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» LymeNet Flash » Questions and Discussion » Medical Questions » The Microscopy Thread (Page 15)

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Author Topic: The Microscopy Thread
Lymedin2010
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At time = 49:00, biofilm of Bartonella. These are the vacuoles that form, they are Bartonella biofilm!!!

https://youtu.be/uYEHsRRxrQw?t=2944

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Lymedin2010
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Again look at her rbc at time 4:35, exactly as the chart. RBC has a vacuole, which is the Bartonella biofilm, & you can see when she zooms in the Bartonella moving within the vacuole.

https://youtu.be/Bv7dKi4Wcaw?t=275


Compare it to this chart alone & you will notice.

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

 -


Then go into that Bartonella video & pickup all the clues on Bartonella biofilm & all the vasculature problems it creates because of this.

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TNT
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quote:
Originally posted by Lymedin2010:
At time = 49:00, biofilm of Bartonella. These are the vacuoles that form, they are Bartonella biofilm!!!

https://youtu.be/uYEHsRRxrQw?t=2944

This is interesting. So, he's saying that the cleared and vacuolated RBCs with the faint dot in them are actually infected with Bartonella inside a vacuole? of biofilm? I find it strange that in that diff-quick stain picture there are no typical dots on the peripheral of the RBCs as would normally be expected. I've definitely seen those vacuolated RBCs with the faint dot. But in my blood I see more of the peripheral dots on the RBCs.... which is typical Bartonella according to the literature.

This is a good example of why I don't like Dr. M's approach. He is hyper-focused on the genus Bartonella when in fact there are several pathological Rickettsias, of which he does NO testing for. Clinically and pathologically the Rickettsias are almost exactly alike, yet if you don't test positive for one of the Bartonellas, he concludes that your "Bart" symptoms (or your small vessel disease symptoms) must be from something else. What about the other Rickettsias that cause the SAME VASCULAR SYMPTOMS???!!! This is also a good example why one cannot treat based on tests alone, but must look at the symptom picture. Unless you are going to test for all infectious Rickettsias, and he does not.

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Lymedin2010
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Fainted? They pretty big in the live video & the bart move within the vacuoles or if they burst then freely in the rbc. Big in the diagram above & you see the same thing in the diagrams below.

We already knew this one, but let's go through it.

FIRST CONFIRM BARTONELLA CAN BE IN BLOOD:
1 of 4:

" Many Bartonella spp. have been shown to multiply and persist in red blood cells, sharing common persistence and dissemination strategies."

" may explain the peculiar tropism of Bartonella for red blood cells"

" In conclusion, it appears that specific (polar flagella) and common (deformin, invasion-associated locus) mechanisms have been developed by the different Bartonella spp. to adhere to and invade the red blood cells."

" The ability of Bartonella to persist in erythrocytes for long periods may have been driven by evolutionary constraints, as it increases its transmissibility by blood- sucking arthropods. This persistence, which explains the prolonged bacteraemia in symptomatic and asymptomatic subjects, and the occurrence of disseminated disease in the homeless (chronic bacteraemia) or AIDS patients (bacillary angiomatosis), may also be favoured by the intra-erythrocytic localisation which partially protects Bartonella from the immune system."

" A bacterial protein named deformin also appeared to be involved, at least for B. bacilliformis,in the formation of pits and trenches in the red cell membranes [11, 21], that may favour both colonisation and entry into the cell [17]. "

" Apart from their tropism for red blood cells, a second typical pathogenic feature of Bartonella spp. is their ability to trigger angiogenesis. Such pathological angiogenesis is observed in bacillary angiomatosis and peliosis [28 – 31]."

http://www.benbrew.com/lb/bartonella17.pdf

*******************
FIRST CONFIRM BARTONELLA CAN BE IN BLOOD:
2 of 4:

" Progression of the chronic and relapsing Bartonella spp. intraerythrocytic bacteremia in a mammalian reservoir host. After initial inoculation, for example, of bacteria in arthropod feces that are superficially scratched into the skin, the bacteria reside and persist in the still-enigmatic primary niche (lag phase). Bacteremia is initiated several days postinoculation by a rapid appearance of high numbers of bacteria in the bloodstream (arrow 1), with bacteria binding to and subsequently invading the erythrocytes. Intraerythrocytic bacteria replicate until reaching a steady number, which is maintained for the remaining life span of the infected erythrocytes. The primary niche is believed to seed additional erythrocyte infection waves at regular intervals (arrows 2–4) until a specific antibody response clears the infection by blocking the erythrocyte invasion. However, bacteremia may peak (arrow 5) after prolonged periods (weeks to months) of abacteremia (dormant phase) presumably by clonal expansion of antigenic and/or phase variants, which are seeded into the bloodstream from the primary niche. During the long-lasting intraerythrocytic bacteremia, efficient transmission of the intraerythrocytic pathogen to other susceptible hosts is mediated by blood-sucking arthropods, such as fleas and lice. Details are described in the text (see 'Progression of Bartonella spp. infection in the reservoir mammalian host')."
http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2012.00324.x/full


********************
FIRST CONFIRM BARTONELLA CAN BE IN BLOOD:
3 of 4:
*****" Below means very important, as an average of 8 can be within a rbc & within the vacuole. I have seen video of ~9 of them in a rbc.

" This is contrary to the existing premise that cultures of blood from healthy individuals should be sterile. Cats can be infected and become bacteremic with Bartonella species such as B. clarridgeiae and B. henselae."
" Arthropod vectors, including ticks, fleas, and lice, have been proposed for almost all the Bartonella species; and transmission of the organisms to people may also occur by scratches or bites from reservoir hosts, in particular, cats."
" With all other known bacteria, prolonged bacteremia is associated with signs of septicemia in the host. Bartonella bacteremias in the natural hosts, however, can be asymptomatic. This is contrary to our present understanding of bacteremia and goes against the idea originated by Koch that bacteria do not occur in the blood of healthy animals or humans (12). Bartonella may be the single bacterial genus capable of producing asymptomatic bacteremia in mammals and, thus, may be an exception to Koch’s postulate. Using confocal microscopy, we have shown that B. henselae occurs within naturally infected asymptomatic cat erythrocytes (Fig. (Fig.2)2) (83) and that B. quintana occurs in human erythrocytes (unpublished data). As the Bartonella species are intraerythrocytic and, hence, might be less exposed to the immune system, their hosts may become adapted to the chronic bacteremia."
*****"Recently, the kinetics of the colonization of B. triborum in rat erythrocytes has been reported (87). The organism multiplies until there are an average of eight Bartonella species per cell and thereafter remains in the cell for the life of the erythrocyte. It was suggested that this nonhemolytic intracellular colonization of erythrocytes is a bacterial persistence strategy that preserves the Bartonella species for potential transmission by arthropods. The host, then, could contaminate blood-feeding arthropods such as ticks, fleas (50), sand flies, or lice (14, 76), which could then subsequently infect a new host."
"In this acute stage of infection, the Bartonella species may be observed in erythrocytes. The level of erythrocyte parasitization can reach 100%, resulting in severe anemia and, occasionally, death. Death could also occur because of opportunistic infections (specifically, salmonellosis) following infection-induced immunosuppression. The fatality rate without treatment can be 40% (63)."
"Transmission electron microscopy (54) and confocal microscopy (Fig. (Fig.2)2) (83) have shown that B. henselae occurs within the erythrocytes of bacteremic cats, and such infections can persist for up to a year (54)."
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC119901/

********************
FIRST CONFIRM BARTONELLA CAN BE IN BLOOD:
4 of 4:

"Domestic cats were experimentally infected with culture propagated Bartonella henselae by intradermal (ID) and intravenous (IV) routes. Cats were more efficiently infected by the ID (88 cats) than by the IV (216) route. Bacteremia was detected 1–3 weeks following inoculation and lasted for most cats for 1–8 months. However, one naturally infected cat was observed for 24 months and was found to be cyclically bacteremic, with bacterial levels varying one hundred fold or more from one period to another."
http://www.sciencedirect.com/science/article/pii/S0147957196000252


********************
NEXT PROOF OF VACUOLES (the bart biofilm) & bartonella intravacuolar multiplication?

Look at the halo around these stained Bartonella within the RBC. And we have seen more than one & know a few can grow within the vacuole. As it grows the vacuole can get bigger. This is a big WOW to me, as not only is bart protected WITHIN the rbc, but can potentially be protected from abx WITHIN the vacuole/biofilm. We hear about the biofilm in the lengthy video I posted on Bart & vasculature ( https://www.youtube.com/watch?v=uYEHsRRxrQw&feature=youtu.be&t=2944 ) , only in the vascular it is not quite as easily discernable as it is in this stain & in the live video we have.

https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=119901_cd0120191002.jpg

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC119901/


Figures 2 & 3 on this page also show diagrams of the intra-erythrocytic vacuoles.
http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2012.00324.x/full

********************
And now for the kill...Invasome-mediated engulfment is possible, which then makes CONTROLLED Intravacuolar multiplication (average 8-10 barts per vacuole & per rbc), possible in order to mediate the prevention of RBC & endothelial cell lysis & death.
Diagram 5 on link below.
http://onlinelibrary.wiley.com/doi/10.1111/j.1462-5822.2012.01806.x/pdf

_____________________________________

Thin & deteriorated rbc's in Malaria references:
Compare the above Lymies blood video of the bartonella that is external to the rbc's (in the blood plasma) & one can see how THIN & unrecognizable the rbc's can become. The same phenomena of rbc deterioration can be seen in Malaria & we can keep this in mind for Babesia & this Bartonella video.

