Improvement of an Isolation Medium for Actinomycetes

By comparing differences among the effects of several isolation mediums on soil actinomycetes, we attempted to find a isolation medium that could result in actionomycetes with high diversity. Soil samples were isolated by the dilution plateing method, and genomic DNA was extracted using phenol-chloroform method. After amplification by 16S rDNA PCR, PCR products were sequenced and undertaken phylogenetic analysis in order to determine the ownership of isolates. The results showed that the isolated strains belonged to 15 genuses; as for the established medium in the present paper, ZSSE was easier to isolate rare actiomycetes with rich diversity, and could replace routine agars such as Gause`s No.1, HV and ISP5; CHV was easier to isolate micromonospora.

Actinomycetes were bacterium that could form branches, and such branches were mycelium formed by thallus.They were generally saprophytic, and widely distributed in nature, and only a small number of them coexisted with plants.They distributed in soil, air and water in the form of spores or mycelium, especially in slightly alkaline or neutral soil with low water content and rich organic matters.
Ever since Wakksman and Umezwa found the usage diversity of actinomycetes, actionmycetes have been applied as a producing strain for antibiotics, vitamins, enzymes and enzyme inhibitors, and thus have been a microbial population with huge application values.
Acquiring new strains was a necessary condition for modern actinomycetes resource development, and therefore, studies on the separation methods tended to be very critical.There have been great progresses made in selective isolation of rate actinomycetes by researchers at home or abroad in recent years (Jiang, 2006, PP. 181-183;Li, 2002, PP. 105-108;Li, 2003, PP. 114-117;Si, 2004, PP. 61-65).However, a large number of actinomycetes couldn't be separated, and as seen from modern molecular biology studies, there were at least 90% unknown actinomycetes that couldn't be isolated existed in nature (Duan, 2007, PP. 32-33).Accordingly, in the present paper, we investigated the differences among effects of several mediums on isolating soil actinomycetes, and attempted to find a separation medium for isolating actinomycetes with high species diversity.

Soil
Soil samples were sampled from different regions in Shanxi Province and all collected 15cm away from the earth's surface (Table 1).

Medium
Preparation of soil extracts: 400g dried soil without miscellaneous stones were added to 960mL tap water, completely agitated, and sterilized by autoclaving at 121℃.After standing a while, the resultant solution was filtered twice by filter cloth and filter paper subsequently, and sterilized at 121℃ for 20 min.The filtrate was used to prepare soil leaching juice agar medium.

Soil treatment
Soil samples were dried in ventilated dark room for 7 days.
1.2.2Separation method 50mg/L nalidixic acid and 100mg/L nystatin were added to six mediums as an inhibitor of bacteria, viz.HV, CHV, ZSSE, Gause`s No.1, soil extracts agar and glycerol-asparagine.Samples were diluted in a gradient, and 10 -6 and 10 -7 of stock solution was coated in the plates.the plates were incubated at 28 ℃.Actinomycetes colonies with different forms were obtained at different periods, and colonies of all separated plated were counted.

Separation effects of mediums
As seen from Figure 1 and Table 2, among six mediums, the number of non-actinomycetes in plates from Gause`s No.1 was the least while the size of colony was biggest.The number of non-actinomycetes in plates from HV and CHV was the most.The number of actinomycetes in plates from ZSSE was the most, and CHV followed.Aerial mycelium of actinomycetes in soil extracts agar and glycerol-asparagine grew slowly, and several colonies were tough to distinguish from others.

Discussions
Although there were great differences in genus among the obtained rare actinomycetes resulted from different soil samples, genus diversity of the isolated actinomycetes increased with the enrichment of vegetations.Generally, the strains isolated from Gause`s No.1 were mainly common Streptomyces, other rate actinomycetes were tough to isolate, and other bacteria excluding actinomycetes grew less; the strains isolated from ZSSE, HV, CHV, soil extracts agar and glycerol-asparagine were mainly other bacteria, and such bacteria could be mainly gram-positive.Accordingly, the added inhibitors failed to work effectively; the strains isolated from glycerol-asparagine were mostly Streptomyces, the resultant colonies formed aerial mycelium slowly, and thus it was hard to distinguish the rare actinomycetes; genus from HV and had many sorts of actinomycetes, and especially for soil extracts agar, the number of bacteria was so higher that large colony was easy to select other bacteria after long incubation; CHV could enhance the number and diversity of Micromonospora, but decreased the diversity of other actinomycetes; ZSSE resulted in a rich species diversity of actinomycetes with the largest sorts and number of rare actinomycetes, and its colony size was uniform.The growth of bacteria couldn't basically hamper the selection of actinomycetes colonies.
Taken together, ZSSE medium established in the present paper possessed the nutritional characteristics of both Gause`s No.1 and soil leaching juice, and was easier to isolate rare actinomycetes with rich diversity.It overcame the shortcomings that general mediums couldn't take both specificity and diversity into account, and could basically replace Gause`s No.1, HV and glycerol-asparagine to isolate actinomycetes; compared to soil extracts agar, bacteria biomass was less, and it was easier to select actinomycetes.CHV medium was more suitable for the separation of micromonospora.