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TRNG I HC S PHM H NI 2 KHOA SINH KTNN

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NGUYN TH LAN

PHN LP, TUYN CHN CHNG VI KHUN ACETOBACTER XYLINUM C KH NNG TO MNG BACTERIAL

CELLULOSE C C TNH DAI MNG

KHA LUN TT NGHIP I HC Chuyn ngnh: Vi sinh vt hc

H Ni 2011

Kha lun tt nghip K33 Khoa Sinh KTNN

GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan ii

MC LC

Trang

PHN M U

1. L do chn ti 1

2. Mc tiu ca ti 2

3. Ni dung nghin cu 2

4. ngha ca ti 2

PHN NI DUNG

CHNG 1. TNG QUAN TI LIU

1.1. V tr v c im phn loi ca Acetobacter xylinum

trong sinh gii: 3

1.1.1. V tr phn loi ca Acetobacter xylinum 3

1.1.2. c im phn loi ca Acetobacter xylinum 3

1.2. c im v c ch hnh thnh mng bacterial cellulose 7

1.2.1. c im cu trc ca mng bacterial cellulose 7

1.2.2 C ch hnh thnh mng bacterial cellulose 8

1.3. Tnh hnh nghin cu Acetobacter xylinum trn th gii 9

v Vit Nam

1.3.1 Tnh hnh nghin cu Acetobacter xylinum trn th gii. 9

1.3.2 Tnh hnh nghin cu Acetobacter xylinum Vit Nam 11

CHNG 2. VT LIU V PHNG PHP NGHIN CU

2.1. Vt liu v thit b 12

2.1.1. Vi sinh vt 12

2.1.2. Thit b v ho cht 12

2.1.3. Mi trng 13

2.2. Phng php nghin cu 13


GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan iii

2.2.1. Phng php phn lp vi khun Acetobacter 13

2.2.2. Phng php nghin cu c im hnh thi nui

cy ca cc chng A.xylinum 14

2.2.2.1. Phng php quan st hnh thi t bo trn

knh hin vi 14

2.2.2.2. Phng php quan st hnh thi khun lc [1] 14

2.2.3. Phng php nghin cu c im sinh l - sinh

ho ca cc chng Acetobacter xylinum 15

2.2.3.1. Phng php kim tra hot tnh catalase [11] 15

2.2.3.2. Phng php kim tra kh nng oxy ho

ru ethanol thnh acid acetic [11] 15

2.2.3.3. Xc nh kh nng oxy ho acid acetic [11]. 15

2.2.3.4. Phng php xc nh kh nng tng hp cellulose 16

2.2.4. Phng php xc nh trng lng kh ca mng BC 19

2.2.5. Phng php xc nh kh nng thm ht nc

ca mng BC (g/100cm2) 16

CHNG 3. KT QU V THO LUN

3.1. Phn lp chng Acetobacter xylinum 17

3.1.1. Lm giu mu nguyn liu 18

3.1.2. Phn lp vi khun Acetobacter 19

3.1.3. Tuyn chn cc chng A.xylinum to mng

Bacterial Celullose (mng BC) 21


GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan iv

3. 2. Tuyn chn cc chng c kh nng to

mng BC dai, mng 24

3.3. Nghin cu mt s c tnh sinh hc ca cc chng

Acetobacter xylinum tuyn chn 27

3.3.1 Nghin cu c im hnh thi t bo hc ca cc

chng Acetobacter xylinum 27

3.3.2. c im nui cy ca cc chng Acetobacter xylinum 28

3.3.3. c im sinh l - sinh ho ca cc chng

Acetobacter xylinum 29

3.3.3.1. Kh nng ng ho ngun cacbon ca cc chng


3.3.3.2. Kh nng ng ho ngun nit ca cc chng


3.3.4. nh hng ca nhit n kh nng sinh trng

ca 2 chng R1 v B4 31

PHN KT LUN 33

TI LIU THAM KHO 34


GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan v

DANH MC HNH BNG BIU

HNH Trang

Hnh 1.1. T bo vi khun A.xylinum 4

Hnh 1.2. Si cellulose ca mng BC v si cellulose ca thc vt 7

Hnh 1.3. S hnh thnh nhng di ribbon trong mng BC ca A.xylinum 9

Hnh 3.1. Qui trnh phn lp vi khun A.xylinum 17

Hnh 3.2. Mt s mu mng thu c sau khi lm giu nguyn liu 19

Hnh 3.3. Chuyn ha ethanol thnh acid acetic ca vi khun 20

Hnh 3.5. Mt s loi mng do cc chng vi khun hnh thnh trn

b mt mi trng dch th 23

Hnh 3.6. Kh nng to mng cellulose ca cc chng A.xylinum 23

Hnh 3.7. Cc loi mng bacterial cellulose (BC) 26

Hnh 3.8. Hnh dng t bo ca hai chng vi khun A.xylinum 27

Hnh 3.9. Khun lc A.xylinum 28

BNG

Bng 1.1. c im sinh ha ca chng vi khun A.xylinum 5

Bng 1.2. Cc c im phn bit Acetobacter v Gluconobacter 6

( so snh vi Pseudomonas )

Bng 3.1. Mt s c tnh ca mng BC 25

Bng 3.2. c tnh khun lc vi khun Acetobacter xylinum 28

Bng 3.3. Kh nng ng ha ngun cacbon ca chng R1 v B4 30

Bng 3.4. Kh nng ng ha ngun nit ca 2 chng R1 v B4 31

Bng 3.5. nh hng ca nhit n kh nng sinh trng

ca chng R1 v B4 32


GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan vi

DANH MC T VIT TT

T vit tt T y

A. xylinum

BC

R1

B4

BHN2

cs

Cty

Acetobacter xylinum

Bacterial cellulose

Acetobacter xylinum R1

Acetobacter xylinum B4

Acetobacter xylinum BHN2

Cng s

Cng ty


GVHD: ThS. Nguyn Th Thy Vn SV: Nguyn Th Lan 1

M U

1. L do chn ti

Bng l mt tai nn thng gp trong lao ng v sinh hot hng ngy.

Ngoi tn thng da, trng hp bng nng cn li di chng n kh nng

vn ng, thm m v sc khe ca ngi bnh. Vit Nam, ring vin bng

quc gia H Ni mi nm tip nhn khong 3000 bnh nhn bng do cc

nguyn nhn v mc khc nhau. Hn na s bnh nhn bng l tr em v

s bnh nhn nhp vin b bng su. TS. Nguyn Vit Lng trng phng

hnh chnh tng hp ng thi l ngi iu hnh cc ti nghin cu ca

Vin Bng Quc gia cho bit: Trc y nhng ca bng su rt d t vong

do hi thiu nhng loi thuc c hiu. Thuc nhp khu rt him v t.

