Lecture – History of Microbiology and Virology

 

• Because most of the organisms that cause infectious disease are too small to see with the naked eye, early man did not understand the biological basis for disease. It took the combined efforts of many scientists working in diverse and unrelated fields to help us determine exactly what kinds of microscopic organisms cause which human and agricultural diseases.
• Antony van Leeuwenhoek (1632-1723) – lived in Holland
  • A cloth merchant – created simple lenses to examine cloth for imperfections
  • His hobby was to grind better lenses that allowed up to a 300 fold magnification. 
  • He examined “rain, well, sea and snow water” and described with pictures the “wee animalcules” that he observed to the Royal Society of London
• First evidence of small living things – bacteria, protozoa, algae, yeasts, sperm, eggs, RBC, etc.
  • We now know that his 300x magnification was not great enough to see bacteria, but he obviously described organisms that were bacteria. Perhaps he used a technique of dark-field illumination to get better resolution.
• 1800’s – invention of the compound light microscope. The use of two lenses increased magnification up to 2000x.
• 1838 – 600 distinct of microbes were recognized and hand drawn pictures published in the Atlas by Ehrenberg.

 

• Spallanzani (1729-1799) – lived in Italy

 

• At this time people were not sure where new livings beings came from. It had been observed that flies could be generated from a piece of meat left out and that mice would eventually appear in a pile of old rags. This was known as the theory of “spontaneous generation” (life from non-living material).
• Spallanzani was able to debunk the theory of spontaneous generation. He managed to isolate a single “critter” (aka one of Leeuwenhoek’s animalcules) in a water drop. He was able to observe it divide into two identical replicates of the same organism. Though the real credit for putting to rest the theory of spontaneous generation goes to Louis Pasteur.

 

• Pasteur (1822-1895) – lived in France

 

• When Pasteur was a young boy he saw a rabid wolf biting the people in his village – which the villagers said was because the devil had possessed the wolf and that it was “God’s will”. This spurred him to want to study the biological basis of disease.
• In 1837 Pasteur was studying the problems with fermentation in a beer brewery. If there were big round globules seen under the microscope in the fermentation liquid (yeast) then the result was ethanol. If there were smaller rods (bacteria) then the result was lactic acid (sour smelling). If there were different shaped bacteria then the result was butyric acid (rancid smelling).
• If the fermentations that had the bad smells were consumed they caused people to get sick. He made the hypothesis that the bacteria caused disease. He was the first person to put forth this theory – and no body believed it! He eventually determined that cleanliness was the key to preventing the introduction of bacteria into the vats and the production of bad beer.
• His experiments to finally disprove the theory of spontaneous generation was to boil broth in a special swan necked flask that would allow air to gain access but not any particulate matter (like bacteria or yeast). The broth in these containers has remained clear and sterile up to this very day!

 

• Edward Jenner (late 1700’s – 1800’s) – lived in England?

 

• Made the observation that cow maids who milked the cows had pox marks on their hands (from the cowpox virus) but never had the horribly disfiguring scars on their faces from having suffered through a bout of smallpox. He reasoned that being exposed to cowpox (a mild illness) protected the cow maids against infection with the much more virulent smallpox disease.
• In 1796 he scratched the arm of 8-year-old James Phipps and placed some pus from a cowpox pustule from an infected cow maid into the wound, inoculating the boy with cowpox. Two months later he inoculated the child with a combination of cowpox and smallpox (challenging the immune system). The boy remained healthy, suggesting that prior exposure to the cowpox had protected him against the smallpox.

 

• In 1876 there was no widely accepted “germ theory” – the idea that microorganisms can cause human and animal disease.

 

• By 1900 the field of bacteriology (the study of bacteria) was well established and several bacteria that could cause human or animal disease were identified.

 

• Robert Koch (1834-1910) – lived in Germany

 

• Koch was a local country physician who was frustrated by inability of current treatments to prevent and cure human disease. He was impressed by the work of Lister, an English surgeon, who discovered that wearing clean gowns and sterilizing operating tools nearly eliminated patient infection and death.
• At this point in history, folk myths on how to prevent disease were common. In Russia they believed that you could ward off a scourge by hitching four widows to a plow and having them draw a furrow around the village at night.
• In 1876 the disease anthrax was killing sheep and cows and having a major economic effect. Koch took blood samples from dying animals and saw microscopic rods in the blood.
  • He then took the cow blood and injected it into a mouse. The mouse died. He then observed the same rods in the blood of the dead mouse. His hypothesis was that the rods were causing the disease.
  • At this point he realized that he needed to get a pure culture of the bacteria. He designed a hanging drop system to grow bacteria in isolation away from contaminating bacteria from the environment. He used a hanging drop system.

