- Email: [email protected]
Microbiological assay of folates in 96-well microtiter plates
38
FOLICACID
[41
of folate per liter of RBCs was obtained even though RBC folates are composed mostly of 5-methyltetrahydrofolate monoglutamate, and of tetra-, penta-, and hexaglutamates (molecular weight of 849, 979, or 1109, respectively).8,9 Although such estimations of folate content are likely to be reasonable approximations when comparing samples, it is obvious how inaccuracies can occur because of the vast differences in affinity between different folate compounds for folate-binding protein, 4'1° which is used in limiting amounts in competitive binding assays; and because of differences in the molecular weight of measured folate coenzymes compared to the folate used in the standard curve. The RBC or whole-blood folate assays are conceptually excellent reflections of tissue folate stores because of the relatively long half-life of RBCs. However, these assays have been criticized because of inaccuracies of competitive binding assays. It is hoped that the RBC/tissue folate assay described here provides a degree of accuracy such that clinicians and investigators will once again rely on RBC or whole-blood folates to assess tissue folate sufficiency.
Acknowledgment The authors thank Academic Press for providing permission to adapt this chapter from Santhosh-Kumar et al. Anal. Biochem. 225, 1-9 (1995). 8 T. Tamura, Y. S. Shin, M. A. Williams, and E. L. R. Stokstad, A n a l Biochem. 49, 517 (1972). 9 j. Perry, M. Lumb, M. Laundy, E. H. Reynolds, and I. Chanarin, Br. J. Hematol. 32, 243 (1976). 10 p. C. Elwood, M. A. Kane, R. M. Portillo, and J. F. Kolhouse, J. BioL Chem. 261, 15416 (1986).
[41 Microbiological Assay of Folates i n 96-Well Microtiter Plates By
DONALD W. HORNE
Our understanding of the dietary requirement for, and the metabolism of, the vitamin folic acid rests, mainly, on our ability to determine the amount present in serum, erythrocytes, and other tissues. Several procedures are currently in use for estimating folate concentrations; these include microbiological, radiochemical, and electrochemical assays. These proce-
METHODS IN ENZYMOLOGY,VOL. 281
Copyright © 1997by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/97 $25
[4]
MICROTITER PLATE ASSAY OF FOLATES
39
dures have been reviewed by Tamura, t who concluded that the microbiological assay is still the method of choice for the assay of the complex mixture of folates present in biological samples. Two developments, which we have refined, make the microbiological assay of folates more reproducible, much easier, and much less time consuming than in the past. These developments are the use of glycerolcryoprotected Lactobacillus casei as the inoculum for the assay2-4 and the use of 96-well microtiter plates and microcomputer analysis of the data. 5'6 The 96-well microtiter plate assay procedure is described in this chapter. We also point out a problem, apparently inherent to clear-wall plates and turbidimetric assays, which results in overestimation of folate concentration of samples in the two perimeter rows of 96-well plates (rows A and H according to standard designation). We present a solution, which is the use of microtiter plates with opaque, black walls. Bacteria Cultures and Reagents Lyophilized cultures of L. casei subspecies rhamnosis (ATCC 7469): American Type Culture Collection (Rockville, MD) Folinic acid calcium salt pentahydrate [(6RS)-5-formyltetrahydrofolate]: Fluka BioChemika (Ronkonkoma, NY) L-Ascorbic acid, sodium salt 2-Mercaptoethanol Glycerol Microtiter plates: Microtest III, Falcon No. 3072 (Fisher Scientific, Norcross, GA) or UV96PSB black-wall 96-well plates and US10 polystyrene lids (Polyfiltronics, Rockland, MA) Folic acid (L. casei) medium: Difco (Detroit, MI) Instruments Microplate absorbance reader: We use a Bio-Rad (Hercules, CA) model 2550 EIA reader Microcomputer interfaced with reader: We use Macintosh SE and BioRad MacReader software; not essential for the assay 1 T. Tamura, in "Folic Acid Metabolism in Health and Disease" (M. F. Piccaino, E. L. R. Stokstad, and J. F. Gregory, III, eds.), pp. 121-137. Wiley-Liss, New York, 1990. 2 N. Grossowicz, S. Waxman, and C. Schreiber, Clin. Chem. 27, 745 (1981). 3 S. D. Wilson and D. W. Horne, Clin. Chem. 28, 1198 (1982). 4 S. D. Wilson and D. W. Horne, Methods Enzymol. 122, 269 (1986). 5 E. M. Newman and J. F. Tsai, Anal. Biochem. 154, 509 (1986). 6 D. W. H o m e and D. Patterson, Clin. Chem. 34, 2357 (1988).
