Microscope hypochondria - “hollow” erythrocytes =? Babs ??

Attempted my first blood smears with a Giemsa-type quick stain. I am looking for evidence of Babesia. Still learning how to consistently do proper thin smears, haven’t done oil immersion before, etc..

Can anyone hypothesize a theory why I am seeing occasional small clusters of red blood cells that look like actual donuts/bagels, with a see-through bright hole in center? Seems to only occur in close proximity to a white blood cell, but rather sporadic/infrequent on my smears (<1%).

iphone6 camera doesn’t do this justice. These central dots are visibly much brighter when looking with eyes, no matter how I adjust the condenser lighting, and some of the “holes” appear to have a tiny purple-stained inclusion (my camera image can’t capture that)... so, I probably need to spend some time playing with immersion oil.

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Veterinary images of large-species Babesia seem to maybe depict this, but they’re only describing the stained Babesia inclusions, so I’m not sure if this is a possible presentation of Lysis? My pics are 40x@10x plus iPhone digital zoom, from earlobe capillary smear (Volu-Sol stain).

Can’t find any reference materials that describe this, and malaria lysis imagery is much different. Maybe it’s just my imagination and this is totally normal somehow — it just seems odd to only occur in tight clusters.

yea-ok - well done for getting stuck into microscopy

its a learning curve that requires quite an investment in terms of technique and also in training yourself to recognise what you are looking at.
but i have found it to be extremely helpful in guiding my own self diagnosis and treatment

you are wise to beware of "hypochondria" and confirmation bias though - as its an area where there is a bit of a dearth of really good reference materials - and on the other side plenty of people with wild beliefs posting content on the internet about what they are convinced they can see under the microscope.

a healthy scepticism will serve you well.

i have recently travelled this road of educating myself and searching out good reference materials -for blood borne tick infections - so will try to help with these to shorten your path - see further below

ref your images - as blue lyme says - you really do have to use a 100x objective and oil immersion to get any definition at the scale of things inside red blood cells. And even then it will likely take some optimisation of setup and staining etc to get enough definition to draw any conclusions.
the reason is at the scales we are starting to get to the limits of what is possible with transmission light microscopy - simply because of the wavelength of visible light starts to be around the same size as the objects we are trying to see.

the images you posted do look a little unusual - and are worth further investigation - but as they stand its just not possible to distinguish if they are normal red blood cells, anaemic red blood cells- or some other morphological irregularity of rbc, red blood cells with vacuoles inside them from bartonella, or RBCs with some other organism inside - like babesia. clumping is more suggestive of bartonella - so i would be looking into that carefully if it were me.

if you havent read it already - my thread on microscopy for Babesia and bartonella on this forum ( see link in my signature) documents my own journey down this same path in some detail and includes many images - both reference images and my own slides - so i would advocate giving that a careful read - it will likely save you a good deal of time.

reference images for bartonella and a lot of useful information on its behaviour are in this presentation by two of the leading researchers / clinicians in the bartonella world
https://www.youtube.com/watch?v=njywmmpjiku

this preso from a Dr Lindner a conventional MD who has found Babesia in the majority of his fatigued patients including his own daughter - using giemsa capillary blood smear from the ear lobe - shows some excellent reference images for babesia in capillary blood smears - and presents his findings to his state senator.
https://www.senatorbrooks.com/2022/09/15/hhs-092022/

question - you mention Giemsa stain - which is the standard for looking for blood born pathogens - and which causes differential staining with pink, purple, blue depending on the chemical make up of organisms / artefacts - but your slides look more orange. was it a giemsa type stain you are using ?

ah - understood - the lindner stuff is a great resource - - thank you for bringing it to my attention

i am trying his method and will post my latest findings / experiments shortly

ref - stain - i am mixing my own Gimesa from powder and then filtering prior to us but have also had some issues with getting dark enough stains - on my last run i used a 12minute stain exposure - which worked pretty well - there doesn't seem to be any downsides for the longer exposures

i also use an old 1970's lab microscope

i did away with the old transformer and 12v halogen bulb and fitted a LED chip and dimmer

it is super bright - and surprisingly - even though its not advertised as a dimmable LED chip - it is dimmable with a Chinese motor speed controller / /dimmer to around 40-50% up to 100% brightness - which is fine

it works near perfectly - the only issue is there is occasionally some flicker that causes mild bands to appear on the digital camera image

if you like i can share the parts i used for the LED conversion

Polarizing film with darkfield ? I saw you mentioned that on lymenet thread, tried to look into it, and I am confused what exactly you’ve constructed or what that combination can accomplish?

I should’ve played with oil before posting this thread. I might need new oculars and a better phone camera if I start to find anything that is truly worth sharing. Purple stain comes through the oil objectives much better than dry 40x. Time to re-read some references materials. Definitely seeing sharp purple imagery under the 100x! Using cooking oil for now.. =). Indeed, thicker oil is probably best.

Bart seems impossible to visually “confirm” from what I’ve read, though we can definitely see it. Not convinced thats what I’m looking at, but it seems plausible.

