Penicillium and Strain Isolation Procedures

Fig. 1 - Fleming's Plate

Sir Alexander Fleming discovered the effects of Penicillin in 1928. Purely by chance, he noticed that Staphyloccus bacteria would not grow in the vicinity of cultures of the fungus Penicillium notatum that were growing on experimental petri dishes.

Fleming suggested that that the fungus was producing a chemical that prevented the development of cell walls in bacteria (see figures1 and 2). He named this chemical penicillin (see figure 3), and it soon became apparent, in the prevailing climate of the 1940s, that it had the potential to be used as a drug to combat bacterial infections resulting from war injuries. A method needed to be devised quickly to allow penicillin to be produced on a large scale.

Mass Production of Penicillin

Fig. 2 - Penicillium Culture

It soon became clear, however, that Penicillium notatum was not suited to large scale production. Scientists Howard Florey and Ernst Chain attempted to resolve this by exposing the fungus to X-rays to produce mutant strains that could be cultured more successfully.

This, combined with the use of huge aerated fermentation tanks containing corn-steep liquor, and strain isolation techniques, allowed them to achieve a ten-fold increase in penicillin production. Moreover, the discovery of Penicillium chrysogenum, a new and more productive strain of the fungus, resulted in an increase in the yield of penicillin by a factor of 1000. Chain also developed a method of crystallising and concentrating the drug, with the result that by D-Day ,June 6, 1944, there was enough penicillin to meet demands.

What is Strain Isolation?

Strain isolation is a method commonly used by microbiologists to isolate, purify and culture a desired strain of a particular microbe. It can be as simple as picking and re-streaking the microbe on a nutrient agar plate until a pure culture is obtained (the 'dilution' method).

Other methods include investigating the nutrient requirements of the desired microbe and providing only those nutrients on successive culture plates until eventually only the microbe in question remains (the 'selective medium' technique). Fleming used a combination of both these methods to isolate and purify his cultures of Penicillium notatum. Florey and Chain also employed similar methods to culture their mutated Penicillium strains.

Fig. 3 - Chemical structure of Penicillin G

Isolation of Microbes Using the Dilution Method

The original ‘dilution’ method pioneered by Joseph Lister attempts to isolate a single cell of the desired microbe so that it can be cultured on a nutrient medium. Successive dilutions of the microbe in nutrient broths were the earliest technique used to do this, but it has now largely been replaced by a dilution method using a solid substrate.

Here, a sterile needle draws the sample across a culture plate several times. The needle is re-sterilised and a small amount of the sample is taken from the original streaks and spread at right angles to them. This procedure is repeated until the final streaks represent a diluted quantity (ideally single cells) of the sample microbe, which is then cultured.

Isolation of Microbes Using the Selective Medium Technique

This method utilizes the fact that individual microbe species prefer specific nutrients on which to grow. By choosing a particular nutrient medium some cultures will be encouraged to grow while others are inhibited.

Examples include the isolation of the bacterium Pseudomonas fluorescens on a medium containing ammonia and lactate, and the purification of Steptomycetes bacteria using glycerol and arginine substrates. Penicillium chrysogenum has itself been isolated and purified by Rafi and Rahman using either glucose or yeast agar media.

Producing, Isolating and Testing Mutated Strains of Penicillium

New strains of Penicillium mould are still being produced by mutating selected colonies which are then isolated and tested for their effectiveness. Although Florey and Chain used X- rays, mutagens can also include ultraviolet (UV) irradiation or chemicals such as colchicine and ethyl methanesulphonate (EMS).

Penicillin is extracted from colonies of these strains and plated out in the presence of Staphylococcus bacteria. The zones of growth inhibition that result are measured and compared to those of a standard penicillin sample to give an indication of the penicillin’s strength. An alternative method of determining penicillin concentration is to use HPLC (High Performance Liquid Chromatography) assaying techniques.

Fleming's discovery, coupled with Florey and Chain's work in purifying and concentrating penicillin, earned each of them the Nobel Prize in Physiology or Medicine in 1945. Their techniques have been further developed by other scientists in recent decades, with the result that penicillin production is now efficient and economical.

References

Bowden, M.,2002, Howard Florey and Ernst Chain, "Pharmaceutical Achievers," chemheritage.org, accessed 30/3/2010

Mailer, J. and Mason, B.,2001, "Penicillin: Medicine's Wartime Wonder Drug and its Production at Peoria, Illinois," Illinois Periodicals Online, niu.edu

Rafi, M. and Rahman,2002, "Isolation and Identification of Indigenous Penicillium chrysogenum Series," fspublishers.org, accessed 1/5/2010