Growth of Tetrahymena in Carbohydate-Free High-Glutamate Media

SYNOPSIS. In chemically defined media, pH 6.4–6.8, supplementation with glycerol permitted growth equal to that with a carbohydrate supplement. In a pH 5.4 medium described in detail, containing L-glutamic acid 0.25%, supplementation with L-serine (essential for this strain), especially when further supplemented with proline, likewise permitted growth nearly equal to that with carbohydrate. These results are discussed in relation to the question of which components of peptone act as substrates and the resemblances of the Tetrahymena pattern of substrate utilization to that of metazoa.     

Antagonists of Growth Inhibition of Crithidia by Allopurinol, a Guanine Analog

SYNOPSIS. Allopurinol, 4-hydroxypyrazolo[3,4- d ] pyrimidine, a relatively non-toxic xanthine-oxidase inhibitor used to treat gout, inhibited growth (ID_0–90 1.0 μg/ml) of the trypanosomatid flagellate of insects Crithidia fasciculata in minimal media in respect to biopterin, folic acid and purine. Allopurinol inhibition was antagonized by thymidine (4.0 μg/ml) when a ) biopterin (≥ 1.0 ng) was present alone; traces of folate entering from the inoculum or contamination of chemicals probably sufficed here to satisfy the folate requirement; b) biopterin (5% pure) 1.0 ng was added together with either pteroylglutamic acid (PGA) 4.0 ng or hypoxanthine 0.01 mg. With biopterin absent, full growth was permitted by PGA 10 ng + hypoxanthine 0.03 mg, and completely inhibited by the standard concentration of allopurinol, but in this instance thymidine did not release the inhibition.
Allopurinol is therefore useful for investigating pteridine function via inhibition analysis with the singularity that folate acts uniquely in trypanosomatids as precursor of biopterin, and the complication that both pteridines catalyze multiple biosyntheses. The biosynthetic step between folate and biopterin is postulated to be a site of inhibition of allopurinol in Crithidia.     

Cryoprotectants for Crithidia fasciculata Stored at -20 C, with Notes on Trypanosoma gambiense and T. conorhini

SYNOPSIS. Cryoprotectants were tested in both complex and semidefined media for the trypanosomatid Crithidia fasciculata. Near log-phase or end-of-log-phase cultures were frozen for 24–48 hr at ∼ -20 C, then warmed in air to room temperature. Immediate motility was correlated with viability. The best protectant of the 83 tested was glycerol at ∼ 10% (w/v). Survival without cryoprotectant was rare. Outstanding cryoprotectants (perhaps also useful solvents for drugs poorly soluble in water) were: ethylene glycol; 2,2'-dioxyethanol (diethylene glycol); 1,2,4-butanetriol; 1,4-cyclohexanediol; dimethylsulfoxide; propylene glycol; and N -acetylethanolamine. Several sugars were active, e.g., D-arabinose, sucrose, and sorbitol. Trypanosomes tolerated cryoprotectants much less; tolerance was better in growth media than in suspension media. Trypanosoma gambiense was grown in blood-enriched media + 2-2.5% glycerol, suspended in 20% (w/v) glycerol. then frozen; this permitted 3-week survival. T. conorhini survived 4 weeks after growth in media containing glycerol 2.5%+ ethylene glycol 4%+ rutin 1.0 mg per 100 ml.     

Crithidia guilhermei: gelatin- and haemoglobin-degrading extracellular metalloproteinases

The extracellular metalloproteinases of the insect trypanosomatid Crithidia guilhermei were characterized through the incorporation of different protein substrates (gelatin, casein, haemoglobin, and bovine serum albumin) into SDS-PAGE. Two gelatinases (60 and 80 kDa) showed ability to degrade casein as well and a 67-kDa enzyme presented the broadest specificity since it was also able to degrade casein and haemoglobin. Besides the 67-kDa extracellular proteinases detected on haemoglobin-SDS-PAGE, a 43-kDa haemoglobinase was only observed with this substrate. All C. guilhermei proteinases were incapable of using bovine serum albumin. C. guilhermei was also grown in four different culture media and the best proteinase production was reached using yeast extract-peptone medium containing glucose as the major carbon source. The results point to the importance of the use of distinct culture media and proteinaceous substrates on the characterization of extracellular proteolytic activities in trypanosomatids, since alterations in growth conditions and methods of detection could lead to distinct proteolytic profiles.     

