Use of Microorganisms as Analytical Tools

1690 A N A L Y T I C A L C H E M I S T R Y

at about 150 nil. belov the initial value. Some dexterity of operation is required to avoid taking too much time during the titration. Some samples may be colored enough to require this technique.

The stability of the hydrovylamiiie reagent ni th time is somewhat variable. A simple means of verifying its stability is to analyze a sample of pure vanillin. The indicated purity should fall betn-c.en 09.7 and 100.3%.

E S T I ~ I A T I O h ~ OF ACETOV 4YILLOiVE COYTENT

A simple modification of the procedure enables the total e x - bonyl content of sanqiles to 1~ dctpimined. .4 similar sample size and volume of reagent me taken and the mivture is refluxed for about 1 hour. After cooling to room tempprature, the reaction mivture is titrated n ith the O.5-lr alcoholic hydrochloric acid to the same end point. After correcting for a reagent blank carried through the same procedurel the amount of hydrovyl- amine required for the total carbonyl less that required for the vanillin is calculated as acetovanillone. This reaction time probably could be shortened, b u t no demonstration has been made.

OTHER ALDEHYDES AND KETONES

If the crude vanillin has been derived from angiosperms such as birch, p-hydroxybenzaldehyde and syringaldehyde may be

present. Limited tests shomed that these will be titrated quan- titatively as vanillin. Acetosyringone (4’-hydroxy-3’5‘-dimeth- oxyacetopheiionc) apparently behaves like acetovanillone.

LITERATURE CITED

Brauns, F. E.. “Chemistry of Lignin,” Academic Press, Sew

Bryan, C. C., private communication. Bryant. W. 11. 13 , and Smith, D. M., J . Am. Chenl. Soc., 57, 57

Guerithw, E., and I.angcnnu, E. E., ASL. C H E x . , 25, 12 (1933). Leger. F., and IIibbert, €I., J . Am. Chen . Soc., 60, 565 (1938). Nitchell, .J., “Organic ;hialysis,” vol. 1, Interscience, Sew

AIitchell, J., and Smith, D. 11.. AXAL. C H m r . , 22, 746 (1950). gar. H., and AIcCarthy, J., Ibid., 23, 994 (1951).

Reavillc. E. T., aiiti Shrew, 0. W., I b i d . , 27, 565 (1955). Smith, IT. T.. Jr., Wayner, E. F., and Patterson, J. 31., Ib id . ,

Ytillirian. I t . C.. and Reed, R. AI.. Perfumery Essent. Oil Record,

Stone. J. E., and Blundell, 31. J., ANAL. CHEM., 23, 771 (1951). \’avon, G., arid AIonthPard, P., BUZZ. soc. chim., 7, 551 (1940). Weinberger, W., IND. ENG. CHEM., XXAI.. ED., 3, 365 (1931). Wse. L. E.. and Jahn, E. C., “Wood Chemistry,” 2nd ed., vol.

York, 1952.

(1 93.7).

York, 1953.

26, 155 (1954).

23, 275 (1932).

1, Iteinhold, New York, 1952.

RECEIVED for review March 14, 1955. Accepted September 13, 1955.

8th Annual Summer Symposium-Role of Reaction Rates

Use of Microorganisms as Analytical Tools DALE A. HARRIS Research Laboratories, Chemical Division, M e r c k & Co., Inc., Rahway, N. J,

Microorganisms occup) a definite place in the field of analytical analysis. They can be used successfully to assay various antibiotics, vitamins, and amino acids with an acceptable degree of accuracy even when crude and unknown types of materials are involved. In general, they are inexpensive to run, are not difficult to handle, and can be adapted for large-scale operations. They have shortcomings, particularly with interfering substances causing assay drifts w-ith the vitamin and amino acid assays. In many cases they represent the only satisfactory analytical tool available for use in specific chemical, stability, biological standardization, nutritional, and microbiological studies.

