protocols in molecular parasitology

Culturing and Biological Cloning of llypanosoma brucei Mark Carrington 1. Introduction Any biochemical analysis is usually made easier by the availabil- ity of large numbers of cells to be analyzed, and one of the reasons for the position held by Trypanusoma brucei as the best character- ized parasite is the relative ease with which it can be cultured in the laboratory. The ability to culture cloned populations derived from individual trypanosomes before and after an antigenic switch is vital in investigations into the mechanism of antigenic variation. Genomic DNA prepared from such cloned populations used to analyze variant specific glycoprotein (VSG) genes by Southern blotting led to the discovery of the genomic rearrangements involved in antigenic varia- tion (I-5). This chapter will describe the growth of trypanosomes in labora- tory rodents. The techniques that this involves start with the growth from a frozen stabilate, which may be a field isolate, and the prepara- tion of further stabilates from infected blood. The basis of the preparation of large numbers of trypanosomes (1 x 109-5 x lOto cells) from blood is the retention of blood cells on a DEAE-cellulose column because of their surface negative charge, while trypanosomes pass through (6,7). These cells then provide the basis for further study, such as the preparation of DNA or RNA (see Chapter 8), the purification of the VSG or other protein, and metabolic labeling. From Methods m Molecular Biology, Vol 21’ Protocols m Molecular Parasitology Edlted by John E Hyde Copynght 01993 Humana Press Inc , Totowa, NJ 1 2 Carrington Populations that are homogeneous for a single VSG are prepared by cloning an individual trypanosome. This is achieved by infecting a mouse with a single trypanosome; the VSG expressed usually remains homogeneous for several syringe passages through lethally irradiated mice (8,9). To investigate antigenic variation it is necessary to establish con- ditions in which a relapse peak of parasitemia will occur. This can be achieved by infecting a rabbit, which leads to a chronic relapsing parasitemia (8), or by infecting a rat with a small number of para- sites, in which case a second peak will occur (9). Parasites cloned from a relapse population will express a different VSG from the iso- late used to infect the ammal. The metabolic labeling of trypanosomes has been used to investi- gate the kinetics of synthesis of VSGs (10-12) and the covalent modi- fication of the mature C-terminus with a glycosylphosphatidylinositol (GPI) moiety, specifically in this context the identification of the fatty acyl component as myristate (13,14). VSG metabolically labeled with [3H]-myristic acid has been used as a substrate to identify GPI-spe- cific phospholipase C (15-18). Procyclic form trypanosomes representing the life cycle stage found in the midgut of the tsetse fly vector can be readily propagated in culture using the culture medium SDM-79 (19). One aspect not cov- ered in this chapter is the growth of bloodstream trypanosomes in cul- ture (see ref. 20 for a recent use of this technique). It is worth noting that for most purposes growth in rodents is the only practically fea- sible protocol as bloodstream forms are not as amenable to culture as procyclics. 2. Materials 2.1. Growth and Maintainance of Bloodstream Trypanosomes 1. Trypanosome dilution buffer (TDB): 20 mM Na2HP04, 2 mM NaH2P04, 80 mA4 NaCl, 5 mM KCl, 1 mM MgS04, 20 mit4 glucose, pH 7.7. 2. TDB + FCS: TDB containing 10% (v/v) heat inactivated fetal calf serum. 3. TDB containing 20% (v/v) glycerol. 4. Citrate glucose anticoagulant (CGA): 100 mM tri-sodium citrate, 40 mA4 glucose, pH 7.3. Trypanosoma brucei 3 5. Separation buffer (SB): 57 mMNa2HP0,, 3 rniV NaH,PO,, 44 mMNaC1, 4 mA4 KCl, 5 mit4 glucose, 80 mM sucrose, pH 8.0. 6. DEAE-cellulose (preswollen Whatman DE52): This should be resus- pended m SB and equilibrated to pH 8.0 with orthophosphoric acid. 7. Disposable 1-mL, 2-mL, and 20-mL syrmges, 15 x 0.5 mm (25-g), 25 x 0.6 mm (23-g), and 25 x 0.8 mm (21-g) needles. 