in recent years on high purity metals in the electrochemicals field, increased demand for extremely sensitive analytical procedures to determine trace contaminants may give sufficient impetus to the development of microbial assays. In the next decade, the challenge will undoubtedly be taken up and highly sensitive microbial assays for trace elements Fill be developed with the precision and accuracy of the vitamin and antibiotic assays. The second large advance will be in the estimation of small molecules like amino acids and vitamins in situ. This development will come on the heels of our break-through on the use of phagotrophic protozoa as assay tools. Already there are instances in the litera-
ture in which protozoa were used to determine amino acid content of intact proteins. Although the present methods leave much to be desired, a better understanding of protozoan nutrition and refinements in methodology will resolve the problem. There will be in hand new assays by which determinations will be made without prior resort to complicated extraction procedures. Lastly our ability to determine with ease minute quantities of high molecular compounds will be achieved through the researches in the field of immunology. This field has made remarkable advances in the last decade and has much to offer the analyst. Many are familiar with some such techniques, which are commonly employed in medical labora-
tories. As increased emphasis is placed by the chemist on the synthesis of complex molecules intimately associated with life processes such as proteins, the demand for imnzunological procedures will increase. The next decade will find a wide variety of immunological methods in use in the analytical laboratory. This symposium was arranged to review some of the methods available in the area of analytical microbiology. The papers discuss the theoretical considerations, application, and limitations of some of the methods employed in the fields of the speakers.
RECEIVED for review January 12, 1959. Accepted April 2 , 19.59.
Microbiological Assay of Amino Acids, Vitamins, and Antibiotics Application of Tube Methods HENRY
W. LOY and WILLIAM W. WRIGHT
Food and Drug Administrafion,
U. S.
Deparfment o f Health, Education, and Welfare, Washington
b The use of microbiological tube assays requires attention to many details not ordinarily encountered in analytical chemistry. The microorganism and the medium in which it grows are used as reagents. Considerations in the study, development, and application of microbiological tube methods include reference standurds, test organisms, media, sample preparation, assay tube preparation, measurement of response, and technique. Reference standards should be pure and authentic, test organisms should be sensitive to the substance under test, the media should b e of known composition, the sample should be measured precisely and prepared SO as to liberate the active component and eliminate interfering substances, the assay tubes should be prepared without harming the sample, and the measurement o f response should be done accurately b y titrimetry, turbidimetry, gravimetry, or other suitable technique.
I
advantages of microbiological tube methods are rapidity] accuracy, and sensitivity (9, 10). The analytical chemist develops and uses MPORTANT
every tool and every reaction that will bring sensitivity, precision, and selectivity to the measurement of the substance in question. In the application of analytical microbiology, the living cell gives not only a high degree of sensitivity, but a biological specificity because of the particular properties of the metabolic process of the cell ( 8 ) . Thus, the test organism and the medium in which it g r o w are used as reagents. The reaction is the response in the metabolic process brought about by the substance under analysis. The end point is determined by titration of a metabolite such as lactic acid, or by measurement of growth by, for example, turbidimetry or gravimetry. In dealing with the organism as a reagent, the selection and maintenance of activity of a suitable microbiological culture are important. In the reaction] not only the specificity of the substance under analysis, but the avoidance of any foreign material that may affect the growth or metabolism of the living organism, is desirable. Thus, the development of methods for microbiological assay parallels the development of any analytical chemical procedure. The increased use of microorganisms
25, D. C.
