Borrelia Spirochete Morphology in Peripheral Blood
Various morphological forms of spirochetes can be observed in blood. This experiment aimed to document some of these forms observed within 5 days of preparing a mini-culture on a microscope slide. Eleven donors with Lyme borreliosis and/or Myalgic Encephalomyelitis provided a fingertip blood drop which was mixed with BSK-H culture medium. Spirochetal forms were observed from each donor mostly within in 48 to 72 hours. Supporting evidence for the spirochetal nature of the agents is provided including micrographs from long-term culture experiments that included 5 of the current donors; and additionally, results from polyclonal fluorescent antibody staining specific for Borrelia species and Borrelia garinii from 3 donors.
There is controversy surrounding some aspects of Lyme borreliosis, a spirochete infection which is transmitted by the bite of a tick and caused by Borrelia burgdorferi and related strains. The areas of disagreement include testing: which often depends upon a Western Blot detecting antibodies whose presence some believe may be variable (1), treatment: 100mg doxycycline twice daily is considered to eradicate the infection in one month (though alternatives, intravenous and longer treatments are sometimes given) (2); persistence: it is claimed that ongoing symptoms after treatment are not due to continued infection but are related to tissue damage, reinfection or other explanations (3); location of spirochetes: some consider that borrelia spirochetes are rarely found in blood (4).
Direct detection of spirochetes is possible using darkfield microscopy, a method that uses oblique illumination sometimes described as 'light staining' and in which the subject shows brightly against a dark background. Darkfield microscopy is used for the diagnosis of syphilis which is caused by another spirochete, Treponema pallidum, by observing the very thin spirochetes in tissue samples (5). Though microscopy alone cannot identify the exact species of a spirochete, it can locate their presence and allows unusual species or variants to be observed. In a study of 90 patients which compared testing methods, Tylewska-Wierzbanowska and Chmielewski (6) concluded that: "There is no correlation between the level of antibodies (ELISA), the number of protein bands (Western blot) and the presence of spirochetes in body fluids (culture and PCR), indicating that in addition to serological testing the use of PCR and cultivation in the diagnosis of Lyme borreliosis should be recommended."
Lyme borreliosis (LB) and myalgic encephalomyelitis (M.E.) patients have active communities sharing knowledge and support through online groups. Friends made through these resources were invited to participate by providing a few drops of fingertip blood to see if spirochetal forms could be cultured and observed with darkfield microscopy. Two different kits were designed to be sent by mail allowing donors to provide samples with minimal inconvenience.
The dimensions given for borrelia burgdorferi spirochetes vary considerably. Russian researchers found that the length varied from 3 to 74 microns with average size ranging from 10.7 to 24.8 microns (7). The bacteria are generally too thin for observation with conventional light microscopy (8). Spirochetes are often thought of as long bacteria with a helical or waveform appearance, though other forms have been identified. There are various spheroplast forms whose size and shape can vary considerably. Mursic et al state: "The role of different atypical bacterial forms, spheroplasts and L-forms in infectious disease is considered an important problem in microbiology. Spheroplasts and growth into L-forms occur in many bacterial species in liquid and on various solid media. These forms are products of partial or complete removal of the cell wall by enzymatic digestion (..) and partial or incomplete inhibition of cell wall synthesis..." (9)
As well as L-forms with a reduced or absent cell wall there are 'mutant' types of borrelia spirochetes. Some have lost their flagella and motility and are seen as straighter than wild types and may have lower metabolic activity (10). Atypical forms of borrelia include elongated bodies that are comparatively thick (11), they can appear bacillus-like, may be long and thin with a thicker region, can be looped or twisted or have a looped end (12).
Spirochetes can undergo transformation into a chain of round-bodies sometimes referred to as a 'string of pearls'. Prior to transforming into this distinctive form, spirochtetes can sometimes be seen to have signs of segmentation along their length. Lyme Info (www.lymeinfo.net) cite Hindle, E. (1912) who wrote on 'spirochaeta gallinarum' (13): "The spirochaete gradually assumes the appearance of a chain of beads ( ) contained within the transparent cell-wall." And further cite Ewing (1907) on Spirochaete pallida: (14): "The organism may appear as a chain of granules which outline a complete spirochaete."
