Biological specificity of disease

Koch’s contribution to medical biology continues when he further established the fact of “Biological Specificity”. Now what actually Biological Specificity” means? It actually shows that a particular disease can be produced by a particular bacterium only. Other bacteria either do not produce any disease or may produce another form of disease following inoculation. He established this by trying to produce “Anthrax” disease from different types of spore forming bacillus like hay bacillus.
In the same time another interesting thing happened. J. Joubert, a person with profound medical knowledge collaborated with Pasteur and they together also started working with anthrax, a serious problem at that time. They were unaware of Koch’s advancement but the conclusions made by them were similar to that of Koch, further confirming Koch’s theory.

Koch’s postulate

Robert Koch thus has established the relationship between a specific microorganism and a specific disease. The criteria for this can be generalized as: -

1. The microorganism must be present in every case of the disease;
2. The microorganism must be isolated from the diseased host and grown in pure culture;
3. The specific disease must be reproduced when a pure culture of the microorganism and inoculated into a healthy susceptible host and
4. The microorganism must be recoverable once again from the experimentally infected host.

These criteria has been generalized as Koch’s postulate to show respect to Koch’s work.

Koch and Anthrax

After the discovery that anthrax can be transmitted from infected to healthy animal through inoculation of blood serum, Robert Koch started thinking about cultivating the causative agent of anthrax. To achieve this goal he performed a simple experiment. He took the spleen out from an infected animal and chopped it into fine particles & then dropped the particles in drops of sterile serum. He looked at it hour after hour and observed the tiny rods of bacterium forming long filaments. In between these filaments he also observed ovoid, refractive bodies somewhat different from the original bacteria (rod shaped). He mentioned that these are spores germinated from the original bacteria due to adverse environmental conditions. He was the first to isolate spores of bacteria. He eventually introduced the spores into fresh serum & again observed the formation of rods, filaments & spores. Likewise eight successive transfers of spores into fresh serum has been carried out and after that the spores were introduced in a healthy animal which rendered the animal ill. Finally the rod shaped organisms could again be isolated in culture.

Robert Koch

The proof of bacteria causing anthrax (etiology) was provided by a German country doctor Robert Koch in 1876. He was nowhere near a scientist and had no laboratory. He worked in his home with very primitive instruments and few small animals. He showed that mice cane be infected with by transferring materials (serum) from diseased animals and this he had done up-to a series of 20 mice by successive inoculation. Strikingly in every transfer the same symptoms were observed. He then performed an experiment to observe the bacterium or rather the causative agent as whether it was due to a bacterium was not clear till then.

Bacteria as causative agents of infectious disease

The fact that bacteria can be a potential agent for causing infectious disease came from the study on Anthrax, a serious disease of domestic as well as wild animals, which can be transmissible to humans. From 1850 - 1865 many researchers identified a rod shaped bacteria in enormous amount in the blood stream of infected animals i.e. animals suffering from anthrax. One of them was C. J. Davaine, who showed that the rods are invariably present in diseased animals but undetectable in healthy ones and also the disease can be transmitted to healthy animals by introducing the blood containing the rods shaped elements (bacteria).

Microorganisms as potential threat in surgery

After the invention of anesthesia, surgical treatment became an important part in medical science. Anesthesia gave the doctors the liberty to perform operations of longer duration and complexity. But this increase in time of operation leads to another problem. As time of operation increased the potential of microbes attack on exposed wounds also increased many folds. As a result many people suffered from post operative sepsis and even died. But the actual reason behind sepsis was not understood at first by the doctors. Pasteur by that time already showed the presence of microorganisms in air and developed many ways to prevent organic infusion from getting contaminated by air microbes during his study with “Spontaneous generation theory” (consult earlier posts). Joseph Lister, a young British surgeon was very much impressed by Pasteur’s work & thought that surgical sepsis might be the result of microbial infection of the tissue exposed during operation. He then continued developing many techniques to maintain aseptic condition during operation. Those include sterilization of the surgical instruments, aseptic dressings of the wounds and spraying of antiseptic agents in operation room to minimize air microorganism contamination. These methods gave instant success in reducing surgical sepsis many folds. People were not convinced about his theory but its striking success made every surgeon to follow the aseptic procedures just like canning process was successful in preserving food for long without knowing the reason behind it. This success is also an indirect proof of germ theory of disease.

