nitrate, while the nitrite color is brownish. Quantitative Determination of Sulfur. Total Sulfur in Soil and Organic Materials. — The total sulfur content of a soil or crops is determined by the bomb combustion method. Procedure. — The materials are first finely ground. Place 5 grams of soil, or 1 gram organic material, in the bomb cup and add 12 grams sodium peroxide for the soil, 3 grams for the organic niatter, 1 gram magnesium, powder, thoroughly mix using caution not to scatter the sodium per- oxide. Place the cover on the bomb cup and make tight with the lock nut. Heat until bottom of bomb is red. Cool in water. Transfer fusion to beaker using warm 118 SOIL BIOLOGY water, acidify with concentrated hydrochloric acid (34 cc. for 12 grams peroxide), adding 10 cc. in excess of neutrahty. Evaporate to dryness on steam bath; take up with 1-1 HCl and again evaporate to dryness a second time. Take up with hot water and filter hot. The filtrate and wash- ings are heated to boiling and 15 cc. of a ten per cent solu- tion of barium chloride added with constant stirring. Place on steam bath and keep warm six hours. Filter and wash free of chlorides, dry and burn to constant weight, adding sulfuric acid after the first weighing and again burn to expel excess acid. Weigh as barium sulfate. 7.04 mgs. of BaS04 — 1 mg. S. Another more convenient and a rapid method is that of ' the sulfur photometer. With the sulfur in solution ready for precipitation proceed as below. Make slightly acid with hydrochloric and make up to 250 cc. in a graduated flask. Mix and take out 10 cc. to which 90 cc. of water is added. Place the 100 cc. in an Erlenmeyer flask, add 0.3-0.5 gram of barium oxalate powder (equal parts barium chloride and oxalic acid), cork immediately, and shake occasionally for 20 minutes. Adjust the graduated tube in the water in the crystallizing dish so the rounded end is under water (| inch of water). Use the electric fight.* Darken the room with the black shades. Place some of the solution in the separatory funnel, admit solution in tube until the last tip of the cone of the light just disappears. Remove tube, read in milli- meters the depth of liquid. Repeat reading three times, using same solution. Refer to the chart and report as milligrams of solution per 100 grams of soil and as pounds per acre. This method is accurate to 0.2 or 1 per cent and gives excellent results for sulfur in soils. Read the standard solution and report it with the results obtained. Sulfates in Soils. — Place 100 grams of soil in a 400 cc. shaker bottle, add 200 cc. of water acidified with hydrochloric, 5 cc. per 1000, and shake for 7 hours in the CHEMICAL METHODS 119 mechanical shaker, filter and make up to 250 cc. and pro- ceed as in the total sulfur determination, using sulfur photometer. Qualitative Test for Sulfur. Organic Sulfur. — To 1 cc. of the stock solution pre- pared for organic nitrogen test, add acetic acid and then lead acetate. A black precipitate indicates sulfur. Inorganic Sulfate. — Add barium chloride to the solution. A white finely divided . crystalline precipitate indicates sulfate. Hydrogen Sulfide. — For the gas use moistened lead acetate paper; add lead acetate in acetic acid solution. Determination of Phosphorus, Carbon, Dry Matter, Acidity, and Magnesium. Total phosphorus, carbon (total organic and inorganic), and magnesium in soils. Organic materials and raw rock phosphates. The methods given in "Soil Fertility Laboratory Manual," by Hopkins and Pettit, are used for these deter- minations. Determination of Calcium. Proceed as in the manual referred to above with the exception of titrating the oxalate against N/10 potassium permanganate instead of weighing as CaO. 1 cc. N/10 KMn04 = 2 mgs. Ca. Determination of Iron. Total Iron. — The total iron is determined by reduc- ing all the iron present to the ferrous iron and then oxidiz- ing to ferric iron by A^/10 permanganate. Procedure. — Place 20 cc. of the solution to be analyzed in a 250 cc. beaker, add 50 cc. water and 15 cc. concen- trated sulfuric acid. Add zinc dust and heat if neces- 120 SOIL BIOLOGY sary. Test a drop for complete reduction by placing it on the porcelain plate in contact with ammonium thiocyanate. Red coloration indicates ferric iron. If no ferric iron is present cool and titrate with N/10 potassium perman- ganate. 1 cc. iV/10 KMn04 = 0.0056 Fe, = 0.0072 FeO, = 0.0080 FeaOs. Carbon Dioxid. — The carbon dioxid evolved during decomposition of organic residues is determined by collect- ing it in standard potassium hydroxid solution contained in fermentation valves. Remove the valve, wash the contents with 200 cc. hot water into an Erlenmeyer flask, add 3-5 cc. of a ten per cent solution of neutral barium chloride, shake, let stand a few minutes and titrate the excess potassium hydroxid with standard acid preferably of equivalent strength. 