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摘要:目的 为了解人芽囊原虫对外界条件的适应性，观察培养条件改变对其形态和繁殖方式的影响。 方法 从腹泻患者粪便中检查并分离培养获得人芽囊原虫，分别用locke氏-鸡蛋双相培养基（LES）和改良1640培养基（RPMI-1640）培养，改变培养条件，观察人芽囊原虫形态和繁殖方式。 结果 在两种培养基中均可见到人芽囊原虫的四种主要形态:空泡型、颗粒型、阿米巴型和包囊型，其繁殖方式以二分裂为主，可见少量出芽生殖。当从LES培养基突然变为RPMI-1640培养基时，人芽囊原虫出现短时间（48～72h）内生长停滞，随后出现多种形式的繁殖方式，可见较多的二分裂、出芽生殖、三分裂、多分裂、裂体生殖，持续24h后恢复为二分裂为主。 结论 培养条件改变能够引起人芽囊原虫繁殖方式的改变，出现短暂性多样化繁殖方式，人芽囊原虫对环境的适应能力很强。
关键词:人芽囊原虫;繁殖方式;多样化;暂时性;培养基
Temporary coexistence phenomenon of diversified reproductive mode of Blastocystis hominis.
ZHANG Hong-wei，HE Li-jun，LI Wen，et al
（Henan Provincial Center for Disease Control and Prevention，Zhengzhou450003，Henan，P.R.China）
Abstract:Objective To understand the adaptability of Blastocystis hominis（B.hominis）to environment，observe effect of culture condition change on reproductive mode of B.hominis. Methods B.hominis was isolated from patient with diarrhea and cultured in different culture medium including Locke-egg-serum medium culture（LES）and modified RPMI-1640culture medium（RPMI-1640）.When culture condition was changed between LES and RPMI-1640，morphous and reproductive modes were ob-served. Results All of4form including vacuolar form，granular form，ameba form and cyst form were found in each culture medium，separately，and main reproductive mode is binary fission，a few of gemmation was exist.When culture condition was changed abruptly from LES medium to RPMI-1640medium，different reproductive mode including binary fission，ternary fission，multiple fission and gemmation were found after growth retardation of24～72hours.This phenomenon continued less than24hours and binary fission remained reproductive mode then. Conclusion Reproductive mode of B.hominis can be effected by culture condition change，temporary diversified reproductive mode appear.B.hominis has very strange adaptability to environment.
Key words:Blastocystis hominis;Reproductive mode;Diversify;Temporary;Culture medium;LES medium;RPMI-1640medium
Blastocystis hominis（B.hominis）was once de-scribed as a yeast，and the yeast designation became firmly entrenched in textbooks and in the minds of parasitologists.The ultrastructural study ［1］ provided the first indisputable evdence that the organism was not yeast，and classified Blastocystis in the subphylum Sporozoa on the basis of pro-posed methods of division，in addition to morphological and culture characteristics.A new class，Blastocystea，and a new order，Blastocystida，were suggested.Further ultra-structural studies have supported these conclusions.The or-ganism is now recognized as an emerging intestinal protozoan parasite restricted to humans and other primates，causing intestinal disease with potentially disabling symptoms ［2］ .Studies on morphous ［3～5］ ，diagnosis and treatment ［6］ ，pathogenicity and molecular biology ［7］ of B.hominis had been done.But researchers were particularly bemused by the organism's amazing multiplicity of forms.Failure to find intracellular or tissue invasive B.hominis，a life cycle，or intermediate hosts contributed to the continuing enigma.This report describes the adaptability of B.hoministo envi-ronment，and observes effect of culture condition change on reproductive mode of B.hominis.
1 Materials and methods
1.1 Isolation of B.hominis B.hominis were isolated from the feces of a35years old man with2weeks intermit-tent diarrhea.Vacuolar form，granular form，and ameba form were observed in stool by light microscopy examina-tion.200μl stool specimen of patient was inoculated in Locke-egg-serum culture medium（LES medium）and cultured at37℃.Transfers（200μl cultures）were done at3-and4-day intervals，at which time the cell mass was approximately5×10 8 B.hominis cells per tube.
1.2 Preparation of culture mediums
1.2.1 LES medium The medium formula is1part beat-en whole egg to3.6parts Locke's solution（NaCl，8g;Ca-Cl 2 ，0.2g;KCI，0.2g;MgCl 2 ，0.01g;Na 2 HPO 4 ，2.0g;NaHCO 3 ，0.4g;KH 2 PO 4 ，0.3g;H 2 0，1000ml）.After being filtered through gauze，medium（4ml）was pipetted into tubes（18by125mm）and coagulated in flowing steam for10min at a30°slant，followed by the addition of4ml of Locke's solution and autoclaving at15lbs/in 2 steam for15min.Before using，1ml of inactive new born calf serum and Ampicillin（1mg/ml）were added to medium.
1.2.2 RPMI 1640culture medium（RPMI1640medi-um）RPMI1640Medium（10.4g/L）（Invitrogen company）was prepared according to instruction.Before using，1ml of inactive new born calf serum and Ampicillin（1mg/ml）were added to4ml medium.
