24. Thực tập vi sinh vật chuyên ngành - Pgs.Ts.Nguyễn Xuân Thành PDF

Trng i hc Nng nghip H Ni Gio trnh thc tp vi sinh vt chuyn ngnh 1
B GIO DC V O TO
TRNG I HC NNG NGHIP H NI
***

NGUYN XUN THNH (Ch bin) V TH HON
NGUYN TH BNH - INH HNG DUYN

Ch bin & hiu nh
PGS.TS NGUYN XUN THNH

THC TP VI SINH VT
Chuyn ngnh Chuyn ngnh Chuyn ngnh Chuyn ngnh

H Ni - 2007


MC LC

Ni dung Trang
Bi s 1: Trang thit b cn thit trong nghin cu vi sinh vt 1
Bi s 2: Phng php c nh tiu bn v nhum t bo vi sinh vt 12
Bi s 3: Chun b dng c v mi trng nui cy vi sinh vt 16
Bi s 4: Nui cy vi sinh vt 26
Bi s 5: Phng php ly mu phn tch vi sinh vt 31
Bi s 6: Phng php phn tch vi sinh vt 33
Bi s 7: nh gi c tnh sinh hc ca vi sinh vt 37
Bi s 8: Phn lp tuyn chn Azotobacter t t 40
Bi s 9: Phng php ly mu v phn lp tuyn chn vi khun
Rhizobium
42
Bi s 10: Phng php xc nh nhanh trao i cht vi sinh vt 46
Bi s 11: Vi sinh vt trong mi trng 50
Bi s 12: Phng php xc nh nm men, nm mc, to v
nguyn sinh ng vt
53
Bi s 13: Qu trnh chuyn ho nit di tc dng ca vi sinh vt 60
Bi s 14: Chuyn ho lu hunh di tc dng ca vi sinh vt 63
Bi s 15: Vi sinh vt phn gii ln (phospho) 65
Bi s 16: Enzym trong qu trnh trao i nit, phospho, lu hunh 67
Bi s 17: Xc nh sinh khi vi sinh vt t 75
Bi s 18: Sinh trng ca vi sinh vt 83
Bi s 19: Thm quan kin tp v vi sinh vt 86
Ph lc 88
Ti liu tham kho 91

Bi s 1
TRANG THIT B CN THIT TRONG NGHIN CU VI SINH VT
Mc ch yu cu:
+ Nm c nhng my mc, trang thit b cn thit trong nghin cu v
vi sinh vt.
+ Bit s dng thnh tho mt s my mc thng dng ca phng nghin cu.
+ Hiu c tm quan trng ca cng tc tiu c, kh trng.
+ S dng thnh tho knh hin vi
+ Phn bit cc dng hnh thi ca vi sinh vt
Ni dung :
+ Gii thiu nhng trang thit b cn thit trong phng nghin cu vi sinh vt.
+ Gii thiu cc dng c v nguyn liu cn thit nghin cu vi sinh
vt: dng c lc, kh trng, dng c quang hc, dng c o lng, mi trng
nui cy.
+ Thao tc vn hnh v s dng cc trang thit b trong phng nghin cu
vi sinh vt
+ Cu to v s dng, bo qun knh hin vi
+ Quan st hnh thi vi sinh vt
I. MY MC
1. T nui cy vi sinh vt (Incubator)
T nui cy hay cn gi l t nh n l thit b quan trng dng trong cng
tc nghin cu vi sinh vt, v nhit trong t c th thay i t 0
0
90
0
C tu
theo mun ca ngi nghin cu v nhit trong t sau khi c xc nh
th lun lun trng thi n nh trong sut thi gian nui cy.
1.1. Cu to
Cu to v t nui cy c 2 lp: lp trong l kim loi dn nhit gi nhit
bn trong ca t, lp ngoi l kim loi dy hn v c bc pha trong bi
mt cht cch nhit (amiant). Gia lp trong v lp ngoi l khong trng gi
cho nhit trong t t b bin i.
Trong t c b phn cm nhit bo nhit ln xung cho r-le hot
ng v qut gi c lp phn gia thn iu ho nhit bn trong.
Phn ngoi t nui c h thng bng in t iu chnh nhit theo
yu cu ca nghin cu. Pha trn t c lp van an ton, nu nhit trong t
vt qu dao ng bin , van an ton s t ngt.
1.2. Cch s dng
ng mch in, bm nt m cng tc t (c th gi vi giy n khi xut
hin n bo trn bng in t). Sau bm nt t nhit v thi gian (set
up) theo yu cu nui cy, iu chnh nhit v thi gian bng n nt tng

ng mi tn ln hoc xung. Nhit trong t m tng dn v t ti nhit
xc nh.
Nhit s c duy tr trong sut thi gian nui cy nh sn. Trn
bng in t lun xut hin ch s bo nhit thc t trong t. Khi thi gian
nui cy, t s pht ra ting bo hiu v r le t ngt t ng tt ch lm
vic.
Nu mun nui cy lin tc lu di c th khng cn t ch thi gian,
ch t nhit . Khi no mun kt thc th bm nt tt cng tc ngun.
1.3. Khi s dng my cn ch nhng im sau y
+ Hiu chnh thit b trc khi s dng.
+ Phi ni t vi dy t v kim tra in th ca my vi in th ni
t my xem c ging nhau khng, trng hp khng ging nhau phi dng
bin th.
+ Khi s dng t m ln u, phi kim tra b phn iu chnh nhit
xem c chnh xc khng, nhit trong t c u khng.
+ Ca t lun lun phi ng kn, tr khi ly hoc cho nguyn liu vo
nui cy nhng cng khng c m ca t rng v lu.
+ Lun lun phi m bo cho t m c kh ro, sch s, phi cho t
hot ng thng xuyn nht l nhng hm tri m. Khi lm cc cht dch
nui cy hoc lm bn trong t, phi lau chi v st trng ngay.
+ Nn t trong t 1 cc nc v trng trong qu trnh nui cy gip
cho qut gi hot ng tt.
+ Khi khng dng, tt cng tc in v rt phch cm in ra.
2. T sy kh (Drying oven)
2.1. Tc dng
Dng t sy kh kh trng cc dng c thy tinh, s nh ng nghim,
xi lanh, hp lng, cc, phu, ci chy s..., cc kim kh nh dao, ko, panh v
cc dng c khc khng c nc khc nh bng, bng, vi... Tr vt liu lm t
cao su v mi trng nui cy khng c kh trng bng t sy kh.
Nguyn l cu to ca t sy kh cng gn ging nh t m, ch khc l c
th tit trng nhit 175 - 200
o
C.
2.2. Cch s dng t sy kh
Cc dng c phi ra sch, kh, bao gi cn thn trc khi cho vo t, sau
khi sp xp cc th vo trong t ri ng kn ca v ng cc l thng kh. Bt cng
tc in, t ch lm vic cho t (iu chnh nhit v thi gian ging nh vi
t nh n). Thng thng dng c nui cy v phn tch vi sinh vt c kh trng
160-180
o
C trong 2 gi. Vi cc dng c, nh: pipt, xi lanh, khng c sy
qu 60
o
C v nhit cao lm gin n thu tinh dn n mt chnh xc ca dng
c. Lu khi sy khng nn t dng c st thnh t v y nhit thng cao

hn nhiu d lm chy giy gi hoc nt bng, cng khng nn xp dng c qu kht
nhau khng kh c th lu thng c v lm nng u cc vt cn kh trng.
Sau khi ngt mch in, ch nhit h dn xung bng nhit phng th
mi uc m ca t ly dng c sy ra. Dng c ly ra phi trn gi g,
trn giy hoc vi, khng c trn gch men, trn sn gch hoc sn xi
mng v dng c ang nng gp lnh s d v v lm nh hng n tnh v trng
ca dng c.
3. Ni hp hi nc cao p (Autoclave)
3.1. Nguyn l
Ni hp hi nc cao p (hnh 1) lm bng kim loi chu c nhit cao
(t nht l 135
o
C) c th dng in, dng ci hoc dng than un cho nc si,
hi nc s nn dn li trong ni v nu tip tc cho nc si th p lc
trong ni s tng dn, p lc cng tng th nhit ca hi nc trong ni cng
cao, nh vy gia nhit t
0
ca hi nc v p lc P ca n c lin quan vi
nhau, nhng khng phi theo mt t l ng thng (xem th).
Khi p lc k ch s 0 c ngha l: p lc P trong ni hp = p lc P khng kh.
Cho nn khi p lc k ch 1kg/cm
2
th chnh l ch hiu s gia p lc bn
trong vi p lc khng kh tn ti trong ni.
1kg/cm
2
= P (trong ni) - P (khng kh trong ni)
Do p lc P trong ni khng ph hp vi P p lc k hay ni mt cch
khc, nhit trong ni khng tng ng vi nhit ca p lc k. V vy
mun cho nhit trong ni ph hp vi p lc k th phi loi b ht khng kh
trong ni ra (1kg/cm
2
= 1atm = 1 phoud).
Bng 1. So snh mi lin quan gia p sut ghi trn p k ca ni hp
biu th bng atmosphere v nhit trong ni loi ht khng kh
p
sut
(atm)
Nhit
(
o
C)
p
sut
(atm)
Nhit
(
o
C)
p
sut
(atm)
Nhit
(
o
C)
p
sut
(atm)
Nhit
(
o
C)
0,0 100,0 0,5 112,5 1,0 121,0 1,5 127,0
0,1 102,5 0,6 114,5 1,1 129,9 1,6 128,0
0,2 105,0 0,7 116, 1,2 124,0 1,7 129,0
0,3 107,5 0,8 117,0 1,3 125,0 1,8 130,0
0,4 110,0 0,9 119,0 1,4 126,0 1,9 131,0
2,0 132,0


Hnh 1. S ni hp p lc

loi b ht khng kh trong ni ra c 2 cch:
a) ng kha thot hi tng p lc trong ni ln n khong 0,3 atm
ri x cho thot ht hi ra, sau kha li v cho tng p lc.
b) M kha thot hi v un cho n khi hi nc bt u thot ra thnh
mt lung hi trng kh mnh, kh u th ng li v cho tng p lc.
3.2. Cch s dng ni hp cao p
- nc vo ni hp vi lng va (xem vch ngang ghi trn ng
thy tinh hoc bnh cha lp bn ngoi ni hp). Ch nc phi ngp dy may
so trong ni nu l ni hp xch tay.
- Cc dng c em hp phi c bao gi k, i vi cc bnh v ng mi
trng c nt bng phi bc bng giy du hoc giy nhm trnh hi nc
ng lm t nt.
- Khi sp xp dng c vo ni hp khng nn st nhau qu, vt nng
xung di vt nh ln trn.
- y np, kha cht cc c theo tng i i xng nhau khi vnh,
khi h, khi tho kha cng phi lm nh vy. ng van iu p v van x hi.
- M mch in hoc t nhin liu cung cp nhit cho ni, n nt m
cng tc ni (nt on), t ch lm vic (nhit v thi gian hp) cho ni

bng cch iu chnh mi tn ln xung. Ch lm vic lun th hin trn
bng ghi in t. Sau khi hp theo nhit v thi gian nh sn, r le s t
ngt, ni pht ra ting ku bo hiu qu trnh hp kt thc.
Vi ni hp xch tay phi lun lun c mt theo di kim ch p lc trn
ng h p lc k trong khi hp, loi ht khng kh trong ni theo 2 phng
php nu trn. Khi t ti mc cn thit th iu chnh ngun nhit duy tr
p lc khng i trong mt khong thi gian cn thit.
Nhit v thi gian hp kh trng ph thuc vo vt em kh trng v
mc ch nghin cu. Ngi ta thng hp nhit 121
o
C trong khong 20
pht th nha bo ca mt s loi vi khun cng s b tiu dit.
- Khi t ti thi gian cn thit th ngt in (n nt off) hoc rt ht
nhin liu ra v i cho p lc h dn xung 0
o
C, nhit trong ni gim hn
ri mi c m np ly dng c kh trng ra. Ch trnh h p lc t
ngt bng cch m van x hi ra qu mnh s lm rn nt hoc v dng c.
Cng khng nn ni hp ngui lnh mi ly dng c ra, v lc ny np ni s
mt cht vo ming m cao su rt kh m.
- Cc dng c ly ra khng c nn gch men, nn , nn xi mng (v
dng c ang nng gp lnh s v, nt) v m bo tnh v trng cho dng c.
4. T lnh (Freezer)
T lnh l mt thit b quan trng dng gi v bo qun ging vi khun,
virus v bo qun cc loi huyt thanh, cc loi vacxin, cc mi trng dng
nui cy...
T lnh 0
o
- 4
o
C dng gi ging vi khun.
T lnh -15
o
C n -30
o
C dng gi ging virus.
5. My ly tm (Centrifugal machine)
5.1. Cng dng
- Tp trung y ng cc phn t cn nghin cu cha trong mt bnh phm
hay cht mang.
- Tp trung vi khun nui cy trong mi trng lng tch ring vi
khun.
- Lm trong mt cht lng cha nhiu phn t c vn c.
- Tch hng cu ring vi huyt tng.
5.2. Cch s dng
My ly tm thng thng c mt trc quay trn, v pha trn ca trc c
mt hnh sao nhn cc ng cha cht lng cn ly tm. Cc ng ny c mc
vo sao bng mt ci quai. Khi quay cc ng s gin ra thng gc vi trc quay
ng v cc ht lng xung theo ng trc ca ng v dn v pha y. Vi
kiu my ny, tc ti a l 4500 - 6000 vng trong mt pht.

Cc my ly tm gn y c b phn thng bng t ng, c my thi
gian t ng, c ng h ch tc v.v..., khi ly tm ch cn vn cc nt theo
mun.
6. Nhng thit b cn thit khc
- My ht chn khng (vacuum gauge)
- My m khun lc (colony counting)
- My o pH (pH meter) hay thang o pH (pH paper set)
- My ct nc (Deionizers)
- My nh mu (Mixers)
- My m t bo (cell counting)
- My lc (shaker)
- Phng hoc bung v trng (clean bench, laminars)
II. CC DNG C V THIT B PHNG TH NGHIM
1. Thit b quang hc
- Knh hin vi quang hc (Microscopy)
- Knh hin vi chp nh
- n soi knh hin vi
- T quang nn en
- Thc o vt knh v thc o th knh
- Knh lp hai mt eo trn
- My projector
- My Over head
- My nh
- n t ngoi (UV lamp)
2. Thit b o lng
- Cn k thut (technical balance)
- Cn tiu ly c lng knh
- Cn tiu ly xch tay
- Cn bn
- Cn phn tch in
- Nhit k treo tng v cc loi nhit k o nhit khc
- ng h in t m pht, giy
3. Cc loi dng c khc
- Cc loi dng c thy tinh: Phin knh, hp lng, ng nghim, l, bnh,
phu, cc ng ong, xi lanh, pipet, que gt, n cn...

- Cc loi dng c kim loi: Dao m cc loi, ko thng, ko cong, panh,
que cy, n x, hp tiu c, ca xng...
- Cc loi dng c men, s: Khay men, ci chy s, xoong ni pha
ch mi trng.
- Cc loi dng c cao su, vi, bng, bng: Gng tay m v ra
dng c, ng, bng thm nc, bng khng thm nc, vi gc, vi lc, vi
mn.
- Cc loi ha cht ch mi trng, ra dng c thy tinh v st trng
tiu c.
- Cc loi thuc nhum v thuc th phn ng sinh ha.
- Cc loi ging vi khun v virus, cc loi khng huyt thanh chn
on, cc loi khun t chn on.
III. KNH HIN VI
Knh hin vi l mt dng c quang hc rt cn thit nghin cu hnh
thi vi sinh vt v nghin cu nhng vt ht sc nh m mt thng khng th
nhn thy c, c nhiu loi knh hin vi khc nhau.
* Cu to knh hin vi quang hc
Knh hin vi quang hc gm c 2 b phn:
1. B phn c hc
1.1 Chn knh hay knh
Dng knh hin vi trng thi cn bng, cu to t kim kh c bit.
1.2 Thn knh
Ni lin vi chn knh, c to t loi kim kh c bit. Thn knh gn
ton b cc b phn ca knh hin vi.
1.3 ng knh
L mt ng kim kh rng hnh tr lp trn tr knh, u trn ca ng knh lp
th knh, pha di ca ng knh l bn xoay dng lp cc vt knh. Tc dng
ca ng knh l khi vt nh c phng i ln th nht bi vt knh, th a vt
nh qua ng knh ti th knh phng vt nh ln th 2. Nh vy vt nh ta
quan st thy chnh l nh o.
1.4 Khay knh hay a knh
C th hnh vung hay hnh trn l ni t tiu bn quan st, gia c l
thu quang a nh sng t b t quang knh ln tiu bn. Trn khay knh c
b phn kp tiu bn cho vng v b phn gi l xa di chuyn tiu bn theo
hai chiu khc nhau, t tri sang phi, t trc ra sau v ngc li tm vt
nh. Ngoi ra, khay knh cn c th di chuyn theo cc chiu bng cch vn hai
c hai bn khay knh.
1.5 c iu chnh

Gm c c iu chnh ln (c s cp) v c iu chnh nh (c vi cp). c s cp
dng iu chnh tiu im, c vi cp dng iu chnh cho nh vt r nt.
2. B phn quang hc:
2.1. Gng phn chiu
t pha di khay knh gm c hai mt, mt mt phng v mt mt lm,
dng ly nh sng.
2.2. T quang knh
c lp vo pha di khay knh bi mt c c nh, dng tp trung nh sng
vo tiu bn.
2.3. B phn chn sng
C hnh ging nh con ngi t pha di t quang knh c th m rng
hay hp dng iu ha nh sng vo tiu bn.
2.4. Vt knh
L mt h thng quang hc rt quan trng v phc tp, gm mt s thu
knh, n trc tip phng i nh tht ca vt xem, kh nng phng i ca vt
knh ph thuc vo tiu c tc l ph thuc vo bn knh cong ca thu knh;
thu knh cng cong, tiu c cng ngn th kh nng phng i cng ln. Vt
knh khi dng c lp vo bn xoay pha di ng knh. C hai loi vt knh:
+ Vt knh kh: l vt knh c phng i thp x8; x20; x40; dng
xem ti, xem vi khun di ng, xem khun lc, hay xem k sinh trng hoc
xem cc tiu bn t chc...
+ Vt knh du: l vt knh c phng i cao x 90; x100; x 120, n
c mt vng khc mu u vt knh phn bit vi vt knh kh.
Vt knh kh v vt knh du khc nhau cht m nh sng phi i qua
tiu bn (phin knh) v vt knh. vt knh kh cht i qua l khng kh m ch
s khc x (chit xut) ca khng kh l n = 1 rt khc vi ch s khc x ca
thy tinh n = 1,52; do cc tia sng khi ra khi tiu bn s b phn x v phn
ngoi ca chm nh sng khng lt c vo vt knh.
vt knh c phng i ln ngi ta dng du bch hng (huile de
cdre) c ch s khc x n =1,51 xp x vi ch s khc x ca thy tinh n = 1,52
t vo gia tiu bn v vt knh. Lc ny thy tinh v du bch hng l mt
mi trng gn nh ng nht, nn nh sng khi i qua thy tinh s khng b
khc x m chiu thng vo vt knh.


Tiu im

Tia sng T quang knh

2.5. Th knh
Gm c hai thu knh lp vo hai u ca mt ci ng nh lp trn u ng
knh, mt thu knh hng v mt ngi xem v mt thu knh hng v vt quan
st, knh trn l knh phng i nh tht do vt knh thu c, knh di l knh th
trng lm sng t th trng do m ta nhn thy r nh c phng i.
Th knh c phng i cng cao th khong cch gia hai thu knh cng
ngn (tiu c ca th knh cng ngn) v ngc li. phng i ca th knh
thng c 4 s: x5 ; x7 ; x10 ; x15.
Mun bit phng i ca vt quan st ( phng i ca knh hin vi),
ngi ta nhn phng i ca vt knh vi phng i ca th knh. V d
dng vt knh du x90 v th knh x15 th phng i ca vt quan st hay
phng i ca knh hin vi s l:
90 x 15 = 1350 (ln)


Hnh 2: Cu to knh hin vi

1. knh 8. Th knh
2. n chiu 9. ng knh
3. B t quang knh 10. Thn knh
4. Vng bo him 11. c s cp
5. Khay knh 12. c vi cp
6. Vt knh 13. Cng tc
7. Bn xoay 14. B phn iu chnh khay knh
3. Cch s dng knh hin vi
3.1. Kim tra knh hin vi
t knh vo v tr lm vic, cm in hoc quay gng phn chiu v
pha nh sng, t knh trn bn cho ngay ngn t th c li nht cho ngi quan
st. Khi quan st tiu bn cn s dng c 2 mt, mt tri dng quan st, mt phi
dng ghi chp hoc v, khng nn nheo mt mt li xem, v nh th rt d
mi mt v au u. Cn luyn tp c th xem knh c bng c hai mt.
3.1. Quan st tiu bn ti vi vt knh kh
Khng dng t quang knh v b phn chn sng, nht l i vi vt knh
c phng i thp (38), khi ngun sng hp th dng gng phng vi vt
1
2
3
4
5
6
7
8
9
10
11
14
13
12

knh c phng i thp, dng gng lm vi vt knh c phng i cao
(340), khi ngun sng rng th dng gng no cng c. H thp tt cng t
quang knh v t m b phn chn sng.
3.2. Quan st tiu bn nhum vi vt knh du
Lun lun s dng t quang knh, nng cao t quang cho st vo tiu bn.
Khi s dng t quang knh cn ch my im:
t phin knh ln khay knh v c nh, dng vt knh c phng i
thp c nh trong th trng trc. H thp vt knh cho st gn tiu bn (khi
h vt knh mt nhn ngoi trnh mnh lm v tiu bn). Theo di trong
ng knh, ri t t vn c s cp ln, n khi trng thy nh (thng c hnh
chp) th ngng vn c s cp v bt u s dng c vi cp, vn ht sc chm
n khi thy nh r nt th thi (c th vn ti hoc vn lui). Sau khi iu
chnh tiu im vi vt knh phng i thp th quay vt knh ra, nh mt
git du bch hng vo im nh soi trn tiu bn, khng git du lan rng
ra, xoay u vt knh du vo, v vn vt knh du st xung tiu bn ngm vo
git du, ch mt nhn ngoi ng vn st qu s v phin knh, n khi
thy chp nh, tc l nh trng thy nhng cha thy r, lc ny iu chnh
c vi cp cho n khi nh vt r nt trong th trng.
4. Cch bo qun knh hin vi
+ Khi ly knh t trong hp knh hin vi ra, dng tay phi nm chc, ko
knh ra theo hng nm ngang, khng ng vo thnh hp, sau dng tay
tri chn knh mang i (bao gi cng phi dng 2 tay khi di chuyn). Nu
mang i xa phi c nh chc chn trnh b lc.
+ Khng c s tay vo u vt knh v th knh, nu bn c th dng
vi mm hoc giy lau knh lau. Vt knh du dng xong ly vi mm mn
hay giy dai mn lau sch du bch hng u vt knh, sau tm xylon lau
cho ht du (xylon c tc dng lm tan du bch hng). Cui cng lau li mt
ln na bng vi mm, mn hay giy mm.
+ Khi dng xong phi xoay cc b phn ca knh v ng v tr quy nh,
khng c vt knh nm trong trc knh nh lc quan st m phi t ng
l m hoc xoay vt knh ra hai bn v vn cho p st xung a knh, t quang
h thp xung, gng phn chiu xoay dc thn knh. Ton b knh u coi nh
trng thi ngh.


* Cu hi n tp bi s 1:
1. Trang thit b, my mc chuyn dng cho nghin cu VSV?
2. Nguyn l vn hnh v cch s dng ni hp hi nc cao p (Autoclave)?
3. Cu to v cch s dng knh hin vi?
4. Cu to v cch s dng my m khun lc?
5 Trnh by phng php v cch tnh kch thc t bo VSV?

Bi s 2
PHNG PHP C NH TIU BN V NHUM T BO VI SINH
Mc ch yu cu:
+ Hng dn hc vin lm tiu bn vi sinh vt t cc mu vt.
+ Nm vng phng php nhum n, nhum giemxa v phng php
nhum Gram.
+ Nhn dng hnh thi vi sinh vt v phn bit vi khun Gram dng,
Gram m
Ni dung:
+ Phng php lm tiu bn vi sinh vt
+ Pha ch thuc nhum v cc phng php nhum: nhum n, Gram,
Giem xa, Wright.
+ Quan st mt s tiu bn hnh thi vi sinh vt: cu khun, trc khun,
cu trc khun
I. PHNG PHP LM TIU BN V NHUM T BO VI SINH VT
1. Mc ch ca c nh tiu bn v nhum t bo VSV
T bo vi sinh vt gn nh l khng mu, do quan st bng phng
php xem trc tip rt kh, v vy cn phi lm tiu bn ri em nhum mu.
Nhum vi sinh vt c 4 mc ch:
- nghin cu hnh thi, cu to c bit ca vi sinh vt nh gip m,
nha bo,
- phn loi vi sinh vt cn c vo tnh cht bt mu Gram, tnh cht
khng cn, khng toan.
- d phn bit v quan st c cc vi cu to trong t bo VSV.
- bo tn tiu bn trong mt thi gian di, chp nh.
2. Phng php lm tiu bn vi sinh vt nhum
2.1. Chun b phin knh
- Chn phin knh trong, sch, khng m, khng c du m, c
ngm trong cn, khi dng lau kh bng vi mm v h qua trn ngn la n
cn.
- Khoanh din pht vi sinh vt bng cch dng bt ch m khoanh mt
vng mt di phin knh.
2.2. Pht mu vt
- Nu ly mu vt l vi sinh vt t ng canh trng dch th th sau khi
kh trng que cy v ngui, ly mt git mi trng nh ln phin knh ch
khoanh trn bng bt ch m, ri dn mng ra trong din khoanh.
Cn ch thao tc khi ly vi sinh vt pht knh: Tay phi cm que cy,
nung que cy bch kim v a ton b phn kim kh ca que cy qua ngn

la n cn 2 - 3 ln, lm trc khi ly vi sinh vt v sau khi pht knh xong;
tay tri cm ng mi trng vo lng bn tay v cm nghing ng bng 5
ngn tay. Dng ngn tay t ca bn tay phi m nt bng, sau khi quay nt
bng mt vng trong ming ng cho trn (kp nt bng vo gia ngn tay t v
bn tay, hay gia ngn tay t v ngn tay eo nhn) h ng mi trng trn
ngn la n cn, v u ng nghim lun lun st ngn la n n cn,
cho que cy vo su trong ng mi trng ly ra mt git mi trng, rt que
cy ra, h ming ng nghim v ng nt bng li, cho ng nghim vo gi, sau
cm phin knh chun b sn, mun cm phin knh cho vng th ngn tay
ci gi mt cnh di ca phin knh, ba ngn tip gi cnh i din, ngn t
mt di phin knh, gi cho phin knh khng di chuyn trong khi pht.
- Nu l vi sinh vt t canh trng c (mi trng thch): th dng que cy
bch kim ly mt t vi sinh vt mt khun lc, (khng nn ly nhiu vi sinh vt
v pht dy qu s kh xem v kh phn bit hnh thi ca vi sinh vt). t que
cy ln phin knh c sn mt git nc ct hay nc sinh l hoc nc tht
v trng lm huyn dch vi sinh vt, trn u vi sinh vt trong git nc ri
dn mng ra.
- Nu dng mu pht knh th c th ly mu tnh mch ra tai (i
vi ng vt sng) hoc mu tim (i vi ng vt m khm). t git mu ln
phin knh, ri dng u mt phin knh khc, c cnh nhn v thng hoc cnh
ca mt l knh t nghing mt gc 30 - 45
o
vi phin knh git mu lan
khp cnh ri y nh v u ti u kia ca tiu bn, mu theo phin knh ch
khng phi b phin knh y i, tiu bn tt nu mu c dn u trn phin
knh thnh mt lp mng.
- Nu dng ph tng lch, gan, thn, hch, phi... th ct mt ming nh,
thm bt nc bng bng hay giy thm, ri chm nh trn phin knh 3 - 4
ch, khng mnh trn phin knh, ch thm nh nhng, hoc c th ko lt
nh ming ph tng trn phin knh thnh vt di cng c.
- Nu dng m m, ty xng pht knh th ly que cy ly m ch c
nhiu m nht, ri dn mng ra trn phin knh, nu m kh th trc khi dn, nh
mt git nc sinh l v trng trn phin knh, l tng t vi ty xng.
2.3. Sy kh tiu bn : C 2 cch
- t kh nhit phng th nghim.
- H cao trn ngn la n cn, khng st tiu bn vo ngn la, nng
qu thn vi sinh vt s co qup li, protit trong nguyn sinh cht ng nhanh,
nh hng n hnh thi t bo.
2.4. C nh tiu bn
+ C nh tiu bn c 3 mc ch:
- Git cht vi sinh vt vic s dng khng gy nguy him.
- Lm cho vi sinh vt gn cht vo phin knh, khi ra nc khng b tri i.
- Lm cho vi sinh vt bt mu tt hn.

