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Darkfield Microscope for Lyme ( borrelia ) detection
Arch Pathol Lab Med. 1983 Jul;107(7):384-6.
Detection of Borrelia in acridine orange-stained blood smears by fluorescence microscopy.
Sciotto CG, Lauer BA, White WL, Istre GR.
Tick-borne borreliosis (relapsing fever) can be an important, unsuspected cause of febrile illness. The diagnosis is generally made by identifying Borrelia spirochetes in stained peripheral blood smears. Since Borrelia may be difficult to detect with Romanowsky stains, an alternative method, using acridine orange (AO), was used to screen blood smears. Duplicate blood smears of seven patients were examined with the AO technique and Romanowsky stains. In all seven cases spirochetes were easily identified with the AO-stained smears compared with only five cases with Romanowsky stains. In a double-blind laboratory experiment, six of ten duplicate smears from a single patient with mild spirochetemia were positive by AO, whereas only two of ten were positive by Romanowsky stain. We concluded that the AO stain is simple, rapid and more sensitive than Romanowsky methods for detecting cases of low-level spirochetemia.
APPLIED MICROBIOLOGY, p. 540-543 -1974 Vol. 28, No. 4
Biology of Borrelia hermsii in Kelly Medium
HERBERT G. STOENNER
More than 800 Borellia hermsii in mouse plasma were required for establishment of growth in an artificial medium (Kelly), but only a single organism of a fully adapted strain (25th subculture) was required for a successful subculture. As judged by generation time, maximal concentration in culture, and length and motility of the organism, the process of adaptation extended through at least 11 subcultures. Because the organisms regularly died shortly after the logarithmic growth phase, transfers at 7- to 10-day intervals were required to maintain continuous cultures.
Spirochetes in the medium and citrated mouse plasma were counted under a cover slip with a dark-field microscope. As delivered from a 0.005-ml calibrated capillary pipette (Kimble microcapillary pipette), 0.0025 ml of medium was deposited on a slide, surrounded by a ring of a mixture containing petrolatum and plasticine (2:1, wt/wt), and covered with a cover slip, and sufficient pressure was applied to extend the drop to a diameter of about 8 mm. Counts were made of dilutions containing 3 to 12 organisms per 0.0025 ml; the entire area was searched for borreliae at a magnification of x 125. All counts were made in duplicate and averaged. It should be emphasized that cover slips, slides, and capillary tubes must be cleaned thoroughly. A method was devised for culturing a single spirochete in sterile capillary tubes (90 mm long, 0.4 mm inner diameter). In this method, the number of spirochetes in vigorously growing cultures (5 to 7 days old) or mouse plasma was first established by direct count.
Parazitologiia. 2001 Jan-Feb; 35(1): 3-8.
Comparative evaluation of the efficacy of Borrelia indication in ixodes ticks (Ixodidae) using dark field microscopy and polymerise chain reaction (PCR)
Nefedova VV, Korenberg EI, Nesterenko LN, Gintsburg AL, Kovalevskii IuV, Gorelova NB.
Indication of Borrelia (B. burgdorferi sensu lato) in 205 adult unfed I. persulcatus ticks from a natural focus was carried out simultaneously by methods of PCR and dark-field microscopy of vital preparations. PCR method revealed Borrelia prevalence in a considerable number of ticks, in which Borrelia were not found by microscopy of 250 microscopic fields in a preparation from each individual tick.
At the same time, PCR method didn't give positive results for approximately 8% of ticks, which contained rather high concentration of Borrelia (more than 10 per 100 microscopic fields). In general, PCR method doesn't have advantages in comparison with a microscopy of vital preparations for study of the Borrelia prevalence in ticks.
Journal of Clinical Microbiology, May 1999, p. 1361-1365, Vol. 37, No. 5
Prevalence of Borrelia burgdorferi in Ixodes ricinus Ticks in Urban Recreational Areas of Helsinki
Juha Junttila,1 Miikka Peltomaa,2,* Hanna Soini,3 Merja Marjamäki,3 and Matti K. Viljanen3
Lyme borreliosis, an infection caused by the tick-borne spirochete Borrelia burgdorferi, is a major health problem for populations in areas of endemicity in the Northern Hemisphere. In the present study we assessed the density of ticks and the prevalence of B. burgdorferi sensu lato among ticks in popular urban recreational areas of Helsinki, Finland. Altogether 1,688 Ixodes ricinus ticks were collected from five areas located within 5 km of the downtown section of Helsinki, and 726 of them (303 nymphs, 189 females, and 234 males) were randomly chosen for laboratory analysis.
The midguts of the ticks were divided into three pieces, one for dark-field microscopy, one for cultivation in BSK-II medium, and one for PCR analysis. Ticks were found in all the study areas; their densities varied from 1 to 36 per 100 m along which a cloth was dragged. The rate of tick infection with B. burgdorferi sensu lato varied from 19 to 55%, with the average being 32%. Borellia afzelii was the most predominant genospecies in all the areas, and no B. burgdorferi sensu stricto isolates were detected. Only two ticks were concurrently infected with both B. afzelii and Borrelia garinii.
Dark-field microscopy gave more positive results for B. burgdorferi than did cultivation or PCR analysis. However, the agreement between all three methods was fairly good. We conclude that Lyme borreliosis can be contracted even in urban environments not populated with large mammals like deer or elk. The disease should be taken into account in the differential diagnosis of certain symptoms of patients from these areas, and the use of measures to improve the awareness of the general population and health care officials of the risk of contracting the disease is warranted.
DF microscopy. One of the three parts of the tick midgut was placed on a microscope glass in a drop of BSK-II medium and was covered with a cover glass. The number of spirochetes in the midgut sample was estimated by DF microscopy (Laborlux D; Leitz, Nürnberg, Germany) by examining 100 fields at a magnification of ×400. Typical movement, morphology, and size were used as the identification criteria for the borreliae.
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