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[149] Microbiological methods for determining magnesium, iron, copper, zinc, manganese, and molybdenum
[149]
MICROBIOLOGICAL METHODS FOR METALS
1035
the Beckman flame spectrophotometer are given in the instruction manuals which should be consulted before the analysis is planned. Since the line positions of the spectrophotometer shift with changing conditions, these must be checked daily and also whenever the wavelength setting is changed. The 100 % standard solution is used to set the instrument for maximum brightness. It is necessary to standardize the flame spectrophotometer before each run and to check a known point on the standard curve frequently during the determination. After every two or three readings the 50 % standard solution is checked again to detect any change in operating conditions of the instrument. The flame background must be subtracted from all readings; this is determined by means of a distilled water blank and should be checked frequently. This procedure is repeated for each substance being determined, the instrument being reset and the proper solutions used to standardize the spectrophotometer in each case.
[149] Microbiological Methods for Determining Magnesium, Iron~ Copper~ Zinc, Manganese, and Molybdenum By D. J. D. NICHOLAS Microbiological methods for determining trace metals have the advantages of specificity, reproducibility, and sensitivity at low levels of metal over chemical or physical methods of analysis. Disadvantages include the necessity for rigorous pure culture techniques and the frequent use of standard growth curves to check possible mutation of the organisms. This article describes methods for determining Mg, Fe, Cu, Zn, Mn, and Mo in biological materials, using the mold Aspergillus niger (Mulder strain). Other strains of A. niger may also be used for assays of metals. 1,2
Principle A. niger (h/i. strain) requires N, P, S, K, Mg, and the trace metals Fe, Cu, Zn, Mn, and h/io for optimum growth. 1-6 In the absence of one of 1 D. J. D. Nicholas, J. Sci. Food Agri. 1, 339 (1950). D. J. D. Nicholas, Analyst 77, 629 (1952). s E. G. Mulder, Arch. Microbiol. 10, 72 (1939). 4 D. J. D. Nicholas and A. H. Fielding, Ann. Rept. Long Ashton Research Station (Bristol, England) 126 (1947). 5 D. J. D. Nicholas and A. H. Fielding, J. Hort. Sci. 26, 125 (1951). 6 R. A. Steinberg, Botan. Rev. 5, 327 (1939).
1036
DETERMINATION OF INORGANIC COMPOUNDS
[149]
these essential elements the growth of the fungus is retarded, as shown by reduction in dry weight yields and sporulation. Moreover, an increase in an essential element from deficiency to sufficiency levels when all others are present at optimum amounts results in a specific and quantitative increase in growth. In this way a standard growth series is prepared for any of the essential mineral nutrients and assayed by taking dry weight yields of the pads. For the bioassay of test material a known amount of it is added to a culture solution that contains all the essential elements other than the one to be determined. The growth of the fungus under these conditions depends on the amount of test element that it derives from the material added. The growth measured by dry weight yields of the mats is referred to a standard series for the element, grown under the same conditions.
Experimental Methods Water Supply. Distilled water is prepared by being distilled twice from Pyrex glass to reduce the metal content. The minimum metal in micrograms per milliliter (p.p.m.) detected by the bioassay method is Cu, 0.001, Zn 0.005, Fe 0.002, Mn 0.002, Mo 0.00001. Water from a tinnedcopper still is therefore unsuitable for assay of metals other than Mg and Zn. The metal content of water is markedly reduced by distillation either once or twice from Pyrex glass. There is no further reduction by a third distillation. Water of low conductivity prepared from standard ion exchange resins is also suitable for trace metal assays, but the Mo content may be too high for the sensitive A. niger assay for the element. Glassware. Hard-glass 500-ml. Erlenmeyer flasks are thoroughly cleaned by washing several times with hot water and an organic detergent and then with concentrated nitric acid followed by single-distilled and finally with twice-distilled water from Pyrex glass. Alternatively a 0.5 % w / v solution of disodium ethylenediaminetetraacetic acid is used instead of the acid wash, and flasks containing this solution are steamed in an autoclave to remove metals. Glassware and water supply may be checked for metals by shaking with 0.01% w / v solution of dithizone (diphenylthiocarbazone) in redistilled CC14. Metal dithizonates are indicated by a change from green of dithizone to red. Preparation of Culture Solutions Free .from Trace Metals. A suitable culture solution (1 1.) for A. niger is as follows: Macronutrients--dextrose, 50 g., KNOa, 5 g., KH2PO4, 2.5 g., MgSO4"7H20, 1 g., and Ca(NOa)2, 0.5 g.; micronutrients--FeCla'6H20, 20 nag., ZnSO4.7H20, 20 mg., CuSO4"5H20, 1 mg., MnSOt-5H~O, 3 rag., Na~MoO4"2H~O, 1 mg.; glassdistilled water to 1 1.
