J. Anim Sci. 2008. 86:1395-1401. doi:10.2527/jas.2007-0238
© 2008 American Society of Animal Science
Improved in vitro procedure for maintaining stock cultures of three genera of rumen protozoa1
B. A. Dehority2
Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster 44691-4096
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Abstract
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To maintain stock cultures of rumen protozoa, studies were initiated to explore possible methods for keeping the protozoa viable without feeding every day. Cultures of Entodinium caudatum, Epidinium caudatum, Enoploplastron triloricatum, and Entodinium exiguum were used to study the effect of not feeding for 1 or 2 d. The study lasted 88 d, and although bacterial concentrations decreased when cultures were not fed for 2 d (over the weekend), they recovered quickly with subsequent daily feedings. The exception was Enoploplastron triloricatum, which showed a gradual decline over the entire study. Addition of streptomycin to the media had little effect on maintaining bacterial concentrations in all cultures except Entodinium caudatum, in which the overall mean concentration was greater (P < 0.01). No differences in pH or bacterial concentrations were found between cultures fed daily and those held without feed for 2 d, with or without streptomycin. For maintaining protozoal cultures (10-mL volumes) over a long period without feeding on weekends, the following schedule is proposed: transfer and feed 0.12 mL of 1.5% ground wheat–1% orchardgrass on Monday; feed 0.12 mL of 1.5% ground wheat–1% orchardgrass on Tuesday, Wednesday, and Thursday; transfer and feed 0.5 mL of 1.5% ground wheat–1% orchardgrass on Friday; do not feed on Saturday and Sunday.
Key Words: Entodinium caudatum • Entodinium exiguum • Enoploplastron triloricatum • Epidinium caudatum • in vitro culture • rumen protozoa • streptomycin
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INTRODUCTION
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The principal constraint to culturing rumen protozoa is that only small amounts of substrate can be added daily. Otherwise there is an overgrowth of the accompanying bacteria with a subsequent lowering of pH to a level of acidity that is inhibitory or lethal to the protozoa. Thus, it seemed feasible that cultures would not have to be fed daily if additional substrate was added and antibiotics were included in the medium to inhibit bacterial growth.
In preliminary studies from this lab (B. A. Dehority, unpublished data), the level of substrate was increased, antibiotics (penicillin, 2,000 U/mL, and streptomycin, 130 U/mL) were added, and Epidinium caudatum and Entodinium caudatum cultures were still viable after 3 to 4 d without feeding. However, even after returning to the normal daily feeding schedule and transferring several times, the protozoa died out after several weeks. Subsequent studies indicated that penicillin was toxic at a concentration of 100 U/mL, whereas streptomycin at 130 U/mL was not.
The present study was undertaken to investigate whether a feeding schedule could be established that would maintain stock protozoal cultures without feeding on weekends (Saturday and Sunday). Presumably, timing could be altered to accommodate holidays falling midweek.
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MATERIALS AND METHODS
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Ruminal fluid used in media was collected from cannulated animals following a protocol approved by the Ohio State University Animal Care and Use Committee.
Protozoal cultures used in this study were established by picking individual cells under a microscope (100x magnification), as described by Dehority (1998)
. The Ent. caudatum culture originated from a single cell picked from rumen fluid of a Gerunuk antelope housed at the St. Louis zoo (St. Louis, MO). A clone culture of Epi. caudatum was isolated from steer rumen fluid. The Enoploplastron triloricatum culture was begun by picking 3 cells from sheep rumen fluid. A culture of Entodinium exiguum was established from a mixed culture with Epidinium. The larger Epidinium cells were sedimented by centrifugation, and 0.5 mL of the supernatant containing only Ent. exiguum cells was inoculated into a fresh tube of medium.
Cultures were grown and maintained in either medium M or medium SP and were fed 0.12 mL of a 1.5% ground wheat–1.0% orchardgrass mixture or 1.5% ground wheat–1.0% alfalfa mixture daily (Dehority, 1998). Both the wheat and orchardgrass were ground to pass through a 40-mesh screen (425-µm openings) and suspended in distilled water. All media preparation, feeding, and transfers were carried out under a stream of O2-free CO2 (Dehority, 1969, 1998). Cultures were incubated in a 39°C incubator at an angle of 10°. Using a wide-mouthed pipet, unless otherwise noted, all transfers were made by transferring 5.0 mL of the culture to a tube containing 5.0 mL of fresh medium plus 0.12 mL of the substrate suspension. After transferring the 5 mL of culture, 3 mL of the old culture was transferred to a tube containing 3 mL of 50% formalin (18.5% formaldehyde) for counting.
