TRNG I HC CNG NGH SI GN

KHOA CNG NGH THC PHM

BI GING THC HNH H CAO NG

Nm hc 2010-2011

NI QUI PHNG TH NGHIM

Mt s vi sinh vt c s dng trong cc bi th nghim c th gy bnh cho ngi
v ng vt, v th cc ni qui c ban hnh ngn nga nguy c nhim bnh cho
sinh vin v cn b phng th nghim. Bt k c nhn no khng tun th tt cc ni qui
hay gy nguy hi cho ngi khc u khng c php vo phng th nghim. Khi c
bt k thc mc no cn phi yu cu s hng dn ca gio vin hoc cn b phng th
nghim.
1. Cc qui nh chung
+ Sinh vin vo phng th nghim phi mc trang phc bo h (o khoc trng) c
bng tn (th sinh vin)
+ Sinh vin phi tham d 100% cc bui th nghim
+ Sinh vin phi n ng gi, nu n tr qu 15 pht, sinh vin khng c php
vo phng th nghim v c xem nh vng mt khng l do
+ Nu v bt k l do bt kh khng no sinh vin khng tham d c bui th
nghim, sinh vin phi bo trc (hoc vo bui th nghim) cho cn b cc trch
nhim
+ Khi lm h hng cc trang thit b/dng c ca phng th nghim, sinh vin c
ngha v phi hon tr li
+ Sinh vin phi c k bi trc khi vo th nghim v khng c mang ti liu
th nghim vo phng
+ Khi lm /trn cc dung dch hoc lm b dng c thy tinh phi bo co cho
cn b phng th nghim v xin kin gii quyt.
+ Sinh vin phi nm vng cc thao tc v trng.
+ Gim thiu s hnh thnh kh dung khi thao tc.
+ Ra tay trc v sau khi th nghim.
+ Khng c n/ung/nghe nhc/c sch-bo trong phng th nghim.
+ c k cc ni qui/qui nh c ca phng th nghim.
+ V sinh bn/gh/k v cc dng c trc v sau khi th nghim.
+ b rc thi ng qui nh.
+ Khng ngm cc dng (vit, king) trong ming hay gn vo tai.
+ c v k tn vo cc qui nh/ni qui chc chn sinh vin c v hiu.
+ Tr y dng c sau khi hon thnh xong bi th nghim. Dng c phi c

ra sch.
+ V sinh phng th nghim theo yu cu ca ngi ph trch
2. Cc yu cu an ton
+ Ct tc, mc cc phc trang bo h (o khoc trng, gng tay chng nhit) v
dng dng c/thit b ng lc, ng ni.
+ Nghim cm dng ming ht pipette.
3. Trong cc tnh hung khn cp
+ Lu v tr cc trang b cp cu khi cn (dng c y t, bnh cu ha, vi nc,
in thoi v s in thoi cp cu).
+ Bo co cc tnh hung khn cp ngay lp tc cho gio vin hng dn hoc cn
b phng th nghim.
+ Bnh tnh khi c tnh hung khn cp.
PTN Cht lng Thc phm

PHN I: I CNG V VI SINH VT HC

Bi 1: CC THAO TC C BN TRONG PHNG TH
NGHIM VI SINH
1. Mi trng , pha ch v chun b mi trng
1.1. Mi trng nui cy vi sinh vt:
phn lp, nui cy hay bo qun ging vi sinh vt, ngi ta phi s dng cc
mi trng dinh dng c (hoc lng). Mi trng dinh dng khng ch cha cc
thnh phn cn cho s pht trin ca v sinh vt m cn phi m bo cc iu kin l
ha thch hp cho s trao i cht ca vi sinh vt vi mi trng bn ngoi.
V vy, thit lp mi trng cn phi bit r nhu cu ca vi sinh vt v cc cht
dinh dng v cc c im trao i cht ca chng. Cn lu l nng cc cht ha
tan trong mi trng phi cn bng p sut thm thu ca t bo vi sinh vt th mi m
bo c s pht trin ti u ca chng
Mi trng thng t tn theo ngi sng to ra chng (v d mi trng
Kzapek, mi trng Hansen) hay theo cc thnh phn dinh dng c trng ca mi
trng (v d mi trng dch trch gi u, khoai ty)
1.2. Nguyn tc pha ch mi trng:
Nguyn tc c bn nht pha ch mi trng l phi m bo cc nhu cu c bn
ca vi sinh vt (ngun C, N v cc khong). Ngoi ra cn c mt s nguyn tc sau:
a. Ty theo nhu cu nghin cu hay hc tp m pha ch mi trng ph hp:
- Nu mun nui cy vi sinh vt quan st hnh thi th phi nui cy trn
mi trng c
- Nu mun tm hiu s trao i cht ca vi sinh vt thi dng mi trng
lng
- Mi trng ch cha cao tht, pepton dng nui cy vi khun hoi sinh
b. Ty vo nhu cu dinh dng c bit ca vi sinh vt b sung thnh phn khc
nhau no :
- Cn nui cy vi sinh vt phn gii cellulose cn b sung cellulose vo mi
trng
- Cn nui cy vi sinh vt chuyn ha N, cn b sung cc hp cht cha N
vo mi trng
- Hay b sung cc khng sinh vo mi trng nhm nui cy cc vi sinh vt
c kh nng khng li khng sinh

1.3. Phn loi mi trng

i. Da vo thnh phn
- Mi trng t nhin: Cc loi mi trng l cc hp cht t nhin nh:
khoai ty, cm go, b khoai m, ph phm ch bin tht, dch sathnh
phn mi trng thng phc tp v khng n nh
- Mi trng tng hp: s dng cc loi ha cht hu c hoc v c tinh
khit pha ch mi trng vi t l chnh xc
- Mi trng bn tng hp: s dng c cc ha cht tinh khit ln cc thnh
phn t nhin.
ii. Da vo tnh cht vt l
- Mi trng lng
- Mi trng rn: thng c t 1,5-2% agar hoc gelatine
- Mi trng bn lng: c khong 0,3-0,7% agar
iii. Da vo cng dng:
- Mi trng c trng hay chn lc: mi trng c cha mt thnh phn
c bit ch ph hp vi 1 hoc 1 nhm vi sinh vt no . V d mi
trng dng phn lp vi sinh vt chuyn ha m, chuyn ha ln
- Mi trng kim nh: dng xc nh mt tnh cht no ca vi sinh
vt. Ngi ta thng b sung mt hp cht c bit c s bin i c th
nhn thy c trong qu trnh nui cy vi sinh vt nh cc cht ch th
mu.
1.4. Cch pha ch mi trng
Pha ch mi trng l mt khu quan trng do vy cn phi chnh xc. Gm cc
bc sau:
- Cn cc thnh phn mi trng
- Nu l mi trng lng: pha cc thnh phn vo nc, ch th t pha
ch
- Nu l mi trng c: ha tan cc thnh phn vo nc ri thm 1,5-2%
agar v un n khi agar tan chy.
- Lc: thng lc mi trng qua vi hoc bng
- Chnh pH: ty theo yu cu vi sinh vt hoc yu cu nghin cu, thng
iu chnh pH ph hp. Thng dng cc loi ha cht nh: H2SO4,
H3PO4, KOH, NaOH

- Phn phi vo dng c cha: nu l bnh cha th cho vo khong 2/3 th

tch bnh, nu l lm mi trng thch da th cho vo khong 10-15ml/da,
nu lm thch nghing th cho vo 1/4-1/5 chiu cao ng nghim.
- Tit trng mi trng: thng dng phng php ch yu l nhit m vi
p sut cao (121oC, 1atm) nhm tiu dit tt c cc bo t cng nh cc vi
sinh vt khng mong mun c sn trong mi trng .
Thc hin pha ch mi trng
Mi nhm thc hnh pha ch hai mi trng dng nui cy vi sinh vt nh
sau:
- Mi trng 1 (mi trng dch trch gi u):
+ Gi u: 200g
+ Glucose: 10g
+ Trypton: 5g
+ Yeast extract: 1g
+ Agar: 15g
+ H2O: 1000 ml
- Mi trng 2 (Mi trng dch trch khoai ty):
+ Khoai ty: 200g
+ Saccharose: 50g
+ Pepton: 5g
+ Yeast extract: 1g
+ Agar: 20g
+ H2O: 1000 ml
Cch thc hin: gi u hoc khoai ty un vi H2O si trong 20 pht, lc ly
dch trong, b sung cc thnh phn cn li. Sau tip tc un n khi agar tan hon
ton. Phn phi vo cc bnh cha v em tit trng 121oC trong 15 pht.
2. Cc dng c s dng trong vi sinh vt hc

2.1. Dng c dng cy chuyn

Cc loi dng c c s dng cy chuyn u nhm mc mc ch chuyn vi
sinh vt t mi trng c sang mt mi trng mi cho cc nhu cu khc nhau: cy
chuyn, nhn ging, phn lp vi sinh vt.

