Microbiological Process Report

Analytical Microbiology
IV. Gravimetric Methods
J. J. GAVIN'
Food Research Laboratories, Inc., Long Island City, New York
Received for publication July 2, 1957

PRINCIPLE been confirmed by comparison with rat growth and

In these methods, the response of the test organism thiochrome procedures (Hamner et al., 1943).
to graded concentrations of the substance being as- An indication that fungi could be used in analytical
sayed is determined, after a suitable incubation period, procedures for the determination of trace metals was
by measurement of the amount of growth in terms of given by Niklas and Toursel (1941). Nicholas (1952)
dry cell weight. Under the conditions of assay, this has advocated the use of Aspergillus niger and Penicil-
weight is proportional to the concentration of the lium glaucum for the determination of trace metals in
limiting factor. By comparing the response of the test biological materials. He has claimed that his method is
organism to the sample being assayed with that of the better than chemical procedures for determining the
same organism to a known standard preparation, the amount of copper, zinc, molybdenum, and manganese
potency of the sample may be determined. in soils because of its greater sensitivity, higher accu-
racy at low concentrations of metal, and close agreement
TYPES OF GRAVIMETRIC METHODS of the results with crop performances.
All gravimetric methods can be placed in one cate- In a majority of the methods in this group, mutants
gory. As fungi have been used almost exclusively in of Neurospora are used as test organisms. These have
these methods for the assay of growth factors, the pro- been used to assay pyridoxin (Stokes et al., 1943a;
cedure followed in each case is similar. Graded con- Bonner and Dorland, 1943); choline (Horowitz and
centrations of the sample being assayed are added to a Beadle, 1943; Luecke and Pearson, 1944a, b; Hodson,
series of flasks containing liquid nutrient medium. A 1945); inositol (Beadle, 1944); p-aminobenzoic acid
standard series is prepared in the same manner. After (Agarwala and Peterson, 1950); biotin (Hodson, 1945;
sterilization, growth from a conidial inoculum, either Tatum et al., 1946); lysine (Doermann, 1945); leucine
dry on an inoculating needle or suspended in saline, is (Regnery, 1944; Ryan and Brand, 1944); adenine
introduced into each flask of the series. Following incu- (Mitchell and Houlahan, 1946); cytidine and uridine
bation at some predetermined temperature for a given (Loring et al., 1948).
period of time, the mycel al crop is harvested and dried It should be noted that, while gravimetric methods
to a constant weight. The values for the mycelial have been confined to the assay of growth factors and
weights from flasks of the standard series are plotted to fungi as test organisms, they could be adapted for
against the respective dose concentrations. Interpola- substances other than growth factors and for different
tion of the mycelial weights for the sample series on this test organisms. There has been little interest in devel-
curve permits calculation of the concentration of the oping gravimetric methods using bacteria or yeasts
assayed factor in the sample. because other existing methods give excellent results
Assay methods have been reported for a variety of in a shorter period of time, with fewer manipulations.
growth factors. In the first application of a quantitative However, they might have usefulness in the assay of
microbiological assay procedure to vitamin determina- inherently colored products.
tion, Schopfer (1935a, b) described a method for the MEASUREMENT OF RESPONSE
assay of thiamine using Phycomyces blakesleeanus as the
test organism. Several investigators (Meiklejohn, 1937; The determination of amount of dry cellular material
Sinclair, 1938; Bonner and Erickson, 1938; Burkholder formed in each flask involves three steps, namely,
and McVeigh, 1940) modified and applied this method harvesting, drying, and weighing; none of which present
vith some success. The accuracy of the method has any particular problems. The mats which are formed
1 Present address: Department of Research Therapeutics, may be harvested in a single operation using a stiff wire
Norristown State Hospital, Norristown, Pennsylvaniia. needle, taking care to wipe up any pieces of mycelium
80
19581 ANALYTICAL MICROBIOLOGY. IV 81
that might adhere to the sides of the flask. Sporulation Substance to be Assayed
which interferes with harvesting may be repressed For a gravimetric procedure to be applicable for the
either by the inclusion of zinc sulfate in the basal me- assay of a particular factor, that factor must have cer-
dium (Stokes et al., 1943a) or by swirling the flasks tain properties. These are: (1) The ability to affect the
twice daily to promote submerged growth (Ryan, growth of some fungus in a manner which is reflected
1950). After harvesting, the growth is pressed dry be- by changes in the dry weight of the mycelium formed
tween filter paper or paper towels, rolled into a pellet, by the organism. (2) Solubility in water, or some solvent
and dried to a constant weight. It can be conveniently miscible with water which will not interfere with the
handled during the drying and weighing on porcelain growth of the organism at the concentration used. (3)
spot plates. It should not cause flocculation or precipitation in the
Modifications of this procedure have been proposed. culture medium that will affect the measurement of the
The contents of each flask may be collected on filter weight of the mycelium (this is particularly true when
paper in a Buchner funnel (Tatum et al., 1946) or a filtration techniques are used to harvest the mycelium).