in the identification and quantitative determination of a variety of nutritional and inhibitory substances has been an important factor in the development of our biochemical and nutritional knowledge. By means of estensive collaborative studies, many of these procedures have been standardized into precise quantitative methods that have been included in official compendia (3, 6, 17, 27). The methods described in the Antibiotic Regulations of the Food and Drug Administration ( 5 ) have been developed in collaboration with interested members of industry. Other organizations also have employed collaborative studies for the development and improvement of methods, so that the area of analytical methods study is one of constant change and improvement. Microbiological tube methods provide excellent examples of efforts in this respect, and a scientist with a background in analytical and biochemistry may be considered best qualified to obtain the high degree of accuracy required in the application of these methods. Analytical microbiology has brought together the biochemist and the bacteriologist in considering mutual problems with respect t o the growth and VOL. 31,
NO. 6, JUNE 1959
971
metabolism of microorganisms. The effect of this union upon increased understanding of microbiological intermediary metabolism is important. The nutritional requirements of organisms were first satisfied in a crude manner; combinations of tissue extracts and hydrolytic products were discovered by trial and error to be suitable. The advance of nutrition science to the point of identification of the principal nutrients required in plant and animal nutrition, and the efforts of the biochemist in making these available in pure or relatively pure form for further study, now make it possible to prepare nutrient media of known composition and purity. This permits tabulation of detailed and exact nutrient requirements of many microorganisms, information that is necessary for their use as test organisms in analytical methods. Growth factors that have attracted the widest interest and study are the vitamins and amino acids. A substance that meets the definition of a vitamin or other essential nutrient for one species may be readily synthesized by another, and therefore not required to be supplied from the external environment. Mutant strains of organisms have been developed in vhich the ability to synthesize has been destroyed in one respect or another, thus increasing the number of nutrients required for survival, The pattern of amino acid requirement of some single-cell organisms resembles those of higher animals. A number of interesting relationships have led to a better understanding of the complex synthesis of the nonessential amino acids and of protein synthesis. For example, the role of pyridoxal as a coenzyme in transamination reactions has been studied effectively in the lactobacilli. The Association of Official Agricultural Chemists is now engaged in a collaborative study of microbiological tube methods for the assay of 12 amino acids with a view to their adoption as “official” ( 2 1 ) . hficrobiological tube methods have been devised for almost every antibiotic in general use. In the Antibiotic Regulations (b), turbidimetric procedures are described for bacitracin, nystatin, streptomycin, and the tetracyclines. The U. s. Pharmacopeia provides turbidimetric procedures for bacitracin, neomycin, streptomycin, the tetracyclines, and tyrothricin. E. coli has been used as a test organism in the turbidimetric assay of chloramphenicol and polymyxin (2Q); and a dihydrostreptomycin-resistant mutant of Staph. aureus has been used for the analysis of novobiocin (11). Considerations that must be given attention in the study, development, and application of a microbiological method
972
ANALYTICAL CHEMISTRY
fall into a specific pattern. These may be discussed appropriately under the following headings: reference standard, test organism, medium, preparation of sample solution, preparation of assay tubes, measurement of test organism response, and technique. REFERENCE STANDARD
Because of the variation in environmental conditions that cannot be avoided from one assay to another, it is essential that each assay include tubes containing graded amounts of a reference standard. For this purpose reference standards for seven B vitamins (calcium pantothenate, cyanocobalamin, folic acid, nicotinamide and nicotinic acid, pyridoxine hydrochloride, riboflavin, and thiamine hydrochloride), 12 amino acids (L-arginine monohydrochloride, L-cystine, L-histidine monohydrochloride, L-isoleucine, L-leucine, L-lysine monohydrochloride. L-methionine, L-phenylalanine, L-theonine, Ltryptophan, L-tyrosine, and L-valine), and 14 antibiotics (bacitracin, chloramphenicol, chlortetracycline, dihydrostreptomycin, erythromycin, neomycin, novobiocin, nystatin, oxytetracycline, penicillin G, polymyxin, streptomycin, tetracycline, and tyrothricin) are distributed by the U. S. Pharmacopeia. These are available a t small cost and should be used in preference to substances obtained through commercial channels. The Food and Drug Administration furnishes working standards of the certifiable antibiotics, and several noncertifiable ones that the U. S. Pharmacopeia does not provide. Standards of known purity must be used for assays in which U. S. P. Reference Standards or FDA n-orking standards are not available. TEST ORGANISM
Cultures of various test organisms are obtainable from the American Type Culture Collection, 2112 h l St., N. W., Washington 25, D. C. In the assay of essential nutrients, the most obvious requirement is that the test organism utilize in its metabolism the nutrient under study, and fail to grow in a nutrient medium from which the substance under assay has been omitted. For assaying antibiotics and a variety of antimetabolites and toxic materials, including spray residues, the organism must be inhibited by the given substance, preferably in extremely small quantity, and it must give graded responses of partial inhibition to graded amounts of the substance (8, 10).
