| H || | te MANA TH I] I Ps PMNdo cS af eee meh AUT TR Ua TBO Puls Uae ev THESIS FOR DEGREE OF M. S. HANNAH VIRGINIA LANGWORTHY 1915 THESIS 1" Cc. ee. fv A A ~~. ¢ Wo. ~ . ~ b es Lo" “9 a 4 7? ‘ ? tf ANA — . ne c¢ Yu Ae ‘ , ee - ~ vi ( o — : 4 yp fy 1 Ob OAL EO x ¢ o wt Grateful acknowledgment is meade of the kind assistance and encouragement received from Dr. Ward Giltner, under whose direc- tion this work was carried on. The Factors Influencing the Longevity of Micro8rganisms when Subjected to Desiccation. es eel THESIS The Factors Influencing the Longevity of Microorganisms when Subjected to Desiccation. 1. Properties of the organism which probably depend on species differences. (a) Spore-formation. (vo) Capsule formation. (c) Peculiarities of cell composition. <.- Physiological diffetences in organisms resulting from treatment before drying. (a) Temperature of cultivation. (bo) Nutrition. (c) Age of culture. (d) Virulence. 3. Nature of the medium in which the orgenism is suspended before drying. (a) Its possible plasmolyzing effect. (bo) Its content of protective or water-retaining sabstance. 4. Physical structure of the substratum upon which drying occurs. (a) Smooth, non-absorbent surfaces. (bd) Textile fibres or Pabrics. (c) Soil. 5. Effect of physical agencies. (a) Light. (bd) Temperature. (c) Variation in humidity. 102130 PART I. The Factors Influencing the Longevity of MicroSrganisms When Subjected to Desiccation. (With special reference to the factors influencing longevity of bacteria in soil) A general discussion of the factors influencing the longevity of microorganisms should involve all the unfavorable conditions to which they may naturally be subjected, in the course of their existence, as lack of available food material, accumulation of their own bi- products or those of organisms with which they are grow- ing, the injurious effect of temperature, direct sunlight and insufficient moisture. The sum total of these repre- sents the machinery by which nature's defensive activity is exercised, but since the importance of each of these is controlled by a great number of lesser factors, this Giscussion has been restricted to the subject of desicca- tion with the idea of giving it a more detailed consider- ation than would be possible with the more general topic. The factors influencing the length of time an organism will live, when subjected to drying, ere here divided into five main groups, with sub-divisions as in- dicated in outline on page 1. Properties of the organism which probably depend on species differences. 2. Physiologicel differences in organisms resulting from treatment before drying. 3. Nature of mediim in which organism is sus- pended before drying. 4. Physical structure of sub-stratum on which drying occurs. 5. Effect of such physicel influences as light, temperature, and variations in humidity. 1. Properties of the organism itself, which may have an effect upon its resistance, such as (a) spore- formation, (b) capsule formation, (c) peculiarities of cell composition, to which resistence of certain non- spore-bearing species is attributed. (a) Such spore-forming organisms as B. mesen- tericus, and the bacilli of anthrax and tetanus, have been known to live in the air dry condition for many years, tetanus spores heving been known to produce the disease after eleven years or more of drying in soil. lpnis fact is of especial significance since the disease is commonly . acquired by introduction into wounds of such dry materials 88 garden soil and street dust. Chapin. Btates that te- tanus spores may retain their vitality for sixteen years, so that it is not surprising that lands have been known to remain infected for several years. The spores of B. an- thracis remain alive in dry garden soil at lesst fifteen years. Briscoe* in regard to the resistance of certain bacterial spores, says: "We have found thet the spores of B. subtilis dried on are agar slant and remaining in this state for eight years, gave growth when seeded into broth". ConnS states that spore-besring bacteria may be dried without injury, for their spores protect them from destruction. “According to Swan", states Lafar®, “spores of B. megatherium dried on a coverglass retained their vitality and germinating power for more than three years. The seat of this high resistance has already formed the object of numerous researches. One school looks for it in a peculiar modification of the spore-plasma, for in- stance in the presumably low water content thereof, as Suggested by Lewith. Others again attribute to the spore membrane an exceptionally low heat-conducting power, and & very slight degree of permeability to noxious substances", 7 says the spore is a resting stage which serves to Jordan tide the species over a period of dryness, femine, or un- suitable temperature. In this resting state the living matter of the spore may remain dormant for years or even for decades. Fraenke1® states that the continued or temporary influence of dryness and moisture ,heat and cold, is well borne by the spore. Sternberg? makes the state- went that spores in a desiccated condition preserve their vitality for a great length of time. Lonnisl© states that the resistance of spores to all unfavorable externel in- fluences is extremely great, and that drying and high temperatures which cannot ordinarily be endured by vegitive forms are of practically no effect upon the spores. (bo) Certain bacteria possess gelatinous walls or - 4 « Capsules. Considerable evidence is offered in the litera- ture in support of the belief that such a structure makes the organism less readily affected by heat and chemicals, and that it also retards the removal of moisture. Since the protection offered by such a structure is by no means comparable to that of the spore, the relation of capsules to nonspore-bearers is here considered, exclusively. Jordan’? states that most of the vegetative forms of bacteria are rather quickly killed by ordinary air dry- ing "if the actual body substance is not protected by a gelatinous cepsule". Lafar® says of Streptococcus mesenter- ioides (sometimes referred to as Leuconostoc mesenterioides), “Liesenberg and Zopf were unable to discover any spores, and in any case their presence would be unimportant, since the organism already possesses, in its mucinous envelope, an excellent means of protection against adverse circum- Stances”". Owing to this envelope it was able (according to Liesenberg and Zopf), to withstand three and one-half years desiccation in the air, whereas the naked modification, i.e. the form developed on a medium containing no sugar, and having no capsule, succumbed efter a much shorter exposure. Lohnisll describes a fluorescent organism