Gooch crucible (Barton-Wright, 1953). The disad-
vantage in the use of paper is that the mycelium must Culture Media and Assay Solutions
be removed from it before drying and weighing. The major problems in gravimetric assay procedures
A simple and convenient alternative has been recom- are related to the technique of incorporating the sample
mended by Ryan and Brand (1944), and Siegel (1945). solutions into the culture medium. The basal media
The flask contents are filtered through tared fritted used in these procedures are relatively simple in com-
glass crucibles, washed with water, and dried to a con- position. Most of the test organisms will grow well on
stant weight directly in the crucible. In this manner, a medium containing a source of carbon, inorganic
rapid processing of a series of flasks is possible without nitrogen, several inorganic salts, and the factor re-
handling of the individual pellets, resulting in improved quired for growth.3 Although growth on such a medium
precision of the measurement (approximately t2 per is satisfactory for the analysis of pure compounds, it is
cent). not optimal and may be affected by a variety of sub-
The conditions recommended for drying the myce- stances.
lium have varied from laboratory to laboratory. For The nitrogen metabolism of the test organisms may
example, while most investigators used temperatures be a source of difficulty in gravimetric assays. For
from 80 to 100 C for 2 to 4 hr, conditions have ranged example, the response of Phycomyces blakesleeanus to
from vacuum drying over anhydrous calcium chloride thiamin will vary with both the concentration and
for 18 hr2 (Ryan and Brand, 1944) to drying at 109 C source of nitrogen (Meiklejohn, 1937; Sinclair, 1938;
for 12 to 16 hr (Agarwala and Peterson, 1950). The Burkholder and McVeigh, 1940) (table 1). Further, if
specific conditions used for any given assay are rela- a single source of nitrogen, such as asparagine, is uti-
tively unimportant if constant weight, an essential lized in the basal medium at its apparent optimum
requirement for any quantitative gravimetric proce- concentration, nitrogenous constituents of complex
dure, is attained.
biological samples may stimulate additional growth of
this organism (Sinclair, 1938). It may be advisable to
The mats may be weighed on any suitable analytical develop modifications in the medium (or include suit-
balance. The sensitivity of the measurements will, of
3 In addition, biotin must be added to the basal media
course, be dependent upon the characteristics of the
used in assays employing Neurospora mutants.
particular balance used.
TABLE 1
FACTORS WHICH INFLUENCE GRAVIMETRIC METHODS Comparison of the effect of different sources of nitrogen
on the growth of Phycomyces*
The factors which influence gravimetric methods
Source and Per Cent of Nitrogent
are, as with other types of methods, many and varied.
Certain of these are applicable to the use of fungi as Expt. a
As-
para- Glycine Glutamine
test organisms. Others, however, are peculiar to the use ~g in

of Neurospora mutants. As it is unlikely that this pro- > .04 0.2 0.5 0.75 0.2 0.4 0.6 0.8 1.0 1.2
cedure will be applied to bacterial or yeast analysis, jAg
reference to these organisms will be omitted in the fol- 1 0.1 45.046.050.753.843.8 53.2 56.2 - - -
lowing discussion. 0.3 87.4 81.1 86.2 90.5 66.4 110.0 115.7- -
2 0.5 101.0 128.2 136.6 175.0 -
2When this procedure is used, it is recommended that the 3 0.5 101.41 30.61136.8 - 160.0
mycelial pellets be placed on aluminum foil or wax paper as
they will adhere to glass or porcelain surfaces under the stated * Sinclair, 1938.
conditions. t Dry weight of fungus in mg.
