Production in the Past

How was penicillin first made?

It took over 5 years to turn the Penicillium notatum mould into the penicillin we know today. It was a long, tedious often frustrating task requiring the brilliant minds of many scientists led by Howard Florey.

The production of penicillin by Florey's team was undertaken in stages:

Stage 1. Growing the Mould

Howard Florey and Ernest Chain began to investigate Alexander Fleming’s Penicillium notatum mould. They knew the mould produced an antibacterial substance and wanted to find out more about it. The mould grew slowly and had very special needs. The scientists began to grow it in different media containing marmite, malt extract, meat and yeast extracts. They studied the rate of growth of the mould and thought about what effect it would have on different bacteria. This work on the rate of growth continued while all the other experiments were taking place, so they could continue to increase the production of penicillin.

Stage 2. Testing

Florey, Chain and the team began to take the juice from under the mould and test it on bacteria. Norman Heatley designed and made a special dish so they could easily test how mould affected different bacteria. They became convinced that penicillin could kill harmful bacteria. How to extract and purify the penicillin from the brown juice was their next challenge.

Stage 3 Extraction

The penicillin was very unstable and short-lived. The scientists worked hard to find a better way of extracting it. At first, the mould juice was simply filtered through parachute silk. Chain found a way to extract the penicillin from the mould juice by passing it through different solvents. He worked out a way to put the mould juice through a series of chemical steps. Each step isolated, purified and concentrated the penicillin in the liquid. This worked, however not as well as they would have liked. At this stage Heatley devised a clever trick called the solvent transfer process.

" Heatley’s method of reversing the chemical path which Chain had followed and of extracting the penicillin from the mould juice into ether and then passing it back through a chemically neutral buffer material produced a superior extract..." [ Source: Bickel ]

This meant that they were able to obtain more penicillin from the mould. They managed to obtain 100 grams of brown powder containing penicillin which could be used to conduct further experiments.

The first extraction plant consisted of inverted bottles with separating funnels and to minimise destruction of the penicillin during water to solvent extractions they needed to do it in cool conditions. They used a cramped store room or the roof of the laboratory. This was very uncomfortable work especially in the middle of winter. The second extraction plant was developed a year later.

Stage 4 Experimentation

Florey's team began to experiment with this brown powder containing penicillin, to see what effect it had on other organisms . Chain dissolved it in water and injected it into two mice. The mice showed no ill effects from the injected powder. That was amazing as the powder was almost 99% impure. At this time, Chain thought it was all penicillin, however any one of the impurities could have killed the mice. The outcome was one of the many strokes of good luck to go the team’s way. The team now began to experiment with the powder to see if it was toxic. They increased the strength and tested it on blood, hormones, cells, etc.

Stage 5 The Mice Experiment

One morning Florey injected a lethal dose of bacteria Streptococcus pyogenes into 8 mice. Four infected mice were given injections of penicillin. Florey and his laboratory assistant James Kent watched the mice carefully. Later that night Heatley arrived and continued to keep watch over the mice. By 3.30 am the 4 mice that did not receive any penicillin were all dead. The bacteria had killed them. All the mice that had received the penicillin were healthy. This proved that penicillin killed deadly bacteria without harming animals. Florey said 'It looks like a miracle".

Stage 6 Increased Production

Now the race was on to make enough penicillin to begin testing it on humans. They needed to grow a lot of mould so they used any container they could find. A flat container was ideal so the mould could grow over a large surface and they could drain the mould juice from under it. They tried biscuit tins, trays, dishes, pans and lids, however bed pans were the best. Thus they designed and made special pottery flasks shaped like bed pans. Heatley made a trolley that held six flasks that could be tipped so the mould juice could be drained off without killing the mould.

During this time the scientists had problems with contamination and at times the penicillin yield from batches of mould was virtually nothing. They continued to work to solve these problems.

The second extraction plant was made almost entirely from junk. It included a bath, milk cans, a milk cooler, a letter box, aquarium pumps and various taps and valves. The mould was grown in culture vessels, filtered then cooled and passed through the solvents to extract the penicillin and purify it. The solvents were separated and the penicillin freeze dried.

