Experiment Research On The Morphology Of Geotrichum Silvicola And Physiology Experiments

Objective: Geotrichum sivicola, a new strain in Geotrichum, was isolated from a children's kerion-like lesions and a patient's blood suffered with toxic epidermal necrolysis one after another in our department. The morpholgy of the blood isolation has been partly studied.In our research, the morphology characteristics of the blood isolation on the five kinds of culture media were observed to identify more suitable medium for its growth. Further more, by the conventional physiology experiments, we could understand the physiological characteristics of Geotrichum sivicola, which could provide assistance for the identification of laboratory work and was of great significance for clinical diagnosis on the infection of Geotrichum sivicola.Methods1 Morphology observation on the five kinds of media1.1 General colony observation Put the blood isolation at the ordinary temperature and inoculate it respectively on the media of Sabouraud's Agar (SDA), Patato Glucose Agar (PDA), Malt Extract Agar (MEA), Corn Meal Agar(CMA) and Czapek's Agar(CZA) after three days, and culture it at 37℃and 27℃for two weeks, then observe it's development and measure the diameters of colonies every day, finally draw growth curves.1.2 Direct examination Put some samples taken from the five kinds of media colonies every day on the glass slide, cover them with slip when dissolved in the solution of cotton blue stain, then observe it under light microscope.1.3 Slide culture Inoculate the blood isolation on the micro- culture of the above five kinds of media at 37℃and 27℃for two weeks, and observe the growth of hypha and spores every day.2 Physiology experiments2.1 Germ tube test Pick some colonies into 0.5 ml human serum at 37℃for 3 h, then observe the germ tube.2.2 Chlamydospore formation test Inoculate the blood isola- tion onto the Corn-Tween 80 agar and culture it at the ordinary temperature for 24-72 h, then observe the chlamydospore.2.3 Nitrate assimilation test Make up fungal suspension solution and add it into the Nitrogen source assimilation agar base, then add some nitre and peptone on the medium surface. Culture at the ordinary temperature and observe it every day.2.4 Cycloheximide tolerance test Make up fungal suspension solution and inoculate it on the SDA with cycloheximide and the one without cycloheximide, then culture it at the ordinary temperature for 3 days. Observe the result.2.5 Urease test Inoculate the blood isolation on the Urea agar, and culture it at the ordinary temperature for 1 week, then observe the medium color. 2.6 Sugar fermentation test Inoculate the blood isolation into the Sugar fermentation broth base agar and culture it at 37℃, then observe the result.2.7 Carbon source assimilation test Use the Test Kit of API 20C AUX, analyse the result using the API 20C AUX code book.Results1 Morphology observation on the five kinds of media1.1 General colony observation1.1.1 Draw growth curves The fungus growed faster in the first week on the PDA, SDA and MEA than CMA and CZA .The growth was better at 27℃than 37℃.1.1.2 The colony diameterThe comparison under the two temperatures on the same medium showed that the colony diameters at 27℃was larger than 37℃in the two weeks(P<0.05).The comparison between random two media at 27℃showed that the colony diameters on PDA was the largest in all the media, and the diameters on SDA, MEA were larger than that on CMA,CZA(P<0.05). No difference between PDA and SDA on the first day(P>0.05).The comparison of colony diameters between random two media at 37℃was similar to 27℃,and the difference was significantly(P<0.05).1.1.3 General colony observationWhen cultured for 1 day, it began to grow presented with whitish farinose, hairy, membranousand flat, often dry. Then it gradually transformed into wettish, sticky yeast-like type colony.1.2 Direct microscope examinationAt 27℃: it showed plentiful rectangular, tubby arthroconidia with different cubage, round or oval microspores and respectable budding cells and germ tubes, chlamydospores and branched hyphae with septa could be seen.At 37℃: there could be seen numbers of rectangular arthroconidia, fewer chlamydospores, budding cells and germ tubes, hyphae.1.3 Slide cultureAt 27℃: SDA: plentiful branched hyphae with septa, abundant rectangular, oval arthroconidia, budding cells and germ tubes often presented, arthric hyphae extended from germ tube soon disarticulating into rectangular arthroconidia. PDA: it was similar to SDA. MEA: large numbers of arthric hyphae and rectangular arthroconidia, more budding cells and germ tubes. CMA: thinnish septate hyphae could be seen, dispersed or short-chained rectangular, oval spores, fewer budding cells. CZA: it was similar to CMA on the whole, but more thinnish hyphae.At 37℃: SDA: large numbers of rectangular, oval arthroconidia and round conidia, gradually increasing budding spores and germ tubes, multilateral sprouting could be seen. Hyphae were infrequent often with di- or trichotomous. PDA: it was similar to SDA. Blastospores and germ tubes were more than SDA. MEA: respectable arthric hyphae, rectangular arthroconidia, budding cells, germ tubes. CMA: some thinnish branched hyphae with septa. Blastospores and germ tubes were fewer than 27℃. CZA: budding cells and germ tubes were less than CMA.2 Physiology experiments2.1 Germ tube test negative result2.2 Chlamydospore formation test positive result2.3 Nitrate assimilation test negative result2.4 Cycloheximide tolerance test positive result2.5 Urease test weak positive result2.6 Sugar fermentation test negative result2.7 Carbon source assimilation test using the API 20C AUX code book, it showed the strain was Geotrichum penicillium.Conclusion1 The blood isolation grows faster and better on PDA, SDA and MEA, which shows that the three media are the more suitable ones for Geotrichum silvicola, and provides a reference for choicing suitable medium to culture and identify the strain.2 The results of culturing at the two temperatures indicates that the strain grows better at 27℃than 37℃.3 Urease test can be looked as the identification guidance among the genus of Geotrichum.4 The results of API 20C AUX and urease test could provide the guidance to identify the strain and Trichosporon.