In Memoriam - Louis S. "Buddy" Diamond, 1920-2009

 

Louis Stanley Diamond, known to many as 'Buddy', who died on the 6^th of September 2009, was a major figure in the history of parasitology research. Although best known for his fundamental and lasting contributions to the study of Entamoeba histolytica, the causative agent of amoebic colitis and amoebic liver abscess, his impact on research into many parasitic protists was significant. He was an early member of the Society of Protozoologists, the forerunner of the International Society of Protistologists, was its President in 1983-4, and was honoured with a Festschrift in 1995.

Buddy was born and raised in Philadelphia, the son of a doctor. His interest in protists began at an early age, when at the age of 14 seeing a Paramecium in a High School Zoology class inspired him to save up and purchase his own microscope. He went on to the University of Pennsylvania where the influence of David H. Wenrich and Robert M. Stabler led him towards the parasitic protists; this was also where he first encountered E. histolytica. Graduating in 1940, he moved on to the University of Michigan where he obtained a Master of Science degree in Parasitology. He served in the US Army Air Force and later the US Army as a medical technician, spending much of his time screening thousands of stool specimens for parasites, and published his first paper, on a rapid staining technique for intestinal protists (Diamond 1945). During this time he encountered two important people in his career: Franklin G. Wallace, later to be his PhD supervisor, and Leon Jacobs, who brought him the National Institutes of Health. Buddy continued his military service in the Army Reserve until 1980.

His dissertation at the University of Minnesota under the guidance of Franklin Wallace was on the trypanosomes of frogs, describing species and establishing life-cycles. In 1951 Buddy moved on to the US Fish and Wildlife Service in Maryland, where he worked mostly on the trichomonads of waterfowl, and then the Animal Parasite Laboratory of the US Department of Agriculture while writing up his PhD thesis, which took until 1958! In part this was due to the pressures of work and having a growing family, but mostly it was due to Buddy's dislike of writing; he was always very critical of his own scientific writing ability. While at the USDA, Buddy developed the first of the many parasite cultivation media for which he became well known, TYM (trypticase-yeast extract-maltose; Diamond 1957), frequently referred to simply as 'Diamond's medium'. This medium, used primarily but not exclusively for the axenic cultivation of trichomonads, is so reliable and produces such abundant growth that it is still used widely to this day, and the paper in which it is described has been cited many hundreds of times. Having finally completed his PhD, he was soon recruited by Leon Jacobs to the newly established Laboratory of Parasitic Diseases at the NIH in Bethesda, Maryland, where he was to remain for the rest of his career.

The NIH had a long history of work on E. histolytica. Both Charles W. Rees and Leon Jacobs had attempted and failed to obtain axenic cultures over the preceding decades, although monoxenic cultures with either bacteria and Trypanosoma cruzi as the associated organism had been generated. Within a year and a half, Buddy had obtained the first ever axenic cultures of E. histolytica. He modestly claimed later that serendipity had a lot to do with it - his choice of strain, his previously experience of developing media, the existence of monoxenic cultures - but the fact remains that he had achieved success where many great amoebiasis researchers had failed. Indeed, many thought axenic cultures might be impossible. His results were published in Science (Diamond 1961) and the 'modern era' of Entamoeba research began.

Over the following decades, the complex diphasic medium in which axenic cultures were first obtained was replaced by a series of alternative monophasic media that were simpler to make and use, and gave higher yields (Diamond et al. 1978). These opened up amoebiasis research to biochemical, immunological and eventually molecular investigations. Modifications of his media made the same range of studies possible for Trichomonas and Giardia. Researchers in Buddy's laboratory at the NIH were the first to study the DNA of E. histolytica (Gelderman et al. 1971 a,b) and he lived long enough to see the publication of the organism's genome sequence (Loftus et al. 2005). Indeed, although he finally retired from the NIH in 1994, he was heavily involved in the initial planning for the genome project in 1998.