8. Siliconized cavity microscope slides; the slides are placed m a desicca- tor together with 5 mL 2% (v/v) dichlorodimethylsilane in 1,2,3- trichloroethane and the desiccator is evacuated for 1 min. The slides are then removed and baked at 100°C for 1 h. 9. Laboratory rats and mice. 10. A facility to lethally irradiate laboratory mice (850-900 rads); the mtce must be irradiated during the 24 h prior to infection. 11 Glass capillary tubes (50-100 pL vol), Crystaseal (glass sealant) (Hawksley Ltd, Lancing, Sussex, UK), and plastic cryopreservatron tubes mto which the capillaries will fit. 12. Neubauer improved hemocytometer and microscope to estimate parasiterma. 2.2. Metabolic Labeling of Bloodstream Trypanosomes 1. Modified Eagle’s Minimal Essential Medium minus methionine: Eagle’s Minimal Essential Medium (Gibco 320- 190 formulation) without methion- me and without vitamms. To this are added: Eagle’s Minimal Essential Medium nonessenttal ammo acids to 1X concentration, 0.03% (w/v) glucose, 0.1% (w/v) bovine serum albumin, 1% (w/v) glucose, 25 mM HEPES, pH 7.4 (see ref. II). 2. [35S]-methionme, >lOOO Ci/mmol. 3. RPM1 1640 medium supplemented with 1% (w/v) fatty acid free bovine serum albumin, 25 mM HEPES, pH 7.4. 4. [9, 10-3H]-myristic acid, >50 Ci/mmol. 5. 2% (w/v) sodmm dodecyl sulfate (SDS). 6. 1 mg/mL bovine serum albumin. 7 20% (w/v) trichloroacetic acid. 8. 10% (w/v) trichloroacetic acid. 2.3. Culture of Procyclic Trypanosomes 1. SDM-79 medium (19). Unfortunately this is not commercially avail- able so the components are given in Table 1. 2. Hemm stock: 2.5 g/L hemm dissolved in 50 mM NaOH; autoclave to ensure sterility Carrington Table 1 Components of SDM-79 Medium for the Culture of Procychc Trypanosomes Component Amount/L MEM (Earle’s) powder (Gibco 072-1400A) 7.0 g Medium 199 powder (Gibco 071-l 100A) 2.0 g 50X MEM essential amino acids (Gibco 043- 1130H) 80mL 100X MEM nonessentral amino acids (Gibco 043- 1140H) 60mL Glucose log HEPES (sodium salt) 8.0 g MOPS (free acid) 5og Sodium hydrogen carbonate 2.0 g Sodmm pyruvate 100 mg L-Alanme 200 mg L-Argmine 100 mg L-Glutamine 300 mg L-Methronine 70 mg L-Phenylalanine 80 mg L-Proline 600 mg L-Serine 60 mg Taurine 160 mg L-Threonine 350 mg L-Tyrosine 100 mg Guanosine 10 mg Folic acid 4mg Glucosamine hydrochloride 50 mg p-Aminobenzoic acrd 2w Biotin 02mg All components should be tissue culture grade After all the components have dissolved, adjust the pH to 7 3 with 2M NaOH and filter sterilize. Add 3 mL/L hemm stock and 10% (v/v) heat inactrvated fetal or newborn calf serum to complete the medmm The medtum has a shelf life of about 2-3 mo at 4°C before addltlon of serum and <1 mo after 3. Fetal or newborn calf serum that has been screened to ensure it sup- ports the growth of procyclic trypanosomes. 4. Sterile plastic tissue culture flasks, 25-225 cm* size, depending on the volume of culture needed. 5. 27°C incubator and facilities for sterrle manipulatrons. 6. 1%mL cryopreservation vials. 7. 60% (v/v) glycerol (tissue culture grade). Trypanosoma brucei 5 3. Methods 3.1. Growth of Trypanosomes in Laboratory Rodents The proper methods and instructions on exsanguination of mice and rats should be determined by consultation with the appropriate local authority. Permission and training to perform the other proce- dures on animals outlined here should also be obtained. The doses of parasites given in the methods have been used for the following mice and rats: (BALBK x CBA) Fl mice 20-25 g, CFLP mice 20-30 g, and CFY rats 300400 g. It is not necessary to use these strains, but the initial inoculum of trypanosomes may have to be varied in order to achieve the required growth rate, because even within one strain of rats the growth rate of trypanosomes expressing different VSGs varies. 3.1.1. Infection of a Mouse with Trypanosomes from a Frozen Stabilate 1. Remove the stabrlate from liquid nitrogen, thaw rapidly in a 37°C water bath, break off the sealed end, add to 1 mL TDB, and mix. 2. Estimate the number of trypanosomes/mL using a hemocytometer (see Note 1). 3. Infect two mice, one with 5 x 105, and one with 1 x lo6 trypanosomes by intraperitoneal Injection of the appropriate volume using a I-mL syringe and 15 x 0.5 mm (25-g) needle (see Note 2). If a cloned pop- ulation of trypanosomes is being propagated, a lethally irradiated (850 rads) mouse should be used to prevent any chance of an immune response. 