One of the main problems encountered in the analysis of antibiotics is due to the frequent use of mixtures of two or more antibiotics in one product. Test organisms are seldom so selective that
they are susceptible to one antibiotic only. However, the level a t which the organism is inhibited by one antibiotic may be such that i t is not affected by commonly encountered amounts of a second antibiotic. To clarify this problem Arret et al. ( 1 ) determined the “interference thresholds” for ten antibiotics in two widely used tube methods. They also recorded the “sensitivity thresholds,” the minimum amounts of single antibiotics producing measurable growth inhibition. There must be a suitable means of measuring growth response or inhibition of growth of the test organism. In the case of lactobacilli, for example, lactic acid is a normal by-product of growth, and titration of the acid formed has been a simple and reliable measure of the degree of response of the test organism. Nore recently, means have been improved to relate graded increases of the substance under assay to cell numbers, as measured by turbidity readings. When analytical microbiology evolved, biochemists thought in terms of the specificity of animal response to growth factors. This specificity relates to the metabolizable isomers of naturally occurring substances as well as to the active forms of nutrients that occur in a variety of bound complexes, so that specificity is a characteristic that can be related to living tissue as opposed to the inanimate chemical means of measurement. An example is the specific requirement of S . faecalis for the L forms of the amino acids. With some reservations, this is in general comparable to animal requirements. Even so, although S. faecalis will not respond to hydroxylysine in the complete absence of L-lysine, there is a reduction in the lysine requirement when both acids are present ( 2 0 ) . Further, with the yeast S. carlsbergensis there may be variation in the utilization of the different forms of vitamin Bs as they occur naturally (18, 19). This differs from the ability of the animal to utilize these forms with equal biological value. Nevertheless, the specificity of living cells is a desirable feature that is frequently found and has given microbiological techniques a distinct advantage over chemical measurements. Consideration must be given to the sensitivity and stability of the test organism selected for use. To provide a suitable basis for measurement, the organism must respond to increments of the substance under assay to give a response curve that will provide differences within a critical range of precision. This sensitivity can often be increased by frequent subculture of the test organism, during which the response curve assumes a steeper slope. The improvement observed with S. carls-
bergensis in response to pyridoxime is an example. I n considering the stability of the test organism, an example is the study that has been conducted for about the past 4 years by a special panel of the U. S. Pharmacopeia. This panel has determined a t intervals the activity of the culture of L. leichmannii distributed by the American Type Culture Collection. This culture, carried in a fortified milk broth, over a period of time, deteriorated in its total response to vitamin BIZ. Means of restoring its sensitivity &-ere not successful and the U.S. Pharmocopeia panel recommended that the American Type Culture Collection draw from its stock of lyophilized material to prepare a new culture for distribution. It was shown that the stability of this organism may be maintained Kith lyophilization of a highly sensitive culture, and distribution in the lyophilized form was approved. Similar studies have been conducted during the past year with other cultures that are used in assays for amino acids and other B vitamins, and good results have been obtained. The reasons for the change in cultures with respect to their sensitivity and activity in these microbiological methods is little understood, and many factors that cause this loss of response have not been determined. Cultures used for antibiotic assay occasionally become less susceptible. It has then been necessary to prepare new cultures from lyophilized material. One of the problems in assaying mixtures of antibiotics is that the test organism may be inhibited by several antibiotics. I n such cases it has been possible to develop artificial resistance to a given antibiotic by