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Selecting a Giemsa stain is tricky, no clue what is best or where to order from. Some Giemsa blood smear photos are much more impressive than others. Small quantity pre-mixed kit on amazon made it very easy to get started, along with a set of small rectangular polypropylene Coplin jars that hold 5 slides (or up to 10 loaded back-to-back). I’ve spent less than $200 so far... cheaper than an unreliable serology test.

yea-ok said...
Bart seems impossible to visually “confirm” from what I’ve read, though we can definitely see it. Not convinced thats what I’m looking at, but it seems plausible.

bartonella itself is very small indeed - in the region of 1um so smaller than can really be seen with light microscopy - and although it can be stained with some more specialist stains - eg silver stain - it does not stain with giemsa - so that's where that statement often found in older medical tests, that it cannot be seen with light microscopy, comes from.

however, once we understand that bartonella forms a "vacuole" around itself within red blood cells - a vacuole being a space much larger than the bacteria itself, and separated from the rest of the material inside the red blood cell by some kind of pseudo-membrane - such that the normal contents of the red blood cell are displaced and the bartonella bacteria lives inside protected, then we can see that bartonella infection can be visualised - even if the bacteria themselves cannot. This vacuole formation by bartonella is very well documented in relatively recent papers on the molecular methods the organism uses to do this - just not visually in microscopy text books yet.

these vacuoles have features that allow them to be distinguished from noise or other causes, and as a result once found, we can use them to identify bartonella rather easily.
-the vacuoles do not stain with giemsa - but the RBC's material does - allowing vacuoles to be visualised as clear spots inside the darkly stained RBC's.
-very few other organisms infect and live inside red blood cells
-most that do, eg malaria, Babesia etc - DO NOT form vacuoles - but live directly in the contents of the RBC
-the vacuoles in bartonella typically form in distinctive ring formations around the inner edge of the RBC membrane
-this pattern is distinctive - nothing else in the medical text is known to form that pattern.
-other morphological changes in RBC's occur in concert with vacuoles - that help confirm the diagnosis
>lack of central pallor in infected RBC's
>a halo or ring of clear gel like substance around infected RBC's
>clumping of infected RBC's - not homogenously mixed with rest of blood

ref your images - its just not possible to say yet with the current resolution
bartonella CAN look like that - ie a single v large vacuole in the centre of the RBC's - but without better resolution its just not possible to distinguish this from mildly anaemic RBC's, a certain number of which often appear in any normal sample. i would say its suspicious though and worth looking into further.

in terms of improving image resolution - things that should help
1, darker staining - longer durations of stain time - improves contrast
2, better illumination - together with above - again to improve contrast - and allow optimum condenser settings
3, proper immersion oil - BTW - its not the viscosity that's critical for image quality but the refractive index - the concept is to make it the same refractive index as glass - so there are not light bending effects at each boundary - slide glass to air - air to objective lens
4, optics - there looks to be some dirt in the optics ( see blurred dark lines and other artefacts in the image ) - you can identify these by rotating or flexing the various optical components and seeing which ones move relative to the image.
cleaning the optics can be tricky - hard to do perfectly

iphone 6 has a 8mega pixel camera ( the rear facing one ) - which is good enough for this purpose
there are slight issues with focussing ability - as it was not designed for use as we are using them - but i would say its not the limiting factor in your images at present

the above are the things i would pursue for diagnosis of bart and babesia

darkfield microscopy is of limited use for these - giemsa is much better in my view

ref darkfield for lyme spirochetes - after a careful study of this approach i belive it is overrated
mainly because the prevalence of spirochetes in blood in chronic lyme is very low indeed (even PCR that can reliably detect 1 spirochete in 0.5ml of blood - detects lyme only 50% of the time in patients known to be infected)

if you have done some smears you have some appreciation of how many slides you would have to view - and how many fields of view per slide it would take to view 0.5ml of blood and find a single spirochete
and there are lots of other string like thing of around the same size - but of human cellular origin - that serve to muddy the waters

hope its of some help

Post Edited (Garzie) : 11/30/2022 6:29:21 AM (GMT-8)

Interesting about Bart vacuoles. I’ll have to read up. I’ve indeed found something that looks a lot like babesia, with some proper looking Maltese crosses and everything. Occasional night sweats last time I took disulfiram, otherwise I wouldn’t believe what I am seeing.

I’ve done about a dozen smears, found what I was looking for without too much effort. Low quantitative numbers, but definitely seeing things that only have an infectious explanation. Purple rings don’t show up on my photos as well as i’d like, but nearly all of the “purple specs” in these particular photos are indeed definite rings when observed by eye using 100x@10x. These were all from finger prick, but nothing I interpret as biofilm colonies like I see in my earlobe smears.

(links vanished... moderated, or broken edit. See Garzie commentary.)

I need new oculars for sure. My camera auto-focuses on eyepiece surface scratches when I adjust for better kohler illumination/contrast. Pitfall of buying used...