which produces Slime when growing on certain plants, which slime makes it possible for it to combat Successfully destruction through drying. Revislz in discussion of results with cultiva- tion of organisms of the coli type in soil for a period ranging between twelve and sixteen months, remarks: *The two types of Organisms which developed a mucilaginous type of growth were the ones which survived longest", In another articlelS he suggests that the Slime formed by or- geanisms of the coli type may add to the water-abserbing and retaining capscity of the soil, and therefore promote the longevity of that organism. L8nnisl9 says, "not only the spores, but also the bacteria with slimy walls endure the effects of desiccation very well". Lafar® emphasizes the importance of making dis- tinction between organisms like Str. mesenterioides, which surrounds itself with a gelatinous envelope, and orgenisms which carry on a slimy fermentation, i.e., conversion of Sugar outside the cell into mucinous matter without them- selves being enclosed in capsules. Jensenl4 uses the terms ‘ capsule-formation and slimy fermentation interchangeably and regards the process as protecting the organism against desiccation. Buchanan's articlel® on the gum of Ps. radicicola offers a very comprehensive review of the literature on the nature and morphological origin of bacterial slimes. Certain investigators, as he says, describe gum formation as the result of a true fermentation of carbohydrates, by bacteria. Of these, some call it an extracellular synthesis, while others contend that the gum formation is a true synthetic process, but not necessarily due to an extra cellular fer- ment. But, to quote directly, "In the majority of cases, the slimes and gums have been determined to be the result of e& swelling or solution of the cell wall or bacterial capsules". He suggests that there is a continuous movement outward of substances elaborated by the protoplast, anda growth in thickness and area of the capsule and wall by inter- polation. If this material deposited is capable of swelling in water a definite capsule is developed; and if soluble in water, either immediately or gradually, a viscid solution may develop. Most of the bacterial gums reported in the liter- ature are described as carbohydrates of the fermula (CgH 1005 )n. Bacterial slimes classed as dextrans are described by Brdutigem, Kramer, Ritsert, Scheibler and many othersl®. Lipman, Greig-Smith, Maassen,and Laxal5 found levulan to be the specific gum of several slime-forming bacteria. Schmidt-Mihlheim, Hueppe, Emmerling, Greig-Smith, Laurent, Ward and Seilerl5 describe bacterial gums having the char- acteristics of galactans. A few nitrogenous bacterial gums are mentioned, but they appear to be less common than those of a carbohydrate nature. The protective action of these gums has been ascribed to their water retaining capscity. (c) Exclusive of organisms with such special protective structures as spores or capsules, it appears to be true that certain species are more resistant then others. To quote Chesterl6, "Neisser found that the organ- isms of typhoid and diphtheria were the most resistant, cholera, influenza, bubonic plague and gonococci, the least, and the pus-forming cocci, meningococcus and tubercle bacil- lus of intermediate resistance. “Briscoe* credits the tubercle bacillus with a greater resistance than most non- spore-besring organisms, and says: “As regards desiccation tubercle bacilli appear to take an intermediate position between spore and nonspore-bearrers". To quote further, "This power of resistance is no doubt due, in part at least, to the content of the waxy or fatty substance found largely in the outer layer of the tubercle bacillus. The presence of this wexy material gives them their well-known character of “acid-proof" power when stained". There are doubtless other characteristics of a cell, in addition to those mentioned, spores, capsules or high fat content, which may render it less readily injured by desiccation, but with our present insufficient knowledge of the bacterial cell it is difficult to explain why B. typhous or Bact. diphtherial, with none of the previously mentioned faculties, should exhibit so much greater longevity than Bact. pestis, the gonococcus and many other nonspore- formers. — Be Physiologicel differences in organisms, resultir from treatment before drying as (a) temperature of cultiva- tion, (b) nutrition, (c) age of culture and (d) virulence. (a) Although but little work has been done to demonstrate that organisms grown on a favorable medium are more resistant to desiccation than those developed on a less favorable nutrient material, the nutrition has been shown to affect the resistance of the cell to moist and dry heat, and it might reasonably be inferred that the same would be true of its resistance to drying. In certain cases the medium is undoubtedly important. Streptococcus mesenterioides®, as previously stated, has been found to resist desiccation for a much longer period if developed on a saccharine medi, then on one which contains no sugar; in this case the effect of the medium is only indirect, as it is the capsule devel- oped by the organism in the presence of proper sugars, which makes it resistant. - 8 « (b) Ficker's experiments1& with the cholera vibrio deal with the influence of the temperature of growth upon the ability of the organism to endure desiccation. He says: "Doubtless, the temperature at which the organisms are cultivated and their ability to resist drying at dif- ferent temperatures, stand in a certain relation. Contrary to expectation, the drying at a higher temperature does not always produce a more rapid, and the drying at a lower temp- erature a more gradual effect. Experiments show that the organisms cultivated at 37° and occurring on the dryer agar surface are better prepared for the rapid removal: of moisture occurring in the desiccator at 37° than are the organisms grown at lower temperatures. They suffer no such sudden removal of water if dried at a temperature closely approach- ing that at which they were cultivated. “However, the cultures grown at 15° or 22° and dried at 15° were found to live considerably longer than the cultures developed at 37°, the conclusion, therefore, being that cultivation at a temperature below the optimum produces the individual with the greatest resistance to desiccation. Copy of Ficker's table, Showing the relation of temperature of growth to the temperature at which the organ- iam is desiccated. Desiccator kept at 37° 22° 15° 57°; Removal after Removal after Removal after 18 hrs. + 2 days + 4 days 0 2 days 0 5 days + 6 days 0 5 days 0 4 days O | 6 days 0 Cholera ! Culture 220 | 18 hea. O < days + 4 days + grown 2 : 2 days 0 5 days + 6 days + days at o days oO 4 days + 9 days O 6 days O 15° | 18 hrs. Q 4 days + 2 days 0 6 days + 5 days 0 9 days 0 e 9 « As the method by which these results were secured is not apparent from the table, Ficker's explanation of methods employed is quoted here directly: "From agar plates of the cholera organism kept two days at 15°, 22° and 37°, equal quantities were smeared directly in thin streaks on covereglasses, which were kept in the desiccator at 15°, 22° and 37°. Since the cultures grown at different tempera- tures did not contain comparable masses of bacterial growth, for proof of ability of the organism to develop, two cover- glasses were transferred at intervals to each of four tubes of peptone solution". Ficker suggests that the differences in the proto- plasm of cultures grown at a ldwer temperature and the cell substance of the form developing in least time at the opti- mun temperature of growth, may hold true in other respects; that there may be imperceptible differences which possess nevertheless, a far-reaching significance. To quote directh, "It has never been proven that the vegetation developing at the optimum temperature and with the greatest rate of growth is also more advantegeously situated in the struggle with life-menacing influences, or that it displays all the capacities of its species any more than with plants the development is best in all physiological respects even when the most luxuriant vegetative development occurs". (c) The literature offers quite contradictory information as to the effect of the age of the culture upon its vitality. Von Wahi? in discussing resistance of spores from cultures of different age, says the age of the culture is immaterial so long as mature spores are present in abundance. - 10 - Ficker's results!9 with the drying of cholera vibrio cultures of different age indicate that cultures one or two days old endure desiccation better than older cultures, but of these two the forty-eight hour culture is less sensitive to drying at 37° than is the twenty- four hour culture. The results of Kitasato and Bercknoltz, quoted in the same article, show about the same resistance in cultures from one to five days old. Cultures older than these showed a marked decrease in resistance. This, ss Ficker]9 claims to have demonstrated, was due not only to the fact that there were fewer living organisms present in the same mass of an old culture, but these surviving organ- isms possessed in themselves less vitality than did the vibries from younger cultures. Chapin“ states that old cultures die sooner than fresh ones, and that different strains have different powers of resistance. Chester16 states that fresh cultures of Ps. radicicola, by containing a larger nunber of active organisms are better for inoculat- ing cotton than old cultures. By "“ffesh" cultures are meant those which have just attained their maximum growth. (d) Ficker19 also demonstrated, in the case of cholera vibrio that » virulent strain was more resistant than an avirulent strain. S- The nature of the medium in which the organism is suspended before drying, in regard to (a) its possible plasmolyzing effect and (b) its content of protective or water-retaining substances. It has been demonstrated both experimentally and -ll- practically that the medium with which the organisms are surrounded, before being subjected to desiccation, has an influence upon their resistance. (a) Ficker's experiments!® showed that transfers of old cholera vibrios from the surface of agar to distilled water resulted in a disturbance of the turgor of the cell which was so injurious as to make its death, when desic- cated, occur much sooner than it did if suspended in physio- logical salt solution before drying. With young cultures the reverse was true. Suspension in tap water or distilled water before drying appeared to have the same effect, but desiccation after suspension in physiological salt solution was quickly injurious. He explains this on the basis that as the air drying process resulted in increase of concentra- tion of the salt solution, the cell was subjected to both plasmolysis and desiccation. The explanation is not com- plete, however, for a broth of the same salt content as the physiological salt solution was favorable to both young and old cultures. (bo) Fickerl8 found the cholera vibrio to retain its vitality longer when dried after suspension in milk or vroth than in distilled water, tap water, physiological salt solution, serum or saliva. He considers it remarkable that the bacteria died out so quickly, dried in saliva, since,as he writes, one might expect the slime of the saliva to pro- tect the enclosed bacteria for some time. The saliva was filtered for this experiment, however, so that there is a possibility that the "large cell elements" otherwise offer- ing sure protection against drying, were excluded. These experiments imply a certain protection gained by presence of -~12- nitrogenous or albuminous constituents. Ficker also showed that a greater longevity resulted when the organisms were cultivated on a solid medium and suspended in fresh broth or milk than when they were grown in those liquids and dried ‘on cover glass films prepared directly from the cultures in which they developed. No Goubt the presence of their own biproducts wae injurious, in the Jatter case. (A portion of these, at least, was filtered out in the case of the sus- pension in fresh liquid). This injury must have been an appreciable one, to outweigh the shock, to the organisms of transferance from the medium upon which they grew, into a fresh solution. Numerous examples are cited of long preservation of organisms, ina dry state, when surrounded by nitrogenous or albuminous material. Chapin@ says, "The thicker the layer of infectious material, the longer ig its virulence likely to be maintained. This thickmess depends largely upon the native of the medium. Ina dried watery medium, bacteria may die quickly, while they may survive long in sputum or foeces. Heim@9 found the pneumococcus, concerning whose resistance there is much dispute, to live as much as one and one-third years, and retain its virulence dried in blood or pus on silk threads. His experiments show that this method will practically guarantee the virulence of this organism for at least six months. This method, tested out on other pathogenic organisms, was found practical, although not to the same extent with all as with the pneumococcus. Burger< recovezed pneumocotoi from oe handkerchief seven days after it had been in use. Wood found that sputum containing - 13 « pneumococei might, under favorable conditions, preserve their virulence for thirty-five