Microbiological assay methods

Nearly all microbiological assays for pterinoids have been by means of Streptococcus faecalis, Lactobacillus casei, Pediococcus cerevisiae and, for biopterin, Crithidia fasdculata. The subject of microbiological assays was reviewed (Hutner etal, 1958) therefore this section is brief. 

The need is acute for a simple, comprehensive microbiological assay for folio compounds. One way would be to assay PABA after liberating it with acid or alkali. Several potential assay bacteria have been mentioned, e.g. Acetdbactor suboxydans and L. plantarum 17-5. In using Tetrahymena for assay purposes one is substituting enzymatic liberation of FA compounds for chemical hydrolysis of FA to PABA the two methods may be complementary. The response of a thermophilic Tetrahymena (Holz et ai., 1959) to known bypas g compounds is under investigation. The results encourage the hope that a practical procedure is not too remote (Holz, 1958). 

Familiarity with an assay organism usually uncovers quirks demanding side investigations this applies even to the classical S. faecalis. When it was transferred a few times on yeast extract-acetate-glucose, in 6 weeks it responded hardly or not at all to PGA, yet it responded to something in yeast (Jones and Morris, 1949). This observation remains unexplained. MasiasR (1956) has noted variations in response to FA depending on whether acetate or citrate pH buffer was used. Despite the fact that most of the folic compounds in blood seem to be unavailable to S. faecalis, e.g. 

Utilization of 6-Fobmtl-FH Poltolutahateb bt Streploeoeeus faecalis and Pedioeoccus eerevisiae 

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Table XII. Microbiological Assay Methods First Used to Determine Amino Acids...

It may be determined by various chemical methods, but its analysis in body fluids lends itself to a microbiological assay method. 

Malabsorption Syndrome, with Special Reference to the Effects of Wheat Gluten (Frazer), 5, 69 Mellituria, Nonglucose (Sidbury), 4, 29 Microbiological Assay Methods for Vitamins (Baker and Sobotka), 5, 173 Organic Acids in Blood and Urine (Nordmann and Nordmann), 4, 53 Paper Electrophoresis Principles and Techniques (Peeters), 2, 1 Paper Electrophoresis of Proteins and Protein-Bound Substances in Clinical Investigations (Owen), I, 238 Parathyroid Function and Hyperparathyroidism, Biochemical Aspects of (Nordin), 4, 275... 

Vitamin Br, Absorption, Distribution, and Excretion, Physiology and Pathology of (Grasbeck), 3, 299 Vitamins, Microbiological Assay Methods for (Baker and Sobotka), 5, 173... 

Overall, microbiological assay methods have been the most sensitive of all assay systems and the ability to measure residues in the ppb to ppm range is common and has been for over 20 years (1 ). However, most of the residue assay systems lack specificity and require confirmation by spectral systems for a proper identification of the individual antibiotic or the antibiotic family. 

Microbiological Assay Methods for Vitamins Herman Baker and Harry Sobotka... 

It is an essential condition of biological assay methods that the tests on the standard preparation and on the sample whose potency is being determined should be carried out at the same time and, in all other respects, under strictly comparable conditions. The validation of microbiological assay method includes performance criteria (analytical parameters) such as linearity, range, accuracy, precision, specificity, etc. 

Conventional plate assays require several hours incubation and consequently the possibility of using rapid microbiological assay methods has been studied. Two such methods are ... 

Gentamycin is a widely used antibiotic but presents a number of undesirable side-effects when administered in high doses. Thus, it is vital to be able to monitor serum levels accurately. In the past this has been achieved using a number of assay methods other than HPLC. However, recently an HPLC method which compares favourably with microbiological assay methods has been described (Marples and Oates, 1982). This system utilises pre-column derivatisation with o-phthal-aldehyde to facilitate fluorimetric detection with subsequent resolution on a Spherisorb 5-ODS reversed phase support using isocratic conditions with methanol-water-EDTA (Fig. 11.9.4). Baseline resolution of tobramycin and four gentamycin components was achieved by this method. 

Barea JM, Navarro E, Palomares A, Montoya E (1974) A rapid microbiological assay method for auxins, gibberellic acid and kinetin using yeast. J Appl Bacteriol 37 171-174... 

In addition to the reviews on vitamin B12 which have been published in previous volumes of Vitamins and Hormones (Jukes and Stokstad, 1951 Shive, 1951 Folkers and Wolf, 1954), there are several others, notably the treatise by Jukes, Williams et al. (1954). Other reviews are concerned with the chemistry and nutritional roles of vitamin B12 (Smith, 1954) and its involvement in the metabolism of nucleic acids (McNutt, 1952). Microbiological assay methods were described in detail by Hoff-J0rgensen (1954). 

HPLC techniques present several potential advantages over earlier spectro-photometric, fluorometric, and official microbiological assay methods, which are very tedious and often difficult to perform. These advantages include simple sample preparation, direct analysis of compounds without any derivatization, and potential simultaneous determination of several compounds in a single run, in addition to a reduction of analysis time, excellent precision, and relatively good sensitivity. 

This method is based on the same principles as the many microbiological assay methods that use lactic acid bacteria (44), except that titrimetric determination of response cannot be used. One-half the maximum growth is obtained in tubes containing about 0.1 m/xg. of Bis per milliliter. The method described in this section is a modification of the methods of Davis and Mingioli (11) and Burkholder (7). 

The amino acid contents of different preparations of peanut protein (calculated to 16% of nitrogen) are given in Table III. The analyses of the protein isolated by the method of Arthur and others (7,10) were reported by Murphy and Dunn (154). They obtained the values by microbiological assay methods. The analyses of the other preparations were compiled from various literature sources by Guthrie and others (92,100). 

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