Tt c cc dng c dng cy chuyn u phi m bo c v trng bng nhiu

cch khc nhau, nhm trnh vic a vo mi trng nhng vi sinh vt khng mong
mun. thng s dng phng php tit trng bng nhit kh 140-150oC trong t nht 1
gi. Cc dng c k trn u ch yu dng chuyn i vi sinh vt t mt mi trng
lng sang mi trng khc. chuyn vi sinh vt t mi trng rn sang mi trng
khc, ngi ta ch yu s dng que cy vng/ thng hoc que cy mc.

Que cy thng (a) v que cy vng (b)

Khi s dng que cy, thng que cy s c tit trng trn ngn la n cn
hoc n Bunsel. Khi tit trng phi m bo u que cy hoc bt c thnh phn no
ca que cy phi c tit trng hon ton bng cch t nng .
2.2. Cc phng php phn lp v cy chuyn vi vi sinh vt:
Phn lp vi sinh vt l vic phn tch cc chng vi sinh vt trong mi trng t
nhin v c lp chng nhm chn la ging vi sinh vt thun khit cho nhng mc ch
khc nhau. phn lp vi sinh vt, ngi ta thng tin hnh nui cy vi sinh vt trn
cc mi trng chn lc nhm u tin s pht trin ca mt loi vi sinh vt no .
Nu nh khng c c mi trng c trng th thng ngi ta s nui vi sinh
trn cc mi trng rn v lm cch no tch ri cc t bo vi sinh vt v cho chng

pht trin ring r trn mi trng rn to thnh khun lc (colony) vi hnh dng c
trng v vic chn la s c tin hnh trn cc khun lc ny.

Mt s hnh dng khun lc mc trn mi trng rn: hng 1 (nhn t trn xung),
hng 2 (nhn ngang), hng 3 (dng ra khun lc)

Cc phng php cy

Cch cy trong ng thch nghing

Cy trn a mi trng thch bng que cy vng

Cy trn a mi trng thch bng que tri

Cc thao tc chnh khi cy v sinh vt

Cc khun lc mc ring r, d chn la

Thc hnh:
S dng mi trng pha ch phn trn, tin hnh lm cc dng mi trng
thch a, thch nghing v tin hnh cy mt s ging vi sinh vt do phng th nghim
cung cp. Nui nhit thch hp v nh gi kt qu sau 48 gi nui cy.

Bi 2:KNH HIN VI QUANG HC NN SNG

Mt knh hin vi tt l mt dng c rt quan trng trong phng th nghim vi sinh.
C nhiu loi knh hin vi khc nhau, trong loi thng dng nht l knh hin vi
quang hc nn sng (bright-field light microscope). Knh hin vi ny c nhiu thu knh
v c mt ngun nh sng trng. Chng phng i v chiu sng cc vt th nh b nh
vi khun v cc vi sinh vt khc m chng ta khng th nhn thy bng mt thng.
Knh hin vi loi ny s c chng ta s dng trong ton b mn hc ny. Nu s dng
thnh tho knh hin vi, sinh vin c th thu nhn c nhng thng tin chnh xc v
hu ch v cc tiu bn hoc mu nui cy vi sinh vt. Vic nghin cu trong phng th
nghim vi knh hin vi s gip cho chng ta c c nhng n tng su sc v cc
hnh thi rt nh b ca s sng, nhng hnh thi ch quan st c khi chng c
phng i nhiu ln.
1. Cc b phn ch yu ca knh hin vi quang hc nn sng v chc nng ca
chng
a. Hy nhn vo knh hin vi trc mt v so snh n vi hnh v. Knh hin vi c
t trn mt chn (base) vng chc v c mt b phn tay cm (arm) chng
ta c th di chuyn knh hin vi n v tr khc (Lu : khi cm hoc mang knh
hin vi i ni khc, lun phi s dng c hai tay; mt tay nm ly b phn tay
cm trong khi tay khc vo chn ). Khng bao gi c nhc knh ln bng
cch nm vo cc thu knh.
b. Hy nhn vo bn cha tiu bn, chng nm gia h cc thu knh bn trn v
mt b phn cung cp nh sng bn di. Bn cha tiu bn c mt l trn v
tr trung tm. N cho php nh sng t bn di i xuyn qua v n c cc
thu knh bn trn. Tiu bn cn quan st s c t trn bn cha tiu bn, ni
m c nh sng chiu t bn di ti. Lu n nm iu chnh bn cnh bn
cha tiu bn, chng dng di chuyn tiu bn qua tri/phi hoc trc/sau trn
bn cha tiu bn . Bn cha tiu bn loi ny gi l bn cha tiu bn c kh
(mechanical stage)
c. Mt n chiu c t trong chn . Anh sng s i xuyn qua t quang Abbe
(Abbe condenser). T quang c cha cc thu knh. Chng c tc dng tp trung
cc tia sng vo tiu bn. T quang c ca sp (iris diaphragm, shutter) dng
iu chnh lng nh sng. Mt thanh gt (rotating knob) c gn km theo
iu chnh ca sp. T quang c th c nng ln hay h xung nh mt nm

d.

e.

f.

g.
h.

iu chnh (adjustment knob). Khi h knh t quang xung s lm gim lng tia
sng chiu vo tiu bn (iu ny thng khng c thc hin khi nghin cu vi
sinh vt). Cch tt nht l gi t quang v tr cao nht v ch iu chnh lng
sng bng cch ng/m ca sp.
Pha trn bn cha tiu bn, c gn vi tay cm, l mt ng trn (tube) c cc
thu knh phng i. Bn di ng trn l mt c xoay (rotating nosepiece) c
gn vi ba hay bn vt knh (objective lenses). Khi xoay c xoay, mt trong s
cc vt knh s c t ng vo v tr l trn trn bn cha tiu bn. Bn trn
ng trn l th knh (ocular lens, eyepiece) (knh hin vi c th c mt th knh,
loi hai th knh cho php chng ta c th nhn bng c hai mt).
Ty loi knh hin vi ang s dng m c xoay v bn cha tiu bn c th c
nng ln hay h xung bng nm s cp (coarse adjustment knob) v nm th
cp (fine adjustment knob). Trong mt s loi knh hin vi, chng c t ti hai
v tr ring hoc s c t ci ny bn trn ci kia. Khi s dng phi vn nt s
cp mt cch nh nhng nng hoc h bn cha tiu bn. u tin, iu chnh
cho bn cha tiu bn c nng ln ti a gn st vi vt knh, ng thi phi
a mt nhn t pha bn ngoi trnh vic vt knh m thng tiu bn, t
lm hng vt knh. Nt th cp s lm di chuyn bn cha tiu bn mt cch rt
chm chp v vy khng th thy s di chuyn ny khi nhn bn ngoi. Ta s
dng nt th cp khi mt ang nhn vo th knh v iu chnh nh nhng
chnh r nt nh ang quan st.
Lu ch c h bn cha mu vt i xung khi a mt vo quan st th knh
trnh trng hp vt knh m thng tiu bn; ch khi ngi k thut vin
quan st t bn ngoi mi cho php nng bn cha tiu bn ln
Khi xoay nt th cp qu nhanh c th lm cho nt xoay b kt cng, khi
khng c c xoay tip m phi thng bo ngay cho gio vin hng dn.
Tng s ln phng i ty thuc vo vt knh v th knh ang dng. Nhn vo th
knh, sinh vin s thy mt k hiu 10X, c ngha l phng i 10 ln. Nhn vo
cc vt knh s thy k hiu 4X, 10X, 40X v 100X, tng ng vi
phng i 4, 10, 40, v 100 ln. Vt knh low-power cn c gi l vt knh 10x
hay 16mm. Vt knh high-dry (high-power) cn c gi l vt knh 40x hay
4mm. Vt knh du cn gi l vt knh 90x, 100x hay 1.8mm. Khi phng i
tng, kch thc ca u vt knh s nh dn v cho php t nh sng i qua.
l l do m sinh vin cn phi iu chnh v tr ca t quang v ca sp chn sng

khi dng cc vt knh khc nhau c th nhn tiu bn c r rng hn. T

quang tp trung nh sng ln mt vng nh bn trn bn cha tiu bn, cn ca
sp iu chnh lng sng i vo t quang. Khi s dng du soi knh, du soi knh
s c t v tr gia tiu bn v vt knh. Do du soi knh c tc dng khc x
ging nh thy tinh nn s gim thiu lng tia sng b mt i. Th knh c t
trn u ca mt ng kim loi s phng i nh c truyn t vt knh. Kt qu
l phng i chung nhn c s l tch s phng i ca vt knh vi
phng i ca th knh. V d, khi s dng th knh 10x v vt knh 43x th
phng i chung l 10 x 43 = 430 ln.