HE literature is full of references on the use of micro- T organisms in assaying antibiotics, vitamins, and amino acids. Although various niicroorganiqmp, including bacteria, fungi, yeasts, and protozoa have been studied, the bacteria have demonstrated the widest degree of uqefulness This paper i:, con- fined to their use in the variow ~nicrobiological anal) tical techniques. Bacteria are extremely small, unicellular plant like, micro- scopic organisms which differ from true plants in that, n i th one known exception, they lack chlorophyll (9). They reproduce by binary fission and their size generally varies from 0.2 t o 5 microns. Cell growth, or the measurement of some manifestation thereof, is the basis for all microbiological assays. I n the growth of a bacterial culture, a series of phases, characterized principally by variations in the growth rate, may be demonstrated. I n general,

the folloiving definitions (23) illustrate the sequence of events found in a typical bacterial sigmoid growth curve.

Lac PHASE. The growth rate is essentially null. ACCELER.4TION PHASE. The growth rate is slow but increasing. EXPONESTIAL PHASE. The growth rate is constant. The

cells are.dividing regularly and at maximum speed in a geometric progression.

RETARDATIO~ PHASE. The growth rate is decreasing. Here the bacterial cells cease to multiply a t maximum rates and cell divisions become less and less.

STATIOSARY PHASE. The gron-th is essentially null again. Actually the bacterial population remains almost constant, but theoretically there is an equilibrium betv-een dying and newly formed cells.

PHASE OF DECLINE. The growth rate is negative. Ifere the cells are dying a t a constant rate or nearly so.

The first two phases may be shortened by the use of young, actively groviing cells, a principle frequently used in microbiological assays. Many physical and chemical factors may also alter one or more of the above phases, sometimes eliminating them. The bacteria commonly used for microbiological assay work have a generation time-Le., the time required for one cell to divide into tivo cells-of usually less than 1 hour (26).

Bacteria are adaptable to the assay of a number of vitamins and amino acids necessary in animal nutrition because, in general, all forms of life share certain common metabolic processes in their basal cellular biochemistry (16). However, bacteria may also be used for the assay of antibiotics which do not, in general, exert cellular metabolic effects in higher animals. The apparent discrepancy between the response of the cells of higher animals and of certain microorganisms appears to be due to the cellular organization in the two classes of organisms (16 ) .

V O L U M E 27, NO. 11, N O V E M B E R 1 9 5 5

Of the many practical advantages in the use of microbiological assays (16) the rapidity with which the test bacteria react is certainly impressive. This, together with the fact that only small quantities of the test substances are needed to obtain a specific and measuralile response, makes possible a reistirely small scale field of opwations for performing a reaaonahly large number of assays.

ANTIBIOTIC ASSAYS

Microbiological assays for the quantitative estimation of antibiotics have had widespread use (10, 13, 19, 25, 28). They have served as efficient guides in chemical isolation a.nd puri- fication studies, in pharmacological studies, in routine production and control activities, and in microbiological research and development work. There are certain advantages for these bioassays. A-lp:~rt from the sensitivity factor, the bioassay may often be a p p k d as well to an unknown as to a known antibiotic (10). Also, althoug!i the antibiotic material to be investigated is often chemically heterogeneous and of variable compositioii, a microbio1ogic:il method can usually be applied without preliniinary fractionation. This generally holds true for the assay of materials in body fluids as d l . On the debit side, however, is the fact that a microbiological method does not necessarily differentiate between two or more antibiotics.

There are certain general methods Tvhich have been 7hox-n t o be applicable to fermentation broths as well as chemically isolated preparations of varying degrees of purity. These include the dilution, diffusion and turbidimetric techniques. The dilution method represents the all or none end-point type of response whereas the diffusion and turbidimetric methods represent the graded response.