Nu bnh nhn b bng nh, din tch vt bng nh cc t bo xung

quanh t pht trin dn ph kn vt thng. Nhng nu l vt bng su th

buc phi cy ghp da. Song song vi vic cy ghp da l vic tm kim cc

loi da t thn, da d loi v cc loi mng sinh hc thay th da tm thi trong

iu tr. Mc d da t thn l vt liu l tng nhng trong nhiu trng hp

khng p ng hoc th trng bnh nhn khng cho php ly Vic tm

kim cc loi da d loi v mng sinh hc thay th cho da tm thi tr

thnh phng php hiu qu hn c. Tuy nhin cc loi da d loi nh da ch

ti, ng kh; da ln ti, ng lnh li c nhng nhc im nht nh nh

vic s dng th b ng, bo qun v x l phc tp, kh c th s dng

rng ri trn lm sng. Chnh v vy, vt liu che ph tm thi t cc loi

mng sinh hc l la chn s mt, c vai tr rt quan trng trong iu tr

bng. Mt trong cc loi mng sinh hc v ang c quan tm ngy nay

l mng cellulose vi khun.

Cellulose vi khun c cu trc ho hc ng nht vi cellulose thc vt

nhng li c mt s tnh cht ho l c bit nh bn c hc, kh nng



thm ht nc cao, ng knh si nh, tinh khit cao, polymer ln...

rt thch hp lm mng tr bng. Th gii c nhiu nghin cu v mng tr

bng t cellulose vi khun. Vit Nam cc nghin cu v vn ny cn rt

t, mi ch c nghin cu ca nhm tc gi Nguyn Vn Thanh (2008) v

nhm tc gi inh Th Kim Nhung (2009) c cng b. Cc loi mng tr

bng ca chng ta hin nay phn ln l nhp ngoi vi gi rt t .

T nhng l do ni trn, chng ti quyt nh chn ti nghin cu:

Phn lp, tuyn chn chng vi khun Acetobacter xylinum c kh nng

to mng Bacterial Cellulose ( mng BC ) c c tnh dai, mng

2. Mc tiu ca ti

- Phn lp cc chng vi khun Acetobacter xylinum (A.xylinum) t mt

s ngun nguyn liu Vit Nam.

- Tuyn chn cc chng A.xylinum phn lp c kh nng to mng

Bacterial Cellulose dai, mng.

- Nghin cu mt s c tnh sinh hc ca cc chng tuyn chn.

3. Ni dung nghin cu

- Phn lp cc chng vi khun Acetobacter xylinum.

- Tuyn chn cc chng A.xylinum c kh nng to mng Bacterial

Cellulose (BC) dai, mng.

- Nghin cu mt s c tnh sinh hc ca cc chng A.xylinum.

4. ngha ca ti

- ti nghin cu la chn cc chng A.xylinum c kh nng to

mng BC dai mng. C trin vng ng dng trong ch to mng tr bng.



NI DUNG

CHNG 1. TNG QUAN TI LIU

1.1. V tr v c im phn loi ca Acetobacter xylinum trong sinh gii:

1.1.1. V tr phn loi ca Acetobacter xylinum

Tn gi: Acetobacter xylinum l mt tn gi chnh thc theo h thng

danh php quc t 1990.

Nm 1957, theo Bergeys manual of determinative bacteriology

A.xylinum c xp vo chi Acetobacter, thuc h Pseudomonadaceae, b

Pseudomonadales, lp Schizomycetes [13].

n nm 1974, theo Bergeys manual of determinative bacteriology,

A.xylinum li c coi nh l mt loi ph ca Acetobacter aceti, thuc chi

Acetobacter v c nhm vo nhng chi khng r ngun gc [14].

Nm 1984 Theo Bergey [4], Acetobacter xylinum c xp vo chi

Acetobacter thuc h Acetobacteraceae. H vi khun ny gm 2 chi l

Acetobacter v Gluconobacter.

Ngy nay, tn ti nhiu quan im khc nhau trong vic phn loi vi

khun acetic ni chung v A.xylinum ni ring. Vn ny vn ang gy

nhiu tranh ci, i hi cn c nhiu hn nhng nghin cu tip theo.

1.1.2. c im phn loi ca Acetobacter xylinum

* c im hnh thi - t bo hc

A.xylinum c dng hnh que, thng hay hi cong, kch thc khong

2m, t bo ng ring l hoc xp thnh tng chui, khng c kh nng di

ng, khng sinh bo t. Cc t bo c bao bc bi cht nhy to vng

nhn c cha hemicellulose.



Vi khun A.xylinum thuc nhm vi khun Gram m, hiu kh bt buc,

ho d dng. T bo ca chng thng tm thy trong gim, dch ru, nc

p hoa qu, trong t.

* c im nui cy

Trn mi trng thch a, vi khun A.xylinum hnh thnh khun lc

nhn hoc x x, ra mp khun lc bng phng hay gn sng, mu trng hoc

trong sut, khun lc bng phng hoc li ln d tch khi mi trng. Vi

khun A.xylinum khi nui cy trong mi trng lng iu kin tnh, chng

s hnh thnh trn b mt mi trng mt lp mng BC [18]. Ngc li trong

iu kin nui lc, cellulose hnh thnh dng ht nh vi kch thc khng

u nhau v phn tn trong mi trng dinh dng to ra nhng c tnh hnh

thi khc hn cellulose trong iu kin nui cy tnh [13].

*c im sinh l sinh ho

+ c im sinh l

Vi khun A.xylinum pht trin nhit 25 - 350C, pH = 4 - 6. Nhit

v pH ti u tu thuc vo ging. 370C, t bo s suy thoi hon ton ngay

c trong mi trng ti u. Tuy nhin vn cn nhng kin khc nhau v

nhiu tranh ci v iu kin nhit , pH ti thch cho chng vi khun

A.xylinum sinh trng v to mng.

Hnh 1.1. T bo vi khun A.xylinum [36]

1m



A.xylinum c kh nng chu c pH thp, v th thng b sung thm

acid acetic vo mi trng nui cy hn ch s nhim khun l.