Knh hin vi quang hc nn sng

i. di tiu c (focal length) ca mt vt knh t l vi ng knh ca vt knh .

Lu , trc khi chnh mt vt knh sang v tr thng ng vi l trn trn bn cha
tiu bn, phi m bo vt knh s khng m thng tiu bn. Nu cha chc chc,
tt nht nn h bn cha tiu bn xung tn di cng trc khi chuyn sang s dng
mt vt knh khc.
j. S dng mt mnh vi mm v sch lau nh nhng cc thu knh v pha trn t
quang khi chng b bm bi.
k. Ch duy nht nhn vin phng th nghim mi c ly vt knh hay th knh ra khi
knh hin vi (v mt l do no ).
l. Ch nhng ngi hc cch s dng mi c dng knh hin vi.

nh sng truyn qua tiu bn, du soi

v vt knh

Cch cm di chuyn knh hin vi

2. Thc hnh trn knh hin vi vi tiu bn
a. S dng mt s tiu bn c sn hoc t lm.
b. t tiu bn ln bn cha tiu bn v kp li chc chn. t tiu bn v tr sao
cho tia sng i t t quang xuyn qua trung tm phn c nhum mu.
c. a vt knh low-power vo v tr thng ng v a bn cha tiu bn tht gn
vt knh. Lu : quan st t pha bn ngoi.
d. a mt vo th knh quan st. Nu s dng loi knh n trng (monocular
scope), sinh vin phi m c hai mt (sinh vin s lm quen dn vi vic ch tp

trung vo nh ang quan st trong knh hin vi thay v cc nh khc bn ngoi).

Nu s dng loi knh hai trng (binocular scope), sinh vin hiu chnh hai trng
knh qua li khi nhn vo th knh ch thy mt th trng duy nht. Phi m
bo t quang ang v tr cao nht v chnh ca sp sao cho nh sng xuyn qua
l va quan st. H bn cha mu vt xung t t bng nt s cp cho n
khi thy nhng vt th c mu xut hin trong th trng.
e. S dng nt th cp chnh cho nh r nt nht. Di chuyn tiu bn theo hng
ti/lui v tri/phi. Vt knh low-power cho mt ci nhn ton cnh v tiu bn v
gip sinh vin la chn mt th trng va . quan st r hn cn phi chuyn
sang mt phng i cao hn.
f. Khi la chn c th trng va , xoay vt knh high-dry vo v tr thng
ng. Nu sc nt ca nh cha t, sinh vin chnh li bng nt th cp. Nu
khng thy nh trong th trng, sinh vin hy nhn t bn ngoi ng thi quan
st v chnh cho vt knh gn st vo tiu bn (khng c chm vo tiu bn).
Sau nhn vo th knh, chnh cho bn cha tiu bn h xung t t, u tin
bng nt s cp, sau bng nt th cp cho n khi nh r nt nht. Lu : sinh
vin cn so snh v cu trc ca nh quan st c cc phng i khc nhau.
g. Di chuyn vt knh high-dry sang mt t th hi nghing mt cht ri nh mt
git du soi knh ln trn tiu bn. Sinh vin quan st t bn ngoi v chuyn vt
knh du sang v tr thng ng (trnh chm vo tiu bn). S dng nt th cp
chnh nh cho r nt.
h. Ghi nhn li nh quan st c bng cch v vo trong mt hnh trn mt s t
bo vi sinh vt m sinh vin nhn thy.
i. Sau khi hon tt vic quan st, ly tiu bn ra khi knh hin vi (khng c
du soi knh dnh vo vt knh high-dry)
3. Mt s vn gp phi khi s dng knh hin vi
Vn gp phi

Cch x l
Nng t quang ln

Khng nh sng
khi nhn vo th knh

M ca sp
Kim tra li tiu bn: t sai v tr

Bi hay si vi c nhn
thy trong th trng

Lau nh nhng vt knh v th knh

Gy ra bi cc bt kh trong du soi
knh; kim tra li tiu bn

Nhng ht nh di
chuyn trong mt th
trng m

Phi chn rng ang s dng vt knh du

ch khng phi l vt knh high-dry
m bo rng du soi knh ang
ngp trong vt knh

Bi 3: TIU BN GIT TREO, GIT P QUAN ST S DI

NG CA VI KHUN
Mt s vi khun khng di ng (non-motile) nhng trong mi trng lng chng
thng di chuyn hn lon, gi l chuyn ng Brown (Brownian movement). y l kt
qu chuyn ng ca cc phn t nc t ko cc vi khun chuyn ng theo.
S di chuyn tht s (ng lc t thn, self-propulsion) c thy mt s vi khun
nh mt s c ch khc nhau. Vi khun di chuyn nh tin mao (flagella motion). Cc
xon khun c cc lng mao xung quanh (axial fibrils) s di chuyn theo kiu xon c
(corkscrew-type motion) v kiu un khc (bending-type motion). Mt s vi khun khc
th trt nh nhng (gliding motion).
Cc kiu di ng hay khng di ng c th c quan st bng tiu bn git treo
(hanging drop slide). Tiu bn git treo cng dng quan st hnh dng vi khun
trng thi sng cng nh s sp xp ca cc t bo vi khun khi chng kt hp vi nhau
(xem hnh). Mt vng Vaseline xung quanh g ca ch lm (coverslip) s gip tiu bn
khng b kh.
Tiu bn git treo
1. S dng cc vi sinh vt sau trong th nghim
a. Saccharomyces cerevisiae
b. Lactobacillus acidophilus
c. Bacillus subtilis
d. Staphylococcus sp
e. Mt s chng khc
2. Dng que tm, v mt vng trn nh bng Vaseline xung quanh ch lm ca
phin knh. Khng nn dng nhiu Vaseline.
3. Sau khi lc tht k dung dch huyn ph vi sinh vt, dng que cy vng v
trng t mt git dung dch huyn ph ny ln gia phin knh hnh vung
(coverslip).
4. a phin knh hnh ch nht (depression slide) p ln phin knh hnh vung
(coverslip), sao cho ch lm ca phin knh hng xung di v git huyn ph
vi sinh vt nm lt vo ch lm. Nhn xung nh nhng hai phin knh gn
vo nhau.
5. Lt ngc hai phin knh li v t ln knh hin vi quan st vi vt knh du.

Cch lm tiu bn git treo

Bi 4: QUAN ST NM SI
Nm si (nm mc) thng c nhn din bng cc c im hnh thi c trng
quan st c di knh hin vi. T bo nm thng pht trin thnh h si gi l khun
ty th. Si nm c th c hoc khng c vch ngn. Khun ty mc ln trn b mt c
cht thng l nhng cu trc mang bo t. Cung mang bo t c th phn nhnh hoc
khng phn nhnh. Bo t v tnh ca nm si thng tp trung trong hai nhm: bo t
kn v bo t trn.
Hai ging nm si Aspergillus v Penicillium thuc lp nm bt ton
Deuteromycetes, khng c hnh thc sinh sn hu tnh. H si c vch ngn. Bo t vi
tnh l bo t trn.
Mucor v Rhizopus thuc nhm nm tip hp Zygomycetes. H si khng c vch
ngn v c th c r gi. Bo t v tnh l bo t kn. Bo t hu tnh l bo t tip hp.
Rhizopus c cung mang bo t v khng phn nhnh. Mucor c cung mang bo t
phn nhnh.

Aspergillus sp.

Mucor sp.

Rhizopus sp.

