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.

1 2 3 4 5 6 7
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, ta 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).

1 2 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. Mi 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.

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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.
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