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Auxanographic detection of experimental murine uremia with Cryptococcus neoformans
Zbl. Bakt. Hyg., I. Abt, Orig. A 248, 268-273 (1980) Mycology Unit, Robert Koch Institute, Federal Health Office, Berlin
Auxanographic Detection of Experimental Murine Uremia with Cryptococcus neoformans Auxanographischer Nachweis der experimentell erzeugten Uraemie bei der Maus mit Hille von Cryptococcus neoformans R. A. FROMTLINGt, ANNE M. FROMTLING1, and F. STAIB With 2 Figures' Received June 27, 1980
Abstract A modified blood residual nitrogen plate auxanographic method was applied to the detection of experimental uremia in a murine model. The yeast-like fungus, Cryptococcus neoformans, was used as the indicator. Transient uremia was induced by injection of 0.2 ml glycerol intramuscularly. The low molecular weight nitrogen levels were estimated by measuring the diameter of the auxanogram at intervals of 2 hr for 24 hr and at 32 hr after the glycerol injection. After 4 hr, elevated levels of low molecular weight nitrogen were found. Maximum levels occurred 20 hr post glycerol injection. This method requires only 5,u1 of whole blood per assay. The results can be read after an incubation time of 24 hr at 26°C. The stability of the prepared plates was determined to be at least 96 hr at 4 0C. The ease of use, reliability and versatility of the modified auxanographic method are discussed.
Zusammenfassung Bei der experimentellen Uraemie der Maus wurde in einem Modellversuch der BlutRest-Stickstoff (Rest-N) mit Hilfe einer modifizierten auxanographischen Assimilationsprufungsmethode unter Verwendung von Cryptococcus neoformans als Indikaror bestimmt. Die temporar begrenzte Uraemie wurde durch eine intramuskulare Injektion von 0,2 ml Glycerin ausgelosr, Fur eine Blut-Rest-N-Bestimmung wurden nur 5,u1 Vollblut benotigt, Die Ergebnisse der Auxanogrammplatten konnten nach einer Inkubationszeit von 24 Std./26°C anhand des Auxanogramm-Durchmessers (mm) abgelesen werden. Fur diesen Auxanogramm-Nachweis gegossene Platten einer Charge zeigten anhand von Kontrollen mit Hilfe einer Harnsroff-Standard-Losung noch nach 4 Tagen/ 4°C vergleichbare Ergebnisse. Von Blutproben der Tiere, in Abstanden von 2 Stun den 1
Fellow of the Senator for Health and Environmental Protection, Berlin (West).
Uremia with Cryptococcus neoformans
269
uber einen Zeitraum von 24 Stunden und abschliefsend 32 Stunden abgenommen, wurde der Blut-Rest-N mittels des Auxanogramm-Durchmessersbestimmt. Die erste nachweisbare Rest-N-Erhohung lag 4 Stunden nach der Glycerin-Injektion vor, die hochste, d. h. das Auxanogramm mit dem grolSten Durchmesser nach 20 Stunden. DIe Durchfiihrung, Zuverlassigkeit und vielseitige Verwendbarkeit dieser modifizierten auxanographischen Methode werden diskutiert.
Introduction Uremia resulting from acute or chronic renal dysfunction may lead to death by generalized toxemia or infection (2). Animal models can be of value in the study of the compromised host during uremia. Experimental uremia can be induced in animals by the intramuscular injection of glycerol (7). In 1964, Staib modified the Beijerinck auxanographic method for the detection of low molecular weight (residual) nitrogenous substances in clinical samples (1, 4, 5). While the method was previously used to identify unknown yeasts by their assimilation of known carbon and nitrogen sources (3), he used a known yeast (Cryptococcus neoiormans, Candida albicans) to evaluate unknown nitrogen sources, i. e., human serum (4). In a subsequent experiment, the auxanographic method was applied to the detection of uremia induced in animals infected with C. neoiormans. A higher incidence of ocular involvement was observed in uremic animals than in control animals (6). The purpose of this investigation was to study the development and course of glycerol-induced uremia in a murine model and to assess the applicability of the auxanographic method to monitor the uremic state.
