Microscopy and/or culturing advice

[Guest guest]

My wife and I developed the same GI condition at about the same

time. We're 5 to 6 weeks into it. It hits hard for maybe 10 days

and then tricks us into thinking we're clearing it before hitting us

with another round (seems like antigenic variation common to

protozoans). Most symptoms, as well as colonic ulcers, seem

consistent with Entamoeba, but not entirely. Perhaps its something

else. My wife was inconsistent with Flagyl due to side effects. It

went roughly 1.5 g day one, 750 mg/day for 3 days and 1.0 g day 5.

The diarrhea actually got worse. Possibly a side effect, or perhaps

the treatment was sub-par (definitely so for amoeba) and the

diarrhea was going to get worse anyway. The only other thing I can

think of is that the Flagyl expanded the niche for a pathogenic

fungus (if that's what it is). Avelox monotherapy seemed to work

but that turned out to be a mirage. The symptoms flared up again

while she about 10 days into Avelox.

The docs are trying their best to pretend it's not even infectious.

They also pretend that one ova and parasites test is sufficient when

the literature clearly states that 3 to 6 consecutive samples may

need to be tested to rule out protozoa. So although I've still got

some tests in the pipeline, I have little confidence in the

outcome. I expect we will be on our own with this soon.

I examined my diarrhea under phase contrast (40x objective + 10x

eyepiece) and there are very high numbers of oval-shaped

structures. I don't have a way of measuring them directly, but

based on their size relative to HeLa cells they seem consistent with

protozoal cysts. I streaked the sample on a slide, air-dried it,

fixed in methanol and stained with Giemsa and I didn't seem to

retain many of these oval structures, but some of what is there do

seem to have 2 or more " nuclei " (if that's what they are).

In the past, I've seen at least one species of yeast under the scope

before and they were quite spherical occasionally, I think, with a

bleb off one end. They were also often in chains of two or more

cells and, if memory serves notably smaller than the oval structures

I'm seeing now. So these oval things don't look like the fungus I'm

familiar with, but I know very little about fungal diversity.

Does anyone know if there are fungi that can look like protozoal

cysts? I'll try to make some Lugal's iodine but rumors are that it

sticks to most everything. Will the iodine stain help me

distinguish cysts from fungus?

I don't have acridine orange but I do have ethidium bromide as well

as access to fluorescent microscopy. I could stain intracellular

DNA (e.g., nuclei) then, but only if I first permeabilized the

cells. Anyone have advice on how to use NP40, Triton X 100 or

another detergent to permeabilize cells without obliterating them?

Would freeze-thawing them work? I assume that I would see one

nucleus in fungus and 2 to 4 in protozoa, and could discriminate on

that basis.

Does anyone know how to culture Entamoeba or fungi? I have access

to broth for the culture of E coli and saccharomyces. Are there

simple modifications that I could make to the recipes to grow either

amoeba or fungi? Would the amoeba eat E coli in a normal LB broth?

Is there a way to get amoeba to excyst, amplify and then re-encyst,

so that I can compare by microscopy the amplified cysts with the

ones I'm shedding? I also have a fairly comprehensive collection of

abx, antifungals and antiprotozoals. So I could spike cultures with

various agents to look for susceptibilities and try to discriminate

between fungus and protozoa that way.

I looked for microscopy groups, but didn't find one that

seemed appropriate, that is active and not exclusive to blood-borne

spirochetes. Is there a forum where people might have experience

with this sort of thing?

Thanks for any advice.

Matt

[Guest guest]

> Is there a way to get amoeba to excyst, amplify and then re-encyst,

> so that I can compare by microscopy the amplified cysts with the

> ones I'm shedding?

" Attachment to both host mucin and host cell surfaces is accomplished

by the parasite's galactose-and-N-acetylgalactosamine-binding lectin.

This molecule is necessary to both complement resistance and quorum

sensing, and its homolog in Entameba invadens mediates encystment in

culture (the encystment of E. histolytica itself has not been

successfully reproduced in vitro) [2].

" [2] Coppi A, Eichinger D. Regulation of Entamoeba invadens

encystation and gene expression with galactose and N-

acetylglucosamine.