The principle on which the bioassay of antibiotics is based is that equivalent doses of a given antibiotic exert equal inhibitory effects on similar populations of a given species of bacterium under established standardized conditions (28). Thus, the measurable response can be correlated with the dosage of test material used by a mathematical relationship ivhich can be represented graphically by a dosage response curve.

SERIAL DILUTION METHODS

I n the standard serial dilution method (10, 88), a number of tubes, ea,ch containing initially the same number of bacterial cells and the same volume of liquid nutrient medium, are dosed with varying amounts of antibiotic. The tubes are then incubated until sufficient growth of the uninhibited bacteria has occurred. Depending on the assay conditions, this incubation period may vary from a few hours to several days. The end-point or threshold concentration is taken as the lowest concentration of a.nti- biotic that prevents growth of the test bacterium as evidenced by a lack of turbidity in the broth. A similar set of tubes containing known amounts of the test material is run as a control, and for tests requiring more than 3 to 4 hours, sterile tests preparations are required. Because an all or none response is being dealt with, a definite value for the end point cannot be given, only a range. The range can be narrowed by more intermediate tubes, but the degree of sensitivity desired must be equated against the increased time, effort, and cost. This bioassay technique can he made accurate and figures such as the following have been reported (30) n-ith a penicillin assay where the concentration of test material decreased by 107, increments:

.&ESayS

1 C 8.9% s td . dev. 3 z t 6.3% s td . error 5 i 4.5% std. error 8 =t 3.4% std. error

16 i 2.3% std. error

Serial dilution assays may not only be carried out in liquid nutrient medium but also in solid nutrient medium (32). Where- as the results obtained are less accurate than in broth and the

1691

procedure is somewhat more cumbersome, they do have the advantages tha t sterile preparations are generally not required and that the threshold concentrations may be obtained simultaneously against several different bacteria.

DIFFUSION hlETHODS

3t xidely used bioassay technique for anti- usion or cylinder-plate method. In this pro-

cedure R solid nutrient medium is seeded with a test bacterium. The solution of the antibiotic to be assayed is brought into contact with the solid seeded medium either by the means of metal cylinders or paper disks placed on the surface or in holes cut in the solid medium itself. After an incubation period of usually 18 to 24 hours, the diameters of the inhibition zones are measured. The resulting zone value is essentially a measure of the equilibrium condition between two factors-namely, out8ward diffusion of antibiotic and growth of the test bacterium which tends to cover the nutrient surface. If a standard is set up a t the same time using known amounts of the same ant,ibiotic, when possible, a dosage response curve can be plotted from ryhich the strength of the unknowx can be determined.

The paper disk technique is capable of high accuracy although some workers feel it is not as sensitive to such low dilutions as the other plate diffusion methods ( I O ) . Volunies as small as 0.02 ml. of liquid can be used per disk, however. With the cylinder plate technique, the concentration of the substance in the solution to be assayed determines the inhibition zone whereas Ts-ith the paper disk technique it is the amount of the substance deposited on the disk vhich is the controlling factor, provided it is freely soluble.

There are many more or less controllable factors which are capable of affecting the inhibition zone sizes obt:tincd in the diffusion methods, These include the folloiving as outlined by Lees and Tootill (19):

Choice of test organism Condition of test’ organism Density of seeding of test organism on assay plates Formulation and condition of assay medium Depth of seeded agar in assay plate Potency of test solution Volume of test solution applied to the plate Area of seeded agar to which test solution is initially applied Time of application of potent solution Temperature of incubation of assay plate Length of incubation cycle

While all of these factors are extremely important, certain of them are of particular interest. The basic assumption one makes in an antibiotic assay is that, the zone size produced by the antibiotic solution under test is determined solely by the antibiotic itself. If, however, the solution is a mixture of two or more antibiotics, then the response of the bacterium varies and, thus, does not result in a valid response. Where these other “contaminating” factors are known or suspected effort,s should be made to incorporate them into the assay medium.

