Microbiological Process ReportAnalytical Microbiology

IV. Gravimetric Methods

J. J. GAVIN'

Food Research Laboratories, Inc., Long Island City, New York

Received for publication July 2, 1957

PRINCIPLEIn these methods, the response of the test organism

to graded concentrations of the substance being as-sayed is determined, after a suitable incubation period,by measurement of the amount of growth in terms ofdry cell weight. Under the conditions of assay, thisweight is proportional to the concentration of thelimiting factor. By comparing the response of the testorganism to the sample being assayed with that of thesame organism to a known standard preparation, thepotency of the sample may be determined.

TYPES OF GRAVIMETRIC METHODSAll gravimetric methods can be placed in one cate-

gory. As fungi have been used almost exclusively inthese methods for the assay of growth factors, the pro-cedure followed in each case is similar. Graded con-centrations of the sample being assayed are added to aseries of flasks containing liquid nutrient medium. Astandard series is prepared in the same manner. Aftersterilization, growth from a conidial inoculum, eitherdry on an inoculating needle or suspended in saline, isintroduced into each flask of the series. Following incu-bation at some predetermined temperature for a givenperiod of time, the mycel al crop is harvested and driedto a constant weight. The values for the mycelialweights from flasks of the standard series are plottedagainst the respective dose concentrations. Interpola-tion of the mycelial weights for the sample series on thiscurve permits calculation of the concentration of theassayed factor in the sample.Assay methods have been reported for a variety of

growth factors. In the first application of a quantitativemicrobiological assay procedure to vitamin determina-tion, Schopfer (1935a, b) described a method for theassay of thiamine using Phycomyces blakesleeanus as thetest organism. Several investigators (Meiklejohn, 1937;Sinclair, 1938; Bonner and Erickson, 1938; Burkholderand McVeigh, 1940) modified and applied this methodvith some success. The accuracy of the method has

1 Present address: Department of Research Therapeutics,Norristown State Hospital, Norristown, Pennsylvaniia.

been confirmed by comparison with rat growth andthiochrome procedures (Hamner et al., 1943).An indication that fungi could be used in analytical

procedures for the determination of trace metals wasgiven by Niklas and Toursel (1941). Nicholas (1952)has advocated the use of Aspergillus niger and Penicil-lium glaucum for the determination of trace metals inbiological materials. He has claimed that his method isbetter than chemical procedures for determining theamount of copper, zinc, molybdenum, and manganesein soils because of its greater sensitivity, higher accu-racy at low concentrations of metal, and close agreementof the results with crop performances.

In a majority of the methods in this group, mutantsof Neurospora are used as test organisms. These havebeen used to assay pyridoxin (Stokes et al., 1943a;Bonner and Dorland, 1943); choline (Horowitz andBeadle, 1943; Luecke and Pearson, 1944a, b; Hodson,1945); inositol (Beadle, 1944); p-aminobenzoic acid(Agarwala and Peterson, 1950); biotin (Hodson, 1945;Tatum et al., 1946); lysine (Doermann, 1945); leucine(Regnery, 1944; Ryan and Brand, 1944); adenine(Mitchell and Houlahan, 1946); cytidine and uridine(Loring et al., 1948).

It should be noted that, while gravimetric methodshave been confined to the assay of growth factors andto fungi as test organisms, they could be adapted forsubstances other than growth factors and for differenttest organisms. There has been little interest in devel-oping gravimetric methods using bacteria or yeastsbecause other existing methods give excellent resultsin a shorter period of time, with fewer manipulations.However, they might have usefulness in the assay ofinherently colored products.

MEASUREMENT OF RESPONSE

The determination of amount of dry cellular materialformed in each flask involves three steps, namely,harvesting, drying, and weighing; none of which presentany particular problems. The mats which are formedmay be harvested in a single operation using a stiff wireneedle, taking care to wipe up any pieces of mycelium

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ANALYTICAL MICROBIOLOGY. IV

that might adhere to the sides of the flask. Sporulationwhich interferes with harvesting may be repressedeither by the inclusion of zinc sulfate in the basal me-

dium (Stokes et al., 1943a) or by swirling the flaskstwice daily to promote submerged growth (Ryan,1950). After harvesting, the growth is pressed dry be-tween filter paper or paper towels, rolled into a pellet,and dried to a constant weight. It can be convenientlyhandled during the drying and weighing on porcelainspot plates.

