JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1976, p. 360-371 Copyright © 1976 American Society for Microbiology
Vol. 4, No. 4 Printed in U.S.A.
Examination of Various Cell Culture Techniques for CoIncubation of Virulent Treponema pallidum (Nichols I Strain) Under Anaerobic Conditions P. L. SANDOK, S. T. KNIGHT, AND H. M. JENKIN* The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 Received for publication 24 March 1976
Treponema pallidum (Nichols virulent) was incubated with and without cells in cell culture medium reduced to -275 mV Ecai, pH 7.3, under deoxygenated conditions. Five to ten percent of the treponemes attached to cells and remained motile for at least 120 h in cell-treponeme systems of co-incubation. Virulent treponemes could be detected after 120 to 144 h in the supernatant fluids of celltreponeme co-incubation cultures and in cell-free tubes containing medium harvested from aerobically cultivated mammalian cells. Medium supplemented with ox serum ultrafiltrate, pyruvate, and sodium thioglycolate and gas mixtures containing H2 and CO2 enhanced treponemal survival. Increases in treponemal numbers were observed using dark-field microscopy but were not substantiated using the rabbit lesion test. Continuous passage ofthe treponeme was not achieved in vitro. a
Bacteriological systems for the investigation of growth factors for cultivation of Treponema pallidum in vitro have met with little success (9, 33). Therefore, an alternative approach to the problem of treponemal growth is needed. Other investigators are reexamining the possibility that oxygen may play a role in the physiological requirements of the microorganism for growth (3, 5, 8, 12, 13, 22). Oxygen does not appear to completely inactivate the microorganism in vivo (10, 12). It has been shown that T. pallidum may take up oxygen (5), that the presence of 10 to 20% 02 mixed with N2, H2, and CO2 may stimulate the incorporation of certain amino acids (3), and that the presence of oxygen may not be detrimental to the growth of the microorganism in vitro (13). Nonetheless, with two exceptions (13, 22), most workers have found it difficult to examine the direct interplay of 02 with T. pallidum in vitro without killing most of the virulent treponemes within 24 to 36 h (3, 8, 33, 34). Although it has been reported that treponemal growth occurred in aerobic cell culture systems, only a small proportion of microorganisms may be used for serial passage. Virulence retention of aerobically cultivated treponemes appears to be very sporadic (13). This is not surprising since neither the redox potential nor the presence of toxic by-products of oxygenation (i.e., peroxides and superoxides) may be adequately controlled under aerobic conditions in vitro in contrast to in vivo conditions (4, 8, 26, 27). Problems associated with treponemal sur-
vival in vitro which may be due to peroxidation of treponemal and cellular constituents or metabolites and the formation of superoxide anions in the medium may be circumvented in anaerobic systems of cell-treponeme cultivation. The bulk ofcurrent evidence suggests that T. pallidum in vitro requires the maintenance of a low redox potential in the medium besides a number of nutrients for survival (9, 18, 25, 32, 33). Earlier we established a set of conditions wherein a rather modest enhancement of treponemal survival could be consistently obtained in anaerobic cell and cell-free (spent cell culture medium) cultures (25). This report is an extension of our earlier findings and a description of modifications which have enhanced the survival of the virulent treponeme. The utility of anaerobic cell-treponeme cultures as a basal system for further investigations on the growth requirements for T. pallidum is reported in a series of studies on the effects of established parameters on treponemal survival in vitro. Evidence is presented that certain nutrients (i.e., pyruvate, ox serum ultrafiltrate [OSU]) and gases (i.e., CO2 and H2) enhance treponemal survival in spent cell culture medium in much the same way that has been documented for bacteriological systems of incubation (33).