days. Many of the earlier writers claim a considerable longevity for the tubercle bacillus in dried sputum. Fischer! found that the orga.isms lived from one hundred twenty-six to one hundred eighty-six days in tuberculous sputum dried on glass. Sormani®* found the bacilli to live two months in dried tuberculous sputum. Cadeac“9 obtained evidence of tubercle bacilli living eighty to one hundred fifty days in a dried tuberculous lung. Villemin, Koch, DeThoma and Moffuci credit that organism with a life of from one to nine months in dry sputum. Briscoe’, in discussing resistance of tubercle bacilli, says: "When exposed to desiccation, pure cultures of the germs, in thin layers are found to be dead in a few days. In sputum and other foul material they appear to live longer than other nonspore-bearers". In another place he says: "In the presence of foul material tubercle bacilli live from a month to a year or more. ... It would be expected that tubercle bacilli protected by the mucoid material, as found in sputum and diseased tissues, in which these germs more frequently occur, and also by their abund- ance of naturally waxy constituents would be protected against drying and injuries from the presence of foul material". As Briscoe's conclusions are based not only on his individual research, but a most comprehensive survey of previous inves- tigations, they may be regarded as well founded. Diphtheria bacilli remain virulent for montis dried in the false membranet Chapin® says: "Vaccine virus when dried in the crust which forms from the vesicle retains its virulence for ~o> SE ~ 14 - a considerable time. A thin layer of the same lymph on a quill does not remain active, when exposed to the air, for more than a week or ten days. The ivory points covered with vaccine matter, which were so much used a few years ago, were usually guaranteed to keep three weeks and often did remain virulent a month or more. But there was usually more than one layer and the thickness of the material was further increased by the presence of blood and leucocytes". Whether the protective action of these albuminous and water retaining substances is, as Chapin suggests due merely to the greater thickness of the layer of dry substance, or to its water retaining capacity or nitrogenous constitu- ents, has not been definitely determined. From the results of my own experiments on drying Ps. radicicola in quartz sand, after suspension in different solutions, it would seem that the protective power of the material is not due merely to ita increasing the thickness of the film. 4. Physical structure of thesubstratum upon which drying occurs, showing comparisons between longevity of organisms on (a) smooth, non-absorbent surfaces, (b) textile fibres or fabrics and (c) soil. Organisms have been foind to die out much more rapidly dried on glass, marble, porcelain or other smooth, had surfaces than when dried on threads, cloth, cotton or in soil. To quote from Marshal1$, "Hansen found that yeast cells dried on cotton were still alive after two to three years, while if dried on platinum wire some died in five days and others lived as long as one hundred @ays. Compressed beer yeast mixed and dried with powdered charcoal kept as long as ten years; Ps. radicicola dried on a cover-glass or filter paper died within twenty-four hours; on seeds this same organism was still alive after fourteen days, and in the dried nodules of legumes a few cells were able to reproduce after more than two years". Chesterl§ says that Ps. radicicola when dried in thin films on glass perishes very rapidly, but that it may live eleven to sixteen days on cotton. Harding and Prucha“9° have shown that Bact. com- pestris may live for as much as thirteen months on cabbage seeds, but dried on cover slips is dead at the end of ten days. Briscoe® Says that this difference is no doubt large- ly due to the difference in the hygroscopic moisture re- tained by these substances. He found tubercle bacilli to live only eight to twelve days when dried in thin smears glazed paper slips. B. coli, B. vilaceus and B. prodigio- sus, according to his experiménts, were even more sensitive, dried under thoseconditions. Billings and Peekham“® found that B. typhosus, B. coli, and Staph. aureus endured desiccation on silk threads for as much as five months witho.:t loss of vitality. Other investigators found those orgenisms to have a much lower resistance, on the same material. Abel© observed of the plague bacillus that it lived fourteen days on a cover-glass and thirty days on threads, pieces of linen and parts of organs. Germano? working with the same organism observed a longevity of twenty-five to thirty days, when it was dried on vieces of wool or silk. - 16 « Rosenaut claims that the bacillus of bubonic plague may be harbored in bedding and clothing, hence the necessity, in case of such diseases for destruction or at least thorough disinfection of garments, carpets or rugs possibly contaminated. Koch and Gaffky!8 say that the cholera vibrio may live only a few hours, dried on glass, but four days on fabrics. Tubercle bacilli have been known to retain their virulence thirty-nine to seventy days in a folded handker- chief, or carpet, or woolen cloth kept at room temperature in diffused light, Noetel2’ experimented to determine the longevity of tubercle bacilli on clothing. The organ- isms were conclusively demonstrated on: Coat and vest worn daily, Jacket and hose worn daily, An old coat, Coat, hose and plush vest not worn for three weeks, Wool jacket and old hose not worn for five weeks. Chapin® although minimizing the importance of in- fection by fomites, cites a few authentic cases in which disease has resulted from cléth, and other absorbent mater- ials, infected a considerable time previous. The two spore- formers most important as disease-producers, tetanus and anthrax have frequently been shown to be transferred on sum materials as lamp wick, wool, hair and dust. Chapin also mentions a case in which typhoid fever was proved to have been caused by use of army blankets, infected at least seven months previously. Living bacilli were demonstrated on sev- eral of the blankets at that time. In another instance~ an ~ 19 « individual acquired diphtheria by contact with a garment on which a laboratory assistant had spilled a portion of a bouillon culture of that organism, two days before. Lehmann and Neumann® state that Strep. pyogenes has been known to retain its virulence one and one-third years, dried en silk. This organism is usually regaréed as sensitive. Von Wahil? claims that spores dried on metallic or other hard, smooth surfaces are less resistant than spores dried on wadding or silk. (c) The evidence obtainable from