Modifications of this procedure have been proposed.The contents of each flask may be collected on filterpaper in a Buchner funnel (Tatum et al., 1946) or a

Gooch crucible (Barton-Wright, 1953). The disad-vantage in the use of paper is that the mycelium mustbe removed from it before drying and weighing.A simple and convenient alternative has been recom-

mended by Ryan and Brand (1944), and Siegel (1945).The flask contents are filtered through tared frittedglass crucibles, washed with water, and dried to a con-

stant weight directly in the crucible. In this manner,

rapid processing of a series of flasks is possible withouthandling of the individual pellets, resulting in improvedprecision of the measurement (approximately t2 per

cent).The conditions recommended for drying the myce-

lium have varied from laboratory to laboratory. Forexample, while most investigators used temperaturesfrom 80 to 100 C for 2 to 4 hr, conditions have rangedfrom vacuum drying over anhydrous calcium chloridefor 18 hr2 (Ryan and Brand, 1944) to drying at 109 Cfor 12 to 16 hr (Agarwala and Peterson, 1950). Thespecific conditions used for any given assay are rela-tively unimportant if constant weight, an essentialrequirement for any quantitative gravimetric proce-

dure, is attained.The mats may be weighed on any suitable analytical

balance. The sensitivity of the measurements will, ofcourse, be dependent upon the characteristics of theparticular balance used.

FACTORS WHICH INFLUENCE GRAVIMETRIC METHODS

The factors which influence gravimetric methodsare, as with other types of methods, many and varied.Certain of these are applicable to the use of fungi as

test organisms. Others, however, are peculiar to the use

of Neurospora mutants. As it is unlikely that this pro-

cedure will be applied to bacterial or yeast analysis,reference to these organisms will be omitted in the fol-lowing discussion.

2 When this procedure is used, it is recommended that themycelial pellets be placed on aluminum foil or wax paper as

they will adhere to glass or porcelain surfaces under the statedconditions.

Substance to be AssayedFor a gravimetric procedure to be applicable for the

assay of a particular factor, that factor must have cer-tain properties. These are: (1) The ability to affect thegrowth of some fungus in a manner which is reflectedby changes in the dry weight of the mycelium formedby the organism. (2) Solubility in water, or some solventmiscible with water which will not interfere with thegrowth of the organism at the concentration used. (3)It should not cause flocculation or precipitation in theculture medium that will affect the measurement of theweight of the mycelium (this is particularly true whenfiltration techniques are used to harvest the mycelium).

Culture Media and Assay SolutionsThe major problems in gravimetric assay procedures

are related to the technique of incorporating the samplesolutions into the culture medium. The basal mediaused in these procedures are relatively simple in com-position. Most of the test organisms will grow well ona medium containing a source of carbon, inorganicnitrogen, several inorganic salts, and the factor re-quired for growth.3 Although growth on such a mediumis satisfactory for the analysis of pure compounds, it isnot optimal and may be affected by a variety of sub-stances.The nitrogen metabolism of the test organisms may

be a source of difficulty in gravimetric assays. Forexample, the response of Phycomyces blakesleeanus tothiamin will vary with both the concentration andsource of nitrogen (Meiklejohn, 1937; Sinclair, 1938;Burkholder and McVeigh, 1940) (table 1). Further, ifa single source of nitrogen, such as asparagine, is uti-lized in the basal medium at its apparent optimumconcentration, nitrogenous constituents of complexbiological samples may stimulate additional growth ofthis organism (Sinclair, 1938). It may be advisable todevelop modifications in the medium (or include suit-

3 In addition, biotin must be added to the basal mediaused in assays employing Neurospora mutants.

TABLE 1Comparison of the effect of different sources of nitrogen

on the growth of Phycomyces*

Source and Per Cent of Nitrogent

As-Expt.a para- Glycine Glutamine

~g in

> .04 0.2 0.5 0.75 0.2 0.4 0.6 0.8 1.0 1.2

jAg

1 0.1 45.046.050.753.843.8 53.2 56.2 - - -

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 -

3 0.5101.41 30.61136.8 - 160.0

* Sinclair, 1938.t Dry weight of fungus in mg.