These possible variations do not mean that everything that is long and thin in a blood-drop is a spirochete. Amongst other possibilities to consider are fibrin strands, which can appear to wriggle with Brownian motion - BSK does not prevent the development of fibrin strands and neither does EDTA completely. Basket and smudge cells are sometimes seen and these disintegrating lymphocytes can create a lot of detritus of unusual shapes. A skin puncture can result in samples containing damaged cells and non-blood fluids as well as external contamination. Collagen fibers and decaying cell membranes have been suggested as an explanation for some spirochete-like agents (15).
When spirochetal forms become visible in a wet-drop blood slide, they can usually be observed for a few days before the majority have developed into the characteristic 'string of pearls' which then break-up into shorter chains and individual coccoids. The resulting forms would then require techniques such as PCR or antibody staining to identify them as being spirochetal. Even though the window of opportunity for observation is reasonably broad its starting point can be quite variable. Sometimes spirochetal forms can be observed immediately upon preparation of a slide, yet even with the same donor on different occasions it may take hours or days for them to grow. If a donor is taking antibiotics this might also be a factor that could impede growth.
Therefore it is desirable to create conditions which encourage the growth of any spirochetal forms present to improve the chances of observation. Borrelia burgdorferi is microaerophilic preferring the presence of oxygen at low levels (16). Mysterud and Laane (17) achieved good results in isolating spirochetes in blood culture by using a solution of sodium citrate which encouraged the growth of spirochetes. Additionally, the mini-culture environment that they created on the slide gradually developed regions where oxygen levels were favourable for spirochete growth.
Materials and Methods
Donors gave Informed Consent to participate in the experiments and for their data to be published. All experiments were conducted in accordance with the Declaration of Helsinki 1975.
For these experiments Barbour-Stoenner-Kelly Medium (BSK) with 6% rabbit serum (Sigma-Aldrich) was chosen as an additive to encourage spirochete growth, dilute blood components that could impede spirochete growth and separate the blood cells for ease of observation with a darkfield microscope.
For the first phase of the experiment a kit was sent to donors consisting of a hardboard slide mailer containing 3 pre-cleaned glass microscope slides and 50mm coverslips, a lancet and a cleaning wipe, along with a return stamped and addressed envelope (SAE). Donors put a small fingertip drop of blood in the centre of each slide, placed the coverslip over it and returned the slides in the mail. Upon return of the slides a drop of BSK was added at the edge of the coverslip which was drawn into the sample by surface tension. The long sides of the coverslip were then sealed with adhesive. First observations with the microscope were made 24 to 48 hours after the sample was prepared and again on following days for up to a total of 5 days.
The second kit tested additionally included a sterile pipette and a tiny tube of BSK as well as a tube of adhesive. To prepare the sample the donor produced a blood drop as with Kit 1, then added a small drop of BSK to the sample. This was stirred for a few seconds with the pipette tip before the coverslip was placed over it and then blotted. The long sides of the coverslip were then sealed with adhesive.
The stock BSK was tested for contamination with control slides in which the culture medium was exposed to the atmosphere on a slide for one minute and stirred with a pipette-tip (as occurred with Kit 2) before a 50mm coverslip was placed and sealed; then stored for 48 hours and examined for contamination. Some particles were noted, a few very sparse bacteria and a very few elongated agents were observed which did not have spirochete characteristics.
Videos of 640x480 pixels at 30 frames per second were recorded from the microscope as well as still images at 1600x1200 pixels with a Fuji Finepix F10 digital camera mounted onto the microscope eyepiece. Images and videos were carefully edited by filtering noise and adjusting levels and some were cropped. Clips from each individual donor's sample were compiled into short videos which comprise the main data for the experiment.
Of eleven donors in total, seven donors provided samples for Kit 1. Ten donors provided samples for Kit 2 including 6 of the donors of Kit 1. Two donors additionally provided samples for fluorescence microscopy experiments.
