Chapter 2: Materials and Methods
The original axenic C. elegans culture was obtained from the lab of Dr. Nancy Lu. This culture has been maintained in non-shaken HS-YE-HLE liquid cultures at room temperature since 1995.
Three solutions was used in the maintenance and testing of C. elegans: (1) Stock cultures of the nematodes was maintained on a soy-yeast-heated liver extract media (HS-YE-HLE. (2) An osmotic buffer solution, M-9, was used to wash nematodes free of nutrients in the HS-YE-HLE solution before they was introduced into the CbMM media for testing purposes. (3) Nematodes was tested in the defined-culture media CbMM.
Stock Culture Media
HySoy-Yeast Extract (HS-YE): HS-YE was composed of 40.0 grams of HY-Soy Powder (Quest International) and 10.0 grams of Yeast Extract (Difco) per liter of solution.
Heated Liver Extract (HLE): One to two pounds of frozen beef liver was purchased from a local market. The liver was thawed and rinsed with water then cut into 1 inch squares and drained. The liver was stored at 4 C for approximately 24 hours so auto-lysis could take place. Liver was placed in an equal volume of water and homogenized in a blender until all of the large clumps had been disrupted. The homogenate was filtered through two layers of Miracloth cloth and held on ice until all of the liver had been processed. The homogenate (600 ml) was placed into a 1 liter beaker and heated in a 60 C water bath. A final temperature between 52 - 53 C was maintained for exactly 6 min. The beaker was then placed into ice water until all of the homogenate was prepared. The homogenate was placed into screw capped centrifuge tubes and centrifuged at 37,000 to 39,000 x g for 30 min. The brake was not used. The supernatant fluid was collected and filtered through two layers of Miracloth. The extract was pre-filtered through a 90 mm 1.0 micron Gelman Science 61664 type A/E glass fiber in a Buchner vacuum filter apparatus. This material was filter sterilized using Nalgene 125-0045 sterilization filter units.
HySoy-Yeast Extract- Heated Liver Extract: HS-YE-HLE (Tomlinson and Rothstein, 1962) was prepared using a ratio of 1 part HLE to 9 parts HS-YE.
TEA Stock Solution: The 10 % TEA (w/v) stock solution was composed of exactly 9.2 ml of Triethanolamine (2,2,2-Nitrilotriethanol; 97 %) and 90.8 ml water. The solution was stored at room temperature.
M-9 Buffer: M-9 was composed of 6.0 grams of Na2HPO4, 5.0 grams of KH2PO4, and 0.25 grams of MgSO4*7H2O in 1 liter of water. The solution was autoclaved at 110 C-122 C, 15 psi for 30 min.
CbMM Defined Media
Preparing CbMM Media: The 2 X CbMM was prepared using the following method: All of the solutions were brought to room temperature. The solutions were placed in a 1000-ml beaker on a magnetic stirrer/heating plate while being stirred. Two amino acid solutions and the nucleic acid solution was combined the in the 1000-ml beaker. Each amino acid beaker was washed with no more than 3.0 ml water to complete the transfer of the solutions. The mixed salt solution was added into the 1000 ml beaker quantitatively using no more than 2.0 ml rinse water. Exactly 6.25 ml of the two vitamin solutions were each pipetted into the mixture. The pre-weighed dry ingredients (glutathione, choline and myo-inositol, cytochrome c, glucose or K-acetate) were then added. Each dry component was allowed to dissolve prior to adding the next dry component. No more than 1 to 2 ml of rinse water was used to transfer each dry component. Exactly 10 ml of beta-sitosterol was added and the medium was warmed slightly to 30 C for about one hour. The heating facilitated the complete solution of all components. The pH was adjusted to 5.9 + 0.1 with 10 % KOH. The media was then transferred into a 500-ml volumetric flask and brought to volume with water. The 2 X solution of CbMM was filter sterilized using several 0.22 micron pore size sterile filter units. CbMM was stored in a refrigerator at 2 C in dark until used.
TEA Soluble Components Stock Solution (TSC): A 250 ml solution of TSC was composed of: 0.1500 grams of biotin, 0.1500 grams of dl-thioctic acid (lipoic acid), 0.3000 grams of niacin (nicotinic acid), 0.3000 grams of p-aminobenzoic acid, 0.3000 grams of pteroylglutamic acid (folic acid), 13.00 ml of TEA stock solution. After the dry chemicals had dissolved, the solution was brought to volume with water in a 250-ml volumetric flask. Aliquots of 8 ml were placed into screw capped vials and stored frozen at -20 C.