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able "blanks") to adapt these procedures to specificproblems.The addition of amino acids to the basal medium

may cause a variety of effects. To avoid stimulation byamino acids which might be present in samples, Agar-wala and Peterson (1950) added casein hydrolysateand asparagine to the assay medium used for the deter-mination of p-aminobenzoic acid with Neurosporacrassa. Doermann (1945) reported both a sparing effectand inhibition by amino acids in the lysine assay witha mutant of the same organism, as arginine acted as aspecific inhibitor while asparagine and glutamic acidstimulated growth. Strauss (1951) found that methio-nine will inhibit the pyridoxinless mutants under certainconditions. This inhibition may be reversed, however,by the inclusion of threonine in the basal medium(table 2). Table 3 illustrates the diversity of antagonis-tic amino acid relations in Neurospora mutants.

Substitution of amino nitrogen compounds for growthfactors may occur. Dimethylaminoethanol has thesame activity as choline for the cholineless mutant ofNeurospora crassa (Jukes and Dornbush, 1945). Me-thionine has partial activity for this same organism and

TABLE 2Methionine inhibition of Neurospora crassa 44602 and its reversal

by threonine*

DL-Threonine (mg)Methionine

0 1 2 3 4 5

mg

0 13.9t 15.5 14.4 15.3 15.5 15.10.4

D-Methionine 7.2 7.3 8.2 10.8 13.5 14.7L-Methionine 6.1 5.6 11.2 15.3 17.7 18.5

0.8D-Methionine 6.3 7.0 9.5 13.2 13.5 15.1L-Methionine 3.9 3.1 5.5 9.2 15.6 11.8

1.0D-Methionine 7.2 8.0 10.6 11.9 13.9 15.6L-Methionine 4.2 4.0 6.6 8.2 11.6 11.2

* Strauss, 1951.t Recorded dry weight (average of 2) of mycelium produced

in 72 hr on 20 ml M/15 phosphate medium, pH 7.0, containing4 mg (NH4)2SO4 nitrogen per 20 ml.

TABLE 3Some amino acid antagonisms in Neurospora*

Growth Factor Growth Inhibitor

Isoleucine + valine Excess of eitherLysine ArginineD-Alpha-aminoadipic acid Arginine, asparagine, glutamic

acidGlycine or serine AsparagineMethionine or threonine Excess methioniiieNone (reversed by arginine) CanavanineHistidine "Complete" mediumAdenine Indole

will interfere with the assay if present in excess of 4,ug per ml of culture medium (Horowitz and Beadle,1943).

Certain other compounds will alter the response ofthese test organisms. The ratio of sucrose to leucine inthe assay medium directly influences the amount ofgrowth of the leucineless mutant of Neurospora(Regnery, 1944). If thiamin is added to the basalmedium for the pyridoxin assay, the test organism,Neurospora sitophila, will grow better at limiting orsuboptimal concentrations of vitamin B6, thus increas-ing the sensitivity of this assay (Tatum et al., 1946;Harris, 1952; Hodson, 1956).

Natural substances, such as yeast or liver extracts,tissue extracts and so forth, may cause either inhibitionor stimulation (Mitchell and Houlahan, 1946; Mitchell,1950). The inhibition may be due to amino acid antag-onisms as mentioned above while the stimulation maybe attributed to nonspecific sparing effects by sub-stances in the extracts.The pH of the medium is an important consideration.