Mol Biochem Parasitol. 1999 Jul 30;102(1):67-77. "

Thats from my research mentorship paper, written on the quick and for

my mentor's eyes only, so check the ref. I referenced some papers

that I read only in abstract (a felony).

Are you sure you need to permeabilize even for high concentrations of

acridine orange? I used, if I recall, 1% SDS, 1% NaOH for non-

obliterative permeabilisation (for minipreps, in order to obtain

selective permeability to plasmids but not chromosomes). That seemed

like alot of SDS to me but what do I know. I also have the idea

somehow that aldehydes can do this job... or at least they are used

in tissue fixation/prep for sectioning. The Brorsons have applied AO,

so they might have a ref to a protocol.

[Guest guest]

Thanks a bunch. It would have taken me way too much time to track

down the re-encystment issue. The oval structures are so abundant I

can not imagine they are incidental. But my incomplete

understanding is that many people are carriers of entamoeba, so I

wouldn't want to culture amoebae and falsely assume that the

resulting trophozoites grew out from these numerous oval

structures. That's why I wanted to re-encyst them--for comparison.

Do you know if you can plaque out the amoeba on a lawn of E coli?

Amoeba eat bacteria right? In that case I would be able to " titer "

the amoeba. Incidental amoeba should yield very low titers. But if

these oval structures are amoebic, then the titer should be quite

high. Is that unrealistic? Would this have to be done under

anaerobic conditions?

I don't have AO. My understanding is that AO crosses membranes,

EtBr doesn't. So you have to permeabilize the cell membrane if

you're using EtBr. I forgot about SDS/NaOH but the concentration

and timing you use is for E coli. I don't know how tough E coli is

compared to a protozoal cyst. I suppose I could subject aliquots to

various levels of abuse and find the aliquot that is permeabilized

but not obliterated. I've looked at Broronson's AO work, as well as

a paper on the Q fever spirochete and AO. I'd love to have that

stuff. Awhile back I froze-thawed my own cells and exposed them to

EtBr. Occasional cells had multiple, very bright occupants.

Alarmed me at first, because I figured they were invasive bacteria.

But I estimated their numbers and I think they were just

(metaphase?) chromosomes. Took me awhile before I could accept that

idea. If true, it gives you an idea of the power of AO (or the less

desirable EtBr) to light up interesting cellular inhabitants.

The health center just called with the results. Negative on the Ova

& parasite test. (No " we'll need to repeat this test to be sure " ;

no " we'll need you to come in again to get to the bottom of this " ).

I tried to corner her on what was tested. She said giardia and

cryptosporidium and really had a hard time pronouncing

cryptosporidium so I think she was reading it off the report. I

asked twice if entamoeba was included. I think she would have said

yes if it was. I don't think the former Soviet Union guarded their

secrets more closely than our doctors.

>

> > Is there a way to get amoeba to excyst, amplify and then re-

encyst,

> > so that I can compare by microscopy the amplified cysts with the

> > ones I'm shedding?

>

> " Attachment to both host mucin and host cell surfaces is

accomplished

> by the parasite's galactose-and-N-acetylgalactosamine-binding

lectin.

> This molecule is necessary to both complement resistance and

quorum

> sensing, and its homolog in Entameba invadens mediates encystment

in

> culture (the encystment of E. histolytica itself has not been

> successfully reproduced in vitro) [2].

>

> " [2] Coppi A, Eichinger D. Regulation of Entamoeba invadens

> encystation and gene expression with galactose and N-

> acetylglucosamine.

> Mol Biochem Parasitol. 1999 Jul 30;102(1):67-77. "

>

> Thats from my research mentorship paper, written on the quick and

for

> my mentor's eyes only, so check the ref. I referenced some papers

> that I read only in abstract (a felony).

>

> Are you sure you need to permeabilize even for high concentrations

of

> acridine orange? I used, if I recall, 1% SDS, 1% NaOH for non-

> obliterative permeabilisation (for minipreps, in order to obtain

> selective permeability to plasmids but not chromosomes). That

seemed

> like alot of SDS to me but what do I know. I also have the idea

> somehow that aldehydes can do this job... or at least they are

used

> in tissue fixation/prep for sectioning. The Brorsons have applied

AO,

> so they might have a ref to a protocol.