Prolonged Survival of Tetrahymena at 0.5 C in Citrated, Lecithinized, Defined Media

SYNOPSIS. A fully defined medium aside, also one with acid-hydrolyzed casein, both pH 6.2–6.4 permitted survival of Tetrahymena pyriformis (syngen 1, mating type II) for no more than a week at 0.5 C, despite fortification with crude soy lecithin. Lecithin had permitted prolonged survival in crude media (pH 7.2), probably due largely to its phytosterols and antioxidants. Metal imbalances appeared responsible for the poor survival in defined media because additional citrate, histidine or Fe permitted monthlong survival. Al or Ni in the presence of increased citrate were also favorable. The casein-hydrolysate medium and a fully defined medium, both fortified with lecithin, and with additional citrate or Fe, permitted survival at 0.5 C for at least 2 months. Krebs-cycle intermediates, uridine, thymidine, adenosine, and guanosine acted as growth substrates in nearly carbohydrate-free media, which suggests trial of their survival-promoting properties for cold-stored cultures. Parallels are noted for survival in the cold between Tetrahymena and erythrocytes and sperm.     

Factors Affecting the Salinity Tolerance of the Freshwater Cryptomonad Chilomonas paramecium

ABSTRACT. The capacity of the freshwater cryptomonad Chilomonas paramecium to develop a tolerance for seawater in the growth medium was investigated as part of a research program exploring potential microbial food sources for estuarine bivalve mollusks. By gradually increasing the percentage of estuarine seawater included in a freshwater culture medium over the course of 10 years, strains were obtained that tolerate from 16 to 32% seawater (highest salinity 10.5 ppt), achieving equivalent final densities with similar gross biochemical composition. However, after subculture in seawater-containing media for over 20 years, growth rates in more-saline media remained appreciably slower than in low-salinity media. Reduction of C. paramecium growth rate by seawater was found to be exacerbated in media with an initial pH of 3.5 as compared with pH 4.0–5.0, suggesting either a specific H⁺ effect upon metabolism of the medium carbon source (lactic acid) or a general cation effect upon nutrient uptake or cell metabolism. By contrast, depression of growth rates at high salinity was ameliorated by eliminating sodium-phosphate enrichments in growth media. This suggests that cations in the phosphate salt were contributing to cation-mediated growth inhibition. Results indicate a potential for C. paramecium , cultured in moderately saline media with no phosphate enrichments, to be used as a carbohydrate supplement for laboratory and hatchery feeding of estuarine bivalve mollusks.     

Effect of pH of the Medium on the Availability of Chelated Iron for Chlamydomonas mundana

SYNOPSIS. Nutritional and cultural factors influenced growth of Chlamydomonas mundana at different H-ion concentrations. Growth at acid pH (5.5–6.0) primarily depended on the acetate and chelated iron concentration and on the method of preparation of the medium. The organism had increased Na, Ca, and Mn requirements at an acid pH. Sensitivity to increased levels of phosphate was greater at low than at high pH. The requirement for chelated as compared with non-chelated Fe was qualitatively easier to demonstrate at low pH. The procedure followed in preparing the growth media was more important with higher levels of chelator and Fe and with media of low pH. All 6 synthetic chelators studied were more effective, as measured by growth, if combined with iron before addition to the media. At high pH (7.0–7.5), the organism was much more adept at utilizing over-chelated Fe. This latter finding, and the greater sensitivity at low pH to trace-metal imbalance in ill-prepared media, indicate that the organism absorbs Fe much more efficiently at neutral or slightly alkaline pH.     