growing it in the presence of increasing subinhibitory concentrations. Such artificial resistance is produced most easily with streptomycin (4). Although cross resistance such as that between streptomycin and dihydrostreptomycin does occur, the mutant usually remains susceptible to other antibiotics. However, a culture of Staph. aureus that was artificially made resistant to dihydrostreptomycin, was also made much more susceptible to novobiocin than the parent culture (11). A culture made artificially resistant may lose its resistance unless it is maintained in a medium containing the particular antibiotic. However, when it is lyophilized, i t cannot be cultured directly from the lyophilized state in a medium that contains the antibotic. It must be grown first in an antibiotic-free medium before transfer to the antibiotic-containing medium. MEDIUM
I n developing a basal medium to be used in the assay for an essential nu-
trient, the nutritional requirements of the test organism must be known. A fairly common source of nitrogen is a solution of acid-hydrolyzed casein from R hich the growth factor under study has been removed by adsorption. When supplemented with tryptophan, sometimes cystine, and occasionally methionine, the amino acid composition is usually satisfactory. For amino acid assay, a combination of pure amino acids is used with omission of the one under study. S. faecalis and several of the lactobacilli have a requirement for about 18 of the 22 amino acids. The amino acid requirements of S. carlsbergensis is discussed by Parrish, Loy, and Kline (19). Dextrose of high purity is normally used as a carbon source. The composition of the mixture of inorganic elements required by the test organism is important. I n the study of organic growth factors, the trace element requirements of the organism must be present. Khere trace element requirements of the organism are studied, meticulous care is essential to provide inorganic salts that do not contain the element under study. The medium must contain a suitable buffer, and for this purpose sodium acetate is usually adequate. A citrate, phosphate, or other buffer is required in some cases. The required B vitamins are generally added in the crystalline form; hence it is easy to assay for any one, simply by omitting it from the basal medium. In examining the suitability of a medium for the assay of an essential nutrient, suitable growth of the organism must be obtained in the presence of the substance under assay, and little or no growth should occur in its absence. Other requirements include uniform response to graded levels of the assay substance and uniformity in response between assays. An example of studies conducted for the satisfactory solution of problems of this nature is that of the U. S. P. Study Panel for Vitamin Biz (14). The composition of the medium must be examined for possible interactions during the sterilization process. For example, amino acids in the presence of dextrose undergo the browning reaction to form a combination that may not support growth of the test organism ('7). The stability of any required reducing agent and the influence of the medium upon the substance under assay must also be studied. Liquid media for antibiotic tube assays are generally capable of supporting luxuriant growth of the test organism. However, to increase the susceptibility of an organism to a given antibiotic, it is occasionally necessary to limit the nutrients t o the point where the rate of growth is actually somewhat decreased.
PREPARATION
OF SAMPLE SOLUTION
An organism used in an analytical method generally utilizes or is inhibited by the free form of the factor under assay. Extraction of a growth factor from a complex material must be made to separate it in the free form. Pantothenic acid as it exists in coenzyme A cannot be utilized by L. plantarum. Because acid or alkaline hydrolysis is not acceptable in this case, hydrolysis must be conducted by using a duel enzyme system (19, 13, 24-96). I n the assay for an essential nutrient, where the test substance is stable, dilute acid is usually employed for efficient extraction of the substance, and for hydrolysis of starch or other carbohydrates that may stimulate growth of the organism. Esters of certain antibiotics are not as active as the free antibiotics. Chloramphenicol palmitate is inactive in vitro and must be hydrolyzed in the body before it is fully active. Triacetyloleandomycin has only a fraction of the