1 cc. N/2 KOH = 11 mgs. CO2. MECHANICAL METHODS. Collecting Soil Samples for Biochemical Analy- sis. — The collection of soil samples for biochemical studies should be carried out in the same manner as sam- pling for soil analysis which consists of taking 16-20 borings per tenth acre plot, by means of the soil auger or soil tube. This ensures a representative sample. Duplicate samples are taken to a depth of 6| inches, 20 inches, and 40 inches as desired. The borings are mixed and the required amount placed in a properly labeled Mason fruit jar. This makes the work comparable and meets the practical requirements. Collecting Soil Samples for Bacteriological Analysis. — A different problem is presented in sampling a given area for bacteriological analysis than that of sampling for soil analysis. The irregular results obtained from samples which include the surface two inches have caused them to be discarded in taking the sample. It has also been found that the soil auger is responsible for con- tamination, especially when the subsurface and subsoil are sampled. The sampling is accomplished by removing the surface two inches with a sterile spatula and then with a sterile spatula dl*awing the sample. Place it on a sterile oilcloth or in a sterile soil pan, mix, and place the composite sample in a sterile container. This does not disturb the soil materially, and suffices for surface samples. A special soil tube which is constructed of brass, pointed at one end^ and which is plugged with cotton at the other end, has recently been recommended. Such a tube made longer than the one proposed by Noyes (Jour. Am. Soc. Agron. (1915) 6, 239) should prove valuable for obtaining samples 121 122 SOIL BIOLOGY deeper than 6f inches. For studying the bacterial flora at greater depths than the surface 6f inches the pit method has been employed. It consists in digging a pit to the required depth to which it is desired to obtain the last sample. The sides of the pit are cut down with a spade as sharply as the soil will permit, and then they are sterilized either by direct flaming or scraped with a sterile spatula. The samples are then removed from the sides at right angles to the vertical axis. This may be done with a sterile soil tube or a sterile spatula. The sample is treated as in the other method outlined from this point. The container used may be either a sterile cotton- plugged bottle or a sterile fruit jar. The spatulas and other apparatus are sterihzed by flaming in the field. (An alcohol lamp is used.) A sterile agate pan is a suitable substitute for the oilcloth. The samples should not be taken when the wind is blowing over a light breeze unless the soil is moist on the surface. Early in the morning, 6-9 o'clock, is usually the best time for taking samples. The error due to contamination of the sample in the field is very slight if the above conditions are fulfilled. The soil samples are used in the desired experiments immediately after collection. Preparation of the Soil Samples. — Soil samples for biochemical analysis of field experiments are not dried for the determination of ammonia. For the moisture and nitrate determinations the samples are dried in an electric oven at 108° C. for 8 hours. Samples collected for bac- teriological analysis are immediately used for inoculation or incubation. Samples intended for laboratory practices (to be used from December to April) are air-dried; sieve to remove stones and large roots and place in barrels or soil bins, for use later. Some soils are pulverized, but in no case are they ground as in soil analysis. Dry soil is satisfactory MECHANICAL METHODS 123 for demonstrating the principles involved in most soil biological studies. A supply of fresh, moist soil is always maintained in the greenhouse which serves any time as a source of fresh inoculating material. Sampling of Crops. — The use of common farm materials instead of artificial materials, such as casein, blood meal, etc., is taken as the standard in these studies. Samples of hays, straws, stover, and such like should be collected in the field so that their history may be accu- rately known. Decomposition is related to the stage of development of crops and it is, therefore, of advantage to know the condition of sample. Samples should be air- dried and ground if used in beaker experiments. Grind- ing to pass a 2-mm. or 10-mesh sieve is sufficient. A card catalogue of all soil samples, limestones, phosphates, and crop samples is a great convenience. The analysis is entered on the card together with the other necessary data. Farm and green manures are collected and used whenever possible. Shaking. — A mechanical shaker is convenient for use in preparing soil infusions and soil extracts. Preparing a Soil Infusion. — The bacteria are satis- factorily removed from the soil particles by shaking the soil with water. Place 100 grams of soil in a 400 cc. sterile shaker bottle and add 200 cc. sterile distilled water. Place a clean rubber in the bottle and shake for 5 minutes. Allow solution to settle 15 minutes and then use as desired by means- of sterile pipettes. Ignition of Soil. — Ignited soil is used in culture solu- tions, especially for nitrite and nitrate bacteria. Place the soil in an iron or nickel crucible and burn at a red heat until