4. Follow the mfection by estimatmg the density of trypanosomes in the blood obtained from tail bleeds. The parasitemia should reach l-5 x lO*/mL after 3 d if a rodent-adapted laboratory strain is being used. 3.1.2. Preparation of a Frozen Stabilate 1, Infect a mouse as in Section 3.1.1.; if a cloned population of trypano- somes is being propagated, a lethally irradiated (850-900 rads) mouse should be used. 2. Allow the parasitemia to develop for 3 d; it should reach l-5 x lO*/mL. 3. Anesthettze the mouse and exsanguinate into a 2-mL syringe contain- ing 0.2 mL CGA. It should be possible to recover l-2 mL of blood from a 30-g mouse. 6 Carrington 4. Transfer the blood to a tube on ice and add an equal volume of ice-cold TDB + 20% glycerol. 5. Fill glass capillaries with this mixture and seal one end with Crystaseal and place the capillaries into a plastic cryopreservatlon tube on ice. 6. Once sufficient stabilates have been made, cool the tube(s) slowly by placing them in the gas phase above llquld mtrogen in a Dewar flask. After at least 3 h submerge the tube(s) for long-term storage. 7. After 1 wk check the viability of the stabllate by infecting a mouse as m Sectlon 3.1.1. 3.1.3. Large Scale Preparation of Trypanosomes from Blood 1. Infect a mouse and follow the level of parasltemia as in Section 3.1.1 If a cloned population of trypanosomes 1s bemg propagated, a lethally irradiated (850-900 rads) mouse should be used. Within 3 d the den- sity of trypanosomes m blood should be >l x lO*/mL. Exsanguinate and estimate the density of trypanosomes, then dilute to 3 x 107/mL with TDB. 2. Rats are anesthetized prior to infection with 3 x lo7 trypanosomes by mtraperitoneal mjectlon using a l-r& syringe and a 0.6-mm (23-g) needle (see Note 3). Follow the parasltemla by viewing blood from tall bleeds under a microscope. Three days after infection the parasltemla should be >3 x lO*/mL. 3. Prepare a DEAE-cellulose column before collectmg the blood. This can conveniently be poured m a 50-mL syringe using glass wool to block the flow of column matrix out of the bottom. The volume of DEAE- cellulose used will be determined by the number of rats, but as a guide a 20-mL column is usually sufficient for three rats. Equilibrate the col- umn by passing through 5-10 vol of SB. 4. The rats are exsangumated using a 20-mL syringe containing 2 mL CGA. It 1s usually possible to recover lo-15 mL of blood from a 300-g rat. Ensure that the CGA and blood mix to prevent clotting. Transfer the blood to a glass centrifuge tube on ice; leave on ice until the blood has been collected from all of the rats. 5. Centrifuge the blood (750g for 10 mm at 4°C); there should be a discrete whitish layer comprlsmg mainly trypanosomes overlaymg the sedl- mented red blood cells. Remove and discard the serum from above the trypanosomes and carefully layer ice-cold SB on top of the trypano- some layer. Usmg a Pasteur plpet and a gentle swirling action, resus- pend the trypanosomes with minimal disturbance of the red blood cells. Transfer the suspension to a fresh centrifuge tube on Ice. Trypanosoma brucei 7 6. Repeat the centrlfugation and resuspension. Two cycles are usually enough to remove most of the erythrocytes and serum protein. Keep the suspension on ice. 7. Apply the suspension to the DEAE-cellulose column; keep the column flowing by adding SB to the top as necessary. Only the trypanosomes will pass through the column, leukocytes and erythrocytes are retained on the column. Estimate the yield of trypanosomes using a hemocytometer. 3.2. Cloning and Generation of Antigenic Variants 3.2.1. Cloning of Bloodstream Form Trypanosomes 1. The parasitemia m the infected animal is followed and blood is col- lected when there are more than 1 x lo* trypanosomes/mL. A fraction enriched m trypanosomes is prepared by centrifuging 1 mL of blood in a mlcrofuge (12,OOOg) for 1 min, removing the serum and resuspendmg the whitish trypanosome layer in 1 mL TDB with mimmal disturbance of the red blood cells. 2. Estimate the density of trypanosomes and dilute to 1 x 103/mL with TDB + FCS. Place one l-pL drop m the cavity of a cavity slide and inspect using a microscope. The whole drop should be wlthm the field of view. If two observers agree that there is only a single trypanosome within the drop, add 0.3 mL TDB + FCS. Recover this into a I-mL syringe and Inject intraperitoneally mto a lethally irradiated mouse. 