C th c nh bng nhng phng php sau y:
+ C nh bng nhit : H phin knh trn ngn n cn bng cch a i,
a li khong cch 10-15 cm 3 - 4 ln, nu h nng qu s lm bin dng
hnh thi vi khun.
+ C nh bng cht ha hc:
- Nh vi git cn nguyn cht hay cn 96
o
5 - 10 pht
- Nh vi git cn mtylic 2 - 3 pht
- Ngm tiu bn vo axton 5 pht
+ C nh bng hi foocmalin 3 - 5 pht
II. Phng php nhum tiu bn
2.1. Thuc nhum n v cc phng php nhum n
2.1.1. Dung dch fucxin (fuchsine) trong axit phnic
+ Chun b thuc nhum
Fucxin kim 1 g
Cn nguyn cht hay 96
o
10 ml
Axit phnic kt tinh 5 g
Nc ct 100 ml
Nghin fucxin vi 5ml cn trong ci sch, quy u, t t 2/3 lng
nc vo, quy u, xong cho thm axit phnic, trn u cho vo l kn 24
gi, em lc qua giy, trng cc bng 1/3 nc ct v 1/2 cn cn li, dung dch
ny l dung dch fucsin c, khi dng nhum n hoc nhum Gram th em pha
long dung dch ny gp 10 ln vi dung dch axit phnic 5%.
Dung dch fucxin 10 ml
Dung dch axit phnic 5% 90 ml
+ Phng php nhum n
a) Nh thuc nhum ln tiu bn c nh 1 - 2 pht, c khi n 10
pht ty theo loi thuc nhum.
b) Ra nc, vi nc t t chy xung mt u phin knh cm hi
nghing n khi nc trong l c.
c) Thm kh bng giy thm hay bng hi nng.
d) Quan st trn knh hin vi
2.1.2. Dung dch xanh mtylen trong axit phnic
+ Chun b thuc nhum
Xanh mtylen 1 g
Cn nguyn cht 100
o
10 ml
Nc ct 10 ml

Cch pha ging nh fucxin trn.
+ Phng php nhum cng ging nhum fucsin.
2.2. Phng php nhum Gram( nhum kp)
2..2.1. Chun b thuc nhum
a) Dung dch tm gentian trong axit phnic
Tm gentian 1 g
Cn 96
o
10 g
Nc ct 100 ml
b) Dung dch fucxin trong axit phnic
Fucxin kim 1 g
Cn 96
o
10 g
Nc ct 10 g
Cch pha 2 dung dch ny ging nh dung dch fucxin ni trn
c) Pha dung dch lugol
Itua kali (KI) 1 g
It tinh th (I) 0,5 g
Nc ct 150 ml
Nghin ituakali vi mt t nc ct, sau cho it tn nh vo lc
cho tan ht, cui cng cho nc ct, lc u, 24 gi ri em lc. ng vo
chai mu. Khng nn pha nhiu v d b bin cht.
d) Pha dung dch ty mu cn axtn.
Cn nguyn cht 5 phn
Axtn 1 phn
Nu khng c axtn th dng cn nguyn cht hoc 90
o
cng c.
2.2.2. Phng php nhum Gram
1) Nh dung dch tm gentian ln tiu bn 1 - 2 pht.
2) Ra nc nhanh, vy kh nc.
3) Nh dung dch lugol 1 pht (tiu bn c mu nu en).
4) Ra nc nhanh, vy kh nc.
5) Nh cn axtn t u phin knh, nghing phin knh cho cn chy
qua ch pht vi sinh vt.
6) Ra nc nhanh.
7) Nh dung dch fucxin long 1 pht.
8) Ra nc

9) Thm kh - h kh - xem knh.
Vi khun Gram dng bt mu tm, vi khun Gram m bt mu hng.
* Cn ch : Bc ty mu bng cn axtn rt quan trng. Nu ty
khng k th d nhm ln vi khun Gram m vi vi khun Gram dng v
ngc li, nu ty lu qu th vi khun Gram dng mt mu tm cho nn khi
nhum mu fucxin th cng bt mu .
Quan st trn knh hin vi nhn thy: hng cu nhum mu nu hng,
nhn bch cu nhum mu tm.

* Cu hi n tp: Bi s 2

1. Trnh by 3 phng php c bn trong nghin cu v VSV?
2. Th no l Gram? nh by s sai khc gia nhum n v nhun kp?
3. Cc bc tin hnh ch to tiu bn?

Bi s 3
CHUN B DNG C V MI TRNG NUI CY VI SINH VT

Mc ch, yu cu:
+ Bit c v chun b dng c nui cy VSV
+ Nm vng cch pha ch mi trng nui cy VSV
+ Hiu c phng php kh trng cc loi dng c v mi trng nui
cy VSV
Ni dung kin tp:
+ Chun b v ra dng c cn thit pha ch mi trng nui cy VSV
+ Hc cch bc gi cc dng c thng dng v nt bng cho ng nghim,
pipet,
+ Cch pha ch mi trng thch nghing v a thch
I. CHUN B DNG C
1. Cc dng c thng c s dng trong nghin cu vi sinh vt
- a petri (hp lng)
- ng nghim, bnh tam gic, bnh cu, chai thu tinh
- Pipt, xi lanh, que gt, que cy
- Lam knh, lamen
2. Yu cu
Cc dng c phi sch v mt ho hc v vi sinh vt hc (cc dng c phi
c v trng).
3. Cch x l dng c trc khi ra
- i vi dng c thu tinh mi cha s dng, cn ngm nc l hoc
dung dch H
2
SO
4
long 24 gi. Ra li bng x phng v nc nhiu ln cho ti
khi dung dch ra c pH trung tnh.
- Cc dng c qua s dng, nht l cc VSV gy bnh trc khi ra
nht thit phi c kh trng bng hi nc p lc git cht cc t bo, m
bo an ton cho ngi ra, khng cho mm bnh c nhim vo mi trng mi.
- i vi cc VSV khng gy bnh cho ngi v ng thc vt, ch cn
tho nt bng, xp vo ni hoc chu nhm chuyn dng, nc x phng,
dm dng c ngp kn nc, un si 15-30 pht. Gom cc cn bn vo ti nilon,
buc kn ri mi b.
- Dch nui VSV trc khi b cn thm vi git formalin, lc mnh
git cht t bo.
- S dng dung dch sunfo-cromic ngm ty cc vt bn trn dng c
thu tinh.
4. Cch ra dng c

Chn chi ra thch hp vi tng loi ng hoc bnh, pha u nn m
dy chun phn li st khng chc thng y ng nghim hoc y bnh.
Dng ming nhm thm nc ra hoc bng thm cn lau sch cc k
hiu ghi trn thu tinh.
Dng chi hoc ming ra thm dung dch nc ra c k pha trong
ng hoc bnh hay a petri ti khi sch ht cc vt bn. Dng khn mm c k
pha ngoi. X nc lm sch cht ty ra. Trng li bng nc ct. Dng ngc
dng c vo gi ng cho rc ht nc. Lm kh nhit phng hoc phi
nng hoc sy kh 80-105
o
C. a Petri nn ra v xp theo b d lp li vi
nhau.
Cc phin knh v l knh dng xong nn ngm ring vo dung dch ty
ra hoc st trng. Dng khn mm c ra, trng nc ct, thm kh, hong li
trc khi ct. Cc l knh rt d v, cn thn trng hn v nn ra di vi nc
chy nh.
Vi cc pipet dng ht dch VSV: sau khi s dng cn ngm ngay vo
ng nc st trng, khu b nt bng, ngm tip vo dung dch ty ra 1 ngy
ri chuyn sang bnh ra pipet t ng qua m. Trng nc ct, hong kh. Nu
ra trc tip di vi nc, cn iu chnh sao cho dng nc chy qua bn
trong pipet.
Vi cc pipet dng cho cc phn ng ho hc, khng cn ngm nc st
trng, t pipet di vi nc chy x bt ho cht bm dnh bn trong,
ngm vo ng nc x phng mt ngy, ra sch ri trng nc ct. Pipet ch
nn hong kh nhit phng. Nu sy nhit cao, thu tinh gin n lm
sai lnh th tch.
* Pha dung dich ra
a) Dung dch sunfo-cromic:
Bicromat kali: 50 g
H
2
SO
4
c: 500 ml
b) Dung dch kim trong alcol:
NaOH vin: 120 g
Nc ct: 120 ml
Alcol 95
o
: 1000 ml
5. Chun b dng c kh trng
Dng c trc khi kh trng phi c ra sch, lm kh v gi giy
bo m tnh v trng sau khi sy.
Mi pipet c gi trong di giy di c chiu rng 4 - 5cm. u pipet
c dng ht bng ming c t nt bng mt t bng, nt bng cn va
phi, nu cht qu s kh ht dch v kh ly ra khi ra. Pipt c gi bt u
t pha u nh git, qun giy dn dn vo theo kiu xoy trn c cho n khi
ht pha u c nt bng. Phi qun giy cho st kht vo pipet. Sau khi qun

giy, ta phi gi cho khi bn v khi rch bng cch buc thnh tng b cng
kch c hoc cho vo ng ng pipet lm bng kim loi hay bng ba cng.
Cc que gt cng c gi ring tng ci v sau cng b li nh pipt.
Cc a Petri c gi thnh tng chng, mi chng khong 4-5 b.
Cc chai l, ng nghim v ng Burri dng nui cy vi sinh vt nht
thit phi c y nt (nt nha hoc kim loi hay cao su nhn to chu nhit
hoc bng nt bng). Nu lm nt bng phi dng bng khng thm nc (bng
m). Nt bng cn lm ng kiu cch thun tin khi thao tc th nghim, c
th lm nt bng trn hoc nt bng c bc vi mn.
Ly bng theo lp, tu vo kch c ming bnh, chai l v ng nghim
ly lng bng ph hp. C 2 cch lm nt bng thng dng:
+ Nhi bng vo gia, dn u ra xung quanh thnh hnh trn, ly u mt
ngn tay t vo gia, cc ngn ca bn tay kia gi u pha ngoi ri y st
ln ngn tay t gia to thnh nt bng.
+ t ming bng va ly (theo hnh ch nht) trn mt bn sch, cun
trn li theo chiu di n ht, ri gp lm i to thnh nt bng.
Xoay u nt va to ra vo ming ng hoc bnh, su khong 2-3 cm, iu
chnh sao cho khng to thnh rnh trn nt bng ngn chn s tp nhim t
khng kh, vut u phn cn li bn ngoi ri bn cht li nh hnh ngn la.
Nt bng t yu cu cn va phi, khng cht qu, cng khng lng qu, d
dng ly ra khi thc hin cc thao tc nui cy VSV.
Vi cc bnh mi trng thch c th bao giy bc thay cho nt bng.
6. Kh trng cc dng c thu tinh
Phng php c bn kh trng cc dng c thy tinh l phng php
kh trng bng sc nng kh (bng nhit). Cng vic ny c thc hin trong
t sy (Drying oven) nhit 160 - 180
0
C trong vng 2 gi. Khi c th tiu
dit c t bo dinh dng ln cc bo t ca VSV.
Cc dng c kh trng c bo qun trong ti polyethylen ct gi
ch kn, trnh bi bm. Ch bc giy ngay trc khi s dng. Sau khi kh trng,
cc que gt, que cy ch nn s dng trong vng 24 gi, hp petri trong vng 3
ngy, ng nghim, bnh nn khong 7-10 ngy nu bo qun tt. Nu lu,
dng c phi c kh trng li trc khi s dng.
Mt s dng c nh que gt, que cy, ng nghim, pipet c th kh trng
bng hi nc p lc cao (s dng ni hp cao p), thng thng 121
o
C/30.
Kh trng xong nn s dng ngay.
II. CHUN B CC MI TRNG NUI CY VI SINH VT
Cng nh cc sinh vt khc, sinh trng v pht trin cc t bo vi sinh
vt cn cc cht dinh dng thch hp. Khi nui cy nhn to, ngi ta lm cc
loi thc n cung cp cho tng nhm vi sinh vt khc nhau. Dng thc n

ny c gi l mi trng nui cy. Tu tng ging VSV khc nhau m c mi
trng nui cy chuyn tnh khc nhau.
1. Cc yu cu v mi trng nui cy
- y cc cht dinh dng ph hp vi tng kiu trao i cht ca
tng nhm vi sinh vt, khng cha cc yu t c hi, mi trng nui cy phi
c m bo cho s sinh trng bnh thng ca vi sinh vt.
- V trng tuyt khi nui cy ch pht trin mt loi VSV mong
mun.
2. Cch gi tn mi trng
Mi trng thng c gi theo tn ngi ch to cng thc (v d mi
trng Hansen, MacConkey) hoc theo ngun dinh dng chnh c trong mi
trng (mi trng tinh bt-thch, malt-thch, glucoza-pepton,)
3. Phn loi mi trng
Mi trng c phn loi theo thnh phn ho hc, ch dinh dng
v cng dng
3.1. Phn loi theo thnh phn ha hc
3.1.1. Mi trng c thnh phn xc nh (mi trng tng hp) - defined
medium
Mi trng c ch to t cc cht c thnh phn c xc nh r rng.
V d mi trng A c cha 5g glucoza, 1g (NH
4
)
2
SO
4
, 2g K
2
HPO
4
, 1 lt nc.
Nu nh b sung 1 lng nht nh axit amin c th no (Lyzin) vo th mi
trng A vn c gi l mi trng c thnh phn xc nh.
Mi trng c thnh phn xc nh c s dng nghin cu cc nhu
cu dinh dng hoc cc con ng sinh ho c bit ca vi sinh vt
Tuy nhin, khi nim mi trng xc nh cng ch c tnh cht tng i
bi v cc ho cht s dng pha ch mi trng ngoi thnh phn chnh c
xc nh, cn c cc nguyn t vi lng tp nhim khng c xc nh.
Ngun nc pha ch mi trng cng cn c ch trng. Vi loi mi
trng ny nht thit phi s dng nc ct tinh khit pha ch.
3.1.2. Mi trng thnh phn khng xc nh
(mi trng t nhin hoc bn tng hp)- Undefined medium, complex medium
Mi trng c cha cc hp cht c thnh phn khng xc nh r rng
nh cc cht chit t m ng thc vt hay t bo nm men.
Ngoi cc cht chit t nhin, mi trng cn c c cc ho cht c thnh
phn xc nh. V d nh mi trng A trn c b sung thm pepton.
3.2. Phn loi theo ch dinh dng
3.2.1. Mi trng ti thiu (Minimal medium)
Loi mi trng cung cp nhu cu ti thiu ca VSV, khng d tha. V
d nu mt loi VSV no cha thng tin di truyn sinh tng hp tt c cc

loi axit amin cn thit ca chng th khng cn thm axit amin vo mi trng.
Hoc nu VSV bnh thng cn 2 loi axit amin, ch cn b sung 2 loi m
khng cn thm bt k loi no khc.
Tu tng trng hp, mi trng ti thiu cho tng loi VSV khc nhau
cng khc nhau. Nhiu mi trng ti thiu ch dng nui cy mt phm vi
tng i hp cc VSV.
3.2.2. Mi trng hoc giu (All purpose hoc Rich medium)
Mi trng cha hng lot cht dinh dng vt xa nhu cu ti thiu ca
VSV. Mi trng giu tr gip sinh trng ca nhiu loi VSV. Trong mi trng
giu, VSV sinh trng v pht trin nhanh v tt hn so vi mi trng ti thiu.
S d nh vy l v cc ngun dinh dng m VSV cn c th ly d dng t cc
hp cht nh axit amin, axit bo, vitamin, nucleic c sn trong mi trng.
3.3. Phn loi mi trng theo cng dng
3.3.1. Mi trng chn lc hay mi trng tuyn chn (Selective medium)
Mi trng ny m bo cho s pht trin ca cc VSV m ta mong
mun v c ch s pht trin ca cc VSV ngoi mun.
Mi trng chn lc ch yu dng phn lp cc chng VSV thun
khit t t nhin hoc dng nui cy tch lu(enrichment). C 2 cch lm
tng tnh chn lc ca mi trng:
- Cho thm cht c ch cc VSV khng mong mun nhng khng nh
hng ti sinh trng ca cc loi VSV ta nh nui cy hoc tuyn chn. Cc
cht c ch bao gm cc loi thuc nhum nh tm kt tinh (crystal violet), cc
cht khng sinh, cc cht chng nm, NaN
3
... Nng cao ca mui, ng tc
ng n p sut thm thu ca t bo c s dng nh mt nhn t chn
lc VSV.
- Loi b mt cht m cc VSV khc cn ti khin chng khng pht trin c.
3.3.2. Mi trng phn bit (differental medium)
Mi trng phn bit cho php nhiu loi VSV pht trin v phn bit
nhanh chng loi ny vi loi khc. V d mi trng c cha bromcresol (cht
ch th mu) tr gip sinh trng ca nhiu loi VSV nhng da vo kh nng
chuyn mu ca cht ch th c th nhn bit ngay loi no c kh nng hnh
thnh axit t ng.
3.3.3. Mi trng chn lc - phn bit (Selective - differental medium)
Mi trng ny bao gm c 2 chc nng chn lc v phn bit. N gip
cho vic chn lc mt nhm nh VSV ng thi phn bit chng vi nhng
nhm VSV khc.
3.4. Phn loi mi trng theo trng thi vt l
3.4.1. Mi trng lng (dch th) -Liquid medium
Thng c s dng nui cy VSV nhm thu nhn sinh khi, cc sn
phm trao i cht (enzim, khng sinh, vitamin) hay pht hin cc c im

sinh ho v gi ging v bo qun nhiu loi VSV khng pht trin c tt
trn cc mi trng c.
3.4.2. Mi trng c (c th) - Solid medium
lm ng mi trng, ngi ta thng s dng thch (agar) i khi s
dng gelatin (keo da, xng ng vt) hoc s dng silicagel (ch to t thu
tinh lng v HCl).
Mi trng c dng phn lp ging thun khit, m s lng VSV,
gi ging VSV, nghin cu hnh thi khun lc, hot ng i khng
Thch l mt loi polysaccharit chit t mt loi to bin. V khng b
VSV phn gii nn thch l cht lm ng mi trng kh l tng. Trong
nc, thch nng chy gn 100
o
C v ng c khong 43
o
C. Thch thng
c b sung vo mi trng vi s lng khong 16-20g/l. Trong mi trng
trung tnh, hi axit hoc hi kim, thch vn gi c kh nng to gel kh bn
vng. Trong mi trng axit pH <5,0 thch b thu phn trong qu trnh kh
trng, mt tnh to gel sau khi ngui.
3.4.3. Mi trng mm
Thng s dng gi chng. Thch c b sung vi s lng 6-10g/l
3.4.4. Mi trng bn lng
Thm thch vo vi lng 2-5 g/l. Do nng oxi xm nhp vo bn
trong mi trng bn lng ch mc nht nh nn mi trng ny c s
dng nui cc vi khun ho kh (microaerophilic).
3.4.5. Mi trng xp
Mi trng xp c ng dng trong sn xut (VSV hc cng nghip).
Cht xp nh tru, cm c trn vi cc thnh phn dinh dng khc.
4. Chun b mi trng
Cn c k cng thc cu to mi trng, chun b cc ho cht v dng
c lin quan
4.1. Pha ch
Nhn vo cc cng thc mi trng, ln lt cn ong cc thnh phn.
nh du vo cng thc cc thnh phn cn hoc ong. Nu cng thc c
thch th nn ong nc cho vo ni ng, cn thch cho vo nc thch
ngm d tan khi c un si v ln lt cn ong cc thnh phn khc.
Nu mi trng phi un nu, ngoi th tch nc theo cng thc nn b
sung mt lng nh nc b li th tch bay hi khi un (khong 15-20ml/l)
Mi ho cht xc bng mt tha ring khi cn. Cho ho cht hoc cc
thnh phn c cn ln a c giy cn mt cch t t ti khi va . Nm
vng cch s dng cc loi cn.

Vi cc thnh phn c hm lng nh nn pha dung dch m (stock
solution), tnh hm lng c trong 1 n v th tch suy ra th tch cn ly b
sung vo mi trng.
Cc loi bt go, ng, cm c cn v cho vo nc lnh, va un va
khuy cho ti khi bt chn.
Cc thnh phn km bn nhit nh cht khng sinh, vitaminphi c
kh trng theo phng php ring (thng s dng mng lc khun)
i khi mt s thnh phn c cn ring, kh trng ring v ch c
trn li vi nhau sau khi kh trng trnh phn ng to thnh kt ta khi
ton b thnh phn c kh trng chung (xy ra khi c mt ng thi cc mui
phtphat v MgSO
4
).
4.2. un mi trng
Mi trng dch th: Nu cc thnh phn tan u trong nc th khng
cn un.
Mi trng c: t ni ln bp va un va khuy u bng a thu
tinh. Khi mi trng si mt lc, thch tan ht l c, trnh un lu nc bay
hi s lm cn mi trng. Ht bt hoc lc trong nu cn.
4.3. iu chnh pH
Nu mi trng dng dch th trong sut, khng mu khng cha cc
cht dnh nht nh tinh bt, c th o pH bng my o.
Mi trng c mu, cha cc cht dnh nht khng c o pH bng my
(in cc d b hng), nn s dng giy o pH. Cng c th nhn bit pH qua
mu sc ca mi trng do cc cht ch th mu to nn.
Nhn chung, nu cc thnh phn mi trng c cn ong chnh xc, cc
ho cht t tiu chun, sau khi pha ch mi trng s t c gi tr pH cn
c. Nu pH sai lnh qu ln cn xem li cc khu v phi pha li mi trng.
Nu sai khc khng ln, c th iu chnh bng dung dch kim (KOH, NaOH)
hoc axit (HCl,

CH
3
COOH) long hoc cc mui theo ch dn cng thc. Vic
iu chnh pH cn thn trng sau khi chnh xong th tch mi trng khng b
thay i ngoi phm vi cho php.
* Lu : Khi pH mi trng thp hn 6,0 - 6,5 th s xy ta vic pepton
ho gelatin v sau khi kh trng, mi trng s khng ng li c. Khi pH
thp hn 5,0 s xy ra s thu phn v thch mt kh nng to gel.
Nu mi trng c phn ng kim th khi kh trng s xut hin kt ta
st, xut hin vic caramen ho ng v ng tr nn khng hp thu c i
vi vi sinh vt. trnh nhng hin tng ny, cc mi trng c dng
nui cy cc vi sinh vt a axit hoc a kim phi c tin hnh kh trng
pH trung tnh v sau khi hp p lc mi axit ho hoc kim ho mi trng.
Ngoi ra c nhiu thnh phn ca mi trng thng c kh trng ring
nhng ch pH khng nh hng ti chng, sau mi a vo mi trng
mt cch v trng vi s lng thch hp.