all the organic matter is completely oxidized. Large quantities may be burned in a kiln and stored for use. Centrifuge. — A rapid separation of bacteria and soil can be made by use of the centrifuge. It is also valuable in obtaining extracts containing enzymes and the Uke. 124 SOIL BIOLOGY Filtration. — ' Soil solutions are filtered for chemical determinations and for making soil extract media. A battery of suction filters is of great advantage in obtaining clear soil extracts. A rapid filtration can be made through glass wool or absorbent cotton. Most soil biological media are filtered through cotton. The Berkefeld and Pasteur- Chamberland filters are indispensable for obtaining bac- teria free filtrates. Cleaning Glassware. — It is absolutely necessary that all glassware shall be perfectly clean. Acids, alka- lies, and organic matter do not permit equal distribution of the solutions to be examined. The test tubes, Petri dishes, and flasks are cleaned in the following way: Boil in soap and water for 10-15 minutes or immerse in the following hot cleaning solution and leave over night: Potassium bichromate 60 parts Concentrated sulfuric acid 460 parts Water 300 parts (Add acid slowly with constant stirring.) Wash with water, rinse with distilled water, and invert to dry. Test tubes may be easily dried in the hot-air oven. The tumblers are washed in the ordinary way with tap water, rinsed with distilled water, and inverted to dry. It is well to plug the test tubes and flasks with cotton when clean and dry. (See the instructor about rolling plugs.) For most glassware which is used for chemical work simple washing in tap water with a cleanser, then with distifled water and finally rinsing with distilled water is sufficient. Dipping in weak hydrochloric acid and then rinsing in distilled water is efficient for a great deal of the glassware employed in the determination of nitrogen. Cleaning cover-slips and slides requires especial atten- tion since the success of flagella staining and obtaining MECHANICAL METHODS 125 good permanent preparations depends to a great extent upon clean slips and slides. The following method has given best results in this laboratory. Wash in distilled water, boil 10 minutes in strong nitric acid, remove and wash in distilled water (do not handle the slips or slides with your hands or dirty forceps), and then wipe dry. Place in absolute alcohol in containers used only for this purpose. Test the cover-slips and slides with distilled water to see if they are clean by dj-awing the film over the surface at will. If alkali is used caution must be exercised as hot alkali or strong alkali dissolves the glass, producing an etched appearance. Autoclave. — The autoclave represents sterilization by moist heat under pressure. It is by far the most satis- factory means of sterilization for most media, solutions, and materials that will stand heating under pressure. The autoclaves 4B and 2B are connected with high- pressure steam. To operate, proceed as follows: Open cocks under doors and lower cock under autoclave. Turn on slowly the high pressure steam cock (upper cock under autoclave), one-fourth turn at first, increasing gradually later. The table below is a guide to the use of the autoclaves. Material Pressure, pounds Time. Water 12 12 15 15 10 15 15 10 min. . 30 min. Urea, dextrose Sand 15 min. Soil 6-8 hours In sterilizing soil, wait until all the air is expelled from it t)efore closing the autoclave. For such masses use the maximum thermometers in the interior of the mass during sterilization. 126 SOIL BIOLOGY TABLE OF AUTOCLAVE — TEMPERATURES AND PRESSURES. Steam pressure, pounds Temperature. C. F. 100 109 115.5 118.0 121.5 126.5 131 134.5 212 5 228 10 240 12 244 15 250 20 260 25 268 30 274 Hot-air Oven. — The hot-air sterilizer has four com- partments. The burners are in the lower compartments. To operate turn on the gas by pulling the levers at either end. Light burners and close doors. When the ther- mometer in the door registers 350° F. (20-25 minutes), pull the levers at each end shut. Usually the oven is loaded with all the glassware to be needed for a long time or with duplicate sets and allowed to run 5-8 hours. Sterilization of Glassware. — Invert the clean Petri dishes and place them in the round seamless tin boxes* assigned for this purpose. Place the cover on the box and sterilize by heating in the hot-air oven at least 1 hour at 350° F. The boxes are inverted before opening to prevent possible contamination of the Petri dishes. Pipettes are handled as follows: Plug the mouth end with cotton and then place them in the horizontal copper boxes used in sterilizing them. Heat in the hot-air oven as above. It is not always necessary to plug the pipettes if commonplace caution is exercised in their use after sterilizing. Tumblers, cylinders, and other glassware which cannot * A 24-ounce round seamless tin box used in the manner above has been foimd to give excellent satisfaction. The boxes should be heated several hours before using the first time, owing to their being lacquered. MECHANICAL