Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 480-488 ISSN: 2319-7706 Volume 3 Number 2 (2014) pp. 480-488 http://www.ijcmas.com Original Research Article Culture of Hemitrichia serpula on wide range of agar medium P.Phate* and R.L.Mishra Department of Botany J.S.M. College Alibag, District Raigad 402 201, India *Corresponding author ABSTRACT Keywords Indigenous corn flour agar; myxomycetes; relative humidity; Among the known species of myxomycetes, Hemitrichia serpula is one of the most distinctive myxomyceteous genus that do not fall in the list of 10 % spore to spore cultured species. This is the first attempt to culture the selected specie on a wide range of agar medium. The aim is to study the life cycle on culture plates and also the impact of temperature and relative humidity on the growth. In the present study a new media named Indigenous Corn flour agar was formulated and found to be excellent for the growth of said species. Introduction Myxomycetes are the small, homogenous group of fungus-like organisms, with approximately 700 species known worldwide. They are the nature s most extraordinary creatures and are often known as True slime molds or Acellular Slime Molds. Myxomycetes are typically collected as fruiting bodies and plasmodia and occur wherever the conditions are suitable for them on earth s surface. Only Physarum and Didymium (Aldrich & Daniel, 1982) serves as an excellent model system for study but give only a partial view of the biology of the Myxomycetes and hence aim is to get a wide range of Myxomycetes into agar culture which can be further used as material for student research project and such material will also serve as primary research organisms in laboratories around the world. 480 The main aim of the present study is to discuss the cultural aspect of Hemitrichia serpula on different agar medium and also the effect of relative humidity and temperature. Most of the easily cultured species are of the order Physarales and no extensive studies have been carried out on the nutritional requirements of the slime molds, so aim is to find out the nutritional requirement of the selected species i.e., Hemitrichia serpula. Materials and Methods Collection The sampling was done in the period 2010-2013 from different localities like Alibag, Aakshi and Jirad of Raigad District (Maharashtra) throughout the year. All the localities were geo referenced. The Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 480-488 specimens were immediately glued along with substratum in plastic boxes, match boxes or cardboard boxes but plastic boxes were found to be best for storage purpose. The specimens were air dried to prevent contamination with other organisms and unique numbers were given to the specimens to carry out the work with ease. A paper containing the details like name of specimen, date of collection, place of collection, habitat etc. was mounted on each plastic box. To study the external morphology the specimens were observed under stereomicroscope and photographs were taken. Yeast Extract Agar, Carrot Agar, Indigenous Corn flour Agar, Oat Meal Agar and Extract Agar were tried to observe the details of growth of species. Extract agar media was prepared by soaking the natural substrate of H. serpula (50 gm in 1liter DW) for 24 hrs and filtering the supernatant through cheese cloth. The volume was then makeup with DW up to 1 liter and then agar was added. A new Indigenous Corn flour agar recipe was formulated in the present study and tried to observe the growth of the species. The bottom of the petriplates was divided into four quadrants with the help of fine marking pen. The spores were then inoculated in each of the quadrant by gently touching the forcep to the surface of the agar so that some spores remain submerged and some left on the surface. The areas of spores inoculated in each quadrant of the petriplates were marked in the form of small circles to check the germination regularly and easily. The plates were incubated in stability chamber at various ranges of temperature and relative humidity. Slide preparation Temporary slides were prepared to study the internal morphology like nature of peridium, columella, capillitium, spore colour, spore ornamentation etc. of the specimens. For semi permanent preparation of slides, mounting media like Amann s lactophenol medium and Hantsch s fluid (Martin and Alexopolous, 1969) were used. A microscope equipped with an ocular micrometer and oil immersion objective was used for measurement of spore size, capillitium diameter etc. Photomicrographs were taken showing the details of internal structure. Spore germination was found in days to few months and was observed by inverting the petriplates, still closed, on the stage of a compound microscope from which the clips had been removed. The petriplates were generally found associated with filamentous fungi. In order to clean the culture, sub - culturing was carried several times by transferring the clear blocks of agar containing the myxoamoebae and swarmers to a new agar plate. Another method used for sub-culture was to find the clear area of agar containing the myxoamoebae and swarmers, adding a drop of water on that area and transferring them with the help of capillary tube and touching the same tube in new petriplates repeatedly releasing the same. Identification For identification of material up to species level, the literature of Lakhanpal and Mukerji (1981) was referred. Culture The spores from the plasmodiocarpus type of fruiting body were collected with the help of alcohol flamed forcep or needle. Wide range of agar media like 0.75 Water Agar (0.75 WA), 1.5% Water Agar (1.5 WA), Asparagine Mannitol Agar, weak 481 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 480-488 Some 1.5 WA culture plates with spores were flooded with 2ml DW on the surface of agar to observe the role of distilled water in spore germination and plasmodial formation. relative humidity for germination of spores and young plasmodium development in Hemitrichia serpula is 25 C and 