Materials and Methods Organism. Cryptococcus neojormans, strain W 71, isolated from pigeon excreta in Wiirzburg, Germany, was maintained at 4 °C on Sabouraud-dextrose agar. Animals. Male, albino mice (NMRI) weighing 18-20 grams were used. Animals were caged individually. Preparation of auxanographic plates. Auxanographic plates for the detection of low molecular weight nitrogenous substances were prepared according to the method of Staib (4). The nitrogen assimilation medium (2.0 % dextrose, 0.1 % KH2P04, 0.05 0/ 0 MgS0 4 , 2 % Bacto-Agar) was cooled to about 45°C after autoclaving (115°C, 15 min.), A solution of 2 X 107 organisms/ml saline was prepared from a 48 hr culture of C. neoformans grown on Beerwort agar (Schultheiss-Brauerei AG, Berlin (West)) at 26°C. One ml of yeast suspension was pipetted into the center of a nine em petri plate (Greiner GmbH., Berlin (West)). Immediately, 10 ml of warm medium were poured into the plate and the plate was agitated for several seconds to insure homogenous mixture of organism and agar. Medium was allowed to solidify at room temperature. Excess moisture was evaporated by inverting open plates in a 37°C incubator for 1 hr. Plates were then stored at 4 °C until 1 hr prior to use. Induction of uremia. Preliminary studies with groups of mice deprived of water for 24 hr prior to 90 % glycerol injection (7) showed an increase in auxanogram diameter for up to 3 days, accompanied by increasingly severe symptoms of uremia from which most of the animals did not recover. Animals injected with 45 Ofo glycerol developed a
270
R.A .Fromding, A.M .Fromding, and F.Sraib
minimal increase in auxanogram diameter and mild symptoms of uremia, both of short duration (less than 48 hr). Water deprivation had no effect on animals receiving 45 % glycerol. Based on these observations, conditions were chosen which resulted in development of severe but transient uremia. Uremia was induced by injection of 0.2 ml glycerol (90 0/0) intramuscularly in the hind leg of each mouse (6). Animals were given food and water ad libitum. Detection of uremia . Blood was obtained from the mice by cutting the tip of the tail. Five Itl of blood were collected with a capillary pipette and placed in the center of an auxanograph ic plate which then was incubated at 26 °C for 24 hr. Diameter of yeast growth was measured in mm. Kinetics of development of experimental murine uremia. Level of low molecular weight nitrogen was determ ined by the auxanographic method in 24 mice 2 min. prior to induction of uremia. Following glycerol injection, a group of four mice was tested by auxanogram every two hour s for 24 hr and at 32 hr. Degree of uremia was expressed as the change in diameter calculated by subtracting the value of the pre-treatment auxanogram from the post-treatment value for the same individual animal. Mean change in diameter was calculated from each time-group. Urea standards (ranging from 20 to 320 mg % ) were tested at 0, 24, 48, 72 and 96 hr to test the stability (shelf-life) of stored plates and to test the sensitivity of the organism to increasing nitrogen concentr ation.