METHODS 127 be safely subjected to dry heat or steam sterilization can be effectively sterilized by letting them stand in (1-300) solution mercuric chloride for 20 minutes and rinsing with sterile water. Glass bottles, lipless Jena glass beakers, or tile pots can be used in place of tumblers and permit of sterilization in the autoclave. Sterilization of Seeds. ' — It has been found extremely difficult to completely sterilize seeds for use in experiments where sterile conditions are required in the beginning or throughout. A satisfactory method is wanting for all kinds of seeds. However, in a great deal of the work, com- plete sterilization is not necessary, especially is this true of legume studies. To recognize the possibilities and elimi- nate the necessity of complete sterilization will often spell success in these experiments. A solution of mercuric chloride (1-500) for 10 minutes gives an efficient sterilization against legume, non-symbi- otic nitrogen fixers, nitrite, nitrate, ammonifiers, and many other organisms but does not insure kiUing mold spores. Solutions of mercuric chloride, hydrogen peroxide, cop- per sulphate, bromine, silver nitrate, and suKuric acid sometimes give rather unsatisfactory results owing to the persistence of air bubbles on and inside the seeds. Hutchinson and Miller used a solution of mercuric chloride in a vacuum apparatus. This obviated the trouble from air bubbles. Treatment with a 5 per cent solution of chloride of fime for three hours has given good results at this laboratory. Harrison and Barlow suggest a method which is suited to special investigations. Pods are picked from plants while they are yet green. They are then washed in 1-1000 mercuric chloride for one hour and dried in folds of sterile cotton. The pods are then burned by holding in a flame with sterile forceps, after which they are opened and the seeds placed in folds of sterile cotton. When dry they 128 SOIL BIOLOGY are removed to plugged sterile test tubes by means of sterile forceps. Large seeds with tough seed-coats are taken in forceps, dipped in alcohol (95 per cent) and passed through a low flame. Cowpeas and soybeans have been successfully treated in this way in this laboratory. Sterilization of Nodules. — The nodules are effec- tively sterilized by treatment with 1-500 mercuric chloride for 3 minutes, washing in sterile water, and then crushing in another portion of sterile water in a sterile container with sterile glass rod. Sterilization of Parts of Plants. — It is sometimes necessary to sterilize the stem or leaf of a plant for inocu- lation purposes. It has been found that 1-1000 mercuric chloride rubbed into the leaf or stem will not only give very satisfactory sterilization but seems to penetrate the tissue and tends to keep the cells sterile. In inoculating experi- ments this fact should be considered as it may destroy the inoculation. For further methods consult Irwin Smith's "Bacteria in Relation to Plant Diseases, I." Sterilization of Soil. — It is often necessary to sterilize a soil for experimental purposes. This subject has been studied by many workers and the present methods are included below. It is important for the student of soil biology to note the changes which have been found to occur in the steriliza- tion of a soil, as they account for the irregular behavior observed in many experiments. It will be recalled that sterile conditions have been stated as being very unsatis- factory for plant growth. Some of the reasons are to be found in the following paragraphs. The three common methods are: 1. Moist heat (autoclave). 2. Dry heat (hot-air oven). 3. Volatile antiseptics. MECHANICAL METHODS 129 Moist Heat — The autoclave is the only satisfactory- means of attaining complete sterilization aside from fire. The changes which occur increase with a rise in tem- perature. The chemical changes brought about by steam under pressure in the soil are many and complex, but only a few are of importance. These are the nitrates, nitrites, am- monia, and precipitation reactions, such as formation of insoluble calcium, phosphorus, and iron compounds. Lyon and Bizzell concluded that 30 pounds pressure for two or four hours reduced the soil nitrates and nitrites to ammonia. They also found a great increase in the am- monia content after sterilization. This ammonia origi- nates chiefly from organic matter. Schreiner and Lathrop found a notable increase in many forms of organic nitrogen, an increase in water soluble constituent and in acidity upon treating at 30 pounds pressure for three hours. Thus it is seen that steam sterilization greatly increases the soluble matter of a soil and changes the organic matter more than either of the other methods. The biological effects are evidenced by complete death of all forms of life in the soil and the deleterious influence exerted on plant growth for 2-3 months after treatment. Some investigators have noted very great beneficial results to plant growth after sterilization by this method, planting, however, after the soil has been weU aerated. Sometimes this