PHNG PHP PHN LP X KHUN CHI STREPTOMYCES PGS.TS.

Bin Vn Minh Trng i hc S phm H Hu X khun c tn khoa hc l Actinomycetes, l mt nhm vi khun tht (Eubacteria) phn b rt rng ri trong trong t, nc, bn ao v trong cc c cht hu c khc, nhng tp trung nhiu nht l trong t . phn lp x khun chi Streptomyces ngi ta thng dng phng php thch a vi cc mi trng dnh ring cho x khun nh cc mi trng Gause, mi trng Krasilnicov, mi trng tinh bt sulfate m Trong bi vit ny chng ti gii thiu cch phn lp x khun thuc chi Streptomyces t t vn hoc t i khi dng mi trng Gause-1. 1. Chun b a. Dng c, thit b Bung cy v trng; cn k thut 1ci; gi ng nghim 1 ci c 10 ng nghim; a thy tinh 1 ci; que trang 1 ci; que cy 2 ci, n cn 1 ci, bp in 1 ci, soong 2 lt 1 ci; besher loi 500 ml 1 ci; loi 250 ml 1 ci; pipt loi 1ml 1 ci, loi 5 ml 1 ci; ng nghim 30 ng; chai tam gic loi 250 ml 3 chai; ko ct giy 1ci; hp Petri 20 hp/ nhm; khn lau 2 ci; bt la 1 ci.

Hnh 1: 1. Que trang bng thy tinh; 2. que cy mc; 3. que cy thng ; 4. cn in t mini; 5. My ly tm; 6 t cy v 7. ni hp p lc hi nc

b. Nguyn liu v mu vt - Bng khng thm nc 100 g; nc ct 2 lt; cn t 100 ml; giy thm 1 hp; giy bo c gi u ng nghim v hp Petri; dy buc bng nilon - Mi trng Gause-1 thch a 20 hp, thch nghing 30 ng nui cy x khun - Chai tam gic loi 250 ml cha mi trng Gause thch pha ch trc - t vn, hoc t i, mi loi 10 50g - Cht c ch vi khun : dung dch K2Cr2O7 0,4% * Mi trng Gause-1 iu chnh (g/l): 20g tinh bt; 3g MgSO4.7H2O; 3g K2HPO4; 1g KNO3 ; 0,5 g NaCl; 0,01g FeSO4.7H2O; 20 g agar, nc ct 1 lt. Hp kh trng 121 0C trong 30 pht. 2. Nguyn tc phn lp - Gieo cy mu x khun pha long trn mi trng dinh dng c trng ; nui dng trong iu kin thch hp cho cc khun lc mc tch bit nhau. - Cy tch t khun lc x khun mc tch bit sang ng mi trng dinh dng thch nghing thu nhn chng x khun thun khit. 3. Cc bc tin hnh a. Pha ch mi trng Gause-1 thch a, thch nghing - Cn, ong chnh xc tng thnh phn mi trng