The above description of his research career perhaps gives the impression of a narrow focus on the cultivation of E. histolytica, but this is far from the truth. His professional interests ranged widely within amoebiasis research and into various luminal and other protistan parasites. He never lost sight of the fact that E. histolytica causes a potentially fatal disease that kills thousands annually, mostly in developing countries. This led to long associations with researchers in both India and Mexico, in particular. His links with India spanned most of his career and he visited the country 19 times to foster collaborations with Indian scientists, including the prominent gastroenterologists Om Prakash of the All Indian Institute of Medical Sciences in Delhi and Kamal Jalan of the Kothari Institute for Gastroenterology in Kolkata. Likewise young Indian researchers were welcomed to his lab and long relationships, both personal and professional, developed with several. His links with Mexico started in the early 1970s when he was invited to participate in one of the first "Seminars on Amebiasis", a conference series that continues to this day. Buddy quickly became heavily involved with these events and was co-organiser of the 1975 meeting celebrating the centenary of Fedor Lösch's publication of the first description of amoebic dysentery. With Drs. Bernardo Sepúlveda and Adolfo Martínez Palomo, Buddy helped develop a vibrant amoebiasis research community in Mexico that is still going strong with a new generation of investigators.

Due to his modesty when it came to his achievements, it is rarely recognised that Buddy was the first to cryopreserve protists successfully (Diamond 1964, 1995); where would we be without this tool? He was also an innovator in other ways. His doctoral thesis work was published in a journal that existed only in microfiche form (Diamond 1965), then the way of the future! He was also cited in Science as an early user of e-mail to keep long-distance research collaborations going (Palca 1990) and he was always encouraging to those who wished to apply new technology to the study of his favourite parasites!

Many researchers passed through Buddy's laboratory at the NIH over the years, some having the privilege of spending years learning from him. To those fortunate enough to stay there for extended periods he became a mentor and an inspiration. His generosity and willingness to share his knowledge, technical expertise and materials, as well as his enthusiasm and encouragement for young researchers is, we believe, responsible for the spirit of cooperation that characterises the amoebiasis field to this day.

To be welcomed into Buddy's lab was also to be welcomed into his extended family. The remarkable Diamond household in the 1980s and 1990s was multi-generational , where Buddy and Mildred, his wonderful wife of 70 years, lived with their daughter and grandchildren, and with Mildred's father, each on a different floor of the same house but all eating together as a family, and with their frequent guests, every day when possible. Children of visitors often became surrogate grandchildren and those relationships took on a life of their own away from the research links.

Buddy did have his idiosyncrasies. He was in his lab at 6AM every day in order to beat the traffic. Fortunately, he did not expect those working with him to keep to this timetable, except if you happened to be staying at his house! This habit of rising early actually saved his and Mildred's lives when, in 1985, an earthquake of magnitude 8.1 struck Mexico City while they were visiting for a "Seminar". Fortunately, it struck at 7:19 AM while they were already at breakfast - their hotel room was destroyed. Buddy was obsessive about cleanliness in the lab and cleaned it daily himself, as he did not trust the cleaning crew to do it well enough. This quirk probably dates from the time before laminar flow hoods, when axenic culture work had to be done on a bench in a 'clean' room. He was meticulous, perhaps even obsessive, when it came to his cultures. Every parasite isolate was subcultured in duplicate or triplicate with inocula of different sizes and at strict time intervals, each resulting culture was inspected under the microscope and the results recorded on a pad, and all the data were then transferred to a hard covered book where information was recorded on growth, which tube was selected for subculture, the inoculum sizes, and the type of medium used. What at first seemed obsessive to new arrivals gradually became logical when the ability to look up information on cultures that had been cryopreserved years or even decades earlier demonstrated its value. The attention to detail when it came to his cultures was the basis of his success and is the main reason why many researchers used to more 'forgiving' cells struggle to culture E. histolytica. What Buddy emphasised to everyone he trained was that if your cultures are not 'happy', how can you trust any results you obtain with them?

In what little time he allowed himself outside of work and family, Buddy's passion extended to bird-watching and the ballet. The former has rubbed off on more than one person he mentored! In Buddy's case it involved treks at least twice a year to Hawk Mountain in Pennsylvania to help identify and count the migrating birds of prey, but it also extended to side trips to watch birds on his travels to India and elsewhere.