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

https://www.youtube.com/watch?v=s3DSB1xXuec
___________________________________________
Another Bart video:
https://www.youtube.com/watch?v=NbMojXbWrfE


A great Bartonella video from the NorVect lectures:
https://www.youtube.com/watch?v=mLK48ecsn-s

[ 01-13-2017, 08:55 AM: Message edited by: Lymedin2010 ]

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TNT
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Great job with your research, Lymedin. I agree with much of what you are saying. I just have a hard time getting from A to B if A is simply a visual without any kind of proof such as fluorescent DNA typing in those live blood videos (those youtube videos) or a Giemsa or Wright stain with the same morphology. We make similar conclusions with borrelia, but we have authoritative references with live videos of borrelia to back up very distinct morphology.

Going back to Dr. M's diff-quick slide in the video (at 48:10 in the video you posted: https://youtu.be/uYEHsRRxrQw?t=2944 ) where he claims the big area (much of the frame) that looks like a water mark is a biofilm. He has much training and a lot of microscopy experience, but I find it hard to believe that is a (translucent) biofilm. If biofilm is circulating in the blood stream and going through capillaries, most times it is going to appear in very thin strands about the diameter of a RBC (like I show in my blood, and like what Dr. Fry shows in his stained smears of biofilm). I really think what he is showing is contamination or precipitate.

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Lymedin2010
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Someone notable has confirmed the very first Bartonella video.


It looks like the rbc's are coagulated by the fibrin strands & are clumped. It is the Bart in the cells that is causing the accumulation of fibrin & making a makeshift biofilm using the host resources. Fibrin strands are normally used in blood coagulation & so it seems it is just eliciting coagulation really. I think that is the blood from the affected capillary area.


The previous slide shows an awesome fibrin collection in a capillary & one can easily see how the circulation can be blocked & cause issues and pain. I too have the damn circulatory issues & pains.

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TNT
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quote:
Originally posted by TNT:
quote:
Originally posted by Lymedin2010:
At time = 49:00, biofilm of Bartonella. These are the vacuoles that form, they are Bartonella biofilm!!!

https://youtu.be/uYEHsRRxrQw?t=2944

This is interesting. So, he's saying that the cleared and vacuolated RBCs with the faint dot in them are actually infected with Bartonella inside a vacuole? of biofilm? I find it strange that in that diff-quick stain picture there are no typical dots on the peripheral of the RBCs as would normally be expected. I've definitely seen those vacuolated RBCs with the faint dot. But in my blood I see more of the peripheral dots on the RBCs.... which is typical Bartonella according to the literature.
I just listened to that spot in the video again and he actually does reference the "red cells that have these round forms along their margin."

The thing is, I don't see the round forms along the margins of the RBCs, I only see the dots inside the middle of some of the red cells inside those clearings (or vacuoles).


quote:
Originally posted by Lymedin2010:
Someone notable has confirmed the very first Bartonella video.

Like I said, I'm not saying it's not Bartonella, but I'm curious who confirmed that those youtube videos do show Bart??
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TNT
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quote:
Originally posted by Lymedin2010:

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

So, you are saying those "flipping" objects in the plasma are Bartonella organisms.....

.....Ok, I'm starting to see how they could be that. Only, I'm not sure about the ones inside those ghosted cells being Bart. They look too much like trapped immune components--especially the way they move and their size. But, the flipping objects, sure, they are the right size, and it would make sense comparing it to my own blood. Only thing, in my blood, they may be another Rickettsia such as Anaplasma that I'm seeing instead.

Here is one of my videos depicting similar objects that I thought could possibly be apicomplexans, but is more likely to be a Rickettsia such as Anaplasma, especially since I have found (in my stains) morulas in my WBCs and dots on the outside perimeters of the RBCs.

https://www.youtube.com/watch?v=nUZaE-NFlKw

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TNT
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Here is another example as a close-up:

https://www.youtube.com/watch?v=IcVMe35q7XE

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TNT
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I'm just wondering why we don't have an example of a Wright or Giemsa stain of Invasomes? It seems like there should be a few examples of it, although I realize good Bartonella research is just beginning.

[ 01-01-2017, 02:26 PM: Message edited by: TNT ]

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thatdudefromkansas
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String of Pearls out of RBC's video will be coming shortly.

When you observe this phenomenon, consider that there is a great likelihood that it is from the breakdown of the RBC's over time, and not related to any bacteria or other organism.

What are you guys opinions on it?
Check out my video first when I post it.

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thatdudefromkansas
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https://youtu.be/QZ0mv6nHRO8


I'm pleased with the resolution of my new objective as well.

Markedly different than the achromatic one I had before.

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TNT
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quote:
Originally posted by thatdudefromkansas:
https://youtu.be/QZ0mv6nHRO8


I'm pleased with the resolution of my new objective as well.

Markedly different than the achromatic one I had before.

EXCELLENT!!! That is extremely good resolution at that magnification! I would say that is one redeeming quality of the Amscope...that it can accommodate Nikon & Zeiss objectives. The only thing I can use on my AO is AO.

I would not consider all string of pearl phenomena simply a result of RBC degradation. I don't see that very often as my samples age. I see mostly bursting, oozing, or ghosting as the RBCs degrade. Furthermore, S13 showed us a really good example of string of pearls morphing into a spirochete (or typical string) in a time-lapse back on page 2 (9-9-14).

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TNT
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quote:
Originally posted by S13:
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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TNT
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Also, I believe Dr. Alan MacDonald talks about the string of pearl spirochete morphology in his videos. I think the string of pearls that result from RBC break-down are bigger-sized pearls than the spirochetal string of pearls we usually see.
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thatdudefromkansas
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TNT, exactly.

There is a great differeceived between that and what I just posted in the video.

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BorreJaakko
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Hello all, now I got my used microscope, Olympus BX-41 with mostly Plan objectives, as well as Carson HookUpz 2.0. I was hoping to connect my dSLR to the microscope but it seems too much hassle or too expensive -- the microscope only has dual head. So I am using my phone for now.

The microscope supports dark field but I believe a seperate condenser is needed, so I am now starting with bright field. I am new to all this microscopy stuff so all this stuff about condensers etc. is new to me. I ordered some sample specimens of blood and various tissues, which has helped me to see that at least I am not screwing up the sample creation.

I have few questions, some of them quite elementary:

I looked LymedIn200's great video about making a blood smear, using oil around. But now lately he has been talking about looking for better ways to make the smears. Any hints?

Also, he uses oil to cover the smear. Is this "oil immersion" when you do not put oil inside? What does it mean in practice that the objective is "oil immersion", like my 100x plan objective seems to be? Do I need a special "oil immersion" condenser? Is it any use the 100x objective at all as now I do not have a oil immersion condenser at all.

Also, how do you guys fit the extra bright LED lamp under the microscope condenser. It seems there is no room in my microscope to fit anything between the light source itself and the condenser.

I remember seeing some instructions on how to use freezing/fridge to expedite red blood cell dying so that sprichoetes would be visible in only few hours instead of 5 days. But I lost a link. Does anybody have a link or know how this is done?

Thanks, I really appreciate your replies and hope that I can now witness the suckers in my blood first-hand.

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TNT
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quote:
Originally posted by BorreJaakko:
I have few questions, some of them quite elementary:

I looked LymedIn200's great video about making a blood smear, using oil around. But now lately he has been talking about looking for better ways to make the smears. Any hints?

I recommend using immersion oil just as his tutorial video shows. But, after putting your coverslip on your drop of blood, don't seal with oil right away; wait a number of hours-even overnight- and then seal with immersion oil. This allows the blood at the edges to dry, providing a seal which prevents the oil from contaminating your sample as easily.


quote:
Originally posted by BorreJaakko:
Also, he uses oil to cover the smear. Is this "oil immersion" when you do not put oil inside? What does it mean in practice that the objective is "oil immersion", like my 100x plan objective seems to be? Do I need a special "oil immersion" condenser? Is it any use the 100x objective at all as now I do not have a oil immersion condenser at all.

I recommend using immersion oil "Type B." Type A is too fluid and runs everywhere. If your objective is an "oil immersion" lens, you will maximize your resolution by using (immersion) oil for that lens. DO NOT USE IMMERSION OIL WITH NON-OIL LENSES BECAUSE YOU WILL RUIN THOSE LENS WITH OIL. ONLY USE OIL WITH SPECIFIED OIL LENSES!!!

