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» LymeNet Flash » Questions and Discussion » Medical Questions » The Microscopy Thread (Page 14)

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Author Topic: The Microscopy Thread
thatdudefromkansas
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Lymedin, pretty smoothe operation there, haha.


I'm gonna be posting a video shortly.

I had sent off some blood slides to Dr. MacDonald to have stained, but then he stated he had retired and, as such, was unable to continue that work. So he was not able to get around to staining my slides. It was for MS research and was provided free of charge.

If he is active again, and doing that work....drop him my name and let him know I still have MS, haha. I'm not gonna bother him again.
It'd be nice to have a definitive answer, though.
I could even get him a CSF sample.
I can provide you details in a message.

Also, you can find online courses to enroll in sometimes relevant to microbiology, etc.
www.edx.org is a good place. They have courses taught by professors from MIT, Harvard, etc, free to take.

Anyways, I made a short video. Slow internet here in Kansas, like the life here, haha.
Its a video of a gemma with a spirochetal body (tennis racket) showing the development of a second body, perhaps something like transverse fission occurring (one bacteria becoming two, asexual), perhaps simply a single organism reverting to an atypical spiral form, or perhaps it is a small cyst with a second spirochete emerging (though it is rather flat and discoid, making me think it's either a single organism, or the transverse fission).

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Lymedin2010
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Dude, I would love to see the video. Thanks for the edu link.


I will have to find the gemma conversion video again, may take some time.


Check out this spiro a fellow Lymie captured, as it moves & looks exactly like what I see in the ticks that I observed, at least for some of the spiros. Some are longer & some are shorter too & there is no standard size it seems.


This is the same exact form & movement we see in the CytoViva video & what I see in my blood from time to time. Granted there will also be false-spirochetes in our blood that can many times come pretty damn close to this, but will not have the complete vitality & movement of a true spiro.

https://www.youtube.com/watch?v=yBKXCXo2wB8


Here is CytoViva again:
https://www.youtube.com/watch?v=DnsuiSKGDRs


And at 1:19 in my tick video:
https://youtu.be/RnFR4Ca5MVo?t=79

All very similar.

And then the cyst forming one in my blood:
https://www.youtube.com/watch?v=OXJTGo59gqM&t=2s

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thatdudefromkansas
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https://youtu.be/YMdunXbr2eI
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Lymedin2010
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Thanks.

I have a collection of Tennis rackets in ticks, I never posted them though.

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TNT
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quote:
Originally posted by Lymedin2010:
And then the cyst forming one in my blood:
https://www.youtube.com/watch?v=OXJTGo59gqM&t=2s

quote:
Originally posted by thatdudefromkansas:
https://youtu.be/YMdunXbr2eI

EXCELLENT, BOTH OF YOU!!
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TNT
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You guys are referring to "gemmas." How are gemmas different than cysts?
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Lymedin2010
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I think Gemmas are immediate cysts & cysts can grow larger & can in itself have various morphologies from perfectly round, to oval, to wavey, and at times with a spiro protruding out.


On another note,
Do you want to check ticks under a microscope & then want to confirm presence of Bb?

"

CARE PLUS 38404 LYME BORRELOISE TICK TEST

Quickly and accurately indicates presence of the Borrelia bacteria in the tick, which may cause Lyme disease.
No waiting time for laboratory results.
95,8% reliability within 10 minutes.
Includes checklist.
Provides important information for your doctor, as a result of which any necessary treatment can be applied quickly – this increases the likelihood of success.

You must act quickly after being bitten by a tick. We know earlier is certainly better when it comes to the removal of the tick, detection and treatment of Lyme disease. Use the Care Plus® Tick Test to immediately determine whether the engorged tick is carrying Borrelia bacteria, which can cause Lyme disease. This (self-)diagnosis test looks like a pregnancy test and provides a 95,8% reliable result within 10 minutes. (study 2011)

This self-administered test and the checklist help provide information early on to you and to your healthcare practitioner. In the event of a positive result, and when you experience any symptoms, we recommend to consult your doctor and take the filled in checklist with you. On the checklist you note valuable information about the bite (date, site on your body, area), the tick itself (size), skin reddening, test result Tick-Test and various possible symptoms. While it’s not a substitute for clinical advice, this test in combination with the checklist can help motivate people, who otherwise might have waited, to seek medical attention, especially if they have a positive result for bacteria in the tick. Contents: Tube, liquid, wooden stick, pipette, test cassette, leaflet/checklist.

User instructions:
Step 1. Remove the tick.
Step 2. Place the tick in the empty tube.
Step 3. Crush the tick on the bottom of the tube with the wooden stick.
Step 4. Use the pipette to add 10 drops of liquid to the tube.
Step 5. Close the tube and shake it vigorously.
Step 6. Use the pipette to suck some liquid from the tube.Note: do not transfer any parts of the tick itself, as these can block the test field.
Step 7. Put 5 drops of sample solution into the funnel shaped opening of the Care Plus® test cassette and lay down the test cassette in horizontal position.
Step 8. The test result will be visible within 10 minutes.

Negative test result

A line appears in the control area (C). This means that the test has been carried out correctly. If no line appears in the control area (C), the test result is invalid and the test procedure must be repeated using a new test.
No line appears in the test area (T). This means that no Borrelia bacteria have been found in the tick.

