Plasmodium Falciparum - Science topic

Lowering the extension time may help.

Some of the pesky problems that are not addressed by the in-silico analysis are related to the behaviour of the compounds in aqueous solutions: poor solubility of the compounds, aggregation, unspecific interactions with cell surfaces and sample tubes.

You can use the method suggested by Silvia Di Santi. You should change the media every 24hr. If the parasitemia is high, split some and keep it below 5%, we usually keep it around 2-3% unless we want to perform any antimalarial assay. Most importantly, you should check the parasitemia level using microscopy every 2-3 days intervals at least and provide fresh RBCs while splitting. For higher parasitemia, you can use inactivated serum or plasma from AB+ donors.

Below is our article for a brief explanation and synchronization method as well:

Please what did you later do to get the cells to grow? Please how do I also get P.falciparum culture? I want to test the effect of my research drug on the growth of plasmodium cells in vitro

Thanks to you both, Elemaran and Masoumeh.

Yes please Hao Wang

Thanks!! Divya Das and Katie A S Burnette for your response,

I have chosen the ring stage because symptoms of malaria start to appear in this stage.

It is possible. Mainly gametocytes and ring forms are found in peripheral blood film and heparine tube does not interfere with the morphology these structures.

We normally dissect the midguts in 1% NP-40 in PBS to permeabilise the tissue for 30 min followed by the staining with mercurochrome for at least another 30 minutes. Also we only use 0.1% mercurochrome for staining. Hope that helps although my answer comes quite late.

How was your experiment? Did It work?

Harrison - sorry for the very late reply here, but just noticed this question on RG for CLC so I thought I'd answer it. I'm the product manager for CLC Genomics Workbench @ QIAGEN btw.

You shouldn't have any issue downloading data from NCBI/GenBank within CLC, and there's no limit on genome size you can work with, Plasmodium falciparum included. (Actually - the largest genome ever assembled was done using CLC's de novo assembler 😉) which version of CLC Genomics Workbench are you using?

Plasmodium falciparum 3D7 is the reference genome, correct? based on the info at GenBank - this genome corresponds to assembly ID - GCF_000002765.4

With the latest version of CLC GxWB (v12) - this shoudl be really easy to do. From the menus -

The results should be 14 chromosomes. You can either select all of them at once and click "Download and Save" or - if your university or insittution is on limited bandwidth or being throttled by NCBI, you can try downloading them one at a time.

Here's a screenshot:

How big is max scan area? Usually it is about 100 um, depends on the scanner. You can scan such area (not sure about true non-contact mode, perhaps tapping mode (especially - phase contrast) would be more informative), pick some infected cells (should be possible to distinguish with proper scan size in pixels - 1024 and more) and scan them with proper scan size.

Good luck!

Hi Irmayani Irmayani,

I do believe you can easily access such info from the manufacturer's website. Kindly find a link below for a copy of the protocol

Best,

Hi, there are some automated systems connecting different tissue culture flasks to change media but it's generally lab made systems with pumps... not sure there are any commercials systems available...

good luck

Dear Kokouvi,

Thanks for your candid advice.

Sorry M. A. A. Al- Fatlawi this link does not work. Is it for a PhD position? I'm already a PhD student at the IIB, Uni of Liverpool

You have me confused at many levels. To the main question, apicoplast (an organelle) does not have cell surface proteins. It is the main parasite cells that do have surface proteins; e.g. merozoite surface proteins on merozoites (the blood stage of Plasmodium) used for RBC invasion, and PfEMP on trophozoites and schizonts (which are inside RBCs) for clustering RBC for ultimate destruction.

Apicoplast apparently is not involved in cellular invasion - perhaps you misread Wikipedia article. It synthesises fats and isoprenoids for cellular metabolism. Those that are actually involved in invasion are apical secretory organelles including micronemes and rhoptries. If you look for the molecular mechanism of invasion, merozoite surface proteins is the answer.

Hello, Gibson assembly method, were evaluated for constructing P. falciparum vectors.

See attachment for more info.

Good luck!