Qui trnh
+ S dng cc nm si sau trong th nghim: Mucor, Aspergillus, Rhizopus
+ Quan st hnh thi khun lc, mu sc si nm trn hp petri.
+ Dng mt ming bng keo trong, t mt c keo dnh ln khun lc nm mc.
Sau ly ra v p st vo phin knh sao cho bng dnh dnh cht vo phin
knh.
+ Quan st di knh hin vi

Bi 5: TO VT BI V NHUM N
Tiu bn git p em li rt nhiu thng tin nhng chng cng c nhng nhc im
nht nh. Vi sinh vt di chuyn trong dch lng bi chuyn ng Brown hay t chuyn
ng nn rt kh khn trong vic quan st. Chng ta c th thy c hnh dng v hot
ng ca vi sinh vt nhng khng th xc nh chnh xc hnh thi ca chng (c bit l
nhm vi khun). Do vy, xc nh chnh xc hnh thi ca vi sinh vt ngi ta thng
s dng phng php nhum mu (quan st vi sinh vt cht)
Vi khun c thnh t bo vng chc duy tr hnh dng ca mnh. V vy, chng ta
c th phn loi vi khun cn c theo hnh dng ca chng. Vi khun c ba dng c bn:
hnh cu (spherical, round), hnh que (hnh gy, rod) v hnh xon (spiraled). Vi khun
hnh cu gi l coccus (s nhiu l cocci). Vi khun hnh que gi l bacillus (s nhiu l
bacilli) hay ch n gin gi l rod. Vi khun c dng xon c ti thiu hai n ba ng
cong trn t bo c gi l spirillum (s nhiu l spirilla). Cc vi khun c t bo di
v ngon ngoo vi cc vng xon (lng hoc cht) c gi l spirochetes.
Cch thc m cc t bo to thnh nhm th c trng cho tng ging hoc loi vi
khun. Cc t bo ng thnh tng cp (diplococci), ng thnh chui (streptococci),
ng thnh tng cm (staphylococci), hoc ng thnh mt cm bn t bo (tetrads), v
i khi chng cng ng thnh tng t bo ring l.
Cc vi khun hnh que (bacilli) thng dng mt t bo ring l, nhng cng c khi
xut hin dng mt cp ni tip nhau (diplobacilli) hay ng thnh mt chui di
(streptobacilli). Mt s loi c khuynh hng ng thnh mt cm cc t bo hnh que
song song (palisade) hoc to thnh hnh ch V, X, Y khi chng nhn i v phn chia.
Mt s loi to thnh nhiu hnh dng v kch thc khc nhau (a hnh th,
pleomorphism).
Cc xon khun thng xut hin dng mt t bo ng ring l v thng khng
kt thnh nhm.
1. To vt bi v lm tiu bn nhum n
To vt bi vi khun (bacterial smear) l lm kh t bo vi khun trn phin knh.
Cc bc thc hin c bn l (1) vi khun c tri ln phin knh sao cho cc t bo
nm thnh mt lp mng, (2) t bo vi khun khng b ra tri trong qu trnh nhum v
(3) vi khun khng b bin dng.
S dng phng php nhum n s to s tng phn gia vi khun v cnh nn.
Phng php ny c dng thu thp thng tin v hnh dng, kch thc v s sp

xp ca t bo. Bc k tip l t phin knh ln gi, ph ln vt bi phm nhum, ch

mt vi giy. Cc phm nhum c bn thng dng l crystal violet (thi gian nhum 20
30 giy), carbolfuchsin (thi gian nhum 5 10 giy) hoc methylene blue (thi gian
nhum 1 pht).
To vt bi
i. Dng bt lng (khng xa) khi ch tn vi khun mt u phin knh. Dng
bng keo trong dn ln dng ch va ghi
ii. Lc k dung dch vi khun. Vi thao tc v trng chuyn 1 hoc hai vng cy
vi khun vo gia phin knh. Tri u trn din tch khong inch. Nu to
vt bi t mi trng rn th cho mt git nc vo gia phin knh v dng
que cy thng cho mt lng nh sinh khi vo git nc. Trn u sinh khi
vi git nc. Tri u trn din tch khong inch.
iii. H phin knh trn ngn la c nh v git cht vi khun.
Nhum n
i. t phin knh ln bn (hay ln gi).
ii. Nhum vi phm nhum (alkaline methylene blue trong 1~1.5 pht,
cabolfuchsin trong 5 ~10 giy, hoc crystal violet trong 20 ~30 giy)
iii. Ra tri thuc nhum bng nc trong vi giy.
iv. Thm kh bng giy thm. Khng c ch xt ln vt bi.
v. Quan st bng knh hin vi vi vt knh du
vi. C th s dng ba loi phm nhum khc nhau vi cng mt loi vi sinh vt
c s so snh.

To vt bi vi sinh vt

Hnh dng c bn v s sp xp ca cc t bo vi khun.

(a) Cocci. 1.Diplococci (cp); 2. Treptococci (chui); 3. Staphylococci (chm nho);

4.Tetrads (nhm 4 t bo). (b) Bacilli (hnh que). 1. Streptobacilli (chui); 2.
Palisades; hnh V, X v Y; 3. Bacilli to bo t (bo t nh, trn, rng, khng b
nhum mu, gia hay mt u ca t bo); 4. Mt bacillis a hnh th (chiu di v
rng khc nhau). (c) Xon khun. 1. Spirilla (hnh un cong hay xon ngn); 2.
Spirochetes (di, cun xon cht hay lng lo)

Vi khun c nhum bng Crystal Violet. (a) Bacillus subtilis (x1000). (b)
Spirillus volutans (x 1000). (c) Micrococcus luteus (x 1000)

Mt s hnh dng thng thng ca vi khun

Bi 6: NHUM GRAM
Vo nm 1884, Christian Gram, mt nh nghin cu bnh hc ngi an Mch,
khm ph ra mt phng php nhum vi sinh vt bng phm nhum pararosaniline.
Thng qua vic s dng theo trnh t hai loi phm nhum, c 2 mu khc nhau, ng ta
nhn thy vi khun c chia thnh 2 nhm. Nhm th nht gi mu phm nhum
u tin: crystal violet (nhm vi khun gram dng). Nhm th hai b mt mu phm
nhum u tin sau khi c ra bng mt dung dch ty mu ri c tip tc nhum
bng phm nhum th hai l safranin hay carbon fuchsin (nhm vi khun gram m).
Dung dch iodine c dng nh l mt cht cn mu (mordant) (c nhim v gn phm
nhum ln/vo mt cht bng cch kt hp vi phm nhum to phc khng tan)
sau ln nhum u tin.
C ch ca k thut nhum ny cha c hiu mt cch tng tn. Tuy nhin,
ngi ta vn cho rng l do c s khc bit v thnh phn ha sinh ca thnh t bo
vi khun. Thnh t bo vi khun Gram dng th c nhiu peptidoglycan (phc cht ca
protein- ng) v cc peptidoglycan ny c lin kt cht ch vi nhau t gip cho
t bo c th khng li cht ty mu. Thnh t bo vi khun Gram m c thnh phn
lipid nng cao cho nn chng c th ha tan trong cht kh mu (alcohol,
acetone,) v b ra tri cng vi crystal violet.
Nhum Gram l mt trong s cc cng c hu dng nht trong cc phng th nghim
vi sinh v c s dng rt thng xuyn. Phng php nhum Gram c ci bin
bng cch thay i nng phm nhum, thi gian nhum v thnh phn ca cht ty
mu. Phng php ci bin ca Hucker, c s dng trong bi th nghim ny, hin
nay ang c s dng rng ri. Vic la chn cht ty mu ty thuc vo thi gian
mong mun hon thnh cc bc nhum. S dng ethyl alcohol 95%, dng trong bi th
nghim ny, cho php sinh vin tp lm quen vi cht ty mu bi v n c tc dng rt
chm. Acetone l cht ty mu c tc dng nhanh nht trong khi hn hp (50:50) ca
ethyl alcohol 95% v acetone th mc trung bnh. Hn hp acetone-alcohol thng
c dng trong cc phng th nghim.
Vt liu v ha cht
Dung dch Crystal violet
+ 2 g Crystal violet ha tan trong 20 ml ethanol 95%
+ 0.8 g Ammoni oxalate ha tan trong 80 ml nc ct
+ Trn 2 dung dch trn li vi nhau, gi 48 gi ri lc. Bo qun trong l
sm mu, s dng trong vi thng

Dung dch Iodine

Ha tan 1 g Iodine trong 3 ~ 5 ml nc ct, thm 2 g KI. khuy cho tan
hon ton, thm nc ct cho 300 ml . Bo qun trong l sm mu
Dung dch ty mu
+ Ethanol 95% hoc hn hp gm 70ml ethanol 95% v 30 ml aceton
Dung dch Safranin
+ Chun b sn dung dch Safranin O 2.5%, trc khi dng pha vi nc ct
theo t l 1:5 (v/v) c dung dch 0.5%
Qui trnh
i. S dng cc vi sinh vt sau th nghim
- Staphylococcus sp.
- Bacillus subtilis
- Lactobacillus acidophilus
- E. coli.
- Streptobacillus sp.
ii. Chun b vt bi v c nh vi sinh vt
iii. Dng dung dch crystal violet ph ln vt bi. yn trong 1 pht
iv. nghing phin knh 45o, ra di vi nc cho tri ht phn thuc nhum d
(git cui cng chy khi phin knh khng cn mu)
v. Ph ln vt bi dung dch iodine. yn 1 pht
vi. nghing phin knh 45o, ty mu bng dung dch alcohol 95% cho n khi git
cui cng chy ra khi phin knh khng cn mu (thng thng khong 10 20
giy). y l bc quan trng nht. Nu b ty mu qu mc, mt s vi khun
Gram dng s b mt mu tm v s tr thnh Gram m sau khi nhum mu ln
th 2
vii. Ngay lp tc ra phin knh di vi nc
viii. Ph ln vt bi dung dch Safranin. yn 1 pht
ix. Ra di vi nc
x. Thm nh bng giy thm. H kh phin knh trc khi quan st di knh hin
vi
xi. S dng du soi knh v vt knh du quan st
Lu :
- Lun dng ging vi khun tr bi v mt s ging vi khun Gram dng gi
s mt kh nng gi mu ca phc cht violet-iodine v s tr thnh Gram m khi

quan st. V vy, ch nn s dng cc t bo nui cy trong vng 24 gi.