40
FOLICACID
[4]
Memowell microplate memory device [Matrix Technologies (Lowell, MA); a light-box device for keeping track of pipetting position on the plates] or similar device; not essential for the assay, but recommended. Glycerol-Cryoprotected Lactobacillus casei Glycerol-cryoprotected L. casei for the inoculum is prepared as described by Wilson and Horne 3 as modified by Horne and Patterson. 6 Dissolve 9.4 g of folic acid L. casei medium (Difco) and 50 mg of sodium ascorbate in 200 ml of distilled water. Add 120 ng of (6RS)-5-formyltetrahydrofolate and sterilize by filtration (0.22-/xm pore size). Suspend the lyophilized L. casei, in its shipping vial, in 1 ml of the medium. Transfer 0.25 ml of the suspension to the remaining medium and incubate at 35-37 ° for about 18 hr. Cool in an ice bath and add an equal volume of cold, 80% (v/v) glycerol (sterilized by autoclaving at 121° for 15 rain). Store 4-ml aliquots in sterile tubes at about - 7 0 °. Preparation of Tissue Extracts Rat pancreas cytosol is used here as an example. Male Sprague-Dawley rats are anesthetized with ketamine and xylazine [80 and 11 mg/kg, respectively, intraperitoneally (i.p.)], and the pancreas of each is removed, trimmed of fat, weighed, and homogenized in 3 vol of ice-cold 0.25 M sucrose, 10 mM HEPES (pH 7.4), 10 mM 2-mercaptoethanol, and 10 mM sodium ascorbate. All further steps are carried out at 4°. The homogenate is centrifuged at 18,000 rpm in a Sorvall (Norwalk, CT) RC5B centrifuge for 30 min. The supernatant is removed and 0.2 vol of 5× extract buffer {10% (w/v) sodium ascorbate, 1 M 2-mercaptoethanol, 0.25 M HEPES, 0.25 M CHES [2-(N-cyclohexylamino)ethanesulfonic acid], pH 7.85} is added. [We use 0.2 vol of 5 x extract buffer so that the final concentration of extract buffer ingredients will be - 1 × (see below) to protect labile folates during heating and subsequent conjugase treatment.] The solution is heated in a boiling water bath for 5 min and centrifuged in an Eppendorf microcentrifuge for 5 min to remove precipitated protein. The supernatant is treated with rat plasma conjugase (3/-glutamyl hydrolase) to hydrolyze folylpolyglutamates to the monoglutamates.4'7 For routine assay of total folates by the microbiological assay, we extract tissue by mincing in 1 x extract buffer [2% (w/v) sodium ascorbate, 0.2 M 2-mercaptoethanol, 0.05 M HEPES, 0.05 M CHES (pH 7.85)], heating in 7D. W. Horne, D. Patterson, and R. J. Cook,Arch. Biochem. Biophys. 270, 729 (1989).
