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Pages 193-220:
| Working together
back in 1931 and using one of the smaller Rife microscopes having a magnification
and resolution of 17,000 diameters, Dr. Rife and Dr. Arthur Isaac Kendall,
of the department of bacteriology of Northwestern University Medical School,
were able to observe and demonstrate the presence of the filter-passing
forms of Bacillus typhosus. An agar slant culture of the Rawlings strain
of Bacillus typhosus was first prepared by Dr. Kendall and inoculated into
6cc. of "Kendall" K Medium, a medium rich in protein but poor in peptone
and consisting of 100 mg. of dried hog intestine and 6 cc. of tyrode solution
(containing neither glucose nor glycerine) which mixture is shaken well
so as to moisten the dried intestine powder and then sterilized in the
autoclave, 15 pounds for 15 minutes, alterations of the medium being frequently
necessary depending upon the requirements for different organisms. Now,
after a period of 18 hours in this K Medium, the culture was passed through
a Berkefeld "N" filter, a drop of the filtrate being added to another 6
cc. of K Medium and incubated at 37 degrees C. Forty-eight hours later
this same process was repeated, the "N" filter again being used, after
which it was noted that the culture no longer responded to peptone medium,
growing now only in the protein medium. When again, within 24 hours, the
culture was passed through a filter - the finest Berkefeld "W" filter,
a drop of the filtrate was once more added to 6 cc. of K Medium and incubated
at 37 degrees, a period of 3 days elapsing before the culture was transferred
to K Medium and yet another 3 days before a new culture was prepared. Then,
viewed under an ordinary microscope, these cultures were observed to be
turbid and to reveal no bacilli whatsoever. When viewed by means of dark-field
illumination and oil-immersion lens, however, the presence of small, actively
motile granules was established, although nothing at all of their individual
structure could be ascertained. Another period of 4 days was allowed to
elapse before these cultures were transferred to K Medium and incubated
at 37 degrees C. for 24 hours when they were then examined under the Rife
microscope where, as was mentioned earlier, the filterable typhoid bacilli,
emitting a blue spectrum, caused the plane of polarization to be deviated
plus 4.8 degrees. Then when the opposite angle of refraction was obtained
by means of adjusting the polarizing prisms to minus 4.8 degrees and the
cultures illuminated by a monochromatic beam coordinated in frequency with
the chemical constituents of the typhoid bacillus, small, oval, actively
motile, bright turquoise-blue bodies were observed at a magnification of
5,000 diameters, in high contrast to the colorless and motionless debris
of the medium. These observations were repeated eight times, the complete
absence of these bodies in inoculated control K Media also being noted.
To further confirm their findings, Drs. Rife and Kendall next examined 18-hour-old cultures which had been inoculated into K Medium and incubated at 37 degrees C., since it is just at this stage of growth in this medium and at this temperature that the cultures became filterable. And, just as had been anticipated, ordinary dark-field examination revealed unchanged, long, actively motile bacilli; bacilli having granules within their substance; and free swimming, actively motile granules; while under the Rife microscope were demonstrated the same long, unchanged, almost colorless bacilli; bacilli, practically colorless, inside and at one end of which was a turquoise granule resembling the filterable forms of the typhoid bacillus; and free swimming, small, oval, actively motile, turquoise-blue granules. By transplanting the cultures of the filter-passing organisms or virus in to a broth, they were seen to change over again into their original rod-like forms. At the same time that these findings of Drs. Rife and Kendall were confirmed by Dr. Edward C. Rosenow, of the Mayo Foundation, the magnification with accompanying resolution of 8,000 diameters of the Rife microscope, operated by Dr. Rife, was checked against a dark-field oil-immersion scope operated by Dr. Kendall and an ordinary 2-mm. oil-immersion objective, X 10 ocular, Zeiss scope operated by Dr. Rosenow at a magnification of 900 diameters. Examinations of gram- and safrinin-stained films of cultures of Bacillus typhosus, gram- and safrinin-stained films of cultures of the streptococcus from poliomyelitis, and stained films of blood and of the sediment of the spinal fluid from a case of acute poliomyelitis were made with the result that bacilli, streptococci, erythrocytes, polymorphonuclear leukocytes, and lymphocytes measuring nine times the diameter of the same specimens observed under the Zeiss scope at a magnification and resolution of 900 diameters, were revealed with unusual clarity. Seen under the dark-field microscope were moving bodies presumed to be the filterable turquoise-blue bodies of the typhoid bacillus which, as Dr. Rosenow has declared in his report (Observations on filter-passing forms of Eberthella typhi - Bacillus typhosus - and of the streptococcus from poliomyelitis, Proc. Staff Meetings Mayo Clinic, July 13, 1932), were so "unmistakably demonstrated" with the Rife microscope, while under the Zeiss scope stained and hanging-drop preparations of clouded filtrate cultures were found to be uniformly negative. With the Rife microscope also were demonstrated brownish-grey cocci and diplococci in hanging drop preparations of the filtrates of streptococcus from poliomyelitis. These cocci and diplococci, similar in size and shape to those seen in the cultures although of more uniform intensity, and the characteristic of the medium in which they had been cultivated, were surrounded by a clear halo about twice the width of that at the margins of the debris and of the Bacillus typhosus. Stained films of filtrates and filtrate sediments examined under the Zeiss microscope, and hanging-drop, dark-field preparations revealed no organisms, however. Brownish-grey cocci and diplococci of the exact same size and density as those observed in the filtrates of the streptococcus cultures were also revealed in hanging-drop preparations of the virus of poliomyelitis under the Rife microscope, while no organisms at all could be seen in either the stained films of filtrates and filtrate sediments examined with the Zeiss scope or in hanging drop preparations examined by means of the dark-field. Again using the Rife microscope at a magnification of 8,000 diameters, numerous nonmotile cocci and diplococci of the streptococcus and poliomyelitis viruses, they were shown to be of fairly even density, size, and form and surrounded by a halo. Again, both the dark-field and Zeiss scopes failed to reveal any organisms, and none of the three microscopes disclosed the presence of such diplococci in hanging-drop preparations of the filtrate of a normal rabbit brain. Dr. Rosenow has since revealed these organisms with the ordinary microscope at a magnification of 1,000 diameters by means of his special staining method and with the electron microscope at a magnification of 12,000 diameters. Dr. Rosenow has expressed the opinion that the inability to see these and other similarly revealed organisms is due, not necessarily to the minuteness of the organisms, but rather to the fact that they are of a nonstaining, hyaline structure. Results with the Rife microscopes, he thinks, are due to the "ingenious methods employed rather than to excessively high magnification." He declared also, in the report mentioned previously, that "Examination under the Rife microscope of specimens containing objects visible with the ordinary microscope, leaves no doubt of the accurate visualization of objects or particulate matter by direct observation at the extremely high magnification obtained with this instrument." |