Positive test result

A line appears in the control area (C). This means that the test has been carried out correctly. If no line appears in the control area (C), the test result is invalid and the test procedure must be repeated using a new test.
A line appears in the test area (T). This means that Borrelia bacteria have been found in the tick. In the event of a positive result, we recommend to consult your doctor and take the filled in checklist with you.

Take note: the intensity of the lines may vary, but this does not change the test result in any way.

Tip: Even if the Tick Test indicates a negative result, always visit your doctor if a red ring (Erythema migrans) develops around the bite area or if you experience any symptoms."

http://www.ebay.com/itm/CARE-PLUS-38404-LYME-BORRELOISE-TICK-TEST-/302106608411?hash=item4656f5031b:g:iW4AAOSw8gVYALMr

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TNT
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So, the gemmas are the most juvenile stage of the cyst? Ok. I just wanted to make sure I had my morphology correct because of these videos I just uploaded. About the time you think you have all the morphologies of borrelia figured out, BAM, you're hit with another one! I can't wait til Lida Mattman's 4th edition arrives!!

Lymedin, I've seen that test kit advertised, and wondered how accurate it really was. But, not because I'm going to be collecting and messing with any ticks, that's for sure!


Video of spirochete with darkfield:

https://www.youtube.com/watch?v=jEeiffqaIps


Video of (gemma) cyst with darkfield:

https://www.youtube.com/watch?v=2ktZv_VOBUU


Video of Rickettsia bacteria with darkfield:

https://www.youtube.com/watch?v=oJgI3lr2ng8

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TNT
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I have a video with ketes, gemma cysts, and Rickettsia bacteria all in the same frame but I have to edit the sound on that one. I will post it as soon as I'm able to.
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TNT
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Have a few interesting darkfield pics of gemma cysts inside some leukocytes. Will post them later as well.
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thatdudefromkansas
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TNT, what is your microscope setup?
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TNT
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It's an American Optical 10 series with an oil darkfield condenser. Those darkfield videos I just posted are with a funnel stop in the 100x objective, so my darkfield may not be as precise as it could be on those videos. I recently got a 100x plan objective with an iris, but have not done any darkfield with it yet. Also, those videos were with a scope that currently has a left hand stage control, which made it difficult for me to use, thus the shakiness in the video.

See the pics of my setup a couple pages back.

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thatdudefromkansas
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Ok, cool.

I've been using my Amscope T490. Oil condenser, Plan 100x Oil with iris.

Thinking of upgrading to a Plan Apochromatic 100x Oil with Iris, but those seem to be quite expensive. I want to go with a Nikon, Zeiss, or Leica.

Gotta find a good deal on eBay first.

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thatdudefromkansas
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https://youtu.be/tfWNVaXMcBw

check out from about 45 seconds onward.

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TNT
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Dude! Those are those granules I was talking about! The smaller ones look identical to the ones I've seen in my blood. It looks like they (the larger ones) are some type of pregnant gemma.

Are you off ABX right now by any chance?

Great video!

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thatdudefromkansas
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I am.

And I have noticed a significant increase in the presence of these bodies as well as the spirochetal ones upon stopping, along with increased symptoms.

Had to take a break due to GI stuff from the (I presume) clarithromycin.

Interesting, I saw a large number of these granular bodies. It looked more like a cluster of spirochetal bodies attached to each other or something else.

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thatdudefromkansas
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I have also purchased a PlanApo 100x oil objective with iris to use.

Hopefully it will bring some resolution to the aberration you see with y current objective, and will also provide me with higher resolution so this stuff is a little clearer.

I'm also messing with the digital zoom on my camera at 400x to see if i can get increased resolution at around 2,000x.

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thatdudefromkansas
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Currently watching and recording a spirochete emerging from a cyst. Will have that video up later. Batter on camera is getting low, but hopefully I can record long enough to get the spirochete separating.
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TNT
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How'd it go, dude, did your camera battery hold out?
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thatdudefromkansas
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Waiting on my slow internet to finish upload.

Nope, didn't hold out.
Would have taken quite a while, and the file would have been massive anyways.
That would have taken days to upload with my current internet speed.

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thatdudefromkansas
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https://youtu.be/SVobHYLvTns


later in the video is a digital zoom closeup.

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mustardseed2
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I started doing some staining. This showed up fairly bright. Any idea what this could be? OR just an artifact from a poor stain?


 -

I seems to have a lot of random stuff like this kicking around:

 -


 -

[ 12-19-2016, 10:52 PM: Message edited by: mustardseed2 ]

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Lymedin2010
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Looks like artifacts/inclusions to me.
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thatdudefromkansas
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New objective lens came in.

It's Plan Apo, as opposed to Plan Achro, and I see a big difference.

I think it was well worth the price.
Minute details of the bacteria are more readily visible, as well as surface detail of granules.

More use will give a better understanding of all the visual advantages. I have a test video I will share whenever it has uploaded.

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TNT
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quote:
Originally posted by mustardseed2:
I started doing some staining. This showed up fairly bright. Any idea what this could be? OR just an artifact from a poor stain?