1.3 Observation of morphous and reproductive mode of B.hominis with Changed culture conditions 200μl cul-tures of B.hominis in LES mediumwas transferred into RP-MI1640Medium and LES medium separately，and cultured at37℃.Morphous and reproductive mode of B.hominis was observed.After B.hominis was transferred twice in RPMI1640Medium，200ul cultures of B.hominis in RPMI 1640Medium was transferred into LES medium and RPMI 1640Medium separately，and cultured at37℃.Morphous and reproductive mode of B.hominiswas observed.
2 Results
2.1 Morphous and reproductive mode of B.hoministrans-ferred in same culture medium when B.hominis was transferred from LES medium to LES medium or from RPMI1640Medium to RPMI1640Medium，Morphous and repro-ductive mode of B.hominis was stable.All4forms of B.hominis（vacuolar form，granular form，ameba form and cyst form）could be found in2culture mediums separately（see fig1）.The main forms were vacuolar form and granu-lar form.Ameba form and cyst form could be found at fist24～48h after transfer，Ameba formwas appeared later than cyst form.The main reproductive mode of B.hominis was binary fission，a few of gemmation was exist.
After over one week's grow，vacuolar form and granu-lar form could only be found in culture mediums and fission of B.hominis had stagnated or stopped.At this time，in-tensive active small granular substances were found in some of granular form cell.Continuous observation found that cell ruptured and active granular substances overflowed and dif-fused into culture medium at last（see fig.2～10）.But new parasites could not be found after continuously culture or transferred to new culture medium.
Fig1 Morphous of B.hominis（Iodine stain，×400）（略）
1.vacuolar form;2.granular form;3.ameba form;4.cyst form
2.2 Morphous and reproductive mode of B.hominis with Changed culture conditions when B.hominis was trans-ferred from LES medium to RPMI1640Medium，temporal growth retardation of B.hominis had lasted for about48～72h，and fission and increasing density of B.hominis could not be found in RPMI1640medium.Then multiple repro-ductive mode of were found coexistence，including binary fission，ternary fission，tetra-fission，plasmotomy，en-dodyogeny，gemmary and schizogony（see fig.2）.As for plasmotomy，parent cell primarily divided into some consec-utive parts，and then became a few of daughter cell with dif-ferent size.Gemmary could be easily observed，and one or two daughter cell appeared on the surface of parent cell.Both endodyogeny and schizogony were not common.En-dodyogony produced two large progeny within the interior of parent cell.When schizogony occurred，progeny filled the parent cell.Numbers of progeny range from a few to hun-dreds were released to the culture medium until the cell bursts，and the organisms diminishing inversely in size as numbers increase.Progeny of widely varying size and devel-opment occupied in the same parent cell.Progeny cell could be vacuolar form or granular form.The coexistence phe-nomenon of diversified reproductive mode of Blastocystis Hominis lasted less than24h，and then ternary fission，tetra-fission，plasmotomy，endodyogeny，gemmary and schizo-gony were disappeared.The main reproductive mode was still binary fission.But when B.hominis was transferred from RPMI1640medium to LES Medium，on the contrary， this coexistence phenomenon of diversified reproductive mode of Blastocystis Hominis was not observed.Morphous and reproductive modes of B.hominis were same as that of transfer in same culture medium.
Fig2 Reproductive mode of B.hominis（Iodine stain，×400）（略）
1.binary fission;2.ternary fission;3.tetra-fission;4.plasmotomy;5.endodyogeny;6，7.gemmary;8，9.schizogony10doubtful sporulation. 3 Discussion
Diagnosis of B.hominis mainly depends on morphous by microscopic examination，alter of morphous is very im-portant in clinic.But external environment has great effec-tive on the growth of B.hominis.B.hominis grows to greater numbers and more consistently on egg slant medium with horse serum than on Diamond TPY medium ［8］ .B.hominis grows rapidly in RPMI1640medium，density is higher and survival time is longer than that in LES medi-um ［9］ .The results demonstrate that different culture medi-ums affect the growth of B.hominis.Studies on ultrastruc-ture show that outside environment also influence the mor-phous and structures，such as osmotic pressure，drugs and metabolism condition，and so on ［4，10］ .
In our study，binary fission，ternary fission，tetra-fission，plasmotomy，endodyogeny，gemmary and schizo-gony coexist when B.hominis was transferred from LES medium to RPMI1640Medium，this coexistence phe-nomenon of diversified reproductive mode of B.hominiswas appeared temporarily and last less than24h.Our results show that reproductive mode of B.hominis changes greatly with alteration of culture condition.On the other hand，B.hominis shows well adaptability to external environment.When culture condition changes abruptly，growth temporari-ly suspends，then multiple reproductive mode appears to adapt and rapidly fit with the alteration of environment，at last predominant binary fission recovers.
At the last stage of culture，we observe a phenomenon which is similar to sporation.Intensive active small granular substances fill in some of granular form cell or local in the center of cell.Small granular substances appear irregular movement within the cell.When cell ruptures，active gran-ular substances overflowed and diffused into culture medi-um.Butwe could not prove sporation of B.hominisbecause new cell were not found after continuously culture or trans-ferred to new culture medium.
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