3. After 3 d exsangumate the mouse (see Section 3.1.2.) and estimate the parasitemla. Use the blood to infect a further lethally Irradiated mouse (see Section 3.1.1.). Continue in this way until the parasitemia is above 1 x lo* on the third day after infection. This usually occurs m the sec- ond mouse and rarely requires the use of a third serial passage. 4. If antlbodies are available, the homogeneity can be checked by lmmu- nofluorescence microscopy (ref. 9; see also Chapter 31). The population is usually more than 99.9% homogeneous with respect to the VSG (9). If antibodies are not available then they should be raised against VSG puri- fied from the cloned population (8,21). If homogeneous, the VSG and anti- serum should produce a single preclpitin arc m an immunodiffusion assay. 3.2.2. Creating Relapse Populations 3.2.2.1. ESTABLISHMENT OF A CHRONIC INFECTION IN A RABBIT This method was used to generate the cloned antigenic variants first isolated from the MITaR 1 serodeme (8). When a rabbit is infected a chronic relapsing parasitemia occurs, and samples of blood taken at 8 Carrington intervals of more than 1 wk should contain a series of different anti- genie types. Individual trypanosomes can then be cloned and expanded in mice. 1. Infect a lethally irradtated mouse, follow the parasitemia and exsan- guinate after 3 d (see Section 3.1.2.). Determine the density of trypan- osomes in the recovered blood using a hemocytometer. 2. Use these trypanosomes to inject a rabbit; an inoculum of 1.5 x lo* trypanosomes has been successfully used with a 2.5-kg New Zealand White. This should not need to be adjusted for other breeds, however the time taken for the first peak of parasrtemia to occur may vary. 3. At intervals, collect approx 2 mL of blood from an ear vein and mea- sure the parasitemia If the parasitemia is >l x 10*/n& clone immedi- ately (see Section 3 2 1.) If necessary, the population can be expanded by infecting a lethally irradiated mouse before cloning mdividuals. 3.2.2.2. INFECTION OF RATS WITH A SMALL INOCULLJM This represents an alternative method and has been used to gener- ate cloned populations in the ILTaR 1 serodeme (9). If a rat is infected with a small number of trypanosomes it usually survives the first peak of parasitemia; the second peak is lethal but comprises novel anti- genie types. 1. Infect a lethally irradiated mouse, follow the parasitemta and exsan- gumate after 3 d (see Section 3.1.2.). Determine the density of trypano- somes in the recovered blood using a hemocytometer. 2. Dilute the trypanosomes with TDB to 100 cells/ml and inoculate a rat intraperitoneally with 0.1 mL. 3. Follow the parasitemia to ensure that one peak occurs and exsangui- nate the rat when the second peak rises above 1 x lo* trypanosomes/mL of blood. 4 Clone individual trypanosomes from this blood (see Section 3.2.1.). 3.3. Metabolic Labeling of BZoodstream Form Trypanosomes 1. The trypanosomes are separated from blood usmg DEAE-cellulose (see Section 3.1.3.). 2. After passage of the trypanosomes through the DEAE-cellulose, the cells are washed once in labeling medium; recover the cells from the column eluate by centrifugation (750g for 10 mm). Discard the super- natant, resuspend the cells in labeling medium and centrifuge again (750g Trypanosoma brucei 9 for 10 mm). Resuspend the cells in the relevant labeling medium at the desired density. The medium is determined by the type of metaboltc labeling (see Note 5). 