[149]
MICROBIOLOGICAL METHODS FOR METALS
1037
The pH of the basal culture solution is 3.8; 50 ml. is used for each culture. The macronutrients contain sufficient of the micronutrients to provide for optimum growth of the mold. Methods used to remove them from a solution of macronutrients in 200 ml. of distilled water are summarized in Table I. There is no need for purification when Mg is to be assayed. TABLE I REMOVAL OF TRACE METALS FROM A SOLUTION OF INORGANIC MACRONUTRIENTS AND DEXTROSE
Trace metal removed Copper
Zinc and iron
Molybdenum Manganese
Method used for removal Add 2 ml. 10% w / v copper sulfate to 200 ml. culture solution, adjusted with hydrochloric acid to pH 3, and precipitate as a sulfide with hydrogen sulfide (15 minutes) from a Kipp's apparatus or a tank or cylinder. Let precipitate stand for 15 minutes, and filter through Whatman No. 42 paper into a clean Erlenmeyer flask. Eliminate hydrogen sulfide by boiling and agitating solution for about 20 minutes, and check removal with lead acetate paper. Adjust 200 ml. solution to pH 5.5 with 5% w / v sodium hydroxide, and shake four times with 30-ml. portions of 5% w / v 8-hydroxyquinoline in chloroform in a liter Pyrex glass separating funnel. Extract excess quinolates with three 30-ml. portions of redistiUed chloroform and then with similar quantities of redistilled ether. The latter removes chloroform. Eliminate the ether by boiling the solution on an electric hot plate. Zinc and iron are also removed at pH values above 5.5, but in alkaline solution phosphates are precipitated. The same as the coprecipitation method for the removal of copper. Coprecipitation of manganese and copper diethyldithiocarbamates at pH 5.5. Add 10 ml. 0.1% w / v sodium diethyldithiocarbamate in water and 5 ml. 5% w / v copper sulfate solution. Set aside for 30 minutes, filter or centrifuge precipitate, and adjust filtrate to pH 3 with HC1. Add a further 5 ml. 5% w / v copper sulfate, precipitate copper sulfide with hydrogen sulfide from a cylinder, and proceed as described for copper.
Residual metal after purification, ~/50 ml. 0.05
0.01
0.0005 0.01
1038
DETERMINATION OF INORGANIC COMPOUNDS
[149]
Only microgram quantities of micronutrients are added to the culture solution so t h a t C P or AR, grade materials are usually satisfactory. Should purification prove to be necessary, as it m a y be for Fe, Zn, and Cu, the following procedures are used. E x t r a c t a standard CuSO4"5H20 solution as a Cu dithizonate at p H 2.8, using 0.01% w / v dithizone in redistilled CC14. Transfer the copper dithizonate and excess dithizone into a 200-ml. beaker, and evaporate the CCI, on an electric hot plate. Add 10 ml. of redistilled nitric acid to digest the residual copper dithizonate, and evaporate to dryness. Add triple-distilled water, boil for 15 minutes, cool, and make TABLE II METAL ASSAY SERIES FOR
Magnesium Iron
C')pper Zinc Manganese Molybdenum
A. niger
Metal, -~/50 ml. basal culture solution
Standard deviation
0, 50, ~ 100, 150, 200, 300, 400,- 500 o, 0.1,- 0.25, 0.5, 1, 2.5, 5,~ 10 o, 0.05.~ 0.10, 0.20, 0.40, 0.6, 0.8,~ 1.0, 1.5, 2, 4 O, 0.25," 0.5, 1, 2, 3, 4, 5," 10 0, 0.01,~ 0.025, 0.05, 0.1, 0.5, 1,~ 5 0, 0.0005,~ 0.001, 0.002, 0.003, 0.004, 0.005, 0.0075,a 0.01
_+25 _+0.1 _+0.05 _+0.25 -+0.01 _+0.005