LEDs I’m considering are on eBay, shipped direct from China for $2-$3 each, listed as “7w dimmable g4 12v” — I initially found these things on lightinthebox.com listed as 5w, but buying a $20 package of them plus shipping deters me, not knowing how well it will work. Phillips patented some clever circuitry for this many years ago, and I have used Phillips full-size led bulbs on old dimmers with no trouble, so it is definitely possible— Phillips retails a 2watt dimmable g4 bulb for ~$10. These things are all 4-5cm, so relocating the g4 socket base by an inch is required. Several slightly different versions floating around listed at 5w-7w, they all appear to have a similar tiny integrated circuit chip with capacitor, and a single square/rectangular LED. My scope is from 2007, haven’t dismantled yet to confirm it is a magnetic transformer or how it’s wired up.

For spirochetes, thatdudefromks uploaded at least a couple of undeniable kete videos. Someone else made a YouTube with supposed live ketes visible under Brightfield (phase contrast?), using a dilute Acridine orange stain mixed with vinegar+sugar+salt — https://m.youtube.com/watch?v=czcqglrm3ji (Upon rewatching, might not be spirochetes at all, but the video description is interesting if the raw AO method is even remotely valid). Darkfield could be my next goal, seriously might try to 3d print a dovetail adapter to retrofit an amscope condenser.

Post Edited (yea-ok) : 12/1/2022 11:28:13 PM (GMT-8)

yea-ok said...

I’ve done about a dozen smears, found what I was looking for without too much effort. Low quantitative numbers, but definitely seeing things that only have an infectious explanation. Purple rings don’t show up on my photos as well as i’d like, but nearly all of the “purple specs” in these particular photos are indeed definite rings when observed by eye using 100x@10x. These were all from finger prick, but nothing I interpret as biofilm colonies like I see in my earlobe smears.

OK - well done - you are making progress with the image quality.

and i think you are starting to see how tricky it is to get really good images and then, even when images are optimised - interpretation is a real challenge.
You are wise to beware of confirmation bias as mentioned in your first post.
many people assume everything that looks in any way like what they think they are looking for is infection, then others deny microscopy is any use for diagnosis of TBD at all.
As is often the case - the truth is somewhere in between.

Microscopy can be very useful in diagnosing these diseases and in fact has some distinct advantages - like it does not require species specific antigen's like serology - so its not species specific which is a big advantage for something like babesia where there are thought to be 300 species in the wild - but commercial serologic tests for only 2 or 3 of these. On the other hand there are many other things that appear as rigs and dark and light spots on microscopy slides, the list of things that can do this is actually rather long. the key is in proper interpretation - and that is a hard won skill.

ref the images - i have taken a good look and will provide some feedback based on my many hrs of doing this and not an expert as such - but just being a little ahead of you in the journey in terms of the hrs spent educating myself.

image 1
that is a suspicious object - not quite typical of babesia though - a bit too irregular - could be babesia - but could also be a number of other non infectious things - eg debris, platelet, optical artefact etc - my best estimate 60/40 tht its something else - dont worry, if its Babesia there will other better examples

image 2
those groups of dots are platelets - they have a clear cytoplasm around them which is obscuring the outline of the RBC's underneath them - making the RBC's look like they have gaps in their outline / are ruptured. the are aggregating a little more than normal - so something is likely triggering this - which could be a pathogen - so that would give me a reason to keep looking.
reference images of platelets are available with either a google search on Giemsa platelets or "platelet clumping" or Schaller's lab guide has some tips on how to differentiate them from microbes - often in individual cases its simply not possible.
https://tinyurl.com/2hh4rhe6

image 3
again - these are platelets - note the clumping, clear cytoplasm, irregular shape of the core


image 4
multiple dots /objects in the middle circle on the image - again platelets
upper and lower circled objects -both indeterminate
top one - could be Babesia, could be debris, could be an artefact or residue of the stain, could be optical central object
bottom one has some clear cytoplasm around it so likely platelet ( they often stick to RBC's like this) - but could be babesia

overall it looks like there is a great deal of dirt and debris in the scopes optics - this on its own is scattering light and dark spots across the entire image.
the scope needs cleaning
mine was actually much the same when i got it - and i had to dismantle and clean each individual optical element carefully with alcohol and re-assemble. its doable DIY - but fiddly
one of the issue with older scopes believe it of not is that fungus actually grows on the optics - and needs to be removed.
the scratches on the lenses are not idea but are actually less of a problem - as they appear in lines and can be distinguished from true objects in the slide a bit more easily.

overall i would say so far only a few somewhat suspicious objects - no images that are indicative of bart at all (Note- this is not unusual as it typically occurs in infrequent clumps and many normal looking slides are the rule eg 10 normal slides and 1 slide with only one clear example)

better lighting will help - and i will post the inexpensive parts i am using below - but the dirty optics are perhaps the biggest roadblock right now.

post some images of the biofilm colonies - that would be interesting to compare

keep at it - its a learning curve!!