 -

I seems to have a lot of random stuff like this kicking around:

 -


 -

I agree with Lymedin, I see no particular morphologies in those pics. As a tip: When you put stain on top of your dried smear, make sure you sufficiently flood the top of the slide. That way it will not begin to dry before you rinse. This will minimize the possibility of artifacts.
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TNT
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quote:
Originally posted by thatdudefromkansas:
New objective lens came in.

It's Plan Apo, as opposed to Plan Achro, and I see a big difference.

I think it was well worth the price.
Minute details of the bacteria are more readily visible, as well as surface detail of granules.

More use will give a better understanding of all the visual advantages. I have a test video I will share whenever it has uploaded.

I can't wait to see some images & video with it!
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thatdudefromkansas
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quote:
Originally posted by TNT:
quote:
Originally posted by mustardseed2:
I started doing some staining. This showed up fairly bright. Any idea what this could be? OR just an artifact from a poor stain?


 -

I seems to have a lot of random stuff like this kicking around:

 -


 -

I agree with Lymedin, I see no particular morphologies in those pics. As a tip: When you put stain on top of your dried smear, make sure you sufficiently flood the top of the slide. That way it will not begin to dry before you rinse. This will minimize the possibility of artifacts.
Yep, make sure you flood the top of the slide.
I always flood the top of the slide just to the point of the stain's surface tension breaking and flowing over the edges, if that makes sense. You can work with it however you desire, especially if you want to conserve resources, but I always completely fill the top of the slide.

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TNT
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Has anyone seen this video and do any of you know anything more about this website (lyme-n.com)? It appears that you have to join the membership to view the website.

Here's the video:

https://www.youtube.com/watch?v=d9hY670JQK8

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thatdudefromkansas
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https://youtu.be/4arWiJ4Iw8U


New objective test.

I'll have to see about finding a larger specimen to film.

The resolution is certainly much higher.
That last portion is digital zoom at 3000x.

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thatdudefromkansas
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Interesting video.

I have sent off an email to them. We'll see what they say.

Is that a collagen coated slide or something?
It appears those spirochetes are maybe on a different plane than what you can see in the background (you can see lots of movement out of focus).

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thatdudefromkansas
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http://fightinglyme.weebly.com/lyme-n-blog/lyme-n-website-up


It appears to be a website for a study.
However, it appears as though the website came down. Perhaps because of outside pressure from medical community, etc.

Here's a youtube channel.
https://www.youtube.com/channel/UCXlWHWaMMnG_FelaXy6s_Dw

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thatdudefromkansas
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http://www.healingwell.com/community/default.aspx?f=30&m=3742123

Discussion of Lyme-N treatment on the healingwell.com lyme forum.

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thatdudefromkansas
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It's a nebulizer treatment based off of organic and inorganic compounds.

60 day treatment is 3,800 dollars. Yikes.


My instincts tell me it is likely a moneymaking scheme, as reviews I have read online by people who have gone through the treatment report no changes.

Allegedly, it is a nebulizer treatment with a compound that allows penetration of the blood/brain barrier.
I'd be interested to see what the ingredients are. Seems like a nebulized solution of something that is nebulizer simply as a gimmick to make it appear more legitimate or something.
Of course I could be wrong, but that is what I am getting from the stuff I have been reading online in the short time I have been reviewing it.

On a side not, I need to get a fresh blood sample under the microscope. I have noticed that the BSK creates a noticeable change on light refraction in samples. So samples in the BSK do not appear as bright/clear as samples of whole blood...which makes sense.

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TNT
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In this video it doesn't appear that the spirochete is a Bb spirochete. It's too BIG. Notice the comparison bar. It's an incredibly long and thick Bb spirochete if it is one.

Maybe a Relapsing Fever spirochete....

Sorry, forgot to post the video link:
https://www.youtube.com/watch?v=jR_-Vk1KUz8

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thatdudefromkansas
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Perhaps.

It wasn't clear. It says Lyme....but are they using that as an all-encompassing term?

Also........would it's appearance in those videos suggest that it is a laboratory specimen?

How often, even in video produced by reputed laboratories/pathologists/scientists, do you see BB take a form like that? You'll still see videos of BB in the spiral form, but even then, the wavelength is generally much longer and less rigid.

These videos give the appearance of something much closer to treponemes and not borrelia.

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thatdudefromkansas
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You could also give benefit of the doubt to the micrometer bar in the video being an estimate, and begin WAY off.
It'd be an easier estimate next to an RBC. However, that could be generated by software/camera that is fairly accurate.

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mustardseed2
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quote:
Originally posted by thatdudefromkansas:
quote:
Originally posted by TNT:
quote:
Originally posted by mustardseed2:
I started doing some staining. This showed up fairly bright. Any idea what this could be? OR just an artifact from a poor stain?


 -

I seems to have a lot of random stuff like this kicking around:

 -


 -

I agree with Lymedin, I see no particular morphologies in those pics. As a tip: When you put stain on top of your dried smear, make sure you sufficiently flood the top of the slide. That way it will not begin to dry before you rinse. This will minimize the possibility of artifacts.
Yep, make sure you flood the top of the slide.
I always flood the top of the slide just to the point of the stain's surface tension breaking and flowing over the edges, if that makes sense. You can work with it however you desire, especially if you want to conserve resources, but I always completely fill the top of the slide.

