Dear Negin,

apologize for my further request to tell about the source / bibliographic reference/ a recipe,  or

"why" you "find  that we can fix cells and tissues in 0.1M sodium cacodylate containing 4% formalin and 2.5% glutaraldehyde."

are you sure you are using 4% "formalin" ? which equals only 1.6% formaldehyde (the latter of which = 4% formaldehyde I guess you meant to really use).

And on the other hand, you naturally CAN fix E.sp. microcyst with the same fixative you use usually for cells and tissues...but if it is the best fixative for it you will see when you examine your specs as ultrathin sections in the TEM (guessing you will prepare/process  your specs for  and observe them in a transmission electron microscope (TEM) and not in a Scanning EM). The only thing you would need to have in mind is that you will dilute your fix-working solution when mixing it with the M199 or RPMI medium....so you would have to calculate)...

Please respond by answering my honest questions so also others can help you straightway too.

Dear Negin,

apologize for my further request to tell about the source / bibliographic reference/ a recipe,  or

"why" you "find  that we can fix cells and tissues in 0.1M sodium cacodylate containing 4% formalin and 2.5% glutaraldehyde."

are you sure you are using 4% "formalin" ? which equals only 1.6% formaldehyde (the latter of which = 4% formaldehyde I guess you meant to really use).

And on the other hand, you naturally CAN fix E.sp. microcyst with the same fixative you use usually for cells and tissues...but if it is the best fixative for it you will see when you examine your specs as ultrathin sections in the TEM (guessing you will prepare/process  your specs for  and observe them in a transmission electron microscope (TEM) and not in a Scanning EM). The only thing you would need to have in mind is that you will dilute your fix-working solution when mixing it with the M199 or RPMI medium....so you would have to calculate)...

Please respond by answering my honest questions so also others can help you straightway too.

Negin,

     As Wolfgang stated ,make sure you are adding a 2x fixative concentration to equal vols. of cells in suspension.  I generally will slow rock fix this for 10 min, then do a light centrifuge to a pellet and continue to fix and process the pellet.  We also found some (i.e. E.coli) samples do better with a lower osmolarity (20-50 mM cacodylate), so take note if you notice any membrane shrinkage.  Good Luck.

Michael Delannoy

Negin, there is no problem regarding a late response, anyway.

Since I have helped during the last 2 years also some other Iranian colleagues I know that it isn't really simple to access ResearchGate anytime.

So I only can tell you that I've found some specific literature for you (also "separate" fixatives for TEM and/ or SEM).

So, if it is easier for you to send me an e-mail address to be able to communicate easier than via ResearchGate, please let me know ( [email protected]  until 30th of November 2015). Best wishes and regards, Wolfgang

Thanks for your helpful guidance!

Dear Negin,

you are welcome. You should get my reply if e-network lines are open.

Best regards, Wolfgang

Dear Dr.Torabi

I referred you to materials and methods section of this article(specially section 2.4.):

Effect of Different Terpene-Containing Essential Oils on the Proliferation of Echinococcus granulosus Larval Cells

Interdisciplinary Perspectives on Infectious Diseases

Volume 2014 (2014), Article ID 746931, 7 pages

I have some question. Why you should use this fixative for microcycst of Echinococcus in liquid culture medium too? Why you could not harvest cells and remove medium?

2. Materials and Methods

2.1. Plant Material

The essential oils were kindly provided by Dr. Liesel Gende and Dr. Martín Eguaras (Laboratorio de Artrópodos, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata). The R. officinalis essential oil was extracted as reported by [5] and the M. piperita and M. pulegium as reported by [7].

2.2. Cell Culture and Drug Treatment

E. granulosus cell culture was obtained using previously reported methods [17]. In brief cells were cultured at 37°C in medium 199 (Gibco BRL) supplemented with 10% FBS, 10% hydatid fluid, reducing agents (5 × 10−5 M 2-mercaptoethanol and 100 μM L-cysteine), 2 mM L-glutamine (Bio-Rad, USA), 4 mg mL−1 glucose (Sigma, USA), 1 mM sodium pyruvate (Sigma, USA), and antibiotics (penicillin, streptomycin, and gentamicin 100 μg mL−1). E. granulosus cells were cultivated for at least 4 weeks. Some cultures were subcultured once a week and others were maintained without subculture to promote the formation of cellular aggregates growing in suspension. Thymol (Sigma) was dissolved in dimethyl sulphoxide (DMSO) at a drug concentration of 10 mg/mL. Essential oils of M. piperita, M. pulegium, and R. officinalis were dissolved in 10 mL of distilled water and were emulsified with propylene glycol (PG) to 5% v/v. Albendazole (ABZ) (Sigma-Aldrich) was used as reference drug and the solution was prepared by dissolution of 10 mg of pure standard drug in 1 mL of DMSO. Then, thymol, ABZ, or each essential oil was added to the medium resulting in final concentrations of 10, 5, and 1 μg/mL. Cells incubated in culture medium containing 10 μL DMSO or PG served as controls. Each experiment was repeated three times.

2.3. Growth Inhibitory Assay on Isolated Cells

E. granulosus cells were seeded into 24-well microplates to achieve an approximate density of 5 × 105 cells well−1 in 1 mL medium. For this experiment cell cultures after 24 h of subculture were used. Thymol or the different essential oils were added in serial concentrations and cultures were incubated for 7 days. At days 0, 2, 5, and 7 viability was assessed by trypan blue dye (Sigma) exclusion using a hemocytometer. During the experiments, cultures were followed microscopically to determine the appearance of morphological alterations.

2.4. Drug Effect on Isolated Cells and Cell Aggregates

For this experiment cell cultures after 24 h of subculture or four-month-old cultures containing big amount of cell aggregates were used. At days 2, 5, and 7 of treatment, samples were harvested (aggregates were recovered in 1.5 mL plastic tubes and individual cells were treated for the whole procedure attached in the slides), fixed with 2.5% (v/v) glutaraldehyde in 0.1% (v/v) sodium cacodylate buffer for 48 h at 4°C, and then washed several times in cacodylate buffer. The specimens were later dehydrated by sequential incubations in increasing concentrations of ethanol (50% 10 min, 70% 10 min, 80% 10 min, 90% 10 min, 95% 10 min, 100% 10 min twice) and finally immersed in hexamethyldisilazane for 18 h. They were sputter-coated with gold and inspected on a JEOL JSM-6460 LV scanning electron microscope (Japan) at 15 kV.

Thanks Dr.Nasiri.