Hi,

in principle Nested PCR is very effective. From this perspective and based on literature you can even dilute higher and should suceed. Therefore the higher dilution should be better as this will also reduce the amount of all other components form the first amplification like Primers etc

Best Sven

Danielle Snider I feel that a good way to do synchronisation is to closely watch your parasites, make slides every 6 hours to judge the population. If population is made of rings trophozoites and schizonts, I would suggest to stick to only one method i.e. sorbitol (5% Sorbitol for 10-15 minutes at RT or 37 ), gently shake tube and spin at low g values. Wash supernatant that is mainly ruptured cells and rbc's multiple times. add fresh blood maintain a hematocrit of 1% and allow parasites to grow at 37 under gassed environment.

Two sorbitol treatments can be given in tandem cycles to achieve a tight synchrony.

good luck dear David L. Murphy

Dear Jude,

Thanks a lot for your reply. As I am no expert in this field and the question came up in one of our group discussions recently, I think, I would still need to wrap my head around the bigger picture before having a scientific discussion, as it is not directly connected to my work. Anyhow, our general question was based on the following literature:

1.) Virgilio L. Lew, Teresa Tiffert, and Hagai Ginsburg,

Excess hemoglobin digestion and the osmotic stability of Plasmodium falciparum–infected red blood cells (BLOOD, 15 MAY 2003 + VOLUME 101, NUMBER 10, 10.1182/blood-2002-08-2654.Supported)

2.)Mailin Waldecker, Anil K. Dasanna, Christine Lansche, Marco Linke, Sirikamol Srismith, Marek Cyrklaff, Cecilia P. Sanchez, Ulrich S. Schwarz, Michael Lanzer,

Differential time‐dependent volumetric and surface area changes and delayed induction of new permeation pathways in P. falciparum‐infected hemoglobinopathic erythrocytes (Cellular Microbiology 2017; 19: e12650 DOI 10.1111/cmi.12650)

Best,

Julian

What does it mean by parasites didn't want to grow? Are they growing up to a certain stage and die? Do you see more gametocyetse ?

Please elaborate on your protocol.

Please try using the following protocol for Albumax (the article is attached for your referance)

Incomplete medium

RPMI 1640 - 10.4 g (1 sachet)

1M Hepes - 25 mL

0.1 M Hypoxanthine - 1 mL (0.1 M hypoxanthine - Dissolve 1.361 g of hypoxanthine in 1M NaOH and make up to 100 mL)

Albumax I - 5 g

Milli-Q water (ultra pure water) - 1 L

Filter sterilize the incomplete medium.

When you want to use the medium, prepare the complete medium by adding sodium bicarbonate to a final concentration of 1.77 mM. (3 mL of 5% (wt/vol) sodium bicarbonate to 100 mL of incomplete medium-Filter sterilize the sodium bicarbonate solution)

Also, please make sure that your red blood cells are free of leukocytes. In my personnel experience, some donor RBCs do not support the growth of malaria parasites. When you get RBCs, make sure your donors are not pre-exposed to malaria parasites. Test different lots of RBCs.

What is your starting hematocrit ? Usually, malaria parasites grow well in 5% hematocrit of 5 mL culture. You can start in small 25 cm2 flasks with a non-vented cap.

I assume you gas the cultures after every media change. That is also a necessary requirement for parasite growth.

Also, what is the source of your parasites? Is it cryopreserved parasites? If so, you can try a different lot of cryopreserved parasites. How many parasites (parasitemia) you add to the starting culture? If they are cryopreserved, they should be thawed carefully, with minimal lysis. Otherwise, if you collect parasitized blood from a human donor, you should wash them thouroughl and gently. Also to establish cultures, ring-stage parasites are preferred as late stage parasites are not preferred for infections.

If you have a perfect protocol and still it doesn't work, you may have to check for micoplasma contamination and any other contaminations.

well said

Following

Hi Danielle,

I used this protocol and it worked very well for me.

https://academic.oup.com/trstmh/article/108/8/488/2765173

I think the link above has comprehensive information to answer your question

best,

Read the paper on : New Method to Quantify Erythrophagocytosis by Autologous Monocytes ;

What do you mean exactly for "vertical streak"?

Could you maybe attach a picture?

If I properly understood you have kinda a vertical spike in the middle of your band. If it is I had the same problem.

Eventually I found out I just badly prepared my running buffer (either the molarity of Tris-HCl was not 1M or the pH was lower than 8.8) so the gel was "weak".

If you haven't still done it just try to prepare fresh buffer making sure molarity and pH are correct.

Thank you. Your first response solved the problem. The concentration of the PfLDH was too high. As soon as made dilution to about 1000 fold the aptamer was able to bind with a corresponding high charge resistance.