I'm using a veterinary quick dip, so I'm not flooding the slide, just dipping for 5 seconds in the fixative, stain, and counter stain. I'll have to keep trying to get rid of the artifacts I guess.
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thatdudefromkansas
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I'll take a closer look at your slides in the morning and help with the issue, that's what we are all here for.

Also, anyone done any dental or mouth swabs for t. denticola?
Curious if anyone has done any staining or d/f view of them.

I might trying the morning as well, got nothing to do tomorrow.

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thatdudefromkansas
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Check it out, guys.

In fairness to the board, I would ask that you be honest.


I have two pieces of equipment I can get rid of:

I have a DRY darkfield condenser....useful with any objective 40X and below, or a oil 100x as long as it has an iris that will adjust the numerical aperture below 0.90.

I also have an OIL 100x 1.25 objective.

Is there anyone in need of a 100x objective that is adjustable, and is there anyone in need of a DRY darkfield condenser?

If you have a condenser, but want to upgrade to a capable objective at 100x, email me.

If you don't have a darkfield, and want to experiment/use that, email me.

They are both Amscope brands. The Objective will fit any standard microscope annulus. (Outside of Nikon, and I believe Leica, but I could be wrong on the second one, just ask).

The D/F may, MAY, only fit amscope brand microscopes. If you research the Amscope T-490, and the condenser it uses will fit your microscope, let me know. I am not gonna research it myself.

However, these two items may come as being excess for me, and I will not need them.

I also don't intend to sell them, but will share them.

If you have a need, perhaps I can provide it to you and you won't have to spare money that you need for other things in this "hobby" , or in life itself.

I was gonna post earlier, but got busy. Now I'm a bit drunk from enjoying a night of movies and wine!

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thatdudefromkansas
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Mustard, seems like a decent kit.

Seems like it is also tailored towards CBC differentials, and is not a gram stain kit or a Giemsa/Wright Stain.
It gives coloration "similar to", but is that coloration of the RBC's and WBC's similar to a Geimsa/Wright, or also bacterial staining?

If not, DM me directly please!

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Lymedin2010
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Lyme-N does sound like another LD treatment that will probably have mixed results, as the many herbs do.
http://www.mactechimaging.com/LargoBioScience-InformedConsent-Rev20150704A.pdf

Nice video with new APO objective. A better test might be on a stained & fixed slide and then you can compare & contrast the same image & get a better appreciation for your purchase, as APO's are definitely better. If you doing 100x, with a greater depth of field, it will be harder to notice outright, but on a 40x the clarity is more apparent.

Spirochetes can be very long sometimes 30,40,50 & 60 microns long. Dr. MacDonald & Eva Sapi have seen them this long as well & they attest to it. I have seen them that long in my blood, but never that long in a tick. So I think it requires some excess nutrient input to stimulate longer growth. The longest I have seen in a tick is about 15-20 microns.

I put out 4 dental videos from my mouth. I brush with baking soda every so often & so probably end up killing a majority of the spiros. At first the baking soda burned my mouth & it felt like a local mouth herx & weird. After some time those feelings went away, as the spiros dies.
The 4th video is most revealing with some plaque/biofilm & many rods.
https://www.youtube.com/watch?v=eeRsUR-TWD0

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Lymedin2010
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Also, guys I have evidence that the SOP may be RBC cell wall shedding & can shed what look like spiros, so they are false-spiros too. I will have to put a video together eventually, as I have been meaning to do it for quite some time now & it is time consuming to go through all my video to get the perfect representation & proof.


The SOP is definitely another morphology in Borrelia & the big dogs admit to finding them in lab grown & BSK-H cultures. I have seen segmented ones in ticks, so I knew they breakup, but I have never seen a SOP in ticks yet.

For instance I think thatdude's video there is a true spiro, as it moves with some aggression from spiraling motion.

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thatdudefromkansas
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Lymedin,

I see the same SOP stuff in areas of a slide with lots of cellular degradation.

I have written that stuff off for the moment, and don't consider/pursue that as spirochetal.
Would need to see a transition to some other form for that to be a thing for me.

It doesn't mean that doesn't occur with Borrelia, or any other species of spirochete, as many prominent (AND smarter) trained pathologists and microbiologists have noted this form.
They would know what they are seeing, and would know what the different components of blood appear as during different stages of cellular degradation. So it may be cell wall debris.

I stick to the long, undulant forms for viewing, as well as the cyst/L -forms present.

Especially after that video I recorded showing the transition point of the spirochete into that form.

It'll be interesting to see your video to see if it is the same thing I have seen. I might need to prep a slide and let it take its course without sealing the edges of the cover slips and take a look at that again.


Also, you're right on the comparison of objectives. The difference in depth of field is also noticeable, even though the objective NA is only .05 higher than my original one (1.25 original vs. the 1.30 new).
The resolution is much better simply due to the greater degree of correction and higher quality glass, etc.
I don't like that the new one does not have a spring, though. So until the specimens settle on the slight, I see a lot of movement of the entire plane.

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thatdudefromkansas
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I've looked up different culture methods as well.

I have reviewed Ichelson's culture media from the 1950's. There is also a US patent that describes the entire culture method utilizing it.