Results The diameter of the auxanogra m was proportional to the concentration of urea standa rd used. The size a nd densities of the au xanogram s were not significantly different in plates used 1 hr after preparation in comparison with those plates which were refrigerated for up to 96 hr (Table 1). The increase of dia m eter and density of yeast growth with increasing urea concentration was easily detectable. Standards w ere prepared solely to test th e reproducibility and sensitivity of the auxan ographic pl ates. No attempt w as made to correl at e urea standard concentration w ith low molecular weight nitrogen concentration in blood on the ba sis of similar app ea ra nce of auxanograms. Table 1. Diameter of urea stand ard auxanograms with Crypto coccus neoiormans (mm). Five ,ul of standard solution were placed on each plate Time (hr)
20
Urea concentration (mg %) 160 80 40
0 24 32 48
16 16 17 18 15 16
20 21 20 20 20 21
72
96
23 23 22 22.5 23 22
26 27 26 27 26 26
320 31 32 30.5 32 31 31
Within 4 hr post-glycerol injection, an increase in auxanogram diameter w as ob served (Fig. 1). The size and density of the zone of growth continued to increase until 20 hr post-glycerol in jection. Photographs of typical auxanograms
Uremia with Cryptococcus neoformans E
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Fig. 1. Change in low molecular weight nitrogen concentration (uremia) as measured by the modified auxanographic technique. Each value is the mean of 4 animals. showing the development of uremia in mice are presented in Fig. 2. The density of c. neoformans growth is greater in these auxanograms than in those prepared from the urea standards. Additionally, the diffusion of red blood cells decreased over the 32 hr course of the experiment. Of the 24 mice used in this experiment, 2 failed to develop uremia as detected by this method. Both of these mice were in the group tested at 12 and 24 hrs, and the drop in mean diameter reflects these data (Fig. 1). Decline in uremia occurred by 32 hr. Previous studies by Staib indicated that low molecular weight nitrogen levels in blood returned to normal by 72 hr in mice in which experimental uremia was induced. Additionally, chemical analysis of pooled serum from two groups of 10 NMRI mice gave the following results: before glycerol injection, urea-nitrogen 28 mg 0/0 and creatinine 1.8 mg 0/0; 24 hr after injection, urea-nitrogen 140 mg % and creatinine 3.0 mg Ofo (unpublished data). Discussion Our present study confirms the observation of Staib and coworkers that changes in residual nitrogen levels in peripheral blood of mice can be detected by the use of this modified auxanographic method (6). The study also shows that diameter of C. neojormans growth is proportional to the concentration of an
272
R. A. Fromt/ing, A. M. Fromt/ing, and F.Staib
Fig. 2. Development of uremia as illustrated by typical auxanograms. Numbers represent hours post-glycerol injection. Five ,ul of whole blood were placed on each plate. Dark central spots are the cellular components of blood. Larger light circles are the growth of C. neoformans dependent on residual nitrogen. Bar = 1 ern.
appropriate nutrient (Table 1). The data in Fig. 1 illustrate the ability of the auxanographic method to detect an increasing course of experimental uremia in mice. Diameter of C. neojormans growth increased from an average of 21 mm for pretreatment mice to an average of 31.25 mm at 20 hr post-glycerol injection. The peak of experimental uremia at 20 hr was followed by a gradual decrease in residual nitrogen levels to the pretreatment value at 72 hr post-glycerol injection. Precision of the intramuscular injection of glycerol is critical for the induction of experimental uremia. Leakage of glycerol from the injection site may account for the negative results seen in 2 of the animals used in this study. An important consideration for the practical application of this technique is the stability or shelf-life of the prepared plates. The data in Table 1 show that the auxanographic plates can be stored at 4 °C for least 96 hr without affecting the results of the test. The collection of blood poses a problem in experiments with a murine model. The auxanographic method described here requires only 5,il1 of whole blood which can be obtained easily with minimum trauma to the animal. Conventional techniques require the separation of serum from whole blood and require larger volumes. Cellular components of blood do not interfere with the detection of residual nitrogen levels using this technique. In the 2 animals that did not develop uremia the decrease in cellular diffusion was comparable with that seen in uremic mice tested at the same time. The acute uremia induced in this model approximates transient uremia which
Uremia with Cryptococcus neoformans
273
easily can remain subclinical, and therefore, remain undiagnosed in experimental animals. The physiologic changes concurrent with acute uremia must be better understood in order to decrease the high potential for infection in uremic animals (6). With the animal model introduced here and the reproducible auxanographic assay, uremic animals can be monitored and specific assays of immunocompetence performed. Since a pathogenic microorganism is used to measure residual nitrogen, this auxanographic method is especially valuable in the assessment of the nutritional value of residual nitrogen for this organism (6). The auxanographic method is not limited solely to the measurement of low molecular weight nitrogenous substances in the blood but also can be applied to extracts of various organs (unpublished data) and thus may help to explain the predilection of certain organisms for a particular organ. Determination of the concentration of such substances in the brain of uremic mice will be the subject of further investigation. Acknowledgements. The authors wish to express their gratitude to Mrs. A. Blisse and A. Passow for their technical assistance.