The size of the inhibition zone is inversely related to the seeding density of the test bacterium. Thus, a compromise must be worked out between zone size and clarity and a suitable linear- dose response curve with a satisfactory slope. The effect of the pII of the medium must be taken into account. Various test bacteria differ in their response to antibiotics depending on the pH of the medium and, thus, it must he buffered accordingly. Likewise the oxidation-reduction potential may be of importance. The lack of sufficient poising capacity may result in a gradient in O/R potential between the agar surface exposed to the atmos- pheric oxygen and the solid medium in contact with the glass bot- tom of the culture dish receiving oxygen solely by the slow proc- ess of diffusion (19). If this factor is critical enough, the test bacterium may show a differential response through the solid nutrient medium, producing, in some cases, a conical rather than


is more sensitive to impurities than most other procedures which may stimulate or inhibit the test bacterium. Colored or turbid samples obviously interfere and introduce errors as do any non- specific changes, such as pH, color, or turbidity, produced in the liquid medium during bacterial growth.

With assays of the graded response type (turbidimetric, diffusion) the probable error can be calculated easily and it is not too difficult to increase the accuracy to any desired scientific and economic degree by sufficient replication. In the case of the dilution, or all or none type procedure, the answer is given only as range. Increasing the number of intermediate dilutions can only narrow this range.

In the early days of antibiotic assay development, the standard error associated with the assay ranged about 5 1 0 to 15% or less with sufficient replication. More recently Lees and Toot- ill (19, 20) have shon-n that with large plates and routine care a standard error of &5% could be obtained, whereas with more precise precautions this could be loviered to *l%.

The following antibiotics can be assayed by the indicated test organisms:

a cylindrical zone. This can be prevented by having a non- seeded base layer of nutrient medium overlaid with a thin seeded layer.

I t is also known that the margins of the inhibition zones depend on the diffusion rate of the particular substance under assay, and this diffusion rate is controlled to some degree by the water content of the solid nutrient medium. Thus, variations in the moisture content throughout the assay medium may lead to local increased absorption of the moisture from the test solution with the resulting effect of increasing the extent of its diffusion. This can be a problem with large glass plates. The inhibition zone of an antibiotic varies inversely with the depth of the solid nutrient medium in the test dish or plate. Thus, differences in medium depth may result in the test solutionsdiffusingat differerit rates. At the same time variations in the area covered by the test solution can affect slightly the zone size obtained. Blthough this is generally not a problem with cylinders or paper disks, it can be a problem with holes cut in the medium or with beads, as diffusion of the test solution from the area of deposition is not a t once wholly horizontal, but also vertical (19). I t can be denion- strated that the application time of two solutions of equalpotency to the assay plates is extremely important.

The size of the inhibition zone is also a function of the growth rate of the test bacterium. Thus, the more rapidly the test organism grows during the incubation period, the smaller the resulting inhibition zones mill be. In the event the plate is allowed to remain at room temperature for any period of time after the test solutions are applied before incubating, the diffusion begins immediately, whereas growth of the test barterium is essentially nil. Relatively larger zones result. This technique can be applied for low potency samples.

The size of the inhibition zone depends to a large extent on the diffusion rate of the test substance, the duration of the lag phase of the test bacterium, and its subsequent growth rate in the ex- ponential phase.

Several interesting techniques have been developed by Goyan, Dufrenoy, Strait, and Pratt (12) and Pratt and Dufrenoy ( 3 7 ) to shorten the incubation period in the pencillin assay. They were able to produce visible latent inhibition zones after 3 hours of incubation by treating the plates with silver salts, exposing to light, and then submitting them to photographic development. The resulting dose-response curve was linear for penicillin concentrations between 1 and 8 microns per ml. More recently ( 2 7 ) it was reported that these latent zones could be revealed by treating the plates with dyes such as bromocresol purple, phenol- sulfone phthalein, Yile blue, Janus green, methyl green, and safranine 0, or reagents such as cadmium acetate, cobalt nitrate, Schiff’s reagent azo-coupling reagent, and ferricyanide followed by ferric sulfate, The curves obtained relating concentration of penicillin to zone diameter were flatter than with the more usual methods having a longer incubation period. This resulted in a wider range suitable for assay purposes, although accuracy probably was somewhat less.

