84 



NATURE, FORMATION, AND ACTIVITIP^S 



organisms, they can be assayed chemically 

 and/or l)iologically (Grove and Randall. 

 1955). 



Chemical Methods 



Chemical methods can be used only after 

 the chemical properties of a given antibiotic 

 are known. Examples of these chemical 

 assays are: (1) use of the blue-colored nin- 

 hydrin reaction to assay antibiotics with 

 free amino groups, such as neomycin; (2) 

 the use of maltol determination to assay 

 streptomycin and mannosidostreptomycin ; 

 and (3) the anthrone test to titer mannosido- 

 streptomycin by determining the amount of 

 mannose present. Stable antibiotics with 

 strong and typical light-absorption spectra 

 can be assayed directly by spectrophoto- 

 metric methods. Chemical methods of assay 

 must be used with caution, since contaminat- 

 ing substances may give false positive values. 

 For example, contaminating amino acids 

 would give a positive ninhydrin reaction and 

 interfere with the chemical assay of neo- 

 mycin by this reaction. 



Biological Alethods 



Biological methods of assay of antibac- 

 terial and antifungal antibiotics are of two 

 general types: (1) dilution and (2) diffusion 

 methods. Dilution assays can be made with 

 or without a standard. It is virtually impossi- 

 ble to make diffusion assays without a 

 standard . 



Dilution assays can be made in both liquid 

 and solid media. The use of solid media 

 permits the assay of a given antibiotic 

 against a number of test organisms with 

 minimal labor. The dilution assay on solid 

 media, one of which was described by Waks- 

 man and Reilly (1945), is a good procedure 

 for use without a standard. Activity is ex- 

 pressed in terms of the minimal amount of 

 antibiotic that will prevent growth of a test 

 organism. The measure can be expressed in 

 micrograms per milliliter or in dilution units. 



Dilution assays in li(iuid media are well 

 suited to statistical analysis; a standard is 

 usually re(|uired. In such an assay, the 

 turbidity caused by the growth of a bac- 

 terium or a yeast is measured and the value 

 of unknown preparations is determined by 

 estimating the amount of unknown neces- 

 sary to permit the same growth as that per- 

 mitted by a known amount of the standard. 

 Factors influencing the results of dilution 

 assays are: (1) the composition of the me- 

 dium (especially its pH and salt content), 

 and (2) the age and number of cells of the 

 test organisms used to inoculate the medium. 

 Diffusion assays are carried out by placing 

 cylinders or filter paper discs containing 

 various amounts of an antibiotic solution on 

 the surface of an agar layer seeded with a 

 test organism. Comparison of the sizes of 

 sterile zones produced by solutions of un- 

 known concentration with those produced by 

 known solutions of a standard permits 

 estimation of the potency of unknown prepa- 

 rations. 



Factors influencing diffusion assays are: 

 (1) the thickness and evenness of the agar 

 layer, (2) the rate of evaporation of water 

 from the agar layer, (3) the composition of 

 the agar medium (especially its pH and salt 

 content), (4) the nature of the solvent con- 

 taining the antibiotic samples (especially 

 pH and salt content), and (5) the method of 

 seeding the agar layer with the test organ- 

 ism, sharper zones of inhibition being formed 

 if a thin agar seed layer is superimposed on 

 a thicker unseeded layer of agar. 



A comparative study was recently made 

 of the relationship between the minimal 

 inhibiting concentration of an antibiotic by 

 the serial dilution method and the diameter 

 of the zone of inhibition by the single-disc 

 method. It was found to correspond to a 

 straight line, with the exception of spiramy- 

 cin for which the values fit a second order 

 equation (Ericsson et al., 19()0). The meas- 

 urement of antibiotic concentrations in 