>

[Guest guest]

I meant tick-borne relapsing fever, not Q fever.

PMID: 10325370 shows the AO-stained spirochetes and is freely

available, if anyone's interested.

I've looked at Broronson's AO work, as well as

> a paper on the Q fever spirochete and AO.

[Guest guest]

You once told me you knew alot about HIV but relatively little about

AIDS. I know alot about the E his lectin light chain and relatively

little about the intermediate and heavy chains. Basically I didnt get

beyond cloning the 5 paralogs, trial-expressing them, and testing out

the fusion tags and some mAbs made in our lab around the time I was

born. It never came to me trying to understand ameba in order to

guide interpretation of data.

I think I've seen them cultured in flasks but other than that I dont

know.

I forgot we were talking cysts. I dont know the architecture of the

cyst wall.

This is worth reading if you havent. It does imply trophos are not

exactly abundant. But it doesnt seem to mention abundant cysts.

>

> Thanks a bunch. It would have taken me way too much time to track

> down the re-encystment issue. The oval structures are so abundant

I

> can not imagine they are incidental. But my incomplete

> understanding is that many people are carriers of entamoeba, so I

> wouldn't want to culture amoebae and falsely assume that the

> resulting trophozoites grew out from these numerous oval

> structures. That's why I wanted to re-encyst them--for comparison.

>

> Do you know if you can plaque out the amoeba on a lawn of E coli?

> Amoeba eat bacteria right? In that case I would be able to " titer "

> the amoeba. Incidental amoeba should yield very low titers. But

if

> these oval structures are amoebic, then the titer should be quite

> high. Is that unrealistic? Would this have to be done under

> anaerobic conditions?

>

> I don't have AO. My understanding is that AO crosses membranes,

> EtBr doesn't. So you have to permeabilize the cell membrane if

> you're using EtBr. I forgot about SDS/NaOH but the concentration

> and timing you use is for E coli. I don't know how tough E coli is

> compared to a protozoal cyst. I suppose I could subject aliquots

to

> various levels of abuse and find the aliquot that is permeabilized

> but not obliterated. I've looked at Broronson's AO work, as well

as

> a paper on the Q fever spirochete and AO. I'd love to have that

> stuff. Awhile back I froze-thawed my own cells and exposed them to

> EtBr. Occasional cells had multiple, very bright occupants.

> Alarmed me at first, because I figured they were invasive

bacteria.

> But I estimated their numbers and I think they were just

> (metaphase?) chromosomes. Took me awhile before I could accept

that

> idea. If true, it gives you an idea of the power of AO (or the

less

> desirable EtBr) to light up interesting cellular inhabitants.

>

> The health center just called with the results. Negative on the

Ova

> & parasite test. (No " we'll need to repeat this test to be sure " ;

> no " we'll need you to come in again to get to the bottom of

this " ).

> I tried to corner her on what was tested. She said giardia and

> cryptosporidium and really had a hard time pronouncing

> cryptosporidium so I think she was reading it off the report. I

> asked twice if entamoeba was included. I think she would have said

> yes if it was. I don't think the former Soviet Union guarded their

> secrets more closely than our doctors.

[Guest guest]

> This is worth reading if you havent. It does imply trophos are not

> exactly abundant. But it doesnt seem to mention abundant cysts.

This

=================================

http://www.pubmedcentral.gov/articlerender.fcgi?

tool=pubmed & pubmedid=14557296

Microscopy

Diagnosis of E. histolytica has historically relied on microscopic

examination of protozoan morphology. Current microscopy- and

histology-based identification frameworks, however, are unable to

differentiate among protozoa with similar morphological features.

Drawings of intestinal amebas (E. histolytica, E. coli, E. hartmanni,

and I. bütschlii) showing their morphologic features are summarized

in Fig. 1.

A separate problem is that the sensitivity and specificity of

conventional microscopy on a single stool specimen for different

species of Entamoeba have been shown in many studies to be less than

optimal (64, 129). A " poor man's " way to distinguish E. dispar from

E. histolytica microscopically is erythrophagocytosis.

Ingested RBCs in the cytoplasm may be visible; this finding is still

considered diagnostic for E. histolytica in patients with dysentery.

It may be used to distinguish between E. histolytica and E. dispar.