In so many ways, the death of Buddy Diamond is the passing of an era. The type of research that formed the core of his career would never be funded today. It involved long, painstaking, unspectacular and often unrewarding work over many years, yet without the availability of his axenic cultures little if any of today's research into Entamoeba, Trichomonas and Giardia molecular and cell biology and "-omics" would be possible. Without his work on cryopreservation, the ATCC protistology collection would not exist and we would be unable to preserve the many strains, mutants and transformants so central to much of our research today. We all feel a great sense of loss now that he is no longer at the end of the phone or reachable by e-mail; we have so many more questions we wished to ask. We have lost not only a great scientist, but a mentor, colleague, and friend.

C. GRAHAM CLARK, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK

FRANCES D. GILLIN, Department of Pathology, University of California, San Diego, School of Medicine, 214 Dickinson Street, San Diego, CA 92103-8416 USA

ALOK BHATTACHARYA, School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110 067, India

SUDHA BHATTACHARYA, School of Environmental Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110 067, India

DAVID MIRELMAN, Departments of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100 Israel

 

References

Diamond, L. S. 1945. A new rapid stain technic for intestinal protozoa using tergitol-hematoxylin. Am. J. Clin. Pathol. 15:68-69.

Diamond, L. S. 1957. The establishment of various trichomonads of animals and man in axenic cultures. J. Parasitol. 43:488-490.

Diamond, L. S. 1961. Axenic cultivation of Entamoeba histolytica. Science 134:336-337.

Diamond, L. S. 1964. Freeze-preservation of protozoa. Cryobiology 1:95-102.

Diamond, L. S. 1965. A study of the morphology, biology and taxonomy of the trypanosomes of Anura. Wildl. Dis. No. 44.

Diamond, L. S. 1995. Cryopreservation and storage of parasitic protozoa in liquid nitrogen. J. Euk. Microbiol. 42:585-590.

Diamond, L. S., Harlow, D. R., and Cunnick, C. C. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72: 431-432.

Gelderman, A. H., Keister, D. B., Bartgis, I. L. & Diamond, L. S. 1971a. Characterization of the deoxyribonucleic acid of representative strains of Entamoeba histolytica, E. histolytica-like amebae, and E. moshkovskii. J. Parasitol. 57:906-911

Gelderman, A. H., Bartgis, I. L., Keister, D. B. & Diamond, L. S. 1971b. A comparison of genome sizes and thermal-denaturation-derived base composition of DNAs from several members of Entamoeba (histolytica group). J. Parasitol. 57:912-916.

Loftus, B., Anderson, I., Davies, R., Alsmark, U. C., Samuelson, J., Amedeo, P., Roncaglia, P., Berriman, M., Hirt, R. P., Mann, B. J., Nozaki, T., Suh, B., Pop, M., Duchene, M., Ackers, J., Tannich, E., Leippe, M., Hofer, M., Bruchhaus, I., Willhoeft, U., Bhattacharya, A., Chillingworth, T., Churcher, C., Hance, Z., Harris, B., Harris, D., Jagels, K., Moule, S., Mungall, K., Ormond, D., Squares, R., Whitehead, S., Quail, M. A., Rabbinowitsch, E., Norbertczak, H., Price, C., Wang, Z., Guillen, N., Gilchrist, C., Stroup, S. E., Bhattacharya, S., Lohia, A., Foster, P. G., Sicheritz-Ponten, T., Weber, C., Singh, U., Mukherjee, C., El-Sayed, N. M., Petri, W. A., Clark, C. G., Embley, T. M., Barrell, B., Fraser, C. M. & Hall, N. 2005. The genome of the protist parasite Entamoeba histolytica. Nature 433:865-868.

Palca, J. 1990. Getting together bit by bit. Science 248:160-162.

 

Selected Bibliography

Diamond, L. S. 1957. The establishment of various trichomonads of animals and man in axenic cultures. J. Parasitol. 43:488-490.