Effective range for assay, coinciding with the maximum differences in mycelial characters inchlding spore cover and dry weights of mats. to volume. This provides a pure copper nitrate solution free from other trace metals. E x t r a c t a standard solution of ZnSO4.7H20 with 0.01% dithizone in redistilled CC14 at p H 7 in the presence of 1 ml. of 0.1% w / v sodium diethyldithiocarbamate (chelates Pb) per milligram of ZnSO4. FeCI~'6H20 is purified by extraction into redistilled ether in the presence of redistilled HC1 at pH3. The ether is removed by heating on an electric hot plate. The iron salt is taken up and made to volume in triple-distilled water. Preparation of Spore Inoculum. A. niger is readily subcultured on nutrient agar slants by a simple spore transfer from the original culture. This procedure minimizes the risk of mutation. The spores from one slant are carefully collected with a sterile platinum wire to avoid contamination and transferred aseptically to 10 ml. of sterile glass-distilled water. A drop of organic detergent m a y be included to disperse the spores. A. niger is grown at 30 ° for 4 days or at 25 ° for 5 days. Preparation of Standard Series for the Metals. Fifty milliliters of basal culture solution is transferred into 500-ml. Erlenmeyer flasks. The necks
[149]
MICROBIOLOGICAL METHODS FOR METALS
1039
of the flasks are closed with inverted beakers and the annular spaces plugged with nonabsorbent cotton wool. This overcomes the risk of contamination from cotton wool plugs. Trace metal standards are added in solution to the purified cultures. It is preferable to contain the added standards in 1 ml. so as to have consistent volume additions. All A. niger standard cultures are prepared in duplicate and are randomized in the
z
"
.
./::~'"'" / < o / :/. " ..-'7 .;" ~ "
t
ooi- Z:/:'/"
::__:L°
J.b?'] / "
•. . . .
200~I!:'"I
f
0 d
/
0 t
20 ,
Ot 01
,
i
50
~ ~
I 50 I 0.2 2 I00
,
I 0.4
I i00 ,
Mg
0!6
| 0.8
4
6
8
' 2OO
3;0
4;0
I
, ,,
i 200
I 500 x i0 -4 7 Mo
110
2.r5 7 Cu
$
]
10 ~
500
20 ~,Zn or Fe ,
i
I 0 0 0 'y Mg
Weight of metal per 50 ml. of solution
FIG. i incubator according to a statistical pattern. The standard errors for the assays are determined periodically as a routine check on the response of the fungus to the metals. A standard series for a metal is always included when an assay for it is being made on test materials. The effective ranges of assay for Mg, Fe, Cu, Zn, Mn, and Mo are shown in Table II and illustrated in Fig. 1. The growth series may be assessed visually by taking into account the development of mycelia and intensity of sporulation and quantitatively by dry weight yields. The pads are filtered into a Btichner funnel, washed free from the culture solution, and weighed in 100-ml. glass beakers, which are then placed in a well-aerated oven at 90° for 12 hours when constant weight is usually reached. They are then cooled in a desic-
1040
DETERMINATION OF INORGANIC COMPOUNDS
[149]
weighed and redried in the oven for another 3 hours, cooled, and reweighed. Copper has a specific effect on spore color which varies from yellow at a low copper level through brown to black at the higher levels. This color change facilitates the assay for the element. When growth curves become erratic it is usually due to residual chelating reagents in the culture solutions. Assay of Metals in Biological Samples. The microbiological method is particularly useful when only small amounts of tissues from normal or metal-deficient plant, animal, or bacterial sources are available. Under these circumstances the trace metal levels to be determined would be below the limits of chemical detection. Weighed amounts of plant or animal tissues are digested in redistilled nitric acid. To 2 g. of fresh tissue or 0.25 g. of dried tissue is added 20 ml. of redistilled HN03. Digestion is continued for 45 minutes on an electric hot plate in a hood; after cooling, 0.5 ml. of pure 70% w/w HCI04 is added, and digestion is continued until the solution is clear. The solution