Sau khi kh trng, pH mi trng c th b thay i i khi phi kim tra li.
4.4. Phn phi mi trng vo cc dng c
Dng c c dng phi v trng. kh trng tt, mi trng ch c
rt ti 1/2 dung tch ca vt cha. Vi cc ng nghim lm thch nghing ch rt
khong 1/4 chiu cao ng v 1/2 vi ng lm thch ng.
Lp mi trng cng dy vic tit trng cng kh, hn na p lc cao
mi trng s si mnh lm y nt v pht ra ngoi.
i vi cc bnh dng nui VSV hiu kh trn my lc ch rt khong
1/5 dung tch bnh. i vi bnh dng nui k kh, sau khi kh trng dn mt s
bnh li vi nhau trong iu kin v trng t c th tch cn thit.
5. Kh trng mi trng dinh dng
Kh trng l mt trong nhng bin php cn thit v quan trng nht
trong thc nghim vi sinh vt hc. Thut ng " kh trng" bt ngun t ting La
tinh vi ngha l s " lm tuyt dc". Trong vi sinh vt hc, kh trng c hiu
l lm cht tt c mi vi sinh vt. Tin hnh kh trng mi trng, dng c, thit
b v cc th khc trnh s pht trin ln ln ca cc h vi sinh vt ngoi lai
vo ging ang nghin cu. Vic kh trng mi trng v dng c l vic bt
buc phi lm khi thc hin tt c cc bi tp.
5.1. Phng php kh trng mi trng dinh dng
Mi trng dinh dng c kh trng ch yu bng cch hp trong ni
hp p lc. Phng php ny da trn nguyn tc lm gia nhit cc vt bng hi
nc bo ho di mt p sut ln hn p sut ca kh quyn. Khi p sut hi
nc tng ln th nhit cng tng theo.
Tc dng phi hp gia nhit cao v p sut bo m cho vic kh trng
thc hin c tt. Khi hp p lc s lm tiu dit c t bo dinh dng ln bo
t ca vi sinh vt. Khi ghi ch kh trng bng cc n v p sut 0,5 ; 1,0;
1,6; 2,0 atm c ngha l ngi ta mun ni n cc p sut b sung.Vic tng p
sut hi nc c to ra trong nhng thit b y kn thnh dy, ng kn cc
ni hp p lc.
5.2. Ch kh trng mi trng
Nhit v thi gian kh trng bng cch hp p lc trc ht c quyt
nh bi thnh phn ca mi trng dinh dng. Cc c cht c cha nhng
cht khng bn i vi nhit 120
0
C phi c kh trng 0,5 atm. Sa, dch
t phn nm men, nc nm men v cc mi trng cha gelatin c kh
trng 0,5 atm trong 15 pht. Cc mi trng c cha ng, chng hn nh
mi trng mch nha, mi trng nc p thc vt c kh 0,5 atm trong 20
-30 pht. Canh tht - pepton v thch - tht - pepton c kh trng 1 atm
trong 20 -30 pht. Mi trng khoai ty nc chit t c kh trng 1,5 atm
trong 30 pht.

la chn ch kh trng phi tnh n pH ca mi trng. Khi mi
trng c phn ng axit, cc hp cht cao phn t trong c th b thu phn
khi hp p lc.
* Vi cc thnh phn mi trng km bn nhit c th kh trng theo
cc cch sau:
- Phng php Pasteur: un nng dung dch 80
o
C/15 pht ri lm ngui
ngay.
- Phng php Tyndal: un si cch thu dung dch trong 30 pht, lp li 3
ln mi ln cch nhau 24 gi.
Hai phng php ny ch c th dit cc t bo dinh dng, khng dit
c cc bo t khng nhit.
- S dng mng lc vi khun: thng dng kh trng cc thnh phn
nh cht khng sinh, vitamin.Kch thc ca l mng lc vo khong 0,2 -
0,45m. Lc khun khng loi c virus.
5.3. Cch lm a thch v trng
Tin hnh mi trng ra a trong iu kin v trng (s dng phng
v trng hoc t cy v trng c lau cn v kh trng bng tia t ngoi).
Tay ngi lm cng phi c kh trng bng cn hoc dung dch st khun.
Cc a petri phi c mi kh trng trong vng 24 gi.
Bnh mi trng mi c kh trng, ngui n 50-60
o
C ri mi ra
a. Khng khi mi trng nng >60
o
C trnh hi nc ng trn np a
v mt thch s dn n d b tp nhim trong qu trnh nui cy. Trc khi
nn quay trn bnh trn u mi trng, trnh lc mnh sinh bt kh.
Sau khi mi trng ra a, nu thy cc bt kh phi dng que cy nung
nng chm v bt kh khi thch cn nng, cha ng. yn cho thch ng
trong a v ngui dn n nhit phng. Xp cc a mi trng thnh tng
chng, bao kn bng giy v trng.
C th lm kh mt thch v kim tra v trng bng cch m h np
a trong t cy thi kh v trng hoc t cc gi petri theo chiu ngc trong
t m 2-3 ngy. Sau , chn la cc a khng nhim VSV s dng.
5.4. Cch lm mi trng thch nghing
Trc tin cn lau sch mt bn ni s t thch nghing, t mt thc
(g, nha) sch cao khong 2-3 cm ln mt bn, kh trng mt bn v thc
bng cn. Tri mt mnh vi hoc giy v trng trm ln mt bn v thc g.
Sau khi kh trng trong ni hp p lc, cn lm ngui cc ng mi trng
nhit phng hoc di qut mt mt lc n khi ch cn khong 50-60
o
C
trnh hi nc ng li nhiu trn b mt thch.
Nh nhng t ng mi trng nm trn lp vi hoc giy v trng, u c
nt k trn thc, iu chnh sao cho mp thch cch xa nt bng 3-4 cm. t cc
ng thnh hng xt nhau, ht mt hng li t tip hng sau gi ln hng trc. i

vi ng thch dng gi ging VSV ch cn t nghing vt mt cht mi
trng gi c m trong thi gian di.
Nu c dng c, c th cho cc ng mi trng c cng kch c vo gi
ng nhiu hng, ri t c gi ng nghim nghing u mt gc trn thc. Do
tnh cht ng u ca cc trn gi s gip cc ng mi trng c nghing u
nh mun. Cch ny gip thao tc t nghing c nhanh v b mt thch
nghing u hn, c th d dng thc hin vi mt s lng ln mi trng.
Trong qu trnh lm thch ng, khng c rung bn hay rung ng
thch. Sau khi thch ng, ng ngui hn mi gi cc ng thch nghing bng
giy v trng, t vo t m 30-37
o
C kim tra v trng v lm kh mt
thch. Sau vi ngy ly ra quan st k b mt thch pht hin cc khun lc
VSV. Soi ng nghim di nh n tm cc khun lc chm trong thch.
Nhng ng thch nghing v trng (khng cha cc khun lc VSV) c dng
lm th nghim v gi ging VSV.


1. Th no l mi trng chuyn tnh?
2. Trnh by S sai khc c bn ca mi trng lng, rn, bn rn?
3. Trnh by cc bc cn thit tin hnh ch to mi trng?
4. Cc dng c, ha cht cn thit ch to mi trng?

Bi s 4
NUI CY VI SINH VT
Mc ch, yu cu:
+ Hiu r yu cu i vi nghin cu VSV l mi dng c v iu kin
lm vic phi tuyt i v trng.
+ Bit c cc k thut nui cy VSV thng dng.
+Bit c mi loi vi sinh vt yu cu mi trng v iu kin nui cy
khc nhau.
Ni dung:
+ Thc hnh thao tc nui cy v trng trn a thch v ng thch
nging.
+ Phng php ria cy phn lp VSV
+ Xc nh mi trng chuyn tnh thch hp cho mt s loi VSV.
I. K THUT V TRNG
Trong phng th nghim, VSV c nui cy nhm d dng xc nh
v kim tra kh nng sinh trng cng nh trao i cht ca chng. VSV c
nhim hoc a vo trong vi dng mi trng nui cy gip gi cho chng
sng, hot ng v nghin cu s sinh trng ca chng. Vic nhim VSV
phi c thc hin trong iu kin khng c cc VSV khng mong mun khc
hoc khng b tp nhim trong mi trng nui cy. K thut v trng c s
dng trong nghin cu VSV nhm loi tr cc s tp nhim.
Tt c cc mi trng nui cy c kh trng trc khi s dng. Vic
kh trng thng c thc hin bng s dng ni hp p lc. Cc dng c
cha mi trng nh ng nghim hay a Petri khng nn m ra cho ti khi thc
s lm vic vi chng, v thm ch sau cng vy.
C hai phng php nui cy trn a petri hay trong ng nghim cung
cp s lng ln VSV trong mt din tch nh v d dng cho vic vn chuyn.
Thch nghing l cc ng nghim cha mi trng c c t nghing
trong khi thch ng li. Thch nghing ging nh a Petri cung cp mt b
mt sinh trng cho VSV nhng ng thch nghing th d bo qun v vn
chuyn hn. Thch cho php lm ng cng tn y ng nghim to ra mt
mi trng thch su. su thng c s dng cho VSV cn t oxy hn
lng ang c trn b mt mi trng. Mi trng thch bn rn ch cha 0,5-
0,7% thch thay cho 1,5% thch c th thng s dng xc nh liu VSV c
di ng c hay khng. Vi khun di ng c s chuyn ng t im nui
cy to ra s xut hin ca cy noen o ngc.
S cy truyn v nhim VSV thng c thc hin vi mt que cy v
trng, c ph mt dy Niken-crom khng b n mn, chu nhit. Trong ,
on cui ca dy c un cong thnh mt ci mc vng, n c gi l mt
que cy trn, nu u dy kim loi thng th gi l qua cy thng. i vi nhng

mc ch c bit, s nui cy c th cng c cy chuyn vi cc ming gc
cotton, pipet, que gt thu tinh hoc xilanh v trng. Nhng k thut ny rt
thng dng trong nghin cu VSV.
Mt que cy trn hay thng c s dng ph thuc vo dng (trng thi)
ca mi trng. Ngi ta c th quyt nh trong khi lm th nghim tu iu kin
v mc ch th nghim.
1. Vt liu th nghim
- ng nghim cha mi trng nc tht
- ng mi trng thch nghing
- ng mi trng thch bn rn
- Que cy u trn, que cy u thng
- Gi ng nghim, thuc nhum Gram
- Nui cy: dch vi khun Lactococcus lactic, Pseudomonas
2. K thut yu cu
Tin hnh th nghim theo th tc sau:
a) Thc hin vi ch mt loi dch vi khun trong 1 ln, ngn chn bt k
s trn ln hoc nhim cho. Bt u vi mt loi nc canh thang, nh nhng
kho y v lc u lng cn.
b) cy vo dung dch nc tht, gi que cy c ging VSV trong tay
thun, mt ng mi trng tay kia.
B1- Kh trng u que cy trn bng cch gi trn ngn la (n gas
hoc n cn) cho n khi nng .
B2- Gi que cy ging nh mt ci bt, un cong ngn tay t phi hp
vi lng bn tay nh nhng rt nt ra khi ng nghim trong khi quay ng.
Khng c t nt ly ra xung mt bn lm vic.
B3- Gi ng nghim nghing mt gc, chuyn ming ca ng nghim
hng v ngn la. Lun gi ng nui cy v ng mi trng mi nghing
gim thiu lng bi bn c th ri vo trong ng. Khng u ng nghim
qu xa hoc dung dch s cy ri khi nt.
B4- Nhng u que cy trn kh trng, c lm ngui vo dung dch
nc tht ly mt vng sc cng b mt dch nui cy. a que cy ra ngoi,
trong khi cm que cy t ming ng nghim v t nt li bng cch quay ng
vo nt. t ng nghim vo gi.
B5- Ly u nt ra khi mt ng nghim cha mi trng nc tht
kh trng, h ming ng trn ngn la. Nhng u que cy c ging VSV vo
mi trng v sau v mt ng t y ng. H ming ng mi trng v
y nt li trc khi t ln gi.
B6- Kh trng li u que cy n khi nng ri ngui. Nhiu khi
ngi ta hay cm nhiu ng nghim trn tay trong cng mt ln cy (hnh).

Khng nm ngoi mc ch gi v cy chuyn gia cc ng nghim nhng phi
thc hin c k thut cy chuyn v trng.
c) i vi mi trng thch nghing, lp li bc 1-4 v cy trn thch
nghing bng cch di chuyn u que cy nh nhng ngang trn mt thch t
y ng ln pha nh, phi cn thn khng chc thng mt thch. H ming
ng nghim trn ngn la v t nt li. Kh trng u que cy, ngui, dn
nhn trn ng nui cy.
d) i vi mi trng thch bn rn, s dng que cy thng, lp li bc
1-4. Cy su vo mi trng bng cch m su u que cy thng vo gia, sau
rt ra. H ming ng nghim trn ngn la v y nt li. t nng que cy,
ngui, dn nhn ng nui cy.
e) Nui cc ng nui cy 35
o
C vi thi gian thch hp.
g) Ghi nhn hin tng xut hin mi ng nui cy, tham kho cc hnh mu.
h) Xem mi v xut hin ca cc ng nui cy. Nhum Gram v so snh chng.
II. K THUT PHN LP VI SINH VT BNG PHA LONG
Trong t nhin, hu ht VSV sinh trng trong mi trng c cha nhiu
loi VSV khc nhau. Vic nui cy hn hp t c s dng trong nghin cu
VSV bi v kh khn trong vic xc nh ring r tng loi v khi chng cng
cc vi sinh vt khc cng biu l cc hot tnh. Nui cy thun khit, ch cha
mt loi VSV n l yu cu trong cc khi nim nghin cu nh c tnh, bnh
pht sinh, trao i cht v kh nng khng khng sinh
Trong nhng nm 1970, Joseph Lister t vn nui cy thun khit
bng cch tin hnh dy pha long ti khi mi mt v mt l thuyt ch cha
mt vi khun. Tuy nhin, s thnh cng l rt gii hn v s tp nhim (c mt
cc VSV khng mong mun) rt ph bin. Nm 1980, Robert Koch t nn
mng cho mi trng c, nh cc nh VSV hc c th tch ri VSV bng
pha long v thu nhn chng trn mi trng c. Mt vi khun c pht hin
da trn s hnh thnh khun lc c th nhn thy c ch cha mt loi VSV.
Hin nay c 3 phng php nui cy thng c s dng phn lp
VSV: ria a, gt a v a. Trong k thut ria a, mt que cy trn c s
dng to vt mu trn nhiu ln trn b mt ca mi trng nui cy rn
trong a Petri. V mt l thuyt, dng que cy trn to vt nhc li cho b mt
thch, VSV ln lt ri khi que cy c phn b ng u trn mt thch,
mi t bo s pht trin thnh mt khun lc. Cy ria a l mt k thut phn lp
ang c s dng ngy nay.
Cy gt v a l k thut nh lng cho php xc nh s lng VSV
trong mt mu c cht. Trong k thut gt a, mt s lng nh mu pha long
xc nh c gt trn a mi trng c bng cch s dng mt que un cong
(hnh dng ging nh mt chic gy hockey). Trong k thut a, mt lng
nh mu pha long c trn vi thch nng chy v trc tip vo a Petri
v trng cn rng. Sau khi nui, VSV sinh trng c th nhn thy l cc khun

lc trn hoc trong a thch . xc nh s lng VSV trong mt mu
ban u, cc a vi s khun lc t 25-250 c la chn. t hn 25 khun lc
l khng ng bi v mt s tp nhim n gy ra t nht 4% sai s. Mt a c
ln hn 250 khun lc th kh m. S lng VSV trong mu ban u c tnh
theo phng trnh sau:
S lng khun lc
VSV/ml =
pha long
1. Vt liu
a Petri cha mi trng thch
ng nghim cha thch nng chy
a Petri v trng, bnh nn 250 ml
Pipet 1 ml v trng, pipet bp bng
Mi trng thch nghing
Dch nui cy: 2 loi vi khun
2. Th tc tin hnh
a. Dn nhn vo y ca 2 a mi trng tng ng vi 2 loi dch nui cy.
b. Kh trng que cy: h nng u que cy, lm ngui ri ly v trng
mt vng que cy dch VSV
c. Thao tc to vt cy c th thc hin vi cc a Petri t trn bn hoc
trn tay.
- Nhc mt mp ca a petri ln v cy phn u tin bng cch to cc
ng khng chng nhau (ng rch rc). Khng c lm xc mt thch
trong khi cy.
- Kh trng que cy, quay a tip v ria qua mt khu vc ca phn th
hai sang phn th ba hoc ria trn phn cn li ca b mt thch, cn cn thn
trnh to cc ng chm vo cc phn cy trc .
- Kh trng que cy trc khi cm li vo gi ng.
d. C th ria cy trn nhiu a vi mt vi loi dch VSV. Sau dn
nhn, ghi tn ngi thc hin, ngy v ngun dch nui cy.
e. Nui cc a va cy 30- 35
o
C trong t nh n cho ti khi pht hin
cc khun lc ring r pht trin (thng t 24-48 h). Ch khi t cc a Petri
trong t phi ngc li cho phn np a xung pha di.
g. Sau khi nui cy, ghi li kt qu
h. Tip tc cy chuyn t mt khun lc n dng sang a mi trng
mi nhiu ln cho n khi thu c ging thun khit. Sau cy t mt khun
lc trn a thun vo ng mi trng thch nghing gi ging.

III. MI TRNG CHUYN TNH
Mi trng chuyn tnh c s dng phn lp cc VSV m s lng l
gii hn trong dch pha long c nhiu VSV u th khc nhau. V d nu dch
phn lp c 1 triu t bo vi khun A v ch c 1 t bo vi khun B, vi khun B c
th b hn ch trong phn ria cy th nht a phn lp. gip phn lp cc
VSV thiu s, cc phng php nui cy lm giu v chn lc c th lm tng
cng s sinh trng ca mt s VSV v hn ch s sinh trng ca cc VSV
khc. Mi trng chn lc cha cc ho cht ngn chn s sinh trng ca cc
VSV khng mong mun m khng nh hng ti s sinh trng ca cc VSV cn
phn lp. Mi trng lm giu cha cc ho cht kch thch s sinh trng ca cc
VSV mong mun. Cc VSV khc s sinh trng nhng s sinh trng ca cc
VSV mong mun s c tng ln.
Mi trng chuyn tnh l mi trng m ch to iu kin cho s pht
trin thun li ca VSV A m khng to iu kin thun li cho VSV B (hay
VSV B khng pht trin hoc pht trin km trn mi trng ny).
Da vo mi trng chuyn tnh m xc nh c VSV A thng qua
vic hnh thnh khun lc ca VSVS .
Mt loi mi trng hu ch khc phn lp VSV l mi trng phn
bit. Mi trng ny cha vi loi dinh dng cho php kho st phn bit
mt loi VSV ny vi cc loi khc bng trao i cht hoc thay i mi trng.
Do c nhiu phng php v nhiu loi mi trng nn phi chn th tc
ph hp vi loi VSV mong mun. V d nu vi khun B chu c mui c
th c thm vo mi trng nui cy. Cc iu kin vt l c th c s
dng la chn mt loi vi khun. Nu vi khun B c kh nng khng nhit,
mu c th c un ln trc khi phn lp. Trong th nghim ny, s xc nh
tiu chun s dng chn mt mi trng nui cy. Hai loi mi trng s
c so snh.
1. Vt liu
a Petri cha mi trng phenylethyl alcohol agar
a Petri cha mi trng trypton agar
Thuc nhum Gram
Hn hp dch nui E. coli v Staphylococcus
2. Th tc tin hnh
Mi trng trypton agar Mi trng phenylethyl alcohol agar
Trypton: 1,0% Trypton: 1,0%
Cao tht: 0,3 g Cao tht: 0,3 g
NaCl: 0,5 g Phenylethyl alcohol: 0,25
Agar: 1,5 g NaCl: 0,5 g

a. S dng mt ci bt nh du chia mi a thnh 3 phn bng cch
nh du di y. Dn nhn mt phn trn mi a cho mi loi dch VSV.
b. Ria mi loi dch VSV trn mt thch nh hnh v.
c. t ngc cc a nui trong t nh n 30-35
o
C. Ghi li kt qu sau
24, 48 v 72h nui cy. Nhum Gram cc khun lc xut hin khc nhau v
quan st di knh hin vi. V hnh quan st c.


1. Cc loi dng c ha cht cn thit ch to mi trng?
2. Trnh by kh trng tng loi dng c, mi trng nui cy VSV?
3. Cc bc tin hnh v cch lm phn lp, tuyn chn v cy truyn VSV?

Bi s 5
PHNG PHP LY MU PHN TCH VI SINH VT
Mc ch:
+ Hiu r phng php v nguyn tc ly mu c cht phn tch VSV.
+ Nm c cc cch bo qun v chun b mu c cht phn tch VSV.
Ni dung:
+ Phng php ly mu c cht trn thc a.
+ Bo qun mu
+ Chun b mu phn tch VSV.
Nguyn l:
Mi mt bin php k thut tc ng vo t hay c cht u gy ra s
thay i sinh vt t v hot ng ca chng trong mi trng. Mc thay i
hot tnh ph thuc vo kch c ca mu c cht v vic x l mu tip theo
(sng, phi kh hoc lm lnh cc mu c cht ti ly t thc a).
1. Xc nh s lng mu v dng c ly mu
1.1 Xc nh s lng mu cn ly:
- Ph thuc vo mc tiu, ni dung nghin cu m nh ra yu cu s lng
v v tr ly mu phi i din.
- Ph thuc vo iu kin c th ngoi thc a, hay khu vc cn ly mu
phn tch
iu quan trng nht ngi ly mu phi thc hin l ly mu sao cho ng
nht v phi i din c khu vc, khoanh t hay c cht cho khu ,
vng .
V d: s lng tng ng cc mu c cht trong cc ng ph thi, rc
thi c ly t khu vc iu tra v c trn ln to thnh mt mu
i din.
1.2. Cc vt liu cn chun b ly mu
- Dng c ly mu ( Dao, ko, li tr, mai thung)
- Cn, bng v trng
- X ng
- Ti ng mu
- Thng lm lnh
1.3. Mu i din
Cc mu ngu nhin c th ch trn u nu chng c ly t mt khu
vc ng nht v i din.

V d: Nu ly mu t phn tch VSV t (cn da vo yu cu ca mc
tiu nghin cu, chnh xc n u, ly bao nhiu mu, din tch rng hp ca tng
khoanh t)
- Din tch
- a hnh
- su theo tng phu din t
- Loi hnh s dng t (thm ch ly theo kiu s dng t)
- Cy trng
- iu kin m
Cc mu t cn c ly khc bit thc s. Nhng khc bit ln trong vng
ln cn phi c loi tr khi mu. Nu chng c tm quan trng c bit th mu
phi c ly ring r.
2. Phng php ly mu v vn chuyn mu
2.1 Nguyn tc ly mu
m bo mu i din, cc im ly mu phi c phn b ngu
nhin trong din tch iu tra. iu ny tt nht c tin hnh theo phng
php ly im theo ng cho nu a hnh ng nht v din tch < 1000 m
2

s ly 5 im i din.
Ly mu khng nn gn b rung hoc ng, v s lng VSV trong
mu t s khng i din cho khoang t chnh vng .
Khi ly mu gt b lp r c b mt (nu c), sau ct vung xung
pha di. C nh vy ly cng nhiu im cng tt. Ly c im no lp tc
phi cho ngay vo ti ng mu trch s nhim tp.
Nu ti yu cu phn tch VSV theo phu din t, th o phu din (nh
phn hc th nhng), sau ly t theo tng tng ring bit phn tch.
Nu rung ly mu ang thm canh cc loi cy trng cn, mt rung
khng ng nh, th phi xem xt c th thc a ly mu sao cho ng nht
v chnh xc. V d: lung khoai, ng, sn, u, lc, rauth khng c ly t
trn mt lung v di rnh lung, m phi ly sn lung, khng c
ly gn gc cy trng.
Ly mu t phn tch tt nht vo thi gian t tng i n nh,
ngha l ngi dn khng tc ng nhiu vo t (thng sau thu hoch)
Ly mu t cc c cht khc, nh: ng rc thi, ph thi, cng cn
phi xem xt rt c th thc a sao cho ly mu chun nht, i din nht khu
vc nghin cu.
Trng lng cn ly khong 200 gam/1 mu
Ly mu phn tch VSV phi m bo nguyn tc l v trng, trch s
nhim tp, ly xong phi cho mu vo phch lnh ng mu (v sinh sn ca

VSV cc nhanh, nu khng bo qun trong iu kin lnh ngay s liu phn tch
s khng chnh xc)
2.2 Vn chuyn mu
Mu phn tch VSV, tt nht ly xong chuyn ngay v phng phn tch
bo qun. Trong qu trnh vn chuyn mu khng c lm thng ti ng
mu v lun lun gi trong phch lnh ( km hm sinh trng v pht trin ca
VSV trong mu)
3. Bo qun mu
Tt nht khi mu v ti phng th nghim cn x l mu v phn tch ngay
(cng vic x l mu phi c thc hin trong phng v trng trch tp khun
vo mu phn tch)
Nu khng kp phn tch, th phi bo qun mu t lnh, nhit < 3
0
C.
Mu c bo qun khng qu 30 ngy, tt nht phn tch cng sm cng tt.


1. Chun b dng c trang thit b cn thit ly mu phn tch VSV?
2. Cc bc tin hnh phn tch VSV trong mu c cht?
3. Trnh by cc vn cn lu tm trong bo qun v phn tch VSV t mu c
cht

Bi s 6
PHNG PHP PHN TCH VI SINH VT
Mc ch:
- Gip cho hc vin bit v thc hnh mt s phng php phn tch VSV
trong c cht
- Nm c phng php phn tch VSV (nui cy trong mi trng dch
th v trn mi trng rn hoc bn rn)
Ni dung:
- Gii thiu phng php pha long (chun b dch cy)
- Nui cy VSV trong mi trng dung dch
- Nui cy VSV trn mi trng rn, bn rn
- Kim tra kt qu nui cy
- Tnh s lng VSV trong 1 gam hay 1 ml dch mi trng.
1. Cc bc phn tch
1.1 Dng c phn tch:
- Bnh tam gic, bnh cu, ng nghim (dung tch: 10ml, 100ml, 250 ml),
Pipt chia 0,1 - 1,0 ml, 2ml, 5 ml, 10 ml), hp nhm, que cy, que gt, bi
thy tinh, giy qu, khay men, cuc, khoan .Tt c cc dng c u phi tit
trng.
+ Mi trng nui cy VSV kh trng (Bi 3)
1.2 Chun b bnh nc v trng lm dy pha long:
Chun b 10 bnh tam gic hoc bnh cu c dung tch 100ml hoc 250 ml.
Cho vo mi bnh 90 ml nc my (thng cho 92 ml, v khi kh trng bc hi
khong 2 ml). em bnh cc bnh c nc tit trng hi 1 atm (121
0
C) qua
30 pht. Ly ra ngui s c dy pha long, nh s th t t 1 n 10.
1.3 Xc nh m mu cn phn tch:
Cn 10 gam mu c cht ti cho vo hp nhm, c th cn c trong lng
hpp nhm khi cha c mu phn tch, sau em sy t sy nhit 105
0
C
qua 4 gi, em hp nhm sy ra cho vo bnh ht m cho n khi ngui. em
cn trng lng, sau sy nhiu ln nh vy n khi trng lng khng thay i,
th tnh m ca mu phn tch theo cng thc sau:
A - B
m X% = ---------- x 100
B - C
X: m %
A: Khi lng hp + c cht cn t.
B: Khi lng hp + c cht sy kh.
C: Khi lng hp.