82 J. J. GAVIN [VOL. 6
able "blanks") to adapt these procedures to specific will interfere with the assay if present in excess of 4
problems. ,ug per ml of culture medium (Horowitz and Beadle,
The addition of amino acids to the basal medium 1943).
may cause a variety of effects. To avoid stimulation by Certain other compounds will alter the response of
amino acids which might be present in samples, Agar- these test organisms. The ratio of sucrose to leucine in
wala and Peterson (1950) added casein hydrolysate the assay medium directly influences the amount of
and asparagine to the assay medium used for the deter- growth of the leucineless mutant of Neurospora
mination of p-aminobenzoic acid with Neurospora (Regnery, 1944). If thiamin is added to the basal
crassa. Doermann (1945) reported both a sparing effect medium for the pyridoxin assay, the test organism,
and inhibition by amino acids in the lysine assay with Neurospora sitophila, will grow better at limiting or
a mutant of the same organism, as arginine acted as a suboptimal concentrations of vitamin B6, thus increas-
specific inhibitor while asparagine and glutamic acid ing the sensitivity of this assay (Tatum et al., 1946;
stimulated growth. Strauss (1951) found that methio- Harris, 1952; Hodson, 1956).
nine will inhibit the pyridoxinless mutants under certain Natural substances, such as yeast or liver extracts,
conditions. This inhibition may be reversed, however, tissue extracts and so forth, may cause either inhibition
by the inclusion of threonine in the basal medium or stimulation (Mitchell and Houlahan, 1946; Mitchell,
(table 2). Table 3 illustrates the diversity of antagonis- 1950). The inhibition may be due to amino acid antag-
tic amino acid relations in Neurospora mutants. onisms as mentioned above while the stimulation may
Substitution of amino nitrogen compounds for growth be attributed to nonspecific sparing effects by sub-
factors may occur. Dimethylaminoethanol has the stances in the extracts.
same activity as choline for the cholineless mutant of The pH of the medium is an important consideration.
Neurospora crassa (Jukes and Dornbush, 1945). Me- The activity of p-aminobenzoic acid is dependent upon
thionine has partial activity for this same organism and pH (Wyss et al., 1944). It is most active at low pH's
and decreases in activity as the pH increases towards
TABLE 2 neutrality. Growth of mutants in the absence of their
Methionine inhibition of Neurospora crassa 44602 and its reversal required factor under defined conditions of pH in the
by threonine* presence of certain nitrogen compounds has been noted
DL-Threonine (mg) for the pyridoxinless mutant of Neurospora sitophila
Methionine
0 1 2 3 4 5
(Stokes et al., 1943a, b) and the arginineless mutant of
Neurospora crassa (Srb and Horowitz, 1944). Samples
mg
should be adjusted to the pH of the basal medium be-
0 13.9t 15.5 14.4 15.3 15.5 15.1
0.4 fore their addition to avoid complications that may
D-Methionine 7.2 7.3 8.2 10.8 13.5 14.7 result from changes in the hydrogen ion content of the
L-Methionine 6.1 5.6 11.2 15.3 17.7 18.5 culture medium.
0.8 From the examples cited above, it can be seen that
D-Methionine 6.3 7.0 9.5 13.2 13.5 15.1 while the nutritional requirements of the test organisms
L-Methionine 3.9 3.1 5.5 9.2 15.6 11.8
1.0 used in gravimetric assay procedures may be "simple,"
D-Methionine 7.2 8.0 10.6 11.9 13.9 15.6 components of the test solutions other than the factor
L-Methionine 4.2 4.0 6.6 8.2 11.6 11.2 under assay may significantly alter the environmental
conditions and cause deviation from the expected re-
*
Strauss, 1951.
t Recorded dry weight (average of 2) of mycelium produced sponse. Some of the problems may be resolved by
in 72 hr on 20 ml M/15 phosphate medium, pH 7.0, containing adequate pretreatment of the samples. Dilution, ex-
4 mg (NH4)2SO4 nitrogen per 20 ml. traction, and/or purification procedures can be em-
ployed and are helpful in minimizing the undesirable
TABLE 3 influences of entraneous materials, resulting in im-
Some amino acid antagonisms in Neurospora* proved accuracy by this method.