May be useful to look at this paper:

Here are some publications that contain information about maintenance of Plasmodium falciparum that you may find useful:

Marcus

Many people talk about the mosquito as being a "vector" and avoid calling in an "intermediate host", considering that sexual reproduction occurs in the mosquito. Other people refer to mosquitoes as definitive hosts of Plasmodium.

The late Norman D. Levine did not like the description "final host" in relation to Toxoplasma and Sarcocystis. He said there is nothing "final" about the carnivore host, transmission being ongoing. He preferred "definitive host".

This may be helpful See link attached: http://www.nature.com/nrmicro/journal/v13/n6/fig_tab/nrmicro3450_F2.html

If you have synchronized your malaria parasite using sorbitol why were you sstill having to collect every 4 hours? If you have rings, 24 hours later you will have (majority) late stage trophs. Unless you are collecting various stages of rings, trophs and schizonts you shouldn't have to collect so often for a synchronized culture. Please let us know how your experiment turned out. 

hi see this link can help  you https://www.ncbi.nlm.nih.gov/pubmed/10697834

also this article

 Out of Africa: origins and evolution of the human malaria parasites

Plasmodium falciparum and Plasmodium vivax

Resolution of a protein in SDS gels depends on size of the protein and the percentage of the gel and how long you let the gel run. Refer the attached picture to get an idea of what percentage to make. Use pre-stained ladder to track the resolution of the bands so that you know how long to let the gel run. 

hello..from NCBI , Primer design 

Thank you dear @Uday Mohanta for this valuable information.

Percoll could be used to remove gametocytes - see for example http://www.ajtmh.org/content/59/4/505.full.pdf (depending a bit on how you set up your Percoll gradient and what you want left over, as you may also find yourself co-purifying away the late-stage asexuals if your culture contains these stages).

 Dear Martin,

I am very interested in the Kapa Hyper Plus Kit. DId you try it? Did it worked?

Also I normally used Kapa HIFI and I wonder whether adding TMAC makes any substantial difference in terms of reducing AT bias in Plasmodium.

Thanks!!

An echo to Dr. Kopfli

1. These under 5 yr children had relatively little acquired immunity compared with  the older individuals. 

2. Parasite density would be  higher in these young children.

Therefore, parasite clearance time might take longer in these group of children.

This means parasite clearance time at Day 3 is likely to be lower.

         By definition, ETF means inadequate decrease in parasitemia ( i.e parasitemia on day 3 ≥25% of count on day 0.)

 I feel that you have found ≥25% parasitaemina at Day 3.  

Thank you.

Thank you.

You could easily get it from MR4, it is BEI resources now (https://www.beiresources.org/Home.aspx)

Hi Marina,

Good choice of extraction methodology, phenol-chloroform will give you the cleanest and best quantity of DNA. However, your yield will depend on a bunch of factors such as:

Having said that, on an average I get about 5-7ug of DNA from a 20mL culture of 3-4% parasitemia and 5% hematocrit using phenol-chloroform.

Hope this helps! Good luck!

Yes: if the gametocytes are not able to exflagellate (i.e. produce mature male gametes) then there will be no mating with female gametes, no zygotes produced and hence no infection of mosquitoes.  Efficient exflagellation is not a guarantee of efficient mosquito infection, but it's a first step!  Good luck...

two more papers attached

joshua

For parasite culture Marias answer should be sufficient. If you want to perform down stream analysis such proteomics or transcriptional analysis you may  need to mature the parasites ex vivo in order to capture all the stages (rings, trophs and schizonts). They have unique profiles which you need to be aware of.

Albumax 2 is used as a substitute for human AB+ serum. Albumax is preferred because it is easier than working with human serum as it needs to be filtered and heat inactivated. 

I am glad that Dr Beier answered this question because I used his protocol, optimized at the MRTC, Mali, for my csp ELISA.

However, I am not sure I understand what is addressed in Lillian's question.

After the 1st infective blood meal, are we searching for live sporozoites in the salivary gland (ie like dissecting the gland and looking for sporozoite using a stain under the microscope in the case of experimental infestation) or are we searching, using tools and techniques like ELISA, for antigenic determinants of PF, evidence of a prior infection or the potential to have infected earlier through subsequent feeding? 

I may be wrong here but let me just share with you my thoughts and some observations I personally made at the field during mosquito collections.