Diamond, L. S. 1961. Axenic cultivation of Entamoeba histolytica. Science 134:336-337.

Diamond, L. S. 1964. Freeze-preservation of protozoa. Cryobiology 1:95-102.

Diamond, L. S. 1968. Improved method for the monoxenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae with trypanosomatids. J. Parasitol. 54:715-719.

Diamond, L.S. 1968. Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae. J. Parasitol. 54:1047-1056.

Lunde, M. N. & Diamond, L. S. 1969. Studies on antigens from axenically cultivated Entamoeba histolytica and Entamoeba histolytica-like amebae. Am. J. Trop. Med. Hyg. 18:1-6.

Gelderman, A. H., Keister, D. B., Bartgis, I. L. & Diamond, L. S. 1971. Characterization of the deoxyribonucleic acid of representative strains of Entamoeba histolytica, E. histolytica-like amebae, and E. moshkovskii. J. Parasitol. 57:906-911.

Diamond, L. S., Mattern, C. F. T. & Bartgis, I. L. 1972. Viruses of Entamoeba histolytica I. Identification of transmissible virus-like agents. J. Virol. 9:326-341.

Weinbach, E. C. & Diamond, L. S. 1974. Entamoeba histolytica: I. Aerobic metabolism. Exp. Parasitol. 35:232-243.

Diamond, L. S., Harlow, D. R. & Cunnick, C. C. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72:431-432.

Gillin, F. D. & Diamond, L. S. 1978. Clonal growth of Entamoeba histolytica and other species of Entamoeba in agar. J. Protozool. 25:539-543.

Gillin, F. D. & Diamond, L. S. 1981. Entamoeba histolytica and Giardia lamblia: growth responses to reducing agents. Exp. Parasitol.51:382-391.

Diamond, L. S. 1982. A new liquid medium for xenic cultivation of Entamoeba histolytica and other lumen dwelling protozoa. J. Parasitol. 68:958-959.

Diamond, L. S. 1986. Entamoeba histolytica Schaudinn, 1903: from xenic to axenic cultivation. J. Protozool. 33:1-5.

Bhattacharya, S., Bhattacharya, A. & Diamond, L. S. 1987. Comparison of repeated DNA from strains of Entamoeba histolytica and other Entamoeba. Mol. Biochem. Parasitol. 27:257-262.

Bracha, R., Diamond, L. S., Ackers, J. P., Burchard, G. D. & Mirelman, D. 1990. Differentiation of clinical isolates of Entamoeba histolytica by using specific DNA probes. J. Clin. Microbiol. 28:680-684.

Diamond, L. S. & Cunnick, C. C. 1991. A serum-free, partly defined medium, PDM-805, for the axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and other Entamoeba. J. Protozool. 38:211-216.

Bhattacharya, A., Ghildyal, R., Prasad, J., Bhattacharya, S. & Diamond, L. S. 1992. Modulation of a surface antigen of Entamoeba histolytica in response to bacteria. Infect. Immun. 60:1711-1713.

Diamond, L. S. & Clark, C. G. 1993. A redescription of Entamoeba histolytica Schaudinn, 1903 (emended Walker, 1911) separating it from Entamoeba dispar Brumpt, 1925. J. Euk. Microbiol. 40:340-344.

Diamond, L. S. 1995. Cryopreservation and storage of parasitic protozoa in liquid nitrogen. J. Euk. Microbiol. 42:585-590.

Clark, C. G. & Diamond, L. S. 1997. Intraspecific variation and phylogenetic relationships in the genus Entamoeba as revealed by riboprinting. J. Euk. Microbiol. 44:142-154.

Pimenta, P. F., Diamond, L. S. & Mirelman, D. 2002. Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925: differences in their cell surfaces and in the bacteria-containing vacuoles. J. Eukaryot. Microbiol. 49:209-219.

Clark, C. G. & Diamond, L. S. 2002. Methods for cultivation of luminal parasitic protists of clinical importance. Clin. Microbiol. Rev. 15:329-341.