Types of Penicillin and Assay

Although we refer to the drug penicillin as a definite product, it should be noted that molds are known to produce at least six different penicillins. Four of these, commonly referred to in this country as F, G, X, and K, are produced under natural culture conditions by members of the P. notatum-chrysogenum group. They represent different chemical compounds and possess different physical and chemical properties. Two or more of these penicillins may be produced in the same culture solution and may, in varying proportions, be contained in the final dried product of commerce. Whereas penicillin F was the first penicillin to be studied and crystallized, experience soon showed that penicillin G was chemically more stable and hence more easily recovered. Furthermore, the culture (NRRL 832) first employed for the submerged production of the drug yielded penicillin mostly of type G. For these reasons, commercial penicillin soon came to represent primarily penicillin of this type.

The several penicillins differ in their inhibitory effect upon susceptible bacteria, some being relatively more effective against particular species than others. Besides, they differ markedly in their behavior within the animal body. These differences render some penicillins more effective than others in combating disease. For example, pure sodium penicillin K when tested in vitro against Staphylococcus aureus shows an activity of 2200-2300 u/mg. in contrast to pure sodium penicillin G, which contains 1667 u/mg. When tested in vivo, however, penicillin K is rapidly destroyed and adequate blood levels are difficult to maintain. It is not a satisfactory drug.

On the other hand, penicillin X, which is chloroform-insoluble, and hence can be obtained free from the other types without great difficulty, has been found to be more effective against streptococci, pneumococci, and gonococci than commercial penicillin containing mostly penicillin G. Believing that the production of penicillin X might be of importance from a clinical point of view, we have successfully developed a mold that produces substantially increased amounts of this type of penicillin. From a culture that produced penicillin X in yields approximating 15 percent of the total, an ultraviolet-induced mutation has been developed that produces penicillin X in 50-percent yields. It remains to be seen whether penicillin X will attain significance as a distinct drug.

Before October 1944, penicillin was measured in terms of the Oxford unit the amount of penicillin which, when dissolved in 1 cubic centimeter of water, gave the same inhibition as an arbitrary standard, established by Floret' and associates, which produced zones of inhibition averaging 24 mm. in diameter. In October 1944 a conference was held in London under the auspices of the Health Organization of the League of Nations for the purpose of establishing an international standard and an international unit of penicillin. Pure sodium penicillin G was adopted as the international standard and the international unit was defined as the specific penicillin activity contained in 0.6 microgram of the international penicillin standard. Pure sodium penicillin G, therefore, by definition contained 1,667 units per milligram. An international standard was subsequently prepared by F. H. Stodola and J. L. Wachtel, chemists at the laboratory, by the recrystallization of pooled samples of sodium penicillin G contributed by manufacturers in the United States and Great Britain. Two strains of Staphylococcus aureus were designated as standard test organisms, namely the Food and Drug Administration No. 209P (NRRL B-313) and the strain employed by Heatley (NRRL B-314).

In the assay, or measurement, of penicillin potencies, the so-called Cylinder plate method is generally used. Modifications and improvements have been made since 1941 by W. H. Schmidt, in charge of penicillin assays at the laboratory, and others, but the method remains basically the same as that originally developed by Dr. Heatley. A plate containing nutrient agar is warmed to 37 to 40 C., evenly flooded with a suspension of the test bacteria in warm agar ( approximately 45 C.) , and allowed to solidify. On the surface of this plate are placed a number of hollow cylinders, which make a water-tight seal. Into these are pipetted small amounts of the solutions to be tested. A part of the cylinders in every plate contains a standard of known penicillin content for purposes of comparison.

The plates are then incubated overnight at the optimum temperature for the test bacterial species. The penicillin contained in the cups diffuses into and through the underlying agar, inhibiting the growth of the bacteria in circular zones. The zones of inhibition for all samples are then measured in millimeters, and the potencies of the unknown are determined by comparing their average diameters with those produced by the samples of known penicillin content. The method is reasonably accurate for measuring aqueous solutions containing from 1 to 4 units per milliliter. To attain these levels of concentration, the samples to be tested are diluted with sterile phosphate buffer of pH 6.0.

A modification of the above method, now in common use, involves the use of paper disks of standard dimensions that are dipped into the penicillin solutions to be tested and placed on the surface of seeded agar plates. The plates are then incubated and zones of inhibition measured and evaluated as in the cylinder-cup technique. Two other methods of assay have been successfully used, the serial dilution method and the turbidimetric method.