Tnh h s kh kit k:
1
k = ----------
1 - x
1.4. Chun b dy pha long
Mun m c s lng vi sinh vt d dng, khi phn tch cn pha long
c cht n mt nht nh. Cn lu rng lm dn mt VSV phi pha
long t l lun lun = 1/10
- Cn 10g c cht cho vo bnh th nht c 90 ml nc v trng, lc 15
- 20 pht, trn my lc 150 ln/ pht. Nh vy chng ta c dung dch pha
long 10
-1
, ngha l t l 1:10.
- Dng pipet 10ml ht 10 ml dung dch nng 10
-1
cho vo 90 ml
nc ng bnh th 2, lc u, chng ta c nng pha long 10
-2
, t l 1:100.
- Tip tc lm nh vy i vi cc bnh th 3, 4n khi chng ta c
c dy pha long cn thit, t l 10
-3
, 10
-4
,

10

Ch : cc pipet u phi kh trng, mi mt nng pha long phi
dng 1 pipet ring.
C th chun b dy pha long bng ng nghim c cha 9 ml nc, hoc
bnh tam gic c cha 45 ml nc v trng . ri em kh trng nh lm vi
bnh tam gic. Nu lm bng ng nghim, th ch cn 1 gam c cht kh, hoc 5
gam c cht kh. Ngha l ta vn to c dy pha long t l 1:10. phn tn
u dch c cht trong ng nghim dng pipet ht dch a ln a xung nhiu
ln nhng khng thi kh.
2. Nui cy
Khi c dung dch pha long ri, dng pipt 0,1 ml ly t bnh pha
long no trong dy pha loang cy vo trong mi trng chun b sn cho
tng nhm hoc tng ging VSV nh phn tch. Mi nng t nht phi cy 3
- 5 ln nhc li (s ln nhc li cng nhiu, th kt qu c tin cy cng cao).
Cn cy t nht 3 nng pha long lin tip. Cy xong dung que gt thy tinh
v trng dn u dch cy trn mt mi trng (nu mi trng rn hoc bn rn
trn a mi trng thch). Cn cy vo trong mi trng dung dch trong cc
ng nghim hoc bnh dung dch dinh dng ch cn lc nh l xong.
Ch tt c cc hp lng hoc cc bnh mi trng th nghim u phi
nh s th t theo nguyn tc sau: s mu nghin cu nng pha long - s
ln nhc li.
V d: 5.4.3 - ngha l mu phn tch s 5; cy nng pha long th 4;
cy a mi trng c s ln nhc li th 3.
Sau khi cy xong cho a, hoc bnh nui cy vo trong t nui, nui nhit
thch hp cho tng chng ging VSV khc nhau trong thi gian nht nh (c th
t 48 - 72 gi, c ging phi nui lu hn mi hnh thnh khun lc)

Cng mt dung dch c cht pha long c th phn tch nhiu mt nh
nm, x khun v cc loi vi khun bng cch cy vo mi trng thch hp.
Mi trng c th l dch th, c th l thch bng v t nhng khun lc
mc trn mi trng thch bng v nhng c trng ca mi trng dch th,
chng ta c th tnh s lng ca vi sinh vt.
3. Tnh s lng vi sinh vt
3.1. Phng php thch bng ( trn mi trng thch )
- Mi t bo vi sinh vt trn mi trng thch hp s pht trin v cho
chng ta mt khun lc. Do s lng khun lc cho ta bit s lng vi sinh
vt trong mt gam c cht.
- Sau khi VSV mc trn mi trng thch a (hp lng), em m s
lng khun lc c hnh thnh bng my m khun lc, hoc m trc tip
theo phng php chia trn a mi trng.
Kt qu c tnh theo cng thc sau:
S = T x 10 x N x K
S: S lng vi sinh vt trong mt gam c cht.
T: S khun lc trung bnh trong mt hp petri
10: S khun lc qui ra 1 ml ( v lc nui cy trong hp lng chng ta
dng 0,1 ml dung dch c cht )
N: S nghch o ca nng pha long
K: h s kh kit (Nu khng quy i t kh sang ti)
3.2 Tnh s lng vi sinh vt trong mi trng lng ( phng php nh tnh )
C nhng loi vi khun khng th dng mt thng quan st khun lc
hoc sn phm sinh ra cng khng c mu g c bit nh gi vi khun hot
ng. Trong trng hp ny phi dng phn ng mu xc nh. C mi ng
nghim c phn ng mu gi l ng ( + ). Da vo cc ng dng tnh, cn c
vo bng Mc.Crady chng ta tnh ra s lng vi sinh vt.
V d:
pha
long
10
-4
10
-5
10
-6
10
-7
10
-8
10
-9

S ng
dng
3
+
3
+
3
+
2
+
1
+
0

- Tm s ch tiu: 321
S ch tiu l con s c 3 hng s. Hng s u l s biu hin nng
long nht cc ng nghim u dng. Hai s tip theo l s ng dng 2
nng tip theo sau.
- em s ch tiu ny tra bng Mc.Crady ta s c s lng vi khun tng
ng: 15

- T con s ny ta tnh ra s lng vi khun trong mt gam c cht theo
cng thc:
S = t x 10 x n x k
S: S lng vi sinh vt.
t: S lng vi khun tra bng
10: S lng vi khun qui ra 1 ml
n: S nghch o ca nng pha long
k: H s kh kit

S= 15 x 10 x 10
3
x 1,52 = 2,28 . 10
5
t bo/1g


1. Trnh by phng php pha long?
2. Nhng dng c cn thit lm dy pha long l g?
3. Nguyn tc c bn nht ca phng php pha long nui cy VSV l g?
4. Phng php v cch tnh s lng VSV trn mi trng c, mi trng
lng?

Bi s 7
NH GI C TNH SINH HC CA VI SINH VT

Mc ch:
- Gip cho hc vin hiu r tm quan trng phi nh gi c tnh sinh
hc ca cc VSV
- Bit c cc phng php c bn nh gi c tnh sinh hc ca VSV
Ni dung:
- nh gi thi gian mc ca chng ging cn nghin cu
- nh gi ng knh khun lcc ca chng ging cn nghin cu
- Xc nh kh nng thch ng mi trng pH ca chng ging cn
nghin cu
- Kh nng cnh tranh ca chng ging cn nghin cu
- Hot tnh ca chng ging cn nghin cu
1. Dng c v nguyn vt liu
1.1 Dng c:
a Petri, cn 76
0
, nc v trng, bnh tam gic hoc bnh cu 100, 250,
500, 1000 ml. Bnh tia, lam knh, n cn, thuc nhum, knh hin vi.
Mi trng nui cy chng ging VSV nghin cu
2. nh gi c tnh sinh hc theo tng ch tiu:
Vic nh gi c tnh sinh hc ca cc vi sinh vt c tuyn chn da vo cc
ch tiu sau:
2.1. Xc nh thi gian mc ca cc VSV
Theo phng php nui cy trn mi trng thch bng hoc trong mi
trng dung dch (ty tng ging VSV) t nh n nhit t 28 - 60
0
c (ty
thuc vo c im ca tng loi vi sinh vt).
Theo di tng thi gian khc nhau t 24, 48 ,72, 96, 120, 144, 168
gi.
Tng thi gian trn quan st khi VSV mc c trong thi gian nui t
nh n, th tnh gi. Xc nh xem chng ging thuc nhm mc nhanh hay
mc chm (< 72 gi thuc nhm mc nhanh; > 72 gi thuc nhm mc chm)
2.2. Xc nh kch thc khun lc o trc tip bng thc (mm).
Do nhiu khun lc sau tnh trung bnh trung (tt nht nui 5 ngy
mi o)
2.3. Xc nh kch thc t bo bng phng php do trc tip bng knh
hin vi (m)

C nh tiu bn, nhum t bo sau do bng dng c o chuyn dng di
knh hin vi
2.4. Xc nh kh nng thch ng mi trng pH khc nhau
Bng phng php nui cy trc tip cc mc pH khc nhau, c th l:
pH = 4,0; pH = 5,0; pH = 6,0; pH = 7,0; pH = 8,0. (pH
kcl
)
Cch lm nh sau:
- Pha dung dch m Sorensen 1:
+ Cn chnh xc 9,078 g KH
2
PO
4
1/15 M.
+ Cn chnh xc 11,876g Na
2
HPO
4
. 2H
2
O ho vo 1 lt nc ct c
dung dch Na
2
HPO
4
1/25 M.
Sau pha 2 dung dch ny theo t l nh sau: (i vi 1000ml mi trng).

T l trong dung dch (ml) pH
Na
2
HPO
4
KH
2
PO
4

4,0 0 100
5,0 5 95
6,0 30 70
7,0 80 20
8,0 99 1

pH chnh xc ca mi trng c to ra nh s gip ca dung dch
m photphat c b sung vo mi trng v trng vi s lng 10% so vi th
tch ca mi trng.
- Chun b mi trng chuyn tnh ca cc chng vi sinh vt sau pha mi
trng vi cc nng pH khc nhau, em hp kh trng (1at, 20 pht), ra a
pettri (15 - 20 ml/1 a pettri).
Th nghim c nhc li 4 ln 5 mc pH khc nhau.
- Mi trng dch th c cho vo cc bnh tam gic 100 ml ( 50 ml mi
trng dch th/ 1 bnh tam gic). Cy 1 ml dch khun chuyn tnh ca tng
chng vi sinh vt vo cc bnh tam gic chun b trn ri a ln my lc
(150 vng/ pht, trong vng 48 gi). Sau ly 0,05 ml dch vi khun cy
vo mi trng thch bng ( c chun b trn), dng que gt ( kh
trng) gt u dch vi khun trn b mt thch. Sau em nui trong t nh n
(nhit t 28 - 30
0
c). Sau 48 gi nui cy a ra m s lng khun lc mc
tng nng pH khc nhau.

2.5. Xc nh kh nng khng khng sinh (kh nng cnh tranh)
Theo phng php nui cy trc tip trn mi trng c Steptomixin vi
cc nng khc nhau: 300, 500, 600, 800, 1000 mg/1lt mi trng
(Obtin,1971).
Cch lm nh sau:
Pha thuc khng sinh theo nng nh sn cho vo mi trng
chuyn tnh ca tng chng vi sinh vt ( mi trng c kh trng 1at,
30'). Sau chia ra cc a pettri ri ngui. Cy 0,05 ml dch khun chuyn
tnh ca tng chng vi sinh vt a pettri, em nui trong t nh n (nhit t
28 - 30
0
c). Sau 3 ngy nui cy a ra m v quan st s lng khun lc mc
tng nng khng sinh khc nhau.
2.6. nh gi hot tnh ca cc chng ging VSV
Ch tiu ny ph thuc vo tng VSV khc nhau, m nh gi hot tnh
ca chng khc nhau. V d: Nghin cu v hot tnh phn hy cellul, hot
tnh c nh nit, hot tnh phn huye chuyn ha phospho kh tan
Th nghim v xc nh hot tnh phn hy chuyn ha ln, th: xc nh cng
phn gii ln kh tan
Dng vi sinh vt: T
1
, T
2
.
Nguyn liu: Apatit, photphorit, canxiphotphat.
Vic nh gi cng phn gii ln kh tan theo 2 phng php sau:
o ng knh vng phn gii ca vi sinh vt, cch lm nh sau:
Cn 10g cc cht trn ln lt cho vo tng loi mi trng chuyn tnh.
em kh trng ( 1at, 45') ri ra a pettri (khong 20 - 25 ml/1 a) sau
ngui.
Cc chng vi sinh vt c ho tan thnh dch huyn ph bng nc ct
v trng. Dng que cy ly dch cy im vo mi trng thch bng c
apatit, photphorit, canxiphotphat - Ca
3
(PO
4
)
2
. em nui trong t nh n (nhit
t 28 - 30
0
c). Theo di sau 3 ngy/ 1 ln o ng knh vng phn gii ca
tng chng vi sinh vt.
Xc nh cng phn gii ln kh tan trn mi trng dch th bng
phng php Oniani trn my so mu.
Cn 20g cc cht trn ln lt cho vo tng loi mi trng dch th, kh
trng ( 1at, trong vng 45'), sau ngui.

Cy ging vi sinh vt vo tng mi trng chuyn tnh trn v a ln my lc
(150 vng/pht). C sau 3, 7, 15, 21, 30 ngy th o 1 ln trn my so mu.


1. Trnh by nhng ch tiu c bn nh gi c tnh sinh hc ca cc chng
ging VSV cn nghin cu?
2. Phng php chn v nh gi tng c tnh sinh hc ca VSV tuyn chn
lm ging cho hc tp v nghin cu?

Bi s 8
PHN LP TUYN CHN VI KHUN AZOTOBACTER T T
Mc ch:
- Gii thiu cho hc vin bit cch phn lp tuyn chn chng ging VSV
thun khit t t, m c th trong bi l ging vi khun Azotobacter.
Ni dung:
- Phng php phn lp vi khun Azotobacter.
- Mi trng dinh dng phn lp vi khun Azotobacter.
- nh gi c tnh sinh hc ca vi khun Azotobacter.
- Bo qun ging vi khun Azotobacter.
1. Chun b dng c, nguyn vt liu v mi trng phn lp
1.1. Dng c, nguyn liu
a Petri, cn 76
0
, nc v trng, bnh tam gic hoc bnh cu 100, 250,
500, 1000 ml. Bnh tia, lam knh, n cn, thuc nhum, knh hin vi.
Mu t sng qua ry 2mm.
Bnh cha 45 hoc 90 ml nc v trng
1.2. Mi trng phn lp: s dng mi trng Asby
Gluc 10g K
2
HPO
4
0,5g
MgSO
4
0,2g NaCl 0,2g
K
2
SO
4
0,1g CaCO
3
5g
Thch 15 - 20g Nc ct 1000ml
Cn mi trng, un tan thch cho vo bnh thu tinh c nt mi, kh trng
0,6 - 0,8 atm (105- 110
0
C) trong 30 pht. m (50-60
o
C) phn vo a Petri
20-25 ml/ a (ng knh 9 cm), cng vic ny c tin hnh hon ton trong
phng v trng.
2. Cc bc phn lp vi khun Azotobacter
Cn 5g mu t cho vo bnh tam gic 100 ml c sn 45 ml nc v
trng, cho ln my lc trong 20 pht tc 150 -200 ln/pht.
Dng que cy ly dung dch lc ria cy ln mi trng thch bng. Cy
thnh nhiu ng thng song song hoc cy ria 3 pha vi lng dch gim dn.
Sau em nui trong t nh n 25 - 28
0
C trong 2 - 3 ngy i vi vi khun
mc nhanh. 5 - 7 ngy i vi vi khun mc chm.
Khun lc Azotobacter c mu trng trong, li nhy khi cn non, khi gi c
mu vng lc hoc mu nu sm, bin khun lc u. Vi khun gram m, khng sinh
bo t, di ng nh tin mao.

3. Xc nh hnh thi khun lc, t bo v kh nng di ng ca Azotobacter
- Quan st cc khun lc VSV mc trong a petri. Nhn xt miu t v v
hnh dng cc khun lc ng ring r. Khi miu t cc khun lc khng m np
a petri. Ghi chp cc c im ca khun lc vo bng theo mu sau:
Khun
lc
s
Hnh
dng
ng
knh,
mm

sng,

trong
Mu
sc
B
mt
Nhn
nghing
Mp Cu
trc

cht
Hunh
quang
Hnh
v
khun
lc

- Lm tiu bn git treo quan st s di ng ca Azotobacter. V dng di chuyn.
- Lm tiu bn kh, nhum Gram, quan st hnh thi ca vi khun. V hnh.
phn lp c ging Azotobacter thun khit, dng phng php loi
dn trc tip trn a mi trng Asby. Nu quan st khun lc khng thun
hoc b nhim tp ta loi ngay.
4. Tuyn chn chng ging Azotobacter
Khi phn lp c chng ging Azotobacter thun khit ri, cng vic
tip theo buc phi tin hnh nh gi c tnh sinh hc ca chng ging mi
c phn lp.
nh gi c tnh sinh hc bng cc ch tiu sau: (trong bi thc hnh v
nh gi c tnh sinh hc ca VSV)
- Thi gian mc
- Kch thc khun lc
- Kh nng mc mi trng pH rng
- Kh nng cnh tranh (Khng khng sinh)
- Hot tnh c nh nit phn t
- Kh nng pht trin trn mi trng to cc enzym
5. Bo qun chng ging Azotobacter
Cng tc bo qun chng ging VSV l vic lm rt cn thit v din ra
thng xuyn.
Khi phn lp tuyn chn c chng ging VSV nh mun ri, th
vic thun ha, bo qun l rt qun trng.
Phi cy truyn v nui nhc li rt nhiu ln nhn c ging chng
thun khit. Sau phi thc hin ng nguyn tc trong cng tc bo qun
ging VSV ti phng th nghim. (s gii thiu trong bi thc hnh v bo qun
v gi ging VSV)



1. Trnh by cc tiu tr phn lp, tuyn chn chng ging Azotobacter trong
t? Mi trng nui cy?
2. Nu c tnh sinh hc ca chng ging Azotobacte cn tuyn chn?

Bi s 9
PHNG PHP LY MU V PHN LP TUYN CHN VI KHUN
NT SN (VKNS) RHIZOBIUM

Mc ch:
- Gii thiu cho hc vin bit phng php ly mu nt sn r cy h u
phn lp chng ging vi khun Rhizobium (vi khun c nh nit
phn t cng sinh)
Ni dung:
- Hiu c cc bc phn lp tuyn chn vi khun Rhizobium
- Xc nh c chng ging vi khun Rhizobium mi phn lp
- nh gi c tnh sinh hc ca chng ging Rhizobium mi phn lp
- Bo qun v s dng chng ging Rhizobium mi phn lp
1. Dng c v nguyn vt liu
a. Dng c:
Dao, ko, kp st, ko, hp lng, cn 76
0
, 95
0
nc v trng, bng tam gic
hoc bnh cu 100, 250, 500, 1000ml, dung dch HgCl
2
0,1%. bnh tia, lam knh,
n cn, thuc nhum, knh hin vi,
b. Mi trng Pochon
Manit hay gluc 10g K
2
HPO
4
0.5g
MgSO
4
0,2g NaCl 0,2g
Nc men bia 100ml CaCO
3
1g
Cng 1% 10ml Thch 15 - 20g
Nc ct 880ml
Cn mi trng cho vo bnh thu tinh c nt mi, kh trng 0,6 - 0,8 t.m
(105- 110
0
C) qua 30 pht. m phn vo hp lng 25- 30ml/ hp, cng vic
ny c tin hnh hon ton trong phng v trng.
2. Phn lp vi khun Rhizobium
Ly r cy u c nt sn to, nhiu k sc trng, mu hng thi k
cy ra hoa, mang v phng th nghim phn lp. Bn ngoi nt sn c nhiu tp
khun, phi tit trng trc khi phn lp. Ra sch nt sn, ly ko ct nt sn ra
khi r. Ch khng lm nt sn xy xt. Cho nt sn vo nc trong ra tht
sch, cho vo cn 95
0
trong 3 pht, cho tip vo dung dch HgCl
2
0,1% trong 5
pht, ra bng nc v trng 5 - 6 ln, mi ln 3 - 5 pht. Cho nt sn vo hp
lng c mt t nc v trng. Dng kp st bp nt v lm thnh dung dch nt sn
(c th dng dao lam kh trng ct nt sn). Dng que cy ly dung dch nt sn
cy ln mi trng thch phng. Cy thnh nhiu ng thng song song vi lng
dch gim dn. Sau em nui t nui c 25 - 28
0
C trong 2 - 3 ngy i vi vi
khun mc nhanh. 5 - 7 ngy i vi vi khun mc chm. Khun lc nt sn c
mu trong sut hoc 1/2 trong sut. Bin khun lc u. Nu c cng hay

violet gentian khun lc vn khng b nhum mu. Vi khun gram m, khng
bo t, di ng nh tin mao.
3. Xc nh kh nng c nh nit
3. 1. Th nghim trng cy trong ng thch:
Phng php ny cho php nhn bit c kh nng c nh nit ca vi
khun nt r.
Mi trng dng trng cy gy nhim:
Mi trng Jenhsen (1942) hoc mi trng Thornton (1930)
Mi trng Jenhsen
CaHPO
4
1,0g
K
2
HPO
4
0,2g
MgSO
4
.7H
2
O 0,2g
FeCl
3
0,1g
Nc 1000ml
Thch 8 - 15g
pH = 6,5 - 7,0
Mi trng Thornton
CaHPO
4
2,0g
K
2
HPO
4
0,5g
MgSO
4
.7H
2
O 0,2g
NaCl 0,1g
Nc 1000ml
Thch 8 - 15g
pH = 6,5 - 7,0

C th dng ng mi trng thch nghing hoc ng.
Dng dung dch dinh dng Gibson (1963) 1ml/lit mi trng.

Thnh phn dung dch vi lng nh sau:
H
3
PO
3
0,05g; (NH
4
)
2
MoO
4
0,05g
KCl 0,005g NaBr 0,005g
ZnSO
4
0,003g Al(SO
4
).18H
2
O 0,003g
MnSO
4
0,002g Nc ct 1000ml

Chun b ng mi trng:
ng nghim 150 x 20mm cho ht u nh
ng nghim 200 x 30mm cho ht u ln

Lng mi trng cho vo theo bng sau:
C ng nghim
(mm)
150 x 20
150 x 25
150 x 30
200 x 30
Thch ng
(ml)
8
12
18
25
Thch nghing
(ml)
12
18
30
40
Cho mi trng trng cy theo bng hng dn trn. Nt bng v kh
trng 1at trong 20 pht. Sau t nghing nu cn trng cy trn ng thch
nghing.
Chun b ht ging:
Chn ht ging sch khng b tn thng. Kh trng bng etanol 95%.
Ngm trong 3 pht hoc 0,2% HgCl
2
c axit ha (50ml/lt HCl). Ht c
kh trng phi c ra sch bng nc v trng t nht l 5 ln. Sau em cho
ny mm ( trn giy lc m v trng hoc trn mi trng thch a hoc trn
ct v trng).
Nhim khun:
Nhng ht ny mm c a ra mt a v trng. Cho dch vi khun
vo. Ht c nhim khun c th trng ngay vo ng thch v trng hoc
sau 7 ngy ri mi trng (tt hn l sau 7 ngy) vo ng nghim.
3. 2. Gieo trng cy vo ng nghim
Nhng ng nghim c kh trng em t ln gi st, thay cho nt
bng u ng nghim mi trng bng bc giy en, giy thic hoc tt hn l
bng nt g mm c l khoan dc theo chiu di ca nt g. Cng vic ny phi
tin hnh trong iu kin v trng. c mt l trn giy bc ming ng nghim.
Cho ht u ny mm c nhim khun cy vo ng nghim qua l thng.
C th trng 2 ht, nhng v sau ch 1 cy. Cy mm lc u mc pha trn
nh ng thch ng hoc thch nghing. V sau y vo v tr ng thch
ng, nm trn b mt mi trng. Nu l ng thch nghing, th y cy nm
khong 1/3 mt thch v pha u ng nghim. ng thch ng, r cy s pht
trin trn b mt thch, cn ng thch nghing r s pht trin dc theo mt
thch. Trng cy xong vo gi st trong phng c chiu sng thch hp. C
th chiu sng t trn xung hoc t 2 bn vo.
3.3. iu kin
+ Chiu sng:
Tu tng ging u khc nhau, tui ca cy khc nhau, mc pht trin
khc nhau, m cn cng chiu sng khc nhau ( c th dng bng n n-
ng 60w)
+ Nhit v m:

Nhit t 20 - 30
0
C, m tt nht l 60 - 70%.
+ Chm sc:
Thng xuyn quan st s khc nhau gia ng i chng v ng c
nhim khun.
Nu b sung m nui cy vi nng 70ppm (0,05% KNO
3
), th c
th cho vo mi trng thch trong ng nghim trc khi gieo ht hoc vo lc
cy mc. Tt nht l bn khi cy trng c 5 - 7 ngy.
Nu quan st thy cy xu, khng xanh (c i chng ln nhim
khun) th phi b xung thm m ngay, nhng lng bn khng c cao hn
0,07% KNO
3
, nu cao hn c th b c cho cy v vi khun.
3.4. nh gi kt qu
ng nghim nhim Rhizobium s to c nt sn r cy, sau trng 3 -
6 tun, cn cng thc i chng s khng to nt sn.
nh gi mi lin quan gia thi gian v s pht trin ca cy theo cng
thc sau:
log W
2
- logW
1

Rw =
t
2
- t
1

Rw = mi lin quan gia s pht trin v thi gian
W
1
,W
2
= Trng lng kh ca cy thi gian t
1
v t
2

nh gi mi lin quan gia c nh m ca cy v vi khun qua cc
thi gian theo cng thc:
logN
2
- logN
1

Rn =
t
2
- t
1
Rn = lin quan c nh m
N
1
v N
2
= m tng s ca cy thi im t
1
v t
2

4. nh gi c tnh sinh hc
Bng cc ch tiu sau: (Trong bi thc hnh v nh gi c tnh sinh hc ca
VSV)
- Thi gian mc
- Kch thc khun lc
- Kh nng mc mi trng pH rng
- Kh nng cnh tranh (Khng khng sinh)
- Hot tnh c nh nit phn t
- Kh nng pht trin trn mi trng to cc enzym
5. Bo qun chng ging Rhizobium

Cng tc bo qun chng ging VSV l vic lm rt cn thit v din ra
thng xuyn.
Khi phn lp tuyn chn c chng ging VSV nh mun ri, th
vic thun ha, bo qun l rt qun trng.
Phi cy truyn v nui nhc li rt nhiu ln nhn c ging chng
thun khit. Sau phi thc hin ng nguyn tc trong cng tc bo qun
ging VSV ti phng th nghim. (s gii thiu trong bi thc hnh v bo qun
v gi ging VSV)


1. Trnh by cc tiu tr phn lp, tuyn chn chng ging Rhizobium r cy
h u? Mi trng nui cy?
2. Nu c tnh sinh hc ca chng ging Rhizobium cn tuyn chn?

