Graduate Thesis Or Dissertation
 

The infra-red absorption spectra of the Clostridia

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  • There are six types of Cl. botulinum (Types A, B, C, D, E, and F). The differentiation between these types is based upon the antigenic structure of the toxin. Many strains of these various types have been isolated. In addition, a number of nontoxigenic "type Elike" strains have come to light. It would be of great value to have a rapid method of distinguishing between these strains, or of identifying the type of toxigenic culture. This thesis discusses an investigation into the possibilities of the use of infra-red in an approach to this problem. Smears of bacterial cells were prepared by scraping a small amount of growth from the agar surface of a petri plate, and dissolving it in a drop of water on the surface of a silver chloride disc. In the case of broth cultures, growth to be placed on the discs was first obtained by centrifugation. A second empty disc was then placed over the first with a bit of lens tissue under one edge to prevent the formation of interference fringes. The two discs were then taped together and placed in the IR spectrophotometer, together with a pair of reference discs for the recording of the IR spectra of the whole bacterial cells. 1. Preliminary work indicated that the most reproducible section of the IR spectrum, which would also give unique and somewhat characteristic variation was to be found in the region from 7.0 μ to 10.0 μ. 2. It was further found that the characteristic variations of an organism were magnified to the greatest extent when the smear was of such a thickness that the 7.0 μ to 10.0 μ region of the spectrum had its entire transmittance lying in the range from 20% to 70%. The preparation of smears of the desired thickness became somewhat of an art. 3. The great bulk of the clostridia examined fell into four basic spectral patterns based upon variations within this range. The spectral patterns of the clostridia seemed to be unique from that of several other bacteria which were examined. 4. The age of the culture has a rather profound effect on the IR spectrum of a culture. The variability of the IR spectrum of a culture decreases with age, between two and five days. One pattern, which is seen to predominate at the two day age, completely disappears at the five day age. The changes were seen to be associated with the accumulation of small amounts of glycogen within the cells as the culture ages. These changes seem to be especially associated with Cl. botulinum type E. 5. It is concluded that the technique has little value as an aid in the identification of clostridial species. Most of the pathogenic clostridia could not be distinguished from the non pathogens by this means. Neither could the toxigenic strains of Cl. botulinum type E be distinguished from the non toxigenic "E-like" strains. The technique may have some value in monitoring physiological changes within a given culture or group of cultures, however. 6. Washing the cells before preparing the smears seems to decrease the intensity of the region of the spectrum from 6.2 μ to 8.2 μ. This is the region associated with the types of bonds found in proteins. Changes after washing also occur in the region of the IR spectrum from 8.5 μ to 10.5 μ. This region is associated with bonds found in nucleic acids and polysaccharides. 7. The IR spectra of purified and unpurified spore preparations indicates that the spectra of the spore may vary considerably from the vegetative cell, but no distinctly new types of spectra are seen.
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