- Ha tan cc cht vo nc, un v khuy cho tan - Lm trong mi trng, kim tra th tch v pH - Rt mi trng vo dng c (vi ng nghim th tch, hp Petri chiu cao, bnh tam gic th tch bnh. Lm nt bng loi bng khng thm nc, bao giy bo. - Kh trng ni hp p lc hi nc 1,0 atm/30 pht - Ch mi trng thch nghing v thch a. b. Thu mu, nui tch ly v pha long - Thu mu t hoc cc c cht c x khun t 10 50g, trng hp s x khun c mt t trong mu th phi nui tch ly bng cch mu, b sung cht dinh dng v cc iu kin l, ha cn thit hoc b sung cc cht c ch sinh trng ca cc vi sinh vt i km. - Cn 10 g mu c x khun cho vo bnh tam gic cha 90 ml nc ct v trng, khuy bng a thy tinh cho tan ht mu, t a c dung dch mu pha long 1:10. Rt dch t trn vo cc ng nghim ca my li tm, iu chnh my chy vi tc 2000 vng/pht duy tr chy 15 pht. - Ly 1ml dch huyn ph pha long 1:10 cho vo ng nghim tip theo c cha 9 ml nc v trng, ta c dch huyn ph c pha long 1:100. C tip tc lm nh vy ta c cc pha long tip theo 1:1000; 1:10.000... Ty theo s lng x khun trong t nhiu hay t m ta pha long n nng cn thit, thch hp khun lc mc ln tch ri nhau ra. Thng thng pha long dch t nng 10 6 10 8. c. Phn lp x khun chi Streptomyces theo mt trong hai cch sau: - Cch th nht: cy dch huyn ph ln mi trng c trong hp Petri + Dng pipet v trng nh 0,1 ml dch huyn ph pha long nng 10 6 10 8 ln b mt ca mi trng thch trong hp Petri. + Dng que trang (xem hnh 1) dn u git dch huyn ph trn ton b b mt mi trng hp thch. + y hp Petri, gi li ri lt sp v t vo t m nhit 28 30 0C t 7 14 ngy ri tin hnh quan st v phn bit khun lc x khun vi cc loi vi sinh vt khc. - Cch th hai: trn dch mu pha long trong cc mi trng thch nng chy Dng pipet v trng nh 1ml dch huyn ph pha long nng 10 6 10 8 vo mi trng thch nng chy v ngui n 45 50 0C trong bnh tam gic, lc u ri ra cc hp Petri mi hp khong 15 ml. y hp Petri, gi li ri lt sp v t vo t m nhit 28 30 0C t 7 14 ngy. Sau thi gian thy c khun lc x khun mc tch bit, ta dng que cy mc, mc ly khi thch c khun lc cy truyn qua ng mi trng thch nghing cha sn mi trng Gause -1 trong iu kin v trng. t vo t m nhit 28 30 0C t 7 14 ngy. Ly ra quan st nhn din khun lc x khun, lm tiu bn quan st h si x khun v bo t ca chng. (xem hnh 2, hnh c tnh cht minh ha).

3(a)

3(b)

Hnh 2: 1. Hnh dng khun lc x khun, 2. Hnh dng h si, 3. B mt mng bo t dng phng (3a) v dng gai (3b) di knh hin vi in t.

5. Nhng im cn lu phn lp x khun chng ti dng dch t pha long 10 6 10 8. M i trng Gause-1 l mi trng c thnh phn thch hp phn lp x khun chi Streptomyces. ngn chn s nhim khun t khng kh, cc thao tc phi tuyt i v trng Cc chng x khun sau khi lm sch cn c kim tra li bng cch quan st hnh dng khun lc, hnh dng h si v b mt bo t di knh hin vi. Ti liu tham kho [1] V Th Minh c, Thc tp vi sinh vt hc, NXB i hc Quc gia H Ni nm 2001. [2] Egorov N.X., Thc tp Vi sinh vt, NXB Mir, Maxcva. Nguyn Ln Dng dch, NXB H&THCN H Ni, 1983. [3] Kuster E., and S.T. Williams, 1964. Selection of media for isolation of streptomyces. Nature (London) 202: 928 929.

Method for isolation of actinomycete Streptomyces strain

Abstract: - Actinomycetes belonging to the kingdom Eubacteria, are widely distributed in soil, water, pond sludge and organic substances. To isolate actinomycete Streptomyces, an agar plate method with the Gause-1 medium and serial dilution of soil solution at concentrations from 10 6 to 10 8 were used. - To avoid the contamination from air, the manipulation should be completely sterile. - The cleaned actinomycete Streptomyces strains should be confirmed by observing the shape of colonies, hyphae and spore surface under the microscope. --------------------

Phng php phn lp x khun chi Streptomyces

Tm tt: - X khun c tn khoa hc l Actinomycetes, l mt nhm vi khun tht ( Eubacteria) phn b rng ri trong trong t, nc, bn ao v trong cc c cht hu c khc. phn lp x khun chi Streptomyces ta dng phng php thch a vi mi trng Gause -1 v dch t pha long nng 10 6 10 8. - ngn chn s nhim khun t khng kh, cc thao tc phi tuyt i v trng - Cc chng x khun sau khi lm sch cn c kim tra li bng cch quan st hnh dng khun lc, hnh dng h si v b mt bo t di knh hin vi. ---------------------------------