References 1. Beijerinck, M. W.: L'auxanographie ou la methode de l'hydrodiffusion dans la gelatine appliquee aux recherches microbiologiques. Arch. neerland. sci. 23 (1889) 267 2. Guyton, A. c.: Textbook of Medical Physiology, 5th ed., p.504. W. B. Saunders Co., Philadelphia (1976) 3. Lodder.]. and N.]. W. Kreger-Van Rij: The Yeasts. North Holland Pub!. Comp., Amsterdam (1952) 4. Staib, F.: Das Serum-Reststickstoff-Auxanogramm (mit Sprols- und Schimmelpilzen). Zb!. Bakt., I. Abt, Orig. 194 (1964) 379 5. Staib, F.: Zum Verhalten von Cryptococcus neoformans gegeniiber Serumfraktionen. In: Heite, H.]. (ed.), Krankheiten durch Aktinomyzeten und verwandte Erreger. Wechselwirkungen zwischen pathogenen Pilzen und Wirtsorganismus, p.87. Springer, Berlin-Heidelberg-New York 1967 6. Staib, F., S. K. Mishra, G. Grosse, and Th. Abel: Ocular cryptococcosis. Experimental and clinical observations. Zbl. Bakt. Hyg., I. Abt, Orig. A 237 (1977) 378 7. Thiel, G., D. T. Wilson, M. L. Arce, and D. E. Oken: Glycerol induced hemoglobinuric acute renal failure in the rat. II. The experimental model, predisposing factors and pathophysiological features. Nephron 4 (1967) 276 Drs. R. A. and Anne M. Fromtling, Dept. of Microbiology, Medical College of Virginia, MCV Station, Box 618, Richmond, Virginia 23298, U.S.A. Prof. Dr. Dr. F. Staib, Robert Koch-Instirut (Federal Health Office), Nordufer 20, D-1000 Berlin 65
Auxanographic Detection of Experimental Murine Uremia with Cryptococcus neoformans Auxanographischer Nachweis der experimentell erzeugten Uraemie bei der Maus mit Hille von Cryptococcus neoformans R. A. FROMTLINGt, ANNE M. FROMTLING1, and F. STAIB With 2 Figures' Received June 27, 1980
Abstract A modified blood residual nitrogen plate auxanographic method was applied to the detection of experimental uremia in a murine model. The yeast-like fungus, Cryptococcus neoformans, was used as the indicator. Transient uremia was induced by injection of 0.2 ml glycerol intramuscularly. The low molecular weight nitrogen levels were estimated by measuring the diameter of the auxanogram at intervals of 2 hr for 24 hr and at 32 hr after the glycerol injection. After 4 hr, elevated levels of low molecular weight nitrogen were found. Maximum levels occurred 20 hr post glycerol injection. This method requires only 5,u1 of whole blood per assay. The results can be read after an incubation time of 24 hr at 26°C. The stability of the prepared plates was determined to be at least 96 hr at 4 0C. The ease of use, reliability and versatility of the modified auxanographic method are discussed.
Zusammenfassung Bei der experimentellen Uraemie der Maus wurde in einem Modellversuch der BlutRest-Stickstoff (Rest-N) mit Hilfe einer modifizierten auxanographischen Assimilationsprufungsmethode unter Verwendung von Cryptococcus neoformans als Indikaror bestimmt. Die temporar begrenzte Uraemie wurde durch eine intramuskulare Injektion von 0,2 ml Glycerin ausgelosr, Fur eine Blut-Rest-N-Bestimmung wurden nur 5,u1 Vollblut benotigt, Die Ergebnisse der Auxanogrammplatten konnten nach einer Inkubationszeit von 24 Std./26°C anhand des Auxanogramm-Durchmessers (mm) abgelesen werden. Fur diesen Auxanogramm-Nachweis gegossene Platten einer Charge zeigten anhand von Kontrollen mit Hilfe einer Harnsroff-Standard-Losung noch nach 4 Tagen/ 4°C vergleichbare Ergebnisse. Von Blutproben der Tiere, in Abstanden von 2 Stun den 1
Fellow of the Senator for Health and Environmental Protection, Berlin (West).