Mostly, E. histolytica will be diagnosed on the basis of protozoon

morphology without the presence of RBCs (64). In fact, classical

microscopy does not allow of the invasive protozoon (E. histolytica)

to be distinguished from the noninvasive one (E. dispar) unless

erythrophagocytosis (the presence of ingested RBCs in trophozites) is

seen during microscopic examination. This classical feature has long

been considered the definitive diagnostic criterion for E.

histolytica.

Also, it must be kept in mind that RBCs may be ingested but do not

frequently appear in chronic amebic infections (129). In an in vitro

study, E. histolytica was found to have a significantly higher

phagocytic rate of ingested RBCs than do the nonpathogenic Entamoeba

species (E. invadens and E. moshkovskii) (222). González-Ruiz et al.

(73) reported that the presence of E. histolytica organisms

containing ingested RBCs is a diagnostic indication of active

invasive amebiasis. However in some cases E. dispar is also observed

to contain RBCs (85).

Trophozoites are more frequently observed in fresh stool specimens

that contain mucus, pus, and trace amounts of blood. In wet mounts,

the trophozoite nuclei cannot easily be seen (164). Charcot-Leyden

crystals (products of degenerated eosinophils) and clumped RBCs can

be seen in a wet mount preparation (64, 105, 129). Definitive

diagnosis of intestinal amebiasis requires high levels of skill and

experience (86, 229); inadequate training and diagnostic testing may

lead to misdiagnosis (64; L. Doganci, M. Tanyuksel, and H. Gun,

Letter, Lancet 350: 670, 1997). Motility of E. histolytica in fresh

preparations usually occurs in a linear (not random) fashion, with

the clear hyaline ectoplasm flowing to form blunt-ended pseudopodia,

which guide the endoplasm containing the nucleus (164). If a fresh

stool specimen cannot be examined immediately, it should be preserved

with a fixative such as polyvinyl alcohol or kept cool (4°C).

Occasionally motile trophozoites are seen even after 4 h at this

temperature (170, 229), although the trophozoites generally

disintegrate rapidly in unfixed stool specimens (164).

Stool specimens can be examined either unstained or stained with

Lugol's or D'Antoni's iodine. Iodine stains make the nucleus

perfectly visible. The appearance of chromatoid bodies is the same as

in wet mount preparations (164). Although several other stains,

including Giemsa, methylene blue, Chorazole black E, 's, and

iodine-trichrome, may be used successfully, Wheatley's trichrome

staining or one of the modified iron hematoxylin stains for permanent

smears has been suggested for routine use in the diagnosis of E.

histolytica/E. dispar (63, 64, 138a, 164, 171, 229). Shetty and

Prabhu found that D'Antoni's iodine was much better than saline or

buffered methylene blue for detection of E. histolytica cysts while

saline and buffered methylene blue were equally good for detection of

E. histolytica trophozoites (206). There are several factors that

adversely affect the results of microscopy. These include lack of

well-trained microscopists; delayed delivery to the laboratory

(motility can cease and trophozoites can lyse within 20 to 30 min);

difficulty in differentiation between nonmotile trophozoites and

polymorphonuclear leukocytes, macrophages, and tissue cells;

inadequate collection conditions (a clean, dry, wide-mouth plastic

container not contaminated with urine and water is needed);

interfering substances such as antibiotics (tetracyclines or

sulfonamides), laxatives, antacids, cathartics (magnesium sulfate),

antidiarrheal preparations, (kaolin or bismuth), or enemas (soap);

inadequate number of specimens collected (at least three specimens

are needed); lack of preservation of stool specimens with fixatives

(polyvinyl alcohol, Schaudinn's fluid, merthiolate-iodine-formalin,

sodium acetate-acetic acid-formalin, or 5 or 10% formalin is needed);

and presence of other amebae (E. dispar and E. moshkovskii are

identical and E. coli and E. hartmanni are similar in appearance to

E. histolytica) (64, 114, 229).

[Guest guest]

Maybe you should just fix a slide- and pack it off to Oz to have

Tony's lab look at it.

