This Weeks Citation Classic_______

Trager W & Jensen .J B. Human malaria parasites in Continuous culture.

Science 193:673-5, 1976.
[Department of Parasitology, The Rockefeller University, NY]

Two methods were described for the continuous in for P. coatneyi, RPMI 1640 (a medium developed by
vitro growth in human erythrocytes of the major C. Moore for human white cells) if supplemented
human malarial parasite, Piasmodium ia/clparum. All with Hepes buffer and serum was better than any
stages of the erythrocytic cycle were present in the other medium previously tried. In addition, I had
cultures, and infectivity to Aotus monkeys was dem- good reason to believe that a gas phase with about
onstrated. This was the first report of continuous cul- 5 percent CO and with less oxygen than the 21 per.
ture of any spedes of malarial parasite. [The Sd in- cent present 2in air at sea level would be favorable.
dicates that this paper has been cited in over 850 pub- I then put all of these factors together.
lications.] I inoculated a suspension of human AB erythro-
cytes with a small amount of falciparum-infected
Aotus blood and placed it in two of the homemade
flow vials, which provided for a flow of medium at
2 ml per hour over the settled cells and an atmo-
sphere of 7 percent C0 , 5 percent 2 and a bal-
William Trager ance of N . The vials were2 sampled at one- or two-
The Rockefeller University 2 and the excitement grew as I saw live
day intervals,
New York, NY 10021-6399 parasites and evidence of multiplication. The cul-
tures were diluted with fresh human red cells on the
fourth day and then, as growth continued, every
third or fourth day.
September 25, 1987 It was soon clear that continuous culture had been
achieved, but the flow vials were not very conve-
nient, and it was here that James B. Jensen made his
The cultivation of parasitic organisms has always significant contribution. He had just come to my lab-
been one of my main interests. My first studies with oratory as a postdoctoral fellow when I started the
avian malaria parasites were interrupted from 1943 experiment with the flow vials that had such a suc-
to 1945, when I learned firsthand about human ma- cessful outcome. In trying to simplify the method,
laria while working in the Southwest Pacific as an we thought petri dishes with a daily manual provi-
officerin the Sanitary Corps of the US Army. On my sion of fresh medium would be worth trying. But
return to The Rockefeller University I became inter- how to control the gas phase? Although CO incu-
ested in the possibility of extraceliular culture of ob- bators were already available, we did not have 2 one,
ligate intracellular protozoan parasites, and much of and, furthermore, incubators then on the market had
my research effort during the following 25 years was no provision for maintaining a reduced oxygen ten-
devoted to this goal. In experiments with the avian sion. Fortunately, Jensen had done hisgraduate work
malaria parasite, Plasmod,um Iophurae, I obtained with D.M. Hammond, who taught his stsidehts the
a limited extracellular development and showed that use of candle jars for tissue cultures. We incubated
exogenous sources
1~ ofAlP and coenzyme A were es- our petri disls cultures in candle jars and found
sential to it. that we could get continuous propagation of
Then, in 1975, the World Health Organization and P. fakiparum.
the US Agency for International Development em- Once we knew how to do it, the methods for con-
barked on new programs aimed at developing a ma- tinuous culture of the erythrocytic stages of P. fal-
laria vaccine.Cultivation of the parasites seemed es- ciparum were so simple and flexible that they were
sential, and I returned to the problem of intraeryth- soon taken up in laboratories all over the world. Cell
rocytic development in vifro. In the meantime P. fal- biologists and molecular biologists found the para-
ciparum had been successfully transmitted to the sites a fascinating materiaL Cultures of P. faidparum
South American owl monkey Aotus by WA. are now being used to study the mode of entry of
Siddiqui, providing a laboratory source of the para- the parasite into its host erythrocyte, its effects on
sites. Also, I had found in experiments with P. the host cell, the mode of action of antimalarial
coatneyi, a parasite of the rhesus monkey with a drugs, and the nature of the parasites resistance to
biolo~yvery similarto that ofP. falci pa rum, that the chloroquine and other drugs. It is also used to screen
parasttes developed better in a settled layer of red for new drugs, to isolate and characterize strains and
cells with a continuous slow flow of medium over clones, to study the genetics of the parasite, and to
it than under other conditions, such
5 as the rocking identify immunogenic
7 antigens for ultimate use in
flasks used in most earlier work. I also found that a vaccine.

1. Trager W. Studies on the extraceUalar cultivation of an intracellular parasite (avian mataria). 1. Development of the
organism in erydirocyte extract and the favoring effect of adenosine-tnphosphate. I. Exp. Med. fl:349-66, t950.
(Cited 105 times since 1955.)
2. . Malaria parasites (Plasmodium lophurac) devetoping extracdllutarty in vitro: incorporation of tabeled precursors.
I. Plvtowolo~rt8:392-9, 1971.
3. TragerW & Brohn 8 H. Coenzyme A requirement of malaria parasites: effects of coenzyme A precursors on extracellutar
development in vitro of Plasnx,diu.m Jophwxe. Pz~c.Nat. .4cad. Sd. USA 72:1S34-7, t975.
4. Gelnwi Q M, Siddiqul W A & Schnell I V. Biological basis for susceptibility of Aotus tnvtrgants to species of plasmodia
from man. MElt. Med. 134:780-6, 1969.
5. Trager W. A new method for intraerythrocytic cultivation of malaria parasites (Plasmodium coatneyi and P. lhlcrparwn).
I. Protozoology [8:239-42, 1971.
6. 3ensen .1 B & Trager W. Plasmodium talciponun in culture: use of outdated erythrocytes and description of the candle jar
method. I. P5rasiroio~,63:883-6, 1977. (Cited 190 times.)
7. Trager W. The cultivation of Plasmodium falcipanun: applications in basic and applied research on malaria.
Ann. Trop. Med. Parasirol. (In press.)

CURRENT CONTENTSI!~ @1988 by ISI LSV.37.#]6, .Apr. 18, 1988 15