increase amounts to 4-10 times the crop obtained on the unsterilized soil. The physical characters suffer in a similar way as in the dry heat method which is described below. In order to determine correctly the temperature of the interior of the mass of soil a maximum thermometer should be inserted into the soil. Dry Heat. — The use of dry heat for soil steriUzation changes the chemical, biological, and physical properties of the soil. 130 SOIL BIOLOGY The chemical studies have shown that the nitrogen undergoes changes. The total nitrogen may or may not remain constant according to its state of decomposition or according to the type of soil. Some soils yield ammonia and volatile organic nitrogenous decomposition products when heated much above 110° C. The soluble nitrogen is greatly increased by the heating due to changes brought about in the insoluble forms, and the amount made soluble in this way is related to the moisture content of the soil. Heating at 200° C. changes the organic matter of soils. At this temperature the soluble material is increased from 6 to 10 times. Heating at 100° C. and 250° C. increases the solubility of all the mineral constituents except sodium in both water and fifth normal nitric acid as solvents. At 100° C, there is an increase in water-soluble calcium, magnesium, phos- phorus, sulfur, and bicarbonates. Potassium, silicon, and aluminum increased in half the soils tested while iron de- creased in most instances. Heating at 250° C. or ignition produced similar results. At 150° C. nitrates decomposed while at 200-250° C, practically total destruction of nitrates took place. Am- monia was produced in large amounts at 200° C, At 200° C, 25 per cent of the total nitrogen was lost. The life of the soil is rendered almost extinct for the time being when a soil is heated at 95° C, or above. This temperature kills the vegetative stages of most of the plant life and the active stages of animal life. The spores are not destroyed completely until ignition is reached. The fauna and flora of any soil may be changed at any temperature above 45-50° C. Russell and Hutchinson found from two to four times the crop on a soil heated at 95° C. as on an unheated soil. Many results indicate that plants grow better on heated than unheated soils. Physically the differences appear to be in the increased MECHANICAL METHODS 131 capillary and absorptive power of the soil as determined by Richter in 1896, when he heated a soil to 100° C. for six hours on three consecutive days. Volatile Antiseptics. — This method is being studied with great interest at the present time in many laboratories. At best, it is only partial sterilization acting very similar to dry heat at 98° C. Carbon bisulphide and toluene are used most commonly. Chloroform, ether, xylol, and others may be employed for partial sterilization. Four per cent of toluene is effective. This method of treatment kills the living organisms but does not injure the spores or the encyst forms of life. The life of the soil greatly increases after this treatment and plant growth is more vigorous than on untreated soils. The increased chemical results noted are due to biological changes. Ammonia and nitrate production are greatly increased at first. Unless kept under sterile conditions, these differences gradually subside. Air-drying a soil rapidly or soils which have been dried a long time give similar results when the soil is again placed under normal conditions. This is due to a suppression of the fauna and flora during drying. The rate of multiplication of the bacteria under such conditions makes them the predomi- nant form o life and the consequent yield of ammonia and nitrate is high at first, but later, when the soil remains under normal conditions, the numbers and activities fall to the same level as fresh or untreated soil. Sterilization of Sand. — No permanent changes are caused by sterilizing sand. It should be well aerated after sterilization before being used as a medium for plants. The ignited soil is unchanged by sterilization. 132 SOIL BIOLOGY POT-CULTURE METHODS. The value of data obtained in pot-.culture experiments is recognized by the soil biologist. Under the controlled conditions afforded in the well-equipped greenhouse many facts, unsolvable under field conditions, are established. The following paragraphs are included to assist the student in considering the essentials for successful pot- culture experiments. Containers. — The one-gallon earthen jar, one-gallon battery jar and the four-gallon earthen jars are satisfac- tory containers. A galvanized pot of the Wagner type is excellent as the water may be added beneath the surface. Evidence of zinc poisoning has been obtained in experi- ments of long duration with zinc pots. For accurate studies glass jars are preferable although the cost and break- age are high. The one-gallon earthen jar has a capacity of 10 pounds of soil and 12 pounds of sand. Drainage is provided by placing a glass tube in the bottom. Sand Medium. — Crystal white sand is an excellent medium for studying the elements of plant