[4]
M I C R O T I T E R P L A T E ASSAY OF F O L A T E S
41
a boiling water bath for 10 min, homogenizing using a Polytron (Brinkmann, Westbury, NY), microcentrifuging, and treating with conjugase (as described above). For liquid samples (e.g., serum or plasma) we add 0.2 vol of 5× extract buffer (see above), heat in boiling water bath for 5 rain, and microcentrifuge to remove precipitated proteins. Serum and plasma contain only the monoglutamate derivatives; therefore, conjugase treatment is not necessary. These extraction procedures have been shown to preserve the reduced folates present in tissues and to lead to minimal interconversion of individual derivativesf '9
Assay Procedure Sufficient folic acid L. casei medium (Difco) for the anticipated number of assays is prepared according to the package insert. The medium is heated until just boiling, cooled, and filtered through a 0.22-/zm (pore size) filter. The working buffer is composed of 1.6 g of sodium ascorbate, 0.5 ml of 1 M potassium phosphate buffer (pH 6.1), and 9.5 ml of distilled water. The solution is filtered through a 0.22-/xm (pore size) filter. To prepare a stock solution of folate standard, (6RS)-5-formyltetrahydrofolate, calcium salt is dissolved in distilled water to give a concentration of about 5 raM. The actual concentration is determined by measuring the absorbance at 285 nm (of appropriate dilutions in 0.1 M potassium phosphate, pH 7), using the molar extinction coefficient of 37.2 × 103 cm liter tool-1.1° The solution is then diluted with distilled water to 2 raM; this is the stock solution of the standard. Because L. casei grows only on the (6S)-isomer, the concentration of the active isomer is, therefore, 1 raM. The stock solution is aliquoted into microcentrifuge tubes and stored at - 7 0 ° until needed. The working standard is prepared by diluting 15/zl of the stock solution to a final volume of 5 ml with distilled water. The L. casei inoculum is prepared by diluting the thawed, cryoprotected bacterial suspension 1:2.5 (v/v) with sterile sodium chloride solution (9 g/ liter). This is the dilution of the bacteria we presently use. This dilution is determined by preparing a series of standard curves using different dilutions of the cryoprotected L. casei. We choose the highest dilution of the bacteria that gives the highest absorbance for all points on the standard curve. This dilution is used for routine assays. S. D. Wilson and D. W. Horne, Proc. Natl. Acad. Sci. U.S.A. 80, 6500 (1983). 9S. D. Wilson and D. W. Horne, Anal. Biochem. 142, 529 (1984). 10R. L. Blakley, "The Biochemistryof Folic Acid and Related Pteridines." North-Holland, Amsterdam, 1969.
42
FOLICACID
[41
Eight microliters of the working buffer is pipetted into each well of 96well plates. The standard curve is constructed, in duplicate, from 0 to 180 fmol of 5-formyltetrahydrofolate, with the maximum volume being 60 tzl. The unknown sample volume may be up to 122 tzl. The volume of standards and samples is adjusted to 122 /xl with sterile water, 20 /.d of L. casei inoculum is added, and 150/zl of folic acid L. casei medium is added. The plates are covered with the polystyrene cover and incubated for approximately 18 hr at 37 ° in a humidified oven. The absorbance at 600 nm is read in a microtiter plate reader. (It is not necessary to resuspend the bacteria prior to reading; see Comments.) In our case, the reader is interfaced with a computer that reduces the data. The growth of L. casei on standard 5-formyltetrahydrofolate in both clear-wall (Falcon Microtest Ill) plates and black-wall (Polyfiltronics UV96PSB) plates was determined. While both showed good reproducibility (coefficient of variability was 6.2% or less), black-wall plates gave consistently higher absorbance readings (107% at 15 fmol to 140% at 150 fmol) than clear-wall plates. The reason for this is not known. However, the clear plastic wall may act as a light pipe (similar to transmission in fiber optic cable), thereby transmitting light through the wall in addition to that not scattered by the bacteria. This would lead to lower apparent absorbance. The walls of black-wall plates would not permit this light piping because they are opaque, thereby resulting in greater apparent absorbance than is provided by clear-wall plates. We had noticed that values of folate obtained from bacteria grown in the wells located on the perimeter of clear-wall microtiter plates were about 20% higher than values for the same samples grown in interior wells. This was confirmed by measuring folate in rat pancreas cytosol extract. Each microtiter plate had a standard curve (columns 1 and 2) and in all other wells a 20-t~l aliquot of a 1:200 (v/v) dilution of rat pancreas cytosol extract was assayed. By inspection of the results, it appeared that the values determined in perimeter rows A and H were higher than those of internal rows C - G in clear-wall plates. Therefore, we compared the average folate concentration calculated for each row [rows A - H (omitting values from column 12, since it is on the outside and absorbance values in this column are higher than in columns 2-11)], using one-way analysis of variance (ANOVA). We found that the folate concentrations calculated from rows A and H using clear-wall plates were indeed higher than the values for rows B, C, D, E, F, and G. Using black-wall plates, the results showed no such systematic difference in values from any row. The reason for this is not known for certain. However, the construction of the plate gives some clue. The bottom of the plate over the area of the wells is solid. Looking down on the top of the plate, all of the inside wells have a similar air-to-
[5]
ASSAY OF SERUM, PLASMA, AND RED CELL F O L A T E
43
plastic surface area around the wells. However, the outside wells (those of rows A and H, and columns 1 and 12) have a larger air-to-plastic surface area. One may speculate that more of the light scattered toward the walls would be lost from the outer wells because of the greater air-to-plastic surface. This cannot happen with black-wall plates.
