Friday, November 5, 2010

History of Bacteriology

HISTORY OF BACTERIOLOGY
Bacteria existed long before mankind evolved and bacterial diseases are likely to have coevolved with each species. Many of the bacterial diseases that microbiologists encounter today have been around for as long as humans have e.g Leprosy in the Bible, and in some cases even longer, while others may have developed in recent years. However, doctors and scientists were unaware of the cause of infectious diseases for many centuries. With the beginning of the understanding of microbiology, the importance of bacterial pathogens became apparent.

Early speculations on disease
The idea that disease is caused by something too small to be seen occurs in various scripts before the modern age. In the first century BC the Roman author Varro speculated that some diseases might be caused by tiny animals carried in the air. Varro warned people to avoid marshy places during building work because he thought they contained invisible insects that produced disease by gaining access to the body via the mouth and nostrils.
 
The Greek physician Hippocrates (c 460–377 BC) put forward the notion that disease arose as a result of an imbalance of the four humours that were present in the human body. This imbalance was thought to be caused by miasmas – vapours from either rotting vegetables or rotting bodies, marshy places or polluted rivers (Miasmatic theory). Close contact with any of the above was believed to result in disease. Jacobo Forli dismissed the miasma theory of disease and claimed that it was not possible to get a disease by simply breathing in air alone. He stated that disease arose by breathing in contagious particles that floated in the air, and that these particles created the infection. However, belief in the miasma theory continued from before the first century, through the Roman and Greek periods, right up until around 1500
 
Early diagnostic techniques
Irrespective of whether early doctors subscribed to the miasma theory or the presence of contagious particles, one of the most common diagnostic techniques was uroscopy. The patient was required to produce a urine sample, which would be taken away and examined for appearance, smell and taste. Indeed, sweet tasting urine is a clue to the diagnosis of diabetes. Patients with jaundice will have brown urine and patients with kidney disease may have foamy or red urine. Bloody urine can denote tumours of the urinary tract while white urine can signify infection. Medical practitioners from the earliest times did not overlook these changes. Once the urine examination was complete, the observations were compared with the known colours, smells and tastes recorded on a so-called uroscopy wheel. The doctor would then arrive at a diagnosis and treat the patient accordingly.

From Fracastoro to the golden age

The examination of urine was practised until the time of Girrolamo Fracastoro, an Italian doctor and physiologist who published a paper in 1546 stating that diseases were caused by 'seminaria contagiosa', particles that could be transmitted by direct contact, touch or contaminated fomites. In this respect, Fracastoro’s work is the first evidence of someone thinking through germ theory and the fact that disease could be transmitted by contaminated objects. He also noticed that not all contagious particles were alike, as various diseases attacked different organs. He recorded that smallpox and measles occurred mostly in children, that everyone was attacked once but it was rare for people to have a second attack. Clearly, he recognised evidence of immunity. Fracastoro’s writings have been described as a great landmark in the history of infectious diseases.
 
Theory of spontaneous generation
Fracastoro's views, however, were not universally held. Most scientists and philosophers at that time thought that disease could occur as a result of spontaneous generation i.e some life could arise spontaneously from non living matter (abiogenesis). This theory was based on the observation that meat when left out in the open produced maggots, that mushrooms appeared on rotting wood, that rats and mice emerged from piles of litter and similar phenomena
 
Subsequently, the first serious attack on the idea of spontaneous generation was made in 1668 by Francesco Redi, an Italian physician and poet. At the time, it was widely held that maggots arose spontaneously in rotting meat. Redi, however, believed that maggots developed from eggs laid by flies. He tested his hypothesis by setting out meat in a variety of flasks, some of which were open to the air and some were sealed. As Redi expected, maggots appeared only in the open flasks where flies could reach the meat and lay their eggs.
The results of this experiment did not satisfactory Redi's critics, who claimed that fresh air was needed for spontaneous generation. Redi responded by setting up a second experiment, in which some jars were covered with gauze to allow air to circulate. No maggots appeared in the gauze-covered jars, even though air was present. Redi’s experiments notwithstanding, the belief in spontaneous generation remained strong. Interestingly, Louis Pasteur is charged with producing the first evidence that spontaneous generation does not occur and that bacterial infection comes from the environment, but clearly Francesco Redi was doing similar work in 1688, almost 200 years before Pasteur.
 
Power of the microscope
The debate over spontaneous generation continued for centuries. Around about the middle of 17th century microscopy became an important part of the history of bacteriology. Primitive microscopy was improved by three contemporaries: Robert Hooke in England, Antoni van Leeuwenhoek in The Netherlands, and Marcello Malphigi in Italy.
 
Hook's microscope was pretty advanced for its time, having a lens, eye tube and light source. van Leeuwenhoek’s microscope was almost as good, but it was he who received the praise because he was the first to describe bacteria morphologically and report them in a series of letters to The Royal Society in London. He reported seeing little living animals which he called ‘animalcules’ in water and from scrapings from his teeth. Microscopy revealed a whole new world of organisms that appeared to arise spontaneously.
More early experiments, more debate........
 
In 1745, English doctor John Needham suggested an experiment to test whether or not microorganisms appeared spontaneously after boiling. One of his findings was that if you take a broth (chicken or corn), boil it and then leave it in the open it will flourish with microorganisms. He therefore claimed that microbes developed spontaneously from the fluids. He published his work, believing that he had definite proof of spontaneous generation.
 
