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Classic diagnostic methods: cell culture[In Preparation]The process of chlamydial culture in cell culture is well known, particularly to the older generation of researchers, and will simply be outlined here. Up until the early 1980s, the main method of confirming a diagnosis of chlamydial infection, was the inoculation of clinical material into animals, embryonated hens eggs or (usually) tissue culture cells and the demonstration of characteristic chlamydial inclusions. Generally speaking inclusions were demonstrated in cell culture, either by staining with iodine (Fig 1A: C. trachomatis only), by staining with Giemsa stain (Fig 1B); fluorochrome-labelled poly- or mono-clonal antibody (Fig 1C) or by enzyme immunohistochemistry (Fig 1D). Occasionally C. psittaci were isolated in animals (Fig 2A) and embryonated hens eggs were convenient for the bulk growth of otherwise difficult to grow chlamydiae (Fig 2B). Fig 1. Chlamydial inclusions in tissue culture
Fig 2. Chlamydiae in animals.
The usual tissue culture cells were HeLa 229, L434 mouse fibroblasts or McCoy cells in the case of C. trachomatis and C. psittaci; Buffalo Green Monkey Kidney Cells (C. psittaci and C. pecorum) or HL or Hep2 cells for C. pneumoniae. With the exception of 'fast growing' strains like the LGV biovar of C. trachomatis, it was usually necessary to assist the process of infection by centrifugation of the clinical material onto monolayers of the appropriate cells in tissue culture. Growth of the organisms was also facilitated by the use of anti-metabolites directed against the host cell (cycloheximide; emetine or mitomycin C) or, for the C. trachomatis TRIC biovar, by the use of charged anionic polymers such as Poly - L - lysine or DEAE dextran. Compounds like polyethylene glycol or high energy glucose 6 phosphate also aided the growth of some chlamydiae. After three days it was usually necessary to homogenise the cells and do at least one 'blind passage' in order to ensure one had not missed low level infection. Fields of monolayer cells also had to be searched for tell-tale inclusions. Perversely strains would often decline rather than increase in infectivity on serial passage, an indication that cell culture conditions were far from ideal. This general palaver meant that chlamydial diagnosis by cell culture was performed solely by a few specialist research laboratories. The advantage of it all was that identification of even one chlamydial inclusion was usually sufficient to establish a definitive diagnosis. Even now cell culture is one reference standard against which new diagnostic tests may be compared and it is still the method of choice for medico-legal cases of child sexual abuse etc. The disadvantages were many. The inoculum had to be kept viable until the point where it could grow in cells; this usually meant good transport arrangements and the maintenance of an appropriate cold chain. The tissue culture service was labour expensive to maintain and there were many difficult to quantify variables, including the tissue culture water, the particular clone of cells in use; the quality of the foetal calf serum; the absence of adventitious mycoplasma infection; the quality and sterility of the laboratory ware etc. All of this has been replaced by the demanding, but less variable protocols associated with modern, non viability-dependent diagnostic products with much better sensitivity than was routinely achieved by cell culture. I for one do not mourn the passing of cell culture for the laboratory diagnosis of chlamydial infections. New diagnostic methodologies have greatly widened the availability of laboratory diagnostic methods for chlamydial infection. [MEW] May 2002. NEXT: Immunofluorescence
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