Aseptic Addition Method for Lactobacillus Casei Assay of Folate Activity in Human Serum
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J Clin Pathol: first published as 10.1136/jcp.19.1.12 on 1 January 1966. Downloaded from J. clin. Path. (1966), 19, 12 Aseptic addition method for Lactobacillus casei assay of folate activity in human serum VICTOR HERBERT From the Department of Haematology, The Mount Sinai Hospital, New York, U.S.A. SYNOPSIS An 'aseptic addition' method is described for microbiological assay with Lactobacillis casei of folate activity in human serum. It has the following advantages over the previously reported 'standard' method. 1 The manipulations involved in the assay are halved, by deleting autoclaving of serum in buffers. 2 The use of 1 g. % ascorbate better preserves serum folates than the lower amounts of ascorbate which are the maximum quantities usable in the standard methods. 3 Only 03 ml. of serum is required (0 1 ml. for one sample; 02 ml. for its duplicate). Herbert, Wasserman, Frank, Pasher, and Baker in or after transfer of blood from syringes to acid-washed 1959 reported that folate deficiency could be screw-top tubes). The clots are 'rimmed' with glass rods measured in man using microbiological assay of or wooden applicator sticks, the tubes centrifuged for serum folate activity with Lactobacillus casei. Many five minutes at 3,000 r.p.m. and the supernatant serum aspirated with acid-washed or disposable pipettes andcopyright. other workers have confirmed this work (see review frozen at -20°C. until assay. On the day of assay, the by Herbert, 1965). Various minor modifications of sera are thawed. A 0-1 ml. and a 0-2 ml. aliquot of the methodology described by Baker, Herbert, serum is added aseptically to autoclaved solutions Frank, Pasher, Hutner, Wasserman, and Sobotka containing 5 ml. of double-strength medium (see Table I), (1959) and Herbert (1961) have been reported, 4 ml. of de-ionized water2, and 1 ml. of phosphate buffer including several which appeared in this Journal (containing 1 g. ascorbic acid per 100 ml. buffer) (see Chanarin and Table IL). (Waters and Mollin, 1961; Berry, http://jcp.bmj.com/ 1964; Spray, 1964). It was indicated in the recent past (Herbert, 1964) MAINTENANCE OF ASSAY ORGANISM AND PREPARATION OF that the 'aseptic addition' method for microbiological INOCULA L. casei, ATTC (American Type Culture assay with L. casei of serum folate has a number of Collection) no. 7469 (obtainable for $5 in either lyophil- over 'standard' method. These ized or agar slant culture from American Type Culture advantages the Collection, 2112 M Street, Washington, D.C.) is main- advantages include halving the manipulations tained in the msdium described in Table III and stored involved in the assay by deleting the step involving as a liquid culture at 4°C. We have carried our current autoclaving of serum in buffer; allowing the use of culture in liquid medium for six years, transferring on September 29, 2021 by guest. Protected 1 ml. of 1 g. % ascorbate, which better preserves it every two weeks to fresh maintenance medium, and no serum folates than do the lower amounts ofascorbate mutation has yet occurred. Liquid culture is technically used in the standard method, requiring only 03 ml. simpler than the agar-slant culture used by a number of of serum (0-1 ml. for one sample; 0-2 ml. for its investigators. duplicate), an especially valuable point when infants De-ionized water2 is used for preparation of all media and buffers. It is free of folate and more easily are under study. prepared than distilled water. Furthermore, the heavy metal ions in distilled water have the undesirable ability METHOD to accelerate oxidation of the ascorbic acid in the buffer. PREPARATION OF SERUM SAMPLES Blood is obtained from Five hundred ml. of maintenance medium (Table III) fasting subjects using either acid-washed sterile syringes, is generally prepared at one time and dispensed in 10 ml. disposable plastic syringes, or Vacutainers' no. 3200 to aliquots into 50 screw-capped tubes. The tubes, with ensure freedom from contamination with traces of folate. screw caps loosely affixed, are autoclaved for 30 minutes The blood is allowed to stand for approximately three at 118°C. They are then allowed to cool, the screw caps hours at room temperature (in the original Vacutainer 2Tap water passed through a Barnstead Bantam standard demineral- Received for publication 3 August 1965 izer cartridge no. 0802 (Barnstead Still and Sterilizer Co., Boston, 'Becton, Dickinson and Co., Rutherford, N.J. Mass). 