is evaporated to incipient dryness and, after cooling, 10 ml. of tripledistilled water is added. The solution is boiled for 15 minutes to hydrolyze the lower phosphates. The digest is made to 10 ml. with triple-distilled water, and suitable aliquots in the ratio of 1:2:3 are taken for assay. It is often unnecessary and undesirable to use HCIO4 to digest tissues as HN03 releases the metals in a soluble form suitable for assay from most biological samples. Another suitable procedure is to burn milligram quantities of plant or animal material in a platinum crucible in an electric muffle furnace at about 400 ° . The cooled residue is transferred to the prepared culture solutions for the assays of Mg, Fe, Cu, Zn, h/In, or 1V~o. The use of the A. niger method for determining small amounts of molybdenum in cauliflower leaves is shown in Table III. cator,
TABLE III THE MOLYBDENUMCONTENT OF WATER EXTRACTS AND ASH OF CAULIFLOWERLEAVES
Mo in water extracts • of leaves, 7/g. fresh weight
Mo in ash, 7/g. dry weight
Normal
Mo-deficient
Normal
0 . I to 0 . 2 •D _ 0 . 0 0 2
0 . 0 0 1 to 0 . 0 1 S D _+ 0 . 0 0 1
SD + 0.005
0.5'to 1
Water homogenates of leaves prepared in a glass macerater.
Mo-deficient 0 . 0 3 to 0 . 1 S D _+ 0 . 0 0 2
[78]
A M I N O ACIDS BY GASOMETRIC
NINHYDRIN
METHOD
1041
It is sometimes desirable to determine the trace metal content of protein fractions during enzyme purification. The protein fractions are digested with H N 0 3 before assay, as described previously. A good example of this is a study of the relation between nitrate reductase activity and molybdenum status of fractions of the enzyme from Neurospora crassa and soybean. 7 It was found that the amount of the micronutrient increased with increased specific activity of the enzyme, m Chemical methods would not have detected the Mo changes in the various protein fractions. 7 D. J. D. Nicholas and A. Nason, J. Biol. Chem. 207, 353 (1954) ; see also Vol. II [57]. s D. J. D. Nicholas, A. Nason, and W. D. McElroy, J. Biol. Chem. 207, 341 (1954).
(ADDENDUM)
[75] Gasometric Procedures for A m i n o Acids (See [75],p. 458 for Section I.)
If. Gasometric Ninhydrin Determination of Free Am{no Acids RCH(NHs)COOH
--* R C H O
-b N H 3 -{-COs
Principle. The reaction requires the presence, in an unconjugated state, of both the carboxyl group and a primary or secondary a-amino group.I Glycine, sarcosine,proline,and hydroxyproline yield COs but no aldehyde. The latterthree yield no ammonia. One mole of aspartic acid yields 2 moles of CO2. ~-Alanine yields only 0.16 mole of COs at p H 4.7, however, and none at p H 2.5. One mole of glutamic acid likewise yields but I mole of COs at p H 2.5.N o COs is evolved from amino acids inwhich one of the hydrogens of N H s is replaced by C O R as in peptides and in acetylated or benzoylated amino acids. Except for glutamyl and aspartyl peptides, which have a - - C H ( N H 2 ) C O O H group free (e.g.,as in glutathione), peptides do not give COs, even from their free carboxyl groups. Hence the ninhydrin COs reaction differentiatesamino acids from peptides more sharply than does the above-mentioned nitrous acid method. N o COs is given off ifboth hydrogens of the amide group are substituted, or if the carboxyl group is replaced by an amide or an ester group. Amines, amides, ammonia, and glucosamine give no COs when heated with ninhydrin. Urea gives 0.01 mole of COs in 5 minutes at 100° in the absence of ninhydrin but much less in its presence. At p H 2.5 and for a 7-minute heating period, 1 mole of COs is given per mole of alanine, D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J, Biol. Chem. 141, 627 (1941).