MATERIALS AND METHODS Propagation and harvest of T. pallidum (Nichols virulent strain). The passage of virulent T. pallidum by intratesticular inoculation in New Zealand 360
VOL. 4, 1976
CELL CULTURE ANAEROBIC CO-INCUBATION
rabbits (3.2 to 3.6 kg) has been described elsewhere (9). The testes were harvested as described previously (9, 25) with some modifications. The microorganism was eluted from infected testes in sequential steps in which each testis was further minced. The first eluant was not used for in vitro work. Three to six eluants (3 ml each) were combined, and particulate debris was removed by sedimentation at 250 x g for 10 min at room temperature. At no time was any attempt made to maintain anaerobic conditions during the elution sequence, which took from 20 to 40 min. Source of cells. The rat glial cells (RGC-6) were kindly provided by W. Bondareff (Northwestern University, Evanston, Ill.). The cells were serially passaged in Eagle medium plus 10% newborn calf serum (MEM10) (7). Human prepuce cell cultures were initiated in our laboratory from primary cell cultures derived from fresh tissue mincings. The tissue was obtained from circumcision waste material obtained aseptically from 3- to 6-day-old newborn babies and placed in MEMIO. The tissue was processed within 1 h after surgery. A mixed population of single cells was obtained using routine procedures described by Anderson et al. (1). Primary cell cultures were obtained by incubating the mincings at 37°C (5 to 14 days) in MEM1o supplemented with 100 U of penicillin per ml and 100 ,tg of streptomycin per ml. The medium was changed after 7 days of incubation, and the mincings were discarded. The cells that remained attached to the culture vessel were refed MEM,O and incubated for another 7 to 14 days until confluent monolayers were obtained. The medium was changed every 4 to 7 days or when the pH was 6.8 to 6.9. The primary cell cultures were serially passed to new vessels as described elsewhere (16) without antibiotics. Cell cultures. (i) Cell-free systems of incubation. Cells (3 x 106 rat glial or 106 human prepuce cells) were cultivated aerobically in unreduced medium in 0.95-liter prescription bottles for 48 h at 340C. The supernatant fluids containing cell culture metabolites (spent medium) were aseptically decanted, adjusted to a pH of 7.3 with 0.2 N NaOH, and supplemented with additional nutrients as required. The spent medium was diluted with highly reduced (-400 mV Eca1, pH 7.3) Eagle MEM,o (7) under a stream of deoxygenated N2 (11, 25) to obtain a final redox potential of -275 + 35 mV Ecai, pH 7.3. The redox potential of the medium was tested with a calomel KCl-saturated electrode as described elsewhere (9, 25). The following set of abbreviations are used in the text to designate different cell culture media: PR, prereduced; NF,0, new formulation or modified Eagle (7)- Richter (24) medium. For convenience, reduced media containing aerobically derived metabolites from human prepuce cells have the suffix Hu (e.g., PRNF,oHu and PRMEM,OHu). Reduced media containing aerobically derived metabolites from rat glial cells have the suffix R (e.g., PRNF1OR and PRMEMIOR). Ten milliliters of medium was distributed into sterile anaerobic glass tubes (145 by 15 mm; Bellco, Vineland, N.J.) under N2 and tightly stoppered. Each tube was inoculated with 0.5 ml of the trepone-
361
mal suspension described above to obtain a final suspension of 3 x 106f to 7 x 106 T. pallidumlml. Treponemal inocula present initially in the experiment in excess of 107 treponemes per ml were detrimental to treponemal survival in vitro due to subsequent acidification of the medium (i.e., pH s7.0) after 24 h of incubation. (ii) Cell-treponeme co-incubation system. Cells (rat glial and human prepuce) were cultivated in 0.95-liter prescription bottles until confluent monolayers were obtained. The monolayers were detached from the glass surface with trypsin (16). The final volume of the cell suspension was adjusted to 5 x 104 cells/ml in unreduced medium. One milliliter of the cell suspension was placed into each Leighton tube containing a cover slip (9 by 22 mm) which could be removed for subsequent dark-field examination at x400. Cultures were incubated aerobically overnight at 37°C. After incubation the supernatant fluids