Barb

>

> My wife and I developed the same GI condition at about the same

> time. We're 5 to 6 weeks into it. It hits hard for maybe 10 days

> and then tricks us into thinking we're clearing it before hitting

us

> with another round (seems like antigenic variation common to

> protozoans). Most symptoms, as well as colonic ulcers, seem

> consistent with Entamoeba, but not entirely. Perhaps its something

> else. My wife was inconsistent with Flagyl due to side effects.

It

> went roughly 1.5 g day one, 750 mg/day for 3 days and 1.0 g day 5.

> The diarrhea actually got worse. Possibly a side effect, or

perhaps

> the treatment was sub-par (definitely so for amoeba) and the

> diarrhea was going to get worse anyway. The only other thing I can

> think of is that the Flagyl expanded the niche for a pathogenic

> fungus (if that's what it is). Avelox monotherapy seemed to work

> but that turned out to be a mirage. The symptoms flared up again

> while she about 10 days into Avelox.

>

> The docs are trying their best to pretend it's not even

infectious.

> They also pretend that one ova and parasites test is sufficient

when

> the literature clearly states that 3 to 6 consecutive samples may

> need to be tested to rule out protozoa. So although I've still got

> some tests in the pipeline, I have little confidence in the

> outcome. I expect we will be on our own with this soon.

>

> I examined my diarrhea under phase contrast (40x objective + 10x

> eyepiece) and there are very high numbers of oval-shaped

> structures. I don't have a way of measuring them directly, but

> based on their size relative to HeLa cells they seem consistent

with

> protozoal cysts. I streaked the sample on a slide, air-dried it,

> fixed in methanol and stained with Giemsa and I didn't seem to

> retain many of these oval structures, but some of what is there do

> seem to have 2 or more " nuclei " (if that's what they are).

>

> In the past, I've seen at least one species of yeast under the

scope

> before and they were quite spherical occasionally, I think, with a

> bleb off one end. They were also often in chains of two or more

> cells and, if memory serves notably smaller than the oval

structures

> I'm seeing now. So these oval things don't look like the fungus

I'm

> familiar with, but I know very little about fungal diversity.

>

> Does anyone know if there are fungi that can look like protozoal

> cysts? I'll try to make some Lugal's iodine but rumors are that it

> sticks to most everything. Will the iodine stain help me

> distinguish cysts from fungus?

>

> I don't have acridine orange but I do have ethidium bromide as well

> as access to fluorescent microscopy. I could stain intracellular

> DNA (e.g., nuclei) then, but only if I first permeabilized the

> cells. Anyone have advice on how to use NP40, Triton X 100 or

> another detergent to permeabilize cells without obliterating them?

> Would freeze-thawing them work? I assume that I would see one

> nucleus in fungus and 2 to 4 in protozoa, and could discriminate on

> that basis.

>

> Does anyone know how to culture Entamoeba or fungi? I have access

> to broth for the culture of E coli and saccharomyces. Are there

> simple modifications that I could make to the recipes to grow

either

> amoeba or fungi? Would the amoeba eat E coli in a normal LB

broth?

> Is there a way to get amoeba to excyst, amplify and then re-encyst,

> so that I can compare by microscopy the amplified cysts with the

> ones I'm shedding? I also have a fairly comprehensive collection

of

> abx, antifungals and antiprotozoals. So I could spike cultures

with

> various agents to look for susceptibilities and try to discriminate

> between fungus and protozoa that way.

>

> I looked for microscopy groups, but didn't find one that

> seemed appropriate, that is active and not exclusive to blood-borne

> spirochetes. Is there a forum where people might have experience

> with this sort of thing?

>

> Thanks for any advice.

> Matt

>

[Guest guest]

Barb'

I have been privaledged to look at a few parasitic stool samples

while visiting the lab, but I didn't really pay attention to the way

the sample was fixed, if at all. It may have just been stool only, no

staining. Unfortunately my friend has left her lab and now does

pathology for a surgeon she works alongside.Alkso matts description

of feeling cured on 10 days of avelox only to rebound smells very

fishy to me..I love the guys passion to get to the bottom of this

with his microscope, But would strongly advise he just gets a picture

of what is generally growing. In ,other words does he have any

bacterial organism that dominates his sample (egs. staph,

pseudonomads)... these organisms are the likeley candidates to give

you a good ten days on avelox and then BAM worse- often than before.