the literature in regard to the length of time an organism may live in air- dry soil is neither definite nor complete. To quote from Marshall's Microbiology<4, "Under air-dry conditions each soil grain is surrounded by a very thin film of moisture designated as hygroscopic water...... According to Hall this film is about .75 microns in thickness. Nevertheless it will be seen that the moisture even in air-dry material is deep enough to allow the bacteria a reasonable amount of protection. This will account for the survival of nonspore- bearing bacteria in dry soil for a long time. Indeed in- stances are on record of the isolation of Azotobacter and Nitrosomonas from soils that had been kept in the laboratory for several years", Lohnisl® gays, "Vegetative cells can better endure drying when they are in soil. With spores also this is true. The resistance of spores dried in earth is usually found to ve higher than that of spores dried on cotton, silk, glass, etc." = 18 eo Dugger and Prucha®? found that after rapid dry- ing out of soil cultures there remained a large number of living organisms whose vitality would extend over a con- Biderable period. Nestler®9 investigated an old herbarium and found that even after twenty-three years, ninety-thousand colonies could be obtained from one gram of soil. Azotobacter! remain alive in soil samples if these samples are kept for one hundred sixty days in a desicator and then one hundred forty-eight days in an air-tight con- dition. Germanots results seemed to indicate that the organisms of typhoid and diphtheria did not live as long in soil as on fabrics, although the diphtheria bacillus averaged twenty to forty days longevity in soil, in all trials. Firth and Horocks< found that the typhoid bacillus would live for twenty-three days in dry sand. Phuh1°° found the byphoid bacillus to live twenty-eight days in dry sand, and eighty-eight days in moist garden earth. The bacillus of dysentery, on which he experimented at the same time, lived only twelve days in sand, and one- hundred one days in moist garden earth. Briscoe” found the tubercle bacillus to live two hundred thirteen days in garden soii. But little work has been done to determine the effect of different soil types on the longevity of organ- isms dried in them. The data offered in the literature on this point is not only scanty but far from recent. Modern texts hold that dust does not offer pro- tection to many pathogenic organisms, the dangers due to - 19 ae ordinary dust being much exaggerated according to Rosenaul and Chapin®. They do not attempt to deny that street dust may carry pus-forming cocci and occasionally tubercle bacilli. The latter are commonly found in the dust of rooms occupied by careless tuberculous patients. Washbourn and Eyre claim to have found the pneumoccus in dust from a ward and labora- tory at Guy's hospital, but failed to find it in street dust. Dempster>4 found that the cholera vibrio lived only a short time in perfectly dry soil, but survived for a prolonged period in a soil containing a small amount of moisture. The typhoid bacillus showed a greater tenacity of life in soil than did the cholera vibrio, but entire desiccation proved quickly fatal to it also. Comparison of longevity of these organisms in white sand, grey sand, garden mould and peat showed that with the exception of peat, which apparently contained substances toxic to the organisms, the nature of the soil did not have a direct influence. The vitality of the organisms appeared to de- pend rather on the moisture content of the soil than its composition. “My own experiments, given in Part II, on the longevity of soil organisms in different types of soil, have led me to similar conclusions. The longevity of vegetative cells in air-dry soil is probably, as Lipman=4 suggests, due mainly to the presence of moisture in the hygroscopic form. Undoubtedly the presence of organic colloidal substances with a tend- ency to regain moisture, is of importance also in this connection although as the amount of such substances would be apt to vary with different soils it can hardly be desig- nated as the important factor. Van Buchtelen®5 in speaking of analysis of - 20 - soil solution in the report of the bacteriologist of this college, in 1913, makes certain statements, which, on ac- count of their immediate bearing on this subject deserve direct quotation: "In many cases there was found in the s0il solution a slime. This must be regarded as the first experimental proof of the presence of this substance in soil and it is not impossible that much of the irregular behavior of the life in soil can be explained, to some extent, with a knowledge of this slime. If I may be per- mitted I should like to call your attention to the possi- bility of this substance having an effect on desiccation, diffusion and other processes". It has been suggested that the presence of this substance in soil may be a factor influencing longevity and a few experiments have been started with a view to securing some information as to its value in the soil. It is hardly necessary to add that with a problem which in- volves such a number of factors, none of which are very completely understood, results come very slowly. 5. Effect of such physical agencies as (a) light, (>) temperature, and (c) variations in humidity. (a) Chapin? considers light one of the most important factors to be taken into consideration in regard to the effect of drying upon bacteria. He says: "Germs that are killed in a few minutes in direct sunlight may live for weeks in a dark place or even in diffused light. Twicheel® found that tubercle bacilli lived from one to two months in diffused light, but died in e few hours if exposed to direct sunlight. Migneco® found that when dried - 21 - on a cloth in the sun tubercle bacilli kived from twenty to thirty hours. More recent observers ascribe consider- ably less resistance to this organism. Weinzirl@ claims that it can stand drying in direct sunlight for only a few minutes. The consensus of opinion seems to be that the less the duration and intensity of illumination, the greater the endurance of desiccation. (bd) With regard to the temperature of desicca- tion, Chapin© says, "The higher the temperature, the sooner the germs perish. Ficker!9 found the cholera vibrio to live six days when dried at 15°, but no more than eighteen hours dried at 37°. Verjbitski found plague bacilli to live one hundred thirty days at 4° - 5° or thirty-five days at room temperature. Tidewell2 says that with plague bacilli the colder the climate the greater the persistence of infection. (c) As to relative merits of desiccation in room air and desiccator some fairly positive statements have been obtained. Chapin® says, "As a rule bacteria live much longer when dried in a desiccator than when dried in the open air under natural conditions". Ficker!9 showed that rapid drying of organisms in a desiccator over CaClg or HgS0,4 wae preferable to drying in ordinary room air. This is probably due to the fact that variations are con- tinually occurring in the humidity of the room atmosphere which may have the effect of drying and remoistening the organisms. This process is known to have a very destructive effect, as shown by Ficker's experimentl?, in which the organisms were placed alternately in a desiccator and moist chamber for a couple of hours at a time. The organisms so treated died out much more rapidly than did those which were left in tne desiccator continuously for the same length of time. Lohnis!