were decanted, and each tube received 1 ml of freshly prepared reduced medium with a redox potential of -275 + 35 mV Eca pH 7.3. The cultures were inoculated under a flow of deoxygenated gas with 0.1 ml of a suspension of T. pallidum to obtain approximately 5 x 106 T. pallidum per 5 x 104 cells per culture. Treponemal numbers in an excess of9 x 106 or cell numbers above 105 or below 104 per culture were detrimental to treponemal survival in vitro. The tubes were stoppered and incubated at 34 ± 0.5°C under anaerobic conditions. Media. Highly reduced Eagle medium (-400 mV E,.a, pH 7.3) was prepared on the day experiments were initiated as described elsewhere (25). The effect of toxic components in rubber stoppers to cells was minimized by using nontoxic white rubber stoppers of high quality during all preparative steps and during incubation. The highly reduced medium was diluted, 1:10, with unreduced medium to obtain a redox potential of -275 ± 35 mVEcal, pH 7.3, at the beginning of the experiments. The constitutents of reduced Eagle medium are given in Table 1 under the heading PRMEM,O. The constituents of modified Eagle-Richter medium are shown in Table 1 under the heading PRNF,O. PRNF,O differs from Eagle (7) and Richter (24) media in that it is supplemented with Eagle nonessential amino acids and pyruvate (International Scientific Industries, Cary, Ill.). IMEMZO (24) (Richter improved Eagle medium with a zinc option) was obtained from Laboratory Cell Suppliers, Fort Worth, Tex., or International Biological Laboratories, Rockville, Md. IMEMZO was reduced as described above. The formula for this medium is given in Table 1 under the heading PRI,o. Direct counts. (i) Cell-free systems. Each culture was counted within 1 to 2 h after inoculation and at 24-h intervals thereafter. Samples were removed, and two 0.01-ml droplets of the suspension were placed on microscope slides and covered with square cover slips (18 by 18 mm). Fields for counting under dark-field microscopy were selected randomly. Numbers of motile flexing treponemes and nonmotile treponemes were recorded for each field examined. All estimates of numbers of treponemes in suspension were derived from 5 to 10 fields per cover slip from at least eight cover slips using two to four
362
J. CLIN. MICROBIOL.
SANDOK, KNIGHT, AND JENKIN
TABLE 1. Components of reduced media (mg/liter) Media Components PRMEM1O-
Buffers and saltsd NaCl KCl MgSO4 * 7H20 KH2PO4 Na2HPO4 CaCl2 * 2H20 NaHCO3 NaOH MgCl2 * 6H20 NaH2PO4 12H20 ZnSO4 * 7H20 Fe(NO3)3 * 9H20 HEPESe Organic Glucose Pyruvate Putrescine * hydrochloride Linoleic acid Vitamins' D calcium pantothenate Choline chloride Folic acid i-Inositol Nicotinamide Pyridoxal hydrochloride Riboflavin Thiamin hydrochloride Lipoic acid Pyridoxine hydrochloride Biotin B12 Nicotinic acid Folinic acid Eagle essential amino acids' Arginine Cystine Glutamineg Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophane Tyrosine Valine Nonessential amino acids' Alanine Asparagine Aspartic acid Glutamic acid
6,120 360 180 54 54 126 20 240
4,766 900
1 1 1 2 1 1 0.1 1
105 24 292 31 52.5 52.4 58 15 32 48 10 36 46
PRI,ob
PRNFl,0
6,732 396 40.5 5.4 36.4 251.1 1,982 240 164.7 135 0.13 0.73 4,766
6,732 328.6 239.8 138.5 5.4 251.1 1,982 240
1,890 99 0.14 0.076
1,890 198
1 12.6 0.1 16.4 0.1 0.1 0.1 2.4 0.18 0.9 0.09 1.22 0.9 0.9 124.8 24 292 40.8 52 52 71.1 15 22.1 48 10 36 46
72.4 82.7 124.0
1 1 1 2 1 1 0.1 1
105 24 292 31 52.5 52.4 58 15 32 48 10 36 46
18.9
16 27 24 26 13.5 20.7 18.9
72.4 82.7 124.0
72.4 82.7 124.0
13.5
Glycine Proline Serine Reducing agentsh Sodium thioglycolate L-Cysteine Glutathione
4,766
VOL. 4, 1976
CELL CULTURE ANAEROBIC CO-INCUBATION
363
TABLE 1-Continued Media
Components
PREM,,a
PRI,,b
PRNF,o0
