use Vero, 3T3 or Hella cells! for Toxoplasma culture. For me Hella cells are better tham Vero cells.

Culture aminal cells in DME or RPMI with 5 to 10% fetal serum. them before infection remove medium and add medium WITHOUT serum with antibiotics and insuline ( SEE paper: http://www.ncbi.nlm.nih.gov/pubmed/16271305 ) Add your sample . Incubate at 37ºC, with 5 % CO2.

to make a titration you can use Real-time PCR.

Thanks alot for your nice reply

Hello;

I studied epidemiology of T.gondii some years ago, but now I am working with other parasitic diseases in my PhD. You can find some informations in OIE diagnostic manual.

However, my sister is working with Toxoplasma gondii isolation in Brazil (she is in an M.Sc. degree course in general parasitology) . If u need more details, feel free to contact us!

Lucas M. Cunha (Federal University of Minas Gerais, Brazil).

Mosaab Omar, are you going to determine the virulence of certain Toxoplasma strain on mice, or only propagate the Toxoplasma cells in vivo?

no.i only detect contiminated for toxoplasma gondii in mice by elisa method in TEHRAN CITY-IRAN

Do you want to study seroprevalence and isolate strain from seropositive individuals ?

U can isolate toxoplasma parasite from wild rat brain,

VERO cells are the best option, wich strain are you using?

Grretings

Dear

Thank you very much for your attention to me. I think Vero cell are the best medium for culture and isolation of this parasite but you could culture on RPMI. Also you could injected toxoplasma to pretoneal of rat and isolate these from the exsodate. I hope this suitable for you.

In krachi university,one student has tried some sort of artificial media to culture toxoplasma.Once I have seen his work,pl try to search that thesis on internet.Hope it will solve your problem.

To titrate it, make serial dilution of your inoculum ,then use each dilution to infect either any cell culture or media.

You can collect infected tissues and treat it with pepsin and filtrate it and inject in to mouse S/c route. and observe it for 7-12 days and thereafter. get the sick mouse lung and ground it and inoculate into CV1 cell culture or M617 (macrophage cell line) for isolation of tachyzoites of T.gondii.(you can refer Dr.J.P.Dubey's paper)

afterwards, you can collect tachyzoites from the monolayer pass it through 27 gauge needle and count it using haemocytometer and then you can serially dilute it to know the inoculum. Best luck.

Toxoplasma gondii can not be cultivated without cells, at CINVESTAV Dr. Mondragon works with erythrocytes to grow it.

CV1 (african green monkey kidney cell is the good one to isolate T.gondii from the tissues with RPMI 1640 medium with 10% fetal bovine serum and 5% CO2. Try it. Best wishes.

Just for your information this is an old method but can be useful for you:

1. From a mouse inoculated check his positivity for T. gondii doing a peritoneal lavage with sterile saline solution and noting the presence of tachyzoites in the exudate. From this primary mouse get washed and inoculate other mice intraperitoneally and let chronically to form cysts. If there is high mortality of mice due to the highly pathogenic of strain treat animals with sulfa in drinking water.

2. With approximately one month after inoculation, check the positivity observing cysts in macerated brain in thin layers between the microscope slide and coverslip. (Alternatively it can be seen stained imprints.)

3. Infect orally young cats free of infection with T. gondii . Daily harvest the entire volume of faeces dropped by the cat in individual containers and keep at 28 degrees Celsius and humidity above 80% to sporulate. Examine the stool and track separately from the observation of the first meeting of oocysts, from the peak until elimination in the absence of oocysts. It is very interesting to follow the curve of oocyst production.

4. Following the above methodology you can get the three stages of T. gondii. My email is [email protected] or [email protected]

Hi;

I am working with cutaneous Leishmaniosis. You can find informations in OIE diagnostic manual.

In the April 2012 issue of The Journal of Parasitology is an interesting article by R. Useo et al. 'A New Alternative in Vitro Method for Quantification of Toxoplasma gondii Infectivity'. The article references and describes a cell culture method to produce tachyzoites on fibroblast cells in DMEM supplemented with 2% FCS. It may not be the best method if you are looking for cysts but this article may be a good place to start your search.

Thanks all for your great response and participation. Does anyone have any idea if that toxoplasma pseducyst is formed in the aborted fetus or not? and how the abortion occurs?

We work sometimes with T.gondii. These parasites grow also in Vero cells with DMEN + 2% FBS + 1% of antibiotics (Peniciline-streptomycin-Fungizone). We determine the number of parasites and the viability by trypan blue exclusion using a Neubauer chamber. You can resuspend the tachyzoites in PBS for the experimental infection. Dosage will depend on the T. gondii strain and the animal model you will use.

Thanks alot Elena for your participation

A lot of Toxoplasma tachyzoites can be obtained by culture of RH strain in mouse peritoneal macrophages. After 24 to 48 hrs we will have many free forms. In addition y can release them from the macrophages just pushing of the cells through a 26 needle. Later you can separate the organismos by filtration or whathever you want to do. Some of my procedures and exoerience can be seen in some of my publications.

Thanks very much for your answers.......really i appreciate that

would anyone provide me with a reference of counting Toxoplasma tachyzoites by hemocytometer?

Set up to culture either one of this such as vero cell line, macrophage cell lines or human foreskin fibroblast cell lines. Propagate it for duplication. Then introduce any of the animals' lung, tongue or brain squash (antibiotic treated) into cell line and observe under inverted microscope continously. You would find tachyzoite growth in that. harvest it and do rest of the work whatever may be. Follow strict guidelines for cell culture procedure. You can find lot of procedure from references of Dubey et al., T.gondii, In vitro culture.

We have been working with T. gondii for many years, therefore you will find in our papers some of the procedures for isolation, counting and culture of this parasite, either in mice and hámster macrophages or in kidney fibroblasts. Due to the objectives of our experiments,  we do not like to work with cell lines.