Seems readily recreatable from components you can easily purchase.
Nothing that would require working for a lab, etc.

It requires animal serum, but that MIGHT be substitutable with human serum if you have a centrifuge. Centrifuge your own blood in a vacutainer, or allow gravity precipitation in a syringe to separate it, and use that.
So it wouldn't be exact, but it is doable.

I also want to try something with either gelatin or collagen, or both, in a culture medium.
There are conflicting results with gelatin in studies, but collagen is affective.
I've thought about acquiring a skin punch to use my own biopsied skin tissue for that purpose.
Probably need a prescription for it, though.

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thatdudefromkansas
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With the spirochetes we film....I can see the wave like motion and twisting. You can see that, perhaps, there are still flagella present in the cell. Perhaps they have simply shed the component that gives them rigidity (as a method of eluding detection). But you CAN still sill that movement. As those the flagella are flexing, but lack the ability to provide movement.
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thatdudefromkansas
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Lymedin,

I like that video you posted.

Have you thought about trying a brighter light source?

I use a 1000 lumen headlamp instead of the built in light source.

And I have a 5000 lumen one on the way.

you can achieve higher resolution with brighter light, and see more, especially in brightfield.

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bluelyme
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Dude skin punch ...hardcore ! Nice new vids

Mustard keep the stains coming good work

--------------------
Blue

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Lymedin2010
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An alternative to BSK that is homegrown is welcomed. I have some collagen & my own frozen blood on hand. I have tried some other combos before, but no cigar.

The collagen itself can be mistaken as spiros themselves, that was the problem with the Advanced Lab Culture test and denied approval & on phase 2 they had to degrade the collagen.

I have a really strong feeling that my thin skin will produce some Borrelia in culture. I've been meaning to check it forever & waiting on some good culture medium.

Check out my 63x no oil lens, but there is a caveat to this video as the video was taken without a slip cover & with slip cover it is blurrier (closer to 100x oil). I may have to fine-tune it some & play with the adjustment collar.

Zeiss West Germany Plan NeoFluar 63x/0.90 160/0.11-0.23
https://www.youtube.com/watch?v=GkztFAYUjIs


You can check the 63x w/slip cover vs the 40x in this vid.
https://www.youtube.com/watch?v=_QzUhC8fJTQ

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thatdudefromkansas
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http://beforeitsnews.com/health/2014/04/more-on-cdc-testing-and-advanced-laboratory-lyme-disease-culture-test-2532212.html

I'll have to look up the information for their culture and see what they did and, allegedly, did wrong.

I saw the Dr. MacDonald came out in support of the culture/testing method, and that there was no contamination contrary to what the CDC stated.

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Lymedin2010
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I've been following Advanced Labs from other posts.

http://www.medsci.org/v10p0362.htm

https://www.change.org/p/the-us-senate-calling-for-a-congressional-investigation-of-the-cdc-idsa-and-aldf/u/15826757?recruiter=8219773&utm_source=share_update&utm_medium=facebook&u tm_campaign=facebook_link&fb_action_ids=922327091221946&fb_action_types=og.shares

Contamination claims & then collagen that looks like spiros too.


I need a new slip cover sealant...any ideas?

1) Vaseline: The Vaseline tends to succumb to ambient room temp over time.

2) Elmer's Glue: Works great temporarily, but after it dries it becomes porous. I've even tried applying clear nail polish as atop coating on the Elmer's once the glue dries out, but ultimately the sample tends to dry & we get loss of Brownian motion in samples.

3) Nail Polish: Direct application is too harsh & seeps into the sample from what I have witnessed in live blood preps.

4) Immersion oil: Avoiding that with 40x,but haven not tried JUST doing slip cover edges as of yet.


Any other ideas? Candle wax may be too hot & hot glue gun may impact sample. I need some oil or paste that is robust at room temp AND non-reactive with samples.

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TNT
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Immersion oil works great, Lymedin. I thought that is what you showed us in your "making a slide" tutorial. You did only the edges of the coverslip in the video. That works great for me. The clearance of the 40x (my 45x) is great enough that I don't have any trouble getting my lens in it, even at the edges. I have to be careful right at first, but once the oil spreads out a little and equalizes itself, it's not much higher than the coverslip itself.

If you are worrying about contamination by the oil, don't put the oil on right away, but wait a number of hours, even overnight, until the blood dries enough around the edges to mostly seal itself off. Then seal off with immersion oil. I get no contamination that way.

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Lymedin2010
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All tests below are preliminary & not maximized & tweaked & done a while ago.

Test video w/ Samsung Galaxy S5:
https://www.youtube.com/watch?v=3ehIfJUneK4

https://www.youtube.com/watch?v=5jI31Nk1pI0


Test with camera Sony a6000:
https://www.youtube.com/watch?v=shbAhRgfx1U

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Lymedin2010
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This is the full blown 63x test.

1) 63x w/cover slip, no oil.
https://www.youtube.com/watch?v=e3w4hoJmEJ8


2) 63x verus my 40x & also with manual zoom on the 40x & I think the 40x w/manual zoom is still better.
https://www.youtube.com/watch?v=_QzUhC8fJTQ


3) 63x w/no slip cover on. Damn I wish the lens was this good w/the slip cover on!!!
https://www.youtube.com/watch?v=GkztFAYUjIs

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Lymedin2010
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Yea, that is what I showed in the video & that was a while ago. I have branched out since then trying to find better things.