+ c im sinh ho :

Nm 1950, Prateur chnh thc a ra mt kha phn loi mi cn c

vo cc tiu chun nh: kh nng oxy ha acid acetic thnh CO2 v H2O; hot

tnh catalase; kh nng sinh trng trn mi trng Hoyer [23] Theo quan

im ny, A.xylinum l chng thuc chi Acetobacter, h Pseudomonas, b

Pseudomonadales, lp Schizomycetes. c im phn bit vi cc chng khc

cng mt chi c trnh by bng 1.1 di y:

Bng1.1.c im sinh ha ca chng vi khun A.xylinum

STT c im Hin tng Kt qu

1 Oxy ho ethanol thnh acid acetic

Chuyn ho mi trng cha Bromphenol Blue 0,04% t mu xanh sang mu vng

+

2 Hot tnh catalase Hin tng si bt kh +

3 Sinh trng trn mi trng Hoyer

Sinh khi khng pht trin _

4 Chuyn ho glycerol thnh dihydroxyaceton

To kt ta gch trong dch sau ln men

+

5 Chuyn ho glucose thnh acid

Vng sng xut hin xung quanh khun lc trn mi trng cha CaCO3

+

6 Kim tra kh nng sinh sc t nu

Khng hnh thnh sc t nu _

7 Kim tra kh nng tng hp cellulose

Vng vi khun xut hin mu lam +



Theo Bergey (1984) [4], Acetobacter xylinum c xp vo chi

Acetobacter thuc h Acetobacteraceae. H vi khun ny gm 2 chi l

Acetobacter v Gluconobacter. c im phn bit gia 2 chi c trnh by

trong bng 1.2

Bng 1.2. Cc c im phn bit Acetobacter v Gluconobacter

( so snh vi Pseudomonas )

c im Gluconobacter Acetobacter Pseudomonas

1. V tr tin mao + +/- Tin mao cc

2. Pht trin pH =

4,5 + + -

3. Oxy ha aceton

pH=4,5 + + -

4. Oxy ha axid

acetic - + +

5. Oxy ha lactat - + +

6. Glucose

Gluconat + +/- +/-

7. S dng tinh bt,

lactose - - +/-

8. S dng gelatin - - +/-

9. Sc t hunh

quang - - +/-



1.2. c im v c ch hnh thnh mng bacterial cellulose

1.2.1. c im cu trc ca mng bacterial cellulose

Cellulose cu trc mng BC c thnh phn ha hc tng t cellulose

thc vt nhng cu trc v c tnh li khc xa nhau.

Cellulose c cu to bi chui polyme -1,4 glucopyranose mch

thng, c ng knh si nh hn rt nhiu so vi si cellulose ca thc vt,

ng thi kh nng kt tinh cao (khong 60 %), polimer ha ln nn mng

BC c bn c hc cao, kh nng thm ht nc ln [18]. y l c tnh

u vit hn hn so vi cellulose thc vt. Khi quan st trn knh hin vi in

t thy ng knh ca si cellulose vi khun ch bng 1/100 ca si cellulose

thc vt.[35]

Do c nhng c im u vit nh trn m mng BC c ng dng

rt nhiu trong cc lnh vc cng ngh khc nhau. Mt trong cc lnh vc

ng lu l lnh vc y hc. Trong lnh vc ny, mng BC c ng dng

sn xut mng tr bng [22],

Si cellulose ca mng BC Si cellulose ca thc vt

Hnh 1.2. Si cellulose ca mng BC v si cellulose

ca thc vt [35]



1.2.2 C ch hnh thnh mng bacterial cellulose

*V tr tng hp

V tr tng hp cellulose ca t bo A.xylinum nm gia lp mng

lipopolysaccharide v lp mng sinh cht. [34]. Quan st di knh hin vi

in t khi nhum m bn v tiu bn cho thy: mi t bo c 46 im tng

hp cellulose. Khong cch gia mi im l 12 15nm, kch thc mi

im l 3,5 nm. Khi t bo phn chia, cc v tr tng hp c phn b u

vo hai t bo con [29]

*C ch tng hp

Qu trnh sinh tng hp BC l mt tin trnh bao gm nhiu bc c

iu ho mt cch chuyn bit v chnh xc bng mt h thng cha nhiu

loi enzyme, phc hp xc tc v cc protein iu ho [11], [12].

Qu trnh tng hp c din ra nh sau:

Ti cc im tng hp cellulose, nh c h thng cc enzyme xc tc

m cc phn t ng c chuyn ha thnh cc chui - 1,4

glucopyranose. Vi khun y di cc chui -1,4 glucopyranose t mt u,

cc si ny gi l si y di ( H1.3 1 2 3 ). Ti mt s im tng

hp cellulose khc nhau cng hnh thnh cc chui - 1,4 glucopyranose.

Cc chui mi hnh thnh lin kt vi nhau v vi si y di to thnh si

nh (subfibril) c kch thc 1,5 nm ( hnh 1.3 4). Nhng si nh kt tinh

to si ln hn si v m, tip tc nhng si v m li kt hp vi nhau

to thnh b v cui cng to di ribbon ( H 1.3 5, H 1.3 6, H 1.3 7)



Kch thc ca di ribbon cn nhiu kin cha ng nht, n c th

t t 3-4 x 70-80 nm [33], 3,2x133 nm (Brown v cs, 2000). Nhng di

ribbon t t bo ny khi y ra ngoi s lin kt vi nhng di ribbon khc

bng lin kt hydro hoc lin kt Van de Van to thnh dng st (gel) hay

mt lp mng mng. Kch thc bn ca mng tng ln khi qun th vi khun

sinh trng. Nhng im to mng BC mi c th xut hin, chnh v th m

mng BC c m rng [12], [19]

1.3. Tnh hnh nghin cu Acetobacter xylinum trn th gii v Vit

Nam

1.3.1 Tnh hnh nghin cu Acetobacter xylinum trn th gii.

Do c nhiu ng dng nn A.xylinum thu ht c rt nhiu s quan

tm ca cc nh khoa hc trn th gii v c rt nhiu cng trnh nghin

cu ln nh khp cc quc gia.

Hnh 1.3 S hnh thnh nhng di ribbon trong mng BC ca A.xylinum [22]



Nhng nghin cu v A.xylinum trn th gii tp trung theo hai hng

c bn:

*Hng th nht: ch yu l phn lp, tuyn chn, nghin cu cc c

tnh sinh hc ca A.xylinum t xc nh v tr phn loi ca chng trong

sinh gii.

Tiu biu l cc nghin cu ca Beijerinck [15], Prateur [23] .... trn

c s nghin cu cc c tnh hnh thi, nui cy, sinh l-sinh ha. Cc tc

gi xc nh ngun gc, c im phn loi ca A.xylinum.