Dense Crithidia Growth and Heme Sparing: Relation to Fe,Cu, Mo Chelation*

SYNOPSIS. Heme, intrinsically required by Trypanosomatidae, is unstable, especially in conventional alkaline (pH 7.2–8.0) media. Low solubility of heme in a pH 6.5 basal medium (developed to assay biopterin with Crithidia fasciculata) posed a problem: in media acidified during growth because of glycolysis, heme precipitated, perhaps contributed to acid-limited growth and interfered with densitometric estimation of growth. The remedy was to: replace glucose with less rapidly metabolized mannitol; distribute media in thin layers to promote oxidation of acetate, fumarate, and malate (presumably leaving an alkaline residue); and buffer heavily with histidine + Good zwitterionic buffers, and superimpcse physiological buffering by arginine + asparagine whose catabolism appeared to yield an excess of NH⁺₄ over acid. Thereupon, Fe and Cu deficiencies sharply limited growth in the medium whose main chelators were: (a) 2,3–dihydroxybenzoic + 5-sulfosalicylic acids (which preferentially bind transitional elements at their higher valences; (b) malic and gluconic acids; and (c) histidine. With unconventionally heightened concentrations of Fe, Cu, and Mo (the latter serving as Cu buffer as well as nutrient per se), the hemin concentration could be lowered, widening the margin of safety for heme solubility. Growth then reached 1.4 × 10⁸ cell/ml. This medium may serve to screen for ligands promoting uptake or release of Fe and Cu. The increased growth is a step towards improving the assay medium for biopterin and practical use of Crithidia to assay several B vitamins and essential amino acids for metazoa.     

Use of a Tetrazolium-based Cell Proliferation Assay to Measure Effects of In Vitro Conditions on Perkinsus marinus (Apicomplexa) Proliferation

ABSTRACT. Because the in vitro cell cycle of the apicomplexan oyster pathogen Perkinsus marinus generates cell populations heterogeneous for size and typified by aggregation, both turbidimetric and counting methods for determining population densities and proliferation rates are inaccurate or cumbersome. We show that a commercial, tetrazolium-based cell proliferation assay yields a soluble formazan chromophore upon intracellular reduction by P. marinus . at a rate proportional to cell population biovolume. Using this assay system, we have 1) defined selected culture system parameters which maximize P. marinus in vitro proliferation, 2) assessed selected chemosensitivities, and 3) standardized the assay system for quantification of densities and doubling times of populations propagated with our optimized system. Growth was supported by four tested base media and was maximized in 1:1 DME/Ham's F-12. Temperatures of 10–40° C permitted growth, which was maximized at 35° C. pH 6.0–8.5 permitted growth, which was maximized at 7.0–7.5. Osmolalities of 340–1,930 mOsm supported growth, which was maximized at 790 mOsm. Serum supplements from 1–10% (v/v) did not enhance log phase growth, but enhanced stationary phase metabolic activity in proportion to concentration. Our isolate (ATCC 50439) has a 13 h log phase doubling time when propagated under optimized conditions: 28° C, 800 mOsm, pH 7.0, 1:1 DME/Ham's F-12 medium, 5% (v/v) FBS. It is tolerant of antibacterial agents at concentrations commonly used in vertebrate tissue culture, but is inhibited by several antimycotics at similar concentrations.     

Positive selection of antibiotic-producing soil isolates.

Stepwise discriminant analysis was used to identify the most powerful selective substrates which could be used to formulate media capable of enriching for antibiotic-producing soil isolates. This was achieved by characterizing a collection of 74 soil bacteria, including eubacteria and actinomycetes, according to their ability to produce antibacterial antibiotics and their growth responses to 43 physiological and nutritional tests. The characters which were selective for actinomycetes relative to eubacteria included growth on proline (1%, w/v) and humic acid (0.1%) as sole sources of both carbon and nitrogen, growth on nitrate as a nitrogen source, and growth at pH 7.7-8.0. Growth on proline (1%) and humic acid (0.1%) as sole carbon/nitrogen sources, growth on asparagine as a nitrogen source, and growth in the presence of vitamins were among the characteristics which allowed antibiotic-producing actinomycetes to be differentiated from non-antibiotic-producing strains. Several simple isolation media which incorporated the selective substrates identified by discriminant analysis succeeded in increasing the proportion of actinomycetes isolated from soil samples. Furthermore, the percentage of isolates capable of antibiotic production was considerably increased.     