Water Soluble Components Frozen Stock (WSC): 200 ml of WSC was composed of: 0.2400 grams of thyamine, 0.2400 grams of riboflavin-5'-PO4(Na) *2H2O (flavin mono-nucleotide), 0.2400 grams of pyridoxine hydrochloride, 0.1200 grams of pyridoxamine di-hydrochloride, 0.1200 grams of pyridoxal 5-phosphate, 0.2400 grams of pantothenate (Ca) pantothenic acid hemicalcium salt, 0.1200 grams of pantethine, 0.2400 grams of niacinamide, 0.4800 grams of N-acetylglucosamine and 0.1200 grams of canocobalamine (vitamin b-12). Water was added to bring the solution to volume in a 200-ml volumetric flask. Then the solution was separated into aliquots of 8 ml each and stored frozen.
KOH 10 % Stock Solution: 10 % KOH was prepared by adding 10 grams of KOH crystals to a 100-ml volumetric flask and bringing the volume to mark with water.
Beta-Sitosterol Stock: The sterol solution was prepared as follows: exactly 6.25 ml Tween 80 was added to 0.2500 g beta-sitosterol and 30 ml of distilled water. The solution was then brought to volume in a 50-ml volumetric flask. The solution was placed into small screw top autoclaveable tubes in six ml aliquots and autoclaved for 15 min. @ 122 C, 15 psi with slow exhaust. The tubes were immediately vortexed after removal from the autoclave to combine the two layers present. After cooling the solution to room temperature the solution was then stored at 4 C.
CbMM Media Compound Salt Solution: Salt solution for 10 liters of CbMM was prepared by mixing salt solution B with solution C and then solution A using no more than 1 ml rinse water. The volume of the combined salt solution was determined and the solution split into 10 equal volume aliquots. The solution was then stored frozen at -20C.
Salt SolutionA: Salt solution A was composed of 2.2050 grams of CaCl2 di-hydrate with 0.0650 grams of CuCl2 di-hydrate, 0.2220 grams of MnCl2 tetra-hydrate and 0.1020 grams of ZnCl2 in 100 ml water.
Salts Solution B: Salt solution B was composed of 0.5880 grams of Fe(NH4)2(SO4)2 hexa-hydrate, 4.8600 grams of K3Citrate mono-hydrate and 12.2550 grams of KH2PO4 in 100 ml water.
Salts Solution C: Salt solution C was composed of 6.3030 grams of citric Acid mono-hydrate and 1.7400 grams of Mg(OH)2 in 200 ml water.
Essential Amino Acids (EA): EA for 1 liter of CbMM was composed of: 1.0200 grams of L-valine, 0.1840 grams of L-tryptophan, 0.7170 grams of L-threonine, 0.6230 grams of L-phenylalanine, 0.3890 grams of L-methionine, 1.2830 grams of L-lysine hydrochloride, 1.4390 grams of L-leucine, 0.8610 grams of L-isoleucine, 0.2830 grams of L-histidine and 0.9750 grams of L-arginine in 85.0 ml water.
Nucleic Acid Substitutes (NAS): NAS for 1 liter of CbMM was composed of 0.3652 grams of adenosine 2'&3'-monophosphate (mixed isomers, free acid), 0.3232 grams of cytidine 2' & 3'-monophosphate mixed isomers (free acid, cytdylic acid), 0.3632 grams of guanosine 2' & 3'-monophosphate (mixed isomers, sodium salt, guanylic acid), 0.3681 grams of uridine 2' & 3'-monophosphate (mixed isomers, free acid), 0.1261 grams of thyamine and 0.617 ml of 10% KOH in 85 ml water.
Growth Factors (GF): Individual growth factors were stored individually in dry containers until used (amounts are for 1 liter of 1 x CbMM). Glutathione (reduced), 0.204 grams and cytochrome-c, 0.05 grams were stored individually. Choline dihydrogen citrate, 0.0885 grams and myo-inositol, 0.0645 grams, were stored together.
Energy Source (ES): D-glucose, 32.5 grams, was used as an energy source.