Growth Factor Growth Inhibitor
Incubation
Isoleucine + valine Excess of either The role of temperature is of less importance in gravi-
Lysine Arginine metric procedures than in the methods previously
D-Alpha-aminoadipic acid Arginine, asparagine, glutamic discussed (Gavin, 1957a, b). Here, emphasis is placed
acid
Glycine or serine Asparagine on the selection of the temperature of incubation instead
Methionine or threonine Excess methioniiie of on the control of temperature fluctuations during the
None (reversed by arginine) Canavanine incubation period. Two reasons may be advanced for
Histidine "Complete" medium this difference. The top of the temperature growth
Adenine Indole curve for molds appears to be a plateau rather than a
*
Tatum, 1949. peak, as is the case with most bacteria. This allows
1958] ANALYTICAL MICROBIOLOGY. IV 83
greater latitude for variations in the incubation tem- shape should minimize any variations due to differences
perature. In addition, total growth is measured and in oxygen availability, consideration should be given
minor fluctuations in environmental conditions do not to the selection of the flask size, itself.
affect the over-all amount of mycelial mass formed. During the growth of the test organism, the mycelial
The selected temperature should be close to the opti- mat spreads over the surface of the nutrient medium.
mum for mycelial development by the test organism This causes some modification of the gas exchange rate
as the amount of mycelium formed will vary with between the atmosphere and the medium. The change
major temperature changes. While it is not critical that in rate will not be proportional at all assay levels due to
the temperature be optimal, for at any given tempera- the differences in the amount of surface area occupied
ture the growth will be proportional, it is preferable to by the developing mycelium. The degree to which it is
adjust conditions so that a good growth response is disproportionate is inversely related to the flask size.
obtained. Thus, when small culture vessels are employed, the top
A specific example of improvement in an assay pro- portion of the growth curve tends to level off, limited
cedure by proper temperature selection can be seen in not by the concentration of substance being assayed
the leucine assay of Ryan and Brand (1944). They found but by the concentration of available oxygen. The use
that their assay was complicated by adaption, that is, of larger flasks will correct this condition and reduce
partial or complete ability of the organism to grow variation between replicates.
without the test component. Complete adaption results
in growth that approaches that of the wild type Neuro- ACCURACY OF RESULTS
spora while growth of cultures showing partial adaption
will exceed that observed normally at given concen- Both the precision and the accuracy of gravimetric
trations (table 4). assay procedures are comparable to other microbial
These adaptions have been attributed to back muta- methods of analysis. Replicate values for the same
tion of the leucineless nuclei to the wild type parent sample solution generally agree within 10 per cent,
organism (Ryan, 1946). The frequency of this occur- while recoveries usually range from 90 to 110 per cent
rence is correlated with temperature as is the frequency of theoretical. Most results obtained by this procedure
of mutation. When the incubation temperature in this have been in close agreement with those obtained for
assay was changed from 30 to 25 C, the percentage of similar substances by chemical, physical, and other
cultures which adapted dropped from 14 to 3 per cent types of microbiological methods.
(Ryan, 1946). The procedure is simple, and minor variations in
This particular condition is easily recognized and the technique may not have a great influence on the ac-
aberrant mycelial weights should be omitted from the curacy since the extended incubation period makes the
final calculations. Such complications are not desirable method independent of many factors which affect
in quantitative analysis and should be eliminated when other microbiological methods. Any such advantages
possible. may be over-balanced by the sacrifice of time which is
The precision of these assays is correlated with the necessary for maximum precision and accuracy. Thus,
length of the incubation period. In general, more pre- the gravimetric procedures are most suitable for the
cise assays are obtained with long incubation periods.
A period of 3 to 5 days seems adequate for most of the TABLE 4
vitamin assays using Neurospora as the test organism, Complete and partial adaption of leucineless Neurospora*
while, with Phycomyces, more uniform results are
Mg Mycelium on Different Amounts of Leucine
obtained in the assay of thiamin if a 2-week period is Flask No.
used (Hammer et al., 1943). With amino acid assays, 0.25 mg 0.50 mg 1.00 mg
incubation periods of 7 to 8 days appear to give the 1 9.8t 17.5 32.7
best results. The improved precision is a function of 2 7.3 17.4 32.1
total growth since extension of the growth period per- 3 7.3 44.8t 32.9
mits adequate time for compensation by the test or- 4 7.3 19.0t 31.2
5 16.2t 17.8 33.2
ganism for any unequal environmental conditions 6 20.8t. 17.2 33.5
between the flasks of a series. Consequently, when a 7 7.3 17.5 34.6
long incubation period is used, the only limiting factor 8 7.1 18.0 32.6
is the one being assayed. 9 8.0 20. lt 35.4
10 50.3t 17.3 35.1
Oxygen Relationship
Because the majority of the fungi are strict aerobes, Avg wt 7.4 17.5 33.3
the total amount of growth, at any given assay level, *
Ryan, 1946.
will vary with the amount of oxygen available to each t Partial adaption.
culture. While the use of flasks of the same size and t Complete adaption.