Lower magnification lens typically are NOT oil lenses, but the higher magnification lenses- almost all 100x objectives, and some 40x, 50x, & 60x are oil lenses. Always view your specimen with non-oil lenses first, and work your way up. Once you add oil to the slide, you will not be able to view the specimen with a non-oil lens again.

The way you use an oil immersion lens is to add a drop of immersion oil to the top of the slide and bring your oil lens down until it touches the drop and then focus down from there. Be careful not to make contact with the slide. The only time you will add oil to a condenser lens is if you have a specified oil condenser like an oil darkfield condenser (used for viewing high magnification darkfield). If it is not a specified oil condenser (lens), DO NOT use oil, except on TOP of the specimen (with an oil objective lens).

But, if you have an oil darkfield condenser and are going to view 1000x darkfield, you will place a drop of immersion oil on the top of the condenser lens, move it up til it touches the bottom of the specimen slide. Then, you will also add a drop of oil to the top of the slide and bring your (100x oil) lens down into the oil.


quote:
Originally posted by BorreJaakko:
Also, how do you guys fit the extra bright LED lamp under the microscope condenser. It seems there is no room in my microscope to fit anything between the light source itself and the condenser.

With a BX-41, you may have a difficult time fitting another lamp between your frame/lamp lens and your condenser. This LED lamp may work:
http://www.ebay.com/itm/272446758936?

Or, this specific retro LED for a BX 40 series:
http://www.ebay.com/itm/252501641742


quote:
Originally posted by BorreJaakko:
I remember seeing some instructions on how to use freezing/fridge to expedite red blood cell dying so that sprichoetes would be visible in only few hours instead of 5 days. But I lost a link. Does anybody have a link or know how this is done?

Lymedin2010 shared some details several pages back. You may have to go hunting for that unless he chimes in. Usually it takes less than 24 hours for the spirochetes to come out of hiding. Special techniques, (besides sealing your coverslip), are usually not necessary.
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TNT
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quote:
Originally posted by BorreJaakko:
Hello all, now I got my used microscope, Olympus BX-41 with mostly Plan objectives,...

I forgot to say congratulations!!

And, you got quite the scope for a beginner!

Thanks for joining the club! I'm looking forward to seeing some of your work!!

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Thank you for your info!

quote:
Originally posted by TNT:
I recommend using immersion oil just as his tutorial video shows. But, after putting your coverslip on your drop of blood, don't seal with oil right away; wait a number of hours-even overnight- and then seal with immersion oil. This allows the blood at the edges to dry, providing a seal which prevents the oil from contaminating your sample as easily.

Thanks for the tip. I'll try it immediately.

quote:
I recommend using immersion oil "Type B." Type A is too fluid and runs everywhere. If your objective is an "oil immersion" lens, you will maximize your resolution by using (immersion) oil for that lens. DO NOT USE IMMERSION OIL WITH NON-OIL LENSES BECAUSE YOU WILL RUIN THOSE LENS WITH OIL. ONLY USE OIL WITH SPECIFIED OIL LENSES!!!

Lower magnification lens typically are NOT oil lenses, but the higher magnification lenses- almost all 100x objectives, and some 40x, 50x, & 60x are oil lenses. Always view your specimen with non-oil lenses first, and work your way up. Once you add oil to the slide, you will not be able to view the specimen with a non-oil lens again.

The way you use an oil immersion lens is to add a drop of immersion oil to the top of the slide and bring your oil lens down until it touches the drop and then focus down from there. Be careful not to make contact with the slide. The only time you will add oil to a condenser lens is if you have a specified oil condenser like an oil darkfield condenser (used for viewing high magnification darkfield). If it is not a specified oil condenser (lens), DO NOT use oil, except on TOP of the specimen (with an oil objective lens).

But, if you have an oil darkfield condenser and are going to view 1000x darkfield, you will place a drop of immersion oil on the top of the condenser lens, move it up til it touches the bottom of the specimen slide. Then, you will also add a drop of oil to the top of the slide and bring your (100x oil) lens down into the oil.

Ok, that was VERY helpful. It seems my confusion was about the term "immersion", I did not understand what was immersed in what. I did not figure out that actually both the condenser and the lense itself are immersed in oil. I think I'll stick with non-oil-immersion lenses and condensers until I am more comfortable with using the scope overall.

As with the immersion oil, it seems I may have bought type A. There is only one online store in Finland selling immersion oil online, and on their page they did not say what type it was.

It seems the oil went between the top and bottom glasses, so the fluidity of the oil seems to be too much.

Anyway, I may have to find another place to get type B oil.

quote:
With a BX-41, you may have a difficult time fitting another lamp between your frame/lamp lens and your condenser. This LED lamp may work:
http://www.ebay.com/itm/272446758936?

Or, this specific retro LED for a BX 40 series:
http://www.ebay.com/itm/252501641742

Thank you! This is very helpful, too. I'll look into those.

quote:
Lymedin2010 shared some details several pages back. You may have to go hunting for that unless he chimes in. Usually it takes less than 24 hours for the spirochetes to come out of hiding. Special techniques, (besides sealing your coverslip), are usually not necessary.
I already read the whole thread second time (first time was when I found this discussion), but did not find it... anyway, but if it is in this thread, I'll find it if I will need it.

Good to know that this is not needed. It would be useful though to know how to expedite the process, as my mother is also infected with lyme, so I'll probably wish to view her blood samples too.... and between being a father of a small baby, working full time, and being infected with this disease, I seem to be pressed for time...

[ 01-07-2017, 11:27 AM: Message edited by: BorreJaakko ]

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quote:
I forgot to say congratulations!!

And, you got quite the scope for a beginner!

Thanks for joining the club! I'm looking forward to seeing some of your work!!

Thank you! I am very anxious to see the suckers in my blood, and hopefully at some point can bring something back to this community too.

I have to say that I think the work in this community is nothing short of revolutionary. The progress I have seen just in this thread itself has given such much new hope in battling this disease.

I guess anybody who has gotten so far that they know they have this disease, has enough experience with the medical profession as well as medical industry to know that their understanding of this disease is very limited to say the least. And it seems to me that it is limited because they have a very limited view of how these kind of diseases work in general.

But with enough evidence of the mechanisms how this disease works, even the medical profession and industry cannot keep the truth from coming out. And then there will be a revolution in the medical profession and industry.

So I think the work in this thread is right now on the forefront of the medical research. You guys deserve a medical Nobel prize.

Anyway, it took quite some time to get the scope, as there was only one used lab grade scope being sold in my country.. and I did not want to risk the transportation by mail so I wanted to buy the scope locally.

The asking price for the scope was way too high for me, but luckily there were no other interested buyers for quite some time and seller really wanted to sell it, so we could make a deal that made both of us happy.

Although I am very interested in this microscopy thing, my time is right now very limited, as I have a small baby. (The stress related to her birth was probably what caused the outbreak of my lyme symptoms).

Therefore I am looking for ways for automating the observation of my blood samples. So I would like to set up constant video recording, and then go through the videos afterwards when I have time. Perhaps I am overly optimistic in how easy it is to do this, though...

For this, I would really like to hook up my Canon 7d mk II dSLR into the scope so that the image resolution is as good as possible so I will not miss things. Unfortunately the possibilities for doing that seem VERY expensive. I guess I would need a trinocular head, adapters etc., and that seems to add up to several thousand $'s, and that is way too much....

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TNT
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quote:
Originally posted by BorreJaakko:
It seems my confusion was about the term "immersion", I did not understand what was immersed in what. I did not figure out that actually both the condenser and the lense itself are immersed in oil.

With your current set-up, only your 100x (oil) objective gets immersed. Not your condenser. ONLY if your condenser is an oil condenser will you use oil with it (And, usually only when you are doing 1000x darkfield). You can ruin the condenser if it's not an oil condenser.

Does your scope have a brightfield condenser only, or is it a "turret" condenser? Turret condensers are bigger, usually round, with a big wheel inside that contains annuli for doing phase and darkfield.

Turret condensers look like the condenser with this scope (the 7th picture):

http://www.ebay.com/itm/Olympus-BX51-Phase-Contrast-Microscope-10x-20x-Ph1-Objectives/351936568079?

You can rotate the turret to expose whichever annuli you wish to use at the time. The annuli are removable, so if you have a turret condenser and it doesn't contain, say, the darkfield annulus, you can buy it and install it to enable your scope for darkfield. Same thing for phase, you need the correct annulus for whichever magnification you plan on viewing at. But with phase, you also need the corresponding objective lenses.

What I'm getting at with the above explanation is this. Most turret condensers have an oil condenser lens that allows it to be used with oil for high magnification darkfield. My AO turret condenser does not have an oil lens, so I am not able to do oil darkfield with it. I had to buy a separate condenser just for oil darkfield. So, not all turret condensers are equipped to do oil, just so you are aware. If a turret is equipped, it will have a "well" around the condenser lens to collect oil overflow. That's how you can tell. Notice the lip on the condenser lens on the scope I linked to.

