MN: K THUT THC PHM 3

TI: LN MEN B SU- NG HC CA QU TRNH LN MEN GIN ON

TP. HCM, THNG 10 NM 2013

QU TRNH LN MEN???
1 Ln men l mt qu trnh sinh hc, m phn ng oxy ha kh xy ra trong t bo vsv, trong iu kin khng c cht oxy ha no. Mt phn cht hu c b phn gii thnh cht kh, phn cn li b phn gii thnh cht oxy ha 2 Nghin cu qu trnh ln men l NC c im sinh l-ha sinh v hot ng sng ca mt chng VSV 3

Ty thuc vo c im sinh l ca VSV i vi oxy, ta coi qu trnh l hiu kh hay k kh khng bt buc

CC PHNG PHP LN MEN

i vi ln men k kh thc hin theo phng php nui cy chm

Ln men hiu kh c thc hin nh hai phng php c bn: nui cy b mt v nui cy b su (nui cy chm)

PHN LOI QU TRNH LN MEN

1. Da vo k thut nui cy: Da vo cch np liu + thu sn phm ta c cc loi ln men: Gin on Lin tc Bn lin tc Da vo thnh phn ng nht ca mi trng ta c cc loi ln men: B mt (ni) B su (chm) Bn rn

PHN LOI QU TRNH LN MEN

2. Da vo sn phm chnh: Ln men etilic Ln men lactic Ln men propionic Ln men formic Ln men butiric v ln men axeton-butanol Ln men metan Ln men axetat Ln men xenlulozo

LN MEN B SU TRONG MI TRNG LNG

Tn t mt bng trong xy dng v lp t dy chuyn 1 Chi ph in nng, nhn lc v cc khon ph cho mt n v sn phm thp.

u im
Cc thit b ln men chm d c kh ho, t ng ho

D t chc c x nghip c sn lng ln.

LN MEN B SU TRONG MI TRNG LNG

Nhc im
i hi trang b k thut cao, d b nhim trng ton b Trong ln men chm cn phi khuy v sc kh lin tc

LN MEN GIN ON
VSV sinh trng n chng no mt thnh phn ch yu ca mi trng dinh dng b gii hn

C IM

L qu trnh nui cy VSV m mi trng dinh dng ch c cho vo lc u v khng thm cht dinh dng cho n khi kt thc qu trnh nui cy

Sn phm ca qu trnh ln men c tch ly dn dn v ch ly ra khi qu trnh ln men kt thc

LN MEN GIN ON
GIAI ON TIM PHT

Trong qu trnh nui cy, VSV sinh trng v pht trin theo mt quy lut gm 4 giai on

GIAI ON LOGARIT

GIAI ON N NH

GIAI ON T VONG

NG CONG SINH TRNG

Th hin s sinh trng ca qun th vi sinh vt trong mt mi trng nui cy theo phng php nui cy theo m (batch culture) hoc trong mt h thng kn

GIAI ON TIM PHT (Lag phase)

Trong giai on ny t bo cha c s tng ln v s lng nhng th tch v khi lng tng ln r rt do c s tng cc thnh phn mi ca t bo. Do c s thay i v mi trng sng nn t bo cn mt thi gian nht nh tng hp cc enzyme mi nhm s dng c cc cht dinh dng mi. Giai on tim pht di hay ngn lin quan n bn thn tng loi vi sinh vt v tnh cht ca mi trng. Nu tnh cht ha hc ca mi trng mi sai khc nhiu vi mi trng c th giai on tim pht s ko di v ngc li.

GIAI ON TIM PHT (Lag phase)

Tui ging cy. V d: pha lag s khng c nu dng ng cy gm cc TB ang pha sinh trng logarit

CC YU T NH HNG

Lng ging cy: ni chung lng ging cy nhiu th pha lag ngn v ngc li. Trong cng nghip ln men t l cy ging thng mc 1/10

Thnh phn mi trng: mi trng c thnh phn dinh dng phong ph th pha lag ngn.

Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase)

Trong giai on ny vi sinh vt sinh trng v phn ct vi nhp ti a nu gp mi trng v iu kin nui cy thch hp. Do cc t bo sinh ra ch khc nhau rt t cho nn ng cong sinh trng l mt ng trn nhn ch khng gp khc Qun th t bo trong giai on ny c trng thi ha hc v sinh l hc c bn l nh nhau => thng c s dng nghin cu sinh ha hc v sinh l hc ca vi sinh vt.

Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase)

Sinh trng logarit l sinh trng ng u nu cc thnh phn t bo c tng hp vi tc tng i n nh. Mt khc, nu cn bng dinh dng hay iu kin mi trng thay i s dn n s sinh trng khng ng u.
S sinh trng ca vi sinh vt ph thuc nhiu vo nng cht dinh dng.

Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase)

(a )- nh hng ca s hn ch cht dinh dng i vi sn lng chung ca vi sinh vt. Lc nng cao th sn lng chung s t ti n nh. (b)- nh hng ca s hn ch cht dinh dng ti tc sinh trng.

GIAI ON N NH(Stationary Phase)

C IM
1

ng cong sinh trng i ngang

Nng vi khun trong giai on n nh thng v khong 109/ml

S lng t bo sinh ra bng s t bo cht i

GIAI ON N NH(Stationary Phase)

NGUYN NHN
Hn ch cht dinh dng. Vsv hiu kh thng b hn ch bi nng oxygen. Tch ly cc sn phm trao i cht c hi.

GIAI ON N NH(Stationary Phase)

Thu nh khch thc

Nhng bin i
V nhng bin i khc Sinh ra protein i

GIAI ON T VONG(Death Phase)

S lng t bo c kh nng sng gim theo ly tha

S cht ca t bo c th nhanh hay chm, c lin quan n s t phn hay khng t phn

NGUYN NHN

iu kin bt li ca mi trng: nng cht dinh dng thp di mc cn thit cho hu qu lm gim trao i cht , phn hy dn dn cc cht d tr v cui cng dn n s cht hng lot ca t bo Qu trnh bo qun cc chng VSV

TNH TON QU TRNH SINH TRNG

Nghin cu v sinh l hc TNH TON NHP SINH TRNG

Nghin cu v sinh thi hc VSV

Gii quyt mt s vn ng dng trong sn xut cng nghip

TNH TON QU TRNH SINH TRNG

S lng t bo vi khun tng theo phng thc 2n Ta hy tng tng trong mt bnh kn cha mt lng ln mi trng dinh dng, ta cy vo mt t bo vi khun. Nu thnh phn mi trng hon ton ph hp vi nhu cu ca t bo, vi khun s sinh trng, tng khi lng v th tch,n kch thc nht nh ri phn chia cho 2 t bo. Hai t bo ny tip tc sinh trng v phn chia cho 4, 8, 16, 32,.

TNH TON QU TRNH SINH TRNG

Thi gian 0 20 40 60 80 S ln phn ct 0 1 2 3 4 2n 20 = 1 21 = 2 22 = 4 23 = 8 24 = 16 S lng (N02n ) 1 2 4 8 16 lg10Nt 0,000 0,301 0,602 0,903 1,204

100 120

5 6

25 = 32 26 = 64

32 64

1,505 1,806

TNH TON QU TRNH SINH TRNG

Sinh trng th h ca VSV

TNH TON QU TRNH SINH TRNG

S lng t bo thi gian t: Nt = N0 2n Trong : Nt: s TB sau n ln phn chia trong thi gian t N0: l s lng t bo ban u. n: l s th h hay s ln phn chia. logNt = logN0 + nlog2 S th h n:

Logarit 2 v:

TNH TON QU TRNH SINH TRNG

Trong nui cy gin on, giai on logarit:

Tc sinh trng

Tc sinh trng bnh qun k

k: s th h sinh ra trong n v thi gian

TNH TON QU TRNH SINH TRNG

Xc nh thi gian th h g

Cn c trc tip vo th bn logarit

Da vo hng s tc sinh trng

TNH TON QU TRNH SINH TRNG

Thi gian th h g l thi gian tnh t khi mt t bo sinh ra n khi t bo phn chia hoc s t bo trong qun th tng gp i.
Nu thi gian t = thi gian th h g(t=g) th Nt = 2N0
Thay vo cng thc tnh k ta c:

TNH TON QU TRNH SINH TRNG

Nh vy: Thi gian th h l nghch o ca hng s tc sinh trng bnh qun

Thi gian th h thay i theo chng loi VSV

TNH TON QU TRNH SINH TRNG

Xc nh thi gian th h

PHNG PHP NH LNG VI SINH VT

GII THIU CC PHNG PHP NH LNG VI SINH VT

S hin din ca VSV c th c nh lng bng nhiu phng php nh: m s lng t bo trc tip trn bung m hng cu bng knh hin vi quang hc hoc bng knh hin vi hunh quang.

m gin tip bng s lng khun lc mc trn mi trng xc nh hoc o c ( mt quang) ca t bo

PHNG PHP NH LNG VI SINH VT

Vi sinh vt n bo c kch thc ln nh: nm men, to c th c nh lng bng cch m trc bng bung m trn knh hin vi. u im: Xc nh c ngay lng t bo trong mu.

M TRC TIP BNG BUNG Nhc im: M Khng xc nh c t bo cn sng hay HNG cht, d lm t bo VSV vi cc ht vt th CU khc c trong mi trng v dch huyn ph VSV phi c mt t bo phi ln.

PHNG PHP NH LNG VI SINH VT

m trc tip bng bung m hng cu Pha long mu cn m sao cho trong mi nh ca bung m c khong 5 - 10 t bo VSV. Sau nh 1 git mu ln bung knh v y li bng l knh, quan st di lnh hin vi.