MICROBIOLOGICAL METHODS FOR METALS
1035
the Beckman flame spectrophotometer are given in the instruction manuals which should be consulted before the analysis is planned. Since the line positions of the spectrophotometer shift with changing conditions, these must be checked daily and also whenever the wavelength setting is changed. The 100 % standard solution is used to set the instrument for maximum brightness. It is necessary to standardize the flame spectrophotometer before each run and to check a known point on the standard curve frequently during the determination. After every two or three readings the 50 % standard solution is checked again to detect any change in operating conditions of the instrument. The flame background must be subtracted from all readings; this is determined by means of a distilled water blank and should be checked frequently. This procedure is repeated for each substance being determined, the instrument being reset and the proper solutions used to standardize the spectrophotometer in each case.
[149] Microbiological Methods for Determining Magnesium, Iron~ Copper~ Zinc, Manganese, and Molybdenum By D. J. D. NICHOLAS Microbiological methods for determining trace metals have the advantages of specificity, reproducibility, and sensitivity at low levels of metal over chemical or physical methods of analysis. Disadvantages include the necessity for rigorous pure culture techniques and the frequent use of standard growth curves to check possible mutation of the organisms. This article describes methods for determining Mg, Fe, Cu, Zn, Mn, and Mo in biological materials, using the mold Aspergillus niger (Mulder strain). Other strains of A. niger may also be used for assays of metals. 1,2
Principle A. niger (h/i. strain) requires N, P, S, K, Mg, and the trace metals Fe, Cu, Zn, Mn, and h/io for optimum growth. 1-6 In the absence of one of 1 D. J. D. Nicholas, J. Sci. Food Agri. 1, 339 (1950). D. J. D. Nicholas, Analyst 77, 629 (1952). s E. G. Mulder, Arch. Microbiol. 10, 72 (1939). 4 D. J. D. Nicholas and A. H. Fielding, Ann. Rept. Long Ashton Research Station (Bristol, England) 126 (1947). 5 D. J. D. Nicholas and A. H. Fielding, J. Hort. Sci. 26, 125 (1951). 6 R. A. Steinberg, Botan. Rev. 5, 327 (1939).
1036
DETERMINATION OF INORGANIC COMPOUNDS
[149]
these essential elements the growth of the fungus is retarded, as shown by reduction in dry weight yields and sporulation. Moreover, an increase in an essential element from deficiency to sufficiency levels when all others are present at optimum amounts results in a specific and quantitative increase in growth. In this way a standard growth series is prepared for any of the essential mineral nutrients and assayed by taking dry weight yields of the pads. For the bioassay of test material a known amount of it is added to a culture solution that contains all the essential elements other than the one to be determined. The growth of the fungus under these conditions depends on the amount of test element that it derives from the material added. The growth measured by dry weight yields of the mats is referred to a standard series for the element, grown under the same conditions.
Experimental Methods Water Supply. Distilled water is prepared by being distilled twice from Pyrex glass to reduce the metal content. The minimum metal in micrograms per milliliter (p.p.m.) detected by the bioassay method is Cu, 0.001, Zn 0.005, Fe 0.002, Mn 0.002, Mo 0.00001. Water from a tinnedcopper still is therefore unsuitable for assay of metals other than Mg and Zn. The metal content of water is markedly reduced by distillation either once or twice from Pyrex glass. There is no further reduction by a third distillation. Water of low conductivity prepared from standard ion exchange resins is also suitable for trace metal assays, but the Mo content may be too high for the sensitive A. niger assay for the element. Glassware. Hard-glass 500-ml. Erlenmeyer flasks are thoroughly cleaned by washing several times with hot water and an organic detergent and then with concentrated nitric acid followed by single-distilled and finally with twice-distilled water from Pyrex glass. Alternatively a 0.5 % w / v solution of disodium ethylenediaminetetraacetic acid is used instead of the acid wash, and flasks containing this solution are steamed in an autoclave to remove metals. Glassware and water supply may be checked for metals by shaking with 0.01% w / v solution of dithizone (diphenylthiocarbazone) in redistilled CC14. Metal dithizonates are indicated by a change from green of dithizone to red. Preparation of Culture Solutions Free .from Trace Metals. A suitable culture solution (1 1.) for A. niger is as follows: Macronutrients--dextrose, 50 g., KNOa, 5 g., KH2PO4, 2.5 g., MgSO4"7H20, 1 g., and Ca(NOa)2, 0.5 g.; micronutrients--FeCla'6H20, 20 nag., ZnSO4.7H20, 20 mg., CuSO4"5H20, 1 mg., MnSOt-5H~O, 3 rag., Na~MoO4"2H~O, 1 mg.; glassdistilled water to 1 1.