Even if yours is only a brightfield condenser, you can still do high-magnification brightfield with your 100x oil lens. But, you only use a drop of oil ON TOP of the slide. Nowhere else. And, don't flip any of your non-oil lenses into the oil by mistake! Olympus lenses are not cheap.

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quote:
Does your scope have a brightfield condenser only, or is it a "turret" condenser? Turret condensers are bigger, usually round, with a big wheel inside that contains annuli for doing phase and darkfield.

Turret condensers look like the condenser with this scope:

http://www.ebay.com/itm/Olympus-BX51-Phase-Contrast-Microscope-10x-20x-Ph1-Objectives/351936568079?

You can rotate the turret to expose whichever annuli you wish to use at the time. The annuli are removable, so if you have a turret condenser and it doesn't contain, say, the darkfield annulus, you can buy it and install it to enable your scope for darkfield. Same thing for phase, you need the correct annulus for whichever magnification you plan on viewing at. But with phase, you also need the corresponding objective lenses.

I am not really sure what kind of condenser I have, as I have no experience whatsoever on condensers and the seller did not know anything about microscopes either as this scope was part of an estate that was inherited.

But based on the image you linked I suspect it is a regular brightfield condenser and not a turret.

Here are some pictures. I guess the condenser is the thing in the middle of the first image.

 -

The second image shows that the 100x lense seems to be oil, like you guessed. Others do not have the text "oil" in the name, so I guess they are normal bright field objectives. Other objectvies are Ach 60x (I guess these Ach are the cheapest) 40x PlanC etc.

 -

The third image shows something on top of the lenses, which I took out, I guess this is place to insert the filters?

 -

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quote:

Even if yours is only a brightfield condenser, you can still do high-magnification brightfield with your 100x oil lens. But, you only use a drop of oil ON TOP of the slide. Nowhere else. And, don't flip any of your non-oil lenses into the oil by mistake! Olympus lenses are not cheap.

I really value your help!

As it seems that this is the case, then my question is, if I do brightfield 100x, should I immerse the 100x oil immersion lense in oil or not? Does the oil immersion lense mean it only works with oil?

That would explain why I saw next to nothing on it, when I was trying to use it without oil on top, although other lenses seemed to work fine.

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TNT
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Yes, yours is strictly a brightfield condenser. So, no oil is to ever be used on it. And, yes, it looks like only your 100x objective is an oil lens. That is the norm.

Plan objectives are best, but an Olympus achromat will be just fine. They won't give you a totally flat field of view, but will still give stunning images.

I don't have experience with Olympus scopes, but I am guessing you are correct about that slider being the holder for color filters.

Very nice scope!

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quote:
Originally posted by BorreJaakko:
quote:

Even if yours is only a brightfield condenser, you can still do high-magnification brightfield with your 100x oil lens. But, you only use a drop of oil ON TOP of the slide. Nowhere else. And, don't flip any of your non-oil lenses into the oil by mistake! Olympus lenses are not cheap.

I really value your help!

As it seems that this is the case, then my question is, if I do brightfield 100x, should I immerse the 100x oil immersion lense in oil or not? Does the oil immersion lense mean it only works with oil?

That would explain why I saw next to nothing on it, when I was trying to use it without oil on top, although other lenses seemed to work fine.

That is correct. You will see next to nothing with your 100x oil objective without using oil. Just add a drop of immersion oil (Type A will work for the time-being) and then lower your 100x objective down into the oil and then focus down until you see your image. Be careful not to make contact with the specimen slide.

If you are focused in on your image with your 60x, rotate it out (between the 60x and 100x lenses), add the oil drop to the top of the slide, and then rotate your 100x into the viewing position. It should be very close to being in focus at that point. Just be careful when rotating your 100x into position that it's not too low that it strikes the slide. That's why I prefer to lower the 100x into place rather than rotating it into place.

Always remember to keep the non-oil lenses out of the oil when you are switching between lenses. (It's easy to absent-mindedly rotate a non-oil lens "back" into the oil).

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quote:

Plan objectives are best, but an Olympus achromat will be just fine. They won't give you a totally flat field of view, but will still give stunning images.

Great [Smile]

quote:

I don't have experience with Olympus scopes, but I am guessing you are correct about that slider being the holder for color filters.

Very nice scope!

Thank you!

It seems I was wrong though. The black thing in the middle of the photo is not the condenser. It has a text "Achromat 0.9" on it. Would you happen to know what it is?

The white thing in the second image seems to be the condenser.

It is not showing clearly in the picture, but there is some space between the black thing and the white thing so they are two seperate things.

It seems there are two kinds of darkfield condensers for this Olympus model.

10X —100X dry darkfield condenser (U-DCD)
20X —100X oil immersion darkfield condenser (U-DCW)

Anyway, I think this will be enough for me now, but it is nice to know that there are possibilities for expansion, too.

The only question I have left now is about the lighting. There is some space below the black thing. But there seems to be some ability to focus the light. If I add a LED below the black thing, then I lose this ability. This may mean that LED without focusing may actually be less bright than the current setup...

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quote:
Originally posted by BorreJaakko:


It seems I was wrong though. The black thing in the middle of the photo is not the condenser. It has a text "Achromat 0.9" on it. Would you happen to know what it is?

The white thing in the second image seems to be the condenser.

It is not showing clearly in the picture, but there is some space between the black thing and the white thing so they are two seperate things.

It seems there are two kinds of darkfield condensers for this Olympus model.

10X —100X dry darkfield condenser (U-DCD)
20X —100X oil immersion darkfield condenser (U-DCW)

Anyway, I think this will be enough for me now, but it is nice to know that there are possibilities for expansion, too.

The only question I have left now is about the lighting. There is some space below the black thing. But there seems to be some ability to focus the light. If I add a LED below the black thing, then I lose this ability. This may mean that LED without focusing may actually be less bright than the current setup...

Actually the black thing with "achromat .90" AND the white flip-in lens are both the condenser unit. (The black and white pieces are connected, correct?) Yours just has a flip-in auxiliary lens to give you the ability to make your light coming through more focused at the specimen plane. Flipping the white lens out of the way gives you a broader beam of light up to your specimen.

Sometimes a research grade scope will be equipped with a condenser (usually in the form of a turret condenser) that can be used for both dry and oil darkfield (just like the BX51 scope I linked above). I'm pretty sure that the turret condenser on that BX51 could interchangeably by used on your BX41 scope.

That rotatable collar below your condenser is a diaphram for your light. When the little dot is aligned with the "o," then you have the full beam coming through. If you rotate the dot around to the right you close down the amount of light coming through. Yes, if you insert a separate lamp between the diaphram and condenser, you lose the ability to "dial down" your beam. But, your condenser will still "condense" or focus the beam up to your specimen plane. The $140 retro-LED lamp that mounts to the back of your scope is probably the better LED lamp to go with anyways. It might be unhandy trying to keep the $40 lamp situated on your scope. And, like you said, you lose the ability to "dial-down" your beam if you go with that one. Although, both of those LED lamps do come with a dimmer. It depends on how much money you have to spare.

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BorreJaakko
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Here is an image with 60x magnification, 10x magnification from eye piece and 5x magnification from my phone. It is supposed to be blood smear.

 -

Any comments about level of magnification etc. or smear?

No spirochetes yet, but perhaps tomorrow.

It seems quite difficult to work with the iPhone camera app, because it wants to autofocus exactly at the moment when microscope would be in focus.

Anybody know a good microscope photography app for iPhone?

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Do you have a point and shoot camera you could snap a picture through the oculars (eyepieces) with? It may have better luck focusing. You'll have to zoom in with your point and shoot to get an image, just so you are aware.

I can see the individual RBCs, and the light looks bright with nice white light, so I think a little fiddling will be all that's needed.

I will say it appears like you may have too much blood in your sample. Less blood gives a more defined image. It gives space for the RBCs to spread out under the coverslip. In the sample above, you will not be able to see the spirochetes even if you can get a good focus because the amount of blood prevents the cells from spreading out. You can try pushing down on the coverslip to see if you can push some of the excess out. Pushing on the coverslip also expedites the exit of the ketes from the cells, allowing one to see them sooner.

By the way, there is a way for you to do darkfield with the condenser you already have. You can't do oil with what I am going to show you, but your 60x objective will give you plenty of magnification to see them. Even your 40x is sufficient.

Here is a makeshift way to do dry darkfield with a brightfield condenser:

https://www.youtube.com/watch?v=taKfysZ-LNY

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quote:
Do you have a point and shoot camera you could snap a picture through the oculars (eyepieces) with? It may have better luck focusing. You'll have to zoom in with your point and shoot to get an image, just so you are aware.

Unfortunately I don't have a point and shoot. I'll see if I can find something.

quote:

I can see the individual RBCs, and the light looks bright with nice white light, so I think a little fiddling will be all that's needed.