PHNG PHP NH LNG VI SINH VT

S t bo c trong 1 ml mu ban u c tnh theo cng thc:

N=
Trong :

.. (T bo/ml)

A - s t bo trung bnh trong 1 vung ln. 1000 - h s chuyn i t mm3 sang ml (1 ml = 1 cm3 = 103 mm3) F - h s pha long ca mu trc khi m V - Th tch ca 1 vung ln

PHNG PHP NH LNG VI SINH VT

M TRC TIP BNG KNH HIN VI HUNH QUANG

Nhum VSV bng cc cht pht hunh quang nh: acridin cam (AODC), 4,6-dianidino-2-phenyl indol (DAPI), flourescent isothiocyanate (FITC). u im: Kt qu phn nh ng vi sinh khi.

PHNG PHP NH LNG VI SINH VT

Phng php m khun lc c thc hin bng k thut hp hay hp tri vi cc thit b h tr (my m khun lc, bt m khun lc) c kt qu. PHNG u im: PHP - Cho php xc nh s t bo sng. M - nh lng chn lc vsv. KHUN Phng php: LC 1. Chun b dch pha long mu 2. Chun b cc chui pha long mu 3. Cy mu vo mi trng, mu 4. m s khun lc hnh thnh.

PHNG PHP NH LNG VI SINH VT

My m khun lc t ng

PHNG PHP NH LNG VI SINH VT

C 2 phng php m khun lc a. Phng php cy b mt: Mi trng phi chun b trc 1-2 ngy kh mt. u im: nh lng c cc vsv nhy nhit C th nhn dng c khun lc c trng D dng lm thun chng vsv mc tiu Nhc im: Ch cy c th tch mu nh Ch cho m s khun lc thp

PHNG PHP NH LNG VI SINH VT

b. Phng php a: u im: - Cy c th tch mu ln, xc nh c cc vsv cn dinh dng tip xc t nhiu pha - Cho php m c mt vsv cao, khong 150-300 khun lc Nhc im: - Khng nh lng c nhng vsv qu nhy - Khng xc nh c hnh dng khun lc nt nh - Kh lm thun mt dng vsv.

PHNG PHP NH LNG VI SINH VT

PHNG PHP MNG LC Xc nh s lng vi sinh cc pha long khc nhau. Mu c lc v mng lc c t trc tip ln mt mi trng thch thch hp. => Phng php ny thng p dng vi mu nc v nc thi u im: Xc nh c mt vsv c th Nhc im: Khng thch hp phn tch mu thc phm rn
Phng php lc mng xc nh s lng vi sinh vt

PHNG PHP NH LNG VI SINH VT

PHNG PHP NH LNG VI SINH VT

Cc loi khun lc mc trn mng lc. Theo sch ca Prescott,Harley v Klein (2005) (a)- Tng s vi khun mc trn mi trng tiu chun, Dng ch th mu nhum khun lc cho d im; (b)- Dng mi trng thch hp kim tra nhm vi khun coliform c ngun gc t phn (khun lc bt mu xanh); (c)- Dng mi trng thch m-Endo xc nh vi khun E.coli v cc Coliform khc- khun lc c mu lc; (d)- Nm si v nm men mc trn mi trng Thch - Mch nha.

XC NH KHI LNG T BO

Phng php trc tip l xc nh trng lng kh ca t bo.

Phng php gin tip o mt quang (OD).

XC NH TRNG LNG KH CA T BO

Ly tm thu sinh khi t bo

ra t bo ri lm kh

Cn trng lng kh

Thch hp xc nh khi lng ca t bo nm, nhng khng thch hp i vi vi khun.

O MT QUANG (OD)
O C
c ca dch t bo c th m bng quang ph k v c biu din bng n v hp th. S lng t bo lin quan mt thit vi c ca dch vi khun. T bo phi c khuy trn k trc khi a vo quang ph k o c ca dch treo t bo. y l pp rt thun li. trong thc ta thng o mt quang hc ca dch treo (dch huyn ph). o s lng vi sinh vt bng phng php o c.

O MT QUANG (OD)

O MT QUANG (OD)
Thng qua vic o hp th nh sng c th xc nh c sinh khi vi sinh vt
Khi

Lng t bo tng

Dn n

c tng
Mc tn x nh sng nhiu hn Tr s hp th nh sng tng

Lm cho

o c

O MT QUANG (OD)

Cng thc:

A = ln (I0 / I)
Vi: A : l mt quang ti bc sng . I0 : l cng sng trc khi qua mu. I : l cng sng sau khi qua mu.

XC NH NNG T BO
Cng thc:

C(mg/ml)= A* k * n
Vi: C: nng t bo n: h s pha long k: h s chuyn i bng 5x108