84 J. J. GAVIN [VOL. 6
assay of certain vitamins for which other types of mine in green plants with the Phycomyes assay method.
analytical methods may not be applicable. Am. J. Botany, 27, 853-861.
DOERMANN, A. H. 1945 A bioassay for lysine by use of a
ADVANTAGES AND DISADVANTAGES OF THE mutant of Neurospora. J. Biol. Chem., 160, 95-103.
GRAVIMETRIC ASSAY PROCEDURE GAVIN, J. J. 1957a Analytical microbiology. II. The diffusion
methods. AppI. Microbiol., 5, 25-33.
The advantages of the gravimetric assay procedures GAVIN, J. J. 1957b Analytical microbiology. III. The tur-
are the following: bidimetric methods. Appl. Microbiol., 5, 235-243.
HAMMER, K. C., STEWART, W. S., AND MATRONE, G. 1943
1. The methods are simple and inexpensive. They Thiamine determination by the fungus-growth method
do not require any special or elaborate equipment. and its comparison with other methods. Food Research,
2. The methods are reliable and results are compa- 8, 444-452.
rable with those obtained by other methods of analysis. HARRIS, D. L. 1952 Interaction of thiamine and pyridoxine
3. The methods are precise. in Neurospora. I. Studies of the pyridoxinless mutants.
Arch. Biochem., 41, 294-304.
4. The methods can be used to assay highly colored HODSON, A. Z. 1945 The use of Neurospora for the determi-
solutions. nation of choline and biotin in milk products. J. Biol.
5. In most cases, the age and the size of the inoculum Chem., 157, 383-385.
need not be controlled as growth response is primarily HODSON, A. Z. 1956 Vitamin B6 in sterilized milk and other
a function of the amount of limiting factor and not its milk products. Agr. & Food Chem., 4, 876-881.
HOROWITZ, N. H. AND BEADLE, G. W. 1943 A microbiological
concentration. method for the determination of choline by the use of a
6. The methods in which Neurospora mutants are mutant of Neurospora. J. Biol. Chem., 150, 325-333.
used as the test organisms are specific. JUKES, T. H. AND DORNBUSH, A. C. 1945 Growth stimula-
The disadvantages of the gravimetric assay proce- tion of Neurospora cholineless mutant by dimethylamino-
dures are the following: ethanol. Proc. Soc. Exp. Biol. Med., 58, 142-143.
LORING, H. S., ORDWAY, G. L., AND PIERCE, J. G. 1948 A
1. The requirements for nitrogen metabolism by the method of assay for cytidine and uridine by means of a
test organisms are complex and may be affected by a pyrimidine-deficient strain of Neurospora. J. Biol. Chem.,
variety of nonspecific nitrogenous substances. 176, 1123-1130.
2. The Neurospora mutants have inherent disadvan- LUECKE, R. W. AND PEARSON, P. B. 1944a The microbio-
tages: (a) a separate strain is required for each logical determination of free choline in plasma and urine
J. Biol. Chem., 153, 259-263.
particular procedure, thus requiring maintenance of a LUECKE, R. W. AND PEARSON, P. B. 1944b The determin-
large number of test organisms; and (b) occasional tion of free choline in animal tissues. J. Biol. Chem.,
major changes in metabolism may occur as a result of a 155, 507-512.
single gene mutation with the resultant possibility of MEIKLEJOHN, A. P. 1937 Estimation of vitamin B, in blood
adaption. by modification of Schopfer's test. Biochem. J., 31,
1441-1451.
3. The methods are time consuming. MITCHELL, H. K. 1950 Vitamins and metabolism in Neuro-
4. The methods are not adaptable for large numbers spora. In Vitamins and Hormones, Vol. 8, pp. 127-150.
of samples. Academic Press, Inc., New York, New York.
5. Flasks require considerably more incubation space MITCHELL, H. K. AND HOULAHAN, M. B. 1946 Adenine re-
than tubes. quiring mutants of Neurospora crassa. Federation Proc.,
5, 370-375.
6. The methods have limited application. For routine NICHOLAS, D. J. D. 1952 The use of fungi for determining
analysis, they are employed only in the assay of a few trace metals in biological materials. Analyst, 77, 629-642.
vitamins for which other methods are not available. NIKLAS, H. AND TOURSEL, D. 1941 The determination of
trace elements by means of A spergillus niger. Boden-
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