12 J Clin Pathol: first published as 10.1136/jcp.19.1.12 on 1 January 1966. Downloaded from Aseptic addition methodfor Lactobacillus casei assay offolate activity in human serum 13 TABLE I TABLE III ASSAY MEDIUM MAINTENANCE MEDIUM1 Constituent Amount for Concentration Constituent Amount for Concentration in 4 1. of Double- in Final Volume 500 ml. Medium Final Medium strength (mg. %) (g./100 ml.) Medium Yeast extract (Difco) 3-75 g. 0-75 Hy-ease (1) 40 g. 500 Proteose peptone (Difco) 3-75 g. 0-75 L-tryptophan 0-8 g. 10 Glucose 5 g. 1-0 Adenine (2, 3) 4 ml. 0 5 KH,PO4 I g. 0-2 Guanine HCI (2, 3) 4 ml. 0-5 Tomato juice filtrate' 50 ml. 10 ml. Uracil (2, 3) 4 ml. 0 5 Tween 803 0-5 ml. 0-01 ml. Xanthine (2, 3) 8 ml. 10 L-cysteine HCI 0-5 g. 0-1 L-asparagine H,O 2-4 g. 30 'Adjust to pH 6-8 to 7 with 1 % KOH. This medium, without the L-cysteine HC1 (4) 2 g. 25 L-cysteine, has been previously described. Riboflavin (2, 5, 6) 4 ml. 005 'Canned or vacuum-bottled tomato juice passed through coarse Para-aminobenzoic acid (2, 5) 8 ml. 0-1 filter paper; straw-coloured filtrate adjusted to pH 7 with 10% KOH. Pyridoxine HCI (2, 5, 6) 16 ml. 0-2 3See footnote (8), Table 1. Thiamine HCI (2, 5) 1-6 ml. 0-02 Ca pantothenate (2, 5) 3-2 ml. 0 04 Nicotinic acid (2, 5) 3-2 ml. 0-04 Biotin (2, 6, 7) 1 ml. 0 001 are tightened and the tubes incubated overnight at 37°C. Glucose 160 g. 2,000 The following morning they are examined for clarity Tween 80 (8) 4 ml. 0-005 ml. (indicating absence of bacterial contamination) and, if Glutathione (reduced) 20 mg. 0-25 sterile, are then stored at 4°C. until used. Salt mix (9) 40 ml. 0-5 ml. Na acetate (anhydrous) 160 g. 2,000 At a maximal interval of once every two weeks, 1 drop K,HPO4 4 g. 50 of stored liquid culture is added to 10 ml. of fresh KH,PO4 4 g. 50 maintenance medium, incubated for 18 hours at 37°C., MnSO4H,O (10) 0-8 g. 10 and then stored at 4°C. During the afternoon of the day (1) Salt-free hydrochloric acid hydrolysate of casein, Sheffield Chemicals, Norwich, N.Y. Dissolve in 21. ofde-ionized water with before an assay, 1 drop of the latest stored culture is gentle heating and stirring. added to 10 ml. of maintenance medium and incubated (2) California Foundation for Biochemical Research, Los Angeles, for 18 hours at 37°C. The next morning, 0 5 ml. of this Calif. fresh 18-hour culture is added to 10 ml. of maintenance copyright. (3) Solutions (10 mg./ml.) prepared by adding I g. chromato- graphically pure reagent to 25 ml. de-ionized H,O, gently heating medium and incubated for six hours at 37°C. The with added pellets of KOH until reagent dissolved, making up to inoculum for the assay is prepared by adding 0 5 ml. of final volume of 100 ml. with deionized H,O. Store in refrigerator this fresh six-hour culture to 10 ml. of single-strength with volatile preservative added. (4) Dissolve in KOH before adding. basal medium. One drop of inoculum is added to each (5) Solution (I mg.!ml.) prepared by dissolving 0-1 g: reagent in assay flask. 100 ml. de-ionized H,O. Store in refrigerator with volatile Volatile preservative, consisting of 1 part ethylene preservative added. dichloride, 1 part monochlorobenzene, and 2 parts (6) Store in amber polyethylene bottle (6026, Cole-Parmer Instru- http://jcp.bmj.com/ ment and Equipment Co., Chicago, 111.) to protect from light. 1-chlorobutane, was then added to all stored solutions of (7) Solution (0-08 mg./ml.) prepared by dissolving 8 mg. biotin in assay medium and of buffer by spraying a small aliquot 100 ml. de-ionized H20. Store in refrigerator with volatile from a wash bottle into each solution after each use preservative added. (8) Atlas Powder Co., Wilmington, Del. The viscous commercial before returning to storage. preparation (specific gravity = 1) is diluted 1:10 with water containing some ethanol to reduce foaming. (9) One ml. contains: MgSO4-7H,O, 40 mg.; NaCl, 2 mg.; MnSO4 STANDARDS Three standard solutions of folic acid 4H,0, 2 mg.; FeSO,47H20, 2 mg.; Make 500 ml. by dissolving (pteroylmonoglutamic acid, P.G.A.) were prepared in 20 g. of MgSO4-7H2O 2 g. of NaCl, 1 g. of MnSO4 and 50 ml. aliquots, and stored between usages at -20°C. 4H2O, on September 29, 2021 by guest. Protected I g. of FeSO4 7H2O in de-ionized water, add 1 ml. concentrated (no appreciable deterioration in two months): 10-8, HCI, store with volatile preservative added. (10) Added after pH of assay medium adjusted to 6-6 to 6-8 with 0-1 10-9, 10-10 g. per ml. Crystalline P.G.A., 10 mg., is N H,SO4.