Uremia with Cryptococcus neoformans
269
uber einen Zeitraum von 24 Stunden und abschliefsend 32 Stunden abgenommen, wurde der Blut-Rest-N mittels des Auxanogramm-Durchmessersbestimmt. Die erste nachweisbare Rest-N-Erhohung lag 4 Stunden nach der Glycerin-Injektion vor, die hochste, d. h. das Auxanogramm mit dem grolSten Durchmesser nach 20 Stunden. DIe Durchfiihrung, Zuverlassigkeit und vielseitige Verwendbarkeit dieser modifizierten auxanographischen Methode werden diskutiert.
Introduction Uremia resulting from acute or chronic renal dysfunction may lead to death by generalized toxemia or infection (2). Animal models can be of value in the study of the compromised host during uremia. Experimental uremia can be induced in animals by the intramuscular injection of glycerol (7). In 1964, Staib modified the Beijerinck auxanographic method for the detection of low molecular weight (residual) nitrogenous substances in clinical samples (1, 4, 5). While the method was previously used to identify unknown yeasts by their assimilation of known carbon and nitrogen sources (3), he used a known yeast (Cryptococcus neoiormans, Candida albicans) to evaluate unknown nitrogen sources, i. e., human serum (4). In a subsequent experiment, the auxanographic method was applied to the detection of uremia induced in animals infected with C. neoiormans. A higher incidence of ocular involvement was observed in uremic animals than in control animals (6). The purpose of this investigation was to study the development and course of glycerol-induced uremia in a murine model and to assess the applicability of the auxanographic method to monitor the uremic state.
Materials and Methods Organism. Cryptococcus neojormans, strain W 71, isolated from pigeon excreta in Wiirzburg, Germany, was maintained at 4 °C on Sabouraud-dextrose agar. Animals. Male, albino mice (NMRI) weighing 18-20 grams were used. Animals were caged individually. Preparation of auxanographic plates. Auxanographic plates for the detection of low molecular weight nitrogenous substances were prepared according to the method of Staib (4). The nitrogen assimilation medium (2.0 % dextrose, 0.1 % KH2P04, 0.05 0/ 0 MgS0 4 , 2 % Bacto-Agar) was cooled to about 45°C after autoclaving (115°C, 15 min.), A solution of 2 X 107 organisms/ml saline was prepared from a 48 hr culture of C. neoformans grown on Beerwort agar (Schultheiss-Brauerei AG, Berlin (West)) at 26°C. One ml of yeast suspension was pipetted into the center of a nine em petri plate (Greiner GmbH., Berlin (West)). Immediately, 10 ml of warm medium were poured into the plate and the plate was agitated for several seconds to insure homogenous mixture of organism and agar. Medium was allowed to solidify at room temperature. Excess moisture was evaporated by inverting open plates in a 37°C incubator for 1 hr. Plates were then stored at 4 °C until 1 hr prior to use. Induction of uremia. Preliminary studies with groups of mice deprived of water for 24 hr prior to 90 % glycerol injection (7) showed an increase in auxanogram diameter for up to 3 days, accompanied by increasingly severe symptoms of uremia from which most of the animals did not recover. Animals injected with 45 Ofo glycerol developed a
270
R.A .Fromding, A.M .Fromding, and F.Sraib
minimal increase in auxanogram diameter and mild symptoms of uremia, both of short duration (less than 48 hr). Water deprivation had no effect on animals receiving 45 % glycerol. Based on these observations, conditions were chosen which resulted in development of severe but transient uremia. Uremia was induced by injection of 0.2 ml glycerol (90 0/0) intramuscularly in the hind leg of each mouse (6). Animals were given food and water ad libitum. Detection of uremia . Blood was obtained from the mice by cutting the tip of the tail. Five Itl of blood were collected with a capillary pipette and placed in the center of an auxanograph ic plate which then was incubated at 26 °C for 24 hr. Diameter of yeast growth was measured in mm. Kinetics of development of experimental murine uremia. Level of low molecular weight nitrogen was determ ined by the auxanographic method in 24 mice 2 min. prior to induction of uremia. Following glycerol injection, a group of four mice was tested by auxanogram every two hour s for 24 hr and at 32 hr. Degree of uremia was expressed as the change in diameter calculated by subtracting the value of the pre-treatment auxanogram from the post-treatment value for the same individual animal. Mean change in diameter was calculated from each time-group. Urea standards (ranging from 20 to 320 mg % ) were tested at 0, 24, 48, 72 and 96 hr to test the stability (shelf-life) of stored plates and to test the sensitivity of the organism to increasing nitrogen concentr ation.