6 women and 5 men donated samples. Ages ranged from 37 to 70 years. All donors have had long-term health problems with 8 of 11 being ill for 19 years or longer. 8 donors have at some time been diagnosed with M.E. 10 of 11 donors had been tested for Lyme borreliosis through the NHS and the result was negative (1 not tested). 9 of 11 had private tests that were positive; (2 not tested privately). 7 of 11 had taken antibiotics within one week of preparing their culture sample. The impact of illness varied widely amongst donors with average self-rated physical ability at 32% and mental ability at 44%. See Table 1.
|Gender||Male 5||Female 6|
|Age range / average||37 to 70||average 57|
|Years of illness range / average||5 to 29||average 20|
|Diagnosis||M.E. 1, LB 3||ME + LB 7|
|NHS Lyme borreliosis test||Neg 10||N/A 1|
|Private Lyme borreliosis test||Positive 9||N/A 2|
|Antibiotics taken within last week||Yes 7||No 4|
|Self-rated physical ability range / average||10% to 70%||average 32%|
|Self-rated mental ability range / average||10% to 85%||average 44%|
Donor demographics. LB = Lyme borreliosis. M.E. = Myalgic Encephalomyelitis. NHS laboratory test for Lyme borreliosis is a 2 tier test, part one being an ELISA which if positive or equivocal is followed by a Western Blot. Self rated abilities compared to former health or, if ill for more than 10 years, compared to peers.
Results of Experiment Kit 1
Only 1 set of slides were a complete failure and the donor kindly provided a repeat set which was suitable. All the other sets had at least one slide that was useable. With this kit, problems were mainly found to be related to the amount of blood put on the slide. This is described in the discussion below.
Spirochetal forms were usually observed quite easily with the majority producing results between 2 and 4 days after the sample draw. Figure 1 shows images taken at the same time as the video recording, or excerpted from the video itself. The legend with each picture refers to its corresponding video listed below. 1, 2 and 8 are from the same donor. Particularly recommended are videos 3, 8 (note the waveform spirochete at 10 seconds) and 10.
Figure 1. Results of Kit 1. Showing photographs made at the
same time that video was recorded or a still image extracted from the video.
The legend beneath each panel corresponds to a video file.
Right Click on a number to view the video hosted on Youtube:
Results of Experiment Kit 2
One set of slides showed rapid decomposition of the erythrocytes though one slide from this set was less affected and this was used. Other sets sometimes contained decaying erythrocytes, usually close to the exposed short edge of the coverslip. One set produced results in just 24 hours though most results occurred between 2 and 4 days as with Kit 1.
Some donors had problems achieving a suitable ratio between BSK and blood. A few had problems handling the adhesive to seal the long edges of the coverslip and the glue sometimes trailed across the coverslip (which was easily removed). Some samples were found to be rather deep which made microscopy awkward. However, every set had at least one slide that was useable. Figure 2 shows photographs from Kit 2. In general the results were better than Kit 1. Videos 15 and 16 are from the same donor. Particularly recommended videos are numbers 10, 13, 14, 15, 16 and 20, the latter concluding with a still image showing a spirochete forming 30 individual coccoids within its membrane, like a 'string of pearls'.
Figure 2. Results of Kit 2. Showing photographs made at the same time that video was recorded or a still image from the video. The legend beneath each panel corresponds to a video file.
Right Click on a number to view the video hosted on Youtube
10 11 12 13 14 15 16 17 18 19 20 Play All Videos
Although the experiment aimed to study samples for 5 days, a few slides were examined much later and as expected most had become unusable. However, after 10 days a sample slide from Kit 2 was found to have some interesting spirochete-like agents which were easily observed. In several panels there appear to be encysted (curled-up) spirochetes:
After 36 days a sample slide from Kit 1 was examined and several typical waveform spirochetes had grown in the sample as shown:
Click on a number to view the video hosted on Youtube:
Supporting Evidence from Previous Experiments
Further supporting evidence for the spirochetal nature of some subjects comes from an experiment that included 5 of the same donors that helped with the current experiment. These 5 had previously provided venous blood in a Lavender-top EDTA tube. 1ml of whole blood from these samples was used for long-term culture in 7mls BSK medium with 6% rabbit serum in a 15ml sealed tube kept at room temperature for over 3 months before incubation at 30c. At 5 months the cultures were found to have grown spirochetes with their typical wave and helical forms as shown in Figure 5.Figure 5. Examples of spirochetes as cultured from 5 of the current donor's with long-term BSK culture.
For further details and examples of long-term blood culture in BSK please see:
BSK culture page 1
BSK culture page 2
BSK culture page 3
BSK culture page 4
Further evidence demonstrates that borrelia morphological forms could be detected in 2 of the donors who provided a fingertip blood drop and 1 donor who provided a venous sample (Lavender-top EDTA) for long-term BSK culture. A sample was mixed with KPL's anti-borrelia spp. polyclonal FITC antibody (cat: 02-97-92 Link to product page) and/or Abcam's Anti-Borrelia burgdorferi garinii polyclonal FITC antibody (cat: ab20118 Link to product page).