Non-Essential Amino Acids (NEA): NEA for 1 liter of CbMM was composed of: 0.2720 grams of L-tyrosine. 0.6530 grams of L-proline, 0.1800 grams of L-phenyalanine, 1.4630 grams of L-glutamine, 0.5500 grams of L-glutamate monohydrate (sodium salt), 0.7880 grams of L-serine, 0.0280 grams of L-cysteine hydrochloride monohydrate, 1.6200 grams of L-aspartic acid, 1.3950 grams of L-alanine, 0.7220 grams of glycine and 170 ml of water.
Continual Subculture of Caenorhabditis elegans: Stock cultures were subcultured every month following the small stock culture of nematodes method.
Inoculation Volume For HS-Y-HLE Cultures and for CbMM Assays: Nematode density in stock or inoculation solutions was determined to allow delivery of at least 10 to 20 nematodes into each culture.
Small Stock Cultures of Nematodes: Using sterile technique, 0.5 ml HLE and 0.1 ml of nematodes from previous stock were added into previously autoclaved bottles containing 3 ml of HS-YE. The stock bottles were stored either upright or on edge.
Experimental Tube Setup: All bottles used for cultures were washed once with soap and at least twice without soap in an automatic dishwasher. The glassware was autoclaved for 45 minutes. Experimental volume of CbMM was added to each culture bottle first. The experimental supplement volume of filter sterilized extract was added to each bottle second and the nematode inoculant was added last.
Isolation of C. elegans From Stock Cultures for CbMM Assays: Using sterile technique 5 ml of nematodes approximately 30 days old were removed from an existing culture. The nematodes were washed 4 times in sterile M-9 and 2 times in sterile CbMM before they were used as an inoculum.
C. elegans was cultured in a modified Caenorhabditis brigssae Maintenance Medium (CbMM) or M-9, a non-nutrient buffer (Brenner, 1974). Generally five replicates of 2.5 ml volume were setup in either 1/4 oz screw top rectangular bottles or 1/2 oz square screw top bottles. The standard experimental volume of CbMM was 2 ml. Standard experimental volume of other extracts was 0.50 ml, 0.40 ml or 0.20 ml. Total experimental volume for CbMM experiments was 2.50 ml (Vanfleteren and Roets, 1972).
Nematode populations were determined within 24 hours after culture initiation and every three to four days thereafter until the nematode control populations became too high to count. Prior to counting, the culture bottles were gently swirled to distribute the contents evenly. Nematodes were counted prior, during and after their logarithmic-growth phase (Pinnock, Hieb and Stokstad, 1975). Any movement of the nematode not consistent with brownian motion or associated with movements of the culture fluid was scored as a live nematode. Only living nematodes were counted. Non-moving nematodes were presumed dead and not counted. Nematodes retained inside cadavers were not counted. Eggs were not counted as nematodes. Any contamination of the culture by bacterial or fungal growth was recorded.
Nematode populations and anatomical features were observed using a Nikon binocular microscope model # SMZ-U or a Nikon inverted microscope model TMS equipped with a Nikon Microflex HFZ-DX photomicrographic attachment and a Nikon FX35DX dark box. Anatomical features were recorded using black and white and color photography.
Water was used as the standard control unless otherwise stated. All water was reverse osmosis water from a Nanopure water system providing ultra filtered type I water. Experimental solutions included a phosphate buffer, HS-YE, CbMM, HLE and several test extracts. The effect of test substances were compared to the growth rates, population changes and total populations of controls.
Protein concentration of samples was determined using the Bradford analysis (Bradford, 1976). Protein samples were subjected to SDS-polyacrylamide gel electrophoresis on a 12.5 % gel (Laemmli, 1970). The protein banding patterns were revealed after staining with Coomassie Blue R250. After observation of the banding patterns, the gel was destained and then silver stained (Bloom, Beier and Gross, 1987) to detect less abundant proteins.
Selected fractions were concentrated on the basis of size using Centricon concentrating filters following the standard protocols provided by the manufacturer. The concentrated fractions were used to construct a dose response curve. A haemagglutination assay was performed on selected test substances. Rabbit, pig and human red blood cells were tested as described by Rudiger (1993).
The following equations and abbreviations were used in the calculation and discussion of results:
Standard deviation =
where: n= number of replicates, xi = replicate population and x= average population of the replicate set.
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