Miscellaneous Phenol red 0.1 0.1 0.1 Resazurini 0.25 0.25 0.25 Newborn calf serum 100 ml/liter 110 ml/liter 100 ml/liter Ox serum ultrafiltratei 40 ml/liter 40 ml/liter a PRMEM1,o Prereduced Eagle minimum essential medium (7) + 10% newborn calf serum. b PRI,O, Prereduced improved minimum essential medium (IMEMZO) + 10% newborn calf serum. The unreduced formulation for IMEMZO is given by Richter et al. (24) and is referred to as Richter medium in the text. c PRNF,0, Prereduced medium with a new formulation + 10% newborn calf serum. The formula for PRNF,o was derived by modifying and combining portions of the formulas for PRMEM,o and PRI,. d Buffers, salts, and glucose were prepared as concentrated solutions (10x) and diluted in sterile doubledistilled water. PRNF,0 solutions were sterilized by filtration, and PRMEM,o solutions were diluted and sterilized in an autoclave. e Sterile HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer was added after the media were completed. ' Vitamin solutions (100 x) and Eagle essential (50 x) and nonessential (100 x) amino acids for PRNF,o and PRMEM,0 were obtained, premixed, from commercial sources (Grand Island Biological Co., Grand Island, N.Y., or International Scientific Industries, Cary, Ill.). 9 Fresh glutamine (2 mM) was added to all media before use. h The procedure used for the incorporation of reducing agents is described elsewhere (25). i Concentration of resazurin after combining one part of highly reduced PRMEM,0 (-400 mV EC,9, pH 7.3) containing 2.5 mg of resazurin per liter with nine parts of unreduced media. Resazurin served as a redox indicator during the preparation of highly reduced PRMEM,O. J Ox serum ultrafiltrate was obtained from the Colorado Serum Co., Denver, Colo. It was not used in all preparations of medium.
replicate cultures, which resulted in a minimum of attached treponemes per culture, where the correc250 treponemes counted per treatment. A conversion tion factor (CF) = 3.8 x 103. The correction factor factor (5.29 x 105) to translate numbers of trepo- was derived from the estimated number of cells nemes per field to estimates of treponemes per milli- which may be detected on a cover slip (9 by 22 mm) if liter of medium was determined on the basis of all the cells in the inoculum (5 x 104) become atestimates of the fluid volume of the treponemal sus- tached to the glass surface. Different sets of replipension per microscopic field and checked against cate cultures were counted at each interval of time. results obtained using a Petroff-Hauser counter for Rabbit lesion test and estimation of virulence the same suspensions. Estimates of the standard retention. The procedure used to prepare and to error of the mean were derived from the mean inoculate Dutch Belt rabbits for the rabbit lesion counts of -eight cover slips. The accuracy of this test is described elsewhere (9). The estimated viruprocedure for treponemal enumeration was exam- lence retention for any given set of quadruplicate ined directly as shown in Table 2. All statistical cultures was based on the results of the rabbit lesion tests were performed before conversion of the data to test with samples prepared at each counting interestimates of numbers of treponemes per milliliter of val. Samples at each interval were prepared by dimedium. Numbers of replicate cultures per treat- luting 0.2 ml of a solution containing 50% glycerol ment always equaled or exceeded the minimum plus 50% medium with 0.8 ml of the treponemal number required for the estimation of significance suspension. The samples were mixed thoroughly, using Student's t test (Table 3). slowly cooled to -77°C, and stored before use. Each (ii) Cell co-incubation systems. Direct counts on sample was inoculated intradermally into a total of the supernatant fluids of all Leighton tube cultures four sites on two rabbits. Supernatant fluids or cell were performed as described above. In addition, the culture medium not containing T. pallidum were cover slips were removed with a spatula and placed inoculated in similar fashion on the same rabbits as cell-side-down on a slide. Treponemes attached to 25 controls. The calculation of numbers of virulent to 50 cells were counted, and the number of fields treponemes represents estimates based on the mean counted per cover slip were recorded. Crude esti- day of lesion appearance at all four sites of inoculamates of numbers of attached treponemes per cell tion per sample with respect to a standard curve (9). and per culture were obtained using the following Without exception, variations in the mean date of formulas: no. of treponemes/no. of cells = no. of lesion appearance fell within a range of ±+2 days for treponemes per cell, and no. of treponemes/no. of estimates~-105 virulent T. pallidum/ml and +4 days cells x no. of cells/no. of fields x CF = no. of for estimates
Vol. 4, No. 4 Printed in U.S.A.