I will say it appears like you may have too much blood in your sample. Less blood gives a more defined image. It gives space for the RBCs to spread out under the coverslip. In the sample above, you will not be able to see the spirochetes even if you can get a good focus because the amount of blood prevents the cells from spreading out. You can try pushing down on the coverslip to see if you can push some of the excess out. Pushing on the coverslip also expedites the exit of the ketes from the cells, allowing one to see them sooner.

Great tip! Thank you. I'll do another smear with less blood. I guess I did back-and-forth on the blood, when one is supposed to make it as wide as possible.

quote:

By the way, there is a way for you to do darkfield with the condenser you already have. You can't do oil with what I am going to show you, but your 60x objective will give you plenty of magnification to see them. Even your 40x is sufficient.

Here is a makeshift way to do dry darkfield with a brightfield condenser:

https://www.youtube.com/watch?v=taKfysZ-LNY


This is interesting. I'll look into that too.
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Ok, here are two new images from the blood smear from yesterday. I let it be without oil for perhaps 24 hours. Is this normal that it looks like tree/vine?

 -

 -

Below is also some images from blood smear from today, which had less blood. It has been without oil for about 3 hours.

Almost everything is filled with clean nice white round things (I guess they are the RBC's), but going through the sample for 15 minutes I found a few abnormalities too, they are below among the other photos.

No spirochetes sighted so far. I don't know if it is because Cowden protocol I am taking has been working, because I am doing something wrong, or this has never been Lyme.

 -
 -
 -
 -
 -

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Lymedin2010
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Sorry, my brain hurts & feels like someone poured Clorox in it...I will come back to answer the previous posts on a better day.


For now, check this video out. It is very interesting, almost looks like the spurrs at the end of crenated rbc's. This is not like the Bartonella video, since it does not move in that jerky motion, but in Brownian motion instead. What do you guys think of it, sometimes we see similar in Lyme blood and could be babesia merozoites?

https://www.youtube.com/watch?v=s3DSB1xXuec


Another merozoite video:
https://www.youtube.com/watch?v=FKEGZCZqZ_w

Guys if you look at these 2 videos, you will notice how thin the RBC can get with malaria & perhaps babs & bartonella as well. The rbc cell wall can be VERY thin & unrecognizable. Keep this in mind when viewing the previous videos on the bart & vacuoles. The intravacuole WITHIN the rbc is possible, as the bart video shows, but it is possible for it to burst & degrade the rbc as well and so in some cases when the rbc is too worn out it is difficult to tell whether it is:

1) Vacuole + bart external to any rbc.

OR

2) Bart bursted from intravacuole & still residing within the rbc, which then goes on to degrade rbc to burst.


Another cool malaria video destroying a rbc rapidly.
https://www.youtube.com/watch?v=F9-CT2uDIkw

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Another one sees spirochetes in blood..nice spiros w/good movement:
https://www.youtube.com/watch?v=94c6TpPmbqo

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TNT, look up Anaplasma marginale stained pictures for reference to those objects coming out of your rbc's.

https://microbewiki.kenyon.edu/index.php/Anaplasma_marginale

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TNT
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quote:
Originally posted by Lymedin2010:
TNT, look up Anaplasma marginale stained pictures for reference to those objects coming out of your rbc's.

https://microbewiki.kenyon.edu/index.php/Anaplasma_marginale

Your comment is more in reference to those last videos I posted than to my stains? I pretty much totally agree that those "apicomplexans" are Rickettsia organisms. I have yet to find ANY sign of an apicomplexan such as Babesia or Toxoplasmosis in the stains of my blood. But, plenty of Rickettsias & "Bart"-like stuff, particularly Anaplasma morulas.

I've pretty much concluded that I have more than one strain of Anaplasma in my blood. I have them infecting Neutrophils, Monocytes, AND Platelets. So, maybe even three strains.

Do you think it's more likely they were trying to get in rather than trying to "get out?"

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TNT, it looks to me like they are coming out of your rbc. Sometimes you will see some pitting & trenching when they are going in & I do not see any evidence of that other than maybe one. I do see a very tiny tail on some of them on the rbc end, which may be the rbc phospholipid wall entrails still bonded to the pathogen as it is exiting & pulling out. So there is a tiny tail/bar of phospholipid as it is exiting. Also a lot of rbc's are sticky to the slide & stretched with a fiberous/phospholipid attachment & it may be related to the presence of the pathogen, as there is fairly large number.


BorreJaakko, welcome & nice to have you. Sorry, as your questions are many & I typically give long responses that will take me forever to write up. TNT has done a stand-up job helping out as I have quickly glanced, thanks TNT!!!

Pic 1 & 2- That is what blood looks like when it dries out & is useless for what we are looking for.

3- The cells are too close to one another & it becomes VERY difficult to notice spiros, although not impossible. The larger central object is a wbc.

4 & the rest. You have some wbc's there & some smaller 1 micron or less objects that can be anything really & it is hard to tell as your pictures appear rather blurry & unclear...something is off? Does it look that blurry with your eyes looking through the eyepieces?

Video will always give more detail & tell us about the movement.

"For this, I would really like to hook up my Canon 7d mk II dSLR into the scope so that the image resolution is as good as possible so I will not miss things. Unfortunately the possibilities for doing that seem VERY expensive. I guess I would need a trinocular head, adapters etc., and that seems to add up to several thousand $'s, and that is way too much.... "

http://www.amscope.com/accessories/camera/canon-slr-dslr-camera-adapter-for-microscopes.html

All you need is this & you can use the Canon software for the time-lapse pictures, which you will need to string together in something like Microsoft MovieMaker. You can add this adapter to a trinocular in a standard eyepiece, so just remove one of your eyepieces & add the adapter + cam. I tried this with a Canon 40d, back in 2013/2014 & it was the best & clearest imaging for that time, but now has been masked by 4K. I don't think I have made any of that video available. Your camera may have a built-in time-lapse feature, as I think the newer ones have. Tethering & wireless has made things easier with the newer cameras too.

I love my Amscope 14MP USB cam, as it is very convenient to have both pics & video directly on the PC without any needing to upload from a memory card. It comes at a loss of resolution, but what I post seems to be good enough to see details.

They also make FLAT led bulbs, such as this one. They sell them cheaper than this & I bought one locally, so just check your local or online resources. Get the daylight (5000K) version, as that will allow the pics & cams to pickup the light nicely & effectively, without a yellow hue and you will have more contrast & detail as a result.

http://www.ebay.com/itm/DDSKY-E27-6W-9W-12W-LED-Flat-Bulbs-Extra-Light-Energy-Saving-90-Led-Light-Bu-/192012437590?hash=item2cb4d54856:g:SGcAAOSwx2dYE8O8

NEVER look at a LED directly with your eye through the eyepiece, as it can damage your retinas with the focused light. Always use a cam/vid for this only. I use a CFL bulb when I want to view by eye, which I don't even do anymore since getting the Amscope 14MP cam.

Also, what we do here is for "fun" & "hobby" and not meant to be a diagnosis. We do it out of curiosity & to see what we can learn & how we can inspire the scientific community to look at this further. Sometimes our clues can lead other researches into investing their time, where they might have not thought of before. I think our microscopy efforts collectively have prompted others to look at blood with more consideration & some DNA Probe have also resulted from that. Perhaps our occasional filarial finds inspired the nematode staining quests.


I have now plenty of research links & clues to suggest that Borrelia can be found in our blood, especially in later stages. I will one day post a list in here & it will be a VERY long one.

[ 01-12-2017, 09:42 PM: Message edited by: Lymedin2010 ]

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TNT
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quote:
Originally posted by Lymedin2010:
TNT, it looks to me like they are coming out of your rbc. Sometimes you will see some pitting & trenching when they are going in & I do not see any evidence of that other than maybe one. I do see a very tiny tail on some of them on the rbc end, which may be the rbc phospholipid wall entrails still bonded to the pathogen as it is exiting & pulling out. So there is a tiny tail/bar of phospholipid as it is exiting.

Wow! You see tails on some of them? I never noticed. That could also be flagella. Which video and at what point are you seeing this?
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Lymedin2010
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No, I don't mean that they actually have a tail & I don't think you can see the flagella without higher powered microscopy.

I meant that they are teardroped shape & the tip is on the rbc side with a little bit of the rbc phospholipid sticking & stretching from the rbc. I have a 24" monitor & I can see it clearly the majority of them are exiting.

I would imagine the staining process washes a lot of them off that are in the plasma. Do you see quite this many in your stains or are they simply not staining as many as you see them. It would be nice for you to do concurrent stain & live wet mount, so as to compare them. You show video of the live & then you can compare to your stain to see the number of stained ratio & it may buy you more clues.

How many total stained slides have you done? What is the likelihood of missing any ring-forms at all if they happen to be babs?

It would be really nice to also do some time-lapse on the exit. You can do that with an app on your phone & an adapter.