95% respectively which also suggest that temperature and relative humidity are the major environmental factors which play very important role in the life cycle. Hanging drop method was also used to get the successful germination of spores in cavity slides. The spores germinated in cavity slides were then transferred to the agar plates of different media. Successful germination of spores of H. serpula was found to be in Distilled water in both Hanging drop method and Culture plates. The spores treated with 1% Bile salts (Elliott, 1948) germinated in 5-10 days in 1.5 WA plates. The treatment with 0.001% Tween 80 (Indira, 1969) was found to be ineffective in this case. Different methods were tried to get the successful germination of spores in hanging drop and on culture plates. Some agar plates were inoculated with spores soaked in distilled water for half an hour, some with spores treated with 1% bile salts for 30 minutes and 0.001% Tween 80 for 7-10 min respectively. Similar treated spores were also tried to germinate in hanging drop method. It was found that the germination rate was more in plates with spore suspension (spores soaked in DW for half an hour) than plates where the spores were direct inoculated in four quadrants which suggest the role of water in germination of spores of H. serpula. The above said media were continuously tried to get the successful culture. Some agar plates containing the swarmers and myxoamoebae were also sprinkled with sterilized oat to get the results. From the wide range of media used in the study 1.5% Water Agar, Oat Agar and Indigenous Corn flour Agar were found to be best for the germination, fusion of gametes and formation of plasmodium. The Carrot Agar medium strict to be good for subculture. Asparagine Mannitol agar and weak Yeast extract agar remain ineffective. Degree of growth on Extract agar was found to be less during the studies. The new Indigenous Corn flour Agar media used in the study shows excellent result for both spore germination and plasmodial formation. The specie showed germination in 2-4 days and plasmodium tracks arises in 20 days on said culture plates. The variations in temperature and relative humidity were done to note down the cultural changes. All the results obtained were maintained in the form of photographs. Results and Discussion The observation and results were tabulated (tables 1-5). After trying the various ranges of temperature and relative humidity, it is found that the standard temperature and 482 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 480-488 Table.1 Details of the Georeferenced localities Name of the specie Habitat Year of collection Locality GPS Alibag Dried leaves of Cocus nucifera Hemitrichia serpula 2010-2013 18 38'35" N,72 52'14"E 18 37'47" N,72 73'22"E 18 45'25" N,72 54'14"E Aakshi Jirad Table.2 Spore germination (Temperature - 25 C and Relative Humidity - 95%) Spore germination Hanging drop Culture plate Distilled water + + Tween 80 Bile salts + + Germination: Absent (-) & Present (+) Spore suspension in Table.3 Degree of growth on different culture media at given temperature and relative humidity Growth T- 22 C and RH 85% Medium used 1% Water Agar 1.5% Water Agar Asparagine Mannitol Agar Weak Yeast Extract Agar Indigenous Corn flour Agar Oat Agar Carrot Agar Extract Agar T-25 and RH 95% Spore germination - Formation of plasmodium - Spore germination + +++ - Formation of plasmodium + +++ - - - - - - - +++ +++ - - +++ + +++ +++ + Degree of growth: Absent (-), Medium (+), Good (++), Very good (+++) T- Temperature and RH- Relative Humidity 483 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 480-488 Table.4 Comparison of spore germination time period (days) on different agar Germination time required (days) 1-4 days 2-4 days 2-4 days Medium used 1.5 % Water agar Indigenous Corn flour agar Oat agar Table.5 Time period required (days) for plasmodial formation in Hemitrichia serpula on 1.5 Water agar media and Indigenous Corn flour agar Medium Germination time required (days) Plasmodium formation (days) 1.5% Water Agar Indigenous Corn flour agar 1-4 days 2-4 days 15days 20 days Figure.1 Plasmodiocarpus fruiting body of Hemitrichia serpula on dried leaves of Cocus nucifera. Figure.2 Photomicrographs of H. serpula A- Capillitium and Spores. B- Reticulate spores. 484 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 480-488 Figure.3 Spore germination on 1.5 WA plates at pH 5.6, 25 spores treated with 1% Bile salts. and RH 95% Figure.4 Culture of H. serpula on 1.5 Water Agar plates at pH 5.6, 25 C and RH 95%, ASpore germination and wandering myxoamoebae and swarmers. B Myxoamoebae and swarmers trying to fuse. C Fusion of gametes to form plasmodium. D - Initiation of plasmodium. 485 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 480-488 Figure.5 Subculture results on Carrot agar plates at pH 6.5, 25 C and RH 95%, AMyxoamoebae and swarmers. B - Young plasmodium on bacterial colonies. Figure.6 Culture of H. serpula on Indigenous Corn flour agar at pH 6.5, 25 C and RH 95%, A- Myxoamoebae and swarmers feeding on the agar plate. B- Formation of young plasmodium. C Plasmodium D Plasmodial tracks on agar plates 486 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 480-488 Figure.7 Spore germination on Oat agar plates at pH 6.5, T- 25 During study it was found that, 1.5 WA plates containing the gametes when flooded with distilled water give rise to early plasmodium as compare to the 1.5 WA plates without distilled water which again suggest the role of distilled water in formation of plasmodium in Hemitrichia serpula. No bacterial food material was added to the plates, all the cultures were grown on the original food inoculums grown along with the culture. The above results clear that the one can use high nutrient medium like Indigenous Corn flour Agar and Oat Agar for spore germination instead of routinely using 1.5 Water Agar and 1 % Water Agar. Hence the study somewhat had broadened the range of nutrition of Hemitrichia serpula. 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