The antibodies used here were produced in host animals against dead spirochetes. The antibodies that their immune system generated against the infection were collected and refined. These are then conjugated (combined) with flourescein which glows when it absorbs certain light frequencies (colours). Fluorescein absorbs blue light but it emits green light. With correct filtering it is possible to view ONLY the fluorescence.
When an antibody finds a matching protien in a sample it locks onto it, behaving exactly as an antibody should. This also means that the combined fluorescein is also locked onto the matching target. If enough antibodies lock to a target then with fluorescence filtering the target will glow brightly.
There are 2 other types of fluorescence that need to be considered
in fluorescence microscopy:
1. Background-fluorescence, which occurs because of imperfect filtration, thermal effects and the tendency for many substances to have a tiny amount of fluorescence - including blood plasma.
2. Auto-fluorescence, which occurs when cells have naturally occurring fluorescence at higher levels. This could potentially be misinterpreted as successful fluorescent staining. Auto-fluorescence is particularly notable with lymphocytes (white blood cells) and also occurs with platelets. Furthermore, Dr Lida Mattman considered borrelia to have auto-fluorescent properties.
For these reasons the experiments demonstrated below include control-slides. It will be noted that auto-fluorescence was consistently very low but this is probably due in part to mixing with distilled water to match the similar dilution of stains. When whole blood is not diluted the auto-fluorescence of cells can be considerably higher.
Examples of fluorescent antibody staining from one donor showing fluorescence filtered image on the left of each matched pair and normal darkfield image on the right.:
Please see these pages for the complete fluorescenct antibody microscopy results from 3 donors:
Garinii FITC stain of culture FITC staining borrelia species
Borrelia Garinii FITC stain of blood2 FITC staining borrelia species
Borrelia Garinii FITC stain of blood3 FITC staining borrelia species
Borrelia Garinii FITC stain of blood4 FITC staining borrelia species
KPL's 'Bactrace' FITC stain of blood1 FITC staining borrelia species
KPL's 'Bactrace' FITC stain of blood2 FITC staining borrelia species
Borrelia burgdorferi and Borrelia garinii FITC combined FITC staining
It is not claimed that all of the elongated agents in the images and videos are spirochetal, yet many have characteristics that when correlated with the known morphology of spirochetes and the donor's health and diagnosis; as well as the successful antibody staining of 2 donor's samples and 1 donor's long-term culture, supports the idea that they are in fact spirochetes. The results were variable but it was shown that these forms could be cultured and observed in samples prepared by the donor. The quality of the microscopy imaging was dependent on various factors. The quantity of blood cells, ratio to and mixing with the BSK and depth of the sample all affected the microscopy. Nevertheless, donors who had little or no previous practice managed to prepare slides that produced interesting results.
Problems encountered with Kit 1 were mainly around the size of the blood-drop. An ideal amount would spread to cover 50% to 70% of the area of the coverslip. This was difficult for donors to judge, requiring accuracy to a few microlitres. Too much blood would seal the edges of the coverslip and left no room for the addition of BSK. Too little blood resulted in desiccation of the sample and poor mixing with the BSK.
With Kit 2 BSK was added by the donor making the process more complicated. This slowed down the preparation and meant that blood and BSK were exposed to the atmosphere for longer and increased signs of contamination were noted. Mixing of the blood with the BSK was sometimes incomplete - donors were asked to stir the mixture with the pipette tip for 2 to 3 seconds which proved to be too short, sometimes leaving obvious clumps of blood which resulted in a deep sample. Some of the slides containing rather deep samples for microscopy might have improved with further blotting of the slide prior to sealing the coverslip. The Donor Instruction Sheet requires improvement in these respects.
Donors were people diagnosed with M.E. and/or LB. In these illnesses a phenomenon described as 'sticky blood' can occur. Dr Byron Hyde remarked: "It is well worthwhile for all physicians reading this definition who have an interest in M.E. to examine the Internet for Hughes Syndrome . a vascular syndrome also called Sticky Blood Syndrome, closely parallels the definition of M.E." (18)
Dr Charles L. Crist is an MD with experience of treating Lyme borreliosis, he observes on his website (http://drcharlescrist.net/Borreliosis/Hypercoagulation/ Accessed 26 November 2013.): "Hypercoagulation, or thrombophilia, may be defined as reduced capillary blood flow or a greater tendency than normal for blood to coagulate, or clot. Of approximately 900 borreliosis patients that I have tested, 90 percent have hypercoagulation. Comparatively, only five percent of the general healthy population has hypercoagulation."