Theo cc nghin cu ny, A.xylinum c xp vo nhng nhm phn

loi khc nhau. C quan im cho rng: A.xylinum thuc chi Acetobacter, h

Pseudomonadaceae, b Pseudomonadales, lp Schizomycetes; c nhng quan

im li coi Acetobacter xylinum nh mt loi ph ca Acetobacter aceti,

thuc chi Acetobacter v c nhm vo nhng chi khng r ngun gc. Gn

y nht l quan im xp A.xylinum vo chi Gluconacetobacter, h

Acetobacteraceae. Vn ny cn gy nhiu tranh ci. V th mc d l

hng nghin cu truyn thng nhng ngy nay n vn thu ht c nhiu

nh khoa hc i theo hng ny.

*Hng th hai: Nghin cu v qu trnh sinh tng hp, c im

cng nh ng dng ca bacterial cellulose vi khun A.xylinum.

y l hng nghin cu ch yu nht. Cc nghin cu trong hng

ny khai thc nhng kha cnh khc nhau ca qu trnh tng hp, c im

cng nh ng dng ca bacterial cellulose vi khun A.xylinum.

in hnh l mt s cng trnh nghin cu ca mt s tc gi nh: M.

Schramm v cng s [24].[30],[31] nghin cu v nh hng ca iu kin

nui cy n kh nng hnh thnh mng BC; Neelobon Suwannapinunt v

cng s (2007) nghin cu c tnh vt l ca mng BC [28]; nghin cu v

gen sinh tng hp cellulose A.xylinum c cng trnh nghin cu ca cc tc



gi nh C. Wong v cng s (1990) [32], Dag H. Coucheron (1991; 1993)

[20], [21].

1.3.2 Tnh hnh nghin cu Acetobacter xylinum Vit Nam

Vit Nam nhng vn nghin cu v A.xylinum v mng BC trong

iu tr bng l kh mi m. C rt t cc nghin cu lin quan n A.xylinum,

s hnh thnh mng v ng dng ca mng. Cc cng trnh mi ch bc u

nghin cu v qu trnh to mng, c tnh, cu trc mng lm c s ch to

mng tr bng : nhm nghin cu ca PGS.TS Nguyn Vn Thanh v ThS.

Hunh Th Ngc Lan (2006) tuyn chn chng c kh nng to mng tr

bng c mu trng vi dy 0,5mm v c kch thc khc nhau ty theo

yu cu s dng; nhm nghin cu ca PGS.TS inh Th Kim Nhung khi tin

hnh kho st kh nng to mng ca vi khun Acetobacter xylinum phn

lp tuyn chn c 65 chng vi khun trong c chng BHN2 c trin

vng ng dng tt.

Ngoi ra cn c cc nghin cu ca cc tc gi nh:

Nguyn Thy Hng (2006) nghin cu chn lc dng A.xylinum

thch hp cho cc loi mi trng dng trong sn xut cellulose vi khun vi

quy m ln.

Nguyn Th Nguyt (2008) nghin cu vi khun A.xylinum cho

mng Bacterial cellulose lm mt n dng da,

Nguyn Th Thy Vn (2009) nghin cu c tnh sinh hc v kh

nng to mng Bacterial cellulose ca vi khun A.xylinum v phn lp c

14 chng t mt s ngun nguyn liu Vit Nam.

Mng Bacterial Celullose c vai tr quan trng trong iu tr bng.

Chnh v vy vic nghin cu cc chng c kh nng to mng BC tt nht

p ng c yu cu trong iu tr bng s c ngha thc tin rt ln.



CHNG 2. VT LIU V PHNG PHP NGHIN

CU

2.1. Vt liu v thit b

2.1.1. Vi sinh vt

- i tng nghin cu l cc chng vi khun phn lp t cc mu

mng ln men gim t cc ngun: Bia hi H Ni, dch qu (chui), ru

vang, ngun v c (saccharose v ethanol).

- Chng BHN2 do PTN Vi sinh trng i hc S phm H Ni 2 cung

cp.

2.1.2. Thit b v ho cht

2.1.2.1. Thit b

T m, t sy Binder (c)

Ni hp Tommy (nht)

Box v trng (Haraeus)

My lc TEIO TECH (Hn Quc)

My li tm Sorvall (M)

Cn (Precisa XT 320M - Thu S)

Knh hin vi quang hc LABOMED ( M)

T lnh Electrolux,

Hp lng, ng nghim, bnh tam gic, que trang, lamen, n cn...v

nhiu dng c ho sinh thng dng khc.

2.1.2.2. Ho cht

- Glucose (Cty dc trung ng MEDPLANTEX H Ni Hi

Phng), Saccharose.

- Agar ( Cty TNHH Hi Long Hi Phng)

- Ru ethanol, acid acetic ( Cty ha cht c Giang - H Ni)



- Dihyroxyaceton, Blue Bromophenol 0.04% (xut x: Trung Quc)

-,Pepton, (NH4)2SO4, NaNO3, NaNO2, H2SO4 (xut x: Trung Quc)

- KH2PO4, CaCO3, MgSO4.7H2O, NaCl, NaOH (xut x: Trung Quc)

- Lugol, Fucshin, Tm Gentian, Phenolphtalein (xut x: Trung Quc)

2.1.3. Mi trng

2.1.3.1. Mi trng gi ging (MT1)

Glucose: 20 g (NH4)2SO4 : 3g

Pepton: 5g Thch agar: 20g

MgSO4.7H2O: 2g KH2PO4: 2g

Nc my : 1000ml

2.1.3.2. Mi trng nhn ging (MT2)

Glucose: 20 g (NH4)2SO4: 3g

KH2PO4: 2 g MgSO4.7H2O: 2g

pH: 5,0 6,0 Acid acetic : 2%

Nc da: 1000 ml

2.1.3.3. Mi trng ln men (MT3) Glucose: 20 g (NH4)2SO4: 3g

KH2PO4: 2 g MgSO4.7H2O: 2g

pH: 5,0 6,0 Acid acetic : 2%

Nc da: 1000 ml

2.2. Phng php nghin cu

2.2.1. Phng php phn lp vi khun Acetobacter

Phn lp vi khun theo phng php ca Winogradski v Beijerinck

[11] Phn lp sau khi lm giu.

Ngun vt liu: Mu mng ln men gim t nhin t cc ngun bia hi

H Ni, ru vang, dch qu (chui), ngun v c (saccharose v ethanol).