The Physiology of Astrephomene gubernaculifera1

SYNOPSIS. A comparative physiologic study of 4- and 7-chromosome strains of Astrephomene gubernaculifera was done. The vitamins p-aminobenzoic acid, nicotinamide, biotin, thiamin HCl and vitamin B₁₂ were tested for their ability to support growth. Only vitamin B₁₂ was required for active growth altho the presence of thiamin HCl was necessary to produce structurally typical colonies. Astrephomene will not grow in the absence of an exogenous source of carbon. Of the 36 carbon sources tested, only pyruvate, butyrate, succinate and acetate permitted active growth. Sodium acetate was the best. Strains grew within the initial pH range of 5.0–7.5. At pH values above 8.0 growth declined rapidly. When media were buffered with TES (N-tris [hydroxymethyl]methyl-2-aminoethane-sulfonic acid) at an initial pH of 6.8 growth was enhanced. The organism grew at 15–40 C. Growth in the dark was slightly less than that in the light.     

A rod-shaped,gram-negative,propionigenic bacterium with a wide substrate range and the ability to fix molecular nitrogen

From estuarine mud a rod-shaped, motile, gram-negative, anaerobic bacterium was isolated (strain asp 66). Asp 66 fermented several substrates including glucose, fructose, malate, fumarate, citrate and aspartate. Fermentation products were acetate, propionate and presumably CO₂. Hydrogen was never formed nor utilized. Succinate conversion to propionate was catalyzed by cell suspensions but did not support growth. Asp 66 did not require vitamins and grew well in mineral media with a fermentable substrate. The pH range for growth was from 6.5 to 8.5. Temperature optimum was 27 to 30°C. The strain was able to fix N₂ as evidenced by its growth with N₂ as sole nitrogen source and its ability to reduce acetylene to ethylene. Cell-free extracts of cultures grown under air without shaking contained cytochrome(s) with absorption peaks at 523 nm and at 553 nm. The G+C content of the DNA was 60.8+-1 mol%. The taxonomic position of strain asp 66 is discussed.     

A new pleiotropic mutation causing defective carbohydrate uptake in Escherichia coli K-12: isolation, mapping, and preliminary characterization.

A new pleiotropic mutation, designated cup-1 (for carbohydrate uptake), which impairs the ability of Escherichia coli cells to grow on a large number of phosphotransferase system (PTS) and non-PTS carbohydrates by blocking their entry into the cells, has been isolated, partially characterized, and mapped. The mutants grew poorly even on rich and glucose minimal media. Fast-growing revertants rapidly accumulated in cultures grown on either of the above two media and made stable maintenance of the mutation difficult. Several extragenic suppressor mutations that permitted cup cells to grow on specific single sugars or groups of sugars have been isolated. One such suppressor, which enabled cup cells to grow as well on glycerol minimal medium as their wild-type parent, has been helpful in stably maintaining these cells in this medium. cup-1 has been mapped to 97 min on the standard E. coli map. It cotransduced with a transposon Tn10 inserted clockwise to it and (very weakly) with uxuA. Surprisingly, it failed to cotransduce with pyrB, argI, or valS, three markers located nearby but counterclockwise to it. In F' merodiploids, cup-1 was dominant over its cup+ allele. Cyclic AMP permitted growth of cup-1 cells on some sugars but not all. Apparently, reduced cyclic AMP level and therefore noninduction of several sugar operons is one but not the only effect of cup.     

Effect of carbon sources on the biosynthesis of ergot alkaloids and the activity of carbon metabolism enzymes in Penicillium sizovae

The study was aimed at finding out how different carbon sources influenced the growth of Penicillium sizovae, the biosynthesis of epoxyagroclavine-1 and agroclavine-1 as well as the activity of key enzymes involved in the citric acid cycle, the pentose phosphate pathway and the glyoxylate cycle. The fungal growth was shown to depend on the carbohydrate substrate: it had a two-phase profile when P. sizovae was cultivated on mannitol and glucose, but not on sorbitol. The quantitative content and composition of ergoalkaloids depended on the combination of carbohydrate and organic acid substrates. The overall productivity of the mycelium (epoxyagroclavin-1+agroclavin-1) was highest when mannitol and fumarate were used. A medium with sorbitol and fumaric acid was very selective in terms of epoxyagroclavine-1 synthesis. The high level of alkaloid biosynthesis correlated with the active functioning of the pentose phosphate cycle and with the low activity of the CAC.     