 

 

 

 

sterile environment                                                                    glass coverslip

 

                                                                                                  hanging drop containing bacteria

 

                                                                                                            glass slide                    

 

 

·        To get a pure culture – Koch transferred bacteria from drop to drop (letting the bacteria grow and multiply in between) 8 times. At that point he had a pure culture without any bacteria other than the rods he had observed before.

 

o       He then injected the pure rods back into a healthy mouse and the mouse died of anthrax!

o       He demonstrated that a single kind of bacteria was associated with a particular disease – the true beginning of the “germ theory” of disease. Unfortunately, anthrax can form spores, which are very resistant to killing and can lie dormant in the soil for years.

 

·        He then discovered an easier way to isolate pure cultures of bacteria on the surface potatoes. Different kinds of bacteria forms different colored and shaped colonies, which can be picked and a pure culture isolated.

·        Koch then went on to isolate the bacteria that cause several human diseases including tuberculosis and cholera.

o       There was a skeptic, Pettenkofer, who didn’t think that bacteria caused cholera. He tried to prove this by drinking a solution of cholera bacteria and he didn’t get sick! We may hear of this kind of trick being tried again more recently.

o       What makes some people resistant to a particular disease and others not?

 

·        Koch came up with four postulates that needed to be satisfied before a new stain of bacteria could be linked with causing a particular disease.

 

1.      The organism must be regularly found in the lesions of the disease

2.      The organisms must be isolated in pure culture (hence the need for sterile technique)

3.      Inoculation of such culture of pure organisms into the host should initiate the disease

4.      The organism must be recovered once again from the lesions of the host

 

·        Viruses are much smaller than bacteria (1-2 mm for a common bacterial cell versus
0.3 - 0.05 mm for viruses) and cannot be seen with the light microscope. An electron microscope (EM), which was not invented until the 1930’s, is needed to see the structure of a virus.

 

·        Adolf Mayer (1843-1942) – lived in Germany

 

o       He was an agricultural chemist who was studying a disease of tobacco plants characterized by light and dark areas on the affected leaves. He called the disease “mosaic disease of tobacco” and tried to determine if it had an infectious cause.

o       He ground up infected tobacco leaves and produced a clear, soluble extract. When he injected this extract into healthy plants, they usually became afflicted with the disease.

o       He attempted to culture a bacterium from the infected plant tissue using standard techniques, but could never isolate a pure culture that caused the disease. Koch’s postulates could not be fulfilled.

o       He hypothesized that the bacterium that caused the tobacco mosaic disease was unusual and would need special conditions to be cultured.

 

·        Dimitrii Ivanovsky (1864-1920) – lived in Russia

 

o       In 1892 he was studying tobacco mosaic disease. He put the sap from infected leaves through a very fine porcelain filter (known as a Chamberland filter) and when it was put onto a healthy plant, that plant would still become afflicted.

o       This suggested that it was smaller than any previously identified infectious organism. He suggested that a bacterial toxin might cause the disease since the agent was not able to “strengthen” (replicate itself) in a cell-free medium.

 

·        Martinus Beijerinck  (1851-1931) – lived in The Netherlands

 

o       In 1898 he repeated Ivanovsky’s experiment, but in addition he showed that the sap could be diluted several fold and when added to the leaves of healthy plants could cause disease.  The sap then from these newly infected leaves could also be diluted, which showed that the agent could be “strengthened” (replicate) only on living tissue.

o       This was the first hint of a new kind of infectious organism – which Beijerinck called a “contagium vivum fluidum” or virus. The tobacco mosaic virus (TMV) was the first one identified and has been a highly studied virus ever since then.

 

·        Even though the first virus was identified in 1898 – it wasn’t until the 1939 when TMV was observed using the electron microscope. And the first assay to measure the number of viral particles in a solution (the plaque assay) was not developed until 1917. The “hay days” of virology really came in the 1950’s and 60’s when viruses could be studied at the molecular level. And now we have even more techniques to study and analyze new and old viruses.

·        It is quite possible that if HIV and AIDS had appeared even 10 years earlier our technology would not have been advanced enough to identify and study the virus. In many ways we are quite lucky this virus reared its ugly head late in the 20^th century.