Examination of Various Cell Culture Techniques for CoIncubation of Virulent Treponema pallidum (Nichols I Strain) Under Anaerobic Conditions P. L. SANDOK, S. T. KNIGHT, AND H. M. JENKIN* The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 Received for publication 24 March 1976
Treponema pallidum (Nichols virulent) was incubated with and without cells in cell culture medium reduced to -275 mV Ecai, pH 7.3, under deoxygenated conditions. Five to ten percent of the treponemes attached to cells and remained motile for at least 120 h in cell-treponeme systems of co-incubation. Virulent treponemes could be detected after 120 to 144 h in the supernatant fluids of celltreponeme co-incubation cultures and in cell-free tubes containing medium harvested from aerobically cultivated mammalian cells. Medium supplemented with ox serum ultrafiltrate, pyruvate, and sodium thioglycolate and gas mixtures containing H2 and CO2 enhanced treponemal survival. Increases in treponemal numbers were observed using dark-field microscopy but were not substantiated using the rabbit lesion test. Continuous passage ofthe treponeme was not achieved in vitro. a
Bacteriological systems for the investigation of growth factors for cultivation of Treponema pallidum in vitro have met with little success (9, 33). Therefore, an alternative approach to the problem of treponemal growth is needed. Other investigators are reexamining the possibility that oxygen may play a role in the physiological requirements of the microorganism for growth (3, 5, 8, 12, 13, 22). Oxygen does not appear to completely inactivate the microorganism in vivo (10, 12). It has been shown that T. pallidum may take up oxygen (5), that the presence of 10 to 20% 02 mixed with N2, H2, and CO2 may stimulate the incorporation of certain amino acids (3), and that the presence of oxygen may not be detrimental to the growth of the microorganism in vitro (13). Nonetheless, with two exceptions (13, 22), most workers have found it difficult to examine the direct interplay of 02 with T. pallidum in vitro without killing most of the virulent treponemes within 24 to 36 h (3, 8, 33, 34). Although it has been reported that treponemal growth occurred in aerobic cell culture systems, only a small proportion of microorganisms may be used for serial passage. Virulence retention of aerobically cultivated treponemes appears to be very sporadic (13). This is not surprising since neither the redox potential nor the presence of toxic by-products of oxygenation (i.e., peroxides and superoxides) may be adequately controlled under aerobic conditions in vitro in contrast to in vivo conditions (4, 8, 26, 27). Problems associated with treponemal sur-
vival in vitro which may be due to peroxidation of treponemal and cellular constituents or metabolites and the formation of superoxide anions in the medium may be circumvented in anaerobic systems of cell-treponeme cultivation. The bulk ofcurrent evidence suggests that T. pallidum in vitro requires the maintenance of a low redox potential in the medium besides a number of nutrients for survival (9, 18, 25, 32, 33). Earlier we established a set of conditions wherein a rather modest enhancement of treponemal survival could be consistently obtained in anaerobic cell and cell-free (spent cell culture medium) cultures (25). This report is an extension of our earlier findings and a description of modifications which have enhanced the survival of the virulent treponeme. The utility of anaerobic cell-treponeme cultures as a basal system for further investigations on the growth requirements for T. pallidum is reported in a series of studies on the effects of established parameters on treponemal survival in vitro. Evidence is presented that certain nutrients (i.e., pyruvate, ox serum ultrafiltrate [OSU]) and gases (i.e., CO2 and H2) enhance treponemal survival in spent cell culture medium in much the same way that has been documented for bacteriological systems of incubation (33).