food. It should be washed free of salts with hydrochloric acid and with distilled water until free of acid. When great ac- curacy is required other methods such as ignition and reduction are necessary. Soil Medium. — Soil should be used as the medium ultimately and whenever it will not vitiate the results. The previous history is desirable. It should be sieved and mixed carefully before being placed in the container. Clay soils are more difficult to experiment with owing to their preventing drainage and root development. Car- bonates will greatly decrease this trouble. Moisture. — The optimum moisture content should be established and maintained throughout accurate experi- ments. This in many cases is determined by an empirical method. Weighing the jars at 4-7 day intervals and POT-CULTURE METHODS 133 adding water in equal amounts during these periods to all treatments has proved very satisfactory. Plant Food. — The plant food elements are con- veniently applied in solutions as given in "' Soil Fertility Laboratory Manual," Hopkins and Pettit. Calcium car- bonate and dolomite are applied as the dry salt, 10 grams per gallon jar. Inoculation of Legume Seeds. — Excellent results are obtained when inoculating sand and soil cultures by the following method. Wash the nodules to be used for inoculation with mercuric chloride 1-300 for 10-12 minutes; then wash in sterile water and finally crush in another portion of sterile water. This avoids the trans- ference of an unnecessary number of organisms not con- cerned in the legume experiment. Place a few cubic centimeters (5 cc. per seed if large or with small seeds 10-20 cc. per jar) of this infusion on each seed before covering with sand or soil. This method is the simplest and best for pot-culture experiments. A few nodules are sufficient to make several liters. It is not necessary to sterilize with the mercuric chloride solution in all cases. This method of inoculation has never failed to give excel- lent results at this station. Pure cultures from the laboratory or a soil infusion may be used with good results. Where added soil will not affect the experiment, it may be employed for inoculation. The glue method may also be used. Planting. — A careful selection of seeds to be planted is important. Irregular seeds and those with ruptured coats should be discarded. In many experiments the seeds should be accurately weighed in order to have a check on their chemical composition. A high per cent of germina- tion is desirable. Usually it is advisable to plant a few more seeds than necessary for the final stand so that they may be thinned to a definite number per jar which number depends upon the size of the jar and the kind of plant. 134 SOIL BIOLOGY Crops. — Plants differ in their adaptability to grow under greenhouse conditions and only experience can make fine distinctions as to the best choice of crop. As a rule, annuals are more uniform in their growth than biennials or perennials; this is especially true of the legumes. Cowpeas, soybeans, and the cereal crops are well suited to these methods. In the selection of the crop, the temperature should be considered as well as the light values. The fall is a poor time for the growing of most crops as the light is not intense enough. Warm- weather crops will stand the tem- perature of the greenhouse even in summer. Sudden changes in temperature should be avoided as they are disastrous. Plant diseases and insects are a serious menace to greenhouse experiments and a careful operator should be employed to regulate the temperature and fumigate the house properly. Distilled water sprayed on the plants checks the ravages of red spiders. Sulphur may be found useful as a temporary remedy for mildews. An interesting case of contamination came under the writer's observation in which red spiders carried the cowpea organisms from one jar to another until all became inoculated. Growth of Plants under Sterile Conditions. — This is not a desirable method of experimentation owing to the abnormal conditions which it involves. It is, how- ever, sometimes necessary to grow plants under sterile conditions in order to establish the influence of a definite factor or to determine a specific reaction. Bell jars, WouLfe bottles, beakers, or battery jars covered with cotton will be found of service in this connection. A side tube jar is often valuable for such studies. Erlenmeyer flasks and test tubes can be used by plugging with cotton. Various devices have been tried with partial success. Records. ■■ — Accurate records must be kept of all the POT-CULTURE METHODS 135 facts of importance, such as the history of the soil, plant food* applied, moisture content, kind of seed, number of seeds planted and the number allowed to remain, irregu- larities in growth, injuries due to insects, or from other causes, and of greatest importance, the weights or yields. Photographs should be taken as their worth may prove inestimable. Measurements of growth are often valuable data. The harvested materials are usually air-dried or oven-dried and placed in properly labeled receptacles for storage until ready for analysis. SUGGESTIONS FOR INSTRUCTORS AND STUDENTS PREPARING TO TEACH ACID, ALKALI, AND OTHER STANDARD SOLUTIONS. The number of standard solutions required in a course of this kind is necessarily greater than in courses dealing with but a single field of chemistry or biology. Experience has demonstrated the value of the solutions herein found: - Solution. Use. 1. iV/20 NaOH Titrating media and for use in titrating small amounts of nitrogen. 