- Ngoi ra, cng c mt s chng vi khun l Gram dng yu.
- Cc t bo vi khun Gram dng c th xut hin di dng Gram m, nhng cc
t bo vi khun Gram m th khng bao gi xut hin dng dng Gram dng.
- Khng lm vt bi vi khun qu dy. Vt bi dy i hi thi gian ty mu lu
hn vt bi mng.
- Ty mu cho n khi git dung dch cui cng chy ra khi phin knh khng.
- Vi khun Gram dng c th xut hin di dng Gram m do kh mu bng
ethanol 95% qu mc, v vy cn m bo thi gian ra bng ethanol t 10 20
giy.
- Mt s sai st khc l: (a) que cy qu nng, (b) h nng qu mc khi c nh vi
sinh vt trn phin knh. H qu nng khi c nh vi khun ln phin knh c th
lm v t bo, t mu ca t bo Gram (+) c th b ra tri lm cho t bo
Gram (+) tr thnh Gram (-).
- Nu vt bi vi khun qu dy, thuc nhum thm vo cc t bo khng ng u.
- C th thay Safranin bng mt thuc nhum khc d phn bit vi mu tm.
- Dung dch Iod c th b phn hy nu lu
- Vi khun Gram (+) bt mu tm, Gram (-) bt mu hng

S bt mu ca t bo vi khun khi nhum Gram

Cu to thnh t bo vi khun Gram (+) v Gram (-)

Bi 7: NHUM BO T
Mt s vi khun (Clostridium botulinum, Bacillus subtilis) c kh nng to bo t
(spore, endospore). Bo t l mt th ngh c dng hnh cu hay hnh bu dc hnh thnh
bn trong t bo vo cui thi k sinh trng pht trin. V mi t bo ch sinh ra mt
bo t nn y khng phi l loi bo t c chc nng sinh sn nh nm si. Bo t
to thnh do mi trng khng thch hp cho s sinh trng v pht trin, do thiu cht
dinh dng, do m thp. Khi mi trng tr nn thch hp th bo t li hi sinh v
t bo li c th tip tc phn chia.
Bo t c tnh khng nhit, khng bc x, khng ha cht, khng p sut thm thu.
Bo t c th tn ti trong mi trng thiu cht dinh dng, nhit cao, ha cht m
cc t bo sinh dng khng th tn ti c.
Trong thi k ngh khng thy bo t vi khun th hin bt k s trao i cht no,
l trng thi sng n (cryptobiosis). Ch c mt s chi vi khun c kh nng sinh bo
t: Bacillus, Clostridium, Sporosarcina (G+), Deusulfotomaculum (G-)
Bo t rt kh nhum bi a s cc thuc nhum do mng bo t dy, chc, kh bt
mu v cha nhiu lipid. V th, cn thit phi c nhng phng php nhum c bit
i vi bo t. Vi bt k phng php no, t bo phi c x l nhit acid t
bo cht ca bo t d bt mu. Sau nhum t bo cht ca bo t v t bo vi thuc
nhum c hot tnh mnh ri ty mu ca t bo cht i v nhum n vi mt thuc
nhum phn bit khc. Khi , t bo cht s mang mt mu, bo t s mang mt mu
khc. i khi bo t c nhn thy bn trong t bo. Hnh thi ca n trong t bo v
kch thc b ngang ca t bo mang n. Bo t thng nm trong t bo sinh dng
theo ba v tr:
+ Nm tm t bo: gi l bo t kiu Bacillus
+ Nm lch tm: gi l bo t kiu Clostridium
+ Nu nm cc t bo: gi l bo t kiu Plectidium
+ Cc bo t c th tn ti t do do bi t bo xung quanh n tan r.
1. Nhum bng phng php Carbolic Fuchsin
Ha cht
Dung dch A
+ 10 ml dung dch Fuchsin kim bo ha trong ethanol (khong 10%)
+ 100 ml dung dch acid carbolic (phenol 5% trong nc)
+ Trn u vi nhau (chun b trc khi dng)

Dung dch B
+ 100 ml Ethanol 95%
+ 3 ml HCl m c
Dung dch C
+ 30 ml Methylen blue bo ha trong ethanol (khong 2%)
+ 100 ml dung dch KOH 0.01% trong nc
+ Trn u vi nhau, cng lu cng tt
Qui trnh nhum bo t
i. Lm vt bi trn phin knh
ii. Nh dung dch A ln vt bi, h nh bn di phin knh lm bay hi,
trnh si. Thm dn dn thuc nhum khng b kh cn, gi trong 5
pht. i ngui, thuc nhum i.
iii. Dng dung dch B ra li cho n khi thy va ht mu , ra nc.
iv. Nhum li bng dung dch C trong 2 ~ 3 pht, ra nc, thm kh.
v. Soi knh: dng vt knh du x100. Bo t bt mu , t bo bt mu xanh.
2. Nhum bng phng php Malachite green (phng php Schaeffer-Fulton
hoc phng php Wirtz-Conklin)
Ho cht
- Dung dch Malachite green bo ha (khong 7.6%)
- Dung dch Safranin 0.5%
Qui trnh nhum
i. Lm vt bi v c nh t bo bng nhit
ii. t phin knh ln trn cc nc si. Phin knh c y bng mt t giy
thm c cng kch thc
iii. Tm t t giy thm bng dung dch Malachite green. yn 5~6 pht.
ng t giy thm b kh
iv. Bc t giy thm ra, cho phin knh ngui hon ton. Ra phin knh bng
nc trong 30 giy
v. Nhum vi Safranin trong 90 giy
vi. Ra phin knh bng nc trong 30 giy

Bo t v s hnh thnh ni bo t vi khun

Qui trnh nhum bo t theo phng php Schaeffer-Fulton

PHN 2: CC CH TIU VI SINH VT TRONG

THC PHM
Bi 1: XC NH TNG S VI KHUN HIU KH
1. Nguyn tc:
Vi khun hiu kh l nhng vi khun tng trng v hnh thnh khun lc trong
iu kin c s hin din ca oxi phn t. Tng s vi khun hiu kh hin din trong
mu ch th mc v sinh ca thc phm. Ch s ny c xc nh bng phng php
m khun lc mc trn mi trng thch dinh dng trong mt lng mu xc nh
trn c s xem 1 khun lc l sinh khi pht trin t mt t bo hin din trong mu v
c biu din di dng mt n v hnh thnh khun lc (Colony Forming Unit,
CFU), trong 1 n v khi lng thc phm. Ch s ny c mt s tn gi khc nhau nh
sau: s vi sinh vt hiu kh (Aerobic Plate Count, APC), tng s m trn a
(TotalViable Count, TPC), tng s vi sinh vt sng (Total Viable Count, TVC), s m
a chun (Standard Plate Count, SPC)
Qui trnh phn tch bao gm cc bc cn mu, ng nht mu, pha long thnh
cc dy nng thp phn, chuyn v phn phi u mt th tch xc nh mu ln trn
b mt mi trng rn trong a petri bng phng php tri hp (spread plate) hay
hp (pour plate). U iu kin nhit v thi gian qui nh.
Phng php hp c s dng ph bin trong cc phng kim nghim vi sinh.
iu kin nhit v thi gian thay i ty theo yu cu phn tch v qui nh ca
tng quc gia. Tiu chun ca nhiu quc gia qui nh 30oC trong 3 ngy.
Ch tiu tng s vi sinh vt hiu kh c dng nh gi cht lng ca mu v
vi sinh vt, nguy c h hng, thi hn bo qun ca sn phm, mc v sinh trong qu
trnh ch bin, bo qun sn phm.
2. Mi trng

Mi trng s dng l Plate Count Agar (PCA) c pH 7.0 0.2. Mi trng

c pha ch, phn phi vo cc bnh thy tinh v hp tit trng 121oC trong 15 pht.
Cc bnh hoc ng nghim cha mi trng cha s dng phi c bo qun
trong t lnh 2 8oC. Trc khi s dng phi c un chy v lm ngui khong 45
50oC trong b iu nhit.
Nc ct c cha trong cc bnh thy tinh v hp trng 121oC trong 15 pht.
Sau phi c bo qun trong t lnh 2 8oC.
3. Qui trnh phn tch (mu dng lng)
3.1. Chun b mu trc khi phn tch
Mu c pha long theo dy thp phn bng cch dng pipet v trng chuyn
1ml mu vo ng nghim cha 9ml nc v trng. Trn mu tht k. Dung dch ny c
pha long 10-1. Tip tc chuyn 1 ml mu t pha long 10-1 sang ng nghim cha
9 ml nc v trng th 2. Trn mu tht k. Dung dch ny c pha long 10-2. Tip
tc tin hnh tng t c cc pha long 10-3, 10-4...

ng nui cy
ban u (ng
gc)

Nng

10-1

10-2

10-3

10-4

10-5

pha long

101

102

103

104

105

*Lu :
+ Pipet c nguy c b nhim trong qu trnh thao tc (chm tay, chm mt ngoi
ng nghim, mt ngoi bnh cha), cn phi thay pipet v trng khc.
+ Cc thao tc tin hnh trong iu kin v trng (s dng n cn)
+ Cc dng c (pipet, a petri) phi c tit trng trc khi s dng (sy
150oC/3 gi)
3.2. hp (cy mu)
i.

Dng bt lng du ghi ch ln a petri (tn mu, ngy tin hnh, pha
long)

ii.

Dng pipet v trng chuyn 1 ml dch mu pha long vo gia a petri.

Tng ng vi mi pha long thc hin t nht 2 3 a (tc l 2 3
ln lp li).

iii.

Sau khi cy, vo mi a 10 15 ml mi trng PCA c un chy

v n nh 45 - 50oC.

iv.

Trn u dch mu vi mi trng bng cch xoay trn a petri xui v

ngc chiu kim ng h, mi chiu 3 5 ln ngay sau khi mi trng.

v.

t cc a trn mt phng ngang v yn cho mi trng ng c hon

ton (khong 15 pht).

vi.

6. trong t m nhit 30oC trong 48 72 gi (48 gi ly kt qu s

b, 72 gi ly kt qu chnh thc)

Lu : khi phi hp petri trng thi lt p (phn mi trng trn,

phn np nm bn di)

1 ml
Dung dch
mu
pha long

45 C

PCA
10 -15 ml

a petri v
trng

Lc u a petri
Lt ngc a

ngui

30 C/3 ngy

m s khun lc
v tnh kt qu

Cch tnh kt qu
m tt c s khun lc xut hin trn cc a sau khi . Chn cc a c s
khun lc m c t 25 250 tnh ton. Mt tng vi khun hiu kh:

Trong :
A: s t bo (CFU) vi khun trong 1ml (hay trong 1 g) mu
N: tng s khun lc m c trn cc a chn
Ni: s lng a cy ti pha long th i
V: th tch dch mu (ml) cy vo trong mi a
fi: pha long tng ng
Lm trn kt qu c c, ch gi li 2 s c ngha v biu th kt qu di dng
thp phn gia 1.0 v 9.9 nhn vi 10n (n l s m)

Trng hp c khun lc vi sinh vt mc loang, mi vt loang c tnh l 1

khun lc. Nu s khun lc loang chim hn 1/3 a th phi ghi nhn iu ny v nh
du kt qu nhn c.

TRNG HP C BIT:
Nu pha long cao nht trong dy cc ng nghim ni trn, s khun lc
m c trn 1 a > 250: v d 10-5 ( pha long cao nht) c s khunlc
m c > 250, kt qu c ghi l : > 2.5 x 107 CFU/ml
Nu pha long thp nht trong dy cc ng nghim ni trn, s khun lc
m c trn 1 a < 250: v d nng 10-2 ( pha long thp nht) c s
khun lc m c < 25, kt qu c ghi l: < 2.5 x 103 CFU/ml

BI 2: NH LNG TNG S NM MEN V NM MC

1. nh ngha v nguyn tc
Nm men v nm mc l nhm vi sinh vt rt a dng, cho n nay c hn 4 trm
ngn loi nm men v nm mc c m t. Chng thuc nhm Eucaryote, c vch
t bo l lp v chitin. Nm men v nm mc l cc vi sinh vt d dng, chng tiu th
ngun carbon hu c c cung cp t mi trng bn ngoi t bo.
Hu ht nm mc v nm men thuc nhm mesophiles, mt s t thuc nhm
psychrophiles, hoc thermophiles. Hu ht nm men v nm mc pht trin trong mi
trng c hot nc t 85% tr ln, mt s t loi c th tng trng trong mi
trng c hot nc 60 70%. Nm mc v nm men tng trng c trong
khong pH t 2.0 9.0, trong pH thch hp nht l 4.0 6.5. a s nm men v nm
mc u thuc nhm hiu kh bt buc, mt s c th pht trin trong iu kin vi hiu
kh. Mt s loi c th tip nhn oxi nguyn t t c cht, nhng d dng no, oxi vn
l nguyn t cn thit cho qu trnh pht trin ca nm mc v nm men.
Trong thc phm, s hin din v sinh trng ca chng c th lm thay i cc
tnh cht ca thc phm (mu sc, mi v), gy h hng v c th to thnh mt s
c t gy ng c thc phm.
Mt nm mc v nm men trong mu c xc nh chung di dng tng s
nm men v nm mc bng cc k thut pha long, tri a v m khun lc trn mi
trng Dichloran Glycerol Agar, hay Dichloran Rose Bengal Chloramphenicol Agar
(DRBC). Mi trng Dichloran Glycerol Agar c s dng cho cc loi thc phm c
hm lng nc thp (thc phm kh, ng cc, tiu, cc loi thc phm c du, c
hm lng ng/mui cao). Mi trng DRBC c s dng cho cc mu c hm
lng nc cao (sa, cc sn phm t sa, hp, cc loi rau qu). i vi cc mu
c mt nm mc thp, cc mi trng thng c s dng l Malt Extract Agar
(MEA), Potato Dextrose Agar (PDA) cha 40ppm chloramphenicol hay chlotetraciline.
2. Cch tin hnh
a. Tin hnh pha long thp phn mu ban u

b. S dng k thut tri a tin hnh xc nh ch tiu tng s nm men, nm

mc trong mu
Ghi nhn ln a petri (tn mu, tn k thut vin, ngy)
mi trng vo trong a petri (khong 15ml). Ch cho mi trng ng
c hon ton (khong 15 pht)
Dng pipette cho 0.1 ml mu vo chnh gia a thch
Nhng que thy tinh hnh L vo mt cc cha ethanol. Sau g nh que
thy tinh vo thnh cc loi bt ethanol tha
Kh a que thy tinh gn ngn la n ethanol lm bc chy que thy
tinh. Lm ngui bng cch chm que thy tinh vo phn np bn trong a
thch.
Tri mu vi sinh vt sao cho mu dn u khp b mt mi trng thch.
Nhng que thy tinh vo ethanol, g nh vo cc v t chy que thy tinh.
Lp li thao tc vi a thch k tip. Tng ng vi mi pha long thc
hin t nht 2 3 a (tc l 2 3 ln lp li).
Tin hnh nui cy trong t vi sinh 30oC trong 5 7 ngy
Tin hnh quan st hnh thi i th ca khun lc, m s khun lc nm
men/nm mc v ghi nhn li kt qu.
3. c kt qu
a. Tng t bi nh lng tng s vi khun hiu kh
b. c kt qu t ngy 5 7

Lu : trong thi gian , nm mc c th to bo t v pht tn vo trong mi
trng nui cy, to thnh khun lc mi. hn ch hin tng ny, trong sut
thi gian , khng c chm tay hoc di chuyn cc a cho n khi m kt qu.
Mt khc, khi tin hnh m khun lc cn hn ch vic m a trnh s pht
tn ca bo t vo khng kh, gy nhim vo mu hay mi trng nui cy khc.
Cc a khi em phi mt thch nm pha di, nm hp petri nm trn.