Bi s 10
PHNG PHP XC NH NHANH TRAO I CHT VSV

Mc ch yu cu:
+ Hiu r cc thut ng: trao i cht, hydratcacbon, enzym thu phn.
+ Xc nh s thu phn hydratcacbon v gelatin.
Ni dung kin tp:
+ Thc hin test th s thu phn tinh bt.
+ Thc hin v gii thch test th trn sa giy qu.
Nguyn l chung:
Leewenhoek nhn thy VSV trong ru vang, Pasteur chng minh
rng VSV l cc t chc sng. Nm 1972, Pasteur vit:
Khng th tng tng ni l vt cht hu c ca cc men mi hnh
thnh (vi sinh vt) cha mt nguyn t cacbon n m n khng c ngun gc
t c cht ln men.
Cc phn ng ho hc c quan st bi Pasteur v cc phn ng ho hc xy
ra trong tt c cc t chc sng c gi l trao i cht. Cc qu trnh trao i cht
i hi phi c enzim, c bn cht l protein xc tc cho cc phn ng sinh hc.
Phn ln cc enzim thc hin chc nng bn trong mt t bo gi l enzim ni
(endoenzymes). Nhiu VSV tit ra enzim gi l enzim ngoi bo, chng c gii
phng ra t t bo xc tc cc phn ng xy ra bn ngoi t bo.
Mt s VSV s dng cc con ng trao i cht c bit vi s c mt
ca oxy (ho kh) v cc con ng khc khi khng c mt oxy (ym kh).
Pasteur ni tip:
Ti c th chng minh rng t cc men ny (VSV) sng st vi s c mt
ca oxy t do, chng mt kh nng ln men ca mnh t l vi nng ca kh.
Bi v nhiu VSV c hnh thi t bo v khun lc ging nhau, thm vo
cc yu t nh trao i cht c s dng m t v phn loi chng. Trn
c s cc c cht, mt phn VSV s dng v trao i cht to ra cc dng ca
n, cc test th trong phng th nghim c thit k xc nh cc enzim
ca VSV.
1. Trao i Cacbon
Cc phn ng ho hc gii phng ra nng lng t s phn gii cc phn
t hu c phc tp c gi l d ho. Hu ht VSV chuyn ho hydratcacbon
gii phng CO
2
v nng lng. Hydratcacbon l cc phn t hu c c cha
cacbon, hydro v oxy theo t l (CH
2
O)n. Hydratcacbon c th chia thnh 3
nhm da vo s lng: monosaccharit, disaccharit v polysaccharit.
Monosaccharit l loi ng n c cha t 3-7 nguyn t cacbon, disaccharit

c to nn t 2 n phn t monosaccharit, v polysaccharit bao gm 8 hoc
nhiu hn cc phn t monosaccharit.
Enzim ngoi bo l enzim thu phn chnh bn ngoi t bo, v ph v
cc vt cht ln khi c thm nc thnh cc thnh phn nh hn m chng c
th chuyn vn vo trong t bo. Enzim ngoi bo Amilaza thu phn
polysaccharit, tinh bt thnh cc hydratcacbon nh hn. Glucoza, mt loi
monosaccharit c th c to ra do thy phn. Trong phng th nghim, s c
mt ca cc enzim ngoi bo c xc nh bng cch quan st s thay i c
cht bn ngoi khun lc vi sinh vt.
Glucoza c th vo bn trong t bo v c d ho; mt s vi khun d
ho oxy ho glucoza to thnh oxit cacbon v nc. D ho oxi ho i hi phi
c mt phn t oxy (O
2
). Tuy nhin, hu ht vi khun ln men glucoza m
khng s dng oxy. D ho ln men khng yu cu oxy nhng c th xy ra
trong iu kin c oxy. Sn phm trao i cht cui cng ca qu trnh ln men
l cc phn t hu c nh, thng l cc axit hu c. Mt s vi khun va oxy
ho va ln men, mt s khc khng thc hin c oxy ho v ln men nhng li
ly cacbon v nng lng t cc phng thc khc.
Mt sinh vt b oxy ho hoc ln men c th c xc nh bng cch s
dng mi trng c bn OF ca Rudolph Hugh v Einar Leifson vi loi
hydratcacbon mong mun c thm vo. Mi trng OF l mi trng dinh
dng thch su bn rn cha nng hydratcacbon cao v nng pepton
thp. Pepton s h tr mt phn s sinh trng ca cc vi khun khng oxy ho
v khng ln men. Hai ng nghim c s dng: mt ng m ly khng kh
v mt ng bt kn gi khng c oxy. Mi trng OF cha ch th xanh
bromthymol, s tr thnh mu vng khi c mt axit, chng t xy ra d ho
cacbon. Trong iu kin c alkan, s s dng pepton v khng dng
hydratcacbon c biu l bng mu xanh sm. Nu hydratcacbon c chuyn
ho trong c 2 ng, s ln men xy ra. Mt s vi khun sinh ra kh trong qu
trnh ln men cc hydratcacbon. Mt c th ch s dng oxy ho hydratcacbon
s ch sinh ra axit trong ng nghim m. Mi trng OF c s dng xc
nh s d ho cacbon ca vi khun Gram m Bacillus. S d ho cacbon s c
chng minh trong bi tp ny. Cc test th s rt quan trng trong vic xc nh
vi khun.
1.1. Vt liu
a Petri cha mi trng dinh dng thch - tinh bt
Mi trng thch su gluco-OF
Du khong
Thuc nhum Gram
1.2. Dch nui cy
Bacillus subtilis
Escherichia coli

Pseudomonas aeruginosa
1.3. Th tc k thut yu cu
1.3.1. Thu phn tinh bt
a. eo khu trang, chia mi trng thch-tinh bt thnh 3 phn bng cch
nh du di y a.
b. Cy ria mt ng n vi khun Bacillus, Escherichia, Pseudomonas
trn mi phn.
c. t ngc cc a nui 35
o
C/24h. Sau khi s sinh trng din ra, cc
a c th c ct tr trong t lnh 5
o
C ti giai on th nghim tip theo.
d. Ghi li bt k s pht trin no ca vi sinh vt, sau trn Indon
Gram ln a. Khu vc thu phn tinh bt s hin r, trong khi khu vc tinh bt
khng chuyn ho s nhum mu xanh sm.
1.3.2. Mi trng OF
a. S dng que cy thng, nhim 2 ng mi trng glucoza-OF vi dch
nui cy vi khun ch nh (Escherichia, Pseudomonas hoc Alcaligenes).
b. Nh khong 5ml dch du khong trn 1 ng mi trng. y nt li.
c. Nui tt c cc ng 35
o
C cho ti giai on th nghim tip theo.
d. So snh cc ng mi trng nhim v khng nhim VSV. Ghi li kt
qu nh sau: khun lc c mc hay khng, liu glucoza c s dng? V
loi trao i cht?
S di ng ca vi khun c th xc nh t cc ng mi trng OF.
e. Quan st v ghi li kt qu cc loi VSV m bn khng nui cy (nhn mu sc
a nui cy).
2. Trao i Protein
Protein l cc phn t hu c cha cacbon, hydro, oxy v nit; mt vi
loi protein cng cha lu hunh. Cc n v ph to nn protein c gi l
amino axit. Amino axit gn cht vi nhau bi lin kt peptit, hnh thnh nn mt
chui nh (peptit) hoc mt phn t ln (polypeptit).
VSV c th thu phn peptit hoc polypeptit gii phng ra cc amino
axit. Chng s dng cc amino axit l ngun cacbon v nng lng khi
hydratcacbon khng c sn. Tuy nhin, amino axit c s dng trc tin
trong cc phn ng ng ho.
Cc phn t protein ln nh gelatin, c thu phn bi enzim ngoi bo
v cc sn phm nh hn ca s thu phn c chuyn vn vo trong t bo.
S thu phn gelatin c th c chng minh bng s sinh trng ca vi khun
trong dinh dng gelatin. Dinh dng gelatin c lm tan trong nc m
(50
o
C), ng li (to gel) khi c lm lnh di 25
o
C v ho lng (to sol) khi
gia nhit n khong 25
o
C. Khi mt enzim ngoi bo thu phn gelatin s lm
ho lng v khng ng li c thm ch khi c lm ngui di 20
o
C.

Mt s vi khun c th thu phn protein trong sa c gi l cazein. S
thu phn cazein c th c xc nh trong sa c giy qu. Sa giy qu cha
c vng sa v ch th giy qu. Mi trng b m c, do cazein dng keo
huyn ph v giy qu c mu xanh. Sau khi pepton ho (s thu phn cc
protein sa), mi trng tr nn trong l kt qu ca s thu phn cazein thnh
cc amino axit ho tan v cc mnh peptit. Kt qu ca s d ho cc amino axit
s to ra mt phn ng alkan (mu ta). Sa giy qu cng c s dng xc
nh qu trnh ln men lactoza, trong giy qu chuyn mu hng trong s c
mt ca axit. Lng axit tha s gy ra s ho ng (s hnh thnh sa ng)
sa. Thm vo , mt s vi khun c th lm bin i giy qu lm cho ch th
giy qu tr thnh mu trng di y ca ng nghim.
Ure l mt sn phm loi thi kh tiu ho protein trong hu ht cc ng
vt c xng sng v c bi tit dng urin. S c mt ca enzim ureaza
(gii phng NH
3
t ure) l mt th nghim chn on xc nh vi khun.
Thch-ure cha pepton, glucoza, ure v phenol. pH ca mi trng chun b
l 6,8 ( phenol chuyn thnh vng). Trong khi nui cy, vi khun chuyn ho
ure s sinh ra NH
3
lm tng pH ca mi trng, chuyn mu ch th fuchine
(hng m) pH 8,4.
Chng ta c th kho st hot ng ca vi khun trn dinh dng gelatin,
sa giy qu v thch-ure trong bi tp ny.
2.1. Vt liu
ng nghim cha dinh dng gelatin.
ng nghim cha sa giy qu
ng nghim thch nghing cha thch -ure
2.2. Dch nui cy
Pseudomonas aeruginosa
Proteus vulgaris
2.3. Th tc, k thut yu cu
a. Dn nhn mi mi trng 1 ng nghim l Pseudomonas v 1 ng khc
l Proteus.
b. S thu phn gelatin. Xc nh dinh dng gelatin, dng rn hay
lng? Nhit ca phng th nghim l bao nhiu? Nu gelatin dng rn cn
phi lm ho lng. Sau li lm ho ng.
- S dng que cy thng nhim vo mt ng nghim Pseudomonas v mt
ng khc l Proteus.
- Nui nhit phng v ghi li s quan st 2-4 ngy v 4-7 ngy.
Khng lc mnh ng nghim khi gelatin dng lng.
- Nu gelatin ho lng, t ng nghim trong mt ci cc nghin vi
pht. Ghi li kt qu ch r s ho lng hoc s thu phn bng du (+).
c. Sa giy qu. Miu t s xut hin ca sa giy qu.

- Nhim Pseudomonas vo mt ng mi trng, Proteus vo mt ng mi
trng khc.
- Nui 35
o
C trong 24-48h v ghi li kt qu. Giy qu c mu hng
trong iu kin c axit, mu tm trong iu kin kim v mu trng khi b bin
i. So snh kt qu vi nhng ng sa giy qu khng nhim VSV.
d. Test th ure
- Nhim Pseudomonas vo mt ng mi trng thch nghing ure-agar v
Proteus vo ng khc.
- Nui 35
o
C trong 24-48h. Ghi li kt qu: (+) cho s c mt ca ureaza
(mu ) v (-) cho cc ng khng c ureaza.
- S dng phenol v fuschine xc nh pH mi trng sau nui cy
chng t hot ng ca vi khun c kh nng chuyn ho ure.


1. Trnh by cc tiu tr xc nh nhanh kh nng chuyn ha cc hp cht
ca VSV?
2. Cc loi thuc th, cc test cho tng loi c cht m VSV chuyn ha?

Bi s 11
VI SINH VT TRONG MI TRNG

Mc ch yu cu:
+ Thy c s a dng ca VSV trong mi trng.
+ Hiu c vai tr ca VSV trong cc mi trng khc nhau.
Ni dung kin tp:
+ Miu t hnh thi khun lc VSV s dng cc thut ng din t c
chp nhn.
+ So snh s sinh trng ca VSV trn mi trng c v mi trng lng.
1. Quan h gia vi sinh vt v mi trng
Vi sinh vt c mt khp mi ni, chng c tm thy trong nc chng
ta ung, trong khng kh chng ta th v trn mt t chng ta bc i. Chng
sng trong v trn c th chng ta. VSV sng mi ngc ngch ca h sinh
thi, trn tt c cc dng ca cuc sng v trong hu ht mi trng. Trong a s
trng hp, VSV tn ti khp mi ni l khng c hi. Tuy nhin, trong
nghin cu VSV, cng vic phi c tin hnh ht sc cn thn trnh nhim
bn mi trng v trng v nguyn vt liu bi cc VSV ny.

Vi sinh vt lun hin din trong mi trng sng ca chng ta. Tuy nhin,
s c mt mt s loi vi sinh vt trong cc ni no chng hn trong nc
hoc thc phm c th l khng mong mun. Ngi ta s dng cc VSV gy
bnh xc nh s nhim nc. S c mt v s lng vi khun trong thc
phm c th biu th thc phm b tp nhim trong qu trnh sn xut, cc
vi khun ny c th a n kt qu l lm h hng thc n, gy ng c cho
ngi s dng.
Cc phng php th xc nh cht lng vi sinh vt hc ca nc
c pht trin ngn chn s chuyn giao ca nc cha VSV gy bnh t
ngun phn. Tuy nhin, vic nhn thy cc VSV gy bnh trong ngun nc
cung cp l khng thc t bi v cc mm bnh xy ra s lng nh nn chng
c th khng gp c trong mu. Hn na, khi cc bnh c xc nh th
thng l qu mun ngn chn s xut hin ca bnh tt. V th, s c mt
ca cc VSV ch th c s dng xc nh s nhim bn phn ca nc.
Bnh tt v ng c thc n l kt qu ca s sinh trng ca VSV trong
thc phm. Kim sot s an ton, cht lng ca thc phm cn c quan tm
vi cc test kim tra s c mt cc VSV gy bnh trong thc phm. Trong qu
trnh sn xut (nghin, ra v ng gi), thc phm c th b nhim bn cc
VSV t t v ng vt, ngi sn xut hoc my mc.
Cc vi sinh vt c hi ch l mt phn nh trong ton b qun th VSV.
Hot ng ca hu ht cc VSV l c li trong thc t. Cc hot ng ca VSV
t l tim nng duy tr s sng trn tri t. Qu trnh c nh nit phn t
c m t ln u tin t nm 1893 bi Sergei Vinogradski, ng bc vo
nghin cu cc vng tun hon sinh ho xy ra trong t bi v ng :
Cm kch v nhng pht hin rc r khng th so snh c ca Pasteur.
Ti bt u nghin cu t m vn tuyt hay ca qu trnh c nh nit
khng kh. Ti thnh cng m khng gp qu nhiu kh khn trong vic phn
lp mt loi vi khun k kh hnh que c gi l Clostridium c th thc hin
c chc nng ny.
Vinogradskii t tn vi sinh vt l Clostridium pasteurianum tng
nh Pasteur. Mt s VSV khc li c th phn r cc cht nhim nguy him
trong mi trng.
Trong th nghim ny, chng ta th nui cy mt vi loi VSV. Mi
trng nui cy c th c chun b vi dng ph thuc vo mong mun s
dng. a petri cha mi trng dinh dng c cung cp mt din tch b mt
ln nh gi cc khun lc. VSV s c nhim hoc c a vo trn dinh
dng thch hoc dinh dng canh thang. Cc vi khun c nhim vo mi
trng nui cy s tng s lng trong giai on nui cy. Sau khi nui iu
kin ph hp, mi trng lng tr nn vn c do VSV sinh trng. Trn mi
trng c, khun lc s nhn thy c bng mt thng. Mt khun lc l mt
tp hp t bo c pht sinh t mt t bo VSV n dng. Mc d nhiu loi

vi khun c s pht trin cc khun lc hnh thnh tng t nhau, mi khun lc
xut hin khc nhau thng l mt loi khc bit.
2. Vt liu, dng c
a petri cha mi trng dinh dng thch
ng nghim cha mi trng dinh dng canh thang
ng nghim c nt bng v trng
ng nghim c nc v trng
3. Th tc tin hnh
3.1. Giai on 1
a. Thit k th nghim ca bn. Mc ch l ly mu t mi trng v c
th bn. Hy s dng tr tng tng ca mnh. Sau y l mt s gi :
- C th s dng phng th nghim, phng tm hoc bt k ni no trong
khu vc l mi trng tin hnh.
- Mt a petri c th c m trong khng kh t 30-60 pht.
- Nhim mt a t 1 b mt mi trng chng hn nh sn nh hay gh
lm vic bng mt ming gc trong nc v trng, lau b mt mi trng sau
lau ln b mt a thch.
Sau khi x l, ming gc nn c b vo thng c cht ty u.
b. Nui 2 a trn. Dng 1 ming gc nh miu t trn x l 1 ng
dinh dng canh thang. Sau khi lau trn b mt a thch, t ming gc trong
dinh dng canh thang v li trong qu trnh nui cy.
c. Cc a v ng nghim nn nui nhit xp x nh khi ly mu mi
trng.
d. Nhim 2 a t c th bn, bn c th:
- t mt si tc trn mt thch
- Tin hnh x l bng ming gc m sau khi dng lau mt phn c th
(xem bc 1c).
- Chm cc ngn tay bn vo a thch.
e. Nui VSV t c th bn nhit nh nhit c th.
g. t ngc cc a nui cy nc s t li trn np thay cho trn b
mt mi trng thch, trnh nh hng n kt qu.
3.2. Giai on 2
a. Quan st v miu t kt qu VSV sinh trng trn a. Ghi ch mi loi
khun lc khc nhau xut hin v m t hnh thi khun lc, s dng cc c
trng a ra hnh bn. Xc nh s lng tng i ca cc loi khun lc.
Khi nhiu khun lc hin din, ghi li nhng loi qu nhiu.
b. Miu t s xut hin trong dinh dng canh thang. N vn hay c
ng u? So snh vi ng khng x l. Xem s to thnh cc khi t bo VSV

(gi l kt bng). C mng hay lp mng xut hin ngang trn b mt mi
trng? Nhn xem liu t bo VSV c lng xung y ng nghim hnh thnh
nn cn hay khng?
C th gi li cc ng canh thang cho th nghim khc.

1 Trnh by mi quan h gia VSV v mi trng?
2. S sai khc c bn theo quan st bng mt thng trong tng cc TN n
gin s b bc u nh gi VSV ch th mi trng?

Bi s 12
PHNG PHP XC NH NM MEN, NM MC, NGUYN SINH
NG VT, TO V VI KHUN LAM

Mc ch yu cu:
+ Phn bit c nm men, nm mc, to, vi khun lam v nguyn sinh
ng vt.
+ Hiu r cc c trng phn loi ca cc nhm VSV ny.
Ni dung kin tp:
+ Nui cy v quan st hnh thi ca nm men, nm mc, to v nguyn
sinh ng vt
+ So snh s sai khc gia cc nhm VSV trn.
+ Gii thch hin tng lng hnh nm mc.
1. Nguyn l chung
Sinh vt thuc nhm Eukaryota bao gm: to, nguyn sinh ng vt, nm
v cc ng thc vt bc cao. T bo Eukaryota c quy m in hnh v cu trc
phc tp hn t bo Prokaryota. H gen ca mt t bo Eukaryota c bao bc
trong mt mng nhn gii hn. Hn na, t bo Eukaryota cha mng bao bc
cc c quan t, c cu trc c bit v thc hin cc chc nng c trng.
Vi sinh vt nghin cu trong phn ny l cc loi d dng ho nng sng
t do ngoi tr to v vi khun lam (cc loi t dng quang nng).
Nm men l nhng vi sinh vt c bit kh r. Chng c s dng
rng ri trong cc quy trnh thng mi v c th mua c trong cc siu th
nng bnh. Nm men l loi nm n bo. Leeuwenhoek ln u tin quan
st c nm men trong qu trnh ln men bia.

Nhiu loi to ch nhn thy c di knh hin vi, trong khi nhng loi
khc c th di vi mt. To l nh sn xut oxy v thc n quan trng cho
ng vt nguyn sinh v cc sinh vt khc. Mt vi loi to n bo, nh tc
nhn ca thu triu Gonyaulax catanella v cc loi h hng li c cho
ng vt, k c con ngi khi n phi vi lng ln.
Nguyn sinh ng vt, bt ngun t tn gi lp mao trng c quan
tm nghin cu sm. T nm 1778, Friedrich von Gleichen nghin cu thc
n khng bo bng cch nui nhum cc trng mao.
2. Nm men (Yeasts)
Nm thuc loi t bo eukaryota v c th tn ti di dng n hoc a
bo. Chng l loi d dng v ly dinh dng nh hp th cc vt cht hu c
ho tan thng qua vch t bo v mng nguyn sinh cht. Nm (ngoi tr nm
men) l VSV ho kh. Nm men n bo, nm mc a bo v cc loi v m nh
nm m thuc gii nm. Nm thng a iu kin axit, chu p sut cao v
m thp hn vi khun. Chng c kch thc t bo v hnh thi chi tit ln hn
vi khun. Ngc li vi cc c trng ca vi khun, cc c trng c s nh chi
tit hnh thi v t bo c s dng phn loi nm, vi s tham d cht t
ca cc c trng th cp nh trao i cht v thnh phn khng nguyn. Nm
c cu trc phc tp hn vi khun nhng li trao i cht thay i t hn.
Nm men l loi nm n bo, khng c th hnh si, c dng hnh cu
hoc ovan in hnh. Nm men c phn b rng ri trong t nhin, thng
tm thy trn hoa qu v l cy nh lp ph ngoi dng bt mu trng. Nm
men sinh sn v tnh bng ny chi, l qu trnh trong mt t bo mi hnh
thnh mt u li (ny chi) t t bo m. Trong nhiu trng hp, cc chi khng
th t tch ra, mt chui ngn cc t bo c gi l dng gi khun ty. Khi nm
men sinh sn hu tnh, chng c th sinh ra mt hoc vi loi bo t gii tnh.
Loi bo t gii tnh c sn xut bi mt loi nm men c s dng phn
loi nh s phn chia (ngnh). Hot ng trao i cht cng c s dng
xc nh cc ging nm men.
Nm men l loi ym kh khng bt buc. Hot ng trao i cht ca
chng thng c s dng trong nhiu qu trnh ln men cng nghip. Nm
men thng c dng chun b nhiu loi thc n gm c bnh m, ung
nh ru vang v bia.
Trong phng th nghim, mi trng la chn thch Sabouraud thng
c s dng phn lp nm men. Thch Sabouraud c cc cht dinh dng
n gin (glucoza v pepton) v pH thp, hn ch s sinh trng ca hu ht cc
sinh vt khc. Rt nhiu k thut hu ch vi vi khun c th p dng vi nm
men.

2.1. Vt liu
ng ln men glucoza
ng ln men saccharoza
a Petri cha mi trng Sabouraud
Chai cha mi trng cao nm men -glucoza
Gc bng kh trng, lam knh, ng nghim, xanh methylen, bnh cu, hoa qu
hoc l cy.
2.2. Dch nui cy
Men bnh m
Rhodotorula rubra
Candida albicans
Saccharomyces cerevisiae
2.3. Th tc tin hnh
2.3.1. Nm men
a. Nh nhng ho mt t men bnh m trong mt t nc m trong ng
nghim to ra dung dch trng c.
b. Mi cp sinh vin s s dng mt loi dch nui cy nm men v dch
men bnh m
- Chia mt a mi trng Sabouraud thnh hai na. Ria cy dch nm
men bit trn na mi trng v men bnh m na cn li.
- Nhim mi loi dch VSV vo mt ng ln men glucoza v mt ng ln
men saccharoza.
c. Nui tt c cc mi trng 35
o
C ti khi xut hin du hiu sinh
trng.
d. Nh mt git xanh methylen vo mt t mi loi dch nui cy. Ghi li
kt qu quan st c.
e. Sau khi nm men sinh trng, ghi li kt qu. Kim tra dch nm men
khng nui cy v xc nh kt qu.
2.3.2. Phn lp nm men
a- Ct hoa qu v l cy thnh cc ming nh. t chng trong chai mi
trng cao nm men-glucoza. y ming chai bng mt qu bng. Nui nhit
phng ti khi xut hin s sinh trng. Ghi li s xut hin ca c hai loi
mu sau khi nui cy.
b- Chia a mi trng Sabouraud thnh 2 na. Mi loi mu nui cy trn
mt na a.
+ Lau b mt ci ko ct mu bng ming gc v trng. t ming gc
trn na a mi trng. Mt s vi khun s mc trn mi trng ny.

+ S dng que cy v trng cy ria mt vng que cy dch chit t chai
va chun b phn a.
c. t a cy o ngc, nui nhit phng cho ti khi s sinh
trng xut hin. Chun b dch th vi xanh methylen t cc khun lc khc
nhau. Ghi li kt qu.
3. Nm mc (Molds)
Cc nm a bo dng si c gi l nm mc. Do s a dng v hnh
thi nm mc nn hnh dng rt hu ch phn loi cc nm ny. Mt khun
lc nm mc ln (dng v m) gi l tn v c to thnh t mt khi cc si
gi l h si. Mi si l mt si nm vi si sinh dng pht trin trn b mt
mi trng. Si l lng trong khng kh gi l si sinh sn, c ngun gc t si
sinh dng v sinh ra mt loi bo t sinh sn v tnh. Si ca hu ht cc nm
mc c to thnh t cc t bo phn tch do vch ngang hoc vch ngn. Cc
si ny c gi l si c vch ngn. Mt s nm trong c Rhizopus c si
khng c vch ngn v l mt khi t bo cht duy tr nhiu nhn. Chng c
gi l si nm a nhn.
Nm c c trng v phn loi bi s xut hin khun lc (mu sc,
kch c, vv), t chc si (vch ngn hay a nhn), cu trc v t chc ca bo
t sinh sn. Do tm quan trng ca s hnh thnh v t chc khun lc, cc k
thut nui cy nm mc v kim tra knh hin vi rt quan trng.
Nm, c bit l nm mc rt quan trng trong cng nghip v phng
din lm sng. Cc bo t trong khng kh cng l ngun tp nhim ph bin
nht trong phng th nghim. Mi trng Sabouraud l mi trng la chn ph
bin phn lp nm.
Mt s nm bnh c biu hin lng hnh, chng c 2 dng pht trin.
nm bnh, s lng hnh thng ph thuc vo nhit . Trong bi tp ny,
chng ta s xc nh cc nm mc khc nhau v chng minh s lng hnh. S
lng hnh ca Mucor khng ph thuc vo nhit , n s biu hin ngc li
khi quyt nh iu kin vt l no nh hng ti s sinh trng ca Mucor.
3.1. Vt liu
a. Nui cy nm mc
a Petri cha mi trng Sabouraud
Mi trng Sabouraud nng chy
Dch parafin (mt na l du, mt na l parafin)
a Petri, lamen, que gt thu tinh, pipet
b. S bin i lng hnh
Mi trng Sabouraud: 5ml nng chy 48
o
C
ng plastic 5ml hoc cc giy nh
Li dao lam, bng dnh

3.2. Ging VSV
Rhizopus, Aspergillus, Penicillium, Mucor
3.3. Th tc tin hnh
3.3.1. Giai on 1
a. Lm nhim 1 a thch Sabouraud theo bt c cch no bn mun.
m a trong khng kh (ngoi tri, hnh lang, phng th nghim hoc bt c
ni u) 15-30 pht. Nui nhit phng t 5-7 ngy.
b. Nhim vo 1 a Sabouraud khc loi nm mc nui cy. To 1
ng trung tm a. Nui nhit phng 5-7 ngy.
c. Thc hin nui cy trn lam knh loi nm mc chn.
- Ra sch lam knh v lm kh
- Nh 1 git mi trng thch Sabouraud ln lam knh. Lm phng trn
mt phn lam v i ng li.
- Dng que cy v trng cn thn no b 1 na vng thch, li 1 mp thng.
- Lc nh dch nm mc to li dch huyn ph, v nhim 1 vng que
cy trn mp thng mi trng thch mi to ra.
- t mt lamen trn ming thch nhim nm mc.
- Dng pipet v parafin nng chy dn 3 mp cn li. Khng dn mp
x l nm mc.
- t 1 ming khn giy m di y ca a Petri. t lam knh nui
cy trn que gt thu tinh pha trn khn v y a petri li. Nui cy nhit
phng 2-5 ngy. Khng t ngc a.
- Quan st s biu hin ca cc bo t v tnh trn lam nui cy.
* S thay i lng hnh
a. G nh ng nui cy nm mc to dng huyn ph. Nhim 2 hoc 3
vng que cy dch Mucor. Trn ng bng cch ln trong tay v nhanh chng
ton b vo mt cc rng trc khi mi trng rn li. Cc ong khng cn phi
kh trng trc .
b. t mt ming khn giy m trong mt ci cc ong khc, ln ngc li
trn cc ng thch. Dn bng dnh hoch giy parafin xung quanh mp 2 cc.
c. Nui nhit phng n khi s sinh trng xy ra (5-7 ngy).
3.3.2. Giai on 2
a. Kim tra a nui cy mi loi nm mc, miu t s xut hin v mu
sc ca chng. Sau kim tra di knh hin vi, xem phi nh v mt bn
di.
b. Kim tra s lm nhim a thch Sabouraud v m t kt qu.
c. Kim tra lam nui cy ca mi loi nm mc di knh hin vi . Ghi li
s quan st.