Early evidence of microorganisms causing disease

As early as in 1813 there was evidence that specific fungi can cause diseases of wheat & rye, and in 1845 M. J. Berkley had proved that Potato Blight was also caused by a fungus. There was an incidence of “Great Potato Blight” in Ireland, which was a natural disaster that has immense effect on Irish history. So the importance of these diseases cannot be overlooked. Not only plants, fungal disease of animals were also in picture and in 1836 A. Bassi of Italy had shown the cause of certain silk worm disease was fungus. And few years later J. L. Schonlein showed that fungus can also cause skin disease to human. But unfortunately these discoveries fail to make medical science believe that microorganisms are responsible for major infectious diseases of humans. Actually they underestimated the capacity of microorganisms due to its small size. Everybody thought that an organism as small as bacteria cannot make much harm to a much bigger animal like human.

Microbes and disease

After observing few roles of microbes in fermentation process let us focus on its role in producing various diseases in human or other animal’s body. This is the most known fact about microbes by common people. Here also it was Pasteur who gave the insight. Pasteur was looking in to the spoilage of beer and wine by undesirable microorganisms & termed the fact as “disease” of beer and wine. He also added that many disease of human might also have similar origin i.e. produced by the growth of undesirable organisms in their bodies.

H. Buchner’s contribution in fermentation

After the discovery of fermentation by Pasteur who also showed that fermentation is far less energy yielding process than aerobic respiration, H. Buchner came out with a very important accidental discovery. Pasteur’s work had showed that fermentations are “vital processes” playing important role of basic physiological importance in the life of many cells. H. Buchner in order to prevent grounded yeast cells kept along with sand from decaying added sugar solutions to it. Astonishingly he found that CO2 formation was taking place, which suggest that grounded dead yeast cells can also ferment sugar solution i.e. the cell free extract of yeast can cause fermentation like the live cell. This is a revolution in Biochemistry & led to the belief that every biochemical process be made possible outside the cell. And thus the chemists came well at last. Their earlier theory that fermentation is a chemical process is actually true. The only difference is the presence of biological enzymes making the reaction possible at physiological temperature.

Fermentation: life without air

Pasteur mentioned fermentation as “life without air” as he was the first one to understand that breakdown of organic compounds in absence of oxygen can be a method of obtaining metabolic energy for the microorganisms. There are two types of microorganisms regarding this i.e. that can live/survive without air: - Facultative anaerobes and Obligate anaerobes.

Facultative anaerobes have two types of energy yielding mechanisms. They can follow both aerobic and anaerobic pathways of obtaining metabolic energy. They can hence survive both in absence & presence of oxygen. So when sugar is fermented in absence of oxygen; alcohol & CO₂ are produced whereas when oxygen is present no alcohol formation takes place and CO₂ is the sole end product.

Significance of fermentation

After the discovery of anaerobic life Pasteur understood the actual reason behind fermentation i.e. why the microorganisms carrying out the chemical changes in the organic infusions. Until then this was a mystery. The discovery of anaerobic life just made it possible for him to understand the basis of these chemical changes. The reason is the need of energy to sustain life. And the microorganisms were making the chemical changes to derive energy from the organic infusions either by aerobic or via anaerobic oxidation. In that way they were performing the process similar to respiration in higher organisms.

Anaerobic life

Here comes another historical discovery of Pasteur, the Father of Microbiology – “The existence of forms of life that can live only in the absence of free oxygen” i.e. life in anaerobic form. This conclusion of presence of anaerobic life (at that time it was thought that life cannot sustain without oxygen) was drawn by him while observing the fermentation process of butyric acid. He was studying droplets collected from fermentation vats under microscope & found that the organisms at the margin of the flattened drop quickly became immotile (which were in close proximity of air) whereas that at the center remained motile. He thought that the immobility is due to the death of the organisms & the cause of it must be the contact with air. This was further confirmed when he passed air through fermentation vats of butyric acid fermentation. He found that the rate of fermentation retarded significantly & sometimes even stopped. So he at last concluded that some microorganisms can only live without oxygen and coined the names aerobes and anaerobes to designate them as life with or without oxygen.