The activity of p-aminobenzoic acid is dependent uponpH (Wyss et al., 1944). It is most active at low pH'sand decreases in activity as the pH increases towardsneutrality. Growth of mutants in the absence of theirrequired factor under defined conditions of pH in thepresence of certain nitrogen compounds has been notedfor the pyridoxinless mutant of Neurospora sitophila(Stokes et al., 1943a, b) and the arginineless mutant ofNeurospora crassa (Srb and Horowitz, 1944). Samplesshould be adjusted to the pH of the basal medium be-fore their addition to avoid complications that mayresult from changes in the hydrogen ion content of theculture medium.From the examples cited above, it can be seen that

while the nutritional requirements of the test organismsused in gravimetric assay procedures may be "simple,"components of the test solutions other than the factorunder assay may significantly alter the environmentalconditions and cause deviation from the expected re-sponse. Some of the problems may be resolved byadequate pretreatment of the samples. Dilution, ex-traction, and/or purification procedures can be em-ployed and are helpful in minimizing the undesirableinfluences of entraneous materials, resulting in im-proved accuracy by this method.

IncubationThe role of temperature is of less importance in gravi-

metric procedures than in the methods previouslydiscussed (Gavin, 1957a, b). Here, emphasis is placedon the selection of the temperature of incubation insteadof on the control of temperature fluctuations during theincubation period. Two reasons may be advanced forthis difference. The top of the temperature growthcurve for molds appears to be a plateau rather than apeak, as is the case with most bacteria. This allows* Tatum, 1949.

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ANALYTICAL MICROBIOLOGY. IV

greater latitude for variations in the incubation tem-perature. In addition, total growth is measured andminor fluctuations in environmental conditions do notaffect the over-all amount of mycelial mass formed.The selected temperature should be close to the opti-

mum for mycelial development by the test organismas the amount of mycelium formed will vary withmajor temperature changes. While it is not critical thatthe temperature be optimal, for at any given tempera-ture the growth will be proportional, it is preferable toadjust conditions so that a good growth response isobtained.A specific example of improvement in an assay pro-

cedure by proper temperature selection can be seen inthe leucine assay of Ryan and Brand (1944). They foundthat their assay was complicated by adaption, that is,partial or complete ability of the organism to growwithout the test component. Complete adaption resultsin growth that approaches that of the wild type Neuro-spora while growth of cultures showing partial adaptionwill exceed that observed normally at given concen-trations (table 4).These adaptions have been attributed to back muta-

tion of the leucineless nuclei to the wild type parentorganism (Ryan, 1946). The frequency of this occur-rence is correlated with temperature as is the frequencyof mutation. When the incubation temperature in thisassay was changed from 30 to 25 C, the percentage ofcultures which adapted dropped from 14 to 3 per cent(Ryan, 1946).This particular condition is easily recognized and the

aberrant mycelial weights should be omitted from thefinal calculations. Such complications are not desirablein quantitative analysis and should be eliminated whenpossible.The precision of these assays is correlated with the

length of the incubation period. In general, more pre-cise assays are obtained with long incubation periods.A period of 3 to 5 days seems adequate for most of thevitamin assays using Neurospora as the test organism,while, with Phycomyces, more uniform results areobtained in the assay of thiamin if a 2-week period isused (Hammer et al., 1943). With amino acid assays,incubation periods of 7 to 8 days appear to give thebest results. The improved precision is a function oftotal growth since extension of the growth period per-mits adequate time for compensation by the test or-ganism for any unequal environmental conditionsbetween the flasks of a series. Consequently, when along incubation period is used, the only limiting factoris the one being assayed.

Oxygen RelationshipBecause the majority of the fungi are strict aerobes,

the total amount of growth, at any given assay level,will vary with the amount of oxygen available to each

shape should minimize any variations due to differencesin oxygen availability, consideration should be givento the selection of the flask size, itself.

During the growth of the test organism, the mycelialmat spreads over the surface of the nutrient medium.This causes some modification of the gas exchange ratebetween the atmosphere and the medium. The changein rate will not be proportional at all assay levels due tothe differences in the amount of surface area occupiedby the developing mycelium. The degree to which it isdisproportionate is inversely related to the flask size.Thus, when small culture vessels are employed, the topportion of the growth curve tends to level off, limitednot by the concentration of substance being assayedbut by the concentration of available oxygen. The useof larger flasks will correct this condition and reducevariation between replicates.