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TNT
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I doubt what you are seeing are the flagella, but if it were, you could possibly see them. Definitely with a 24" screen. Remember the flagellated cocci I showed?

https://www.youtube.com/watch?v=bEQnmNKVt6w


There are times I see a relatively high concentration of the individual BLO organisms in my stains. Remember this pic? There are possibly 10 organisms in the frame:

 -


I have done maybe 10-15 stained slides of my own blood (besides stains of other people's blood, not to mention wet mounts). It takes me quite a while to painstakingly look at every square micrometer of a slide, and I have yet to see ANY convincing proof of Babs or Toxo in my blood. Though, I have posted pics of Babs in other people's blood.

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Lymedin2010
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Does this help?

 -

 -

 -

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That is why originally I said I thought it was babesia, because they really look like merozoites exiting the rbc's. I was predicting that you would eventually find the cross or ring-forms within the rbc's with your future stains, but it seems like they don't exist in those forms from your staining, so then Bart or BLO forms it can possibly be.

[ 01-14-2017, 10:37 AM: Message edited by: Lymedin2010 ]

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TNT
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I still am not following why you think it could depict them exiting the RBCs, because it looks to me to be attachment, which is the initial stage of entry. When they leave cells, there are more than one, and it always ruptures the cells (entry doesn't rupture the cells).
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Lymedin2010
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 -

Ahhhh, so then when you added the methanol & stain, it probably retracted the rbc phospholipid trails (temporary tail) & retracted the slight stretching of this organism, from teardrop form (from the exit), into it's proper form. And it is no longer tear dropped in shape in the stained version.

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Lymedin2010
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Because entry would show pitting & exit will show tear drop stretching....a sort of temporary tail. Because it is trying to pull itself out, but yet still sticky to the rbc cell wall.
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Yes, for the longest time I thought they almost had to be apicomplexans. But, I agree with you, as time goes on, the other evidence I see (in my blood), and the more info I learn, it certainly is becoming more evident that they are bacteria.
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Lymedin2010
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Yea, for sure. It is evident & obvious it is "something" at the very least. Especially, if you do not see the same in others.

Of course PCR & testing is best. Even in that long bart video, they said that they have not a lab yet with proper testing.

Way to go, you did really good & helped out others!!!! Great job!!!!

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TNT
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It would be helpful to see some literature on this phenomenon you are describing (the teardrop exit). In response to your observation about them not maintaining the "teardrop" shape in the stains, I have seen a fairly distinct "teardrop" shape at times (in the stains), but not consistently. It's more random.
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Lymedin2010
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Next step, sheep blood agar culture for a few days for possible additional revelations. I think they culture best at 82F, not too far away from room temp, which I bet would culture sufficiently. Given they grow in ticks & other arthropods just as well.
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TNT
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I have actually given culturing a serious thought recently. I read somewhere that Rickettsias culture well in (fertilized) chicken eggs.
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Lymedin2010
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Might be a pain to capture them again from an egg. The agar I believe become visible in the growth areas & you can sample directly & without any egg contaminants.

We spend some time next week tracking info & see what we get.

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Lymedin2010
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Your symptoms because of this might be:
1) CFS
2) Exercise or high activity intolerance.
3) Circulatory issues.
4) Night sweats.
5) Brain fog...not enough O2 to the brain.

???


Ever positive for bart test?

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TNT
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"Ever positive for bart test?"

Slight positive with Galaxy culture (read as a waning infection, ha). But I have not been tested for Anaplasma that I can remember. HGE, but not Anaplasma. Not sure if they would cross over on antibody tests or not, though. I just got tired of spending money on tests that were never conclusive. Especially Medical Diagnostic Labratory's tests, and the local labs antibodies tests (not for Anaplasma).

Microscopy has been more insightful, enlightening, educational, and even entertaining. Not to mention more conclusive. [Wink]

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Lymedin2010
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Most definitely, most of the ones who have done microscopy can tell there is something terribly wrong with their blood & it is telling of a multi-system disease that involves the blood as a super highway.

In case this ever comes up & just to get this out of the way, I don't think these are Howell-jolly Bodies. Your objects appear to be consistently on the outer edge of the rbc's as if they are exiting.

https://images.search.yahoo.com/yhs/search;_ylt=A0LEVirP1XhYWqEAhUMnnIlQ;_ylu=X3oDMTEyNzhtM3JhBGNvbG8DYmYxBHBvcwMxBHZ0aWQDQjI2MThfMQRzZWMDc2M-?p=Howell-jolly+Bodies&fr=yhs-mozilla- 001&hspart=mozilla&hsimp=yhs-001#id=453&iurl=http%3A%2F%2Fstudydroid.com%2FimageCards%2F0b%2Fqb%2Fcard-12398560-front.jpg&action=close

Another great Bart video from NorVect.
https://www.youtube.com/watch?v=mLK48ecsn-s

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Lymedin2010
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BorreJaakko, below is a sample pic from my 50d at the time with the Amscope adapter I linked you. I sold the 50d before the prices had dropped any further & I ended getting a 40d for $100.

Here is the video from 40d, BUT it is much better to watch the original file on my PC as Youtube downgrades the quality. In another video it actually even flipped the video to the vertical mode...dunno why. :
https://www.youtube.com/watch?v=iSTDQN-3Cb0


Here is a pic from a 50d:
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[ 01-14-2017, 03:43 PM: Message edited by: Lymedin2010 ]

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BorreJaakko
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Thank you so much Lymedin2000!

Crap, if only I had known sooner about the AmScope adapter! I already ordered an adapter from lmscope and it was very expensive!

I was trying to Google for adapters, but I could only find custom ones. From these alternatives, lmscope was most attractive.

I don't know if this lmscope adapter will be higher quality than the AmScope though. This is the model I will be getting:

http://www.lmscope.com/produkt22/Datenblatt_DSLRXTW_en.pdf

The adapter is in mail already.

I also purchased a 8 megapixel camera board for rasperry pi, and just got it today. I am just now playing with it.

This camera will give me all the flexibility to control the camera exactly how I want. The software will not be a problem for me as I work with software professionally, so I can develop my own utility.

Rasperry pi camera is quite cheap, so I can leave it to record time lapse or video extended periods.

It can do 2592×1944 at 15fps.

Quality seems reasonably good.

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BorreJaakko
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Lymedin2000, how fast are the spirochetes moving?

What kind of delays you suggest for time lapse imaging?

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Lymedin2010
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I just noticed that they make a Nikon/Canon/Olympus version of the adapter, which fits all 3 cameras with sub-adapters. Very nice & new.
http://www.amscope.com/accessories/camera/canon-nikon-and-olumpus-slr-dslr-camera-adapter-for-microscopes.html

The adapters are VERY sturdy & metallic. Good quality & it does what it is supposed to. The custom ones are very similar & I don't think too far away. The only difference would be the eyepiece type optical & Amscope is good enough to produce quality images for that.


My TL delays are normally 10s & 15s & all depends on the details I want to capture. I can always speed it up, which mostly I do in the software processing. If I want capture lets say a rbc or wbc degrading, I would do 1-5 minutes & again speed up in software, and as much as 15 min intervals. So it depends on what your capturing & what details you are looking for. If you miss something in between & it was hard to capture, you will kick yourself & I tend to go lower with plenty of hard drive space.


Here is an example of real-time movement.
https://www.youtube.com/watch?v=gjCWzt-Xuvs


Here is another Lymie I exchange info with & this one is nice, as it spirals really nicely & aggressively.
https://www.youtube.com/watch?v=1wDV4TliIHg

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BorreJaakko
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I got my Rasperry PI camera. It seems pretty decent, although processing power seems a little limited, which means taking full HD video with high frame rate is not possible.

It seems I did not find anything, until today I cleaned a slide with water and paper and then put a drop of fresh blood on it, and things started happening.

Some white tiny things seem to be attacking the blood cells quite viciously, and the cells become spikey. It seems like some of RBCs are killed and eaten by dozens of them -- alive.

It seems like the video is moving with extra speed, I guess I need to set up the recording frame rate.

https://www.youtube.com/watch?v=W28RLfTVw_g

This raises a question: do you, and if yes, how do you clean your slides?

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TNT
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quote:
Originally posted by BorreJaakko:


Some white tiny things seem to be attacking the blood cells quite viciously, and the cells become spikey. It seems like some of RBCs are killed and eaten by dozens of them -- alive.

https://www.youtube.com/watch?v=W28RLfTVw_g

This raises a question: do you, and if yes, how do you clean your slides?

The little "white" things in your video are immune components called lysozomes. Basically they are the same as the little granules of the white blood cells except they are free in the plasma. They attack pathogens. I've seen many spirochetes being attacked by them. I assume they release enzymes that dissolve pathogens just like the ones inside the WBCs do. I doubt they were attacking your blood cells since they don't attack "self." I've never seen that anyways.

Your video is good. The clarity with that 60x objective is notable. Now all you need to do is seal off your cover-slip with immersion oil and wait to see some ketes. They will surely come out if you are infected. But, you may have to wait and look a little if you are not heavily infected.