The preserved sample was quantitatively transferred to a 15-mL conical, plastic centrifuge tube (graduated with a screw cap) and diluted to a final volume of 11.5 mL. The sample was centrifuged for 5 min at 500 x g, after which 11 mL of the supernatant was drawn off with suction. Two drops of Brilliant Green dye (EMD Chemicals Inc., Gibbstown, NJ) were added (2 g of Brilliant Green and 2 mL of glacial acetic acid, diluted to 100 mL with distilled water), and then the tubes were closed and allowed to stand for 4 h or longer. After staining, the samples were diluted to a final volume of 3 mL with 30% glycerol. Protozoa were counted in a Sedgewick-Rafter chamber (Thomas Scientific, Swedesboro, NJ) as described previously (Dehority, 1984), except if the numbers were extremely low, and then, all cells in a 0.1-mL aliquot were counted (Dehority, 2004). At least 4 aliquots were counted per sample.
The pH of the cultures was measured with an Accumet AB15 pH meter (Fisher Scientific, Pittsburgh, PA), using the 2.0 mL of media remaining after transfer of 5 mL and removal of 3.0 mL for counting. Bacterial concentrations were determined using the most-probable-number procedure described by Dehority et al. (1989). Graphs were prepared and data analyzed using MINITAB procedures (1991, Minitab, State College, PA).
Cultures of all 4 species were split, with one to serve as a control and the second to be treated with streptomycin. Figure 1 shows the pattern of feeding and transfer over an 88-d period. Initially, the cultures were transferred and fed 0.12 mL of a 1.5% ground wheat–1.0% orchardgrass substrate on Thursday (d 0 in Figure 1), 0.4 mL of the 1.5% ground wheat–1.0% orchardgrass substrate was added on Friday, and 115 U/mL of streptomycin added to the second culture tube. The levels of substrate fed over the entire trial are shown in Figure 1. Cultures were not fed on Saturday or Sunday and were transferred and fed Monday. The cultures were fed on Tuesday and Wednesday, transferred and fed on Thursday, and the high level of substrate, plus streptomycin where required, was added on Friday. Instead of continuing to transfer on Thursday (d 22), the cultures were fed 0.12 mL and then transferred and the high feed and streptomycin were added on Friday. This pattern was followed for several weeks, after which time holidays caused a shifting of days for transfer and feeding levels. This can be seen beginning on d 44 (Monday; Figure 1). After that, there were several normal Monday through Friday transfer weeks, followed by a somewhat varied schedule. Streptomycin concentrations added until d 50 were 115 U/mL of medium. On d 50, the concentration was increased to 200 U/mL, and on d 85 it was increased to 400 U/mL.
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Figure 1. Feeding and transfer schedule for studies on the culture of rumen protozoa. Feeding level for 10-mL cultures is shown on the y-axis and indicates the amount (mL) of 1% orchardgrass–1.5% ground wheat suspension fed. Open bars indicate the days on which the culture was fed, and solid bars represent the days on which the culture was both fed and transferred. ^Indicates those days on which streptomycin was added to the second culture tube (see Materials and Methods); *streptomycin concentration in the culture medium increased from 115 to 200 U/mL of medium; **streptomycin concentration increased to 400 U/mL of medium.
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RESULTS AND DISCUSSION
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The concentrations of Ent. caudatum, Epi. caudatum, and Eno. triloricatum, measured at each transfer over 88 d, are shown in Figures 2, 3, and 4. All 3 species exhibited a cyclic-type pattern of growth, but this was more pronounced with Ent. caudatum (Figure 2). Concentrations of Ent. caudatum increased when the cultures were fed daily and decreased when not fed. There appeared to be an advantage to including streptomycin when the cultures were not fed. The overall mean concentration of Ent. caudatum (28 values) without streptomycin was 15,873 ± 7,866 cells/mL and with streptomycin was 24,857 ± 9,044 cells/mL (P < 0.01).
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Figure 2. Concentrations of Entodinium caudatum, cultured according to the scheme shown in Figure 1 . Counts were made at each transfer. The dotted line is for the second culture, which had streptomycin added whenever the culture was not going to be fed for 1 or 2 d.
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Figure 3. Concentrations of Epidinium caudatum, cultured according to the scheme shown in Figure 1. Counts were made at each transfer. The dotted line is for the second culture, which had streptomycin added whenever the culture was not going to be fed for 1 or 2 d.
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Figure 4. Concentrations of Enoploplastron triloricatum, cultured according to the scheme shown in Figure 1. Counts were made at each transfer. The dotted line is for the second culture, which had streptomycin added whenever the culture was not going to be fed for 1 or 2 d.
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Epidinium caudatum concentrations did not fluctuate to the same extent and actually increased over the trial period (Figure 3). Including streptomycin when feed was withheld had minimal, if any, effect on concentration.