2. N/20 HCl Titrating media. Not used very much as most solutions are at the correct reaction. 3. iV/1 NaOH Correcting reaction. 4. N/l HCl Correcting reaction. 5. N/KNO3 Acidity determinations. 6. N/7 HCl Nitrogen determination when amount is sufficient to give more than a dif- ference of 2 cc. in titration. 7. N/7 NH4OH Used as above. 8. Ar/20 H2SO4 Titrating small amounts of nitrogen. 9. iV/1483 KOH Det. P. 1 cc. = 0.2 mg. phosphorus. 10. iV/1483 HNO3 1 cc. = 0.2 mg. phosphorus. 11. N/IO KMn04 1 cc. = 2 mg. Ca, calcivun nitrite. 1 cc. = 0.7005 mg. nitrite, iron deter- mination. Nitrite and iron determinations. Phosphorus determination. Culture media. Culture media. Culture media. Nitrite determination. 136 12. AT/lONazSaOs.... 13. 6 per cent FeCls. . 14. 10 per cent FeCla. 15. 10 per cent NaCl. 16. 10 per cent CaCU 17. 10 per cent KI. . . ACID, ALKALI, AND OTHER SOLUTIONS 137 18. Reduced KOH (300 grams per liter) solution Nitrite and nitrate determinations. 19. Ammonium molyb- date solution .... Phosphorus determination. 20. Saturated ammo- nimn oxalate solu- tion Calcium determination. 21. Nessler solution ... . Ammonia determination. 22. NaOH (2 pounds per hter (26.6 grams K2S) Nitrogen determinations. INDICATORS. The following indicators have been selected from a large number tested as being most reliable. FormuloB of Indicator. Alkali and Acid Color and Use, 1. Sodium alizarin sulfonate, 1 gram AlkaUes red — acids yellow. Alcohol, 60 per cent, 100 cc. . . In all nitrogen determinations where ammonia is distilled. Best indicator where H2S is not (See 6. RosoUc acid.) present. Volatile organic bases do not affect this in- dicator as much as the others. 2. Cochineal, 3 grams AlkaUes violet — acid yellow- ish-red. Macerate in the solution below: Water, 200 cc In nitrogen determinations Alcphol, 60 cc. where sulfides or sulfur dis- tUl over in amoimts suffi- cient to obscm-e the end point of number 1. This indicator is only .slightly affected by HjS or CO2. 3. Lacmoid, 3 grams Alkalies blue — -acids red. Water, 700 cc Alternative for Number 1. " Alcohol, 300 cc. This indicator is affected by HoS. 138 SOIL BIOLOGY 4. Methyl orange, 1 gram Alkalies yellow — acids red. Water, 1000 cc Strong acids. Not affected by H2S or CO2 . . . Sodium silicate, hydrochloric acid, sodium carbonate. 5. Phenolphthalein, 1 gram Alkali red — acid "colorless. Alcohol, 50 per cent, 100 cc. . Phosphorus, media, acidity. Very sensitive to CO2 Valuable for weak alkalies,* carbonates, alkali earths. Useless for NH4S Bicarbonates neutral to this indicator, organic acids. 6. Rosohc acid (commercial), 1 Alkali rose red — acid yel- gram low. Alcohol, 60 per cent, 100 cc . . . Ammonia titration when aluminum or copper are used Useless for acetic acid. Not for reduction. Use sodium affected by ammonia but by hydroxid and sulfuric acid ammoniima salts. as large amounts of am- monium salts interfere. COLORIMETRIC REAGENTS. Soil solutions and other turbid solutions which retain their color upon filtering through the ordinary filters may be decolorized by use of aluminum cream (alkali-free). Two to three cc. per liter usually suffices; repeat if necessary. It is best, however, to avoid as much as possible the use of such materials on account of occlusion. FormulcB of Reagents. Use and Color. 1. Diphenylamine sulfuric acid. . . . Nitrite — brownish blue. Diphenylamine, 1 gram Nitrates — blue. Sulfuric acid (cone), 100 cc Rings on a palate. Small amount of ferric salts do not interfere Delicate test. 2. Brucine suKuric acid Red ring shows nitrates, Brucine, 1 gram Delicate test. , Sulfuric acid (cone), 100 cc. 3. Paratoluidine sulfate Red ring with nitrates. Paratoluidine, 0.5 gram Not delicate. Sulfuric acid (cone), 100 cc. Very well adapted to indicat- ing nitrates when more than 2-3 mgs. of nitrogen present in 25 cc. of solution. CHEMICALS USED BY STUDENTS 139 4. Alpha naphthylamine acetate Nitrites. Very delicate. See and sulfuric acid qualitative test for nitrites, page 116. (a) Alpha naphthylamine, 1 gram. Acetic acid, sp. gr. 1.04, 200 cc. (b) Sulfanilic acid, 1 gram. Acetic acid, sp. gr. 1.04, 250 cc. Use equal amounts of each when making a test. The . alphanaphthylamine should be made up fresh each 2-3 days. A list of chemicals and apparatus have been included as a guide and represent the materials which have been found essential with the present state of development of the course. For special work a larger number of sugars, organic acids, organic salts, stains, and special chemicals than found in the list included are necessary. A greater variety of chemicals and apparatus are necessary in a course of this kind owing to the varied nature of the experiments. CHEMICALS USED BY STUDENTS IN SOIL BIOLOGY. Acid, acetic. Agar agar shredded. ' carbolic. " " powdered. ' chromic. Alcohol, ethyl. ' citric. Alpha naphthylamine. ' hydrochloric. Aluminum metal strips. ' molybdic. " metal powder (coarse) ' nitric. " metal powder (fine). ' oxalic. " and potassium sul- ' osmic. fate. ' picric. Ammonium carbonate. ' salicylic. hydrate, sp. gr. 0.90 ' sulfanilic. " nitrate. ' sulfuric. " oxalate. 