I buy pre-cleaned slides. But, they are usually cloudy so I wash them with soap and running water and let them air dry in a drainer. That removes any bacteria that may be on them out of the box. It's important to have clean slides especially when one is staining blood. Otherwise, you think you see bacteria in the blood when it's just contamination.

Great job!

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BorreJaakko
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quote:
The little "white" things in your video are immune components called lysozomes. Basically they are the same as the little granules of the white blood cells except they are free in the plasma. They attack pathogens. I've seen many spirochetes being attacked by them. I assume they release enzymes that dissolve pathogens just like the ones inside the WBCs do. I doubt they were attacking your blood cells since they don't attack "self." I've never seen that anyways.

Interesting. I thought I saw that happening live, and that it would be visible from the video too.

For example at 0:20 you see those lysozomes "ride" the blood cells. At 1:08 you see a big formation of them, which I figured would be a cell eaten by losozomes. And at 1:30 you see some cells that are completely covered with those lysozomes. Also, I find it interesting that some of the cells have spikes, whereas some of them are nice and round....

quote:

Your video is good. The clarity with that 60x objective is notable. Now all you need to do is seal off your cover-slip with immersion oil and wait to see some ketes. They will surely come out if you are infected. But, you may have to wait and look a little if you are not heavily infected.

Thanks! My problem with sealing off right now is that I bought wrong kind of cover slips... twice! First ones were too long and wide, and it is not feasible to seal them off because the slip is almost as wide as slide, and there is no space for the oil on the side... and the next ones are nice and round, but only 5mm in diameter so they are way too small. So I need to find a place that sells proper size cover slips.

quote:

I buy pre-cleaned slides. But, they are usually cloudy so I wash them with soap and running water and let them air dry in a drainer. That removes any bacteria that may be on them out of the box. It's important to have clean slides especially when one is staining blood. Otherwise, you think you see bacteria in the blood when it's just contamination.

Yeah, I have pre-cleaned ones too, I just thought that maybe it is easiest to just reuse the slides as I don't need the old dried up blood for anything..anyway I'll use fresh ones from now on, thanks for the tip.
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mustardseed2
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Hey guys, hopefully you can help me out again (and this might be relevant to some of the earlier posts from TNT).

Any idea what these round inclusions in the RBC's could be?

They seem too big to be Bartonella.

At the same time, I don't see any typical Babesia forms anywhere in my blood (halo's, crosses, etc).

It leaves me scratching my head as to what these large, round circles are.

Any input would be appreciated.
Thanks!

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TNT
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quote:
Originally posted by mustardseed2:
Hey guys, hopefully you can help me out again (and this might be relevant to some of the earlier posts from TNT).

Any idea what these round inclusions in the RBC's could be?

They seem too big to be Bartonella.

At the same time, I don't see any typical Babesia forms anywhere in my blood (halo's, crosses, etc).

It leaves me scratching my head as to what these large, round circles are.

Any input would be appreciated.
Thanks!

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 -

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Those are Heinz bodies. They can be caused by hemolytic anemia due to unstable hemoglobins, exposure to oxidizing drugs, chemical poisoning, G-6PD deficiency.

https://www.labce.com/spg972957_red_blood_cell_inclusions_and_associated_condition.aspx


Good job on the staining. Blood staining can be extremely helpful in giving us insights into our bodies and clues about what may be going on. That's good you are not seeing any signs of Babesia. Are you seeing any dots on the outside perimeter of the RBCs typical of Bartonella? Any Anaplasma morulas? Any other morphologies?

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mustardseed2
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Thanks TNT! That a great page you posted there, I bookmarked it.

When I started staining, I was convinced I had a Babesia problem. I have a 24/7 headache, chills, night sweats, weakness, tingling, and ringing in the ears. On top of that, I've herxed on Malarone, Artemisinin, Alinia, and Beyond Balance MC-BAB-2.

But alas, haven't seen anything Babesia-like in my blood. A little perplexed by it to be honest.

As for Bartonella, the only dots I see on the perimeter of RBC's are big ones like I posted. I didn't herx at all on high doses of Levaquin for 3 months and Rifampin for 6 months. So I don't think I have Bart.

I haven't seen any anaplasma inclusions in WBC's, but I have seen some rod-like bacteria, similar to the pictures you posted as rickettsia. Too small to be sure though.

I did test postitive for anaplasma through IGENEX. I took several months of Doxycycline, followed by 6 months of Rifampin, so I think I would have hit anaplasma pretty well.

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TNT
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quote:
Originally posted by mustardseed2:
Thanks TNT! That a great page you posted there, I bookmarked it.

When I started staining, I was convinced I had a Babesia problem. I have a 24/7 headache, chills, night sweats, weakness, tingling, and ringing in the ears. On top of that, I've herxed on Malarone, Artemisinin, Alinia, and Beyond Balance MC-BAB-2.

But alas, haven't seen anything Babesia-like in my blood. A little perplexed by it to be honest.

As for Bartonella, the only dots I see on the perimeter of RBC's are big ones like I posted. I didn't herx at all on high doses of Levaquin for 3 months and Rifampin for 6 months. So I don't think I have Bart.

I haven't seen any anaplasma inclusions in WBC's, but I have seen some rod-like bacteria, similar to the pictures you posted as rickettsia. Too small to be sure though.

I did test postitive for anaplasma through IGENEX. I took several months of Doxycycline, followed by 6 months of Rifampin, so I think I would have hit anaplasma pretty well.

Glad I could be of some help! What treatment are you currently on? Are you still on the anti-protozoan meds that you herx on?

That's interesting you are seeing minute rod-shaped objects. Definitely describes Rickettsias. Where are you seeing them? Inside WBCs or in the plasma? Could you post some pictures of them??? It does seem unlikely you could still have a Rickettsia with months of Doxy followed by Rifampin. Perhaps it's still possible if you were not on them together. In Burrascano's guidelines he says Doxy plus Rifampin may be needed.

"However, there are reports of treatment failure even when higher doses and long duration treatment with doxycycline is given. In such cases, consideration may be given for adding rifampin, 600 mg daily, to the regimen."

How long are you canvassing a stained slide? It has taken me quite a while at times before I've found some of the morulas.

Your herxing pattern suggests a protozoan, unless the "herx" was a flare (a worsening) on account of hitting a symbiotic organism. That has definitely happened to me.

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Lymedin2010
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BJ:

1) Great video. What can make it even better is if you increase the contrast & details will begin to pop even further. There might even be sharpness setting on there as well & maybe they have modes such as "vivid" to further enhance the quality. I believe that is a 5MP camera? I think the new Raspberry Pi 2 cameras are 8MP.

2) At time 1:09 it looks like you have an Eosinophil there & sometimes Lysosomes can escape from the wbc & they can be what those white dots dancing around in Brownian motion are. The peroxisome from the wbc escape too & are bigger than the lysosomes. But they can also be platelets or micelles (dissolved fat droplet particles). If you eat a very fatty diet & check your blood 1 hr later, you will see TONS of the micelles in your blood in the plasma.

Your video recording is too jerky & it almost seems like those dancing dots can be pathogens & it will be important for you to fix that in order sometimes to pickup on slight cues as to whether something is normal vs foreign in your blood.

3) I buy new boxes of slides & cover slips & never have to clean them, but if you did then I would wash them off, autoclave them in an oven at high temperatures & then maybe clean them off again with alcohol & cotton. I don't like to do that, as for me it is not worth mistaking an artifact for something interesting in blood, which is precious info for all of us.

You can use one slide & make 3 different samples on it if you like (left, middle, & right) and you can save this way. A lot of times I take a sample & do a simple experiment on the same slide using a 2nd cover & so make a 2 slip-covered slide.


4) I talk about crenation & the spiky rbc's here, as that is a normal occurrence. Sickly blood may have ammonia or other pathogen toxins that can make the rbc's degrade quicker though, but all blood can degrade like this & sometimes super quick based on air exposure on the slide.

https://www.youtube.com/watch?v=kFQom3ssd38&list=PLrV8FYOIQcamUAh2BjIPVcKI-mJ-zaigc

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Lymedin2010
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mustardseed:

WOW, really nice staining work!!!

They look like Howell-jolly Bodies, which are basophilic nuclear material that never got expelled from the cell by the late erythroblast precursors.

Have you ever been scanned, as the spleen normally gets rid of these in normal blood. Do you have Splenomegaly, enlargement of the spleen? I did when they last scanned me a few years ago & I could feel the tenderness on my left side, under my ribs sometimes. Especially when I am taking a heavy combo/dose of abx.

I wonder sometimes if they can also be platelets, just stuck on the outside or on top of the rbc. Your first pic the 1 micron object is nice & pink with stain & I wonder if it can be a platelet on top of the rbc.