Comments Previously, we recommended suspending the bacteria by repeatedly aspirating - 1 5 0 tzl using a pipette. We have found that this step is not necessary, presumably because the light path is up through the well and, because the bacteria have settled to the bottom, essentially equal light scattering occurs whether or not the bacteria have been resuspended. We recommend that black-wall plates be used for the microbiological assay of folates using L. casei and this would, presumably, also apply to assays using Streptococcus faecium and Pediococcus cerevisiae, and to all turbidimetric assays.
Acknowledgments T h e t e c h n i c a l a s s i s t a n c e of R o s a l i n d S. H o l l o w a y is g r a t e f u l l y a c k n o w l e d g e d . This w o r k w a s s u p p o r t e d b y the D e p a r t m e n t of V e t e r a n s A f f a i r s a n d b y N I H G r a n t DK32189.
[5] M i c r o b i o l o g i c a l A s s a y f o r S e r u m , P l a s m a , a n d R e d Cell Folate Using Cryopreserved, Microtiter Plate Method
By ANNE M. MOLLOY and JOHN M. SCOTT The routine analysis of serum and erythrocyte folate has been the subject of numerous reviews, comments, and controversies. The microbiological methods used originally were difficult to set up, difficult to maintain, and slow in obtaining results. Thus many laboratories changed over to newly developed radioassays when they became available. However, the microbiological method remains for most investigators the "gold standard" and the method of choice. What many investigators do not realize is that it has now been refined and automated to such an extent that it is easy to perform, reliable to maintain, and considerably less costly than the radioassay method, particularly where large numbers of samples are involved. Three important changes in the assay technology have contributed to this. First,
METHODS IN ENZYMOLOGY, VOL. 281
Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. ()076-6879/97 $25
FOLICACID
[41
of folate per liter of RBCs was obtained even though RBC folates are composed mostly of 5-methyltetrahydrofolate monoglutamate, and of tetra-, penta-, and hexaglutamates (molecular weight of 849, 979, or 1109, respectively).8,9 Although such estimations of folate content are likely to be reasonable approximations when comparing samples, it is obvious how inaccuracies can occur because of the vast differences in affinity between different folate compounds for folate-binding protein, 4'1° which is used in limiting amounts in competitive binding assays; and because of differences in the molecular weight of measured folate coenzymes compared to the folate used in the standard curve. The RBC or whole-blood folate assays are conceptually excellent reflections of tissue folate stores because of the relatively long half-life of RBCs. However, these assays have been criticized because of inaccuracies of competitive binding assays. It is hoped that the RBC/tissue folate assay described here provides a degree of accuracy such that clinicians and investigators will once again rely on RBC or whole-blood folates to assess tissue folate sufficiency.
Acknowledgment The authors thank Academic Press for providing permission to adapt this chapter from Santhosh-Kumar et al. Anal. Biochem. 225, 1-9 (1995). 8 T. Tamura, Y. S. Shin, M. A. Williams, and E. L. R. Stokstad, A n a l Biochem. 49, 517 (1972). 9 j. Perry, M. Lumb, M. Laundy, E. H. Reynolds, and I. Chanarin, Br. J. Hematol. 32, 243 (1976). 10 p. C. Elwood, M. A. Kane, R. M. Portillo, and J. F. Kolhouse, J. BioL Chem. 261, 15416 (1986).