e. kim tra s bin i lng hnh, b bng dn v dng dao lam ct
ri 2 cc.
g. Dng dao lam ct mt ming mng lp thch x l theo chiu thng
ng. Cn thn t ln lam knh v y lamen ln trn.
h. Quan st di ngun sng mnh v yu. Xem t m ming thch t pha
y n nh, nhiu mt phng thng gc s hin din.
i. Loi b phn tha trong cc v ty u dng c theo ch dn.
4. To (Algae) v vi khun lam (Cyanobacter)
Thuc v s quan tm u tin ca cc nh vi sinh vt hc l vi khun
lam. Vi khun lam c t bo tin nhn v thuc gii Monera, cng vi cc vi
khun khc.
To l tn gi thng thng ca nhm sinh vt nhn tht c kh nng
quang hp nhng cha phn thn, r, l. To c t trong gii Protista. To c
th tm thy trong i dng, nc sch, v thc vt m v t. To c th l
n bo, dng si hay a bo. Chng c hnh thi rt a dng t to nu hoc to
b v to phn pht khng l n cc khun lc to lam hnh cu. To c
phn loi da theo sc t, sn phm d tr, thnh phn ho hc ca thnh t bo
v tin mao.
Trong khi s sinh trng ca cc loi quang nng c tim nng cung cp
oxy v thc n cho cc loi sinh vt khc, mt s to dng si nh Spirogyra, l
mt mi gy phin toi cho con ngi bi v chng cn tr qu trnh lc trong
h thng nc. Cc loi quang nng c th c s dng xc nh cht lng
nc. Nc b nhim cha cc dinh dng tha t nc thi hoc cc ngun
khc c nhiu vi khun lam v t to ct hn nc sch. Hn th na, s lng t
bo to cho bit cht lng nc. Nu c hn 1000 t bo to/ml biu th rng
s tha dinh dng ang hin din.
4.1. Vt liu
Mu nc h
A. Nui iu kin sng 4 tun.
B. Nui trong iu kin ti 4 tun.
C. Thm nitrat v photpho, nui trong iu kin sng 4 tun.
D. Thm sulphat ng, nui trong iu kin sng 4 tun.
4.2. Th tc tin hnh
a. Chun b tiu bn git treo t mt mu nc h. Ly git mu t y
ca bnh cha.
b. Dng vt knh kh kim tra tiu bn phng i cao v thp. Xc
nh s c mt ca to v vi khun lam trong nc h. Tham kho mu sc cho
s xc nh. V hnh cc loi to m bn khng th xc nh. Ghi li mi lin
quan gia s lng ca mi loi to t 4+(phong ph) ti + (ch thy 1 i din).

c. Lp li s quan st v thu s liu vi cc mu nc h cn li.
5. Nguyn sinh ng vt
Nguyn sinh ng vt l cc sinh vt c nhn hon chnh n bo, thuc
gii Protista. Chng c tm thy trong cc khu vc c ngun nc ln. Nhiu
loi nguyn sinh ng vt sng trong t v nc, v mt s l khu h vi sinh vt
bnh thng ca ng vt. Mt vi loi nguyn sinh ng vt l ng vt k sinh.
Nguyn sinh ng vt l cc loi d dng ho kh. Chng sng nh vo
n cc vi sinh vt khc v cc ht vt cht nh. Nguyn sinh ng vt khng c
thnh t bo, mt s (trng roi v trng lng mao) pha ngoi c bao bc mt
lp mng dy c tnh n hi gi l lp da mng. T bo c lp da mng i hi
cu trc c bit ly thc n. Khng bo co rt c th xc nh c trong
mt s mu. S lm y c quan t ny bng nc sch v sau ngc li bi
tit nc t trong t bo, gip cho sinh vt sng c trong mi trng c nng
cht ho tan thp.
Nguyn sinh ng vt c th phn loi trn c s phng thc chuyn
ng ca chng. Trong th nghim ny, chng ta s xc nh cc thnh vin sng
t do ca hai ngnh Protista. Ngnh trng mao gm cc nguyn sinh ng vt di
ng bng s dng chn gi hoc tin mao. Mt s loi s dng c 2 hnh thc
di chuyn ny. Trng amip di chuyn bi ko di phn li ra ca t bo cht
ging nh cc chn gi l chn gi. Cc chn gi bt ngun t cui t bo, phn
cn li ca t bo dng phng cc chn gi v pha trc.
Cc trng roi c mt tin mao hoc nhiu hn. Mc d trng roi l cc
loi d dng, cc sinh vt s dng trong bi tp ny l d dng khng bt
buc. Chng pht trin nh quang hp khi c nh sng v nh d dng trong
iu kin ti.
Cc thnh vin ca ngnh trng mao c nhiu lng mao mc t t bo.
Trong mt s trng mao, lng mao xut hin thnh cc hng trn ton b b mt
ca t bo. Trong cc trng mao sng gn vi cc b mt rn, lng mao ch xut
hin xung quanh ng rnh ming. Thc n c a vo ng rnh ming
thng qua l th t bo (ming) v vo trong hu, ni khng bo cha thc n
hnh thnh.
5.1. Vt liu
Methylxellulo 1,5%
Axit axetic 5%
Pipet
Tm nhn
5.2. Dch nui cy
Amip, trng mt, Paramecium (trong dch nm men - congo )
5.3. Th tc tin hnh

a. Chun b git treo Amip. t 1 git ly t y canh trng ln lam knh.
t 1 mp ca lamen vo trong git nc v cho cht lng chy dc theo
lamen. Nh nhng t lamen trn git canh trng. Quan st s di chuyn ca
amip, v ng di chuyn. Vng no ca t bo cht c nhiu ht nh hn: bn
trong hay bn ngoi nguyn sinh cht?
b. Chun b git treo trng mt. Lm tng t nh trn v v li s di
chuyn ca trng mt. C th nhn thy c mu trng mt (im mt)
hay khng?
Cho 1 git axit axetic thm qua lamen. Quan st biu hin ca trng mt.
c. Chun b git treo ca trng mao v quan st s di chuyn ca n.
d. Cho 1 git methylxellulo ln lam knh. To 1 git treo canh thang trng
mao c nui trn dch nm men - congo . Trng mao s chuyn ng chm
hn trong methylxellulo nht dnh. Quan st trng mao n cc t bo nm men
nhum . Congo l 1 ch th pH. Khi khng bo cha thc n c lm y,
ch th s chuyn thnh mu xanh. C phi do sn phm trao i cht s to ra
iu kin axit trong khng bo hay khng?
m s lng cc khng bo xanh v trong mt trng mao. V hnh 1
trng mao v xc nh cc v tr ca khng bo.


1 Trnh by nhng nguyn tc c bn s b nhn dnh nm men, nm mc,
To, NSV trn mi trng nui cy rn, bn rn?
2. Trnh by mt s phng pghp th nhanh nh gi s b tng nhm VSV
trong mi trng?

Bi s 13
QU TRNH CHUYN HO NIT DI TC DNG CA VSV
(AMN HO, PHN NITRT HO, C NH N
2
)

Mc ch v yu cu:
+ Hiu r vng tun hon Nit trong t, thy c s thay i ho hc
xy ra mi bc trong chu trnh.
+ Gii thch c tm quan trng ca vng tun hon Nit.
+ Phn bit qu trnh c nh Nit phn t cng sinh v t do
Ni dung:
- Cc phng php phn tch VSV trong qu trnh chuyn ha cc hp
cht cha nit trong mi trng
- Thc hnh trc tip cc th nghim v qu trnh chuyn ha nit trong
mi trng
Nguyn l:
Tin hnh cc test th chng minh s c mt ca vi khun amn ho,
phn nitrat ho v c nh nit phn t cng sinh trong t.
Vng tun hon Nit l mt kha cnh nghin cu rng ri v ng dng
quan trng ca vi sinh vt t. Tt c cc sinh vt u cn Nit tng hp
protein, axit nucleic v cc hp cht cha nit khc. S phc hi nit bi cc
sinh vt khc nhau c gi l vng tun hon nit. Vi sinh vt ng mt vai tr
c bn, khng th thay th c trong vng tun hon nit do chng tham gia
rt nhiu phn ng trao i cht khc nhau nhm chuyn ho cc hp cht cha
nit. Khi cy trng, ng vt v vi sinh vt cht i, cc vi sinh vt s phn hu
chng bi s thu phn protein v amn ho.
Thu phn protein l s thu phn cc protein thnh dng amino axit.
Amn ho gii phng amonia do kh amin ho cc amino axit hoc ng ho
ure thnh NH
3
. Trong hu ht mi trng t, amonia ho tan trong nc to
thnh ion amn:
NH
3
+ H
2
O NH
4
OH NH
4
+
+ OH
-

Mt s ion amn s c cy trng v vi sinh vt s dng trc tip
tng hp cc axit amin.
Bc tip theo trong vng tun hon nit l s oxy ho cc ion amn
trong qu trnh nitrat ho. Hai ging vi khun c kh nng oxy ho NH
4
+
trong
hai giai on lin tip c ch r nh sau:
2NH
4
+
+ 3O
2
Nitrosomonas
2NO
2
-
2NO
2
-
+ O
2

Nitrobacter
2NO
3
-

Nhng phn ng ny c s dng sinh ra nng lng (ATP) cho t
bo. Vi khun nitrat ho l cc loi d dng ho nng v nhiu loi b c ch
bi vt cht hu c. Nitrat l mt ngun nit quan trng cho cy trng.
Vi khun phn nitrat ho chuyn ho nitrat v loi b chng khi vng
tun hon nit. Phn nitrat ho l qu trnh bin i nitrat thnh nitrit v kh
nit. S chuyn ho ny c th c trnh by nh sau:
NO
3
-
NO
2
-
N
2
O N
2

Phn nitrat ho cn c gi l h hp ym kh. Nhiu ging vi khun
gm c Pseudomonas v Bacillus c kh nng phn nitrat ho trong iu kin
ym kh.
Nit khng kh c th quay tr li t bi s bin i kh nit thnh
amonia, mt qu trnh c gi l s c nh nit. Cc t bo vi sinh vt c
enzym nitrogenaza c th c nh nit trong iu kin ym kh nh sau:
N
2
+ 6H
+
+ 6e
-
2NH
3

Mt s sinh vt tin nhn sng t do, v d nh Azotobacter, Clostridium v vi
khun lam c kh nng c nh nit. Nhiu loi vi khun c nh nit sng lin kt
cht vi r cc loi c trong t vng r, ni m lng ht tip xc vi t.
Vi khun cng sinh cung cp mt vai tr quan trng hn trong qu trnh
c nh nit phn t. Mt in hnh l mi quan h cng sinh gia Rhizobium v
r cy h u (nh u tng, u xanh, u H lan, c Alfalfa v c ba l), c
n hng nghn loi u khc nhau. Nng dn trng u tng v Alfalfa
ti to Nit trong cc cnh ng ca h. Nhiu loi u hoang di c th
sinh trng trn nhng vng t ngho dinh dng tm thy rng rm nhit
i hoc sa mc kh cn. Loi Rhizobium l c trng cho tng loi cy ch m
chng nhim vo. Khi lng ht v vi khun Rhizobia tip xc trong t, nt sn
r c hnh thnh trn cy ch. Nt sn cung cp mi trng ym kh cn thit
cho qu trnh c nh nit.
Qu trnh c nh nit phn t cng sinh cng xy ra trn r ca cc cy
khng thuc h u. X khun Frankia hnh thnh nt sn trn cy tng qun si.
Bt k s ph v no trong vng tun hon nit cng c th nh hng
quyt nh n s tn ti ca s sng.
1. Qu trnh amn ho
1.1. Vt liu
ng mi trng canh thang pepton
t m
Thuc th Nesler
NH
4
OH
Bn s l trn
1.2. Th tc tin hnh

- Ho t vo nc v trng to dung dch. Ly 1 vng que cy nhim vo
ng mi trng canh thanh pepton.
- Nui nhit phng v lm test th amonia sau 2 v 7 ngy.
- Test th amonia: nh 1 git dung dich thuc th Nesler vo l bn s.
Thm vo 1 vng que cy canh thang pepton cy dch t, trn u. Mu
vng n mu nu ch ra rng c amonia. So snh kt qu vi c nh dung
dch thuc th vi NH
4
OH. S dng ng nghim canh thang pepton khng
nhim dch t lm i chng.
2. Qu trnh phn nitrat
2.1. Vt liu
ng nghim cha mi trng nc tht - mui nitrat
t m
Thuc th nitrat A v B
Bi km
2.2. Th tc tin hnh
- X l mt ng mi trng nc tht - mui nitrat bng dung dch t
nh trn. Nhim vo ng khc P. aeruginosa.
- Nui c 2 ng nhit phng trong 1 tun.
- Kim tra s chuyn ho nitrat. Thm 5 git nitrat A v 5 git nitrat B
vo mi ng nui cy v ng khng x l, lc nh. Mu xut hin trong vng
30 giy l test th dng tnh. Nu ng kim tra khng chuyn mu , thm 1
lng nh bi km, ng th chuyn sang mu l test th m tnh, nu khng
chuyn mu th cng l kt qu dng tnh.
3. Qu trnh c nh nit phn t
3.1. Vt liu
a petri cha mi trng thch nm men-manitol
Xanh methylen, dao lam, cy h u
3.2. Th tc tin hnh
- Ct 1 nt sn t r cy u v ra sch di vi nc chy. Quan st
nt sn.
- Ct nt sn thnh 2 na bng dao lam. Quan st bn trong. Nghin nt
sn gia 2 lam knh v to vt bi bng cch quay 2 lam knh vi nhau.
- Cy ria 1 vng que cy dch nghin trn mi trng thch. Nui nhit
phng khong 7 ngy.
- Lm kh trong khng kh lam knh c vt bi v c nh li bng nhit.
Nhum tiu bn trong 1 pht bng xanh methylen. Ra v quan st di vt
knh du.

- Quan st s sinh trng ca vi khun trn a. Nhum n bng xanh
methylen. So snh hnh thi vi khun trn tiu bn vi tiu bn chun b sn
t nt sn.


1 C ch ca tng qu trnh c nh, chuyn ha nit?
2. Phng php lp lp, nh gi hiu qu ca qu trnh chuyn ha nit di
tc dng ca VSV?
3. Kt qu phn tch, tnh ton hiu qu ca qu trnh chuyn ha nit di tc
dng ca VSV?

Bi s 14
CHUYN HO LU HUNH DI TC DNG CA VI SINH VT

Mc ch v yu cu:
+ Hiu c vai tr ca VSV trong vic m bo vng tun hon lu hunh.
+ V biu vng tun hon lu hunh xy ra trong ct Vinogradskii.
Ni dung kin tp:
+ Quan st s sinh trng ca vi khun chuyn ho lu hunh trong ct
Vinogradskii.
1. Nguyn l chung
Mt trong nhng hng nghin cu vi sinh vt t l vng tun hon lu
hunh. Vi khun lam v mu ta tham gia vo vng tun hon sinh ho lu
hunh. Mc d sc t quang hp ca vi khun lam c quyt nh bi sc t vi
khun , chng vn c th xut hin mu nu do s c mt thm ca sc t quang
hp mu gi l carotenoit. Vi khun quang hp mu ta xut hin mu ta
hoc bi v c s lng ln carotenoit. Vi khun mu ta cng c sc t vi
khun.
Vi khun quang hp s dng sc t sinh ra in t cho tng hp ATP
v s dng lu hunh, cc hp cht cha lu hunh, kh hydro hoc cc phn t
hu c l ngun cung cp in t. Phng trnh tng qut cho quang hp vi
khun l:
CO
2
+ H
2
S
Bacteriocholorophyll
C
6
H
12
O
6
+ S
-2

Mt s vi khun d tr cc ht lu hunh trong hoc trn t bo nh l kt
qu ca s sn xut ion sulfit. Lu hunh d tr c th c dng lm ngun

cung cp in t trong qu trnh quang hp, kt qu l to ra sulphat. Trong t
nhin, sunfit hydro c sinh ra t s bin i sulphat trong h hp ym kh v
s phn r cc amino axit c cha lu hunh. Sunphat c th b bin i thnh
sunphit hydro bi 5 ging vi khun chuyn ho sunpht (r nht l
Desulfovibrio). CO
2
m vi khun quang hp s dng c cung cp bi qu
trnh ln men hydratcacbon trong mi trng ym kh.
K thut nui cy lm giu bao gm phng thc ti to mi trng c
gi l ct Vinogradsky s c dng cho bi tp ny. Chng ta s s dng n
tng cng s sinh trng ca vi khun chuyn ho lu hunh trong iu kin
ym kh. Mt vi loi vi sinh vt c nui cy ph thuc vo s phn ng vi
nh sng v oxy sn c ca chng.
2. Vt liu
Hn hp bn (bn, CaCO
3
, c kh hay giy v CaSO
4
)
ng nghim to hoc ng ong
Que gt
Bn t
m Vinogradsky (NH
4
Cl, Na
2
S, KH
2
PO
4
, K
2
HPO
4
)
Giy nhm, ngun sng
3. Dch nui cy
Pseudomonas aecruginosa
4. Th tc tin hnh
a. Xp cht hn hp bn n 2/3 chiu cao ca mt ng nghim ln hoc
ng ong. Xp cht hn ch bt kh trong ng.
b. Cn thn xp mt lp mng bn t ln trn cng ca lp hn hp bn
u tin.
c. Nh nhng dung dch m xung cnh ca ng ong, ch khng
lm xo trn b mt bn. y ng ong n mc c th.
d. y ming ng bng giy nhm v t pha trc mt ngun sng.
Ngi hng dn c th n nh cc ngun sng khc nhau (v d nh nh sng
nng, hunh quang, hoc xanh).
e. Quan st ng th nghim mi tun mt ln trong 4 tun. Ghi li s xut
hin cc khu vc mu sc. Bn ho kh s c mu hi nu v bn ym kh c
mu en. (Hnh).
g. Sau 4 tun, chun b tiu bn git treo t cc vt xanh hoc tm trong
ng. Quan st di knh hin vi s xut hin ca vi khun v xem c cc ht lu
hunh hay khng?



1 C ch ca tng qu trnh c nh, chuyn ha lu hunh?
2. Phng php lp lp, nh gi hiu qu ca qu trnh chuyn ha lu hunh
di tc dng ca VSV?
3. Kt qu phn tch, tnh ton hiu qu ca qu trnh chuyn ha S di tc
dng ca VSV?

Bi s 15
VI SINH VT PHN GII LN (PHOSPHO)

Mc ch v yu cu:
+ Phng php phn lp, tuyn chn ging VSV phn hu chuyn ha ln.
+ Thy c tc dng vi sinh vt trong qu trnh phn gii Phospho kh tan.
+ Nhn bit c cng phn gii ln di tc dng ca VSV.
Ni dung kin tp:
+ Phn lp chng ging VSV phn gii ln.
+ Phng php b tr th nghim.
1. Vi khun phn gii ln hu c
Ln hu c c th c phn gii bi nhiu loi vi sinh vt. C th dng
mi trng c thnh phn sau phn lp.
1.1. Mi trng phn lp
Lxitin 0,05g MgSO
4
0,3g
(NH
4
)
2
SO
4
0,3g FeSO
4
vt
CaCO
3
5g Gluc 10g
NaCl 0,3g Nc 1000ml
MnSO
4
vt Thch 15 - 18g
Ln hu c c th dng Lxitin hoc axit nucleic.
1..2 Dng c nguyn liu
ng nghim c 9ml nc v trng
ng ht 1ml v 10ml
Hp lng tit trng
ng nghim c thch nghing

1.3 Cc bc thc hin
Cn t 10g. Cho vo bnh tam gic c 90ml nc v trng. Lc 10 pht.
Pha long mu 10
-2
- 10
-5
. Trong iu kin khng c axit nuclic hoc lxitin th
dng lng trng g. Dng dung dch NaClO 2% tit trng trng. Dng nc
ct ri nc v trng ra sch NaClO. Luc trng. Ly lng nghin nh.
Ngm cn gn nc cn. Lm nh th nhiu ln. Lc. Dng axton kt ta.
Dng ru v axtn ho tan v kt ta. Cui cng dng ru ho tan nh
th c th dng c.
Cho mi trng vo ng nghim 1,8 x 18cm mi ng 15ml. Tit trng
120
0
C/15 pht.
Nu chy mi trng, ngui 50
0
C. vo hp lng mi hp 15ml.
Ch mi trng ng li. Dng ng ht tit trng ly 0,1ml dung dch t
nng 10
-3
hoc 10
-4
. Cy ln trn mt mi trng. Dng que thu tinh dn
u khp mi trng. 28 - 30
0
C trong 3 - 4 ngy.
1.4. Kim tra kt qu
Trn mt mi trng s xut hin khun lc mu trng c c hnh trn,
c np nhn. y l khun lc ca vi khun phn gii ln hu c.
Dng que cy ly vi khun lm tiu bn - nhum n. Xem knh. Vi
khun hnh que, hai u trn, ng ring r hoc lin li thnh chui. Ly vi
khun ny tip tc thun ho.
* Ch : C th dng lng trng trc tip, khng qua ra ru v kt
ta bng axtn. Cch lm nh sau:
Ra sch v trng bng NaClO 2% hoc bng cn 95%. Dng nc ct
ri nc v trng ra sch NaClO v cn. Cho lng trng vo bnh tam gic
tit trng. Cho vo 50ml nc v trng nh vo cho u. Cho vo mi hp
lng 1ml nc lng trng. mi trng vo. Lc nh trn u v ng
li. Ly ng ht cho dung dch cn phn lp vo. Mi hp lng cy 0,5ml dung
dch t. 2 - 3 hp lm i chng. 28 - 30
0
C trong 24 gi. Thi gian
nui cy khng c qu lu v d tp vi khun. Khun lc mc. Ly vi khun
lm tiu bn, xem knh. Nu ng nh vi khun phn gii ln miu t trn th
tip tc cy vo thch nghing nhiu ln thun ho.
2. Vi sinh vt phn gii ln v c kh tan
2.1 Mi trng
Saccar 10g MnSO
4
0.03g
(NH
4
)SO
4
0.5g Fe
2
(SO
4
)
3
.7 H
2
O 0.03g
NaCl 0.3g Ca
3
(PO
4
)
2
10g
KCl 0,3g Thch 20g
MgSO
4
0,3g Nc ct 1000ml

2.2 Dng c v nguyn liu
Bnh tam gic Hp lng
ng nghim (NH
4
)
6
Mo
7
O
24
.2H
2
O dung dch
HCl m c v 0,1N
Bnh nh mc 50cc
Dung dch t pha long Bo du
2.3. Cc bc
mi trng vo cc hp lng tit trng. Dng dung dch t 0,1ml
vo san u trn b mt mi trng. 28 - 30
0
C.
2.4. nh gi kt qu
Sau 5 - 7 ngy quan st khun lc v hnh thi vi khun. Khun lc trong
nh, li, ng bin thng. Xung quanh khun lc c vng phn gii trong sut.
Vng ny ln hay b ty vi khun. Thng th vng phn gii b v mun xem
phi lt ngc a petri.
Mun nh gi chc chn hn nn kt hp cht ch gia quan st bng
mt thng v dng dung dch Sulfomolybdatamon kim tra kt qu c ln
d tiu khng. Nu c ln s kt hp vi Sulfomolybdatamon thnh hp cht
photpho molybdatamon mu vng kt ta.
Quan st vi khun: T khun lc c vng phn gii, ly mt t vi khun.
Lm tiu bn v quan st di knh hin vi. Vi khun phn gii hp cht ln kh
tan thnh d tan hnh que. u trn c v nhy b, c bo t, Gram dng.
Mun thun khit th cy vo thch nghing nhiu ln. Mi ln cy u kim tra
di knh hin vi.
phn lp vi khun phn gii hp cht ln v c kh tan thnh d tan c th
dng bo du. cnh bo du v r in thanh c nhiu vi khun phn gii ln kh tan
thnh d tan.
Ly cnh bo du. Ra qua nc my cho sch, nghin nh trong mt t
nc v trng to thnh dung dch. Ly dch bo du cy trn mi trng
thch phng. 28 - 30
0
C trong 5 - 7 ngy, quan st khun lc v t bo vi
khun di knh hin vi.
Ch :
- Nghin bo du thnh dch cho d cy vo mi trng.
- xc nh cng phn gii ca vi khun ta nh lng ln d tiu trn
my so mu.


1 C ch ca tng qu trnh c nh, chuyn ha phospho di tc dng ca
VSV?
2. Phng php lp lp, nh gi hiu qu ca qu trnh chuyn ha phospho
di tc dng ca VSV?
3. Kt qu phn tch, tnh ton hiu qu ca qu trnh chuyn ha phospho di
tc dng ca VSV?

Bi s 16
ENZYM TRONG TRAO I NIT, CACBON, PHOTPHO V LU
HUNH
Mc ch:
- Gii thiu cho hc vin v kh nng sinh enzim ca cc chng ging vi
sinh vt
- Bit cch xc nh enzim ca mt s ging vi sinh vt thng gp
Ni dung:
- Hng dn cho hc vin cc phng php xc nh enzim hay hot tnh
ca VSV
- Nm chc c mt s phng php xc nh hot tinh sinh enzim ca
VSV
I. ENZYM TRONG TRAO I NIT (Nitrat reductaza)
Protein l thnh phn quan trng ca c ht v r cy trng. Chng l
thnh phn chc nng v cu trc c bn ca thnh t bo cy v chim 1/3 nit
tng s trong t. Protein cung cp cho t ly t tt c xc cc sinh vt, ng
thc vt. Protein trong t c phn hu nhanh chng nh nhiu loi vi khun
v nm. Trong qu trnh phn gii, proteaza ngoi bo thc hin vic ct t cc
lin kt peptit to ra cc polypeptit v oligopeptit, kt qu l sau gii
phng ra cc hp cht c phn t lng thp v c tch lu bi VSV. Nh vo
proteaza c gii phng t cc t bo VSV, cc enzym trong t ny c hp
th vo cc keo t hoc gn ho tr vo vt cht hu c t. Cc enzym c nh
ny ch r kh nng khng cao i vi s phn gii protein. Ngc li,
proteaza b c ch bi s lm kh.
Nhiu phng php c s dng phn tch hot ng ca proteaza,
ureaza, amidaza, trong t. Mc d nhiu loi enzym tham gia vo trao i
nit c xc nh, vn c t thng tin v nitrat reductaza. Trong iu kin
ym kh, nitrat c bin i thnh NO
2
-
, N
2
O, N
2
. Trong qu trnh phn nitrat
ho, men d ho nitrat reductaza xc tc giai on u tin bin i NO
3
-
thnh
NO
2
-
trong iu kin ym kh. Nghin cu ca Cooper v Smith (1963) ch r
rng t l gii hn qu trnh phn nitrat ho trong t axit l s bin i NO
3
-
,
trong t kim l s bin NO
2
-
. Trong bi tp ny, mt phng php chnh
xc, n gin v nhy c s dng xc nh nitrat reductaza trong t (theo
Abdelmagid v Tabatabai).
Dng KNO
3
l cht nn, mu t c nui 25
o
C trong 24h di iu
kin m trong ng nghim. Nitrit reductaza b c ch do thm vo 2,4
dinitrophenol. Sau khi nui cy, nitrit gii phng ra c chit xut bng dung
dch KCl v xc nh bng my so mu bc sng 520 nm.
1. Vt liu, ho cht
- ng nghim 180x18mm.

