How do we know Microbiology in perspective to the golden age and beyond

We have learnt an astonishing amount about the invisible world of microorganisms, particularly over the last century and a half. How has this happened? The penetrating insights of brilliant individuals are rightly celebrated, but a great many 'breakthroughs' or 'discoveries' have only been made possible thanks to some (frequently unsung) development in microbiological methodology. For example, on the basis that 'seeing is believing', it was only when we had the means to see microorganisms under a microscope that we could prove their existence.

Microorganisms had been on the Earth for some 4000 million years, when Antoni van Leeuwenhoek started out on his pioneering microscope work in 1673. Leeuwen-hoek was an amateur scientist who spent much of his spare time grinding glass lenses

A pathogen is an organism with the potential to cause disease.

Leeuwen Hoeks Microscope

Figure 1.1 Leeuwenhoek's microscope. The lens (a) was held between two brass plates and used to view the specimen, which was placed on the mounting pin (b). Focusing was achieved by means of two screws (c) and (d). Some of Leeuwenhoek's microscopes could magnify up to 300 times. Original source: Antony van Leeuwenhoek and his little animals by CE Dobell (1932)

Figure 1.1 Leeuwenhoek's microscope. The lens (a) was held between two brass plates and used to view the specimen, which was placed on the mounting pin (b). Focusing was achieved by means of two screws (c) and (d). Some of Leeuwenhoek's microscopes could magnify up to 300 times. Original source: Antony van Leeuwenhoek and his little animals by CE Dobell (1932)

to produce simple microscopes (Figure 1.1). His detailed drawings make it clear that the 'animalcules' he observed from a variety of sources included representatives of what later became known as protozoa, bacteria and fungi. Where did these creatures come from? Arguments about the origin of living things revolved around the long held belief in spontaneous generation, the idea that living organisms could arise from non-living matter. In an elegant experiment, the Italian Francesco Redi (1626-1697) showed that the larvae found on putrefying meat arose from eggs deposited by flies, and not spontaneously as a result of the decay process. This can be seen as the beginning of the end for the spontaneous generation theory, but many still clung to the idea, claiming that while it may not have been true for larger organisms, it must surely be so for minute creatures such as those demonstrated by Leeuwenhoek. Despite mounting evidence against the theory, as late as 1859, fresh 'proof' was still being brought forward in its support. Enter onto the scene Louis Pasteur (1822-1895), still arguably the most famous figure in the history of microbiology. Pasteur trained as a chemist, and made a lasting contribution to the science of stereochemistry before turning his attention to spoilage problems in the wine industry. He noticed that when lactic acid was produced in wine instead of alcohol, rod-shaped bacteria were always present, as well as the expected yeast cells. This led him to believe that while the yeast produced the alcohol, the bacteria were responsible for the spoilage, and that both types of organism had originated in the environment. Exasperated by continued efforts to substantiate the theory of spontaneous generation, he set out to disprove it once and for all. In response to a call from the French Academy of Science, he carried out a series of experiments that led to the acceptance of biogenesis, the idea that life arises only from already existing life. Using his famous swan-necked flasks (Figure 1.2), he demonstrated in 1861 that as long as dust

Swan Necked Flask

Liquid sterilised by boiling.

Liquid allowed to cool.

Dust and microrganisms settle in bend of flask neck. Liquid remains sterile.

Liquid sterilised by boiling.

Liquid allowed to cool.

Dust and microrganisms settle in bend of flask neck. Liquid remains sterile.

Flask tilted, allowing liquid to come into contact with deposit in neck.

Flask tilted, allowing liquid to come into contact with deposit in neck.

Pasteur Swan Neck Flask
Liquid turns cloudy due to microbial growth.

Figure 1.2 Pasteur's swan-necked flasks. Broth solutions rich in nutrients were placed in flasks and boiled. The necks of the flasks were heated and drawn out into a curve, but kept open to the atmosphere. Pasteur showed that the broth remained sterile because any contaminating dust and microorganisms remained trapped in the neck of the flask as long as it remained upright particles (and the microorganisms carried on them) were excluded, the contents would remain sterile. This also disproved the idea held by many that there was some element in the air itself that was capable of initiating microbial growth. In Pasteur's words'____the doctrine of spontaneous generation will never recover from this mortal blow. There is no known circumstance in which it can be affirmed that microscopic beings came into the world without germs, without parents similar to themselves.' Pasteur's findings on wine contamination led inevitably to the idea that microorganisms may be also be responsible for diseases in humans, animals and plants.

The notion that some invisible (and therefore, presumably, extremely small) living creatures were responsible for certain diseases was not a new one. Long before microorganisms had been shown to exist, the Roman philosopher Lucretius (~98-55 bc) and much later the physician Girolamo Fracastoro (1478-1553) had supported the idea. Fracastoro wrote 'Contagion is an infection that passes from one thing to another' and recognised three forms of transmission: by direct contact, through inanimate objects and via the air. We still class transmissibility of infectious disease in much the same way today. The prevailing belief at the time, however, was that an infectious disease was due to something called a miasma, a poisonous vapour arising from dead or diseased bodies, or to an imbalance between the four humours of the body (blood, phlegm, yellow bile and black bile). During the 19th century, many diseases were shown, one by one, to be caused by microorganisms. In 1835, Agostino Bassi showed that a disease of silkworms was due to a fungal infection, and 10 years later, Miles Berkeley demonstrated that a fungus was also responsible for the great Irish potato blight. Joseph Lister's pioneering work on antiseptic surgery provided strong, albeit indirect, evidence of the involvement of microorganisms in infections of humans. The use of heat-treated instruments and of phenol both on dressings and actually sprayed in a mist over the surgical area, was found greatly to reduce the number of fatalities following surgery. Around the same time, in the 1860s, the indefatigable Pasteur had shown that a parasitic protozoan was the cause of another disease of silkworms called pebrine, which had devastated the French silk industry.

The first proof of the involvement of bacteria in disease and the definitive proof of the germ theory of disease came from the German Robert Koch. In 1876 Koch showed the relationship between the cattle disease anthrax and a bacillus which we now know as Bacillus anthracis. Koch infected healthy mice with blood from diseased cattle and sheep, and noted that the symptoms of the disease appeared in the mice, and that rod shaped bacteria could be isolated from their blood. These could be grown in culture, where they multiplied and produced spores. Injection of healthy mice with these spores (or more bacilli) led them too to develop anthrax and once again the bacteria were isolated from their blood. These results led Koch to formalise the criteria necessary to prove a causal relationship between a specific disease condition and a particular microorganism. These criteria became known as Koch's postulates (Box 1.1), and are still in use today.

A bacillus is a rod-shaped bacterium.

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