° states that frequent changes between drying and remoistening are most injurious, but that rapid drying in a space with “rarefied atmosphere", (i.e. in a desiccator), is comparatively favorable. Unpublished experiments of J. Simon have shown that repeated drying and moistening of the soil is much more detrimental to nodule bacteria than keeping the soil constantly dry. Chester!§ in his experiments with Ps. radicicola found that an important condition for successful preservation of the organism in a dry state was to keep the culture sealed from the air and in a dark, cool place. Malassez and Vigna1%6 state that tuberculous Bputun, alternately dried and moistened eight times lived only twelve days, as compared to a longevity of over one hundred days when continuously dry. = 23 «- PART II. Experimental Work. The experimental work, which covers a portion only, of the subject as outlined in Part I was undertaken with the particular aim of determining some of the factors which may have an influence upon the longevity of micro- organisms in soil. As a foundation for this it was of great importance to secure all possible data relating the general subject of desiccation, not only to obtain sugges- tions for further work but to make evident the multiplicity of factors involved and the necessity for their control in carrying on such experiments. So far as can be discovered from the literature, nobody has tried to give a full explanation of the fact that organisms live longer in air-dry soil than when dried on any other material and for the present this is not pos- sible, conaidering our rather incomplete knowledge of the real nature of soil. However, from the review of previous investigations, given in Pert I, it is seen that two prin- cipal factors are suggested; first, the physical structure of soil which makes possible the retention of moisture in the hygroscopic form, and second, the presence of organic colloids, with a tendency to retain moisture. If the factor first named is the one to which - 24 « most importance should be attached, then differences in structure of the soil or other substrata which influence the hygroscopicity would have also an effect upon the survival of organisms dried therein. A portion of the experiments, therefore, have been devoted to the drying of organisms in soils of different types which display different capacities for retaining moisture, and algo on textels which vary in hygroscopicity. Under the second heading are considered the role of the slime found in soil solution by Van Suchtelen and also the possible protective effect of any similar colloidal substances formed as a result of bacterial growth. Before it could successfully be demonstrated that such slimy or mucoid substances were amohg the com- pounds important in protecting the bacteria in soil, it was necessary to secure evidence that slime or gelatinous materials exert a decided protection against desiccation, in soil or out. This may be considered as an explanation for the introduction of experiments apparently irrexevant to the problem in hand. The experiments bearing upon these points are arranged in the sequence given their subjects, in Part I, Buch topics of the complete outline as are not pertinent to the plan being omitted. l. (a) Experiment 1. Object of experiment: To demonstrate the resist- ance of spore-bearing organisms when dried in thin films on glass. - 25 « Plan: Cultures of four spore-bearing bacilli, B. ramosus, B. subtilis, B. mycoides, and a spore-bearer Similar to B. mesentericus vulgatus, isolated from slimy bread, were used for this experiment. The organisms were grown five days on nutrient agar at room temperature, an abundant production of spores, in that length of time, being a certainty. By means of a sterile platinum loop approximately equal amounts of bacterial growth were trans- ferred from these cultures to sterile coverslips (broken in halves to facilitate dropping them in tubes); this material was spread out in a thin layer with the platinum needle and the pieces replaced in sterile petridishes which were then placed in the temperature room (22°-25°C.) in darkness.at At intervals a cover-slip of each organism was transferred to a tube of nutrient broth. Appearance of characteristic growth in the tube of broth was interpreted as proof that the dry spores were still capable of germination. TABLE 1. Days desiccated . . . ..7 39 74 91 100 B. ramosus © «© © «© © « + + + + + B. subtilis . . ....# + + + + B. mycoides . . . .« .« e« # + + + + Bacillus from slimy bread . + + + + + Results: It is apparent from the data above tabu- lated that bacterial spores are not readily destroyed when exposed to desiccation in thin films on Zlass ina dark, well-ventilated place. (This experiment should be continued for years, ) - 26 « (b ) Experiment l. Object of experiment: To compare the effect of desiccation upon slime-forming and nonslime-forming bacteria, (all of which are spore-free) when they are dried in thin films on cover-slips. Plan: The cultures used were: Slime -formers Nonslime-formers Milk bacterium A. B. violaceus. " " B. B. prodigiosus. " " C. B. coli. Bact. aerogenes. Ps. campestris. Sarc. lutea. M. varians. The above organisms were cultivated on nutrient agar for 48 hours, at room temperature. Material from the surface of these agar slants was transferred to clean sterile cover-slips, method of Expt. (a) 1, which were then replaced in the clean petri dishes in which they had been sterilized and stored ina dark, well ventilated place at room tempera- ture. Forty-eight hour cultures were preferred, because with a few of the organisms that was the minimum time in which an appreciable amount of growth would develop. Cultures older than that are supposed to be less vigorous. Since the desiccation was to be carried on at room temperature, it was thought best to cultivate all the organisms at that tempera- ture, even