[41 Microbiological Assay of Folates i n 96-Well Microtiter Plates By
DONALD W. HORNE
Our understanding of the dietary requirement for, and the metabolism of, the vitamin folic acid rests, mainly, on our ability to determine the amount present in serum, erythrocytes, and other tissues. Several procedures are currently in use for estimating folate concentrations; these include microbiological, radiochemical, and electrochemical assays. These proce-
METHODS IN ENZYMOLOGY,VOL. 281
Copyright © 1997by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/97 $25
[4]
MICROTITER PLATE ASSAY OF FOLATES
39
dures have been reviewed by Tamura, t who concluded that the microbiological assay is still the method of choice for the assay of the complex mixture of folates present in biological samples. Two developments, which we have refined, make the microbiological assay of folates more reproducible, much easier, and much less time consuming than in the past. These developments are the use of glycerolcryoprotected Lactobacillus casei as the inoculum for the assay2-4 and the use of 96-well microtiter plates and microcomputer analysis of the data. 5'6 The 96-well microtiter plate assay procedure is described in this chapter. We also point out a problem, apparently inherent to clear-wall plates and turbidimetric assays, which results in overestimation of folate concentration of samples in the two perimeter rows of 96-well plates (rows A and H according to standard designation). We present a solution, which is the use of microtiter plates with opaque, black walls. Bacteria Cultures and Reagents Lyophilized cultures of L. casei subspecies rhamnosis (ATCC 7469): American Type Culture Collection (Rockville, MD) Folinic acid calcium salt pentahydrate [(6RS)-5-formyltetrahydrofolate]: Fluka BioChemika (Ronkonkoma, NY) L-Ascorbic acid, sodium salt 2-Mercaptoethanol Glycerol Microtiter plates: Microtest III, Falcon No. 3072 (Fisher Scientific, Norcross, GA) or UV96PSB black-wall 96-well plates and US10 polystyrene lids (Polyfiltronics, Rockland, MA) Folic acid (L. casei) medium: Difco (Detroit, MI) Instruments Microplate absorbance reader: We use a Bio-Rad (Hercules, CA) model 2550 EIA reader Microcomputer interfaced with reader: We use Macintosh SE and BioRad MacReader software; not essential for the assay 1 T. Tamura, in "Folic Acid Metabolism in Health and Disease" (M. F. Piccaino, E. L. R. Stokstad, and J. F. Gregory, III, eds.), pp. 121-137. Wiley-Liss, New York, 1990. 2 N. Grossowicz, S. Waxman, and C. Schreiber, Clin. Chem. 27, 745 (1981). 3 S. D. Wilson and D. W. Horne, Clin. Chem. 28, 1198 (1982). 4 S. D. Wilson and D. W. Horne, Methods Enzymol. 122, 269 (1986). 5 E. M. Newman and J. F. Tsai, Anal. Biochem. 154, 509 (1986). 6 D. W. H o m e and D. Patterson, Clin. Chem. 34, 2357 (1988).
40
FOLICACID
[4]
Memowell microplate memory device [Matrix Technologies (Lowell, MA); a light-box device for keeping track of pipetting position on the plates] or similar device; not essential for the assay, but recommended. Glycerol-Cryoprotected Lactobacillus casei Glycerol-cryoprotected L. casei for the inoculum is prepared as described by Wilson and Horne 3 as modified by Horne and Patterson. 6 Dissolve 9.4 g of folic acid L. casei medium (Difco) and 50 mg of sodium ascorbate in 200 ml of distilled water. Add 120 ng of (6RS)-5-formyltetrahydrofolate and sterilize by filtration (0.22-/xm pore size). Suspend the lyophilized L. casei, in its shipping vial, in 1 ml of the medium. Transfer 0.25 ml of the suspension to the remaining medium and incubate at 35-37 ° for about 18 hr. Cool in an ice bath and add an equal volume of cold, 80% (v/v) glycerol (sterilized by autoclaving at 121° for 15 rain). Store 4-ml aliquots in sterile tubes at about - 7 0 °. Preparation of Tissue Extracts Rat pancreas cytosol is used here as an example. Male Sprague-Dawley rats are anesthetized with ketamine and xylazine [80 and 11 mg/kg, respectively, intraperitoneally (i.p.)], and the pancreas of each is removed, trimmed of fat, weighed, and homogenized in 3 vol of ice-cold 0.25 M sucrose, 10 mM HEPES (pH 7.4), 10 mM 2-mercaptoethanol, and 10 mM sodium ascorbate. All further steps are carried out at 4°. The homogenate is centrifuged at 18,000 rpm in a Sorvall (Norwalk, CT) RC5B centrifuge for 30 min. The supernatant is removed and 0.2 vol of 5× extract buffer {10% (w/v) sodium ascorbate, 1 M 2-mercaptoethanol, 0.25 M HEPES, 0.25 M CHES [2-(N-cyclohexylamino)ethanesulfonic acid], pH 7.85} is added. [We use 0.2 vol of 5 x extract buffer so that the final concentration of extract buffer ingredients will be - 1 × (see below) to protect labile folates during heating and subsequent conjugase treatment.] The solution is heated in a boiling water bath for 5 min and centrifuged in an Eppendorf microcentrifuge for 5 min to remove precipitated protein. The supernatant is treated with rat plasma conjugase (3/-glutamyl hydrolase) to hydrolyze folylpolyglutamates to the monoglutamates.4'7 For routine assay of total folates by the microbiological assay, we extract tissue by mincing in 1 x extract buffer [2% (w/v) sodium ascorbate, 0.2 M 2-mercaptoethanol, 0.05 M HEPES, 0.05 M CHES (pH 7.85)], heating in 7D. W. Horne, D. Patterson, and R. J. Cook,Arch. Biochem. Biophys. 270, 729 (1989).