I am more interested in fixed slide of tick juice & would love it to be sealed. I may have to use the immersion oil if I don't find a better solution.

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Lymedin2010
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First off, Merry Christmas everyone & I hope that Santa stuffs your stockings with APO objective lenses, 4K recording equipment, & BSK-H culture medium. A fluorescent microscope won't hurt either [Smile]

 -


_________________________________________________

I've had this idea for quite some time now & the damn disease has hampered me from bringing it to fruition. So if you guys want to jump into it before me, be my guest. I did mention this in the past, but I never detailed how one could potentially go about this.

Cheap & easy Xenodiagnosis hack is possible:

1) Collect wild caught ticks.

2) Feed tick your blood externally (never let a tick bite you) & breed successive generations to get closer to sterile tick.

3) Feed final sterile tick your blood externally & wait a few weeks.

4) Send the tick in this bag for testing for $40. "Results are quick and 99.9 % accurate"

https://www.lymeaidkit.com/

Some studies show that the tick progeny do not acquire Borrelia, whilst others show that a small percentage have it. If you breed successive generations, one should get closer to a sterile one. Always best to get a lab-grade sterile one, but I am sure those will be hard to come by.

Alternatively, I am thinking one can use an insect other than a tick to conduct such a test. Perhaps to feed our blood externally to a mealworm or those pet store bought crickets. or a spider.

Watch this if you need inspiration of possibility.
https://www.youtube.com/watch?v=vx11xD55skA


_________________________________________________
Here is another test that is much cheaper, but lower accuracy.
"The study found the clinical accuracy of the Care Plus™ Tick-Test is 95.8%."

http://www.newswire.ca/news-releases/new-tick-test-designed-to-indicate-presence-of-lyme-disease-related-borrelia-bacteria-now-available-in-canada-578618231.html

_________________________________________________
POST UPDATE:

Peter Kemp report bad testing with the Care Plus Test Kit: "Peter Kemp The test is very insensitive and was found to have 0 percent sensitivity (on ticks). It did not work on my own blood except when it was cultured for several days in BSK."


There are many other places to do more reliable PCR tick tests. There is a place that once reported FREE tick testing & on this page there are other tick testing labs. https://sites.google.com/site/marylandlyme/ticks/free-tick-testing

[ 12-29-2016, 07:32 AM: Message edited by: Lymedin2010 ]

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TNT
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quote:
Originally posted by Lymedin2010:
First off, Merry Christmas everyone & I hope that Santa stuffs your stockings with APO objective lenses, 4K recording equipment, & BSK-H culture medium. A fluorescent microscope won't hurt either [Smile]

Great gift ideas!!! Wouldn't that be the Christmas??!! A fully loaded Zeiss Axioskop would be nice! Even a fully-loaded Zeiss Photomicroscope III would do!
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Lymedin2010
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Let me know your partner's email address to send the hints to [Smile]

At time 12:40 in this French documentary, they show us video of Borrelia burgdorferi. Shortly after that, Dr. Eva Sapi shows Borrelia, cyst, & biofilm pictures.

https://youtu.be/GC4TAYa3WAY?t=760

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thatdudefromkansas
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My plan apo 100x oel with iris is a Zeiss.
Certainly a older model, but great.

You can find great deals on eBay for objectives.

There is a significant difference between the stock objectives and others you can upgrade to.

I know Nikon uses their own threading, and Leica might as well.

Most other companies use RMS threading, so any objective will fit your microscope.

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TNT
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Had a sample under darkfield since Friday evening in which I initially saw plenty of ketes (more than I would like to see and more than I have seen in the last couple wet mounts). Now, almost all the free spirochetes have turned into those "granules," the same objects that Dude demonstrated recently, and the ones I previously mentioned that resemble "Everlasting Gobstoppers," (if you've ever watched the movie Charlie and the Chocolate Factory).

I guess the fewer ABX I've been on recently have not been enough. I did not see any granules when the sample first went under the scope. I see a number of perfectly round gemma cysts as well, but the number of gemma cysts appear to have remained the same from when the sample was fresh.

With all the festivities this weekend, I have not been able to do a time-lapse. But, I am determined to capture the conversion of a kete to one of these granules.

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Lymedin2010
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Yea, there sure is a difference with a Zeiss lens in general & the APO's in particular.

Seeing the spiros break up into blebs is awesome, I can never get enough of it. Love it when we capture a cyst transformation.


Below are the pictures of Immuno Stained Borrelia directly from Dr. Sapi's labs. Notice how some of the spiros have BULBOUS (rounded) tips.

 -


Cyst forms:
 -


Biofilm:
 -

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thatdudefromkansas
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TNT, I see those frequently. Especially during a regimine change in antibiotics.

Those are great photos to observe, lymedin.


Hey, anyone open to doing some Skype conversation?
It'd be cool to have a group Skype chat while prepping and looking at slides and whatnot.
Talk about methods, etc. Might be an interesting weekly or monthly thing to do.