An Italian priest, Lazzaro Spallanzani, suggested 20 years later that perhaps the microorganisms had entered the broth from the air after the broth was boiled, but before it was sealed. To test his theory, he modified Needham's experiment. He placed the broth in a flask, sealed the flask, drew off the air to create a vacuum, and then boiled the broth. No microorganisms grew. Proponents of spontaneous generation however claimed that a ‘vital force’ necessary for spontaneous generation had been destroyed by the heat and was kept out of the flasks by the seals.
 
Golden era of microbiology
The French scientist Louis Pasteur finally disapproved the theory of spontaneous generation in 1861. Pasteur showed conclusively that spontaneous generation of life does not occur, and that microorganisms are everywhere, including in the air. To clarify that air and dust were the source of microbes, he filled flasks with broth and fashioned their openings into elongate, swan-neck shaped tubes. The flasks’ openings were freely open to the air but were curved so that gravity would cause any airborne dust particles to deposit in the lower part of the necks. He heated the flasks to sterilize the broth and then incubated them. As long as the flask remained intact, the broth remained sterile but if the neck was broken off so that dust fell directly down into the container, microbial growth immediately commenced. Pasteur was a pioneer of the ‘germ theory’ that disease was caused by living agents. After studying the causes of the souring of milk and wine, Pasteur concluded that these were due to fermentation and also observed that certain bacteria would only grow if oxygen was absent. He also demonstrated that heating could be used to kill microorganisms in milk and wine. This process is called pasteurization and is still in use today.
 
The English physicist John Tyndal while trying to confirm the results of Pasteur’s experiments determined the capacity of bacteria to exist in two forms: a heat labile form likely to be destroyed by heat and a heat resistant form that could survive high temperatures. He found out that intermittent heating at 1000C could eliminate viable microorganisms and thus sterilize solutions thereby validating Pasteur’s disproof of spontaneous generation. This intermittent heating on successive days is known as Tyndalization.
 
The British surgeon Joseph Lister was the first to introduce aseptic techniques after noticing that women became infected after getting examined by doctors coming directly from the autopsy room. He introduced disinfecting of hands and the air prior to surgery. In 1861, he noticed that patients with infected wounds whose dressings were contaminated with Penicillium mould showed greater improvement than those whose dressings were not contaminated. He concluded that the mould must have produced some form of antibacterial agent. Lister corresponded with Pasteur about this and used the term 'antibiosis'. In turn, Pasteur described to Lister how the peasants of Brittany and of the Russian steppes had, for hundreds of years, healed their sores with Penicillium mould. Thus, Pasteur and Lister demonstrated the possibility of using one microbe to kill another, an observation that predated Sir Alexander Fleming’s discovery by over half a century.
 
While the germ theory of disease was becoming established and the destruction of harmful bacteria investigated, it was inevitable that the new science of bacteriology would include a wave of research into isolating bacteria and differentiating them. The undisputed leader in this field was Robert Koch (1843–1910), a small-town medical officer in Poland. He wrote a letter to Ferdinand Cohn, Professor of Botany in Breslau, in April 1876 stating that he had observed the complete life cycle of the anthrax bacillus and that he was prepared to travel to Breslau to demonstrate this to Cohn and his colleagues. Using blood serum from cattle and the aqueous humour from excised animal eyeballs (immediately after slaughter) as a culture fluid, and placed the inoculated preparation under sealed coverslips on a warm stage and studied the progress of the culture hour by hour. He demonstrated the relevance of the organism to the disease by animal inoculation and he showed that the disease was transmissible from one animal to another for over 20 generations. Koch published this work under Cohn's guidance in late 1876.
 
The next person to make an impact on the development of microbiology as a science was Richard Petri, who, in 1887, invented the dish that bears his name. Petri described it as "a slight modification of the culture method”! There was no gold standard culture medium at the time, and people were trying various things. Koch, for example, soaked raw potato in "corrosive sublimate" then sterilised it in steam, split it in two with a sterile knife and allowed the halves to fall apart in a previously sterilised glass dish. The surfaces of the potato were then inoculated. The solidification of broth using gelatin was suggested by mycologist Oscar Bredfeld, who was visiting Koch at that time. Bredfeld suggested using a liquid mixture of bacteria diluted with sterile medium and obtaining growth from a single bacterial cell. Subsequent solidification with gelatin produced early poured plates and gave rise to agar plates.
 
Robert Koch also discovered and cultured the bacteria that caused several diseases, including tuberculosis, during the latter part of the 19th century. He used agar as a solidifying agent, allegedly on the advice of Frau Hesse, the wife of a colleague, to prepare a solid medium on which bacteria could be grown in individual colonies and therefore in pure culture. Furthermore, he put forward his famous criteria – Koch’s postulates – for confirming an organism as the cause of a specific disease. These postulates were elegantly confirmed by his assistant, Loeffler, a few years later when working with diphtheria. Thus, Koch gained a reputation for accurate, detailed and painstaking work.
 
With the introduction of solid media, Koch was able to recover the TB bacillus from sputum. The discovery that he could inoculate animals with the isolated bacteria and cause similar disease followed by reisolation of the infection from the animal revealed an important principle of pathogenicity.

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