Tin hnh phn lp: Vt mu mng c ra qua cho vo ng

nghim cha nc ct thanh trng. Lc u bng my vntex trong 10 pht

thu c huyn ph vi sinh vt. Pha long dch huyn ph cc nng khc

nhau t 10-1 n 10-12. Dng pipet ht 50l mu cc pha long 10-3 n

10-12 nh ln mt mi trng thch v trng trong hp Petri, dng que chang

chang u. Sau trong t m nhit thch hp trong 5-7 ngy. Da vo

s khc nhau ca hnh thi khun lc tin hnh tch mt s khun lc c

trng ra khi mi trng phn lp, cy thun sau chuyn sang thch

nghing bo qun trong t 40C. tin cho vic tuyn chn, chng ti tm

gi mi khun lc c tch trn l mt chng vi khun acetic.

2.2.2. Phng php nghin cu c im hnh thi nui cy ca cc

chng A.xylinum

2.2.2.1. Phng php quan st hnh thi t bo trn knh hin vi

Lm tiu bn cc chng vi khun theo phng php nhum Gram. Sau

soi tiu bn di vt knh du ca knh hin vi quang hc Olympus CH-2

vi phng i 1000 ln.

2.2.2.2. Phng php quan st hnh thi khun lc [1]

Cy cc chng vi khun nghin cu ln cc hp petri c cha mi

trng kh trng, nui trong t m nhit 300C. Sau 7 ngy quan st

khun lc ca cc chng. Khi miu t khun lc cn ghi nhn cc c im

sau y:

- Hnh dng khun lc: Trn, dng amip, dng r cy, dng khng u...

- c tnh quang hc: Trong, bn trong, khng trong, lng lnh, c, hunh

quang.

- Mu sc: Ghi mu sc khun lc v xem c tit sc t vo mi trng hay

khng

- B mt: Bng, x x, gp np, g gh.



- Cu trc khun lc: ng nht, dng ht nh, dng ht ln...

2.2.3. Phng php nghin cu c im sinh l - sinh ho ca cc chng

Acetobacter xylinum

2.2.3.1. Phng php kim tra hot tnh catalase [11]

Nh mt git H2O2 3% ln b mt khun lc, nu thy hin tng si

bt kh th chng vi khun c coi l c hot tnh catalase (catalase +).

Ngc li, chng vi khun khng c hot tnh catalase (catalase -).

2.2.3.2. Phng php kim tra kh nng oxy ho ru ethanol thnh

acid acetic [11]

Nui cy chng vi khun tuyn chn trn mi trng bao gm:

Cao nm men: 10g Ru ethanol : 10%-15% (vol/vol)

Nc my: 1000ml pH : 6,8-7.0

Xanh Bromophenol (BPB) 0,04%: 20ml

Phn phi mi trng vo cc ng nghim (4-5ml), kh trng 1210C

trong 20 pht, ngui khong 30oC cy vi khun nghin cu mi hot ho,

nui 7 ngy trong iu kin thch hp. Quan st thy mi trng chuyn sang

mu vng l dng tnh, khng i mu l m tnh.

2.2.3.3. Xc nh kh nng oxy ho acid acetic [11].

S dng mi trng sau kh kh nng oxy ho acid acetic ca vi

khun tuyn chn.

Thnh phn: Cao nm men: 10g Calcium acetate: 10g

Thch: 20g Nc my: 1000ml

pH : 7,0-7,2

Phn phi mi trng vo bnh tam gic em kh trng 121oC trong

20 pht, ngui khong 40-45oC thch a, cy vi khun tuyn chn mi

hot ho (18-24h) nui 6-7 ngy iu kin thch hp. Quan st hin tng:



Nu xung quanh khun lc c vng trng sa l phn ng dng tnh (acetate

b oxy ho, canxi gii phng ra to mu trng sa), nu khng l m tnh.

2.2.3.4. Phng php xc nh kh nng tng hp cellulose

Nui cy vi khun trn mi trng dch th nhit 28-30oC trong

vng 3-4 ngy, quan st s hnh thnh mng. Kim tra kh nng bt mu ca

mng bng cch nh ln dung dch Lugol v H2SO4 60% n chuyn ho

thnh mu xanh lam.

2.2.4. Phng php xc nh trng lng kh ca mng BC

Mu mng cn xc nh trng lng c lc ln giy lc. Giy c sy

1050C trong 2 gi n khi trng lng khng i v xc nh trng lng trc khi

lc. Giy lc cng vi mu c t trong a petri, lm kh trong t sy cng trong

1050C. Sau 2 gi ngui trong bnh ht m ri em cn li. Trng lng kh ca

mng l hiu s ca trng lng giy lc c v khng c mu mng.

2.2.5. Phng php xc nh kh nng thm ht nc ca mng BC

(g/100cm3)

- t mu mng nghin cu vo hp Petri sy nhit 400C trong 24 gi

[11]. Cn khi lng mng.

- Th mu mng ngp trong nc. Sau 12 gi thu mng, cn khi lng

mng.

- Lng nc ht c l hiu s ca khi lng mng sau 12 gi v khi

lng mng ban u.



CHNG 3. KT QU V THO LUN

3. 1. Phn lp chng Acetobacter xylinum

Nguyn liu c s dng phn lp vi khun A.xylinum gm 4 ngun

c chia lm 2 nhm:

- Nhm c nng ru cao: Ru vang, bia H Ni.

- Nhm c nng ng cao: chui, ngun v c (saccharose v

ethanol)

Qui trnh phn lp vi khun A.xylinum

Hnh 3.1 Qui trnh phn lp vi khun A.xylinum

1.Lm giu mu nguyn liu

2.Phn lp, thu cc chng vi khun t mu mng

7.Kim tra bn cht cellulose ca mng

6.Tuyn chn cc chng c kh nng to mng trn b mt mi trng

5.Tuyn chn cc chng c kh nng oxy ha acetate

4.Tuyn chn cc chng c kh nng oxy ha ethanol thnh acid

3.Tuyn chn cc chng c kh nng sinh acid



Khu 1 trong qui trnh, lm giu mu nguyn liu l cho cc vi

khun trong khng kh c iu kin pht trin, sinh sn, tng s lng trn

mi trng cha nguyn liu ta chun b thu c nhiu t bo vi khun

dng cho cc khu phn lp, tuyn chn tip theo. Mc tiu ca cc khu 2, 3,

4, 5 l phn lp, thu cc chng thuc chi Acetobacter, loi b cc chng thuc

chi Gluconobacter v khu 6,7 l tuyn chn cc chng A.xylinum t cc

chng thuc chi Acetobacter. C th:

3.1.1. Lm giu mu nguyn liu

Bc 1: X l nguyn liu

- Bia, ru vang c pha long; chui c ct nh hoc xay nhuyn

tu loi.

