You cant find any developments or experiments performed in the last 150 years of microbiology?
Many pathogenic agents, such as the chlamydiae genus, the rickettsiae genus, mycobacterium leprae, plasmodium falciparum and the herpes simplex virus cannot be grown in pure culture. This violates Koch's second posulate. The following have all been developed to allow for the detection, isolation and identification of microbes.
Traditional light microscopy has been improved through the use of a number of immunohistochemical stains such as horseradish peroxidase, 3,3'-Diaminobenzidine and nickel and immunofluorescent stains such as Fluorescein isothiocyanate, Rhodamine and Texas Red
Confocal laser scanning microscopy allowed high resolution and in focus images of thick specimens. Transmission and scanning electron microscopy allowed for higher magnifications up to fifty million times and higher resolving power than a light microscope.
Serological assays such as agglutination, precipitation and complement fixation allowed for the detection of infection antibodies in the blood serum of infected patients.
The biggest discovery has been that of nucleic acids. Pathogen discovery on the genetic sequence level is possible because all infectious agents, with the exception of prions, contain nucleic acids which can be detected and are unique between different microbes. Amplification of the nucleic acids by PCR and probe based signal amplification allows the detection of microbial sequences of nucleic acids to occur at the single-copy level.
Using these developments the following infectious agents have been found.
Tropheryma whipplei was found to be the causative microbe of Whipple’s disease and was discovered using consensus PCR. The Sin Nombre virus was discovered by amplifying Hantavirus specific DNA which was extracted from RNA in the infected tissues of patients, it was found to be the cause of Hantavirus pulmonary syndrome.
The coronavirus that causes severe acute respiratory syndrome was identified by creating a DNA microarray to identify and characterise a range of known viruses and novel members of existing families of viruses. The array contained highly conserved viral sequences from GenBank. The SARS outbreak in March 2003 provided an infectious isolate taken from a SARS patient. The isolate underwent reverse transcription and amplification by PCR to produce a cDNA strand. This isolated cDNA strand was hybridised to the array and revealed the presence of an unrecognised coronavirus. Cloning and sequencing the unknown genome and comparing it to known genomes from the coronavirus family confirmed that the unidentified virus was a member of the coronavirus family.