MATERIALS AND METHODS Propagation and harvest of T. pallidum (Nichols virulent strain). The passage of virulent T. pallidum by intratesticular inoculation in New Zealand 360
VOL. 4, 1976
CELL CULTURE ANAEROBIC CO-INCUBATION
rabbits (3.2 to 3.6 kg) has been described elsewhere (9). The testes were harvested as described previously (9, 25) with some modifications. The microorganism was eluted from infected testes in sequential steps in which each testis was further minced. The first eluant was not used for in vitro work. Three to six eluants (3 ml each) were combined, and particulate debris was removed by sedimentation at 250 x g for 10 min at room temperature. At no time was any attempt made to maintain anaerobic conditions during the elution sequence, which took from 20 to 40 min. Source of cells. The rat glial cells (RGC-6) were kindly provided by W. Bondareff (Northwestern University, Evanston, Ill.). The cells were serially passaged in Eagle medium plus 10% newborn calf serum (MEM10) (7). Human prepuce cell cultures were initiated in our laboratory from primary cell cultures derived from fresh tissue mincings. The tissue was obtained from circumcision waste material obtained aseptically from 3- to 6-day-old newborn babies and placed in MEMIO. The tissue was processed within 1 h after surgery. A mixed population of single cells was obtained using routine procedures described by Anderson et al. (1). Primary cell cultures were obtained by incubating the mincings at 37°C (5 to 14 days) in MEM1o supplemented with 100 U of penicillin per ml and 100 ,tg of streptomycin per ml. The medium was changed after 7 days of incubation, and the mincings were discarded. The cells that remained attached to the culture vessel were refed MEM,O and incubated for another 7 to 14 days until confluent monolayers were obtained. The medium was changed every 4 to 7 days or when the pH was 6.8 to 6.9. The primary cell cultures were serially passed to new vessels as described elsewhere (16) without antibiotics. Cell cultures. (i) Cell-free systems of incubation. Cells (3 x 106 rat glial or 106 human prepuce cells) were cultivated aerobically in unreduced medium in 0.95-liter prescription bottles for 48 h at 340C. The supernatant fluids containing cell culture metabolites (spent medium) were aseptically decanted, adjusted to a pH of 7.3 with 0.2 N NaOH, and supplemented with additional nutrients as required. The spent medium was diluted with highly reduced (-400 mV Eca1, pH 7.3) Eagle MEM,o (7) under a stream of deoxygenated N2 (11, 25) to obtain a final redox potential of -275 + 35 mV Ecai, pH 7.3. The redox potential of the medium was tested with a calomel KCl-saturated electrode as described elsewhere (9, 25). The following set of abbreviations are used in the text to designate different cell culture media: PR, prereduced; NF,0, new formulation or modified Eagle (7)- Richter (24) medium. For convenience, reduced media containing aerobically derived metabolites from human prepuce cells have the suffix Hu (e.g., PRNF,oHu and PRMEM,OHu). Reduced media containing aerobically derived metabolites from rat glial cells have the suffix R (e.g., PRNF1OR and PRMEMIOR). Ten milliliters of medium was distributed into sterile anaerobic glass tubes (145 by 15 mm; Bellco, Vineland, N.J.) under N2 and tightly stoppered. Each tube was inoculated with 0.5 ml of the trepone-
361
mal suspension described above to obtain a final suspension of 3 x 106f to 7 x 106 T. pallidumlml. Treponemal inocula present initially in the experiment in excess of 107 treponemes per ml were detrimental to treponemal survival in vitro due to subsequent acidification of the medium (i.e., pH s7.0) after 24 h of incubation. (ii) Cell-treponeme co-incubation system. Cells (rat glial and human prepuce) were cultivated in 0.95-liter prescription bottles until confluent monolayers were obtained. The monolayers were detached from the glass surface with trypsin (16). The final volume of the cell suspension was adjusted to 5 x 104 cells/ml in unreduced medium. One milliliter of the cell suspension was placed into each Leighton tube containing a cover slip (9 by 22 mm) which could be removed for subsequent dark-field examination at x400. Cultures were incubated aerobically overnight at 37°C. After incubation the supernatant