[149]
MICROBIOLOGICAL METHODS FOR METALS
1037
The pH of the basal culture solution is 3.8; 50 ml. is used for each culture. The macronutrients contain sufficient of the micronutrients to provide for optimum growth of the mold. Methods used to remove them from a solution of macronutrients in 200 ml. of distilled water are summarized in Table I. There is no need for purification when Mg is to be assayed. TABLE I REMOVAL OF TRACE METALS FROM A SOLUTION OF INORGANIC MACRONUTRIENTS AND DEXTROSE
Trace metal removed Copper
Zinc and iron
Molybdenum Manganese
Method used for removal Add 2 ml. 10% w / v copper sulfate to 200 ml. culture solution, adjusted with hydrochloric acid to pH 3, and precipitate as a sulfide with hydrogen sulfide (15 minutes) from a Kipp's apparatus or a tank or cylinder. Let precipitate stand for 15 minutes, and filter through Whatman No. 42 paper into a clean Erlenmeyer flask. Eliminate hydrogen sulfide by boiling and agitating solution for about 20 minutes, and check removal with lead acetate paper. Adjust 200 ml. solution to pH 5.5 with 5% w / v sodium hydroxide, and shake four times with 30-ml. portions of 5% w / v 8-hydroxyquinoline in chloroform in a liter Pyrex glass separating funnel. Extract excess quinolates with three 30-ml. portions of redistiUed chloroform and then with similar quantities of redistilled ether. The latter removes chloroform. Eliminate the ether by boiling the solution on an electric hot plate. Zinc and iron are also removed at pH values above 5.5, but in alkaline solution phosphates are precipitated. The same as the coprecipitation method for the removal of copper. Coprecipitation of manganese and copper diethyldithiocarbamates at pH 5.5. Add 10 ml. 0.1% w / v sodium diethyldithiocarbamate in water and 5 ml. 5% w / v copper sulfate solution. Set aside for 30 minutes, filter or centrifuge precipitate, and adjust filtrate to pH 3 with HC1. Add a further 5 ml. 5% w / v copper sulfate, precipitate copper sulfide with hydrogen sulfide from a cylinder, and proceed as described for copper.
Residual metal after purification, ~/50 ml. 0.05
0.01
0.0005 0.01
1038
DETERMINATION OF INORGANIC COMPOUNDS
[149]
Only microgram quantities of micronutrients are added to the culture solution so t h a t C P or AR, grade materials are usually satisfactory. Should purification prove to be necessary, as it m a y be for Fe, Zn, and Cu, the following procedures are used. E x t r a c t a standard CuSO4"5H20 solution as a Cu dithizonate at p H 2.8, using 0.01% w / v dithizone in redistilled CC14. Transfer the copper dithizonate and excess dithizone into a 200-ml. beaker, and evaporate the CCI, on an electric hot plate. Add 10 ml. of redistilled nitric acid to digest the residual copper dithizonate, and evaporate to dryness. Add triple-distilled water, boil for 15 minutes, cool, and make TABLE II METAL ASSAY SERIES FOR
Magnesium Iron
C')pper Zinc Manganese Molybdenum
A. niger
Metal, -~/50 ml. basal culture solution
Standard deviation
0, 50, ~ 100, 150, 200, 300, 400,- 500 o, 0.1,- 0.25, 0.5, 1, 2.5, 5,~ 10 o, 0.05.~ 0.10, 0.20, 0.40, 0.6, 0.8,~ 1.0, 1.5, 2, 4 O, 0.25," 0.5, 1, 2, 3, 4, 5," 10 0, 0.01,~ 0.025, 0.05, 0.1, 0.5, 1,~ 5 0, 0.0005,~ 0.001, 0.002, 0.003, 0.004, 0.005, 0.0075,a 0.01
_+25 _+0.1 _+0.05 _+0.25 -+0.01 _+0.005