ACCURACY OF RESULTS

Both the precision and the accuracy of gravimetricassay procedures are comparable to other microbialmethods of analysis. Replicate values for the samesample solution generally agree within 10 per cent,while recoveries usually range from 90 to 110 per centof theoretical. Most results obtained by this procedurehave been in close agreement with those obtained forsimilar substances by chemical, physical, and othertypes of microbiological methods.The procedure is simple, and minor variations in

technique may not have a great influence on the ac-curacy since the extended incubation period makes themethod independent of many factors which affectother microbiological methods. Any such advantagesmay be over-balanced by the sacrifice of time which isnecessary for maximum precision and accuracy. Thus,the gravimetric procedures are most suitable for the

TABLE 4Complete and partial adaption of leucineless Neurospora*

Mg Mycelium on Different Amounts of LeucineFlask No.

0.25 mg 0.50 mg 1.00 mg

1 9.8t 17.5 32.72 7.3 17.4 32.13 7.3 44.8t 32.94 7.3 19.0t 31.25 16.2t 17.8 33.26 20.8t. 17.2 33.57 7.3 17.5 34.68 7.1 18.0 32.69 8.0 20. lt 35.410 50.3t 17.3 35.1

Avg wt 7.4 17.5 33.3

* Ryan, 1946.t Partial adaption.

culture. While the use of flasks of the same size and t Complete adaption.

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assay of certain vitamins for which other types ofanalytical methods may not be applicable.

ADVANTAGES AND DISADVANTAGES OF THEGRAVIMETRIC ASSAY PROCEDURE

The advantages of the gravimetric assay proceduresare the following:

1. The methods are simple and inexpensive. Theydo not require any special or elaborate equipment.

2. The methods are reliable and results are compa-rable with those obtained by other methods of analysis.

3. The methods are precise.4. The methods can be used to assay highly colored

solutions.5. In most cases, the age and the size of the inoculum

need not be controlled as growth response is primarilya function of the amount of limiting factor and not itsconcentration.

6. The methods in which Neurospora mutants areused as the test organisms are specific.The disadvantages of the gravimetric assay proce-

dures are the following:1. The requirements for nitrogen metabolism by the

test organisms are complex and may be affected by avariety of nonspecific nitrogenous substances.

2. The Neurospora mutants have inherent disadvan-tages: (a) a separate strain is required for eachparticular procedure, thus requiring maintenance of alarge number of test organisms; and (b) occasionalmajor changes in metabolism may occur as a result of asingle gene mutation with the resultant possibility ofadaption.

3. The methods are time consuming.4. The methods are not adaptable for large numbers

of samples.5. Flasks require considerably more incubation space

than tubes.6. The methods have limited application. For routine

analysis, they are employed only in the assay of a fewvitamins for which other methods are not available.

REFERENCES

AGARWALA, S. C. AND PETERSON, W. H. 1950 An improvedmethod for determination of p-aminobenzoic acid byNeurospora crassa. Arch. Biochem., 27, 304-315.

BARTON-WRIGHT, E. C. 1953 The microbiological assay ofthe vitamin B complex and amino acids. Pitman Puib-lishing Corporatio6n, New York, New York.

BEADLE, G. W. 1944 An inositolless mutant strain of Neutro-spora and its use i-n bioassays. J. Biol. Chem., 156, 683-689.

BONNER, J. AND ERICKSON, J. 1938 The Phycomyces assayfor the thiamine (Vitamin BI). The method and its speci-ficity. Am J. Botany, 25, 685-692.

BONNER, J. AND DORLAND, R. 1943 Experiments on theapplication of Neurospora sitophilia to the assay of pyri-doxin in tomato plants. Arch. Biochem., 2, 451-462.

BURKHOLDER, P. R. AND MCVEIGH, I. 1940 Studies on thia-

mine in green plants with the Phycomyes assay method.Am. J. Botany, 27, 853-861.

DOERMANN, A. H. 1945 A bioassay for lysine by use of amutant of Neurospora. J. Biol. Chem., 160, 95-103.