fluids were decanted, and each tube received 1 ml of freshly prepared reduced medium with a redox potential of -275 + 35 mV Eca pH 7.3. The cultures were inoculated under a flow of deoxygenated gas with 0.1 ml of a suspension of T. pallidum to obtain approximately 5 x 106 T. pallidum per 5 x 104 cells per culture. Treponemal numbers in an excess of9 x 106 or cell numbers above 105 or below 104 per culture were detrimental to treponemal survival in vitro. The tubes were stoppered and incubated at 34 ± 0.5°C under anaerobic conditions. Media. Highly reduced Eagle medium (-400 mV E,.a, pH 7.3) was prepared on the day experiments were initiated as described elsewhere (25). The effect of toxic components in rubber stoppers to cells was minimized by using nontoxic white rubber stoppers of high quality during all preparative steps and during incubation. The highly reduced medium was diluted, 1:10, with unreduced medium to obtain a redox potential of -275 ± 35 mVEcal, pH 7.3, at the beginning of the experiments. The constitutents of reduced Eagle medium are given in Table 1 under the heading PRMEM,O. The constituents of modified Eagle-Richter medium are shown in Table 1 under the heading PRNF,O. PRNF,O differs from Eagle (7) and Richter (24) media in that it is supplemented with Eagle nonessential amino acids and pyruvate (International Scientific Industries, Cary, Ill.). IMEMZO (24) (Richter improved Eagle medium with a zinc option) was obtained from Laboratory Cell Suppliers, Fort Worth, Tex., or International Biological Laboratories, Rockville, Md. IMEMZO was reduced as described above. The formula for this medium is given in Table 1 under the heading PRI,o. Direct counts. (i) Cell-free systems. Each culture was counted within 1 to 2 h after inoculation and at 24-h intervals thereafter. Samples were removed, and two 0.01-ml droplets of the suspension were placed on microscope slides and covered with square cover slips (18 by 18 mm). Fields for counting under dark-field microscopy were selected randomly. Numbers of motile flexing treponemes and nonmotile treponemes were recorded for each field examined. All estimates of numbers of treponemes in suspension were derived from 5 to 10 fields per cover slip from at least eight cover slips using two to four
362
J. CLIN. MICROBIOL.
SANDOK, KNIGHT, AND JENKIN
TABLE 1. Components of reduced media (mg/liter) Media Components PRMEM1O-
Buffers and saltsd NaCl KCl MgSO4 * 7H20 KH2PO4 Na2HPO4 CaCl2 * 2H20 NaHCO3 NaOH MgCl2 * 6H20 NaH2PO4 12H20 ZnSO4 * 7H20 Fe(NO3)3 * 9H20 HEPESe Organic Glucose Pyruvate Putrescine * hydrochloride Linoleic acid Vitamins' D calcium pantothenate Choline chloride Folic acid i-Inositol Nicotinamide Pyridoxal hydrochloride Riboflavin Thiamin hydrochloride Lipoic acid Pyridoxine hydrochloride Biotin B12 Nicotinic acid Folinic acid Eagle essential amino acids' Arginine Cystine Glutamineg Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophane Tyrosine Valine Nonessential amino acids' Alanine Asparagine Aspartic acid Glutamic acid
6,120 360 180 54 54 126 20 240
4,766 900
1 1 1 2 1 1 0.1 1
105 24 292 31 52.5 52.4 58 15 32 48 10 36 46
PRI,ob
PRNFl,0
6,732 396 40.5 5.4 36.4 251.1 1,982 240 164.7 135 0.13 0.73 4,766
6,732 328.6 239.8 138.5 5.4 251.1 1,982 240
1,890 99 0.14 0.076
1,890 198
1 12.6 0.1 16.4 0.1 0.1 0.1 2.4 0.18 0.9 0.09 1.22 0.9 0.9 124.8 24 292 40.8 52 52 71.1 15 22.1 48 10 36 46
72.4 82.7 124.0
1 1 1 2 1 1 0.1 1
105 24 292 31 52.5 52.4 58 15 32 48 10 36 46
18.9
16 27 24 26 13.5 20.7 18.9
72.4 82.7 124.0
72.4 82.7 124.0
13.5
Glycine Proline Serine Reducing agentsh Sodium thioglycolate L-Cysteine Glutathione
4,766
VOL. 4, 1976
CELL CULTURE ANAEROBIC CO-INCUBATION
363
TABLE 1-Continued Media
Components
PREM,,a
PRI,,b
PRNF,o0