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TNT
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quote:
Originally posted by Lymedin2010:


Cyst forms:
 -



Lymedin, isn't this picture you posted depicting blebs and not cysts? I'm not arguing, just curious since we've been able to demonstrate the conversion from kete to gemma cyst.....
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Lymedin2010
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Dude, you have my email...email me & yea skype would be awesome. It depends on day & energy, as it is unpredictable with me if/when I have enough stamina. But I love the idea.


TNT, I was going to ask what you guys thought, because to me they look like blebs for sure, as they are too small to be cysts from what I know. I could see the very first pic I posted, top right being a cyst with it's size, but even that seems to be a bleb from a thicker spirochete probably.


I posed the same question to the other guys & we will see what the consensus is.

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bluelyme
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quote:
Originally posted by TNT:
Had a sample under darkfield since Friday evening in which I initially saw plenty of ketes (more than I would like to see and more than I have seen in the last couple wet mounts). Now, almost all the free spirochetes have turned into those "granules," the same objects that Dude demonstrated recently, and the ones I previously mentioned that resemble "Everlasting Gobstoppers," (if you've ever watched the movie Charlie and the Chocolate Factory).

I guess the fewer ABX I've been on recently have not been enough. I did not see any granules when the sample first went under the scope. I see a number of perfectly round gemma cysts as well, but the number of gemma cysts appear to have remained the same from when the sample was fresh.

With all the festivities this weekend, I have not been able to do a time-lapse. But, I am determined to capture the conversion of a kete to one of these granules.

Is the bvt showing any evidentiary progress?
Skype group could be fun ...

--------------------
Blue

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TNT
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quote:
Originally posted by bluelyme:
quote:
Originally posted by TNT:
Had a sample under darkfield since Friday evening in which I initially saw plenty of ketes (more than I would like to see and more than I have seen in the last couple wet mounts). Now, almost all the free spirochetes have turned into those "granules," the same objects that Dude demonstrated recently, and the ones I previously mentioned that resemble "Everlasting Gobstoppers," (if you've ever watched the movie Charlie and the Chocolate Factory).

I guess the fewer ABX I've been on recently have not been enough. I did not see any granules when the sample first went under the scope. I see a number of perfectly round gemma cysts as well, but the number of gemma cysts appear to have remained the same from when the sample was fresh.

With all the festivities this weekend, I have not been able to do a time-lapse. But, I am determined to capture the conversion of a kete to one of these granules.

Is the bvt showing any evidentiary progress?

Yes, it's definitely helping! But, I seem to be in a bit of a slump at the moment. It might be time to switch things up a bit....
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TNT
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Lymedin, your new video today is interesting. The few spirochetes I can see look like SOME of the ones we see in our blood..... straight and a little rigid. I don't notice much undulation.

So, in your opinion, would you consider these true spirochetes?? I'm sure they are, but those in our blood we generally are not as quick to claim they are with absolute certainty.

Sorry for the wording of that last sentence....(under the influence of Lyme brain at the moment).

The referenced video:

https://www.youtube.com/watch?v=vSUdFJnz-mE

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Lymedin2010
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TNT, thanks.

The reason I can say they are spiros, is because when I first observe right after preparation, I do not see them. More likely the RODI shocked them & forced them into cysts. Then after a while the solution balances out in the tick juice + RODI & they start to emerge from the cysts.

More & more come out over time. Sometimes they have a bit of undulation & I have some video & I will post in the future. I already posted a past tick video, where I do a negative of the screen (bluish screen) & you can notice the wave-like spiraling motions in that particular lengthy one.

https://www.youtube.com/watch?v=RnFR4Ca5MVo

I do not see these in many ticks that do not have any spiros. So there were many ticks that did not have these rigid spiros. I also do not see any of the very small & very active zig-zagging spiros & I imagine they might be a different species. Those smaller & zaggers were also dividing with a thin spiro strip between two adult segments & that was interesting to see that in the past.

I also see L-shaped forms & sometimes "Tennis Rackets" as well. I am uploading 002 in the next series of tick juice time lapse & you will see a nice sized tennis racket form there (this will take some time to upload).

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Lymedin2010
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TNT, here is a video with a spirochete with a bit more motility. It comes from the same prep as the previous 2 & there are many other examples too & perhaps even better ones...just too much to go through all to find right now.

Top middle & then goes into quadrant 1.
https://www.youtube.com/watch?v=zRJJrzDtsRw


Then the same spiro as p001, but a few hours later (overnight).
https://www.youtube.com/watch?v=3CRiy0C5bvA


On another note has anyone seen Borrelia with a stain yet?

" The cells are gram negative and stain well with Giemsa and Warthin-Starry stains. Unstained cells are not visible by bright-field microscopy but are visible by dark-field or phase-contrast microscopy. " "A review of reports on the genetic and phenotypic characteristics of strains of the spirochete which causes Lyme disease revealed that these organisms are representative of a new species of Borrelia. We propose the name Borrelia burgdorferi for this species. The type strain of B. burgdorferi is strain B31 ( ATCC 35210). "

http://www.microbiologyresearch.org/docserver/fulltext/ijsem/34/4/ijs-34-4-496.pdf?expires=1483018125&id=id&accname=guest&checksum=8FA08C3A69C298F5430C9577F29933E3

[ 12-29-2016, 08:14 AM: Message edited by: Lymedin2010 ]

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TNT
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Great job, Lymedin!!! in that video of the ketes in the tick. They sure were moving 14 hours later!!!