TUKBIDIJI ETRIC 1\.f ETHOD S

In these techniques, the amount of growth of a test bacterium in a liquid nutrient medium, dosed with various levels of known antibiotic solution, is measured turbidimetrically and plotted in graph form. Several levels of test solution run simultaneously in the same fashion are calculated against the standard. Generally, turbidity or light absorption is measured photoelectrically and the resulting dose-response curve is established by plotting concentration directly against instrument reading. Because the period of incubation is frequently short with this technique, the concentration of inoculum and time and temperature of incubation are extremely important to ensure constant growth rates and, thus, reproducible assays. While this technique is capable of high accuracy, and requires only a short operation time, there are certain disadvantages (10). The procedure

Antibiotic Method Bacitracin Cylinder-plate

Turbidimetric Cblorainphenicol Cylinder-plate Chlortetracycline Cylinder-plate

Turbidimetric

Dehydrostreptomycin Cylinder-plate sulfate Turbidimetric

Erythromycin Cyljnder-plate Neomycin sulfate Cylinder-plate

Turbidimetric Penicillin CJ-linder-plate

Polymyxin B sulfate Cj-linder-plate

Streptomycin sulfate Cylinder-plate Turbidimetric

Tyrothricin Turbidimetric

Test Organism .~lzciococcus j l a u u s .1TCC10240 Staphylococcus aui e u s ATCC10537 Sarcana lutea P.C.I. 1001 S a i c z n a l ~ t e a 1’ C.I. 1001 Micrococcus pyogenes r a r . aureus

B a c i l l u s subtilts 6633 Klebdiella pneumoniae ATCC10031 S a r c i n a lutea P.C.I. 1001 Micrococcus p y o g e n r s Tar. a u r e u s

Klebsiella pneumoniae ATCC10031 Micrococcus pyogenes Tar. a u r e u s

.ITC C 6538-P

ATCC6538P

ATCC6538P Micrococcus pyogenes \ ar. a u r e u s

Baczl lus subtzlzs ATCC6633 Klebsiella pneumoniae ATCC10031 Streptococcus hemolyt icus Lancefield

Group D, ATCC9854

ATCC4617

Specific details of the above assay procedures are given in the United States Pharmacopeia (25 ) .

YITAJIIS ASSAYS

The knowledge gained of the nutritional requirements of various microorganisms has proved to be a very useful approach to t,he study of animal nutrition. The essential nature of inositol, pantothenic acid, p-aminobenzoic acid, pyridoxal, pyridoxamine, biotin, and nicotinic acid was discovered for the nutrition of microorganisms before their essentiality in animal nutrition was established (4, 22, 31). Likewise the studies on folic acid and vitamin B12 were facilitated by the use of microbial assays ( S I ) .

.4s in the case with the antibiotic assays, there are advantages to the microbiological vitamin assays (1). They have the advantages of speed and small requirements of space, labor, and materials when compared to other biologicalmethodsusinganimals or humans. Secondly, they can be used, unlike most chemical methods, before the chemical nature of the vitamin being assayed has been determined. Lastly, they share with other biological assays the property of biological specificity.

Microorganisms, which can be used to assay various members of the known vitaim B complex (1, 4, 14, W2, Si) are now known. This is illustrated below.