Bi 3: NH LNG COLIFORM BNG PHNG PHP MPN

1. Gii thiu v Coliform
Coliforms l nhng trc khun gram m khng sinh bo t, hiu kh hoc k kh
ty , c kh nng ln men lactose sinh acid v sinh hi 37oC trong 24 48 gi. Trong
thc t phn tch, Coliforms cn c nh ngha l cc vi khun c kh nng ln men
sinh hi trong khong 48 gi khi c 37oC trong mi trng lng Lauryl Sulfate v
mi trng Brilliant Green Lactose Bile Salt. Nhm Coliforms hin din rng ri trong
t nhin, trong rut ngi v ng vt. Coliforms c xem l nhm vi sinh vt ch th:
s lng hin din ca chng trong thc phm, nc hay c loi mi trng c dng
ch th kh nng hin din ca cc vi sinh vt gy bnh khc.
Nhiu nghin cu cho thy rng khi s Coliforms ca thc phm cao th kh nng
hin din ca cc vi sinh vt gy bnh khc cng cao. Tuy nhin, mi lin h gia vi
sinh vt gy bnh v vi sinh vt ch th ny vn cn nhiu tranh ci.
Coliforms gm 4 ging: Escherichia vi 1 loi duy nht l E.coli, Citrobacter,
Clebsiella, Enterobacter. Tnh cht sinh ho c trng ca nhm ny c th hin qua
cc th nghim indol (I), Methy Red (MR), Voges-Proskauer (VP) v Citrate (iC)
thng c gi tm tt chung l IMViC. Coliforms chu nhit l nhng Coliforms c
kh nng ln men lactose sinh hi trong khong 24 gi khi 44oC trong mi trng
lng EC. Coliforms phn (faecal coliforms hay E.coli gi nh)l Coliforms chu nhit c
kh nng sinh indole khi c khong 24 gi 44.5oC trong mi trng lng Trypton.
Coliforms phn l mt thnh phn ca h vi sinh ng rut ngi v ng vt mu
nng khc v c s dng ch th mc v sinh trong qu trnh ch bin, bo qun,
vn chuyn, thc phm, nc ung cng nh ch th s nhim phn trong mu mi
trng. E.coli l Coliforms phn cho kt qu th nghim IMViC l ++-- (Indol + ,
Methyl Red + ,Voges-Proskauer -, Citrate -)
2. Phng php MPN (Most Probable Number)
Phng php MPN (phng php s c xc sut cao nht, s ti kh) cn c
gi l phng php pha long ti hn hay phng php chun . y l phng php

dng nh gi s lng vi sinh vt theo s lng vi sinh vt c xc sut ln nht hin

din trong mt n v th tch mu. y l phng php nh lng da trn kt qu
nh tnh ca mt lot th nghim c lp li mt s pha long khc nhau. Thng
thng, vic nh lng ny c thc hin lp li 3 ln pha long bc 10 lin tip,
tng cng 3 x 3 = 9 ng nghim. Qui trnh thc hin nh lng theo phng php ny
l nh sau: cho vo ng nghim c cha mi trng thch hp cho s tng trng ca
i tng vi sinh vt cn nh lng mt th tch chnh xc dung dch mu 3 nng
pha long bc 10 lin tip (v d 10-1, 10-2, 10-3). nhit v thi gian thch hp.
Da vo kt qu biu kin chng minh s tng trng ca vi sinh vt cn kim nh
trong tng ng nghim (thng l cc hin tng sinh hi, i mu, c). Ghi nhn s
lng cc ng nghim dng tnh tng pha long. S dng cc s liu ny v da
vo bng Mac Crady suy ra mt vi sinh vt c trnh by di dng s MPN/100
ml, MPN/1 g mu. chnh xc ca tr s MPN ph thuc vo s lng ng nghim lp
li trong mi pha long: s lng ng nghim lp li cng cao th chnh xc ca tr
s vi sinh vt c th c nui cy trong mi trng lng chn lc v cho kt qu biu
kin thch hp. V d, mi trng BGBL 37oC c th nh lng nhm coliforms,
cng mi trng ny 45oC cho php nh lng coliforms phn hoc mi trng
Mannitol Salt Broth c th dng nh lng Staphylococcus
3. nh lng Coliforms bng phng php MPN
Tun t cy 1 ml dch mu pha long 10-1 vo 3 ng nghim ging nhau, mi
ng cha 10 ml mi trng LST v ng Durham p ngc. Thc hin tng t vi dch
mu pha long 10-2, 10-3 n 10-10 ng nghim trong 48 gi 37oC. ng dng
tnh l ng c sinh kh bt kh trong ng Durham). Ghi nhn s ng dng tnh.
Th nghim khng nh: dng que cy vng chuyn dch mu t cc ng LST
dng tnh sang cc ng cha mi trng BGBL v 37oC trong 48 gi. Ghi nhn s
ng dng tnh ng vi mi pha long
Lu : cn loi b cc ng nghim c cha bt kh trong ng Durham sau khi tit trng
mi trng. Cc ng c kh nng hin din ca Coliforms l cc ng dng tnh c
mi trng LST ln BGBL.

Cc thao tc chnh

4. La chn cc pha long tnh kt qu

Mi mu kim nghim chn 3 pha long lin tip ng vi 1 trong 3 trng hp sau
sao cho thch hp. Tin hnh th t theo cc trng hp v cc bc (a, b,c) sau:
Trng hp 1
a. Chn pha long cao nht (tc l dch pha long c nng mu nh nht) cho
3 ng dng tnh, cng vi hai pha long cao hn tip theo (tc l pha
long ny c nng mu l 1/10 v 1/100 ca pha long th nht c
chn (xem v du)
b. Xem trng hp 3
c. Nu cc dch pha long tip theo ngoi dch pha long cao nht cng cho 3 ng
dng tnh th chn tip 3 pha long cao nht trong c dy (tc l nhng
pha long c nng mu nh nht) (xem bng 1, v d 2)

Trng hp 2
a. Nu trng hp 1 khng th p dng, chn 3 pha long cao nht trong c dy
(tc l nhng pha long c nng mu nh nht), trong s t nht thu
c 1 kt qu dng tnh (xem bng 1, v d 3)
b. Xem trng hp 3
Trng hp 3 (trng hp c bit)
Trong tt c cc trng hp, khi c nhiu hn mt trong 3 pha long c
chn theo trng hp 1 v 2 khng c ng dng tnh, th hy chn pha long no c
pha long thp nht khng c cc ng dng tnh (tc l pha long c nng
mu cao nht) v 2 pha long thp hn k tip trong dy pha long (tc l c nng
mu gp 10 ln v 100 ln pha long th nht chn), (xem bng 1, v d 4 & 5),
tr khi cc ng dng tnh ch tm thy mc pha long u tin c chun b t mu
th. Trong trng hp cui cng ny, cn chn ra 3 pha long u tin tnh MPN,
thm ch lot ng ny bao gm 2 pha long khng c ng dng tnh no
V d la chn cc kt qu dng tnh i vi vic tnh ton MPN

5. Xc nh ch s MPN
S dng kt qu ca phn 4. tra bng 2

Kt qu (MPN/ml) = kt qu trang bng x f/10

f: pha long thp nht c chn (10n)
V d

Bng tra MPN dng cho lot 3 ng nghim 3 nng pha long lin tip

BI 4: NH LNG TRC TIP T BO VI SINH VT

1. nh lng trc tip t bo bng bung m
C th s dng bung m nh lng vi sinh vt c kch thc t bo ln nh
nm men, bo t nm mcvi phng i x100 n x400. Vi t bo vi khun, do
kch thc nh, nn phi s dng bung m Petroff-Hasser. Phng php m trc tip
cn gip quan st c hnh thi t boKt qu m c l tng s t bo c trong
mu, khng phn bit c t bo cn sng hay t bo cht (ch phn bit t bo nm
men cht khi tin hnh nhum vi dung dch methylen blue )
2. Cu to bung m hng cu
Bung m hng cu l mt phin knh dy hnh ch nht, gia l phn lm
phng, ti y c k mt li gm 400 vung nh c din tch tng cng l 1mm2.Bao
gm 25 vung ln; mi vung ln ny gm c 16 vung nh. V th, din tch mt
hnh vung nh l 1/400 mm2 v mt hnh vung ln l 1/25 mm2.
3. Cch tin hnh
a. Pha long huyn ph t bo vi sinh vt sao cho trong mi ln ch t 10 50 t
bo).
b. Lc mnh dch huyn ph t bo ( pha long), dng pippet Pasteur ht dch
2. huyn ph ny.
3. y bung m bng mt phin knh mng
4. Nh nhng dng u pippet (cha mt git huyn ph vi sinh vt), t vo cnh
bung m (ni tip gip vi phin knh mng). Dch huyn ph s i vo bung
m nh c ch mao dn. Bung m c chun b ng khi ch c vng
khng gian nm gia l knh v bung m c ph bi dch huyn ph t bo,
cn cc rnh chung quanh th khng b dnh t.
5. Di chuyn nh nhng khung m dch huyn ph trn y cc khoang. Khi ,
dch nm trong khoang c dy khong 0.1mm
6. t bung m ln knh hin vi, s dng vt knh x10 tm bung m. Chnh
tht r, sau chuyn qua vt knh x40 m

7. iu chnh cng nh sng bng ca sp c th quan st r rng c t bo

ln cc ng k. Tu vo s lng t bo m c th chn cch m tt c cc t
bo c trong trung tm hay ch m cc t bo c trong mt s vung ln i
din. Thng thng ta chn 1 trung tm v 4 nm ngoi ba hoc 5 theo
ng cho (cc nh du X)
8. Bt u m t bo sau khi nh git dch t 3 5 pht; phi m cc t bo nm
trn 2 ng k k nhau c chn ca tng .