Isolation, Cloning and Determination of Biologic Characteristics of Five New Species of Crithidia

SYNOPSIS. Thru the application of technics of serology, cultivation in acid and basic media, host specificity and reaction in foreign hosts it became possible to characterize 10 species of Crithidia and to assign names to 5 of those previously undesignated. Despite a loose host specificity under experimental conditions it was found that each isolate from a different insect host was a different species.     

Crithidia mellificae n. sp. an Acidophilic Trypanosomatid of the Honey Bee Apis mellifera

SYNOPSIS. Crithidia mellificae n. sp. is described from the honey bee Apis mellifera in apiaries of Victoria, Australia. Organisms were isolated and cultured in a modified NNN medium with Cowper-thwaite's medium as an overlay. In contrast to other isolates of Crithidia, C. mellificae grows best at a pH between 4.5 and 5.5 with optimum near pH 5. In Cowperthwaite's medium alone, buffered at pH 4.5–5.5, the organism will survive apparently indefinite passages. The organism has so far as known, no pathologic effects in the hymenopteran host.     

Effects of some antitumor agents on growth and glycolytic enzymes of the flagellate Crithidia

Some antitumor agents known to specifically inhibit certain tumor cell enzymes were examined for activity against glycolytic enzymes and growth of the insect trypanosomatid, Crithidia fasciculata. The cytoplasmic enzymes hexokinase, alpha-glycerophosphate dehydrogenase, malic dehydrogenase, and glucose-6-phosphate dehydrogenase were tested. Agaricic acid (2-hydroxy-1,2,3-nonadecane tricarboxylic acid) was highly inhibitory (50 to 100%) to malic and alpha-glycerophosphate dehydrogenases at approximately 3 x 10(-5)m; 2-(p-hydroxyphenyl)-2-phenylpropane (2 x 10(-4)m), and 5,6-dichloro-2-benzoxazolinone (5 x 10(-4)m) were less effective (50% inhibition) against them. The antiprotozoal agents primaquine (4 x 10(-4)m) and Melarsoprol (8 x 10(-4)m) were 30 to 40% inhibitory. Agaricic acid, 2-(p-hydroxyphenyl)-2-phenylpropane, and 5,6-dichloro-2-benzoxazolinone inhibited growth of Crithidia at less than 10(-4)m. Eight other test compounds from the Cancer Chemotherapy National Service Center (CCNSC) were not toxic to cell growth, although two (4-biphenylcarboxylic acid and 1-[p-chlorobenzyl]-2-ethyl-5-methyl-indole-3-acetic acid) inhibited Crithidia alpha-glycerophosphate dehydrogenase below 1 mm. All of the compounds used specifically inhibited cancer cell alpha-glycerophosphate dehydrogenase. The corresponding enzyme in pathogenic African trypanosomes is important in their terminal respiration. C. fasciculata may be useful in preliminary evaluation of chemotherapeutic agents as potential trypanocides.     

Isolation and partial characterization of mutants of the trypanosomatid Crithidia fasciculata and their use in detecting genetic recombination

Crithidia fasciculata was used as a model trypanosomatid to study the possible existence of genetic recombination in this group of protozoa. The approach was based on the ability to select a variety of mutants on agar plates. Following mutagenesis of wild type cells by nitrosoguanidine or ethylmethanesulfonate, stable mutant phenotypes were obtained. These included mutants resistant to the drugs actinomycin D, 6-azauracil, 6-azauridine, and 5-fluorouracil, auxotrophs and colony morphology mutants. Following mixed growth of pairs of drug-resistant mutants on selective media, isolates exhibiting stable recombinant phenotypes were obtained. The data presented suggest that 1) Crithidia undergoes some type of genetic recombination and 2) Crithidia must be diploid at some time during this process.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.