Vitamin Method Test Organism p-Aminobenzoic acid Gravimetric S e u r o s p o r a crassa (mutant) Riotin Aridimetric Lactobacillus aTabinOSUs -. . . ... Choline Folic acid

Inositol

Nicotinic acid Pantothenic acid Riboflavin

Gravimetric Acidimetric Acidimetric Gravimetric Turbidimetric Acidimetric Acidimetric Acidimetric Acidimetric or

turbidimetric

.Veurospora crassa (mutant) Streptococcus faecal is Lactobacillus casei Xeurospora crassa (mutant) Saccharomyces cadsbergenszs Lactobacillus arabinosus Lactobacillus arrsbinosus Lactobacillus helveticus

Lactobacillus casei

V O L U M E 27, NO. 11, N O V E M B E R 1 9 5 5

Thiamine Turbidimetric Lactobacillus / e r m e n t i Vitamin Ba

Total Gravimetric .Veurospora s i tophi la (mutant) Turbidimetric Saccharomuces carlsbsrgensis

1693

Pyridoxamine + Pyridoxal Turbidimetric Lactobacillus cas&

pyridoxal Turbidimetric Streptococcus /aecaZis

Vitamin BIZ Turbidimetric Lactobacillus le ichmanni i

Specific details of the above assay procedures may be found in the excellent tests of Barton-]$'right (4,) Snell ( S I ) , and others (1, 25) .

Although a number of x-ray mut'ants of the mold Seurospora and certain strains of yeasts can be used, thelactic acidbacteriaare undoubtedly the most useful and most important. I t is an established fact that when the lactic acid bacteria are used for iiiic,robiological assays, the measurement of their response is siiiiple and direct since, with the exception of vitamin B,, the lactic acid produced by the growth and metabolic activities of theye bacteria is directly proportioiial over a ccrtai:i range to the vitamin concentration in the liquid nutrient medium (4). All that is necessary is to titrate this acid produced with sodium hydroxide and prepare a standard curve, Froin this standard curve, the amount of vitamin in the various levels of test solution can be determined by interpolation. This type of assay is thus based on a total response. In certain cases, the growth of the test organism after a short incubation period is measured directly by turbidimetric measurement. In t,his casz, one is measuring the comparative rates of response (31 ). Generally speaking, assays based on a total response are likely to be more reliable than those based on rate of response, because much information remains to be learned about factors present in crude preparations which affect response rates, either to slow them down or to accelerate them, but which generally have litt,le or no effect ontotalresponse.

The assay medium generally includes some natural material such as casein hydrolyzate from which contaminating vitamins have been removed, plus pure vitamins, purines, salts, cystine, tryptophan, fermentable sugar (usually glucose), and a suitable buffer such as sodium acetate. The latter has a stimulating efiect on growth apart from its effect, on pH. Below is list,ed the composition of s typical aseay medium (1):

Biotin Niacin KiHPOi - KHzPOI MgS04.7H20. iVaCI, FeS06.7Hz0, lInSOc.4HzO Glucose anhvdrous Sodium acetate anhydrous

Khile the composition of the above basal medium may vary with the different assays, it is necessary to remember only that it be nutritionally complete for the test organism except for the one vitamin being measured.

The medium is prepared in tubes which are then dosed with the vitamin preparation to be tested. Following sterilization by autoclavirig, the tubes are cooled in a water bath or a t room t,einperature until the temperature within the tubes is absolutely uniform. This precaution is particularly important when turliidimetric measurements of growth are to be macle after 16 to 18 hours. Even slight differences in initial temperature can influence the early growth rate much more markedly than they would tmhe total gioxyth over a 72-hour period. Theconstancy of the incubation temperature throughout the incubator or bath is even more important in the short term incubations, because of its resultant affect on growth rates although it is still important, with the longer assays.

In vitamin assays of natural or crude preparations, the sample treatment frequently becomes of prime importance because the vitamin or unknown growth factor must be in a state readily utilizable by the test organism. Where stability is of no concern, treatment with acid or alkali a t high temperatures is most useful. In other cases, enzymatic digestion must be used when the vitamins or unknown growth factors are destroyed by one of thP

above treatments. Care must be taken that the enzymes them- selves, which are generally crude preparations, do not contain growth factors which interfere in the assay.