Effective range for assay, coinciding with the maximum differences in mycelial characters inchlding spore cover and dry weights of mats. to volume. This provides a pure copper nitrate solution free from other trace metals. E x t r a c t a standard solution of ZnSO4.7H20 with 0.01% dithizone in redistilled CC14 at p H 7 in the presence of 1 ml. of 0.1% w / v sodium diethyldithiocarbamate (chelates Pb) per milligram of ZnSO4. FeCI~'6H20 is purified by extraction into redistilled ether in the presence of redistilled HC1 at pH3. The ether is removed by heating on an electric hot plate. The iron salt is taken up and made to volume in triple-distilled water. Preparation of Spore Inoculum. A. niger is readily subcultured on nutrient agar slants by a simple spore transfer from the original culture. This procedure minimizes the risk of mutation. The spores from one slant are carefully collected with a sterile platinum wire to avoid contamination and transferred aseptically to 10 ml. of sterile glass-distilled water. A drop of organic detergent m a y be included to disperse the spores. A. niger is grown at 30 ° for 4 days or at 25 ° for 5 days. Preparation of Standard Series for the Metals. Fifty milliliters of basal culture solution is transferred into 500-ml. Erlenmeyer flasks. The necks
[149]
MICROBIOLOGICAL METHODS FOR METALS
1039
of the flasks are closed with inverted beakers and the annular spaces plugged with nonabsorbent cotton wool. This overcomes the risk of contamination from cotton wool plugs. Trace metal standards are added in solution to the purified cultures. It is preferable to contain the added standards in 1 ml. so as to have consistent volume additions. All A. niger standard cultures are prepared in duplicate and are randomized in the
z
"
.
./::~'"'" / < o / :/. " ..-'7 .;" ~ "
t
ooi- Z:/:'/"
::__:L°
J.b?'] / "
•. . . .
200~I!:'"I
f
0 d
/
0 t
20 ,
Ot 01
,
i
50
~ ~
I 50 I 0.2 2 I00
,
I 0.4
I i00 ,
Mg
0!6
| 0.8
4
6
8
' 2OO
3;0
4;0
I
, ,,
i 200
I 500 x i0 -4 7 Mo
110
2.r5 7 Cu
$
]
10 ~
500
20 ~,Zn or Fe ,
i
I 0 0 0 'y Mg
Weight of metal per 50 ml. of solution
FIG. i incubator according to a statistical pattern. The standard errors for the assays are determined periodically as a routine check on the response of the fungus to the metals. A standard series for a metal is always included when an assay for it is being made on test materials. The effective ranges of assay for Mg, Fe, Cu, Zn, Mn, and Mo are shown in Table II and illustrated in Fig. 1. The growth series may be assessed visually by taking into account the development of mycelia and intensity of sporulation and quantitatively by dry weight yields. The pads are filtered into a Btichner funnel, washed free from the culture solution, and weighed in 100-ml. glass beakers, which are then placed in a well-aerated oven at 90° for 12 hours when constant weight is usually reached. They are then cooled in a desic-
1040
DETERMINATION OF INORGANIC COMPOUNDS
[149]
weighed and redried in the oven for another 3 hours, cooled, and reweighed. Copper has a specific effect on spore color which varies from yellow at a low copper level through brown to black at the higher levels. This color change facilitates the assay for the element. When growth curves become erratic it is usually due to residual chelating reagents in the culture solutions. Assay of Metals in Biological Samples. The microbiological method is particularly useful when only small amounts of tissues from normal or metal-deficient plant, animal, or bacterial sources are available. Under these circumstances the trace metal levels to be determined would be below the limits of chemical detection. Weighed amounts of plant or animal tissues are digested in redistilled nitric acid. To 2 g. of fresh tissue or 0.25 g. of dried tissue is added 20 ml. of redistilled HN03. Digestion is continued for 45 minutes on an electric hot plate in a hood; after cooling, 0.5 ml. of pure 70% w/w HCI04 is added, and digestion is continued until the solution is clear. The