From parasites biology, for survival purposes, the parasite moves to the point of exit (salivary glands) in a stage infective (sporozoites) to the next host (vertebrate) and induces feeding behaviour in its vectors to enable it continue its development. Once the sporozoite matures, it exits into a vertebrate host and continues its developmental process. Eco-physiologists will call it SURVIVAL STRATEGIES.

I think there is no time of dormancy, reduced activity or waiting as in storage in the salivary glands by sporozoites. This is where the biology of parasite and the biology of vector synchronize into a non consenting agreement to insure parasites survival. 

From the insect biology standpoint, I have never asked myself if there is a limit in the salivary glands for sporozoite load but I believe nature regulates things such that there is no overcrowding of sporozoites in salivary glands. There must be a regulatory mechanism to slow parasite development or enhance exit of sporozoites from the salivary glands. Hence this induced feeding strategy for exit  to the vertebrate host that regulates the gonotrophic cycle in insects.

In case there is no specific host, the insect feeds on any alternative host to free the gland pouch, the space that is needed for the next maturing sporozoites. This is the basis for host substitution as a vector-borne disease control method that has proven effective among rural dwellers in Yoruba land where housing patterns are the" face-me- I- face- you" type with corridors where domestic animals (companion and livestock) pass the night. By this process, anthropophilic vectors gradually develop a zoophilic behaviour. This was also observed among the fulani herdsmen who spend the night covered amid their herds, insects end up exhibiting animal and human blood meal sources.

Finally, I think that if an insect bites once and picks up gametocytes that all develop into sporozoites which exit into the vertebrate host, no live sporozoite will be left if there is no intake of a new batch of gametocytes to develop into fresh sporozoites.

so Clearance of live sporozoites in the case of a single infection is possible but this is impossible in the case of recurrent infections (like in endemic areas) although evidence of infection/ infectivity is once after the first infection via the everlasting presence of antigenic determinants.

I hope I did not confuse you the more Lillian

I have already use the pCAM plasmid with success. You can find it within the link below.

And it's the paper assoicated:

Dear all thanks for your suggestions and sparing valuable time for me

Dear Donald thanks for the article 

I have question that from the literature I came to know that plasmodium infected RBC inhibits the phagocytic activity of monocytes my interest of experiment is to enhance the phagaocytic activity by using exogenous chemicals invitro  for this I would like to check the amount of P. falciparum phagocytosis by THP-1 derived macrophages in presence and absence of exogenous chemical kindly suggest the feasible experiment to perform phagocytosis and to estimate the amount of phagocytosis.

*when I checked literature they suggested to use fresh autologous serum for phagocytosis (Is it from malaria patient?) I dont have access to patient serum so kindly suggest the alternate and I am using combination of fresh ABO blood groups for parasite invasion

Thank you

Hi Marina,

I was going to recommend the same paper by Boyle et al. as Praveen has already suggested. Check out their supplementary information on heparin synchronisation method. Essentially, by adding heparin into your culture, it'll inhibit invasion. So, your 1 h window synchrony can be achieved by adding heparin into your culture until you want them to start invading. You then wash heparin off and leave them in heparin-free medium for an hour before you put the heparin back in. The tip I can give you is that you should start with quite synchronised parasites using sorbitol treatment at relatively high parasitemia. I'd recommend 4-5%. Since you will only allow a portion of parasites to invade within an hour, it'd be difficult to get a decent amount of parasites in one-hour window if you don't start with high parasitemia culture. Please also be sure to time your parasites right when you need to wash the heparin off (at very late-schizont stage of course when you start seeing some parasites beginning to rupture), otherwise you may lose them all if they have all been ruptured while you still have heparin in the medium. Delaying them at room temp to adjust the timing can also be done. If you cannot find the heparin solution as they suggested in their paper, you can use heparin salt from Sigma (H-3149) and use it at 100 ug/mL final concentration. You may want to try different concentrations if it doesn't work well for you at this concentration (i.e. it doesn't completely inhibit invasion).

Sorry for such a long answer but hope it helps. I've been using this method for years so  please feel free to contact me should you need further explanation. 

Cheers.  