GAVIN, J. J. 1957a Analytical microbiology. II. The diffusionmethods. AppI. Microbiol., 5, 25-33.

GAVIN, J. J. 1957b Analytical microbiology. III. The tur-bidimetric methods. Appl. Microbiol., 5, 235-243.

HAMMER, K. C., STEWART, W. S., AND MATRONE, G. 1943Thiamine determination by the fungus-growth methodand its comparison with other methods. Food Research,8, 444-452.

HARRIS, D. L. 1952 Interaction of thiamine and pyridoxinein Neurospora. I. Studies of the pyridoxinless mutants.Arch. Biochem., 41, 294-304.

HODSON, A. Z. 1945 The use of Neurospora for the determi-nation of choline and biotin in milk products. J. Biol.Chem., 157, 383-385.

HODSON, A. Z. 1956 Vitamin B6 in sterilized milk and othermilk products. Agr. & Food Chem., 4, 876-881.

HOROWITZ, N. H. AND BEADLE, G. W. 1943 A microbiologicalmethod for the determination of choline by the use of amutant of Neurospora. J. Biol. Chem., 150, 325-333.

JUKES, T. H. AND DORNBUSH, A. C. 1945 Growth stimula-tion of Neurospora cholineless mutant by dimethylamino-ethanol. Proc. Soc. Exp. Biol. Med., 58, 142-143.

LORING, H. S., ORDWAY, G. L., AND PIERCE, J. G. 1948 Amethod of assay for cytidine and uridine by means of apyrimidine-deficient strain of Neurospora. J. Biol. Chem.,176, 1123-1130.

LUECKE, R. W. AND PEARSON, P. B. 1944a The microbio-logical determination of free choline in plasma and urineJ. Biol. Chem., 153, 259-263.

LUECKE, R. W. AND PEARSON, P. B. 1944b The determin-tion of free choline in animal tissues. J. Biol. Chem.,155, 507-512.

MEIKLEJOHN, A. P. 1937 Estimation of vitamin B, in bloodby modification of Schopfer's test. Biochem. J., 31,1441-1451.

MITCHELL, H. K. 1950 Vitamins and metabolism in Neuro-spora. In Vitamins and Hormones, Vol. 8, pp. 127-150.Academic Press, Inc., New York, New York.

MITCHELL, H. K. AND HOULAHAN, M. B. 1946 Adenine re-quiring mutants of Neurospora crassa. Federation Proc.,5, 370-375.

NICHOLAS, D. J. D. 1952 The use of fungi for determiningtrace metals in biological materials. Analyst, 77, 629-642.

NIKLAS, H. AND TOURSEL, D. 1941 The determination oftrace elements by means of A spergillus niger. Boden-kunde u Pflanzenernahr., 23, 375-380.

REGNERY, D. C. 1944 A leucineless mutant strain of Neuro-spora crassa. J. Biol. Chem., 154, 151-160.

RYAN, F. J. 1946 The application of Neurospora to bioassay.Federation Proc., 5, 366-369.

RYAN, F. J. 1950 Selected methods of Neurospora genetics.In Methods in Medical Research, Vol. 3, pp. 51-75. The YearBook Publishers, Inc., Chicago, Illinois.

RYAN, F. J. AND BRAND, E. 1944 A method for the determi-nation of leucine in protein hydrolysates and in foodstuffsby use of a Neurospora mutant. J. Biol. Chem., 154,161-175.

SCHOPFER, W. H. 1935a Plant test for vitainin B,. Z.Vitaminforsch. 4, 67-75.

SCHOPFER, W. H. 1935b Standardization and possible usesof a plant growth test for Vitamin B,. Bull. Soc. Chim.Biol., 17, 1097-1109.

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SIEGEL, L. 1945 The microbiological determination ofcholine. Science, 101, 674-675.

SINCLAIR, H. M. 1938 The estimation of vitamin B1 in blood.Biochem. J., 32, 2185-2199.

SRB, A. M. AND HOROWITZ, N. H. 1944 The ornithine cyclein Neurospora and its genetic control. J. Biol. Cihem.,154, 129-139.

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