I have about 15 stains and wouldn't know where to start with a stool

sample... I do like getting a picture of the microbial growths out of

my samples though.mine would generally be soft stools dominated by

staph epi....this is generally out of whack as you should be able to

grow heaps of ecoli. I haven't been that lucky with my own ecoli.

tony

> >

> > My wife and I developed the same GI condition at about the same

> > time. We're 5 to 6 weeks into it. It hits hard for maybe 10

days

> > and then tricks us into thinking we're clearing it before hitting

> us

> > with another round (seems like antigenic variation common to

> > protozoans). Most symptoms, as well as colonic ulcers, seem

> > consistent with Entamoeba, but not entirely. Perhaps its

something

> > else. My wife was inconsistent with Flagyl due to side effects.

> It

> > went roughly 1.5 g day one, 750 mg/day for 3 days and 1.0 g day

5.

> > The diarrhea actually got worse. Possibly a side effect, or

> perhaps

> > the treatment was sub-par (definitely so for amoeba) and the

> > diarrhea was going to get worse anyway. The only other thing I

can

> > think of is that the Flagyl expanded the niche for a pathogenic

> > fungus (if that's what it is). Avelox monotherapy seemed to work

> > but that turned out to be a mirage. The symptoms flared up again

> > while she about 10 days into Avelox.

> >

> > The docs are trying their best to pretend it's not even

> infectious.

> > They also pretend that one ova and parasites test is sufficient

> when

> > the literature clearly states that 3 to 6 consecutive samples may

> > need to be tested to rule out protozoa. So although I've still

got

> > some tests in the pipeline, I have little confidence in the

> > outcome. I expect we will be on our own with this soon.

> >

> > I examined my diarrhea under phase contrast (40x objective + 10x

> > eyepiece) and there are very high numbers of oval-shaped

> > structures. I don't have a way of measuring them directly, but

> > based on their size relative to HeLa cells they seem consistent

> with

> > protozoal cysts. I streaked the sample on a slide, air-dried it,

> > fixed in methanol and stained with Giemsa and I didn't seem to

> > retain many of these oval structures, but some of what is there

do

> > seem to have 2 or more " nuclei " (if that's what they are).

> >

> > In the past, I've seen at least one species of yeast under the

> scope

> > before and they were quite spherical occasionally, I think, with

a

> > bleb off one end. They were also often in chains of two or more

> > cells and, if memory serves notably smaller than the oval

> structures

> > I'm seeing now. So these oval things don't look like the fungus

> I'm

> > familiar with, but I know very little about fungal diversity.

> >

> > Does anyone know if there are fungi that can look like protozoal

> > cysts? I'll try to make some Lugal's iodine but rumors are that

it

> > sticks to most everything. Will the iodine stain help me

> > distinguish cysts from fungus?

> >

> > I don't have acridine orange but I do have ethidium bromide as

well

> > as access to fluorescent microscopy. I could stain intracellular

> > DNA (e.g., nuclei) then, but only if I first permeabilized the

> > cells. Anyone have advice on how to use NP40, Triton X 100 or

> > another detergent to permeabilize cells without obliterating

them?

> > Would freeze-thawing them work? I assume that I would see one

> > nucleus in fungus and 2 to 4 in protozoa, and could discriminate

on

> > that basis.

> >

> > Does anyone know how to culture Entamoeba or fungi? I have

access

> > to broth for the culture of E coli and saccharomyces. Are there

> > simple modifications that I could make to the recipes to grow

> either

> > amoeba or fungi? Would the amoeba eat E coli in a normal LB

> broth?

> > Is there a way to get amoeba to excyst, amplify and then re-

encyst,

> > so that I can compare by microscopy the amplified cysts with the

> > ones I'm shedding? I also have a fairly comprehensive collection

> of

> > abx, antifungals and antiprotozoals. So I could spike cultures

> with

> > various agents to look for susceptibilities and try to

discriminate

> > between fungus and protozoa that way.

> >

> > I looked for microscopy groups, but didn't find one that

> > seemed appropriate, that is active and not exclusive to blood-

borne

> > spirochetes. Is there a forum where people might have experience

> > with this sort of thing?

> >

> > Thanks for any advice.

> > Matt

> >

>