140 SOIL BIOLOGY Ammonium diphosphate. " monophosphate. " sulfate. " thiocyanate. Anilin oil. Asparagin. Balsam, Canada. Barium chloride. Barium oxalate powder. Barium hydroxid. Blood meal. Beef extract. Brucine. Calcium carbonate (ground lime- stone) . " acetate. " chloride. " nitrate. " oxide. " triphosphate. " diphosphate. " monophosphate. " sulfate. Calcium sulfate (gypsum). Calcium sulfate (plaster of Paris) . Carbon bisulfide. Casein. Chloroform. Cleanser. Collodion. Copper suUate. Dextrin. Dextrose. Diphenylamine. Dolomite. Ether. Feldspar. Ferric ammonium citrate. " wire. " chloride. Ferrous sulfate. Formaldehyde. Gelatin. Glass wool. Glycerin. Glue (furnitiu-e). Hydrogen peroxide. Iodine. KaoUn. Kieselguhr. Lacmoid. Lead acetate. " oxide. Lysol. Magnesium ammonium phos- phate. " carbonate. " chloride. " metal " oxide. " sulfate. Maltose. Manganese sulfate. Mannite. Mercury metal. Mercuric chloride. Metal, Devarda's alloy. Oil, immersion cedar. Oil, cedarwood. Oil, cylinder, heavy, light. OU, cloves. Oil, paraffin. Faratoluidine. Paraffin, M. P., 52° C. Paraffin, M. P., 40-42° C. Paraffin, M. P., 50-53° C. Peptone, Witte's. Potassium bichromate. Potassium bisulfate (fused). Potassium carbonate. Potassium chloride. Potassium hydroxid. Potassium iodide. Potassium nitrate. Potassium permanganate. APPARATUS 141 Potassium phosphate, mono- basic. Potassium phosphate, dibasic. Potassium sulfate. Potassium sulfide. Rock phosphate. Saccharose. Silver nitrate. Silver nitrite. Sodiimi asparaginate. carbonate. chloride. hydroxid. (metal) . nitrate. nitrite. peroxide. sUicate. sulfate. sulfide. Sodium thiosulfate. Stains, Bismarck brown. Eosin. Fuchsin. Gentian violet. Hsematein. Hsematoxylin. Methyl blue. Methylene blue. Nigrosin Orange G. Safranin O. Versuvin. Indicators : Cochineal. Congo red. Litmus. Methyl orange. Phenolphthalein . Sodium ahzarin sulfo- nate. Starch, potato. " corn. Toluene. Urea. Vaseline. Xylol (xylene). Zinc, granulated. " dust. Wood ashes. APPARATUS. Asbestos. Balance, triple beam. " metric solution scale. " weighing scoops. Beakers, various sizes. Bottles, various sizes. Boxes, copper for pipettes. " seamless tin for Petri dishes. " sUde. Brushes, test tube, flasks. Bulbs, distilling, Hopkins. Bui-ettes, various sizes. Burners, Bunsen, pilot, and micro. Casserole, 210 cc. Cell, blood counting chamber. Clamps, test tube, burette. Corks. Cotton. Crucibles, porcelain, Gooch, iron. Cylinders, graduated, various sizes. Dishes, evaporating. " Petri, dia. larger dish 100 mm. " depth of lower dish 16 mm. Files. Filter cases for 6 sizes. 142 SOIL BIOLOGY Flasks, Erlenmeyer, boiling, filter. " Kjeldahl (500, 1000 cc). " volumetric and KoUe. " various sizes. Forceps, cornet, dissecting. Funnels, glass, Buchner, copper. Glasses, jelly, with tin covers. Glass rods, tubing. Jars, anatomical, staining, speci- men. Lamp, alcohol. Lenses, magnifying, reading glass. Lens paper. Microscopic slides 3X1, concave 3X1. " cover glasses, round, square, several thicknesses. Motors, porcelain, agate. Paper, litmus. " filter, 6 sizes. Pencils, red, blue for glass. Pipettes, 1, 2, 5, 10, 25, 50, 100 cc. " graduated. " stopcock, 10 cc. Plates, porcelain, 12 cavities. Plates, Lafars counting. Platinum foil, wire 0.41 mm. di- ameter. Pump filter. Rings, iron. Rubber stoppers, policemen, tubing. Section lifters. Shears, steel. Sieves, brass nested, set of 5. Spatulas, 3 sizes. Spoons, bone. Stopcocks, Geissler. Supports, iron, funnel test tube. Test tubes, 150 mm. long out- side. Thermometers, several kinds. Towels, barber. Tongs. Triangles. Tripods. Tubes, fermentation (Smith's), fermentation safety valve in top. Watch glass. Wire baskets. Wire gauze. SPECIAL APPARATUS. Aeration apparatus, battery of Colorimeter, Nessler tube, double 16 ' mirrors. Autoclave, 2B, 4B (large). Crocks, earthen, for waste ma- Auger, soil, 1, IJ inch. terial. Balance, analytical, No. 10 and Digestion racks. weights. Distillation apparatus, battery of Balance, analytical. No. 16 and 20. weights. Filters, Berkefeld. Baths, steam. " Pasteur Chamberland. Centrifuge and accessories (elec- Incubators, 37°, 20°, room tem,' trie). perature. SPECIAL" APPARATUS 143 Jars, dialyzers. " sample. Microscopes, regular and binoc- iilar, and accessories. Microtome. Oven, electric, drying and incu- bator (small). " gas, hot-air sterilizer (large). Pans, soil. Plate, electric, hot. Photometer sulfur. Pump, vacuum for aeration ap- paratus. Shaking machine. Stools, laboratory. Trays, laboratory, various sizes. Tables, laboratory. Tube, soil. King.