- 2,4 dinitrophenol 0,9 mM (166,6 mg/l)
- Dung dch cht nn: KNO
3
25mM (2,53g/l)
- KCl 4M (298,24g/l)
- m NH
4
Cl 0,19M, pH=8,5 (10g/900ml, iu chnh pH bng KOH
8,5%, ln th tch n 1000ml)
- Thuc th mu: Ho 2g sunphanil amin v 0,1 g N-(1-naphthyl)-
etylendiamin hydroclorit trong 150 ml nc ct, thm 20 ml H
3
PO
4
c, lm
ngui nhit phng v pha long n 200 ml nc ct trong bnh nh mc.
Dung dch khng mu v ch dng trong ngy.
- Dung dch tiu chun m: NO
2
-
1000g/ml (4,9257g NaNO
2
/l), gi
4
o
C
- Dung dch o chun (NO
2
-
10g/ml) : pha long dung dch m
(5ml/500ml)
- Dy hiu gi chun: 0; 0,2; 0,4; 0,8; 1 g NO
2
-
/ml . Pha cho mi ln o.
2. Th tc tin hnh
- Cn 5g t m vo 5 ng nghim. Dng pipet thm 4ml dung dch 2-4
DNP, 1ml dch nn, trn nhanh ri y np li.
- 2 ng 25
o
C/24h v 1 ng -20
o
C/24h (ng i chng), sau khi
lm tan nhit phng.
- Thm 10ml KCl vo c 2 mu v i chng, lc nh ri lc ngay.
- so mu cho 5ml dch lc, 3ml m NH
4
Cl v 2ml thuc th mu vo
ng nghim, trn u v ng 15 pht nhit phng.
- o mu v i chng 520nm da vo phn ng i chng trng. So
snh vi ng cong hiu chnh chun (x l 5ml dy hiu chnh chun ging
nh dch lc).
3. Tnh kt qu
Tnh lng g N ca dung dch th t ng cong chun
(S-C).20.100
= g N.g
-1
dm.2h
-1

5.5. % dm
Trong :
S: Gi tr mu (g N)
C: i chng (g N)
20: Lng chit xut (ml)
5: c s dch lc (ml)
5: Lng mu m ban u (g)
100.%
-1
dm: H s cho t kh

II. ENZYM TRONG TRAO I CACBON (CMXellulaza - CMCaza)
Xellulo, mt hp cht hu c quan trng nht trong t nhin c quan
tm rt nhiu. Thc vt c cha 4-70% xellulo. S phn gii xellulo bi VSV s
dng t nht 3 h thng enzym khc nhau (enzym ni bo 1-4- glucanaza,
enzym ngoi bo 1-4- glucanaza, 1-4- glucosidaza). Nm (v d Chaetomium,
Fusarium, Polyporaceae, Poriaceae), vi khun (Pseudomonas,), x khun
(Actinomycetes) l cc loi ho kh phn gii xellulo quan trng nht. Trong
iu kin ym kh, xelluloza b thu phn bi vi khun thuc ging
Clostridium. S phn gii ca xelluloza ngoi bo kt thc vi xellobioza.
Trong enzym t, vic xc nh hot ng ca xellulaza t xellulo t
nhin khng ho tan trong nc l rt kh. Cacboxy methyl xelluloza (CM-
xelluloza) x l trc v th thng c s dng. Ging nh xc nh hot
tnh ca xylanaza v invertaza, hot tnh xellulaza c xc nh t cc ng
c gii phng.
S dng CM-xelluloza l cht nn, mu t c 50
o
C/24h v
pH=5,5. Vic gim lng ng c gii phng ra trong qu trnh gy ra s
bin i Kali hexacyanoferrate (III) trong dung dch kim. Kali
hexacyanoferrate (II) phn ng vi ferric (NH
4
)
2
SO
4
trong dung dch axit to
thnh phc hp feric hexacyanoferrate (II) (ph mu xanh) c xc nh bng
my so mu (theo Schinner v von Mersi, 1990).
1. Vt liu, ho cht
- m axetat 2M, pH=5,5: Ho tan 164,06g CH
3
COONa khan/l, pha
long 60ml axit axetic ng bng trong 500 ml. Trn 1000 ml CH
3
COONa vi
190ml axit axetic pha long, iu chnh n pH = 5,5.
- Dung dch nn (0,7% theo khi lng): 7g CMC-Na/1000ml m axetat,
khuy trn my khuy t 45
o
C/2h.
- Thuc th A: 16g Na
2
CO
3
v 0,9g KCN/1000ml.
- Thuc th B: 0,5g Kali hexacyanoferrate (III)/1000ml, gi trong l ti mu.
- Thuc th C: 1,5g ferric amonium sulfat + 1g Natri dodecyl sulfat trong
900ml nc ct, thm 4,2ml H
2
SO
4
c, ho tan 50
o
C. Sau khi lm ngui, ln
th tch n 1000 ml.
- Dung dch tiu chun m c (250 g glucoza/ml): 0,25g glucoza
khan/1000ml
- Dung dch tiu chun lm vic (25 g glucoza/ml): 10 ml dung dch tiu
chun m/100 ml.
2. Th tc tin hnh
- Cn 10g t m vo 3 bnh nn 100ml. Thm 15ml dung dch cht nn
v 15 ml m axetat vo 2 bnh (mu), cho 15ml m axetat vo bnh cn li
(i chng). Lc nh cc bnh, nh du, y nt v 50
o
C/24h.

- Sau khi , nh 15ml huyn dch cht nn vo bnh i chng, lc nh,
lc cc bnh mu v i chng ngay. Pha long 0,5ml dch lc ti 20ml trong
ng nghim.
- so mu, nh 1ml dch nn, 1ml thuc th A v 1ml thuc th B vo
ng nghim, nh du, trn u v 15 pht trong chu nc si.
- Sau khi lm ngui trong chu nc nhit phng 5 pht, thm 5ml
thuc th C, trn v lng 60 pht nhit phng cho pht trin mu. Trong
vng sau 30 pht, o 690 nm trn my quang ph hp ph da vo phn ng
i chng trng.
- chun b ng cong hiu gi chun, ht 0; 0,1; 0,2; 0,3; 0,4; 0,5 v
0,6 ml dung dch lm vic tiu chun vo 7 ng th v pha long n 1ml bng
nc ct. X l cc dung dch ny ging nh lc t. Cc dung dch chun hiu
chnh cha 0; 2,5; 5,0; 7,5; 10; 12,5 v 15 g glucoza.
3. Tnh kt qu
Hot tnh CM-xellulaza c ch ra l tng ng g glucoza trn 1g vt
cht kh v thi gian . Lng glucoza c tnh t ng cong chun.
(S-C).30.40.100
= g GE.g
-1
dm.24h
-1

10.% dm
S: Gi tr o mu (g GE)
C: i chng (g GE)
30: Lng hn hp (ml)
40: H s pha long dch lc (ml)
10: Lng t ban u (g)
100%
-1
dm: H s t kh
III. ENZYM TRONG TRAO I PHOTPHO
Tm quan trng ca photphataza i vi dinh dng ca cy trng
c nhn mnh nhiu ln (Cosgrove 1967, Hayman 1975, Speir 1978, Dick v
Tabatabai 1987,). Trong hu ht cc loi t, s phn chia photpho hu c
cao hn photpho v c. Trong cc este ca axit photpho hu c, s phn chia
ln nht trong t l axit phytanic hoc phytin. Photpho c hp th bi cy
trng i hi s khong ho photpho hu c bi men photphataza thnh
photphat mch thng. Photphataza bao gm cc enzym c sinh ra nhiu trong
iu kin ln d tiu thp. Photphataza c bi tit ra t r cy trng v VSV.
Photphataza ca VSV tri hn trong t.
Tn photphataza miu t mt nhm enzym thu phn este cng nh
andehit ca axit photphoric. C nhiu loi photphataza trong t.
xc nh hot tnh photphataza, c th s dng hoc photphat c
sinh ra trong qu trnh khong ho cc este photphat hu c t nhin hoc cc

thnh phn hu c sau khi khong ho cc vt cht hu c nhn to (v d
naphtylphotphat, phenylphotphat,)
Enzym photphomonoesteraza (cn gi l photphataza) khc nhau trong
cc c cht c trng v pH ti thch ca n. V vy, mt trong nhng s khc
bit l gia photphataza kim v axit trong t. Photphodiesteraza phn hu cc
axit nucleic v c tm thy trong cy trng, ng vt v vi sinh vt. Hot
ng ca Photphotriesteraza ch mi c pht hin nm 1976, nhng cha
c quan tm nhiu. Hot ng ca polyphophataza c quan tm c bit
khi c ngng t, photphat v c c s dng nh phn bn cung cp ln.
Sau khi thm vo dung dch phenylphotphat (mui Natri), mu t c
37
o
C/3h. Phenol c gii phng chuyn mu vi 2,6 dibromchinone clorit v
c xc nh bc sng 614nm (theo phng php ci tin t phng php
ca Hoffmann, 1986).
1. Vt liu, ho cht
- Dung dch nn 0,1 M: 27g Natri phenyl photphat/1000ml nc ct
- m axetat (pH=5):
+ Dung dch 1: 60ml axit axetic ng bng/1000ml nc ct
+ Dung dch 2: 136g Natri axetat/1000ml nc ct
Trn dung dch 1 v 2 theo t l 1:2 v iu chnh pH n 5
- m xitrat (pH=7): 300g trinatri xitrat/1000 ml, iu chnh pH bng HCl
- m boric (pH=10): 12,4 g H
3
BO
3
/100ml NaOH 1M, ln th tch 1000ml.
- Thuc th mu: 200mg 2,6 dibromchinone clorit/100ml etanol (60%v/v)
- Dung dch chun m (1mg phenol/ml): 1g phenol/1000ml, gi trong l mu.
- Dung dch chun lm vic (10g phenol/ml): pha long 10ml dch
m/1000ml
- Dy dung dch chun hiu gi: Ht 0; 5;10; 15 v 20 ml dung dch chun
(10g phenol/ml) vo 5 bnh nn 100ml, thm 5ml dung dch m mong mun,
1ml thuuc th mu v 25ml nc ct. Sau 30 pht, ln th tch vi nc ct,
trn u. Dy chun cha 0,50, 100, 150 v 200 g phenol.
2. Th tc tin hnh
- Cho 5g t m vo 4 bnh nn 50ml, ht vo 10ml m axetat, citrat
hoc boric vo mi bnh.
- Thm 5ml dung dch cht nn vo 3 bnh (mu), ly 5ml cho vo bnh
cn li (i chng), lc nh, y nt v 37
o
C/3h.
- Sau khi , ln th tch, lc u v lc vo cc ng nghim.
- Ht 2 ml dch lc (ph thuc vo hot tnh ca t, lng dch lc c th
thay i t 1-4ml) vo bnh nh mc 100ml cha 5ml m boric. Thm 1ml
thuc th mu v 25ml nc ct, trn u v yn 30 pht. Dy hiu gi chun
cng c x l tng t.

Sau , ln th tch v trn u. o mt mu ca dy chun, mu v
i chng trn my quang ph hp ph 614nm da vo phn ng i chng
trng trong vng 24h.
3. Tnh kt qu
Hot tnh photphataza c biu th l g phenol trn 1g cht kh v thi
gian . Nng phenol ca mu v i chng c tnh t ng cong chun.
(S-C).50.100
= g phenol.g
-1
dm.3h
-1

ml.5.% dm
S: Gi tr o mu (g phenol)
C: i chng (g phenol)
ml: c s dch lc (ml)
5: Lng t ban u (g)
100%
-1
dm: H s t kh
IV. ENZYM TRONG TRAO I LU HUNH
Sulphataza rt quan trng cho s khong ho cc hp cht cha lu hunh
trong t. Chng thu phn cc sulfat hu c, v nh cung cp lu hunh d
tiu cho cy trng. Sulphataza c ngun gc ch yu l t vi sinh vt. Trong t,
chng cng xut hin cc enzym ngoi bo c quan h cht vi cc vt cht hu
c. Trong t nhin, nhiu loi sulphataza khc nhau c mt: arylsulphataza,
alkylsulphataza, steroidsulphataza, glucosulphataza,Arylsulphataza l nhm
enzym c pht hin sm nht v c iu tra nghin cu nhiu. Chng
xc tc s thu phn anion arylsulphat ph v lin kt O-S. Arylsulphataza
trong t c xc nh ln u tin bi Tabatabai v Bremner (1970).
Dimetylsulphit (DMS) ng mt vai tr quan trng trong vng tun hon
lu hunh v c sinh ra ch yu bi vi sinh vt ph du bin. Cc vt cht d
bin i ny b oxy ho quang nng trong kh quyn thnh dimetylsulphoxit
(DMSO). DMSO b ho tan trong nc v c chuyn n cc h sinh thi t
v nc thng qua s kt lng. Trong t, vi sinh vt bin i DMSO thnh
DMS.NADH v DMS.NADPH c dng gim ho tr trong cc phn ng
trong t bo.
n 95% VSV c th chuyn ho DSMO gi cho vic dng phn
ng ny l mt ch tiu nh gi hot tnh ca VSV t.
xc nh hot tnh ca arylsulphataza, sau khi thm dung dch p-
nitrophenylsulphat, mu t c 37
o
C trong 1h. Nitrophenol c gii
phng ra bi hot ng ca arylsulphataza c chit xut v so mu vi NaOH
v xc nh trn my o sng (theo phng php ci tin t phng php ca
Tabatabai v Bremner, 1970).

1. Vt liu, ho cht
- m axetat (0,5M; pH=5,8): 64g Natri axetat trihydrat/700ml, iu
chnh pH ti 5,8 bng khong 2ml axit axetic, ln th tch 1000ml.
- Dung dch nn (0,02M): 0,515g Kali-p-nitrophenylsulphat trong 100ml
m axetat, dung dch ny ch dng trong ngy.
- Dung dch NaOH 0,5M: 20g NaOH/1000ml nc ct.
- Dung dch tiu chun gc (1mg p-nitrophenol/ml): 1g p-
nitrophenol/1000ml, gi 4
o
C.
- Dung dch tiu chun (0,1mg p-nitrophenol/ml): 10ml dung dch chun
gc/100ml.
- Dy tiu chun hiu gi: Ht 0; 1; 2; 3; 4 v 5 ml dung dch tiu chun
vo cc ong, iu chnh n 5ml bng nc ct, thm vo mi cc 25 ml nc
ct. Ht 6ml hn hp ny vo 6 ng nghim ri thm 4ml NaOH. Dy hiu gi
chun c cha 0; 20; 40; 60; 80 v 100 g p-nitrophenol.
2. Th tc tin hnh
- Cn 1g t m cho vo bnh nh mc 50ml. Thm 4ml m axetat v
1ml dung dch cht nn vo 3 bnh (mu), ht ch 4ml m axetat vo 2 bnh cn
li (i chng). Lc nh ri cc bnh 37
o
C/1h.
- Sau khi , cho thm 25ml nc ct vo c bnh mu v i chng, ht
thm 1ml dch cht nn vo bnh i chng. Lc nh, lc dung dch mu v i
chng ngay.
- Trn 6ml dch lc vi 4ml NaOH. o 420nm trn my quang ph hp
ph da vo phn ng i chng trng. c ng cong chun, x l dy tiu
chun tng t nh dch lc mu.
3. Tnh kt qu
Tnh nng p-nitrophenol (pNP) t ng cong chun
(S-C).30.100
= g pNP.g
-1
dm.h
-1

6.% dm
S: Gi tr o mu (g phenol)
C: Gi tr i chng (g phenol)
6: c s dch lc (ml)
100%
-1
dm: H s t kh


1 Cc loi enzym do VSV sn sinh ra trong tng mi trng chuyn ha?
2. Tnh ton kt qu th nghim ca tng loi sn phm nui cy VSV v lng
enzym tng ng c sn sinh?

Bi s 17
XC NH SINH KHI VI SINH VT T
Mc ch:
- Gii thiu cho hc vin hiu c kh nng to sinh khi ca VSV
- Cc phng php xc nh sinh khi VSV
Ni dung:
- Hiu c sinh khi vi sinh vt v nh hng ca mi trng n kh
nng to sinh khi
- Bit c phng php nui cy VSV to sinh khi v nhng tc ng
n qu trnh to sinh khi VSV
1. Nguyn l chung
Trong t, vi sinh vt xut hin vi mt v chng loi rt ln. Trong
, vi khun v nm l cc loi vi sinh vt phong ph nht, nguyn sinh ng
vt v to xut hin vi s lng t hn. Phn sinh khi cacbon trong t c
tm thy c 1-3% cacbon hu c (Sparling 1985). Kho st trn t canh tc cho
thy t l sinh khi vi sinh vt do hot ng trao i cht l 1-5 v 2-8% vt cht
hu c tng ng trong t trng cy v t ng c (Beck v cng s, 1992).
Sinh khi c xc nh da trn s phn tch ton hc cc ng cong h hp
cho thy ch c 2-30% tng sinh khi l hot ng trao i cht.
Hot ng ca vi sinh vt t to nn ph cho t v thc hin cc chc
nng trong h sinh thi:
- Hot ng phn gii: Khong ho c th vi sinh vt, ng thc vt v
cc cht hu c tng hp, huy ng cc cht dinh dng v c v cc nguyn t
vi lng.
- Hot ng tng hp: Sn xut sinh khi vi sinh vt, ng tng hp cc
hp cht mn v cht keo dnh, c nh cc cht dinh dng.
Sinh khi vi sinh vt t l khi lng cc t bo vi sinh vt nguyn vn
trong lng t cho.
Cc phng php khc nhau c s dng xc nh sinh khi vi sinh vt:
- m di knh hin vi
- m trn a mi trng c hoc trong mi trng dch th.

- K thut - xng hi, chit xut-xng hi v o h hp cm ng cht nn.
- Xc nh ATP, axit muramic, D-alanin, kitin hoc cc glucozamin khc,
axit nucleic, photpholipit, lipopolysacharit,
- o t l tng hp AND vi [
3
H] thymidin, t l hp nht ca H
2
32
PO
4
v
[C
14
] axetat.
- Phn tch ph sinh khi vi cht ng v bn vng.
Xc nh sinh khi trong t gp mt s kh khn. Cc phng php nui
cy v m trc tip di knh hin vi c gi tr gii hn. Chng m ch vi
mt lng nh, mt phn nh cha bit ca tng sinh khi vi sinh vt do hnh
thnh cc khi t bo, cng hp ph khc nhau ca cc sinh vt ti ht t
v nhiu loi sinh vt khc nhau c th sinh trng trn mi trng nui cy.
m trc tip di knh hin vi khng phn bit c gia sinh khi sng v
cht. Cc phng php vt l c s lp li tt hn m trn a mi trng v
ph hp cho cc nghin cu so snh. S chuyn i cc n v o sinh khi nn
c thc hin cn trng v i hi cc iu tra su hn. S suy lun t sinh
khi hot ng o c trong phng th nghim n hot ng trao i cht
trong t khng khai ph l khng th c. C phng php m VSV sng
trn a ln phng php vt l u khng tnh ton n phn ln sinh khi tim
tng trong iu kin t nhin.
Nhng nm gn y, cc phng php xc nh sinh khi vi sinh vt gin
tip li quan trng hn. Cc phng php ny i hi thi gian t, v c s lp
li cao. Tuy nhin, khng th phn bit c sinh khi tim tng v hot ng
ca vi sinh vt trong t. Sinh khi vi khun v nm cng khng th phn bit
c. Mt trong nhng phng php c s dng ph bin l phng php
chit-xng hi. Phng php ny cho php nh gi sinh khi vi sinh vt ca t
bng cch o ton b lng vt liu sinh khi hu c c th chit c t cc vi
sinh vt mi b git. Phng php chit xng hi s dng cho t kh v t t
(ngp ng, rung la nc) vi tt c cc gi tr pH ca t. Sinh khi c xc
nh trong t c cha cc cht nn phn hu mnh v t bo ho dung dch
kalisunphat.
2. Xc nh sinh khi cacbon ca vi sinh vt t
* Nguyn tc
Bng cch xng hi mu t, cc t bo nguyn vn c ho tan v cht
hu c vi sinh vt c gii phng. Cht hu c ca t khng th sng th
khng chu nh hng ca vic xng hi. Cc mu t c xng hi cloroform
trong 24h. Cacbon hu c chit bng dung dch kalisunphat 0.5mol/l c xc
nh cho mu xng hi v khng xng hi v hiu s ca cacbon hu c chit
c s dng xc nh lng cacbon sinh khi vi sinh vt.
S xng hi bng cloroform cng nh hng ti h ng vt t. Phn
ng gp cacbon ca cc c th loi ny thng nh (<5%) v c th b qua.

2.1. Vt liu, ho cht v thuc th
- Nu yu cu cc mu t cn phi ng nht th sng cc mu kh
nng gi nc (KNGN) l 40%. Hm lng nc ca mu phi cao hn kh
nng gi nc 30% m bo phn tn u ca cloroform v cho s
xng hi c hiu qu. Cn ch trnh lm t t ng khi v bn. Mu ly
t t ngp nc khng phi sy kh trc khi phn tch.
- ng thu tinh (400 x 200 mm) c phu lc, chai nha 1000ml c np, bnh
ly tm plastic hoc ti nylon, giy lc th, oxit nhm, ht chng tro, a petri.
- Cloroform khng c ru etylic: Lc 70ml Cloroform qua ng thu tinh
vi 50g oxit nhm. Lc 25ml u tin cho giai on 2. Dung dch cloroform
khng c etylic c th gi c 14 ngy trong chai ti mu. Khi c nh sng,
cloroform khng c ru etylic phn hu nhanh to thnh kh phosgen (COCl
2
)
khng mi v rt c.
- Dung dch BaCl
2
1M: 20,8g/100ml.
- Axit HCl 0,1M; NaOH 0,1M
- K
2
SO
4
0,5M: 87.135g/l
- Dung dch ch th: 0,1g phenolphtalein/100ml etanol (60%v/v)
- Phng c kh nng duy tr nhit (25 2)
o
C.
- Bnh ht m c bo v chng n, h to chn khng (bm tia nc
hoc bm in), my lc nm ngang hoc ng, t (-15
o
C n -20
o
C)
2.2. Th tc tin hnh
2.2.1. Chi t- xng hi
* Xng hi: Lt ht bnh m bng giy lc m xng hi mu t. Cn
t nht ba mu t m trong cc t thu tinh (hoc trong a perti), mi mu
cha khi lng tng ng vi 25 n 50g t kh kit. Sau t chng vo
bnh ht m cng vi cc t cha 25ml cloroform khng cha ru etylic, cho
mt t ht chng tro vo cc cloroform v cc t cha vi. To chn khng
cho bnh ht m cho ti khi cloroform bn trong bnh si mnh khong 2 pht.
ng kho chn khng ca bnh ht m v phng (25
o
C 2
o
C) trong
bng ti khong 22 - 24h.
Nu nh khng c mu t th dng mu nh hn nhng gi cho t l
khi lng t v dung mi chit khng i (1: 4). Trong t c cha cht hu
c nhiu hn 20% th tng t l t : dung mi trn 1: 4 (cho ti ti a l 1:30
i vi t cha 95% cht hu c, v d tng thm mc) thu c cht chit.
Ghi li lng t s dng.
Sau khi xng hi kt thc, ly cc cloroform v giy lc ra khi bnh ht
m. Sau tin hnh tch hi cloroform trong mu t bng cch ht chn
khng bnh ht m (lp li 6 ln, mi ln 2 pht) v mu t c th mang i
chit c.