3. a. [35S]-Methionme. The labeling medium used is Modified Eagle’s Minimal Essential Medium minus methionine (see Note 6). After the wash, resuspend the cells at 3 x 107/mL in this labeling medium. Add [35S]-methionme to 100 pCl/mL and incubate at 37OC in a shaking water- bath. This mcubation should not exceed 3 h. b. [9, 10-3H]-Myristic acid: The labeling medium used is RPM1 1640 medium supplemented with 1% bovine serum albumin and 25 mM HEPES, pH 7.4 (see Note 7). After one wash resuspend the cells at 5 x 107/mL m labeling medium, incubate for 15 min at 37°C in a shak- ing waterbath, then add the [9, 10-3H]-myristic acid to 100 pCi/mL and continue the incubation at 37°C m a shaking waterbath. This incu- bation should not exceed 3 h. The [9, 10-3H]-myristic acid is prepared by evaporating the solvent (usually toluene) using a stream of nitro- gen and dissolving the myristic acid in a small volume (1 pIJ10 l&i) of water. This should contain an amount of fatty acid-free bovine serum albumin such that there are equal molar amounts of bovine serum albumin and myristic acid. c. [3H]-Sugars/nucleotides: The same protocol is used as in 3b above except that the desired [3H]- sugar, dissolved m water, is added instead (see ref. 22 for a recent example); in this paper the RPM1 1640, based labeling medium contained 3 g/L glycerol in addition to the bovine serum albumin and HEPES. 4. In all cases incorporation of radiolabel mto macromolecules can be determined by removing 50 pL samples at surtable time points. The sample is added to 50 pL of 2% SDS and immediately incubated at 100°C for 3 min. Add 50 pL of this lysate to 450 pL of 1 mg/mL bovine serum albumin (as a carrier), then add 500 pL 20% trichloroacetic acid. After 10 min at room temperature collect the precipitate by filtration onto glass fiber disks, wash with 10% trichloroacetic acid, dry the disks, add scmtillant, and count the incorporated radiolabel. 3.4. Culture of Procyclic Trypanosomes 3.4.1. Routine Maintenance of Procyclic Cultures The growth of procyclic trypanosomes in culture is straightforward. The cells are subcultured to a density of 1 x lO%nL and will grow to approx 3 x 107/mL. Cell density is estimated using a hemocytometer (see Note 1). The cells can be grown in tissue culture flasks (0.4 r&/cm2 10 Carrington area), spinner flasks, or even conical flasks in orbital incubators. The growth rate varies between different isolates and growth conditions, but doubling should occur between 10 and 24 h. 3.4.2. Preparation of Frozen Stabilates As with bloodstream trypanosomes, procyclic forms can be kept as frozen stabilates in liquid nitrogen. 1. In a 1.8-mL cryopreservation vial mix 0.2 mL 60% (v/v) glycerol and 0.4 mL SDM-79. Add 0.6 mL of a late log phase culture of procychc trypanosomes (1 S-2 X 107/mL). 2. Cool to liquid nitrogen temperature as in Sectton 3.1.2. 3 To resuscitate the culture, thaw the vial rapidly at 37OC and add the contents to 9 mL SDM-79 at 28OC in a 25 cm2 tissue culture flask. The trypanosomes should be motile immediately when the culture is viewed wtth an inverted microscope. 3.4.3. Metabolic Labeling of Procyclic Trypanosomes Procyclic trypanosomes can be metabolically labeled in SDM-79 medium. If the radiolabeled compound is normally present in the medium, for example methionine, then a special batch of SDM-79 should be made without the relevant component. The trypanosomes are washed with the depleted medium by centrifugation (600g for 10 min) and resus- pension in the depleted medium followed by centrifugation again (600g for 10 min) and resuspension at the labeling density. If a depleted medium is not needed, then the radioactive compound can be added directly to a culture. 4. Notes 1. Since trypanosomes are motile it is often difficult to obtain a count using a hemocytometer. An easy way around this is to make dilutions of the cells m TDB containing 0.1% formaldehyde. A Neubauer hemocytometer 1s used to count cells at around 1 x 106/mL so it 1s usual to make at least one IO-fold dtlution of the trypanosome sample. 2. In some protocols for this step the mouse IS held firmly by the scruff of the neck, so it is very important to use a short needle to reduce the chances of the needle passmg through the mouse and into the experimenter’s finger. 3. A common mistake here is to inject the trypanosomes between the skm and the muscle wall of the peritoneum. It is important that the trypano- somes are inJected into the peritoneum. [...]