For the next 20 years Pasteur studied various fermentation processes & came to the conclusion that all fermentation processes are accompanied by the formation of microorganisms. He also concluded that different fermentation processes contain different microbes. Moreover these occurrences of microbes are specific for specific fermentation (as evident by specific end products like alcohol and lactic acids). Pasteur differentiated these organisms microscopically according to shape & size and their favorable environmental conditions. For example, organisms from alcohol fermentation prefer acidic condition whereas that from lactic acid fermentation grows in neutral pH. So to grow lactic acid microbes one should use some neutralizing agents to prevent the acidic effect due to the formation of lactic acid (Pasteur used calcium carbonate i.e. chalk for this purpose).

The most unfortunate thing for the chemists was the fact that Pasteur who was a chemist by training first established that “fermentation processes are result of microbial activity”. Actually a local brewery industry was facing a problem. Instead of alcohol, fermentation of sugar was producing lactic acid in their vats. They asked Pasteur to solve the problem. Pasteur found microscopically that instead of yeast the vat material contained some rod and spherical shaped small objects. He transferred the objects (vat infusion) into new sugar a solution, which results in further development of the same organisms in the solution & also lactic acids instead of alcohol, was produced. Further transformations in fresh sugar solution gave similar results. Pasteur thus named those small organisms “New Yeasts” that were actually bacteria and not yeast. But in Pasteur’s time microorganisms were not classified.

Chemistry had made great advance in early decades of nineteenth century. Urea was synthesized from inorganic source in 1828 which lead to the belief that all organic compounds can be synthesized chemically (earlier it was thought that organic compounds can only be synthesized by living organisms). This was the reason why chemists (J.J. Berzelius, J. Lubig & F. Wohler etc.) thought that all the biological processes are chemical process and has no relation with organisms. The actual truth was something different though. All the biological processes are indeed chemical process, but are controlled by living organisms only through actions of their unique enzymes. So when in 1837 C. Caniard-Latour, Th. Schann & F. Kützing independently proposed that fermentation of sugars to ethyl alcohol and CO₂ was carried out by yeasts, no chemists believed it & opposed the idea. They thought a simple conversion of sugar to alcohol must be a chemical process and has no link with presence of any organisms in the infusion.

Fermentation and Putrefaction

Now there may be a long controversy over spontaneous generation theory but one thing was certain that the presence of microorganisms in organic infusions bring about some chemical changes in the organic infusions itself. These changes primarily are of two kinds: -
i) Fermentation & ii) Putrefaction.

i) Fermentation: - When the organic infusions are of plant origin (i.e. mainly composed of carbohydrates) then their breakdown results in the formation of alcohols & organic acids and are known as fermentation process (in a crude way).
ii) Putrefaction: - When the organic infusions are of animal origin (i.e. mainly composed of proteins) then their breakdown produces ill-smelling compounds and are known as putrefaction process.

Pasteur’s and Tyndall’s experiment proved that microorganisms do not arise spontaneously in properly sterile organic infusions. Thus the doctrine of “Spontaneous generation” during eighteenth - nineteenth century was discarded. But one must keep in mind the fact that primary origin of life in earth did involve a kind of spontaneous generation. Though it cannot be compared with the false doctrine as that was much more gradual & too delicate to describe.

Tyndallization/Discontinuous heating

Tyndall that started thinking about finding a process that will not only kill the bacteria but also their spores during sterilization. This resulted in the development of the process of discontinuous heating which was later termed as Tyndallization. In this process organic infusions were 1st heated for few minutes to kill the bacteria present in it. Then the infusion was kept for sometime in room temperature. This would result in the germination of the spores (heat stable) of the bacteria. Then the infusion was again heated for few minutes to kill the germinated spores (not heat stable). Then again the infusion was kept at room temperature for germination of the spores still left (late germinating spores). After following this cycle for several times one could destroy all the bacteria including their spores present in the organic infusion. Tyndall optimized that boiling for 1 minute for successive 5 cycles (with rest in between) would make the infusion sterile whereas non stop boiling even for 1 hour would not have that effect. But there was a drawback in this system of sterilization as germination time & heat resistance of bacteria vary from species to species and hence one must have a thorough understanding of the bacteria present in the infusion which is very difficult.