'Sticky blood' does not spread well beneath a coverslip and can form a thick film of which the outer edge dries-out quickly. It does not mix readily with the BSK and can result in a sample that is inconveniently deep for microscopy.
In addition to the problems mentioned, persons with M.E. or LB can have significant problems with coordination and fine motor control, impaired eyesight and difficulties with concentration. Some donors had to make special arrangements to post their samples. Given the severity of the illness of some donors it is remarkable how well they managed. It was found that the donors were interested and enthusiastic about trying the experiment and that with little or no previous experience they were often able to prepare a slide suitable for microscopy.
5 of the donors in this experiment had previously provided 2mls of blood in an EDTA collection tube. This method permits control over the preparation of sample slides and enough sample to start long-term cultures and conduct other experiments; making it a far better method than the kits tested here. Nevertheless, this simple experiment gave some who are virtually housebound by their illness an opportunity to participate in an experiment that has produced results.
Variations in the results occurred even from individual donors which suggests that the method is not optimum; it might also indicate that detection of low numbers of spirochetes would be even more variable. The shortest duration of illness amongst the donors was 5 years, with 8 of the 11 ill for more than 19 years. It is unknown whether this experiment could culture spirochetes in people who have been ill for months or even a few years.
The results shown in some videos appear rather shocking. It must be remembered that this is not the natural state of the donor's blood but shows an artificially created environment aimed at encouraging spirochete growth. No attempt was made at counting cells and minimal donor details were collected. Yet an impression was that the severity and length of the donor's illness did not correlate strongly with spirochete numbers in this experiment which looked only at peripheral blood cultured for a limited period. Rather, the ease with which spirochetes were found and their numbers seemed to correlate more with the characteristics of the sample; i.e., a shallow sample with a reasonable density of well distributed cells seemed to be the factors that reliably provided an opportunity to observe spirochetes.
Blood put on a microscope slide undergoes numerous stresses which can damage the cells and create artifacts. Yet 24 hours or more after a slide was prepared, samples often appeared perfectly 'clean'. Cells had a normal appearance and no spirochetes and no collagen fibers or anything else that might be visually interpreted as being a spirochete were observed. The spirochetes have come from somewhere and logic suggests that they either grew from spheroplasts present in the plasma and/or attached to the surface of blood cells; and/or that they were inside cells and emerged from them. It has been suggested that agents like those observed are the product of cell membranes disintegrating and this theory would accord with the progressive increase of the spirochete-like agents over time. However, it does not accord with the fact that within a few days of their appearance, the spirochete numbers decline rapidly having converted into numerous coccoid forms. If these long-agents were artifacts of cell decomposition, one would expect their numbers to increase until the majority of cells had broken down.
Limitations of the experiment
Cell counting was not attempted. No healthy controls were included nor people with recent infection. The spirochete species is unknown.
Donors with long-term illness and diagnosed with Lyme borreliosis and/or M.E. can prepare mini-culture slides from a fingertip blood-drop using a simple postal kit. This allows direct observation of spirochetes reasonably consistently with darkfield microscopy within 5 days. When antibiotics were being taken by the donor at the time of sample preparation it did not prevent the observation of spirochetal forms. Long-term culture and fluorescent antibody staining can further support the conclusion that spirochetes are present. The replication of some of these experiments by qualified persons under laboratory conditions and with healthy control subjects would be of interest to the medical and scientific communities and the many patients that could be affected.
Sincere thanks are due to the donors whose samples and the trouble they took to prepare them made this experiment possible.
Navigate the sections version of this article:
Borrelia Spirochete Morphology Introduction
Borrelia Spirochete Morphology Results
Borrelia Spirochete Morphology Extended Results
Borrelia Spirochete Morphology Supporting Evidence
Borrelia Spirochete Morphology Fluorescent Antibody Experiment
Borrelia Spirochete Morphology Discussion, Limitations, Conclusion and Acknowledgments
Borrelia Spirochete Morphology References
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