... multiplies in the arthropod vector predominantly as epimastigotes (where the kinetoplast lies adjacent to the nucleus), dividing by binary fission in the hind gut and rectum Nondividing, infective trypomastigotes (with a posterior kinetoplast) occur in the triatomine bug hind gut Unlike African trypanosomes, I: cruzi divides intracellularly in the mammalian host and not in the blood Metacyclic trypomastigotes... potentially infected mammals after cleaning the skin sequentially with iodine/70% ethanol and 70% ethanol Add a few drops to each culture Incubate at 23-28°C 3 For infected triatomine bugs, immerse for 10 mm in White’s solution, rinse in sterile saline contammg gentamycin (300 pg/mL) and 5fluorocytosme (300 pg/mL), dry, and dissect aseptically behind the protective screen Mix the intestinal contents... Prepare the following four mixtures of dry reagents by homogenization in porcelain mortars Main base mixture: Grams Final cont in dry mtx m medium, g/L Nap glycero phosphate 5aH20 NaCl Na3P04*l 2H20 KC1 Nas citrate*2H20 Na acetatea3Hz0 Na succinate 1000.0 200.0 250.0 20.0 30.0 39.5 13.5 20.0 4.0 5.0 0.4 0.6 0.79 0.27 Amino acid mixture: L-alanine L-arginine L-aspartrc acid L-asparagme L-cysteine L-cystme... variant surface glycoprotein J Brol Chem 261, 12,147-12,153 Culturing and Biological Cloning of nHypanosoma cmczi Michael A MiZes 1 Introduction Trypanosoma cruzi 1s a protozoan flagellate that is transmitted to mammals by bloodsucking triatomine bugs Transmission is not by the bite of the insect but by contamination of skin abrasions or mucous membranes with bug feces containing infective (metacyclic)... minimal Dispense the medium, seed with parasites, and incubate at 28°C 3.5 Cloning 0fTrypanosoma cruzi Virulent strains of ‘7:cruzi have been cloned by the inoculation of single blood form trypomastigotes into experimental mice ZI cruzi has also been cloned in vitro by using dilution series into liquid media or by plating out diluted suspensions of organisms onto blood agar and subsequently selecting... subsequently selecting single colonies (28,29) Cloning into mice was used to demonstrate that single organisms can establish mammalian infection Dilution series and plating out of colonies onto solid media are unreliable as single organisms are not observed microscopically during the cloning process A simple method applicable to all T cruzi strains that can be grown m vitro involves the seeding of cultures... and transform to amastigotes (having no visible flagellum) that divide within a pseudocyst by binary fission Trypomastigotes emerge from ruptured pseudocysts to reinfect cells or circulate m the blood and be ingested during a vector blood meal From Methods m Molecular Ed&d by John E Hyde Biology, Vol 21, Protocols m Molecular Parasitology Copyright 81993 15 Humana Press Inc , Totowa, NJ 16 Miles There... for Vero cell cultures (16) The samemedium has been used for growing Z cruzi in the presence of a triatomine bug cell line (Triatoma infestans embryo cells, 21) Prepare as described in Section 2.3 1 Set up a Vero cell culture (according to supplier’s instructions) in plastic culture flasks, glass medical flats, or Leighton tubes and maintain until 70% confluent cell growth 2 Add a concentrated suspension... suitable for in vitro drug tests at various concentrations: chambers are removed and cells adherent to the microscope slide base plate stained and examined for viable intracellular forms of T cruzi (23) 3.4 Growth in Defined Media Early defined media for I: cruzi were supplemented with bovine liver catalase It was subsequently shown that this protein extract was contaminated with 25-30 protein bands as... L-glutamine L-histidme HCl Glycine L-isoleucine Glutathione, reduced L-hydroxyprolme L-leucme L-lysine HCl L-methionine 14.25 27.50 27.50 5.00 5.00 7.00 52.25 5.00 13.00 17.50 20.00 0.125 2.50 38.00 37.50 8.75 0.285 0.55 0.55 0.1 0.1 0.14 1.045 0.1 0.26 0.35 0.4 0.0025 0.05 0.76 0.75 0.175 Miles 20 Grams in dry mix L-phenylalanme L-proline L-serine L-threonine L-tryptophan L-tyrosine ethylester L-valine . L-Argmine 100 mg L-Glutamine 300 mg L-Methronine 70 mg L-Phenylalanine 80 mg L-Proline 600 mg L-Serine 60 mg Taurine 160 mg L-Threonine 350 mg L-Tyrosine 100 mg Guanosine 10. the nucleus), divid- ing by binary fission in the hind gut and rectum. Nondividing, infective trypomastigotes (with a posterior kinetoplast) occur in the triatomine bug hind gut. Unlike African. labeling medium, incubate for 15 min at 37°C in a shak- ing waterbath, then add the [9, 10-3H]-myristic acid to 100 pCi/mL and continue the incubation at 37°C m a shaking waterbath. This incu-