those whose optimum was 37° (see Part I, 2c). - 26V_ For test of longevity cover-slips of each organian were transferred at intervals to tubes of sterile nutrient broth, this being done with all precautions to avoid contam- ination. Such tubes were kept at room temperature. The development, within ten days, of characteristic growth in the broth es regards to slime, pigment or other features specific for the particular organism was regarded as proof of its viability. If in any case the growth in broth was not distinctly characteristic, transfers were made to agar Slants to demonstrate the identy of the organism causing the growth in broth with that which was dried on the cover- Slip. Up to the time of the first negative test but one Slip of each organism was transferred to broth at the different trials; thereafter, for at least three times in succession, a slip was transferred to each of two tubes of broth. Data given in Table 2. Results: It cannot be concluded from the results as shown in Table 2, that a slime-forming organism is in- variably more resistant than one which forms no slime, None of the three milk bacteria tested in this experiment showed & resistance equal to that of Bact. aerogenes, M. varians, or Sarcina lutea, but transfers of these three did give growth, at irregular intervals, long after B. violaceus, B. prodigiosus, Ps. campestris and B. coli had given posi- tive and final proof of inability to develop. They may, therefore, be said to occupy an intermediate position, veing neither the most sensitive nor the most resistant - 27 @ | + + + + + + + + | | | | + + + + - -~- - + + +-+- — mm —mmE emg mm ew ei + + + + + + + + + + + + + + + + + + + + + + + + — + + + + + + + + + + + + + + + + + | + + + + + + + + + + + + 4+ + + + + + + + + F + + + + + + +++ + ++ +++ +++ +++ +++. + 4+ + ++ +++ t+ 4+ t+ + FF +t Ft Ft Ht + SUBT AVA’ H BO7UNT’s Stzysodmes Sa SouseFOr98e*40Bg FToo°¢_ susoTstTpord*¢ snoovTota’g 9°30eq ATIN q°30eq ATIK V°3ORq ATIN Dp QS. Hees wht L2T 6TT HIT ROT 26 SB TS TL £9 ES Gh Zh SE BZ Zz GT HT LT 6 9 F DEWOOTSED sfeC ana, *B8T7I0708q sery-920d6 wodn WOTAIBOOTSEepP JO yoesse suy °c ZTEVL - 28 e among the nonspore-forming species, with regard to desic- cation, so far as indicated by this experiment. In certain cases thick smears of the slime-formers proved viable ,when thin smears did not. It may be that the influence of slime is, as Chapin wuggests, only indirect, and that its "protection" is merely the result of its in- creasing the thickness of the surface film. In that case the irregularity in the longevity of slime-formers on dif- ferent cover-slips may be explained entirely on differences in the thickness of the smears, it being difficult to dis- tribute mucilaginous material evenly over a small piece of gless. It is not easy to find an explanation for the unhsual longevity of Bact. aerogenes, M. varians, and Sarcina lutea as evidenced by the results of this experi- ment. As none of them are “acid-fast” this cannot be due to presence of “waxy or fatty constituents"* of the cell. In the case of Sarcina lutea it is not impossible that the peculiar arrangement of groups of individuals in thick packets, may tend toward preservation of at least the in- nermost cells, from complete and rapid desiccation. The validity of this assumption can only be established by ex- tensive experimentation and comparison of the resistance of sarcines with cocci of other arrangement. As to the great longevity of the other two organisms, Bact. aerogenes, and M. varians no explanation has been suggested, or attempted. Resistance to desiccation, in certain nonspore- forming species may be an inherent quality, the basis for - 29 - which cannot be exactly determined. (bd) Experiment 2. Object of experiment: To compare the effect of desiccation upon alime-forming and nonslime-forming strains of a single organism, Ps. radicicola, when dried in thin films on glass. Plan: For this work were used two strains of Ps. radicicola (variety from red-clover nodules) which had been cultivated five days at room temperature on slants of nitro- gen-free ash agar. One of these was the transfer from a culture which had been kept growing on nitrogen-free ash egar for over five months. The growth was vigorous but not slimy. The other was the transfer from a plate colony of this same organism, which plate had been inoculated with s0il in which the organism had been kept for two and a half months. This strain was decidedly mucilaginous. Material from these cultures was smeared on pieces of sterile cover- glass, method of Experiment (a) 1, placed in petri dishes and stored ina dark place at a temperature of 22° - 25°C. Coverslips of these in duplicate were placed, at intervals, in Ashby's solution. The development of typical growth was regarded as proof of viability. Table 3. Effect of desiccation upon slimy and nonslimy cultures of the seme organism. 24 hours 14 days Slimy culture te} + ” " ie Nonslimy culture (a) " ° (dD) + + 4 Bes @6 = 30 - Results: The results of the preceding table demonstrate plainly that Ps. radicicola is more sensitive to desiccation when exposed in thin films on glass than are most of the nonspore-bearing organisms tested in Experiment (b) 1, but they do not indicate that 9 slimy culture is any more resistant than one which is not slimy. Failure to make more frequent tests of the dry smears in Ashby's solution may be accountable in part for this result. As the organism grew so slowly in that medium, failure of the first transfers to develop cloudiness, inside of five days, was mistakenly interpreted as an indication that the smears placed in those tubes contained no living organisms; consequently no further transfers were made until after the appearance of growth in the first four tubes. This was at a time beyond the limit of longevity of this organism. Un- fortunately there was not time in which to repeat this ex- periment, but it seems reasonable to infer that even if transfers between 1 and 14 days had shown growth, the difference could not have been such as to give the mucila- ginous culture any decided advantage over the other, so far as resistance to desiccation was concerned. (b) Experiment 3. Object of experiment: To compare the longevity of three slime-fa@ming, and one nonslime-forming organism, (all spore-free) when these are kept in soil which is per- mitted to dry out gradually. Pian: The cultures used were: Slime-forming Nonslime-foming Kilk bacterium A. B. violaceus. " " A ana C. Ps. radicicola. The two slime-formers from milk,and B. violaceus were grown 48 hours et room temperature on nutrient agar. Ps. radicicola, as it requires a special medium and a longer period in which to develop even moderate growth, was