Results The diameter of the auxanogra m was proportional to the concentration of urea standa rd used. The size a nd densities of the au xanogram s were not significantly different in plates used 1 hr after preparation in comparison with those plates which were refrigerated for up to 96 hr (Table 1). The increase of dia m eter and density of yeast growth with increasing urea concentration was easily detectable. Standards w ere prepared solely to test th e reproducibility and sensitivity of the auxan ographic pl ates. No attempt w as made to correl at e urea standard concentration w ith low molecular weight nitrogen concentration in blood on the ba sis of similar app ea ra nce of auxanograms. Table 1. Diameter of urea stand ard auxanograms with Crypto coccus neoiormans (mm). Five ,ul of standard solution were placed on each plate Time (hr)
20
Urea concentration (mg %) 160 80 40
0 24 32 48
16 16 17 18 15 16
20 21 20 20 20 21
72
96
23 23 22 22.5 23 22
26 27 26 27 26 26
320 31 32 30.5 32 31 31
Within 4 hr post-glycerol injection, an increase in auxanogram diameter w as ob served (Fig. 1). The size and density of the zone of growth continued to increase until 20 hr post-glycerol in jection. Photographs of typical auxanograms
Uremia with Cryptococcus neoformans E
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Fig. 1. Change in low molecular weight nitrogen concentration (uremia) as measured by the modified auxanographic technique. Each value is the mean of 4 animals. showing the development of uremia in mice are presented in Fig. 2. The density of c. neoformans growth is greater in these auxanograms than in those prepared from the urea standards. Additionally, the diffusion of red blood cells decreased over the 32 hr course of the experiment. Of the 24 mice used in this experiment, 2 failed to develop uremia as detected by this method. Both of these mice were in the group tested at 12 and 24 hrs, and the drop in mean diameter reflects these data (Fig. 1). Decline in uremia occurred by 32 hr. Previous studies by Staib indicated that low molecular weight nitrogen levels in blood returned to normal by 72 hr in mice in which experimental uremia was induced. Additionally, chemical analysis of pooled serum from two groups of 10 NMRI mice gave the following results: before glycerol injection, urea-nitrogen 28 mg 0/0 and creatinine 1.8 mg 0/0; 24 hr after injection, urea-nitrogen 140 mg % and creatinine 3.0 mg Ofo (unpublished data). Discussion Our present study confirms the observation of Staib and coworkers that changes in residual nitrogen levels in peripheral blood of mice can be detected by the use of this modified auxanographic method (6). The study also shows that diameter of C. neojormans growth is proportional to the concentration of an
272
R. A. Fromt/ing, A. M. Fromt/ing, and F.Staib
Fig. 2. Development of uremia as illustrated by typical auxanograms. Numbers represent hours post-glycerol injection. Five ,ul of whole blood were placed on each plate. Dark central spots are the cellular components of blood. Larger light circles are the growth of C. neoformans dependent on residual nitrogen. Bar = 1 ern.