Concentrations of Eno. triloricatum are shown in Figure 4. In general, this species was negatively affected by withholding feed over the weekends. Aside from major increases in concentration between d 33 and 36 and d 53 to 57, when fed daily, concentrations decreased over the trial.
Figure 5 presents data for Ent. exiguum, which was not included in the study until d 33. Again, a strong cyclic pattern was observed, suggesting a similarity between species in the genus Entodinium; that is, Ent. caudatum and Ent. exiguum. Streptomycin was essentially without effect.
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Figure 5. Concentrations of Entodinium exiguum, cultured according to the scheme shown in Figure 1. Counts were made at each transfer. This species was only cultured for 55 d (d 33 to 88). The dotted line is for the second culture, which had streptomycin added whenever the culture was not going to be fed for 1 or 2 d.
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One of the principal reasons suggested for requiring in vitro cultures to be fed small amounts daily is that fermentation of excess feed by the bacteria lowers the culture pH and inhibits protozoal growth. Table 1 presents pH data for cultures fed daily for 3 d and those fed only on the first day (2 d without feed). No differences were observed in the range of pH values between the 2 feeding regimens with or without streptomycin.
Bacterial concentrations were measured in the Ent. caudatum and Epi. caudatum cultures with and without streptomycin (Table 2). Compared with the control cultures, there was a decrease in bacterial concentration when streptomycin was added to the culture. However, this did not result in increased concentrations of protozoa. Similar results were obtained in a preliminary trial with Eno. triloricatum (data not shown), in which the feeding schedule was different.
Although not feeding for 2 d generally decreased the concentration of protozoa, 3 of the cultures rapidly increased when they were then transferred and fed daily for several days. The exception was Eno. triloricatum, which tended to decrease over the 88-d period. Concentrations were near zero at that time; however, enough cells were present that it should have been possible to bring the culture back with continued daily feeding.
From the early studies on cultivation of rumen protozoa by Hungate (1943), most investigators have found it advantageous or necessary to add substrate daily (Clarke, 1963; Mah, 1964; Onodera and Henderson, 1980; Marcin et al., 1998). Coleman (1971) stated that none of the species cultivated in his laboratory would survive unless fresh starch was added every day (Ent. caudatum, Entodinium simplex, Epi. caudatum, Polyplastron multivesiculatum, and Ophryoscolex sp.). Entodinium caudatum and Epi. caudatum cultures were able to survive a single day’s omission of starch, but could not be maintained by feeding only every 48 h. In contrast, Broad and Dawson (1975) successfully cultured Ent. caudatum by feeding rice starch every 2 d.
Coleman (1960) added 1 mg/mL (approximately 1,600 U/mL) of penicillin to cultures of Ent. caudatum that contained an excess of rice starch and followed their numbers without any additional feedings. In general, numbers increased over the first 3 to 5 d, but then began to decline, and the protozoa were usually dead after 12 to 14 d. He did divide and dilute one of the declining cultures and observed continued growth for 3 d if he added protozoa-free rumen fluid. Total bacterial concentrations ranged from 103 to 105/mL compared with concentrations of 107 in normal cultures. From these observations, he concluded that penicillin was not toxic to Ent. caudatum; however, he did not report any data on survival after returning the treated cultures to the usual culture techniques. Hino and Kametaka (1977) reached a similar conclusion with Ent. caudatum; that is, penicillin had no harmful effect at concentrations up to 1,000 U/mL.
Somewhat different results have been reported for streptomycin, in that Oxford (1958) found streptomycin to be very toxic to mixed rumen entodiniomorphs at a concentration of 50 U/mL. However, Hino and Kametaka (1977) found no negative effects on Ent. caudatum cultures grown in the presence of 0.25 mg/mL (approximately 185 U/mL) of streptomycin.
Numerous studies have reported the use of antibiotics to inhibit bacterial growth in protozoa cultures to investigate their nutrient requirements and metabolic activities. Various combinations of antibiotics were used, but generally included penicillin, streptomycin, and chloramphenicol (Coleman, 1962; Onodera and Henderson, 1980). Some studies also included neomycin, carbenicillin, aminobenzylpenicillin, cephaloridine, and leucomycin (Hino and Kametaka, 1977; Bonhomme et al., 1982). Bacterial concentrations were markedly decreased to values less than 103/mL. However, in all cases, the protozoa died within 1 to 2 wk.