cultivated five days at room temperature on nitrogen-free ash agar. The growth from six slant cultures of each organ- ism was washed off and suspended in 300 c.c. sterile physio- logical salt solution. This suspension was thoroughly sheken in an attempt to separate clumps of organisms. (That this method of separation is not effectual msy be seen from data given later. Filtering through sterile glass wool would have been preferable, had that method been recon:ended earlier). For the purpose of determining the approximate number of bacteria placed in soil, lc.c. of the suspension of each organism was diluted in physiological salt solution and plated on an appropriate medium. Ten c.c. of the sus- pension of each organism was then added to each of twelve flasks of sterile garden soil which had been prepared in the following manner: The soil was mixed, air-dried, sifted, and placed in 100 c.c. Erlenmeyers, 50 g. to a flask. Each flask was fitted tightly with a one-holed rubber stopper, through which was passed gless tubing 1-1/2 inches long, and plugged with cotton at the upper end. The flasks of soil were sterilized by heating them in the autoclav 45 minutes under 15 pounds pressure. The inoculated flasks were kept on a shelf in the laboratory exposed to diffused light. + + + + + + + - + + + + - + = - - + - ? ? ° ? 6d - lld lld +30d +30d 94d + + + + + + + + + + + 2 + + + + - + + + + +lid Liquefaction complete Organisms from Sand Soil Number of organism Gas in Lactose Chromogenesis Yellow Orange Pink White Fluorescent Brown Grey Durnem's solution Indol produced Ammonia witrates reduced 1 ++ 3 (cont'd). 4 + + 4 5 mT —~ eae ++ ye + + Organisms from Sand Soil. Number _of organism 9 10 11 #2212 #213 ~ 214 Morphology Form (Rod + + + + + + Spherical Dianeter in microns 06 8 8 27 06 8 1.2 il. 9 7 id. Leng th l. Le Lek 9 2. Ze 4. 5. 3. Je 5. Grouping Endospores + + + - + Liotility + + + + Relation to oxygen Aerobic + + + + Facultative + + Boultllion Turbid - - + + + - Ring + - Pellicle + - - + Sediment - - - - - - Agar streak Form of growth opreading + + + + Filiform + Arvorescent + Beaded Topography smooth Contoured + + Rucose + + + + Verrucose Optical characters Opaque + + + + + Translucent + Irridescent Litmus milk Curd - - - - - + Acid - - - - - ~ Alkaline + + + + + + Discolored + + + + + Pepi begins 48n 24h 4 - 48n 72h Pep. complete +4d 48h 7a 74 - Slimy consistency - - - - - - Gelatine Needle form Surface j.rowtn + + + + - + Filiform + + + Beaded Papillate + Arborescent + Liquefaction + + + - + Crateriform iapiform Infundibuliform + + Stratif orm + + Liquefact. canplete Organisms from Sand Wumber of organism Gas in Lactose Chromogenesis Yellow Orange Pink White Fluorescent Brown Grey DungZeam's solution Indol produced Ammonia | Nitrates reduced 9 + + Soil (cont'd). 10 Ll ++ 12 13 + + ++ - 80 -« Organisms from Sand Soil. Number of organism 15 16 1”? 18 19 20 a1 Liorphology Form (Rod + + + + + + Spherical | Diameter in microns -6 1.2 6 8 26 203 8 1.2 2. 1.5 1.1 5 Length 1. Le 1.9 1.5 le lec l. 4. 6. 4, 2.5 3. lel Grouping Strep. Strexstre, Strep Strep. Endospores + + + - - Motility + + + + + - + Relation to oxygen Aerobic + + Facultative + + + + + Bouillon Turbid - + + + + + + Ring + - + - - - Pellicle + + - + - - Sediment + + + + + - + Agar streak Form of growth Spreading + + + + + Filiform + + Arborescent + Beaded Topography Smooth + + + + Contoured + + + Rugose + Verrucose Optical characters Opaque + + + + Translucent + + + Irridescent Litisus milk Curd + - = @ - ~ Acid - - ~ + + ~ Alkaline + + - - - +> Discolored + - - - - - Pep. begins 48n - - = - - Pep. complete - - - - - - Slimy consistency - Gelatine Needle form Surface growth + + + + + - + Filiform + + + + + + Beaded Papillate + Arborescent + Liquefaction + + - + + - - Crateriform + + + sapiform Infundubulbform Stratiform + Liquefaction complete - 81 - Organisms from Sand Soil (cont'd). Number of organism 15 16 17 16 19 Gas in Lactose - - = - - Chromogenesis Yellow Orange Pink Wnite + + + + Fluorescent Brown Grey + + + Dunhem's solution Indol produced Amnoria Nitrates reduced + + a+ a+ a + + 4 - 82 « Organisms from Muck Soil. a Number of orgenism 1 Z 3 4 5 6 Morphology Form (Rod + + + + + Spherical + Diameter in microns a) 23 2 23 4 74 Length . 7 dl. 68 1. 6 9 1.2 1. 1.2 07 Grouping | Sta. Stre. Endospores - - - -- - - hotility - - - + + - Relation to oxygen Aerobic Facultative + .+ + + + + Bouillon Turbid + + + + + + Ring - - - - - - Pellicile - - - + - - sediment ° + + + + + + Agar streak Form of growth Spreading + + Filiform + + + + Arborescent Besded + Topography Smooth + + + Contoured + + + Rugose Verrucose Optical cheracters | Opaque + Translucent + + + + + Irridescent Litmus milk - Curd + - ~ - + - Acié - - + - + - Alkaline + + - + - + Discolored + - - - - - Pep. begins 20d 20d - ° - 48n Pep. complete +30d - - - - 10d Slimy consistency - - - - - + Gelatine Needle form Surface crowth + Filiform + + Beaded + Papillate Arborescent Liquefaction + - - - - + Crateriform + Napiform Infundibuliform Stratiform + Liquefaction complete | 14d 6 + ++ é + - 83 « Organisms from Muck Soil.(Cont'd) Number of organism Ges in Lactose Chromogenesis Yellow Orange Pink White Fluorescent Brown Grey Dunham's solution Indol produced Amnonia Nitrates reduced L Gne -—— GjnG@Enee: o- 3 + 4 ++ ++ - 84 . __ Organisms from Muck Soil. umber of organism 7 Spherical Diameter in microns 25 Leng tn 0? Grouping Endospores Motility Relation to oxygen Aerobic Facultative + Bouill6n Turbid + Ring + Pellicle Sediment _ + Acar streak Form of growth Spreading + Filiform Arborescent Beaded Topography : Smooth + Contoured Rugose Verrucose Optical characters Opaque + Translucent Irridescent Litmus milk Curd Acid Alkaline Discolored Pep. begins 48n Pep. complete Slimy consistency Gelatine Needle form Surface growth Filiform Beaded Papillate Arborescent Liquefaction ; - Crateriform Napiform Infundubuliform Stratif orm Liquefaction complete 9s WMWata se + + 8 + 03 04 a 9 + aert-+ ++ 07 8 Le 10 03 4 1.1 1.1 1.3 + 98+ + nr oe +++ ats e6 @esoeskt*eoe es 11 4 07 8 + 5 @ + + a rr i ee ee | ++ Number of organism Gas in Lactose Chromogenesis Yellow Orange Pink White Fluorescent Brown Grey Dunham's solution Indol produced Ammonia Nitrates reduced - 85 -« 7 + 8 Organisms from Muck So¥l1 (cont'd). 9 LO ll - 86 -« a hanging drop from a 24 - 48 hour agar slant culture. Presence of spores was determined microscopically, by examination of stained and hanging drop preparations. The following tabulation shows on which dates the different organisms were isolated: Date. Muck Sand Sandy loam Clay Clay loam War. 3 1-6 1-7 1-6 1-5 1-6 " 29 -<- B-11 --- 6-8 8-8 Apr. 21 7-9 12-15 7-13 9-16 9-12 May 7 10-11 16-21 14-16 17-19 13-16 Results: