posted
Ive seen the same shapes in my own blood, the thing that resembles the string of pearls:
There is actually a smaller motile borrelia visible (the lower arrow). The upper arrow shows the string of pearls. The left and right arrow show larger "pearls". Some of these strings i see are not made up of pearls, but look like longer worms actually (a single object). One thing that strikes me is that these objects grow quite fast. Basically within 6 hours it goes from seeing nothing (the time the blood is drawn), to these large strings. Im not sure if borrelia is capable of growing this fast? Could there perhaps be other parasites or fungal forms involved with these string like objects?
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Lymedin2010
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I am glad that you share with us the importance of microscopy in chronic infection. Imagine where we would be if LLMD's monitored their patients blood, especially after administering ABX. They can know with some relative degree of certainty whether the ABX are having a positive or negative effect.
I have also noticed in some of the videos that the spirochetes are fairly long. I have seen some jaw dropping long spirochetes & I am amazed that they can grow to that length.
Here is one such spirochete that I captured, but in no way represents some of the longer ones that I have seen. At times I see them 40-60+ microns long. Do you notice that at times some of these spirochetes are longer than what you thought possible? At one point the spiro in the video loops on itself & then two ends spiral into each other. In the video you can see the spirochete line double, thicken & darken where they meet & spiral into each other.
Also, how readily do you find them in WBC's? Early on in my investigation, I would see WBC's with rather large specks. I was always suspicious of their origins & wondered if they could be cycsts.
One fine day I happened to check my blood right after a hot bath soaking. I managed to get my body temp to 102+ that day & after some recouping from the after effects, I made my blood smear & noticed that many of the WBC's had a large quantity of specks. More so than what I have ever seen before this hot bath.
I had left the smear out for a few hours & then checked again & I could not believe it. For the first time ever I so many of the WBC's with spirochetes burrowing out & again some of these were VERY long.
I tried to capture one of them with a hand held camera, the only way I had at that time, but they are difficult to discern in this video.
This might give evidence to the belief that many spiros & cysts lodge themselves on skin surfaces & perhaps heat forces them back into the blood stream, where they can be more readily picked up by WBC's.
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Lymedin2010
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S13, I have seen your work before & I think they are spirochetes as well. You have a beautiful image there showing the spirochetes emerging from the RBC's. Looks like some blebbing from the tips & blebbing from the string of pearls. Even that beautiful more spiral like smaller one.
Whenever possible, I would always do video first. It is so much easier for the naysayers to dismiss these as artifacts without them desiring to investigate this further.
As far as I am concerned, if one sees the bulbous tips, the undulating motion, and sheer abundance of such objects, then that is a dead giveaway. I think it is hard to logically dismiss that as random artifacts & should force the general scientific/medical community to AT LEAST be interested in further investigation.
Also, do you see that many of your RBC's are studded & bumpy? In the very beginning I thought they may have been cysts trying to burrow out, but after finding no evidence of them coming right out of the cells succinctly, I then concluded that it must by the hypertonic nature of the blood being out of the body.
The blood is at 96-98.6 & some h2o evaporation occurs. Over time the blood plasma becomes hypertonic & water travels from a higher concentration to a lower one. So in this respect water rushes out of the internal RBC & finds its way to the blood plasma. As a result the RBC's shrink a bit & appear studded.
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I also made a timelapse from what i think is a spirochete burrowing out of a RBC:
The timelapse is done manually, with intervals of 30mins to an hour. Its interesting to see that the RBC deforms where the potential (bleb-form?) spirochetes burrow from the RBC, and in the final image the RBC takes its normal round shape again.
Also some morphology in the next picture.
Red arrow: shows a long solid worm-like entity initially, which then breaks up in segments (string of pearls?) over time. Green arrow: shows a segmented string of pearls which converts to a more solid spirochete over time. The endpoint remains a bleb or cystform?
There is about an hour between each image.
These spirochetes are weird and "intelligent". Looking at it alive under the microscope is like seeing a very bad horror movie. Especially when you consider this is taking place inside your body!
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quote:Originally posted by Lymedin2010: Also, do you see that many of your RBC's are studded & bumpy? In the very beginning I thought they may have been cysts trying to burrow out, but after finding no evidence of them coming right out of the cells succinctly, I then concluded that it must by the hypertonic nature of the blood being out of the body.
The blood is at 96-98.6 & some h2o evaporation occurs. Over time the blood plasma becomes hypertonic & water travels from a higher concentration to a lower one. So in this respect water rushes out of the internal RBC & finds its way to the blood plasma. As a result the RBC's shrink a bit & appear studded.
Yeah, a lot of RBCs become dehydrated over time and will show bumps and crenation, but that is different from the cyst burrowing out like in the timelapse above. If all RBC's that show these bumps would have cysts inside them, then my blood is extremely infected. Virtually all my rbc's show the bumps over time.
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TNT
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These are just great! I'm fascinated, but totally freaked out. Definitely the worst horror shows I've seen in some of those youtube videos.
Have you ever seen any biofilm?
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Lymedin2010
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Crenated RBC's is what I see in normal folks blood & without any spirochetes, as well as those with Lyme. Here is a "normal" cell after it has been outside the body for some time.
Your beads of string time lapse is precious. On the first picture from the set, I wouldn't have spent much time without seeing clearly defined bulbous tips. Yet the specimen identifies itself as a spirochete via blebbing.
The stringy fibers are just that, fibrin.
Note that the bulbous tips can bleb themselves & that is why I say they must be underdeveloped or modified blebs. Both the blebs & spirochetes (via bulbous tips) can readily penetrate the RBC's & perhaps many other tissues (as they have been found in many varieties of tissues).
He may have some biofilm at the top right clusters of cells. I have seen patches of goo & what appears to be biofilm in my blood. At times they accompany spirochetes & other times they appear to be absent.
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quote:Originally posted by TNT: Have you ever seen any biofilm?
Not that im aware of. The biofilms would be difficult to diagnose from a simple live bloodsample. I think it requires a blood culture with fluorescent markers to identify clear biofilm colonies of spirochetes and blebs / granules. Like what Macdonald has done before.
quote:Originally posted by Lymedin2010: Your beads of string time lapse is precious. On the first picture from the set, I wouldn't have spent much time without seeing clearly defined bulbous tips. Yet the specimen identifies itself as a spirochete via blebbing.
Thank you! Yes, it was not a specific RBC i was tracking with the timelapse, but rather a large area at 400x mag. Within this area there was a lot of growth of these blebs and string of pearls. Literally in the hundreds. Strangely it is only this one area on the slide where it happens. Other areas stay clean without any growth. Somehow the infected RBC's seem to clump up together on the slide? Ive seen these infected areas on more slides before, but it puzzles me what the mechanism is. Perhaps while making the slide, the diseased RBC's stick to a certain area and the healty RBC's flow away to other areas?
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Lymedin2010
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In reference to the biofilm, that is exactly why I use the word "may" & "appears." At the end anything that we simply observe without testing can be discredited & obvious testing is required. We as simple microscopists continue to make observations that hopefully allow us to gather evidence that increases the likelihood of what we believe is there to actually be there.
What I have noticed is when I start a fresh smear, that there are relatively few biofilmed looking areas. The ones that don't have spirochetes and/or blebs I tend to dismiss. The ones that have them, I am more likely to accept & be further suspicious of.
If that same fresh smear is left over time & I make observations a few hours later, then I notice that there is a significant # of WBC's that have lysed & release their lysosomes and internal contents.
Over more time the WBC's lysosomes degrade adjacent RBC's, initially causing them to be sticky at their contact area to the resulting debris & at times the end result causes ghosting of the RBC.
Ghosted RBC's loose their internal content & it can appear rather dramatic at times. At times the end result of ghosted cells looks like candida all mixed in a field that can be mistaken as biofilm.
I found this more evident after doing some time lapse on WBC's & making comparisons from initial cell death to hours later. For this reason what I thought might be candida from my initial findings a few years ago, I now have a reference point of what might be their true origin.
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Lymedin2010
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On another note, I am surprised that we can't simply add a sample of our blood to BSK media & watch the spirochetes flourish? Why do we need methods such as xenodiagnoses, of a sterile tick biting a human to pickup the infection & then testing indirectly. There must be some mechanisms at work, or more likely something missing in the BSK solution?
What if we take a combination of BSK media & rabbits or sheeps blood filtered through the smallest filter possible? Or the heck with it, just take the very infected persons blood & filter it & mix that with BSK media.
Perhaps then we can culture them from our very own blood & use that as a basis for proof.
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Blood added to BSK will allow spirochetes to grow, but so will other things that are much cheaper. BSK is really useful when you want to do long-term culture, perhaps aiming to grow corkscrew spirochetes which I found can take months.
For short-term culture on a microscope slide, normal saline can be bought ready made or prepared at home with distilled water and salt. Sodium Citrate (as used by Mysterud and Laane) is cheap to buy on Ebay and this can also do the job.
Using a duluent separates the cells which makes for easier observation, but as others have remarked, it can also affect the shape of the cells.
Just putting blood onto a microscope slide subjects the cells to numerous stresses which can break them or make them misshapen. Other than doing thin-film smears which dry instantly (videos on how to do this on Youtube); it is questionable how much can reliably be told from blood cells in a wetdrop - though they are still fascinating to observe.
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What i would be interested in, is where to get the BSK medium? Also fluorescent markers like the ones macdonald uses would be very interesting to experiment with!
Since string of pearls is an interesting topic, and i seem to find a lot of in my blood, ive decided to share some videos. The first video is actually the same string of pearls as the one in the first picture from me, a couple of posts back:
Also a rather large colony which ive found, really horrific to see this in your blood. Again, all videos and pictures are taken after several hours of culturing, so you wont see this in freshly drawn blood!:
What you can see is; crenated RBC's (from the dehydration), many string of pearls, some medusa heads, probably lots of cystic forms (the smaller roundish forms? approx 1/2 the size of the RBC?), and perhaps it is clumped together by biofilm, but that is just speculative. Fluorescent microscopy would be very helpfull in this case!
So im starting to think now my blood is literally filled with borrelia cystic forms. Blarghhh... Good thing is that there are hardly any motile spirochete forms.
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Hi S13. I found your photos and videos very interesting. The time-lapse morphology is amazing.
Sigma-Aldrich stock BSK but will not sell to the public as it is shipped frozen with dry-ice. I found a laboratory supplier that ordered it, thawed it out and then sent it on to me.
To prevent drying of the slide, the edges of the coverslip can be sealed with immersion oil (a pipette is handy for this) or I sometimes use Bostik glue which can be removed. These should keep a slide usable for at least a week. To avoid getting immersion oil underneath the coverslip a sample is needed that takes up the whole area. Sealing the coverslip might prevent it from settling-down and giving a nice shallow sample that is easy to focus. Blotting the slide with paper-towel before sealing the edges can help.
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Thank you PeterKemp. Im looking in to more advanced camera system for my microscope, so that i can do automated long term timelapses. Now i have to make each photo by myself each hour or so, which is very time consuming, inaccurate and i get only a very limited amount of photos.
Thanks for the tip on immersion oil. I think it could prove helpful. But is it possible the immersion oil is pulled into the bloodsample?
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Timelapse can be done with a computer program if a camera is connected to the computer via a TV or Video Capture card (PCI card). The program is free and called VirtualDub.
In capture mode it displays the AV output from the camera and can record frames at the required rate. It can also do editing of the recorded videos or videos uploaded from the camera; though it does produce very large AVI files and needs plenty of hard-drive space.
I have made timelapse videos by manually recording a few seconds of video each 30 minutes or so. It is a nuisance to do, but the results have the benefit of showing any motion present which can be quite informative.
Lymedin and myself have both found problems with the scope losing focus when doing timelapse, so it might be that regular checking is necessary. I find this is due to the light being left on. The lamp I use heats up the scope which expands, losing the focus. It probably does not do the microbes much good either!
I have not had contamination problems using immersion oil to seal a slide. If there is enough volume in a sample to cover the whole area beneath the coverslip, the edges will dry out quite quickly and form a barrier. If you try it, make sure to blot the slide before hand to make a shallow sample for observing as drying will be slowed down.
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Lymedin2010
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I have the Amscope MU900 camera & at first it was horrific. When they finally updated the software it was much better at auto detect, but the lag & video refresh was intolerable for this type of work, where we are constantly hunting & changing views on the slide. It would simply take too long for the video to refresh on the monitor.
You would love the time lapse on the Amscope software though, it is very convenient.
Another researcher has told me that clear nail polish can be used to better affix the slip cover, but I have not tried that as of yet.
Here is a link to the BSK from another researcher, but I have never tried to order it.
ATCC Medium: 1914 Revised BSK Medium ATCC currently uses Sigma # B - 8291 BSK - H Complete medium. This medium is purchased in 500ml volumes and stored frozen at 80ºC. The medium is thawed and dispensed as required. If prepared from components: HEPES (Sigma H - 3375)...................................5.64 g Neopeptone..................................................4.7 g Sodium citrate................................................0.7g Glucose........................................................5.64 g NaHCO3......................................................2.0 g
TCYeastolate (BD 255772)..............................2.0 g Sodium pyruvate............................................0.75 g Nacetylglucosamine.......................................0.37 g Bovine Serum Albumin, Fraction V.....................47.0 g CMRL 1066, 10X (w/o Glutamine or NaHCO3)......100.0 ml Rabbit Serum (heat inactivated).........................60.0 ml DI Water........................................................840 ml Dissolve ingredients up to and including bovine serum albumin one at a time in distilled water. Adjust to pH 7.5 with NaOH and filter sterilize. Aseptically add CMRL 1066 and rabbit serum. Mix well and aseptically dispenses into appropriate vessel. Final pH of complete medium should be 7.5-7.6.
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I think it worth remembering that although the BSK formula looks very complicated, borrelia can grow successfully just on a drop of blood and whatever else it can find in a tick's gut.
Ticks regurgitate some of the liquid part of their meal, which I guess probably gets rid of some of any antibodies present and concentrates the cells. Borrelia can convert chitin (which the tick's exoskeleton is made of) into glucosamine; which suggests that it likes/needs it. Prof Brorson found that it also likes extra zinc.
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Lymedin2010
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Do they use a standard media, such as BSK, as a reference point, which allows for all things being equal other than the variables in question? In theory a great concept, albeit it is a pitfall with the complications of Lyme & in particular when making observations of virile drilling spirochetes vs more docile ones and the efficacy of growing spiros directly from human blood. The long growth rate of BB only adds to the complication.
Personally, I was going to obtain rabbit blood from a local butchery & make do with that using micro filtration. If that failed then I would have sought out BSK.
It is interesting about the chitin conversion & perhaps that is why LLMD's suggest glucosamine for joint support. What supplements do you think BB is more likely to deplete in the body & would you supplement at the risk of feeding & propagating infection?
Also, I never quite understood the notion that O2 would kill the spirochetes, especially when they are lodged into the very cells that act as O2 transports???? Isn't this contradictory, unless they enter cyst mode or bleb lengthing (forming blebs along the length of the spiro) upon making contact with in the blood?
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Lymedin2010
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S13, given that you have many spirochetes in your blood, what are your symptoms like?
I have checked the blood of various people who have been bitten & some only have a very few symptoms. For instance one person has only headaches & stomach pain that come & go. They have pains from time to time, but they come & go and it is difficult for them to consider ongoing infection.
Yet when I check their blood, I am left to wonder why they don't have consistent, more, & more pronounced symptoms. It is during these times that I often wonder how important detox pathways are to the manifestation of the disease or perhaps some other factor such as inflammation & immune response?
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Thats the thing, i dont have many spirochetes in my blood. With fresh blood i can hardly see any spirochetes at all! I think they are all cysts and blebs and they emerge after a short while of culturing.
My symptoms are very diverse. Mainly neurological, derealization, brain fog, cognitive difficulties, emotional/rage attacks, severe fatigue, nausea, twitching muscles, weight loss, jaundice, palpitations, SOB, night sweats and a few others. When im doing antibiotics or mhbot i dont have any pain at all. Besides borrelia, bartonella and babesia also play a big role in my disease. The borrelia, im thinking, is just keeping the other infections ongoing without doing too much damage by itself. But knowing what i know now, i will be focusing more on borrelia as well. First the cysts (never had a cyst buster before) and then the cell wall.
With the people you are helping, do you find loads of motile spirochetes in freshly drawn blood?
I think borrelia mostly causes symptoms in already weakened parts of your body. Parts that me be inflamed from previously existing infections or inflammation, or coinfections. If the detox pathways function properly and you eat well enough i think you can compensate a lot of the damage that borrelia does by itself. Like collagen replacement.
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Lymedin2010
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MOST do not have motile spirochetes in FRESH blood, but they come out eventually. Even with my own blood most of the time I find a few motile spirochetes, but every now & then I find a suprising amount from a fresh culture. I have realized that I have not paid much attention to when the blood has been drawn (before/after eating, caffeine consumption, abx usage...etc) & my next goal is to be more mindful, since it could buy clues.
This one person was on 2x ABX & with only 2 major symptoms, headache & stomach pains. When I drew their blood, I was shocked to find so many free swimming spirochetes from a fresh smear. I only drew this persons blood once & it was early on during my microscopy ventures & it just left me bewildered as to why not more symptoms & why so many free swimming? This person does indulge in extensive amounts of carbs & sugars and could be one possible explanation.
Maybe one explantion is that their immune sys is not compromised YET. The carbs & sugars bring out the chetes to the plasma & the WBC's are able to pick them up & rid of them because the immune system is still functioning normally.
I have all of yours except derealization, which I did experience for one day & it was such a crazy & inhumane experience, so I feel for you.
I also don't have emotional/rage attacks, BUT I do have exacerbation of anger. So for instance if I get angry or stressed, then the chemicals that calls for anger or stress get released & since the body does not detox or process them quickly enough, they linger at greater concentrations for longer periods of time.
The end result is that anger in me breeds anger 10x to the point where I can rage and I find that I can get explosive. Before chronic Lyme I was a very subdued individual & people knew me as a laid back guy & knew that nothing bothered me.
This I can also associate with chemical & food sensitivity. So if I eat or breath something & it enters your blood stream, it takes my body much longer to detox & remove the foreign particulates, so I go on feeling sick & toxic for hours after exposure or food intolerance.
Also with sleep. I used to not be able to sleep & it was difficult for me to sleep for years. Now if I lay down & sorta force myself, I can feel the melatonin kick in & I then feel as if I just took a sleeping pill & it knocks me out to the point where I have to sleep.
All these things that I mention above are more pronounced after the mold exposure that I just experienced. Just a few months ago, before the mold exposure, sleep was hard to get to, but falling asleep was seamless & now it feels like I have been drugged to sleep. Again because the concentrations build up too quick & are not handled properly by their respective pathways.
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Lymedin2010
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Do you have any of the following that I have?:
-food allergies -chemical sensitivities & mold sensitivity -internal vibrations -sudden onset of stroke like symptoms -sudden onset of muscle tightness, for me it is the neck, which then leads to brainfog, but it then releases. -jaw tighness -ear ringing -migrating joint pain (it took me years to develop joint pain & only after 1-2 yrs of ABX usage did it become pronounced).
Do you have any one of these too?:
-You engage in physical activity & if you perform it too quick, then you get shortness of breath & at times accompanied heart palps? To me that is a sign that the blood is loaded & parts of the body get devoid of oxygen & call for the heart to compensate.
-At times do you talk to fast & all of a sudden you can get shortness of breath?
-If you don't sleep enough then you get aggravated symptoms & in particular vibrations through the roof?
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Although borrelia prefers oxygen at low levels, inside the red blood cells the oxygen is bound to the heme so it probably does not have much effect on the bacteria. Babesia (which also infects erythrocytes) has very similar oxygen preferences to borrelia, i.e. very low levels.
I have found the same phenomenon of spiros sometimes being present, but mostly needing some culture time before they appear on a slide. One of my pet theories is that the drop in temperature, pressure, movement etc on a slide 'tells' the spiros that they have been ingested by a tick - so it is safe for them to grow.
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Lymedin2010
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So it does not easily "accept" the O2 from the hemoglobin...hmmmm.
So HOB therapy binds more O2 to heme or makes more free floating o2? My oximeter used to read 99% before the load of bacteria grew & now 98% mostly & at times drops to 97%, but that is not such a big change? I know others who run around in their 60%'s due to other medical issues.
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Lymedin2010
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Peter, we have seen the new research on sexual transmission. Knowing all that you know & all that you have seen. Knowing that borrelia can be found, all depending on what stage of the infection one is in, in the RBC's readily & that blood flows through the entire body. What do you feel is taking place?
Is sexual or contact transmission likely? I have seen the studies that describe contact transmission in animals & our microscopy observations lend itself to this likelihood of transmission?
Is Lyme perhaps by itself not as potent, but has a greater impact with co-infections? A person who has been bit, can go on for months, years, & even a lifetime without many symptoms, even if multiply co-infected. So is it not possible if contact transmission occurs that the partner may not necessarily acquire all the co-infections & therefore may take longer or never be symptomatic?
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Hi Lymedin. I have no medical or science training so any ideas I have should be taken with a big pinch of salt.
I'd say that the areas you mention need a lot of urgent research. Borrelia is in the blood. That is something that the CDC/IDSA, PHE and other authorities seem to be avoiding. So either they know that it is in the blood but have reasons for suppressing this information; or they simply haven't bothered trying to locate borrelia in the blood - which seems incredible.
Incredible.
I think that every point you make is credible or even likely - so where is the large-scale research needed to protect the population; to understand P2P transmission, other vectors, infection that starts with virus-sized propagules rather than motile spirochetes?
There are no good answers to these questions. The pittance spent on researching LB suggests to me that the CDC and others have cobbled together their theories and are sticking to them. They either already know the answers, or don't want to know them. We are left trying to figure them out for ourselves and for the sake of others including our loved ones.
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Lymedin2010
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Yea, it truly makes one wonder how the CDC & company do not bother to take a closer look at the blood of those chronically ill, especially in a BLOOD BORNE disease. It is just pure abhorrent idiocy on their part & I don't think they are that stupid and for that reason I think they MUST BE withholding what they really know?
I think the reality might be that they have identified this organisms as being:
-widely spread.
-involving and influential to billions of medical dollars.
-very hard/impossible to kill. Those who are in remission might be pushing time back to a phase where they were bit but asymptomatic & this can go on for months, years, & even lifetimes without any major complications. I have even heard of a story just this year of an older (70's) year lady coming into the hospital with only slight symptoms from a lifetime of Syphilis, but these types of stories are know near & far.
-Can create resistant specimens from ABX treatment readily.
One only need look & there is consistency across the border for what we see in the blood. The blood of those who are not treated look a lot worst & it makes one wonder...how is it that they are alive?
So coming from a psychotherapy background, what prompted you to take that first look? For me it was 2 months of entering chronic stage, realizing what my daughter was developing, the symptoms of many family members, & the people around me. It seemed only logical that I look.
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glm1111
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Excellent thread and info Lymedin! Just thought I would mention what Bryon Rosner has to say about parasites protecting the spirochetes and other bacteria. I will bring up the thread that gigimac posted for those interested in reading what he has to say about the antiparasitics being effective.
Gael
-------------------- PARASITES/WORMS ARE NOW RECOGNIZED AS THE NUMBER 1 CO-INFECTION IN LYME DISEASE BY ILADS* Posts: 6418 | From philadelphia pa | Registered: Jul 2008
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Lymedin2010
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Thanks Gael!
I have heard of the theories of parasites protecting borrelia. I can find borrelia in the RBC's despite aggressive & varied ABX treatment & it looks like borrelia does not need any parasitic help, although it is quite possible given the aggressive & tactical nature of the spirochete.
Very cunning & surprisingly adaptable in a host of creatures. So I wouldn't be surprised if they are found to be protected in masses within the parasites.
Peter, have you done any more MMS experiments? So in essence, MMS had an affect on the spirochetes & many could be found with their motility impacted? Where the blebs affected at all?
posted
Hi Lymedin and All. The MMS experiment used the chemical (chlorine) as a highly toxic antimicrobial. The aim was just to demonstrate that the spirochetes were living organisms and not 'artefacts' which Lyme denialists claim. To test its effect on blebs would be much more complicated and require reculturing the sample; though chlorine is so powerful that it is probably reasonable to speculate that with the levels used they would no longer be viable but neither would human cells.
Propagules are very tough. I attempted some DNA extraction (for PCR) on a sample full of these tiny round-bodies. The bacterial lysis kit I used only worked when I accidentally used 20x the specified amount of enzyme to dissolve the cell wall.
I also tried a microwave protocol which called for 6 minutes at 600 watts under pressure - to break endospores (very tough organisms). I could only simulate pressure by using a tightly sealed tube but gave it 10 minutes at 900 watts. Whilst many RBs shattered, around 25% of the propagules looked perfectly intact under the microscope (though they might actually have been dead).
Some nematodes have extraordinary immune suppression abilities. If memory serves, there were some doctors considering giving people with arthritis a single strongyloides worm which could reduce the inflammation in the joints of a person with arthritis. Just one worm could suppress part of the immune system!
Posts: 30 | From London | Registered: Jan 2012
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So, ive got my new automated timelapse microscopy setup running. Like you said Peter, the image will go out of focus when the lamp stays on for a long time. So i made a system that turns the lamp off in between the frames. That also should preserve the sample.
I was fiddling around a bit, and decided to try your method of sealing the blood sample on the slide with some immersion oil. That works perfectly! I do notice the borrelia bacteria is not forced out of the RBC as fast as before. I think the dehydration in my previous samples triggers the spirochete to mobilize and seek another living area. So for a fast culture i will use the dehydration technique (so no immersion oil seal on the slide) and for long term culture i will use the sealed technique.
I found a bizarre phenomenon today when looking at the long term culture: http://youtu.be/rlAYKAR9X2Q I have never seen this before, and it could very well be contamination from my finger or something. But it looks like fungal growth of the blood. Perhaps a lost candida cell? Each frame of the movie = 5minutes. Total of 7,5hours of growth is shown.
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Im wondering if this picture i have taken a while a go actually shows a budding yeast cell? If so, perhaps that eventually causes the fungal growth from my previous video.
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Hi S13, I use 50mm coverslips and often just seal the long sides which keeps the sample viable for up to a week, but still allows it to 'breathe'. Mysterud and Laane attribute their successful culture on a slide to the fact that certain regions of the sample develop the correct oxygen levels. This is what Dr Lida Mattman said years ago, that at a certain depth in a culture the oxygen level would be ideal for borrelia growth.
The video is very impressive and looks like it could be candida albicans which might have come from the air or could have been present in the sample. I used to find a lot of fungal growths on blood slides, but strangely, after taking long course of abx I rarely see them now.
Your timelapse set-up gives wonderful results. It would be fantastic to see a spirochete actually emerge from a blood cell and morph into a string of pearls. That would really stand the accepted 'wisdom' about borrelia on its head. I have tried to do this, but no luck yet.
Best Wishes, Peter
Posts: 30 | From London | Registered: Jan 2012
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I have smaller coverslides, 18x18mm, so i will try leaving open 1 side for breathing.
I think the fungal forms are probably a result of a suppressed immune system. So by dealing with the bacterial infections, the immune system should become more active, and fungal forms will be eliminated more quickly from the blood. Im still not sure if my fungal hypha is the result of contamination, or if it was present in the sample.
I will do some more experiments with the time lapse setup, so i hope to provide more results soon.
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Lymedin2010
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Here are 2 good candida vids & yours may as well be just that.
_______________________________________ The video reminds me of this Streptomyces coelicolor video. This is a gram-positive bacteria that is typically found in the soil.
Lymedin2010
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Do you guys see smaller versions of the spirochete, as represented by a dumbbell. It basically looks like a rod with bulbous tips on either end, sorta like this but connected together •-•
I see all sorts of these at various sizes & when we consider them all collectively, they can represent the various growth stages.
Also, do you guys see blebs with tails, basically a footed type of bleb? I think these are the blebs forming into the smallest spirochete, a dumbbell, & I am trying to get time lapse proof of this. There are various thickness & thinness of dumbbells too.
It is so difficult to do, since they constantly move in the Z field via Brownian motion and escape consistent focus. With time they also escape the entire field of view & force me to re-position & re-adjust the slide constantly.
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Lymedin2010
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S13, thought of your candida video when I found this article.
"First direct blood test for yeast, T2Candida, gets FDA nod"
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Great video lymedin! More people should know about this. You have almost the same microscope as i have. But i dont have the trinocular option, so i have to use one of my binocular tubes for the camera. Great tip about the led light too! My bulb is old and gives a yellowish color. I think the white led produces a fuller white spectrum right?
I have also seen the dumbbell shapes you speak of. Could be a spirochete i suppose?
In the my series of timelapses, i have captured a string of pearls emerging from a cyst: http://youtu.be/ObT6IHXHVFw Watch in HD to see the details. Im not sure, but could the process be triggered by the touch of the RBC???
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Thank you sir!
You can easily replace with a trinocular head & it would make life much easier for you. Buy used & then when you resell, you will get back most of your money.
Before that I used a soft white CFL, only because that is what I had in the house, but it resulted in yellowish light. I expected to get a white/daylight CFL & fortunately bumped into the Cree LED's. It is cheap enough for you to give them a try in darkfield & it should suite you well.
Oh man, I LOVE, LOVE, LOVE that video! That is exactly what I had wanted to do. So this is all manual time lapse? Do you find yourself having to refocus every few minutes, like I do? I won't have the stamina for 13 hr sessions & hence my lack of microscopy video production.
I wonder if it is really coming from a cyst or a RBC? It certainly is the right size & shape to be a cyst & I think more likely so. Also notice, that as the string of pearl emerges from the cyst, it absorbs the cyst, so that the cyst gets smaller & smaller over time.
Precious work, please keep it up!!!!
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Lymedin2010
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I KNEW they had to bleb out, but could never provide microscopy proof. I knew when I first looked at fresh blood I can only see a few spiros if any & then as time passed more & more spiros emerge. A few hours later I see a boatload & then MYSTERIOUSLY a few hours after that all of a sudden they disappeared from the plasma & there are far fewer left. So instead of many spiros, they become fewer, but there is a ton of dancing debris in the plasma. As if the blood conditions change further & don't allow for the prospering of larger spiros, only the formation of cysts & blebs.
For me it is hard to prove that the dancing dots are cysts or blebs, but with a video like yours people are more willing to listen. They would be more inclined to take a second look, if not anything else. Try to take it even further next time. Let the blebs break up & scatter in the blood plasma. I bet when you do, you will get something like this video I took a few months ago. Your video will show the missing link between the spiros blebbing & them scattering with TONS of blebs/cysts in the plasma over time.