Tyndall’s Experiment

After Pasteur, John Tyndall, an English Physicist and a passionate follower of Pasteur established an important fact that was overlooked by Pasteur. He discovered the presence of “spores” of different microbes in air & in certain organic infusions. Since the spores are more resistant to heat, the organic infusions are often not sterilized after heat treatment giving rise to controversy in favor of spontaneous generation theory. He established that even after prolonged heating of 5½ hr the spores are not killed. So he mentioned that all bacteria have two phases, one thermo-labile phase (destroyed after 5mins of heating) and one thermo-stable phase. His views were supported by a German Botanist Ferdinand Cohn, who also like Tyndall worked with hey bacteria. Since hey is not a very good source of nutrient the bacteria are starved & as a result endospores are produced (spores are dormant phase of bacteria, fungi, lower plants & protozoa to survive the harsh environmental condition. They may also be called reproductive cells as favorable condition gives to the following organisms).

Swan-neck flask of Pasteur

This led to one of the most famous experiment of microbial history, the “Swan-neck flask” experiment by Pasteur. Please refer the figure for the shape of the flask. Now organic infusion inside the flask was made sterile by heating. Indeed there was no microbial growth in the organic infusion as the microbes cannot ascend the bent neck of the flask. But when the neck is broken or the sterile liquid becomes densely populated with microorganisms. Pasteur’s experiments made it clear that the living organisms are evenly distributed throughout the atmosphere. Thus "Germ Theory" was established and gained over "Spontaneous generation" theory.

One of the most important things Pasteur able to do was to demonstrate that air does contain microscopically observable “organized bodies”. What he did was to pass air through a tube plugged with guncotton (served as a filter). He then removed the guncotton & dipped in alcohol and ether mixture. The sediment thus obtained was examined under microscope. It was found that the mixture contains many small, round bodies besides organic matter. These bodies were believed to be the microorganisms everybody was talking about. He then performed two additional experiments which fully proof the fact that of the presence of microorganisms in air. First he passed heated air through preheated organic infusion to find that there is no growth. And 2^nd he placed a piece of germ-laden guncotton a well heated airtight organic infusion to find that growth is appearing through these is as air supply. Thus he proved successfully the presence of microbes in air.

Pasteur’s Experiments

Scientists also observed the fact that the organic infusions kept in open vessel not only generate microorganisms but also undergo certain changes. In mid 19th century the scientists started to think that microorganisms bring about these changes or at least there is a relationship between them. Louis Pasteur, the Father of Microbiology, is the pioneer of this thought process. First he had not given much to the controversy between presence of microbes and spontaneous generation theory. But at last he turned his attention towards this grave problem. He published his work in 1861 as “Memoir on the Organized Bodies which Exist in the Atmosphere”. His work had revolutionized the world of microbiology.

Image from wikipedia.com

Spallanzani successfully proved the case against spontaneous generation theory. But the problem is that faulty experiments were still performed & as a result the controversy remained. Francois Apart in the beginning of 19th century made an interesting experiment. Instead of organic infusions he used food stuffs in the air tight bottle and heated it. Thus he was successful in storing or preserving food stuffs for long time which was earlier impossible. This was a practical application of Spallanzani’s experiment and known as canning or “Apertization” (on behalf of Apart) and used extensively to preserve food in that period.
Then another problem or controversy came in front regarding proving the presence of germs against the spontaneous theory. In late 18th century Oxygen was discovered by Priestley, Cavendish & Lavoisier and soon it was found that it is essential for the existence of living being. So many thought that due to the hermetic sealing used by Spallanzani or Apart; the oxygen supply was cut off & that was the reason of no microbial growth in the organic infusion; not the presence of germs in the air. This is really a big problem and is very difficult to deal with. Later it was proved that if germ free air is passed through the pre heated organic infusion then there will be no decomposition of the organic infusion even in the presence of oxygen.

