Early manuals on the use of penicillin, therefore, recommended injections of penicillin as frequently as every three hours, and dosing penicillin has been described as being similar to trying to fill a bath with the plug out. This is no longer required since much larger doses of penicillin are cheaply and easily available; however, some authorities recommend the use of continuous penicillin infusions for this reason. Resistance
When Alexander Fleming discovered the crude penicillin in 1928, one important observation he made was that many bacteria were not affected by penicillin. This phenomenon was realised by Ernst Chain and Edward Abraham while trying to identify the exact of penicillin. In 1940, they discovered that unsusceptible bacteria like Escherichia coli produced specific enzymes that can break down penicillin molecules, thus making them resistant to the antibiotic. They named the enzyme penicillinase. Penicillinase is now classified as member of enzymes called β-lactamases. These β-lactamases are naturally present in many other bacteria, and many bacteria produce them upon constant exposure to antibiotics. In most bacteria, resistance can be through three different mechanisms: reduced permeability in bacteria, reduced binding affinity of the penicillin-binding proteins (PBPs) or destruction of the antibiotic through the expression of β-lactamase. Using any of these, bacteria commonly develop resistance to different antibiotics, a phenomenon called multi-drug resistance. The actual process of resistance mechanism can be very complex. In case of reduced permeability in bacteria, the mechanisms are different between Gram-positive and Gram-negative bacteria. In Gram-positive bacteria, blockage of penicillin is due to changes in the cell wall.
The reference standard was a large batch of impure penicillin kept in Oxford. The assay was later modified by Floreys group to a more reproducible "cup assay": in this assay, a penicillin solution was defined to contain one unit/ml of penicillin when 339 microlitres of the solution placed in a "cup" on a plate of solid agar produced a 24 millimetre zone of inhibition of growth of Oxford Staphylococcus. : 107 
First International Standard (1944). A single 8 gram batch of pure crystalline penicillin G sodium was stored at The National Institute for Medical Research at Mill Hill in London (the International Standard). One penicillin unit was defined at 0.6 micrograms of the International Standard. An impure "working standard" was also defined and was available in much larger quantities distributed around the world: one unit of the working standard was 2.7 micrograms (the amount per unit was much larger because of the impurities). At the same time, the cup assay was refined, where instead of specifying a zone diameter of 24 mm, the zone size were instead plotted against a reference curve to provide a readout on potency. Second International Standard (1953). A single 30 gram batch of pure crystalline penicillin G sodium was obtained: this was also stored at Mill Hill. One penicillin unit was defined as 0.5988 micrograms of the Second International Standard.There is an older unit for penicillin V that is not equivalent to the current penicillin V unit.