( https://www.youtube.com/watch?v=RnFR4Ca5MVo )


I have not seen them stain with Giemsa, but of course the blood dries within minutes for a Giemsa stain, which doesn't allow enough time for them to exit the RBCs or WBCs.

On some of my first (failed) stains I used a dyed methanol that did pick them up, although the color was not typical. Those pics are on page 5.

The microbiologyresearch link you posted above says "the page requested is not available."

I'm pretty sure Giemsa does not pick up Bb spirochetes like it does Relapsing Fever spirochetes. Another thing I wouldn't agree with from that quote you posted above is that you cannot see Bb spirochetes with brightfield, because you can, although we all know they're more difficult to see with brightfield than with darkfield or phase contrast.

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TNT
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Hey, dude! I found a decent scope for you:

http://www.ebay.com/itm/152288463210?_trksid=p2055119.m1438.l2649&ssPageName=STRK%3AMEBIDX%3AIT


It has a Zeiss 100x Plan objective with an iris that you could sell (since you already have a Zeiss 100x Plan Apo with iris) to put towards getting the couple Phase objectives it appears to be missing.

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Lymedin2010
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Thanks TNT.

Look up the text within the quote, all of it & it will be the first link. Somehow it just does not seem to work when I post in here.


I got some good news for us. I have seen a confirmed case of Bartonella in the blood back in 2014, but the owner did not want to share that video. I've been dying to share it with you guys, but could not.


Now someone else has posted a video on what they thought are vacuoles. BUT when I looked at it, it reminded me so much of the Bart video & so I looked at that original bart vidoeo again & sure enough it is EXACTLY like that.


So here are some points about it:

1) Round or oval, & all depending on which side is displayed as it moves one can see a slight pointy tip (almost teardrop).

2) It moves with more intent & in a jerky fashion and does not move in mere Brownian motion.

3)The original video I saw had at least 9-10 of them, of various thickness & size WITHIN the rbc & they can be seen soooooo clearly with darkfield. Also there were at least 2-3 barts outside the rbc & one thick one in particular. Adjacent rbc's ~3-4 had between 1-3 barts in them as well.

When the video starts you will see a 2 arrows & one bart zipping across from left to right. What he is describing as vacuoles is probably rbc's that have been whittled down & probably have thin walls. Those objects within them are bart as well & they have that same jerky motion.


I am happy I can share this video with you. This person is confirmed with HIGH levels of bart ( 1/1280) they told me after I told them that I think it is bart.

https://www.youtube.com/watch?v=Bv7dKi4Wcaw&feature=youtu.be

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Lymedin2010
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OMG, bart also entering RBC...I am floored!!!

https://www.youtube.com/watch?v=L9Q5Ft8K-eg&feature=youtu.be


Those are VACUOLES that the Bartonella produce, as some are WITHIN the rbc, as her 2nd video showes.


THIS IS BARTONELLA!!!!!

https://www.researchgate.net/figure/221735830_fig1_FIG-1-Common-infection-strategy-of-the-bartonellae-The-drawing-illustrates-the-general


A great video explaining Bart & they show you cappillaries & blood & biofilm on collagen from Bart (Time 47.00).
https://youtu.be/uYEHsRRxrQw?t=2816

[ 12-30-2016, 06:19 PM: Message edited by: Lymedin2010 ]

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TNT
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I'm not sure, Lymedin. I have seen that kind of stuff pretty often and there's no way to actually verify it's Bart. I think we would need more verification than just that the person has high titers for Bartonella. What if they also have a high load of something else that resembles Bart that they haven't been tested for, or even some type of apicomplexan they haven't been tested for.

If those vacuoles do indeed contain Bart organisms, you should be able to see those RBC vacuoles with organisms inside on a Giemsa or Wright stain.

I'm not saying it's not Bart, just that I personally would feel more comfortable with more evidence. In the second video, I don't see the objects actually enter the RBCs. If they did, that would be incredible to see, but still would not differentiate between Rickettsias or apicomplexans (or anything else) unless something specific to the entry was seen that would give us a clue.

But, definitely interesting. Too bad I can't read German.

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Lymedin2010
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Look at the 2nd video & look at the vacuoles WITHIN the rbc. This is Bart!!!
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Lymedin2010
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Time 4:11 smaller vacuole starting to form, a biofilm of Bart.
https://youtu.be/Bv7dKi4Wcaw?t=251


Time 4:35 slightly larger vacuole of bart AND you can see the bart within the vacuole. So vacuole within the rbc & bart with in the vacuole....DEAD GIVEAWAY!!!!!
https://youtu.be/Bv7dKi4Wcaw?t=275


An even larger vacuole growing within the rbc.
https://youtu.be/Bv7dKi4Wcaw?t=201


Time 4:55, vacuole within rbc & bart escaping.
https://youtu.be/Bv7dKi4Wcaw?t=295


Vacuole escaping rbc:
https://youtu.be/Bv7dKi4Wcaw?t=166


Vacuole outside of rbc, as is seen multiple times in the video with Bartonella WITHIN the vacuole.
https://youtu.be/Bv7dKi4Wcaw?t=352

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