appropriate nutrient (Table 1). The data in Fig. 1 illustrate the ability of the auxanographic method to detect an increasing course of experimental uremia in mice. Diameter of C. neojormans growth increased from an average of 21 mm for pretreatment mice to an average of 31.25 mm at 20 hr post-glycerol injection. The peak of experimental uremia at 20 hr was followed by a gradual decrease in residual nitrogen levels to the pretreatment value at 72 hr post-glycerol injection. Precision of the intramuscular injection of glycerol is critical for the induction of experimental uremia. Leakage of glycerol from the injection site may account for the negative results seen in 2 of the animals used in this study. An important consideration for the practical application of this technique is the stability or shelf-life of the prepared plates. The data in Table 1 show that the auxanographic plates can be stored at 4 °C for least 96 hr without affecting the results of the test. The collection of blood poses a problem in experiments with a murine model. The auxanographic method described here requires only 5,il1 of whole blood which can be obtained easily with minimum trauma to the animal. Conventional techniques require the separation of serum from whole blood and require larger volumes. Cellular components of blood do not interfere with the detection of residual nitrogen levels using this technique. In the 2 animals that did not develop uremia the decrease in cellular diffusion was comparable with that seen in uremic mice tested at the same time. The acute uremia induced in this model approximates transient uremia which
Uremia with Cryptococcus neoformans
273
easily can remain subclinical, and therefore, remain undiagnosed in experimental animals. The physiologic changes concurrent with acute uremia must be better understood in order to decrease the high potential for infection in uremic animals (6). With the animal model introduced here and the reproducible auxanographic assay, uremic animals can be monitored and specific assays of immunocompetence performed. Since a pathogenic microorganism is used to measure residual nitrogen, this auxanographic method is especially valuable in the assessment of the nutritional value of residual nitrogen for this organism (6). The auxanographic method is not limited solely to the measurement of low molecular weight nitrogenous substances in the blood but also can be applied to extracts of various organs (unpublished data) and thus may help to explain the predilection of certain organisms for a particular organ. Determination of the concentration of such substances in the brain of uremic mice will be the subject of further investigation. Acknowledgements. The authors wish to express their gratitude to Mrs. A. Blisse and A. Passow for their technical assistance.
References 1. Beijerinck, M. W.: L'auxanographie ou la methode de l'hydrodiffusion dans la gelatine appliquee aux recherches microbiologiques. Arch. neerland. sci. 23 (1889) 267 2. Guyton, A. c.: Textbook of Medical Physiology, 5th ed., p.504. W. B. Saunders Co., Philadelphia (1976) 3. Lodder.]. and N.]. W. Kreger-Van Rij: The Yeasts. North Holland Pub!. Comp., Amsterdam (1952) 4. Staib, F.: Das Serum-Reststickstoff-Auxanogramm (mit Sprols- und Schimmelpilzen). Zb!. Bakt., I. Abt, Orig. 194 (1964) 379 5. Staib, F.: Zum Verhalten von Cryptococcus neoformans gegeniiber Serumfraktionen. In: Heite, H.]. (ed.), Krankheiten durch Aktinomyzeten und verwandte Erreger. Wechselwirkungen zwischen pathogenen Pilzen und Wirtsorganismus, p.87. Springer, Berlin-Heidelberg-New York 1967 6. Staib, F., S. K. Mishra, G. Grosse, and Th. Abel: Ocular cryptococcosis. Experimental and clinical observations. Zbl. Bakt. Hyg., I. Abt, Orig. A 237 (1977) 378 7. Thiel, G., D. T. Wilson, M. L. Arce, and D. E. Oken: Glycerol induced hemoglobinuric acute renal failure in the rat. II. The experimental model, predisposing factors and pathophysiological features. Nephron 4 (1967) 276 Drs. R. A. and Anne M. Fromtling, Dept. of Microbiology, Medical College of Virginia, MCV Station, Box 618, Richmond, Virginia 23298, U.S.A. Prof. Dr. Dr. F. Staib, Robert Koch-Instirut (Federal Health Office), Nordufer 20, D-1000 Berlin 65