Miscellaneous Phenol red 0.1 0.1 0.1 Resazurini 0.25 0.25 0.25 Newborn calf serum 100 ml/liter 110 ml/liter 100 ml/liter Ox serum ultrafiltratei 40 ml/liter 40 ml/liter a PRMEM1,o Prereduced Eagle minimum essential medium (7) + 10% newborn calf serum. b PRI,O, Prereduced improved minimum essential medium (IMEMZO) + 10% newborn calf serum. The unreduced formulation for IMEMZO is given by Richter et al. (24) and is referred to as Richter medium in the text. c PRNF,0, Prereduced medium with a new formulation + 10% newborn calf serum. The formula for PRNF,o was derived by modifying and combining portions of the formulas for PRMEM,o and PRI,. d Buffers, salts, and glucose were prepared as concentrated solutions (10x) and diluted in sterile doubledistilled water. PRNF,0 solutions were sterilized by filtration, and PRMEM,o solutions were diluted and sterilized in an autoclave. e Sterile HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer was added after the media were completed. ' Vitamin solutions (100 x) and Eagle essential (50 x) and nonessential (100 x) amino acids for PRNF,o and PRMEM,0 were obtained, premixed, from commercial sources (Grand Island Biological Co., Grand Island, N.Y., or International Scientific Industries, Cary, Ill.). 9 Fresh glutamine (2 mM) was added to all media before use. h The procedure used for the incorporation of reducing agents is described elsewhere (25). i Concentration of resazurin after combining one part of highly reduced PRMEM,0 (-400 mV EC,9, pH 7.3) containing 2.5 mg of resazurin per liter with nine parts of unreduced media. Resazurin served as a redox indicator during the preparation of highly reduced PRMEM,O. J Ox serum ultrafiltrate was obtained from the Colorado Serum Co., Denver, Colo. It was not used in all preparations of medium.
replicate cultures, which resulted in a minimum of attached treponemes per culture, where the correc250 treponemes counted per treatment. A conversion tion factor (CF) = 3.8 x 103. The correction factor factor (5.29 x 105) to translate numbers of trepo- was derived from the estimated number of cells nemes per field to estimates of treponemes per milli- which may be detected on a cover slip (9 by 22 mm) if liter of medium was determined on the basis of all the cells in the inoculum (5 x 104) become atestimates of the fluid volume of the treponemal sus- tached to the glass surface. Different sets of replipension per microscopic field and checked against cate cultures were counted at each interval of time. results obtained using a Petroff-Hauser counter for Rabbit lesion test and estimation of virulence the same suspensions. Estimates of the standard retention. The procedure used to prepare and to error of the mean were derived from the mean inoculate Dutch Belt rabbits for the rabbit lesion counts of -eight cover slips. The accuracy of this test is described elsewhere (9). The estimated viruprocedure for treponemal enumeration was exam- lence retention for any given set of quadruplicate ined directly as shown in Table 2. All statistical cultures was based on the results of the rabbit lesion tests were performed before conversion of the data to test with samples prepared at each counting interestimates of numbers of treponemes per milliliter of val. Samples at each interval were prepared by dimedium. Numbers of replicate cultures per treat- luting 0.2 ml of a solution containing 50% glycerol ment always equaled or exceeded the minimum plus 50% medium with 0.8 ml of the treponemal number required for the estimation of significance suspension. The samples were mixed thoroughly, using Student's t test (Table 3). slowly cooled to -77°C, and stored before use. Each (ii) Cell co-incubation systems. Direct counts on sample was inoculated intradermally into a total of the supernatant fluids of all Leighton tube cultures four sites on two rabbits. Supernatant fluids or cell were performed as described above. In addition, the culture medium not containing T. pallidum were cover slips were removed with a spatula and placed inoculated in similar fashion on the same rabbits as cell-side-down on a slide. Treponemes attached to 25 controls. The calculation of numbers of virulent to 50 cells were counted, and the number of fields treponemes represents estimates based on the mean counted per cover slip were recorded. Crude esti- day of lesion appearance at all four sites of inoculamates of numbers of attached treponemes per cell tion per sample with respect to a standard curve (9). and per culture were obtained using the following Without exception, variations in the mean date of formulas: no. of treponemes/no. of cells = no. of lesion appearance fell within a range of ±+2 days for treponemes per cell, and no. of treponemes/no. of estimates~-105 virulent T. pallidum/ml and +4 days cells x no. of cells/no. of fields x CF = no. of for estimates