in vitro activity of oleandomycin base, but is hydrolyzed in the body to the more active form. Erythromycin ethyl carbonate must be hydrolyzed in 40% aqueous methanol to liberate the active antibiotic. Benzylpenicillin diethylamino ethyl ester must be hydrolyzed a t p H 8 before it is microbiologically active. A complex sample material may impart into the assay solution substances that can cause stimulation or growth inhibition of the test organism. Soluble protein and fatty substances are examples of materials that stimulate the growth of L. casei in the riboflavin assay, and they must be removed from the assay solution. I n the extraction process, the stability of the substance under assay must be assured. This is illustrated in the stabilization of vitamin B12by the use of sodium metabisulfite (15). For convenience in amino acid determinations, hydrolysis of protein may be conducted in open vessels contained in a vacuum desiccator, thereby avoiding the need for using sealed vials (23). Because of the variety of materials encountered in analytical microbiology, the analyst must use ingenuity in determining the precautions needed to assure complete extraction and stability of the substance under assay. In the analysis of mixtures of antibiotics it is occasionally necessary either to inactivate an interfering component or to separate the antibiotics. Penicillin is inactivated by the specific enzyme, penicillinase. While no such enzymes are available for other antibiotics, other chemical means can be used. Streptomycin can be inactivated with carbazide, dihydrostreptomycin 1%ith barium hydroxide, the tetracyclines by heating a solution a t pH 8 a t 100" C. for 30 minutes, and erythromycin VOL. 31, NO. 6, JUNE 1959
973
b y heating a solution a t p H 2 at 37" C. for 3 hours. Separation by differential solubility is successful in some inst.ances. T o facilitate this process, Weiss, Andrew, and Wright (28) have listed the solubilities of 18 antibiotics and various salts thereof, 32 in all, in 24 solvents. PREPARATION
OF ASSAY TUBES
The order in which the reagents are added is important for the success of the method. Three approaches are in general use. 1. Because many growth factors are heat-stable, they may be sterilized together with the basal media. Aliquots of the standard or sample solution (nonsterile) are placed in clean, but nonsterile tubes, the nonsterile medium is added, and the mixtures are sterilized by autoclaving. After cooling, each tube is seeded with a drop of the test organism suspension. 2. If the factor is heat-labile or reacts adversely with the medium during sterilization, the basal medium is placed in the tubes, sterilized, and cooled. Aliquots of sterile standard or sample solutions are added to the tubes and the mixtures seeded with a drop of the test organism suspension. 3. Because antibiotics are usually heat-labile, aliquots of the sample solution are placed in sterile test tubes. The test organism is incorporated into the sterile medium and this mixture then added to the tubes. MEASUREMENT OF TEST ORGANISM RESPONSE
Both visual and electrometric titration procedures have been used in determining acidity as a measure of response of the test organism. Although turbidity readings of the assay tubes have, for a number of years, been used as a measure of response, only within recent years have suitable instruments become available to ensure precise measurements by the procedure. Photometric instruments with a narrow wave band are essential for this purpose. Directions and precautions to be observed have been described ( 2 , 3, 5 , 16, 62, 2 7 ) . When titrimetric and turbidimetric procedures are compared with respect to the response of the test organism, with nicotinic acid the amount of the vitamin required for the turbidimetric procedure is only one tenth that required for the titrimetric procedure. A somewhat similar, though not such a great, decrease in requirement also occurs in the turbidimetric assay for some other B vitamins and some amino acids. N o difference be-
974
ANALYTICAL CHEMISTRY
tween the requirement for these two types of procedures exists in the case of vitamin BW I n low-potency materials where extraneous turbidity or color is present in the assay solution in a n amount that would interfere with turbidimetric measurements, the titrimetric procedure is required. I n the assay of small quantities of antibiotics, in materials such as animal feeds, where there is substantial turbidity or color in the assay solution, the plate assay procedure is required. TECHNIQUE