- Nguyn liu sau khi x l b sung nc vi lng thch hp.

Bc 2: Ln men etylic

Ln men etylic dch nguyn liu nh nm men c sn trn b mt nguyn

liu. Nguyn liu c cho vo bnh y cht t 5 n 7 ngy. Sau m h

ming ui kh CO2 1ln/ngy ri y cht.

Bc 3: Ln men acetic

Sau 8 ngy b np bnh, dng vi mn y kn ming bnh nhit

thch hp nhng v tr khc nhau. Sau khong 5 n 7 ngy trn b mt mi

trng xut hin mt lp mng dy, mng khc nhau.



Chui Bia H Ni

Hnh 3. 2. Mt s mu mng thu c sau khi lm giu nguyn liu

3.1.2. Phn lp vi khun Acetobacter

Bc 1: Vt mu mng, ra qua bng nc ct. Tin hnh phn lp vi

khun Acetobacter trn mi trng 1 theo phng php ca Winogradski 1,

[2].

Kt qu phn lp c 14 chng, trong :

- 3 chng t ru vang k hiu R1, R2, R3.

- 5 chng t bia H Ni k hiu B1, B2, B3, B4, B5

- 4 chng t chui k hiu C1, C2, C3, C4

- 2 chng t ngun v c k hiu VC1, VC2

Nhn thy nhm vi khun acetic c kh nng hnh thnh mng trn mi

trng dch th, do khi vt mu mng ra bng nc ct v trng loi

b phn ln vi sinh vt khng mong mun 11.Mt khc mi trng 1 dng

phn lp c acid ho bng acid acetic cng gp phn c ch s pht

trin ca cc chng vi sinh vt khc 11.

Bc 2: T cc chng phn lp c tip tc s tuyn Acetobacter

bng cch cy thun trn mi trng 2 c b sung thm CaCO3 (7g/l). Cc

chng Acetobacter c kh nng sinh acid acetic, acid ny phn gii CaCO3



trong mi trng do xung quanh khun lc xut hin vng sng nh trong

sut. Cy cc chng phn lp c ln mi trng 2, sau 5-7 ngy chn

nhng chng c kh nng hnh thnh vng phn gii canxi xung quanh khun

lc ( kt qu thu c 11 chng c kh nng phn gii CaCO3 l: R1, R2, R3,

C1, C2, B1, B3, B4, B5, VC1, VC2 )

Bc ny cha th khng nh c cc chng phn lp c ny l vi

khun acetic. Chnh v vy, chng ti tin hnh kim tra kh nng oxy ha

ethanol thnh acid acetic. (bc 3)

Bc 3: T cc chng chn c bc 2 tip tc tin hnh tuyn

chn Acetobacter theo phng php ca Carr 11.

Theo Bergey 11 vi khun acetic thuc h Acetobacteriaceae gm 2

chi l Acetobacter v Gluconobacter. C hai chi u c kh nng oxy ho

ru ethanol thnh acid acetic. Acid acetic lm mi trng c cha Blue

Bromophenol 0.04% chuyn t mu xanh lc sang mu vng (hnh 3.3). Cc

chng thuc chi Acetobacter c kh nng phn gii tip tc acid acid acetic

thnh CO2 v H2O phc hi mu xanh ca mi trng. Ngc li, cc chng

thuc chi Gluconobacter khng c kh nng phn gii acid acetic nn mi

trng vn gi mu vng.

Hnh 3. 3 Chuyn ha ethanol thnh acid acetic ca vi khun acetic



Hu ht cc chng vi khun phn lp c u c kh nng oxy

ho ru ethanol thnh acid acetic (hnh 3.3). Kh nng ny mnh yu khc

nhau tu chng. Hn na cc chng phn lp c gm 2 nhm c tc

sinh trng khc nhau (nhm sinh trng nhanh v nhm sinh trng chm)

do vic theo di kh nng phc hi mu xanh ca mi trng phn bit

2 chi Acetobacter v Gluconobacter rt kh khn, thiu chnh xc. V th, sau

bc 3 la chn cc chng lm mi trng chuyn mu vng v tip tc kho

st kh nng oxy ho acetate. ( Kt qu thu c 9 chng c kh nng lm

mi trng chuyn mu vng l: R1, R2, R3, B1, B3, B4, B5, VC1, C1 )

Bc 4: Cy cc chng tuyn chn bc 3 ln mi trng c

cha canxi acetate (phng php 2.2.3.3). Theo Bergey, c 2 chi Acetobacter

v Glucobacter u c kh nng oxy ho lactate nhng ch c cc vi khun

thuc chi Acetobacter mi c kh nng oxy ho acetate. V th trong mi

trng c cha canxi acetate, cc chng vi khun thuc chi Acetobacter c

kh nng s dng mui acetate lm ngun ccbon hay ni cch khc chng c

kh nng oxy ho acid acetic thnh CO2 v H2O; gii phng ATP cung cp

cho cc hot ng sng ca t bo ng thi gii phng Ca2+ to vng phn

gii xung quanh khun lc 11 theo phng trnh sau:

Ca(CH3COO)2 + O2 CaCO3 + CO2 + H2O

Kt qu thu c 8 chng vi khun thuc chi Acetobacter, h

Acetobacteriaceae trong s 9 chng vi khun phn lp trn. C th l:

R1, R2, R3, B1, B4, B5, VC1, C1.



3.1.3. Tuyn chn cc chng A.xylinum to mng Bacterial Celullose (BC)

Chi Acetobacter bao gm nhiu loi khc nhau nh Acetobacter

aceti, Acetobacter kiitzingianum, Acetobacter linderni, Acetobacter acetosum,

Acetobacter suboxydans...11. Trong c mt s loi cng hnh thnh lp

mng hoc lp vng trn b mt mi trng lng trong iu kin nui cy

tnh, lp mng ny rt d nhm vi mng Bacterial Cellulose do vi khun

Acetobacter xylinum to ra. V d nh Acetobacter schiitzenbachii c kh

nng to vng dy khng bn vng; Acetobacter kiitzingianum to mng xp

np to trn cc mi trng lng; Acetobacter pasteurianum to vng kh v

nhn nheo...11 V th tuyn chn c cc chng Acetobacter xylinum c

kh nng to mng BC t cc chng Acetobacter phn lp c trn cn

tin hnh theo cc bc nh sau:

Bc 1: Kho st kh nng to mng BC ca cc chng Acetobacter

phn lp trn mi trng 3. Tin hnh nui cy 8 chng vi khun Acetobacter

trong mi trng dch th nhit 300C v theo di kh nng to mng BC

ca cc chng. Kt qu c 6 chng hnh thnh mng trn mi trng lng vi

nhng tnh cht, c im v thi gian hnh thnh mng khc nhau l: R1, B1,

B4, B5, VC1, C1.