As far as the dumbbells, just think about it logically. They break up into cysts & an adult spirochete has to come from somewhere. Even when you look at the smallest spirochete, it had to develop from something smaller.
When I string together the quantities of all that I see collectively it only makes sense that the sequence is bleb-> tailed bleb -> dumbbell spirochete -> spirochete w/slight undulation -> longer spirochete with more undulation -> very long spirochete & a break up into blebs. Of course there are so many variations in between any of stages as well.
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I have not seen the strings break up and scatter yet. I will try to do a longer timelapse to see what happens with the strings over time.
I dont do manual timelapses any more, no that is too time consuming. I have automated the process. Ive made a simple program on my laptop that forces the image-capturing software to take a picture every few minutes. It also turns the light of the microscope on a few seconds before the picture is taken, and turns it off a few seconds afterwards. This way the bulb of the microscope doesnt burn out so fast, and the blood sample is not heated up by the light. And it prevents the image from going out of focus. I think it would be even better with a LED light, since they dont mind being switched on/off all the time.
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Lymedin2010
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So Peter was correct then, the heat DOES force the scope to go out of focus? My discussions with other microscopists lead me to believe that it was the top weight that affected out of focus issues & that the camera + mounting contributed to this effect?
So you see a difference when turning off the light? Prior to keeping the light off when not needed, you had focus issues? And now that you are doing just that, you have managed to resolve focus issues? Also, how old is the blood smear from the first minute you begin the time lapse video recording?
When I press down slightly from the very top of my scope (from the camera), it gets out of focus.
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Yes Peter was right! Its not the static weight of the camera, its the heat that slowly builds up in the frame of the microscope causing it to warp.
And yes i had the same focus issues when i kept the light on. It has completely resolved with switching the bulb off in between pictures.
My microscope also goes out of focus when i press down on the top, but it needs s fair amount of weight to do it. Like 1 lbs of force doesnt make it go out of focus yet, but 4-5 lbs does.
Im not sure, but i think my blood smear was about 10 hours old before i started the time lapse.
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Lymedin2010
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S13, your last video is one of the most impressive videos that I have seen to date. I have distributed your video in various sites & shared it with key people.
I would also like to use your video in my future video with commentary & I will give you & your youtube channel credit.
I will do a lamp/focus test today & hopefully that will inspire me to do something about it. The out of focus issue is such a huge issue when doing time lapse & it is super awesome to know the real source of the issue.
Also, check out these DIC (Differential interference contrast microscopy) blood videos. Really expensive equipment, but I wonder if anyone has done any DIC Lyme microscopy? It really gives it that 3D edge in viewing/recording.
quote:Originally posted by Lymedin2010: S13, your last video is one of the most impressive videos that I have seen to date. I have distributed your video in various sites & shared it with key people.
I would also like to use your video in my future video with commentary & I will give you & your youtube channel credit.
Thank you! Please share it, i think more people need to be aware of this.
Those DIC images look very nice. You really get a sense of the 3D geometry. Expensive equipment probably Posts: 387 | From The Netherlands | Registered: Nov 2013
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Lymedin2010
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"...i think more people need to be aware of this."
Haha, that is exactly why I created this thread. I went from having just a few symptoms & checking my blood for days & hours. I could not find anything & I thought they were right, that the infection was imbedded deep in the tissue.
I checked days & weeks after & slowly started to see things. As I got sicker, I saw more & more of these things in the blood. At that early time, I knew whatever it was, it was responsible for making me & keeping me sick.
It is coming soon, check your blood at 1000X & you can see borrelia in your blood & this will make it available to EVERYONE & no need for scopes. Tie in time lapse apps on the gadgets & bingo!!!
They are using this to check for Anthrax too.
3D Printed Microscope for Mobile Devices that Costs Pennies
The forming of the cyst happened at the edge of the screen which is a bit out of focus (problem of my microscope).
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Lymedin2010
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Another beautiful capture. At the end you can lineup all your captures & a full story emerges.
After a few days of the smear being out, I find surprisingly less spirochetes coming out & more blebs/cysts. And it works out this way a majority of the time.
Are you finding the same thing? Also, are you waiting 20 min between each shot?
For anyone looking, there is a cheap microscope for sale on Ebay, the power supply is not included. The lamp/LED bulb technique I use in my video will more likely be a better substitute for nice clear & bright white light than the darn halogen on the scope.
It has a trinocular port & you can always add the attachment & cam in the future.
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