But when more knowledge was gathered about living organisms, it became evident that this doctrine of Spontaneous Generation was not true. Earlier it was thought that maggots are formed in meat spontaneously. But Italian physician, Francesco Redi showed that the maggots are nothing but larval stages of the flies & will never appear if the meat is protected by placing it in vessel covered with fine gauze. In that case flies are not able to lay eggs on the meat. This experiment is of utmost importance as it gave a much needed proof against Spontaneous Generation theory. For technical reason it was very difficult to prove that microorganisms are growing inside the organic infusion not spontaneously but from their earlier life form already present in air or in the organic infusion. It was in the middle of nineteenth century that the cumulative evidence became sufficient to rule out this theory.
In the middle of 18th century Italian naturalist Lazzaro Spallanzani proved that heating can prevent the appearance of “animalcules” in infusions, although the duration of heating varies. He concluded that air is the carrier of animalcules in well heated infusions, he closed the flasks containing infusion hermetically (earlier others closed the vessel by mechanical plugging which is not sufficient to prevent the entry of air completely) and boiled the infusion. The boiled infusions are free of animalcules but if there is a tiny crack in the bottle the animalcules reappear. This strongly suggests air is the carrier of animalcules, & they are not generated spontaneously.

Origin of microbial world

I have already given you some facts about the discovery of the microbial world and the life of Leeuwenhoek. If you are interested about Leeuwenhoek’s life you will get to know about him in my nest post which will be about his life in short. So keep visiting my blog. I will also post an article on one of the most life threatening diseases of this planet – Malaria soon. But after the discovery of the “Microbial World” the nest thing that comes in mind is the origin of these microorganisms. So our next focus will be “The Origin of Microbial World”.
There are mainly two kinds of belief from the beginning regarding the origin of this wonder world. The first thought was about the belief that “animalcules” were formed spontaneously from nonliving materials. The second thought (Leeuwenhoek is one of the follower) told about formation of microbes from the “seeds” & “germs” of these animalcules, which were always present in the air.
The first thought was known as spontaneously generation or abiogenesis & this doctrine was accepted without question until the Renaissance.

Anton Van Leeuwenhoek Letter

In one letter he described for the first time the characteristic bacteria of the human mouth. He wrote:

I have several gentlewomen in my house, who were keen on seeing the little eels in vinegar; but some of them were so disgusted at the specticle, that they vowed they'd never use vinegar again. But what if one should till such people in the future that there are more animals living in the scum on the teeth in a man's mouth, than there are men in a whole kingdom?

But Leeuwenhoek’s death has ended this observation of microbial world, though everybody has marveled his discoveries. This was primarily because of the lack of skilled professionals for production of high quality simple microscope after Leeuwenhoek as simple microscope of high magnification is very difficult to make. There are compound microscopes available, but they suffer from serious optical defects. This is why ‘Robert Hooke’, a very capable & careful observer of Leeuwenhoek’s time, who was observing the microbial world with his compound microscope, could not see the intricate details seen by Leeuwenhoek simply because of the optical defects of his microscopes. So it took more than a century after Leeuwenhoek’s death (1820) to repeat his experiments with compound microscope after development of optical science. Meanwhile microbiology had also developed in another way i.e. the roles it plays in transformation of matter & causation of disease. Leeuwenhoek is considered the father of microscopy.

References: General Microbiology 5th edition.

He had an extraordinary range & skill of microscopic observations on the microscopic structure of the seeds and embryos of plants & small invertebrate animals. He discovered the existence of spermatozoa & of red blood cells and was thus founder of animal histology. Protozoa, algae, yeasts & bacteria -- all these unicellular organisms were first seen by Leeuwenhoek, often with such accuracy that it is possible to identify individual species from his accounts of them. This microbial world has two distinct features --- diversity and abundance.

Despite the simplicity of the microscopes made by Anton Van Leeuwenhoek, the highest magnification that he could obtain was consequently somewhat less than one third of the highest magnification that is obtainable with a modern compound light microscope. Leeuwenhoek constructed hundreds of such microscopes/instruments.

Leeuwenhoek

Anton Van Leeuwenhoek (1632 - 1723) observed the microbial world/the microscopic world only with the help of a simple microscope (microscope with one, almost spherical lens).

Anton Van Leeuwenhoek (1632-1723) a Dutch merchant is discoverer of the microbial world.

My first post

This blog is all about bacteria. This site will not only deal with bacteria alone. It will cover its interaction with different species along with human being and other microbes. This will also contain all the findings of my own research work regarding bacteria. All recent trends & posts by other scientists etc.