To conduct tube assays successfully, the analyst must have a good background in analytical techniques. The terms used with respect to accuracy of measurement, and other points in the methods where great precision is required, can be interpreted only by one who is well versed in analytical procedures. The greater the precision desired, the more rigid must be the emphasis upon precision in sampling and measuring, and upon cleanliness of glassware and apparatus used. Glassware used in measuring and diluting standard and assay solutions should be within the tolerances established by the National Bureau of Standards. Automatic pipetting machines, if used, should deliver within the accuracy prescribed for precision glassware. All glassware and pipetting machines must be meticulously clean, in order to avoid contamination with micro amounts of substances that may stimulate or inhibit growth of the organism. Difficulties may arise in the extraction process if proper pH adjustments are not made or if there is a lack of disintegration of the sample. Uniform conditions must be observed in the sterilization and subsequent cooling of assay tubes. A lack of uniformity, such as improper autoclaving, or too close packing of the tubes will produce erronenus results. The temperature of the assay tubes during the incubation period must be maintained within the range of =!=0.5"C. Critical consideration must be given to the accuracy of instruments used in measuring t,he response of the test organism. These examples merely illustrate a few of the many steps encountered in microbiological tube methods in which precision is required. I n summary, tube methods in analytical microbiology present problems relating not only to the choice, care, and growth of the test organisms, but also to utilization of the best analytical
techniques available in a chemical laboratory. LITERATURE CITED
(1) Arret, B., Woodward, M. R. Wintermere, D. M., Kirshbaum, Antibiotics a n d Chemotherapy 7, 545-8 (1957). (2) Assoc. Offic. Agr. Chemists, J . Assoc. O j i c . Agr. Chemists 41, 61-71 (1958). (3) Assoc. Offic. Agr. Chemists, Washington, D. C., "Official Methods of Analysis" 8th ed., 1955. (4) DeNunzio, J. C., Bowman, F., Kirshbaum, A., Antibiotics and Chemotherapy 4 , 300-3 (1954). (5) Federal Antibiotic Regulations, Title 21, C. F. R., 141, 146, U. S. Govern-
A.,
ment Printing Office, Washington, D. C. (6) Federal Food, Drug, and Cosmetic Act, U. S. Dept. Health, Education, and Welfare, Food and Drug Administration, U. S. Government Printing Office, Washington, D. C. (7).Friedman, L., Kline, 0. L., J . Nutritton 40, 295-307 (1950). (8) Gavin, J. J., A p p l . Aficrobiol. 4, 323-31 (1956). (9) Ihid., 5, 235-43 (1957). (10) Hutner, S. H.,Cury, .4.,Baker, H., AN.kL. CHEM.30, 849-67 (1958). (11) Kirshbaum, A,, Kramer, J., Arret, B., Wintermere, D. hi., Antibiotics and Chemotherapy 6, 504-10 (1956). (12) Loy, H. W.,Jr., J . Assoc. Oflc. Aar. Chemists 37. 779-81 (1954). (13)"Ibid., 38, 7ioLii (i955j. ' (14) Lov, H. W.,Jr., Haggerty, J. F., Kline: 0.L., Ibid., 35, 161-8 (1952). (15)Ihid., p . 169-74. W.,Jr., Parrish, W. P., (16) Loy, Srhiaffino. ..~~~ S. S.. Ibid.. 39. 172-9 119561. (17).Natioial Fo;mulary, iOth ed.; J. B. Lippincott Co., Philadelphia, Pa., 1955. (18) Parrish, W. P., Loy, H. TV., Jr., Kline, 0. L., J.. Assoc. Ofic. Agr. Chemists 38, 506-13 (1955). (19) Zbid., 39, 157-66 (1956). (20) Peterson, C. S., Carroll, R. W., Science 123, 546-7 (1956). (21) Schiaffino, S. S., J . Assoc. Ofic.Agr. ' Chemists, in press. (22) Schiaffino, S. S., McGuire, J. J., Loy, H. W.,Ibid., 41, 420-3 (1958). (23) Spies, J. P., Coulson, E. J., Chambers, D. C., Bernton, H. s., Stevens, H., Shimp, J. H., J . Am. Chem. SOC. 73, 3995-4001 (1951). (24) Toepfer, E. W., J . Assoc. Ofic. A g r . Chemists 40, 853-5 (1957). (25) Toepfer, E. W.,Zook, E. G., Orr, 51. L., Richardson, L. R., Agr. Handbook No. 29, U. S. Dept. Agr., 1951, U. S. Government Printing Office,
2
Washington, D. C.
(26) Toepfer, E. W.,Zook, E. G., Richardson, L. R., J . Assoc. Ofic. Agr. Chemists 37, 182-90 (1964). (27) U. S. Pharmacopeia, 15th revision,
Mack Publishing Co., Easton, Pa.
(28) Weiss, P. J., Andrew, 51. L., Wright, FIT. W., Antibiotics and Chemotherapy 7, 374-7 (1957). (29) Wintermere, D. hi., Eisenberg, W. H.. Kirshbaum. A.. Ibid.,, 7,. 189-92 (1957). I
,
RECEIVEDfor review January 12, 1959. Accepted March 23,1959.