Unless cryopreservation of rumen ciliates is carried out with specific equipment for controlling the rate of cooling (Nsabimana et al., 2003), it is not a very reliable method for maintaining stock cultures. The author has used a Cryo 1°C Freezing Container from Nalgene (Nalgene, Nunc, Thermo Scientific, Rochester, NY), designed to achieve a –1°C/min rate of cooling. However, recovery of protozoa using this method has been less than 50% and is extremely poor for the larger ciliates. Dimethyl sulfoxide is normally added as a cryoprotectant, and in the author’s experience, when the frozen cultures are revived, bacterial action on the dimethyl sulfoxide produces a sulfur odor, which is extremely unpleasant when feeding and transferring.
The proposed procedure allows protozoa cultures to be maintained during the normal 5-d work week, alleviates any unpleasant odor connected with cryopreservation, and makes them readily available for studies. Obviously, the schedule can be changed to alleviate the necessity to feed over holidays or other scheduling conflicts. Thus, it appears possible to set up a feeding schedule for rumen protozoa that would maintain viable and active stock cultures without having to feed them on the weekend. Based on the data obtained in this study, the schedule shown in Table 3 would be recommended for routine use.
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Footnotes
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1 Salary and research support provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. The author thanks Ruthi Patterson for technical help. 
2 Corresponding author: dehority.1{at}osu.edu
Received for publication April 24, 2007.
Accepted for publication January 31, 2008.
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LITERATURE CITED
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Bonhomme, A., G. Fonty, and J. Senaud. 1982. Obtention de Polyplastron multivesiculatum (cilié entodiniomorphe du rumen) en condition axénique. J. Protozool. 29:231–233.
Broad, T. E., and M. C. Dawson. 1975. Phospholipid biosynthesis in the anaerobic protozoon Entodinium caudatum. Biochem. J. 146:317–328.[Medline]
Clarke, R. T. J. 1963. The cultivation of some rumen oligotrich protozoa. J. Gen. Microbiol. 33:401–408.[Abstract/Free Full Text]
Coleman, G. S. 1960. Effect of penicillin on the maintenance of rumen oligotrich protozoa. Nature 187:518–520.[Medline]
Coleman, G. S. 1962. The preparation and survival of almost bacteria-free suspensions of Entodinium caudatum. J. Gen. Microbiol. 28:271–281.[Abstract/Free Full Text]
Coleman, G. S. 1971. The cultivation of rumen entodiniomorphid protozoa. Page 159 in Isolation of Anaerobes. D. A. Shapton and R. B. Board, ed. Academic Press, London, UK.
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Dehority, B. A. 1984. Evaluation of subsampling and fixation procedures used for counting rumen protozoa. Appl. Microbiol. 48:182–185.
Dehority, B. A. 1998. Generation times of Epidinium caudatum and Entodinium caudatum determined in vitro by transferring at various time intervals. J. Anim. Sci. 76:1189–1196.[Abstract/Free Full Text]
Dehority, B. A. 2004. In vitro determination of generation times for Entodinium exiguum, Ophryoscolex purkynjei and Eudiplodinium maggii. J. Eukaryot. Microbiol. 51:333–338.[CrossRef][Medline]
Dehority, B. A., P. A. Tirabasso, and A. P. Grifo Jr. 1989. Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium. Appl. Environ. Microbiol. 55:2789–2792.[Abstract/Free Full Text]
Hino, T., and M. Kametaka. 1977. Gnotobiotic and axenic cultures of a rumen protozoon, Entodinium caudatum. J. Gen. Appl. Microbiol. 23:37–48.[CrossRef]
Hungate, R. E. 1943. Further experiments on cellulose digestion by the protozoa in the rumen of cattle. Biol. Bull. 84:157–163.[Abstract/Free Full Text]
Mah, R. A. 1964. Factors influencing the in vitro culture of the rumen ciliate Ophryoscolex purkynei Stein. J. Protozool. 11:546–552.[Medline]
Marcin, A., S. Ki
idayová, and V. Kmet. 1998. Wheat protein proteolysis in the monoculture of rumen protozoon Entodinium caudatum. Livest. Prod. Sci. 53:183–190.[CrossRef]
Nsabimana, E., S. Kiidayová, D. Macheboeuf, C. J. Newbold, and J. P. Jouany. 2003. Two-step freezing procedure for cryopreservation of rumen ciliates, an effective tool for creation of a frozen rumen protozoa bank. Appl. Environ. Microbiol. 69:3826–3832.[Abstract/Free Full Text]
Onodera, R., and C. Henderson. 1980. Growth factors of bacterial origin for the culture of the rumen oligotrich protozoon, Entodinium caudatum. J. Appl. Bacteriol. 48:125–134.[CrossRef]
Oxford, A. E. 1958. IX. Some observations on the culture of the cattle rumen ciliate Epidinium ecaudatum Crawley occurring in quantity in cows fed on red clover. N. Z. J. Agric. Res. 1:809–824.
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Abstract
INTRODUCTION