Bc 2: Kim tra bn cht cellulose ca mng theo phng php

2.2.3.4: Nh lugol v H2SO4 60% vo mng to trn mi trng dch th ca

6 chng vi khun trn. Nu mng c bn cht l cellulose n s chuyn ho

thnh mu xanh (phn ng ca hemicellulose).

Hnh 3. 5. Kh nng to cellulose ca cc chng

A.xylinum

Hnh 3. 4. Mt s loi mng do cc chng vi khun hnh thnh

trn b mt mi trng dch th

Mng Vng mng

Nhn, d v Mng dai



Kt qu trong 6 chng Acetobacter c 3 chng c kh nng hnh

thnh mng BC. C th:

- Ru vang: R1

- Bia H Ni: B4.

- Ngun v c : VC1

Ngoi ra, kim tra thm mt s c tnh sinh ho khc ca 3 chng

Acetobacter ni trn nh hot tnh catalase, chuyn ho glycerol thnh

dihydroxyaceton, kh nng sinh sc t nu... 11 c th tm khng nh cc

chng ny u l Acetobacter xylinum.

3. 2. Tuyn chn cc chng c kh nng to mng BC dai, mng

Chng ti s dng mi trng 3 kho st kh nng to mng ca 3

chng: y l mi trng dng nc da thay th cho nc thng l mi

trng sinh dng rt thch hp cho s sinh trng cng nh kh nng to

mng ca cc chng Acetobacter xylinum v n cha nhiu yu t quan trng

cho s sinh trng ca t bo (vitamin, acid amin v cc nhn t khong).

Nui cy cc chng R1, B4, VC1 trn mi trng dch th c cha

nc da (MT3) nhit phng (ma h), quan st kh nng to mng ca

cc chng. Sau 5 ngy quan st mt s c tnh ca mng. Kt qu thu c

bng 3.1.

Hnh 3. 6. Hot tnh catalase



S hnh thnh mng BC ca 3 chng vi khun ny l khc nhau. Mng

BC ca 3 chng c dy mng v c tnh rt khc nhau. Trong thc tin,

chng c dng vi nhiu mc ch khc nhau. Song mc ch ca chng

ti l nghin cu mng BC trong iu tr bng, cn la chn c loi mng

c mng v dai tt nht ph hp vi vic iu tr. Chnh v th loi mng

do chng VC1 to ra s khng c la chn. Ch cn B4 v R1 c dng

cho cc nghin cu tip theo. Hai chng ny em so snh vi BHN2 th hiu

qu to mng v c im mng gn ging nhau.

Bng 3. 1. Mt s c tnh ca mng BC

Mng

BC

Khi

lng

(g/l) (**)

dy

(mm)

Kh nng

thm nc

(g/100cm3)

c im mng

VC1 5,90 0,6 0,7 10,01 Mng mng, d rch, mng

hnh thnh t ngy th 3

B4 3,67 0,8 - 1,3 11,03

Mng mng, dai, b mt

nhn m, mng hnh thnh t

ngy th 3

R1 4,50 1,2 - 1,5 12,40 Mng kh dy, dai, mng

hnh thnh t ngy th 2

BHN2 6,80 2,0 - 4,0 14,06 Mng dy, dai, nhn, mng

hnh thnh t ngy th 2

(**) Khi lng kh trung bnh ca mng BC



Hnh 3. 7 Cc loi mng bacterial cellulose (BC)

B4

R1 VC1

BHN2



3. 3. Nghin cu mt s c tnh sinh hc ca cc chng Acetobacter

xylinum tuyn chn

3.3.1 Nghin cu c im hnh thi t bo hc ca cc chng Acetobacter

xylinum

Quan st hnh dng t bo ca 2 chng A.xylinum phn lp trn

knh hin vi LABOMED vi phng i 100 ln. Kt qu nghin cu cho

thy hnh dng t bo ca 2 chng vi khun nh sau:

Nhn thy, cc chng B4, V1 v BHN2 u c hnh que ng ring r

hoc xp thnh tng chui, khng di ng, bt mu hng ca Fucshin. Mt

khc, cc t bo vi khun di, c kch thc khong 1 - 2m. iu ny c th

gii thch do chng cng thuc loi Acetobacter.

B4

Hnh 3. 8. Hnh dng t bo ca cc chng vi khun A.xylinum

R1

BHN2



3.3.2. c im nui cy ca cc chng Acetobacter xylinum

Khi pht trin trn b mt cc mi trng c th vi sinh vt ni chung

v vi khun ni ring s hnh thnh cc khun lc c trng ca loi . V

vy vic miu t khun lc l mt trong nhng vic cn thit khi nghin cu

c tnh sinh hc ca cc chng vi khun. c im ca khun lc ca 2

chng c trnh by trong bng sau:

Bng 3. 2. c tnh khun lc vi khun Acetobacter xylinum

c im khun lc

Hnh dng c tnh

quang hc Mu sc B mt Cu trc

B4 Trn c

vnh c Vng sm Bng

Dng ht nh

R1 Trn Bn trong Vng sm Bng Dng ht

nh

BHN2 Trn c

vnh c Vng sm Bng

Dng ht ln

Hnh 3. 9 Khun lc vi khun A.xylinum

CHNG R1

CHNG B4 CHNG BHN2



Khi sinh trng trn b mt mi trng c 2 chng vi khun

A.xylinum phn lp hnh thnh cc khun lc khc nhau: khun lc ca

chng B4 c dng ht ln, trn c vnh, mu vng sm v b mt nhn bng.

Chng R1 khun lc li c dng ht nh, trn, mu vng sm v b mt nhn

bng. Tc sinh trng ca 2 chng ny cng khc nhau: R1 (5- 7 ngy),

B4 (3- 4 ngy). Nguyn nhn ca s khc nhau ny c th gii thch l do cc

chng nghin cu c phn lp t a dng cc ngun vt liu khc nhau (R1

phn lp t ru vang, B4 phn lp t bia do vy chng B4 c c im

khun lc kh ging vi BHN2 ch khc l cu trc khun lc nh hn).