[4]
M I C R O T I T E R P L A T E ASSAY OF F O L A T E S
41
a boiling water bath for 10 min, homogenizing using a Polytron (Brinkmann, Westbury, NY), microcentrifuging, and treating with conjugase (as described above). For liquid samples (e.g., serum or plasma) we add 0.2 vol of 5× extract buffer (see above), heat in boiling water bath for 5 rain, and microcentrifuge to remove precipitated proteins. Serum and plasma contain only the monoglutamate derivatives; therefore, conjugase treatment is not necessary. These extraction procedures have been shown to preserve the reduced folates present in tissues and to lead to minimal interconversion of individual derivativesf '9
Assay Procedure Sufficient folic acid L. casei medium (Difco) for the anticipated number of assays is prepared according to the package insert. The medium is heated until just boiling, cooled, and filtered through a 0.22-/zm (pore size) filter. The working buffer is composed of 1.6 g of sodium ascorbate, 0.5 ml of 1 M potassium phosphate buffer (pH 6.1), and 9.5 ml of distilled water. The solution is filtered through a 0.22-/xm (pore size) filter. To prepare a stock solution of folate standard, (6RS)-5-formyltetrahydrofolate, calcium salt is dissolved in distilled water to give a concentration of about 5 raM. The actual concentration is determined by measuring the absorbance at 285 nm (of appropriate dilutions in 0.1 M potassium phosphate, pH 7), using the molar extinction coefficient of 37.2 × 103 cm liter tool-1.1° The solution is then diluted with distilled water to 2 raM; this is the stock solution of the standard. Because L. casei grows only on the (6S)-isomer, the concentration of the active isomer is, therefore, 1 raM. The stock solution is aliquoted into microcentrifuge tubes and stored at - 7 0 ° until needed. The working standard is prepared by diluting 15/zl of the stock solution to a final volume of 5 ml with distilled water. The L. casei inoculum is prepared by diluting the thawed, cryoprotected bacterial suspension 1:2.5 (v/v) with sterile sodium chloride solution (9 g/ liter). This is the dilution of the bacteria we presently use. This dilution is determined by preparing a series of standard curves using different dilutions of the cryoprotected L. casei. We choose the highest dilution of the bacteria that gives the highest absorbance for all points on the standard curve. This dilution is used for routine assays. S. D. Wilson and D. W. Horne, Proc. Natl. Acad. Sci. U.S.A. 80, 6500 (1983). 9S. D. Wilson and D. W. Horne, Anal. Biochem. 142, 529 (1984). 10R. L. Blakley, "The Biochemistryof Folic Acid and Related Pteridines." North-Holland, Amsterdam, 1969.