The basic assumption in microbiological assays is that growth and metabolism of the test bacteria are influenced in the same way by the growth factor being assayed, whether it is in the standard solution or in the unknown solution, and by no other factor in the unknown solution (1). Under these conditions, there is no tendency for the assay values obtained on the test solution to increase or decrease with the dilution level used. This latter condition is known as assay drift and may lead to nonvalid assay resulte. I t can be caused by different factors-for example, in the case of uprard drift substances closely related to vitamins (24), growth factors, or complexes of tjhe vitamin may stimulate the growth of the test bacterium, but to different proportionate degrees from that of the standard vit'amin a t the various dilution levels. In addition, chemically unrelated, nonvitamin substances may alter the response to a vitamin (31). For example, fatty acids may affect the response of Lactobacillus casei to riboflavin rrsulting in inhibition or stimulstiori depending on the concentration. Likewise high levels of thymine and thymidine may wiilace folid acid and vitamin B,,, respectively. Where the drift is due to a known medium inadequacy, it can frequently lie averted by adding the interfering substances to the medium. Khwe there is a downward drift, it generally indicates the preseiice in the sample of some toxic or inhibitory substance [ I ) . A high salt concentration, resulting from medium neutral- ization, may also inhibit the test bacterium.

Various other factors have been investigated for their effect on the dose response curve in the vitamin assays. The critical nature of temperature, both on the amount of growth and, even more important in some cases, on the growth rate, has been emphasized. Furthermore, the requirements of certain bacteria for riboflavin (89) and phenylalanine ( 7 ) may even be altered by temperature differences. I t has been mentioned that the nutrient medium must be buffered, because the pH optima for the lactic acid bacteria can be altered during growth t'hus mak- ing condit,ions unsatisfactory for further growth. Varying the amount of carbon dioxide in the surrounding atmosphere, while generally producing no effect, can in certain insQances affect certain bacterial requirements for phenylalanine, histidine, aspartic acid ( $ I ) , and biotin (14) . The oxidation-reduction potential of the medium may likenise affect certain nutritional requirements. Although the bacteria used in these assays are either microaerophilic (the rod forms, Genus Lactobacil2us) or facultative aerobes (the coccoid forms, Genera Streptococcus and Leuconostoc), they generally are able to grow under the described conditions which promote a rather favorable oxidation- reduction potent,ial. Reports have appeared pointing out that the microbial requirements for pyridoxine (6) and vitamin B12 (15, 17, 18), may be altered significantly by various oxygen texsions.

In recent years several new techniques have been introduced for microbiological vitamin assays (14). One involves the use of cylinder plate or diffusion assays similar to those described for the antibiotic assays ( 2 , 3, 11). Here one obtains zones of exhibition or groivth, and the usual dosage response curves can be plotted. The procedure is faster and lends itself readily to large-scale routine handling, However, the sensitivity is considerably reduced, and 10 to 2000 times as much vitamin is required (14). Closely allied to the diffusion plate t,echnique is the use of bacteria in bioautography. Here a tool is provided for the location of vitamins and their closely related analogs, which have been separated by paper chromatographic techniques. Removal of the indicated active locations by leaching and testing by the usual tube assays has led to quantitative estimation of B12! Blza, and B, mixtures (33, 34).

The accuracy claimed for microbiological vitamin assays is generally i.10 to 15% of the mean. I t has been shovm by


Bessey and Hull (5 ) , however, that much greater accuracy can be obtained with careful control of all the variables and with adequate media. They report a probable error for the mean of zk0.7 to 1.2% with pure materials and It3q;b with crudes.

ARIINO ACID ASSAYS

The microbiological assay of amino acids was the sequel to, and the logical outcome of, the earlier assay studies on vitamins. Basically the same fundamental principles apply. The general assay media and test bacteria are the same as those described for the vitamin assays. The following amino acids can be assayed by the indicated test organism.