Why is your last pic outside the rbc & not within, sometimes the rbc's lyse & release the Howell-jolly Bodies. This one looks more darkly stained & may not be a platelet. Are the rest of your platelets nice & pink in the plasma?


https://images.search.yahoo.com/yhs/search;_ylt=A0LEVirP1XhYWqEAhUMnnIlQ;_ylu=X3oDMTEyNzhtM3JhBGNvbG8DYmYxBHBvcwMxBHZ0aWQDQjI2MThfMQRzZWMDc2M-?p=Howell-jolly+Bodies&fr=yhs-mozilla- 001&hspart=mozilla&hsimp=yhs-001#id=453&iurl=http%3A%2F%2Fstudydroid.com%2FimageCards%2F0b%2Fqb%2Fcard-12398560-front.jpg&action=close

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TNT
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They could be Howell-jolly bodies, and I originally thought that. But, the fact that he is seeing them on the outside of the RBCs would lend to the stronger possibility that they are Heinz bodies. But, it is possible they could be HJ bodies.
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Lymedin2010
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I think Heinz bodies are more irregular in shape & his are nice and more rounded, almost like a perfect circle. Perfect circle because it used to be a nice round nucleus that did not expel. And they are on the slightly larger side too.


At least that is what I think, but you guys be the judge. I think the spleen is overworked in us Lymies, because of all the pathogens and when it suffers our pathogen load gets higher & we get sicker. That has been my own personal account & witness with microscopy & my pains/tenderness in the spleen area & scan.

Sometimes rbc's can also lyse & release them and they can appear external and can be confused with platelets.


Heinz Bodies, which are denatured hemoglobin inclusions or masses WITHIN the rbc, but sometimes the rbc lysis & it can also escape:
https://images.search.yahoo.com/yhs/search;_ylt=A0LEVvaUc39YGysA8mInnIlQ;_ylu=X3oDMTEyNzhtM3JhBGNvbG8DYmYxBHBvcwMxBHZ0aWQDQjI2MThfMQRzZWMDc2M-?p=Heinz+Bodies+Stained+Blood&fr=yhs-m ozilla-001&hspart=mozilla&hsimp=yhs-001#id=18&iurl=https%3A%2F%2Fwww.researchgate.net%2Fprofile%2FJennifer_Neel%2Fpublication%2F8969370%2Ffigure%2Ffig2%2FAS%3A277707435855882%40144 3222020765%2FFig-2-Feline-peripheral-blood-smear-aggregate-reticulocytes-punctate-reticulocytes.png&action=close

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Lymedin2010
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Some girl did microscopy in her father's ALS diagnosed blood & look what they found:

https://www.youtube.com/watch?v=6tA-xmrz7Ns


"This is a darkfield microscopy of my father's blood sample, revealing presence of spirochete like structures. my father has been officially diagnosed with ALS."
https://www.youtube.com/watch?v=pkvwRS5vqYI


Lyme test was negative though & this is what she had to say....."No, my father has been diagnosed only with ALS. We did testing for Lyme, and all tests came back negative, except the microscopy we performed in Romania and they managed to film spirochetes in my father's blood specimen. He does have some of the symptoms that you listed in your post, so he has have a lot of sweating, skin itching, fasciculations, unusual dermatological issues on his face, GI irregularities, constant conjuctivitis (like he has have a sand in his eyes he tells me), bladder disfunction-frequent urination, constipation, bloating. at this very moment he is bed ridden most of the time, he developed a very serious muscle atrophy, his hands are completely paralysed, a lot of atrophy on his legs, but he can still make a step or two on his own. Lately his breathing became bad.... I don't know could I connect all of this with ALS alone."

This sounds like an infection of some sort for sure, as it is not just an atrophy of the main nerve bundles & maybe multiple infected.

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mustardseed2
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TNT,

Since I wasn't getting anywhere on antibiotics or anti-protozoal medications, I've switched gears completely for a few months.

I'm currently doing one month of Diflucan + Ketoconazole nasal spray. This will hopefully hit any candida or other fungus, as I have some consistently nasty oral thrush.

This will be followed by a six week parasite treatment where I'll go through Praziquantel, Ivermectin, Pyrantel pamoate, Albendazole, and Alinia.

My eosinophil count sky rocketed when I tried Alinia, plus I have pictures a few pages back of some stuff in my blood I thought were parasites. It's possible that my herxing on anti-babesia meds was really parasites reacting to those medications (I know Alinia and Artemisinin are anti-helminth, not sure about Malarone)

I usually canvas a slide for about half an hour. I've done probably over a dozen stains at this point, so I'd say I'd be surprised if I found anything new now. Or maybe I don't have Babesia in my finger tips where I'm taking blood from [Razz]

Either way the herxing is weird. I hadn't thought that it could be hitting a symbiotic organism... I'm not even sure what organism would do that when killed.


Lymein2010,

I've never had my spleen tested. I don't have any pain or tenderness. I do blood work every month, but I'm not sure there's anything on there that relates to spleen hmmm.

I'm not sure these 3 are platelets, simply because I find the platelets to be a slightly more irregular, and slightly more diffuse in color.

You are correct to question it though, because I do regularly find them showing up on top of RBC's. It's hard to really show the difference with these pictures, as they're taken with my crappy cell phone camera through the objective.

I'm leaning towards HJ bodies for my pics.

Anyway thank you both!
I love this thread!

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TNT
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quote:
Originally posted by mustardseed2:


My eosinophil count sky rocketed when I tried Alinia, plus I have pictures a few pages back of some stuff in my blood I thought were parasites. It's possible that my herxing on anti-babesia meds was really parasites reacting to those medications (I know Alinia and Artemisinin are anti-helminth, not sure about Malarone)

I usually canvas a slide for about half an hour. I've done probably over a dozen stains at this point, so I'd say I'd be surprised if I found anything new now. Or maybe I don't have Babesia in my finger tips where I'm taking blood from [Razz]

Either way the herxing is weird. I hadn't thought that it could be hitting a symbiotic organism... I'm not even sure what organism would do that when killed.



That's very interesting that your Eosinophil count increased when on Alinia. That definitely says something. Especially since Eos count raises in response to worm/helminth/parasite infection.

Depending on the presentation of your herx, though, I think it's possible that you could be causing a flare of some other symbiotic organism by using the anti-helminth/anti-protozoal meds. What I mean by that is this. Many times these microbes live together symbiotically, or similarly, in such a way that keeps the others in check. When you kill one part of that "community," it allows the proliferation of other competing or perhaps other symbiotic organisms. Take Heartworm infection in dogs as a good example. Veterinarians know that if you treat Heartworm in heavily-infected dogs, it can actually kill the dog. NOT because the die-off (herx) of Heartworm is so great, but because of the MASSIVE release of symbiotic Wolbachia (a Rickettsia) bacteria into the system of the dog. The resulting MASSIVE immune response to the Wolbachia antigen release causes so much inflammation that it kills the dog!

Not the Heartworm, not the die-off, but the immune response to the sudden release of antigenic material kills the dog via a huge immune response. And, if the immune response doesn't kill the dog, I imagine that the unchecked proliferation of the Rickettsia eventually would.

That's why vets use Doxy to treat heartworm in heavily-infected dogs. There is no sudden release of Wolbachia because it kills them first.

So, it's very possible that the antihelminth medication was causing a release of bacteria into your system causing a flare of symptoms relating to a bacterial infection, and not necessarily related to a parasite die-off.

It's the Russian doll effect. That's why order of treatment really does matter. And, a good example of why microscopy can be helpful.

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mustardseed2
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Wow great post TNT, so interesting.

My question would be would this still happen in the presence of antibiotics as well?

For instance, while I was herxing on Malarone, I was also taking zithromax and doxycycline.

When I herxed on Artemisinin, I was also taking minocycline and ceftin.

When I herxed on Alinia, I was taking biaxin and tindamax.

I suppose the herx I was feeling could be from these secondary medications attacking bacteria that was being released?

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TNT
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quote:
Originally posted by mustardseed2:


I suppose the herx I was feeling could be from these secondary medications attacking bacteria that was being released?

That would be my guess. That, or a true parasite die-off herx. I'm not sure exactly how a true parasite herx presents.

But, like I said, your "herx" symptom picture may give some clues. My experience is that true DIE-OFF herxes make me sleepy and flu-like. Flares (worsening of normal symptoms), or especially the onset of new and strange symptoms, although touted by many people (and LLMDs) as true herxes, I feel are really the worsening of some other concurrent infection. Just like the Heartworm model.

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mustardseed2
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I wanted to post one more picture of the rod-like bacteria I mentioned before.

The picture is not the best quality because it's just from my cell phone. Also the RBC's got a little wonky/spiky when I put the cover slip on, so ignore that.

In reality, the thing in question is actually less angular than it appears in the picture. It's definitely an oval, not a circle.

It was moving around in brownian motion, moving through my blood plasma.

Not quite sure what it is.

 -

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TNT
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I see the exact same things in my wet mounts even now. Rod bacteria the same shape and size. I was seeing them in my earliest samples and all along.

Interestingly, I did not see morulas UNTIL I was on Doxy and that followed a few months of Levaquin. I was on both those ABX for a month at the end and AFTER THAT I saw the morulas. I'm still not sure why that was or what it means. I believe I've been infected with Anaplasma from the beginning, though.

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