Hi Nyingchu,

You can also use the bacterial expression vector pET28a(+) with T7lac promoter, which has an N-terminal His tag and kanamycin resistance. You could, for example, use the restriction sites XhoI and NcoI to clone your construct into the expression vector and express it, for example, in KRX cells. But before, make sure that these restriction enzymes do not cut your own construct. For more restriction sites you can use the information about the vector in the internet. Via the His tag you can purify the protein using hexahistidyl affinity chromatography on Ni-NTA-material. Cloning and expression is a daily routine in our lab. Do you have facilities for the expression of proteins and their purification in your own lab? If you have further questions, feel free to aks me...

which marker for lipid peroxidation estimation you used 

Its depends on the objective of your study. Are you looking for annotation or gene expression analysis.

If you are looking for prokaryotic NGS genome annotation follow the below steps.

Get the gDNA raw data

Assembly using VELVET or SOAP de-Denovo

CDS prediction

Fucntional annotation of contigs/CDS using RAST server or Manual BLAST

GO anlaysis

Gene Ontology(GO) mapping for CDS

Biological annotation of individual genes and corresponding CDS

For  gene expression analysis try following steps.

1. Get raw data fastq files.

2. QC using FASTQC or readQC or Groomer tool

3. Trimming filtering using trimmomatic or qtrip or prinseq

4. get reference genome and make Alignment using TOPHAT or Bowtie.

5. Then use gtf file of corresponding genome then transcript assembly and Gene expression analysis using cufflink, cuffcompare, cuffmerge, cuffdiff etc

6. Differential expression analysis using R BIoconductor. Use CummeRbund to analyse cuffdiff results further. CummeRbund is an R package that is designed to aid and simplify the task of analyzing Cufflinks RNA-Seq output.

Read the article by the authors of Tophat and Cufflinks at

Best!!

It is tough to culture this of parasite in mouse as these parasite are species specific and highly adapted to human cells p. bergi may be a choice for rodent cell line the media which they use for the of culture is RPMI media  but the cells in mouse may not support pf 

As artemether is an artemisinine derivative, we can suggest that they have the same molecular target (the K13 propeller domain). But some studies related to the artemether-lumefantrine combination (studied together) shows some Pfmdr1 copy number increase. 

And finally, some years ago, the PfAtpase6 gene were incriminated too. 

In conclusion, actually, mutations on the K13 propeller gene are the best molecular marker admitted by (I suppose) everybody...

I do not know your application but I have used the trehalose based freezing solutions with human epithelial cells.  We thawed the cells the same way with any cryo-media, Fast.  Threhalose is not considered toxic.  Similar to raffinose or other branched carbohydrates.

General procedure:

1. Vials were swirled by hand in a 38 C water bath. 

2.  As soon as the lat remnant of ice disappears, we very gently diluted the crryo-media 10 fold

3. cells allowed the cells to rest for 30mins-1 hour in aerobic conditions. 

The thaw is the highest stress point for cells in the cryopreservation procedure.  Respiration restarts and membranes have to be repaired.  Between the damaged membranes and the superoxide generation, not all cells will make it out of cryo.

Hope this helps

thanks a lot sir for providing me the information

I like both answer and are right.

Thanks you both

 dear petre Ianakiev I need a suggestion as well about my cDNa amplification. I have to amplify a gene of 4000bp with specific primers in order to insert restrction enzyme sequence at the 5' and 3'. I started to RV the RNA and then I tried to amplify the cDNA with the Q5 high fidelity polymarase but I didn't see any band. as above there are larger sequence than this, so I think that is not impossible to amplify a such sequence. Can you or anyone else give me a suggestion? 

Dear  Shillah

may I ask why you are only interested in these haplotypes?

For a suitable paper listing a sequencing strategegy for ama1 you could try

Polley and Conway Genetics. 2001 Aug;158(4):1505-12.

although as this paper shows it's been a while since I worked on population genetics of ama1

Ya, totally support using of Dd2 strain for CQ-resistance.

I would think it might be valuable if done as part of a control program. It might allow you to target interventions or identify areas where interventions have not been used adequately (e. g. perhaps hot spots have inadequate bednet usage). 

Thanks Beatrice

For transportation the RNA is suitable. Since your sample is stored in TRIZol could be dangerous for the transporter and the people around. And in RNA you only have to seal properly the tubes with parafilm.