3.3.3. c im sinh l - sinh ho ca cc chng Acetobacter xylinum

3.3.3.1. Kh nng ng ho ngun cacbon ca cc chng Acetobacter

xylinum

Vi sinh vt c c trng bi kh nng s dng khng ging nhau cc

loi hydrat carbon v ru khc nhau. Khi nghin cu a s cc vi sinh vt d

dng cn phi xc nh nhng ngun hydrat carbon v ru no m bo

c cho loi vi sinh vt cn nghin cu pht trin v nhng bin i g s

xy ra trong mi trng khi chng pht trin 11.

kho st kh nng ng ho ngun cacbon ca cc chng

Acetobacter xylinum phn lp chng ti s dng cc ngun sau y:

Glucose, saccharose, ethanol. Tin hnh th nghim trn 2 chng Acetobacter

xylinum R1, B4. Nui cy 2 chng vi khun ni trn trong mi trng dch th

(mi trng 3) c thay i ngun carbon khc nhau. Nui trong iu kin tnh

nhit 300C, sau 3-4 ngy kim tra s sinh trng ca t bo vi khun, pH

mi trng sau ln men. Kt qu th nghim c trnh by bng 3.3.



Bng 3. 3. Kh nng ng ha ngun cacbon ca chng R1 v B4 so snh

vi chng BHN2

Chng Glucose Saccharose Ethanol

R1 + + +

B4 + + +

BHN2 + + +

(+) : sinh trng rt tt

Kt qu bng 3.3 cho thy kh nng ng ho ngun cacbon ca cc

chng nghin cu l ging nhau.

Theo di s bin i pH mi trng trc v sau ln men ta thy hu

ht cc chng u lm acid ho mi trng, pH mi trng sau ln men gim

do lng acid acetic tch lu trong dch ln men 11.

3.3.3.2. Kh nng ng ho ngun nit ca cc chng Acetobacter xylinum

Tip tc kho st kh nng ng ho ngun nit ca 2 chng A.xylinum

k trn.

Ngun Nit s dng bao gm:

+ Ngun nit hu c: pepton.

+ Ngun nit v c: Amon ( (NH4)2SO4); Nitrat ( NaNO3); Nitrit ( NaNO2).

Cy 2 chng nghin cu ln mi trng dch th (mi trng 3) c thay

i ngun nit khc nhau. Nui trong iu kin tnh nhit 300C. Sau 3-4

ngy kim tra s sinh trng ca t bo vi khun trong mi mu th nghim.

Kt qu th nghim c trnh by bng 3.4.



Bng 3.4. Kh nng ng ha ngun nit ca 2 chng R1 v B4 so snh

vi chng BHN2

Chng Pepton Amon Nitrat Nitrit

R1 ++ + - -

B4 ++ + + -

BHN2 ++ + + -

Ch thch: + + : Sinh trng rt tt

+ : Sinh trng yu

- : Khng sinh trng

Nhn thy 2 chng phn lp c v cng BHN2 u sinh trng rt tt

trong mi trng c cha nit hu c, sinh trng yu hn trong mi trng

c cha nit v c. Hu ht 2 chng nghin cu u khng c kh nng s

dng ngun nit l nitrit. Kt qu cho thy khng c s khc nhau nhiu v

kh nng s dng ngun nit ca 2 chng A.xylinum phn lp t cc ngun

vt liu khc nhau cng nh cc chng A.xylinum c hnh dng t bo v hnh

thi khun lc khc nhau.

3.3.4. nh hng ca nhit n kh nng sinh trng ca 2 chng R1

v B4

Tin hnh cy 2 chng trn trn mi trng thch nghng. Nui nhit

27 350C ( nhit phng), 200C ( iu ha), 450C (t m). ( mi chng

s dng 2 ng nghim / 1 nhit ). Sau 4 5 ngy ta ly ra quan st bng

mt thng mc pht trin ca cc chng. Sau li cy trn thch

nghing ln th 2, nguyn liu dng cy l cc t bo ca ln cy th nht.

Sau li nui cc nhit khc nhau k trn. Sau 4 5 ngy tip tc ly ra

quan st mc sinh trng bng mt thng. Theo di cc mc : khng



sinh trng, sinh trng yu, sinh trng tt. Kt qu th nghim c trnh

by bng 3.5

Bng 3. 5. nh hng ca nhit n kh nng sinh trng ca chng

R1 v B4 so vi BHN2

Chng 200C 27 - 350C 450C

R1 + ++ -

B4 - ++ -

BHN2 + ++ -

(-): khng sinh trng

(+): sinh trng yu

(++): sinh trng tt

Nhn thy 2 chng phn lp v chng BHN2 i chng trn u sinh

trng tt nhit phng (27- 350C), khng th sinh trng c nhit

450C. nhit 200C ch c chng R1 v BHN2 sinh trng c nhng vi

tc rt chm.

Nh vy, c th kt lun c: cc chng A.xylinum phn lp c

thuc loi a m, sinh trng tt nht trong khong nhit t 27- 350C.



KT LUN V KIN NGH

1. Kt lun - T 4 ngun nguyn liu phn lp c 6 chng c kh nng hnh

thnh mng trn mi trng lng.

- Trong 6 chng phn lp c 2 chng ( R1; B4) c kh nng hnh to

mng BC dai, mng.

- Hai chng ny c cc c im hnh thi: t bo c dng hnh que,

hoc hnh elip, ng c lp hay xp thnh tng chui. Khun lc c hnh

trn, mu vng sm, b mt nhn bng. Chng c th sinh trng tt trn mi

trng c ngun cacbon l glucose, saccharose , ethanol v ngun nit hu

c th sinh trng tt hn ngun nit v c. Chng c th sinh trng tt

nhit t 27 350C.

2. Kin ngh

- Tip tc nghin cu nh hng ca mi trng v iu kin nui cy

ti kh nng to mng BC ca hai chng R1 v B4.

- Nghin cu c im mng BC t 2 chng R1 v B4 bc u ng

dng ch to mng tr bng.



TI LIU THAM KHO

1. Nguyn Ln Dng (1976). Mt s phng php nghin cu vi sinh vt

hc, tp 1- 2- 3, Nh xut bn khoa hc k thut H Ni.

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1.1. V tr v c im phn loi ca Acetobacter xylinum trong sinh gii:31.1.1. V tr phn loi ca Acetobacter xylinum31.1.2. c im phn loi ca Acetobacter xylinum31.1. V tr v c im phn loi ca Acetobacter xylinum trong sinh gii:1.1.1. V tr phn loi ca Acetobacter xylinum1.1.2. c im phn loi ca Acetobacter xylinum