42
FOLICACID
[41
Eight microliters of the working buffer is pipetted into each well of 96well plates. The standard curve is constructed, in duplicate, from 0 to 180 fmol of 5-formyltetrahydrofolate, with the maximum volume being 60 tzl. The unknown sample volume may be up to 122 tzl. The volume of standards and samples is adjusted to 122 /xl with sterile water, 20 /.d of L. casei inoculum is added, and 150/zl of folic acid L. casei medium is added. The plates are covered with the polystyrene cover and incubated for approximately 18 hr at 37 ° in a humidified oven. The absorbance at 600 nm is read in a microtiter plate reader. (It is not necessary to resuspend the bacteria prior to reading; see Comments.) In our case, the reader is interfaced with a computer that reduces the data. The growth of L. casei on standard 5-formyltetrahydrofolate in both clear-wall (Falcon Microtest Ill) plates and black-wall (Polyfiltronics UV96PSB) plates was determined. While both showed good reproducibility (coefficient of variability was 6.2% or less), black-wall plates gave consistently higher absorbance readings (107% at 15 fmol to 140% at 150 fmol) than clear-wall plates. The reason for this is not known. However, the clear plastic wall may act as a light pipe (similar to transmission in fiber optic cable), thereby transmitting light through the wall in addition to that not scattered by the bacteria. This would lead to lower apparent absorbance. The walls of black-wall plates would not permit this light piping because they are opaque, thereby resulting in greater apparent absorbance than is provided by clear-wall plates. We had noticed that values of folate obtained from bacteria grown in the wells located on the perimeter of clear-wall microtiter plates were about 20% higher than values for the same samples grown in interior wells. This was confirmed by measuring folate in rat pancreas cytosol extract. Each microtiter plate had a standard curve (columns 1 and 2) and in all other wells a 20-t~l aliquot of a 1:200 (v/v) dilution of rat pancreas cytosol extract was assayed. By inspection of the results, it appeared that the values determined in perimeter rows A and H were higher than those of internal rows C - G in clear-wall plates. Therefore, we compared the average folate concentration calculated for each row [rows A - H (omitting values from column 12, since it is on the outside and absorbance values in this column are higher than in columns 2-11)], using one-way analysis of variance (ANOVA). We found that the folate concentrations calculated from rows A and H using clear-wall plates were indeed higher than the values for rows B, C, D, E, F, and G. Using black-wall plates, the results showed no such systematic difference in values from any row. The reason for this is not known for certain. However, the construction of the plate gives some clue. The bottom of the plate over the area of the wells is solid. Looking down on the top of the plate, all of the inside wells have a similar air-to-
[5]
ASSAY OF SERUM, PLASMA, AND RED CELL F O L A T E
43
plastic surface area around the wells. However, the outside wells (those of rows A and H, and columns 1 and 12) have a larger air-to-plastic surface area. One may speculate that more of the light scattered toward the walls would be lost from the outer wells because of the greater air-to-plastic surface. This cannot happen with black-wall plates.
Comments Previously, we recommended suspending the bacteria by repeatedly aspirating - 1 5 0 tzl using a pipette. We have found that this step is not necessary, presumably because the light path is up through the well and, because the bacteria have settled to the bottom, essentially equal light scattering occurs whether or not the bacteria have been resuspended. We recommend that black-wall plates be used for the microbiological assay of folates using L. casei and this would, presumably, also apply to assays using Streptococcus faecium and Pediococcus cerevisiae, and to all turbidimetric assays.
Acknowledgments T h e t e c h n i c a l a s s i s t a n c e of R o s a l i n d S. H o l l o w a y is g r a t e f u l l y a c k n o w l e d g e d . This w o r k w a s s u p p o r t e d b y the D e p a r t m e n t of V e t e r a n s A f f a i r s a n d b y N I H G r a n t DK32189.
[5] M i c r o b i o l o g i c a l A s s a y f o r S e r u m , P l a s m a , a n d R e d Cell Folate Using Cryopreserved, Microtiter Plate Method
By ANNE M. MOLLOY and JOHN M. SCOTT The routine analysis of serum and erythrocyte folate has been the subject of numerous reviews, comments, and controversies. The microbiological methods used originally were difficult to set up, difficult to maintain, and slow in obtaining results. Thus many laboratories changed over to newly developed radioassays when they became available. However, the microbiological method remains for most investigators the "gold standard" and the method of choice. What many investigators do not realize is that it has now been refined and automated to such an extent that it is easy to perform, reliable to maintain, and considerably less costly than the radioassay method, particularly where large numbers of samples are involved. Three important changes in the assay technology have contributed to this. First,
METHODS IN ENZYMOLOGY, VOL. 281
Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. ()076-6879/97 $25