Regards

Hi,

Our merozoite purification protocol is published in the open access publication "Methods in Malaria Research'  at MR4

See http://www.mr4.org/Publications/MethodsinMalariaResearch.aspx

We also published a detail protocol here:

There is a protocol in the supplementary material

We generally avoid high speed centrifugation

I hope these help

Cheers

James

Artifical mutant alleles have been made with the single mutant forms of dhfr in various systems. It seems the 51 single mutant form of dhfr does not confer resistance to the antifolates - and therefore would confer no advantage to the parasite carrying it, and it would not be selected. In addition the N51I mutation seems to affect the Kcat of the enzyme, which could affect the fitness of the parasite in the absence of antifolate drugs as well.

So we don't see the single mutant because although it could occur by random mutation, the parasite carrying the mutation would have no selective advantage in the presence of antifolates, and would also be worse off in the absence of drug. The parasite would therefore not outcompete wild-type (or other mutant forms), and would either remain at the very low frequency it started at, be lost by genetic drift, or may even not survive at all if the fitness cost in the absence of drug is significant.

These papers may be useful in providing the detail:

CHUSACULTANACHAI, S., THIENSATHIT, P., TARNCHOMPOO,

B., SIRAWARAPORN, W. & YUTHAVONG, Y. (2002). Novel

antifolate resistant mutations of Plasmodium falciparum

dihydrofolate reductase selected in Escherichia coli.

Molecular and Biochemical Parasitology 120, 61–72.

FERLAN, J. T., MOOKHERJEE, S., OKEZIE, I. N., FULGENCE, L.

& SIBLEY, C. H. (2001). Mutagenesis of dihydrofolate

reductase from Plasmodium falciparum: analysis in

Saccharomyces cerevisiae of triple mutant alleles

resistant to pyrimethamine or WR99210. Molecular and

Biochemical Parasitology 113, 139–150.

HANKINS, E. G., WARHURST, D. C. & SIBLEY, C. H. (2001).

Novel alleles of the Plasmodium falciparum dhfr highly

resistant to pyrimethamine and chlorcycloguanil, but

Malarial DHFR-TS as antifolate target 257

not WR99210. Molecular and Biochemical Parasitology

117, 91–102.

SIRAWARAPORN, W., SATHITKUL, T., SIRAWARAPORN, R.,

YUTHAVONG, Y. & SANTI, D. V. (1997b). Antifolateresistant

mutants of Plasmodium falciparum

dihydrofolate reductase. Proceedings of the National

Academy of Sciences, USA 94, 1124–1129.

We always transfect into fresh RBCs (certainly less than 2 weeks from blood draw), even if the parasite look like they're growing ok in older blood.  For a transfection, you want your parasites growing optimally and they are generally acknowledged to grow best in freshly-drawn blood because, as Praveen notes, blood cells age over time and parasites grow in the youngest cells most efficiently.  We also 'feed' them weekly after transfection for the same reason (cells age even faster at 37C than when stored in the fridge).  Because blood batches do differ, Kunal's suggestion to mix a couple of different batches may also help your transfections to succeed.

See reference on optimal transfection procedures:

'A quantitative analysis of Plasmodium falciparum transfection using DNA-loaded erythrocytes.'

Hasenkamp, S., Merrick, C.J., Horrocks, P. Molecular and Biochemical Parasitology 187(2), 117-120 (2013).

Hi Rashad,

as mainly the more mature stages can be found circulating, a common way of detection is using the Pfs25 QT-NASBA. 

Andrea

Many  thanks Surendra Kumar Prajapati

I think  your answer is quite clear for this question

In this case, a gentle cell sorting with SdFFF could be a good solution, as the radiolable eluted in the void volume, while cells are retained by they size and density.

Perhaps something such as elutriation could be also interesting for you.

When should we change the media while culturing Plasmodium falciparum?

Well, I would say that depends on how high the parasitemia is, I normally keep them around 5% and change medium everyday (independent of the stage). If the parasitemia is low (around 1% or below) and they are ring stages is ok to skip media change for one day. 

When should we proceed with splitting the culture?

I normally split when the parasites are in late trophs-schizont stages and the parasitemia is above 1.5% (that also depends on the multiplication rate of the strain being used). In my case the multiplication rate is around 6-8, meaning that if one day they are at 1.5% and I don't split them, next day they will be around 9-12% which is the maximum recommended parasitemia for a continuos culture. If the parasitemia is higher than 1.5% I definitely split them otherwise the culture will crash, I adjust the parasitemia depending on what I'm planning to do with them, if needed for experiment you will need rather high parasitemia but if it is just to keep them up and running you can have low parasitemia and therefore you save effort and m