Cho ba mu i chng m khng xng hi (50g khi lng kh) vo chai
nha PE v dng 200ml dung dch kalisunphat chit ngay.
* Chit: chit tch cacbon hu c, chuyn mu t vo chai nha PE
tht cn thn, cho thm 200ml dung dch kalisunphat, lc chai trn my lc nm
ngang tc 200 vng/pht trong 30 pht hoc my lc ng tc 60
vng/pht trong 45 pht v lc dung mi qua giy lc. Chit i chng khng
xng hi v lc dung mi chit cng nh vy.
Nu nh khng phn tch ngay, bo qun dch chit ca mu t xng hi
v khng xng hi t v gi nhit t -15
o
C n -20
o
C. Trc khi s
dng lm tan dch chit n nhit phng v ng nht chng.
* Ch thch:
1. Trong khi bo qun dung mi chit thng xut hin kt ta trng (c
bit khi gi nhit ng lnh) bi v chng thng c bo ho vi
canxisunphat (CaSO
4
). Khng cn ho tan lng canxisunphat d ny v n
khng tc dng vi bt k ho cht no trong qu trnh tin hnh theo phng
php ny.
2. Cc mng t bo ca cc r non, sng cng b nh hng qua qu trnh
xng hi bng cloroform. Nu nh t c cha nhiu r cy sng cn phi tin
hnh thm mt qui trnh x l trc khi chit theo ph lc.
* Xc nh cacbon trong dch chit
Hm lng cacbon ca vi sinh vt trong mu t c xc nh bng cch
phn tch v hm lng ny c th s dng so snh cc mu t khc nhau.
Nu nh cn s liu v sinh khi vi sinh vt hin ti, th lc cc phn tch
nh vy c nhn vi h s chuyn i c rt ra t cc thc nghim, tng
quan gia khi lng t bo bit vi lng cacbon sau khi xng hi v chit
tch. Tt c cc h s chuyn i s dng u tong quan theo h s ban u
ny.
Xc nh hm lng cacbon trong dch chit bng phng php oxy ho
dicromat hoc phng php phn tch trn my.
2.2.2. Xc nh cacbon sinh khi vi sinh vt bng phng php oxy ho
dicromat
Trong mi trng axit mnh cht hu c b oxy ho v Cr (VI) b kh
thnh Cr(III). Lng dicromat cn li c chun ngc.
- Thuc th b sung:
+ Kalidicromat K
2
Cr
2
O
7
0.0667 M (19.6125g kalidicromat kh/lit nc).
Do bn cht nguy hi ca kalidicromat, phi rt cn thn khi s dng v thi b.
+ Axit photphoric H
3
PO
4
(p = 1.71 g/ml)
+ Axit sunfuric H
2
SO
4
(p = 1.84 g/ml)

+ St (II) amoni sunphat, dung dch chun [(NH
4
)
2
Fe(SO
4
)
2
.6H
2
O] =
0.04M: St (II) amoni sunphat (15.69g)ho tan trong nc ct, thm 20 ml axit
sunfuric, sau cho tip nc ct n mc 1000ml.
+ Dung dch phc sunphat 1.10 phenanthrolin 0.025 mol/l.
+ Hn hp axit: Hai th tch axit sunfuric trn vi mt th tch axit
photphoric
- Thit b b sung:
+ Sinh hn Liebig (Lm lnh bng nc)
+ Bnh y trn 250ml, Buret 10ml, chia 0.05ml, Pipet 2ml
- Cch tin hnh:
Dng pipet ly 2ml dung dch kali dicromat (P
D
) v 15 ml hn hp 2 axit
cho vo 8ml dch chit c lc (P
S
) trong bnh y trn 250 ml. un hi lu
nh ton b hn hp trong vng 30 pht, sau lm lnh v pha long vi
khong 20 ml n 25ml nc qua ng sinh hn trng.
Cng tin hnh tng t nh vy vi mu trng, c cha 8 ml dung dch
K
2
SO
4
.
Xc nh lng dicromat tha bng cch chun ngc vi mui kp
sunphat st (II) amoni dng mt vi git dung dch phc 1, 10-phenanthrolin-
sunphat st (II) lm ch th.
2.2.3. Tnh ton kt qu
Tnh lng cacbon hu c chit ra c bng cng thc (1) v (2)
C(g/ml) = [(V
H
-V
S
)/V
C
]x MP
D
x E x 1000/P
s
(1)
Trong :
V
S
: Th tch dung dch chun dng chun mu (ml)
V
H
: Th tch dung dch chun dng chun mu trng hi lu
(ml)
V
C
: Th tch dung dch chun dng chun mu trng khng hi
lu (ml)
M : K
2
Cr
2
O
7
(mol /lit)
P
o
: Th tch dung dch K
2
CR
2
O
7
b sung (ml)
P
s
: Th tch dung dch mu b sung (ml)
E: 3 (chuyn i t cacbon hu c C[C]thnh CO
2
[C (+ IV)])

C(g/ g t kh) = C(g/ml) x (P
K
/ D
w
+ S
w
) (2)
Trong :
P
K
: Khi lng dung mi chit (g)
D
w
: Khi lng t kh (g)

S
w
: Nc trong t (g nc/ g t kh) .
Tnh lng cacbon sinh khi B
c,
, theo cng thc (3):
B
c
= E
c
/ k
EC
(3)
Trong :
E
c
= (khi lng cacbon hu c chit c t t xng hi) (khi lng
cacbon hu c chit c t t khng xng hi)
K
EC
= 0.38. H s k
EC
c tnh ton t mi lin quan gia kt qu ca
nhng phng php - xng hi v phng php chit-xng hi (12 loi t).
2.2.4. Xc nh cacbon sinh khi vi sinh vt bng phn tch cacbon theo
phng php quang ph
* Nguyn tc
Vi s c mt ca kali persunphat (K
2
S
2
O
8
), cacbon hu c chit c
ca t b oxy ho thnh cacbon dioxit v lng cacbon dioxit c o bng ph
(IR) v (UV).
- Thuc th b sung
+ Kali persunphat (K
2
S
2
O
8.
).
+ Axit photphoric.
+ Natri polyphosphat [(NaPO
3
)
m
], rt tinh khit
- Thuc th Kali persunphat: Ho tan 20 g kali sunphat trong 90 ml nc
ct, dng axit phosphoric iu chnh pH ca dung dch bng 2 v sau cho
nc ct va n 1000ml.
- Thuc th natri polyphosphat: Ho tan 50 g natri polyphosphat trong 90
ml nc ct, dng axit phosphoric iu chnh pH ca dung dch trn n 2 sau
thm nc ct n 1000ml
- Thit b b sung
+ My phn tch cacbon t ng vi b pht hin hng ngoi hoc vi h
thng dng lin tc vi u o so mu.
Trong tiu chun ny vic xc nh cacbon sinh khi vi sinh vt da trn
c s oxy ho cacbon hu c bng persunphat c hot ho tia cc tm.
- Cch tin hnh
i vi phng php oxy ho t ng bng persunphat tia cc tm, trn
5ml phn chit t t bng dung dch kali sunphat vi 5 ml thuc th natri
polyphosphat. Bt k kt ta no ca CaSO
4
u c ho tan trong qu trnh
ny. Thuc th kali persunphat c a mt cch t ng vo bung oxy ho
UV, y s oxy ho thnh CO
2
c thc hin bng tia cc tm. CO
2
c to
thnh c o bng hp th hng ngoi hay bng quang ph UV.

2.2.5. Tnh ton kt qu
- Tnh lng cacbon hu c chit c (C) theo cng thc (4):
C (g/g t kh) = [(V x D
V
) (B x D
V
)] x (P
k
/ D
w
+ S
w
) (4)
Trong :
V: C(g/ ml) ca mu
B: C(g/ ml) ca mu trng
D
V
: pha long ca mu bng natri phosphat (ml)
D
B
: pha long ca mu trng bng natri phosphat (ml)
P
K
: Khi lng dung mi chit (g)
D
W
: Khi lng t kh (g)
S
W
: Nc trong t (g nc/ g t kh)
- Tnh lng sinh khi B
C
s dng cng thc:
B
C
= E
c
/k
EC
(5)
Trong :
E
C
= (ccbon hu c chit c t t xng hi) (ccbon hu c chit
c t t khng xng hi)
K
EC
= 0,35
H s K
EC
c tnh t mi lin quan gia kt qu ca phng php
xng hi v kt qu ca phng php chit xng hi (23 loi t)
C th s dng phng php chit - xng hi kt hp vi phng php
nghin cu phn hu ca cc cht hu c nh du
14
C.
* Qui trnh chit s b vi t c cha nhiu r cy sng
Cho t m (25 n 50 g khi lng kh) vo bnh thu tinh 250 ml c
cha 100 ml dung dch kali sunphat chit trong vng 20 pht trn my lc v
sng (i vi t canh tc kch thc l l 2mm, cn i vi t ng c l
3mm). Ra tht sch r cy (v ht nh) trn sng bng 75 ml dung dch kali
sunphat b sung thm v sy kh, cn chng. Ly tm khong 500 g huyn ph
gm t v dung dch kali sunphat trong bnh thu tinh trong 15 pht. Sau
cht nc lc pha trn. Cho thm 3 git cloroform vo t xng hi. Qui
trnh nh trn.
Nu c cc r cy sng trong t th nht thit phi s dng qui trnh trn.
Hn na, qui trnh nay to cho vic o nit sinh khi vi sinh vt d dng hn
bng vic gim nit v c nn c trong t. N cn gim nhng kh khn khi o
sinh khi vi sinh vt trong t kh v v vy rt tin li cho vic o s dao ng
ca cacbon sinh khi vi sinh vt t v nit trong nm. Sinh khi vi sinh vt o
c khng b chit tch ra khi t bng qui trnh chit s b ny.

3. Xc nh sinh khi nit ca vi sinh vt t
Sau khi xng hi bng cloroform, mu t c chit bng dung dch kali
sunphat. Nit tng s trong dch lc c xc nh v bin i sang nit sinh
khi (Brookes v cs, 1985). Nit tng s c xc nh theo phng php
Kjeldahl.
3.1. Vt liu, ho cht v thuc th
Cc vt liu, ho cht, dng c v thuc th chun b nh xc nh cacbon
sinh khi vi sinh vt trn.
3.2. Th tc tin hnh
3.2.1. Chit - xng hi
Xng hi 150-200g mu t m bng cloroform trong 24h ging nh
phn tch cacbon sinh khi trn. Sau (ngc vi k thut chit-xng hi
trn, cloroform khng c tch ra khi mu), trn t xng hi vi dung dch
kali sunphat theo t l 1:4, lc 30 pht v lc lm mu phn tch (3 ln lp li).
Tin hnh tng t vi mu i chng khng xng hi (3 ln lp li).
Tin hnh phn tch lng nit tng s trong dch lc ngay theo phng
php Kjeldahl. C th ct gi dch lc 4
o
C cho n khi phn tch.
3.2.2. Phn tch nit theo phng php kjeldahl
- Dng c, ho cht b sung:
+ Bnh Kjeldahl (250ml), b ct m Kjeldahl
+ Axit sunfuric H
2
SO
4
(p = 1.84 g/ml)
+ H
2
SO
4
0,05M
+ Hn hp xc tc: 100g K
2
SO
4
+ 10g CuSO
4
+ 1g Se, nghin nh
+ NaOH 32% (w/v)
+ Axit boric 2% (w/v)
+ Dung dch ch th (v d xanh bromcresol, metyl)
- Tin hnh: Cho 0,5g hn hp xc tc v 20ml H
2
SO
4
c vo bnh dch
lc, cng ph cho n khi thu c dung dch trong, ngui nhit phng.
Cho 10ml axit boric v vi git ch th vo bnh hng dch chng ct. Sau
khi thm xt d tin hnh chng ct 3-20 pht chuyn hon ton NH
3
vo
bnh cha axit boric v ch th.
Xc nh N-NH
4
bng chun vi H
2
SO
4
0,05M (im chun l dung
dch chuyn t xanh sang hng. Lng nit trong mu c tnh theo cng thc sau:
(S-C).1,4.100.100
= %N
A. SW.1000
Trong :
S: Lng axit H
2
SO
4
0,05M dng cho mu (ml)

C: Lng axit H
2
SO
4
0,05M dng cho i chng (ml)
1,4: H s chuyn i (1ml H
2
SO
4
0,05M tng ng vi 1,4 mgN)
100: Lng dch tiu th (ml)
A: c s dch tiu th (ml)
SW: Lng t ban u
100.1000
-1
: H s chuyn i (%, w/w)
3.3. Tnh kt qu
Nit sinh khi vi sinh vt c tnh theo phng trnh:
(S-C)
= g N.g
-1
dm
0,54
Trong :
S: Gi tr ca mu (g N-NH
4
.g
-1
dm)
C: Gi tr i chng (g N-NH
4
.g
-1
dm)
0,54: H s K
EN
(mi quan h gia nit sinh khi VSV v nit tng s
trong t).


1 Sinh khi VSV c mi quan h mt thit vi sn phm nui cy v mi trng
ln men?
2. Cc phng php th v cc loi thuc th, cc cng thc tnh cho tng nhm
VSV?
2. Tnh ton kt qu th nghim ca tng loi sinh khi cho tng nhm VSV
chuyn tnh?

Bi s 18
SINH TRNG CA VI SINH VT

Mc ch yu cu:
+ Xc nh cc iu kin nui cy cho mi loi VSV: ho kh, ym kh
bt buc, ym kh khng bt buc.
+ Hiu r 3 phng php nui cy vi khun ym kh.
+ Xc nh 4 pha ca ng cong sinh trng ca mt vi khun in hnh.
Ni dung kin tp:
+ Nui cy vi khun ym kh.
+ o c sinh trng ca vi khun. Gii thch s liu sinh trng ca
vi khun trn th.
+ Xc nh nh hng ca nhit n s sinh trng ca VSV.
1. Nguyn l chung
Vi sinh vt c nhng yu cu v dinh dng, l hc, ho hc v mi
trng phi c p ng sinh trng v pht trin. Kin thc v cc iu
kin cn thit cho sinh trng ca VSV c th gip d dng nui cy cc VSV
v iu khin cc VSV khng mong mun.
Nm 1861, Jean Baptiste Dumas trnh by mt bo co ti vin hn
lm Pasteur. Trong bi bo ny, ng bt u:
S tn ti ca lp trng mao c cc c trng ca cc men l ng ch
, nhng mt c trng cn c quan tm nhiu hn l cc ng vt nh trng
mao ny sng v nhn ln v hn trong s vng mt ca lng khng kh nh
nht hoc oxy t do
Khi chng ta ni khng kh l mt yu cu sinh trng, chng ta cn xem
xt lng oxy trong khng kh. Hn th na, khi chng ta nhc n oxy, chng
ta thng mun ni n oxy phn t (O
2
) ng vai tr l mt ngun cung cp
in t trong h hp ho kh.
Cc iu kin vt l trong mi trng nh hng n s sinh trng ca
VSV l nhit , p sut thm thu v pH.
2. Oxy v s sinh trng ca vi sinh vt
S c mt hay vng mt phn t oxy (O
2
) c th l v cng quan trng i
vi s sinh trng ca VSV. Mt s VSV gi l VSV ho kh i hi c oxy,
trong khi cc VSV khc gi l VSV ym kh li khng s dng oxy. Mt l do
khin VSV ym kh bt buc khng th chu c s c mt ca oxy l chng
thiu catalaza v kt qu l s tch lu hydro peroxyt gy cht t bo. Cc loi
ho kh chu kh c th khng s dng oxy nhng chu c n kha tt, mc d
s sinh trng ca chng c th c tng cng trong iu kin vi ho kh hu
ht cc VSV ny trao i cht theo kiu ln men.

Mt s loi vi khun chu khi mt phn sinh trng tt nht trong kh
quyn c nng CO
2
tng (7-10%) v nng O
2
thp hn. Chng s sinh
trng trong mi trng c su m mt lng nh oxy c th tn mn
vo. nui cy cc VSV chu kh mt phn trn a petri v mi trng khng
ngho i, mt bnh cy nn c s dng. Cc a v ng nghim x l c t
trong bnh ln vi mt cy nn sng. Sau khi y nt, tt nn i khi nng oxy
gim. Cc VSV c kh nng sng trong iu kin c hoc khng c oxy gi l
VSV ym kh khng bt buc.
4 ging vi khun thiu catalaza l Streptococcus, Leucconostoc,
Lactobacillus v Clostridium. Trong Clostridium l ym kh bt buc, cn 3
ging kia ym kh chu kh. 3 loi ny khng c h thng xytocrom sn sinh
ra hydro peroxyt, do khng cn catalaza. Vic xc nh s c hay khng c
mt catalaza l rt hu ch trong nh loi vi khun. Khi mt vi git hydro
peroxyt 3% c cho vo khun lc v catalaza hin din, phn t oxy c gii
phng ra di dng bt kh.
Trong phng th nghim, chng ta c th nui cy vi khun ym kh bt
buc hoc khng bt buc bng cch loi tr oxy t do t mi trng hoc s
dng mi trng bin i. Nhiu phng php nui cy VSV ym kh gii quyt
c c 2 qu trnh ny. Tt c cc phng php nui cy VSV ym kh ch hiu
qu nu nh mu hay cc VSV nui cy c thu thp v chuyn i theo cch
lm gim thiu s biu l ca oxy.
2.1. Vt liu
a petri cha mi trng dinh dng
ng nghim cha mi trng Thioglycolat c ch th
Bnh ym kh
Hydro peroxyt 3%
2.2. Dch nui cy
Alcaligenes
Clostridium
Enterococus
Escherichia coli
2.3. Th tc tin hnh
- Khng lc ng Thioglycolat.
- Dn nhn 4 ng canh thang v nhim mi ng mt vng que cy mt
loi vi khun trong iu kin v trng.
- Nui 35
o
C
- Ghi li s xut hin sinh trng mi ng.
- Dng bt nh du di y chia mi a thch thnh 4 phn. Dn nhn
1 a ho kh v 1 a ym kh.

- Ria cy 1 ng n cc vi khun trn mi phn a.
- t ngc a ho kh nui 35
o
C. a ym kh c t ngc trong
bnh .
- Ngi hng dn s chng minh lm th no bnh tr thnh ym kh.
10 ml nc c thm vo ti iu chnh kh Gas-Pak (xc tc hydro kt hp
oxy to thnh nc, phn ng s dng li khi thm nc vo) t trong bnh vi
ch th xanh methylen. Khi c mt ca oxy ch th c mu xanh, chuyn thnh
trng khi oxy gim i.
- Nui bnh 35
o
C
- Sau khi nui cy, ghi li s sinh trng ca vi khun trn mi a.
- Thc hin kim tra th catalaza bng cch cho thm vi git H
2
O
2
3%
vo cc khun lc khc nhau trn a thch. Test catalaza dng tnh s sinh ra
bt bong bng trng. C th dng knh hin vi xc nh r hn cc bt bng.
Test th catalaza cng c th c thc hin trn lam knh. Vi khun sinh
trng trn mi trng thch mu c th s dng phng php ny.
3. Xc nh ng cong sinh trng ca vi khun: nh hng ca iu kin
nhit
Cc pha sinh trng ca VSV c th c xc nh bng cch c ca
dich nui cy. c khng phi l n v o lng trc tip s lng VSV,
nhng c tng ch ra s sinh trng ca VSV. T s hin din ca 1 triu
t bo/ml c th phn bit c bng mt, my o quang ph c s dng
o xc nh c vi lng VSV nh hn.
a s VSV sinh trng trong mt khong nhit nht nh. Nhit
sinh trng ti thiu l nhit thp nht m loi VSV s pht trin. Cc loi
VSV sinh trng nhanh nht nhit sinh trng ti u ca chng. V nhit
cao nht m chng c th sinh trng l nhit sinh trng ti a.
Vi sinh vt c chia thnh 3 nhm chnh da theo nhit sinh trng
ca chng. Nhit ti u ca VSV a lnh l 15
o
C, ca VSV a m l 25
o
C -
40
o
C. Nhit ti thch ca nhiu loi a nhit l 45-65
o
C, mc d mt vi loi
c kh nng sinh trng trn 90
o
C. Khong nhit a thch ca VSV do di
truyn quyt nh.
Trong bi tp ny, chng ta s v biu ng cong sinh trng ca mt
loi vi khun cc nhit khc nhau a ra khong nhit thch hp ca
loi ny.
3.1. Vt liu
Bnh nn cha dch dinh dng + NaCl 1,5%
ng nghim o quang ph
Pipet 5 ml, 10ml v trng
My o quang ph
3.2. Dch nui cy

Vibrio natriegenes
3.3. Th tc tin hnh
- Cho 4 ml dch dinh dng v trng vo 1 ng o quang ph. ng ny l
i chng chun ho my o.
- Nhim 10ml vi khun Vibrio vo mt bnh nn cha dch dinh dng.
- Xoay bnh trn ln dung dch. Chuyn 4 ml t bnh vo ng o quang
ph th 2, v o s hp ph (Abs.) trn my quang ph.
- Ghi li cc kt qu o c.
- bnh nn nhit mong mun.
- Ghi li s hp ph sau mi 10 pht cho n 60 v 90 pht. Ly bnh ra
khi chu nc cho thi gian ti thiu c th ly mu. Khi ly mu, dng
pipet v trng ht 4 ml cho vo ng o quang ph th 2.
- V th t s liu thu c c ng cong sinh trng ca VSV
theo nhit .


1 Trnh by thuyt sinh trng v pht trin ca VSV?
2. Tnh ton kt qu th nghim theo sinh khi VSV v cc iu kin cn v
ca tng th nghim?

Bi s 19
THM QUAN KIN TP MN HC
Mc ch v yu cu:
+ Nhm trang thit b kin thc thc t cho sinh vin v ng dng cng
ngh VSV trong nng nghip v x l nhim mi trng.
+ Yu cu sinh vin tham gia y cc bui hc ngoi kha theo chng
trnh mn hc.
+ Sau khi hc xong sinh vin phi vit tiu lun v bi hc ngoi kha.
a im hc ngoi kha:
+ Cc phng th nghim VSV hin i chuyn phc v trong ging dy &
nghin cu v lnh vc nng nghip.
+ Cc loi ch phm, phn VSV ang c sn xut v p dng trong
nng nghip.
+ Trung tm VSV ng dng Trng i hc Khoa hc t nhin.
+ Phng nghin cu VSV nng nghip Trng i hc Nng nghip I.

+ Vin Sinh hc Vin khoa hc Vit Nam.
+ Nh my (phn xng) phn hu c vi sinh vt (Sng Gianh tnh Ngh
An, Tin Nng tnh Thanh Ha, Vn in H Ni, Vit Tr tnh Ph Th v
cc tnh trn ton quc).
+ Vin chn nui th y, xng sn xut thc n gia sc, gia cm, thuc th y
cc tnh
+ Cc trang tri, cc cng ty nui trng thu, hi sn trn ton quc.
+ X nghip x l ph thi th cc thnh ph v cc tnh.
Ni dung:
+ Xem cch thit k phng nghin cu v ng dng vi sinh vt.
+ Quan st v tm hiu cc quy trnh sn xut hoc x l nhim mi
trng trn c s ng dng cng ngh vi sinh vt.
+ Tin hnh ly mu kim tra cht lng sn phm cng nh ch phm
VSV.

Bi s 19
THM QUAN KIN TP MN HC
* Cu hi n tp:
Vit thu hoch t kin tp ti cc c s thm quan, kin tp?

PH LC
BNG 1: PHA DUNG DCH M SORENSEN I V DUNG DCH
Na
2
HPO
4
1/15M V KH
2
PO
4
1/15M - S LNG DUNG DCH (Ml)

pH Na
2
HPO
4
1/15
M
KH
2
PO
4
1/15 M
4,53
4,48
5,29
5,59
5,91
6,24
6,47
6,64
6,81
6,98
7,17
7,38
7,73
8,04
8,34
8,67
9,18
0,00
0,10
0,25
0,50
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
9,50
9,75
9,90
10,00
10,00
9,90
9,75
9,50
9,00
8,00
7,00
6,00
5,00
4,00
3,00
2,00
1,00
0,50
0,25
0,10
0,00


BNG 2: TNH S LNG VI SINH VT THEO MAC.CRADY

S lng VSV c xc sut
ln nht khi cy lp li
cc ng nghim
S lng VSV c xc sut
ln nht khi cy lp li
cc ng nghim

Ch
s
2 3 4 5

Ch s
2 3 4 5
000
0,0 0,0 0,0 0,0 132 - - 1,6 -
001 0,5 0,3 0,2 0,2 140 - - 1,4 1,1
002 - - 0,5 0,4 141 - - 1,7 -
003 - - 0,7 - 200 2,5 0,9 0,6 0,5
010 0,5 0,3 0,2 0,2 201 5,0 1,4 0,9 0,7
011 0,9 0,6 0,5 0,4 202 - 2,0 1,2 0,9
012 - - 0,7 0,6 203 - - 1,6 1,2
013 - - 0,9 - 210 6,0 1,5 0,9 0,7
020 0,9 0,6 0,5 0,4 211 13,0 2,0 1,3 0,9
021 - - 0,7 0,6 212 20,0 3,0 1,6 1,2
022 - - 0,9 - 213 - - 2,0 -
030 - - 0,7 0,6 220 25,0 2,0 1,3 0,9
031 - - 0,9 - 221 17,0 3,0 1,6 1,2
040 - - 0,9 - 222 110,0 3,5 2,0 1,4
041 - - 1,2 - 223 - 4,5 - -
100 0,6 0,4 0,3 0,2 230 - 3,0 1,7 1,2
101 1,2 0,7 0,5 0,4 231 - 3,5 2,0 1,4
102 - 1,1 0,8 0,6 232 - 4,0 - -
103 - - 1,0 0,8 240 - - 2,0 1,4
110 1,3 0,7 0,5 0,4 241 - - 3,0 -
111 2,0 1,1 0,8 0,8 300 - 2,5 1,1 0,8
112 - - 1,0 0,8 301 - 4,0 1,6 1,1
113 - - 0,5 - 302 - 6,5 2,0 1,4
120 2,0 1,1 0,8 0,6 303 - - 2,5 -
121 3,0 1,5 1,1 0,3 310 - 4,5 1,6 1,1
122 - - 1,3 1,1 311 - 7,5 2,0 1,4
123 - - 1,6 - 312 - 11,5 3,0 1,7
130 - 1,6 1,1 0,8 313 - 16,0 3,5 2,0
131 - - 1,4 1,0 320 - 9,5 2,0 1,4


BNG 2B: TNH S LNG VI SINH VT THEO MAC.CRADY

S lng VSV c xc sut
ln nht khi cy lp li
cc ng nghim
S lng VSV c xc sut
ln nht khi cy lp li
cc ng nghim

Ch
s
2 3 4 5

Ch s
2 3 4 5
321
- 15,0 3,0 1,7 451 - - - 5,0
322 - 20,0 3,5 2,0 500 - - - 2,5
330 - 25,0 3,0 1,7 501 - - - 3,0
331 - 45,0 3,5 2,0 502 - - - 4,0
332 - 110,0 4,0 - 503 - - - 6,0
333 - 140,0 5,0 - 504 - - - 7,5
340 - - 3,5 2,0 510 - - - 3,5
341 - - 4,5 2,5 511 - - - 4,5
350 - - - 2,5 512 - - - 6,0
400 - - 2,5 1,3 513 - - - 8,5
401 - - 3,5 1,7 520 - - - 5,0
402 - - 5,0 2,0 521 - - - 7,0
403 - - 7,0 2,5 522 - - - 9,5
410 - - 3,5 1,7 523 - - - 12,0
411 - - 5,5 2,0 524 - - - 15,0
412 - - 8,0 2,5 525 - - - 17,5
413 - - 11,0 - 530 - - - 8,0
414 - - 11,0 - 531 - - - 11,0
420 - - 6,0 2,0 532 - - - 14,0
421 - - 9,5 2,5 533 - - - 17,0
422 - - 13,0 3,0 534 - - - 20,0
423 - - 17,0 - 535 - - - 25,0
424 - - 20,0 - 540 - - - 13,0
430 - - 11,5 2,5 541 - - - 17,0
431 - - 16,5 3,0 542 - - - 25,0
432 - - 20,0 4,0 543 - - - 30,0
433 - - 30,0
-
544 - - - 35,0
434 - - 35,0 - 545 - - - 45,0
440 - - 25,0 3,5 550 - - - 25,0
441 - - 40,0 4,0 551 - - - 35,0
450 - - - 1,4 555 - - - 180,0

101

TI LIU THAM KHO

1. Nguyn Ln Dng v cng s. S tay nghin cu vi sinh vt, tp I,II,II. NXB
GD 1978.1979
1. Nguyn Ln Dng v cng s. Thc hnh vi sinh vt. NXB GD 1992
2. Nguyn Ln Dng. Thc tp vi sinh vt (dch gi t ting Nga). NXB GD
1983
3. Nguyn ng v cng s. Thc tp vi sinh vt trng trt NXB.NN 1982
4. Nguyn ng v cng s. Thc tp vi sinh vt NXB.NN 1989
5. Nguyn Xun Thnh v cng s. Thc tp vi sinh vt i cng NXB.NN
1994
6. Nguyn Xun Thnh v cng s. Gio trnh vi sinh vt hc nng nghip.
NXB GD. 2004
7. Nguyn Xun Thnh v cng s. Thc tp vi sinh vt i cng. NXB.NN
2006

24. Thực tập vi sinh vật chuyên ngành - Pgs.Ts.Nguyễn Xuân Thành PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

24. Thực tập vi sinh vật chuyên ngành - Pgs.Ts.Nguyễn Xuân Thành PDF

Uploaded by

Copyright:

Available Formats

Trng i hc Nng nghip H Ni Gio trnh thc tp vi sinh vt chuyn ngnh 1

You might also like