Stage 7 Human Testing

The testing to see if penicillin was toxic to humans continued for a long time. It was not until February 1941 that they were ready to inject penicillin into a person who had an infection. Albert Alexander was chosen to receive the first dose of penicillin. He was a police officer whose body was so badly infected he was near death. He was given penicillin by intravenous drip and within 24 hours he was showing remarkable signs of recovery. In 4 days he appeared to be a lot better, however it was at this time the penicillin ran out. They even filtered his urine to retrieve any penicillin so they could continue the treatment, however Albert Alexander died. Florey was determined not to test penicillin on another adult until they had enough to completely cure the patient.

Stage 8 The American Connection

Britain was at war with Germany, therefore money for research was difficult to obtain. Florey and Heatley traveled to the United States of America to get assistance with increasing the production of penicillin, so they could test it properly on humans. They received assistance and it was in America they found corn steep liquor , that proved to be a better food source for growing the mould. It was still very difficult to grow and manufacture and a search went out for people to bring in any Penicillium notatum they found to see if they could find a better strain of mould. A woman they nicknamed ‘Mouldy Mary’ found a mouldy melon that had a mould on it called Penicillium chrysogenum. This turned out to be a better mould strain as it produced higher yields of penicillin.

Heatley stayed in America and with other scientists, worked on growing the mould deep inside tanks filled with nutrient. This increased the production of penicillin but they still did not make enough to begin human trials.

Stage 9 Back in England

In England, Florey’s team continued to use their extraction plant to produce penicillin. Ernest Chain continued to refine the extraction and purification of the penicillin. In 1943, the British Government gave Florey and the team all the resources they needed. The Sir William Dunn School was turned into a penicillin production factory. A team of women were trained as mould farmers. They cared for hundreds of jars of mould. Increased means of extraction and purification meant there was enough penicillin to treat wounded soldiers. It was an outstanding success and 95% of war wounds treated with penicillin healed.

Work continued on refining the production process and by 1945 enough penicillin was being produced world wide to treat patients with bacterial infections.

Timeline of Penicillin

July 1928
Alexander Fleming discovered the antibacterial properties of Penicillium notatum.
He named it penicillin and experimented with it.

June 1929
Alexander Fleming published his research in the British Journal of Experimental Pathology.
He then gave up because the mould was too difficult to work with and he did not believe it showed any promise.

May 1935
Howard Florey became Professor of Pathology at Oxford University and worked at the Sir William Dunn School of Pathology. He became interested in lysozymes . Florey began to build a team of scientists to work with him on investigating antibacterial substances.

1938
Florey and Ernest Chain read about Fleming’s paper and began investigating and testing Penicillium notatum.

September 1939
World War II began.

March 1940
Ernest Chain injected the raw, brown, impure powder obtained from the Penicillium notatum into mice. They showed no ill-effects from the powder even though it was 99% impure. The team continued to work on the difficult task of extracting the penicillin from the mould.

May 1940
Florey and his team first experimented on mice. The mice were given lethal doses of
bacteria and then injected with penicillin. The treated mice survived, the untreated mice died. The team put all their effort into increasing yields and production of penicillin.

February 1941
Penicillin was first used on a human with severe infections. Albert Alexander, a policeman, began to improve rapidly, however he died because the penicillin ran out before the bacteria were totally destroyed.

June 1941
Florey and Heatley went to The United States of America to ask for assistance to enable them to make enough penicillin to begin clinical trials .

Late 1941
In the United States corn steep liquor was found to increase the yield of penicillin tenfold.

August 1941
The American Government and companies began the commercial production of penicillin.

September 1943
The British Government agreed to fund the production of penicillin.

1943
Commercial production of penicillin began.

October 1943
The first supply of penicillin was given to the British army.

March 1944
Penicillin was manufactured in Australia and there was enough produced to supply the
civilian population as well as the army. This was the first time in the world it was made available to the general population.

1960s
Due to the problem of penicillin resistant bacteria, semi-synthetic penicillin was developed.

1998
Penicillin is still widely used throughout the world to kill bacteria that cause infections.

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