solution is evaporated to incipient dryness and, after cooling, 10 ml. of tripledistilled water is added. The solution is boiled for 15 minutes to hydrolyze the lower phosphates. The digest is made to 10 ml. with triple-distilled water, and suitable aliquots in the ratio of 1:2:3 are taken for assay. It is often unnecessary and undesirable to use HCIO4 to digest tissues as HN03 releases the metals in a soluble form suitable for assay from most biological samples. Another suitable procedure is to burn milligram quantities of plant or animal material in a platinum crucible in an electric muffle furnace at about 400 ° . The cooled residue is transferred to the prepared culture solutions for the assays of Mg, Fe, Cu, Zn, h/In, or 1V~o. The use of the A. niger method for determining small amounts of molybdenum in cauliflower leaves is shown in Table III. cator,
TABLE III THE MOLYBDENUMCONTENT OF WATER EXTRACTS AND ASH OF CAULIFLOWERLEAVES
Mo in water extracts • of leaves, 7/g. fresh weight
Mo in ash, 7/g. dry weight
Normal
Mo-deficient
Normal
0 . I to 0 . 2 •D _ 0 . 0 0 2
0 . 0 0 1 to 0 . 0 1 S D _+ 0 . 0 0 1
SD + 0.005
0.5'to 1
Water homogenates of leaves prepared in a glass macerater.
Mo-deficient 0 . 0 3 to 0 . 1 S D _+ 0 . 0 0 2
[78]
A M I N O ACIDS BY GASOMETRIC
NINHYDRIN
METHOD
1041
It is sometimes desirable to determine the trace metal content of protein fractions during enzyme purification. The protein fractions are digested with H N 0 3 before assay, as described previously. A good example of this is a study of the relation between nitrate reductase activity and molybdenum status of fractions of the enzyme from Neurospora crassa and soybean. 7 It was found that the amount of the micronutrient increased with increased specific activity of the enzyme, m Chemical methods would not have detected the Mo changes in the various protein fractions. 7 D. J. D. Nicholas and A. Nason, J. Biol. Chem. 207, 353 (1954) ; see also Vol. II [57]. s D. J. D. Nicholas, A. Nason, and W. D. McElroy, J. Biol. Chem. 207, 341 (1954).
(ADDENDUM)
[75] Gasometric Procedures for A m i n o Acids (See [75],p. 458 for Section I.)
If. Gasometric Ninhydrin Determination of Free Am{no Acids RCH(NHs)COOH
--* R C H O
-b N H 3 -{-COs
Principle. The reaction requires the presence, in an unconjugated state, of both the carboxyl group and a primary or secondary a-amino group.I Glycine, sarcosine,proline,and hydroxyproline yield COs but no aldehyde. The latterthree yield no ammonia. One mole of aspartic acid yields 2 moles of CO2. ~-Alanine yields only 0.16 mole of COs at p H 4.7, however, and none at p H 2.5. One mole of glutamic acid likewise yields but I mole of COs at p H 2.5.N o COs is evolved from amino acids inwhich one of the hydrogens of N H s is replaced by C O R as in peptides and in acetylated or benzoylated amino acids. Except for glutamyl and aspartyl peptides, which have a - - C H ( N H 2 ) C O O H group free (e.g.,as in glutathione), peptides do not give COs, even from their free carboxyl groups. Hence the ninhydrin COs reaction differentiatesamino acids from peptides more sharply than does the above-mentioned nitrous acid method. N o COs is given off ifboth hydrogens of the amide group are substituted, or if the carboxyl group is replaced by an amide or an ester group. Amines, amides, ammonia, and glucosamine give no COs when heated with ninhydrin. Urea gives 0.01 mole of COs in 5 minutes at 100° in the absence of ninhydrin but much less in its presence. At p H 2.5 and for a 7-minute heating period, 1 mole of COs is given per mole of alanine, D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J, Biol. Chem. 141, 627 (1941).