Amino Acid a-Alanine Arginine Aspartic acid l-Cys tine Glutamic aoid Glyc,ine Histidine Isoleucine Leucine Lysine DL-Met hioniae ~ - h l ethionine Phenylalanine Proline Serine Threonine Tryptophan L-Tyrosine Valine

Method Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acidimetric Acjdjmetric Acidimetric Acidimetric Acidimetric Acidimetric

Test Organism Lactobacillus citrovorum 8081 Streptococcus faecal is Leuconostoc mesenteroides P. GO Leuconostoc mesenteroides P. 60 Lactobacillus arabinosus Leuconostoc mesenteroides P. GO Leuconostoc mesenteroides P. 60 Lactobacillus arabinosus LactobacilIus arabinosus Leuconostoc mesenteroides P. 60 Lactobacillus f e r m e n t ; 36 Leuconostoc mesenteroides P. GO Leuconostoc mesenteroides P. 60 Leuconostoc mesenteroides P. GO Leuconostoc mesenteroides P. 60 Streptococcus Jaecalis Lactobacillus arabinosus Leuconostoc mesenteroides P. 60 Lactobacillus arabinosus

Specific details of the above assay procedure may be found in the excellent text of Barton-Kright (4).

The general methods used for measuring growth-i.e , lactic acid production or turbidity-are the same as those used in the vitiamin assays. ill1 the precautions described apply here with equal importance for the performance of these assays and the preparation of the samples.

If the basal medium is composed only of amino acids essential for the growth of the test bacteria one does not get maximum growth. Thus, the stimulatory amino acids must also be added to give growth comparable to that obtained with acid hydrolyzed casein plu8 tryptophan and cystine. The latter two amino acids must be added, since acid hydrolysis of the casein destroys tryptophan and very little cystine is normally present.

Preparation of the sample is frequently a serious problem, particularly with foodstuff 9. I l any interfering suhstances are frequently present resulting in either abnormal stimulation or inhibition. As such, they affect the rates of growth of the test organism.

I t has been noted by various workers (8, 14) that the relationship of the concentrations of the various amino acids in the medium can be critical, In some cases it may determine the essentiality of a given acid or it may cause the inhibition of the test bacterium. Thus, the role of amino acid antagonisms can be critical and the assayer must establish a medium with sufficiently high levels of amino acids to offset any possible distortion by the addition in the sample of other amino acids.

By controlling all the factors discussed, it has been found that amino acid assays can be run with an error of i5%.

LITERATURE CITED

Association of Vitamin Chemists, Inc., “Method of Vitamin

Bacharach, A. L., Xature, 160, 640 (1947). Bacharach, A . L., and Cuthbertson, R. F. J., Anelyst, 73, 334

(1 948). Barton-Wright, E. C., “11icrobiological Assay of Vitamin B-.

Complex and Amino Acids,” p. 3, Pitman, Xew York, 1952. Bessey, 0. A, and Hull, T. Z., J . Bid . Chem., 155, 71 (1944). Bohonos, K., Hutchings, B. L., and Peterson, W. H., J . Bac-

Borek, E.. and Raelsch, H., J . Biol. Chem., 190, 191 (1951). Brickson, W. L., Henderson, L. M., Solhjell, I., and Elvehjem,

C. A, Ibid., 176, 517 (1948). Bryan, A. H., and Bryan, C. G., “Principles and Practice of

Bacteriology,” 3rd ed., p. 13, Barnes & Yoble, New York, 1945.

Florey, H. W. F., Chain, E., Heatley, X. G., Jennings, 11. A., Sanders, A. G., Abraham. E. P., and Florey, 9. E., “Antibio- tics,” vol. 1, p. 110, Oxford Univ. Press, Sew York, 1949.

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RECEIVED for review June 28, 1955. .2ccepted September 6, 1955.

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Use of Microorganisms as Analytical Tools