PHN 3: NG DNG VI SINH VT TRONG CH

BIN THC PHM
Bi 1: LN MEN RU VANG QU
1. Gii thiu:
Danh t ru vang qu c dng ch loi ru c ln men t dch p tri
cy (nho, du, thm, to...) ca mt s chng nm men. Ru vang qu thu c khng
qua chng ct, c hng v thm ngon ca tri cy t nhin, c cn nh (10 15%)
l loi nc gii kht thm ngon, giu cht b dng, t bit rt thch hp i vi ph
n. Ty theo lng ng kh cn li trong ru vang sau khi ln men xong, ngi ta
phn bit: (a) ru vang kh: 10 g ng/l, (b) na kh: 20 30 g ng/l, (c) na ngt:
45 g ng/l, v (d) ru ngt: 80 110 g ng/l. Trong ru vang c cha cc acid
hu c v v c (acid tartric, acid malic, acidcitric, acid oxalic...). pH ca ru vang
khong t 2.9 3.9.
2. Qui trnh th nghim
1. Ra sch qu (2~3kg)
2. Lm dp qu (nho,...) hoc xay dp (thm, xoi,...) hoc ct thnh lt mng.
Vi chui, nn tin hnh xt lt v sy kh trc . Vi cc loi qu khc, ta
xay dp, vt ly nc qu bng ti vi lc.
3. Cho qu (hoc dch qu) vo trong h plastic loi 5 L ra sch (chn loi c
np vn chc).
4. B sung thm nc (1~2 L) sao cho t 2/3 th tch bnh cha.
5. B sung ng ct trng sao cho dung dch t nng cht kh 25oBx (khuy
u ng tan hon ton).
6. iu chnh v pH 5.0 (s dng giy qu tm v acid citric).
7. B sung mi trng cha huyn ph Saccharomyces oviformis qua nhn
ging trong 30oC /48 gi vi th tch ph hp

8. Kt thc qu trnh ln men chnh khi nng cht kh khng tip tc gim v
hu nh khng c s hnh thnh bt kh trong bnh ln men (khong 5 7 ngy).
Ta c c ru non
9. Lc b xc qu bng vi lc tha.
10. Tin hnh qu trnh ln men ph trong cc chai plastic (chai pet loi 1.251.50
L) nhit 15 18oC (ngn mt t lnh). Thnh thong m h np vn x
bt lng kh CO2 tch ly trong chai (phng trnh phng/n chai)
11. Sau 7 ngy, lc b cn nhiu ln (qua bng gn loi thm nc) c c
ru sng.
12. Tip tc chn trong cc chai plastic nh trn 15 18oC vi thi gian vi
thng c c ru vang chn.
Gi : ty iu kin, sinh vin c th dng 100% dch qu (khng b sung thm
nc) c c hng v ca ru tt hn. c c loi ru vi hng v tt,
sinh vin nn s dng nho v thm lm th nghim. Sinh vin c th s dng ru
sng hoc tip tc chn c ru vang chn vi hng v tt hn
3. Theo di qu trnh ln men qua mt s ch tiu sau
Trong giai on ln men chnh:
a. Nng cht kh (oBx): kim tra 2 ngy/ln.
b. pH.
c. S to thnh bt kh bn trong h nha (ghi nhn kt qu quan st c).
d. cn: mu ru sau khi kt thc ln men chnh c chng ct v xc nh
cn bng phng php chun (hoc phng php dng bnh t trng).
Cc kt qu c ghi nhn vo bng biu v c v th theo thi gian.
.

Bi 2: LN MEN GIM
1. Gii thiu:
Theo khi nim ca T Chc Nng Lng Th Gii (FAO), gim c nh
ngha nh sau: Gim (vinegar) l dung dch acid long m con ngi c th s dng
c thng qua ng tiu ha. Gim phi c sn xut t nhng nguyn liu c
ngun gc t nng nghip cha tinh bt, ng, u tin bng qu ln men chuyn
ng thnh ru sau tip tc ln men acetic chuyn ru thnh acid acetic (thnh
phn ch yu ca gim). y l khi nim c s dng ph bin nht trn th gii cho
ti ngy nay.
Mt s ng dng quan trng ca gim trong cng ngh thc phm l:
Cht gia v lm tng gi tr cm quan ca mt s sn phm thc phm nh: xt, tng
c, tng t, mayonaise, salad trn, rau dm gim (da chut dm gim, rau ci dm
gim, hnh dm gim, hng tho dm gim)
Ph gia bo qun, kh mi tanh ca tht c trc khi ch bin.
Ngy nay, gim ang c a dng ha t nhiu nguyn liu khc nhau. Nht, ngoi
vai tr tng gi tr cm quan, ngi ta cn sn xut mt s loi gim chc nng nh
gim t go lc giu acid amin, gim t c hnh giu acid amin, K, Mg. Hng nm, trn
th gii, sn lng gim tiu th l rt ln.
2. Quy trnh th nghim
i.

Tin hnh lm ru vang qu (da, nho) hoc ru go, ru np than, ru t

dch chit malt thu c ru non.

ii.

Lc b b bng vi lc

iii.

Hiu chnh nng ru v 5% bng nc

iv.

B sung ging vi khun Acetobacter aceti vi t l 10% - 30%.

v.

Cho phn ru non b sung ging vi khun lc vo h (nha hoc thy

tinh) sao cho phn ru chim 50 70% th tch vt cha. Lu , sau khi b sung
ging nng acid ca dch ln men phi t ti thiu 0.5% (tnh theo acid
acetic), nu cha t phi hiu chnh bng acid acetic

vi.

yn trong khong 1 thng. Hng ngy m np h cha mt ln trong khong

thi gian 1 2 pht. Tuyt i khng c lc h cha hoc lm cho lp vng
mng b v hay chm xung

vii.

Kim tra acid nh k hng tun. Nu t nng acid acetic >4.5% l t yu

cu. Ta thu c gim non.

viii.
ix.

Lc k qua mt lp bng gn thm nc hoc ly tm 5000 rpm trong 15 pht

Cho vo chai thy tinh v ng np sau tin hnh thanh trng 80 90oC (un
cch thy) trong 15 30 pht

x.

c c loi gim ngon ta c th tip tc chn gim non iu kin 30oC

trong 1 2 thng tip theo. Sau , lc, ng chai v thanh trng nh trn.

MT S MI TRNG THNG DNG

- M1 (mi trng gi u ng)
Cn 100g gi u ( ra sch), thm 1000ml nc. un si 30 pht. Ly phn dch
trong. Thm nc cho 1000ml. b sung thm glucose vi hm lng 5% (w/v)
- M2 (mi trng khoai ty ng cm): 1000ml
Khoai ty ct nh, ra sch, cn ly 300g. Thm 500ml nc, un si 30 pht. Lc
lty nc trong. Cn 100g cm, thm 500ml nc, un si 30 pht. Lc ly nc trong.
Trn
2 loi dch lc trn v thm sucrose 5% (w/v). B sung nc cho 1000ml.
Khoai ty
30%
Cm
10%
Sucrose
5%
Nc
1000ml
- M3 (mi trng Sabouraud): 1000ml
Pepton
1%
Glucose
4%
Agar
2%
Nc
1000ml
pH 5.6 6.0
- M4 (mi trng Hansen)
Glucose
5%
Pepton
1%
K2HPO4
0.3%
MgSO4.7H2O
0.2%
Agar 2% Nc 1000ml
- M5 (mi trng Potato Dextrose Agar)
chun b cht chit khoai ty, un si 200g khoai ty xt lt, khng gt v vi
1000ml nc trong 30 pht. Lc qua vi tha, gi li phn dch, l cht chit khoai
ty.
Trn vi cc thnh phn khc ri un si ho tan (i vi mi trng Potato dextrose
salt: chun b nh mi trng M5 ri thm: 7.5% NaCl)
Cht chit khoai ty 20% (v/v)
Dextrose
2%
Agar
2%
Nc
1000ml
- M6 (mi trng thch malt)
Ly 250g malt, nghin nh, ho vo 1000ml nc ct. Dng a thy tinh khuy
u v gia nhit t t, n 45 50oC, gi nhit ny trong 30 pht. Sau nng
nhit ln 65 68oC, c khuy, cho n khi qu trnh ng ha xy ra hon ton
(khng thy mu xanh xut hin khi th dch cho vi dung dch Lugol). Lc tch b
qua vi th (hay bng gn thm nc), em hp phn dch 121oC / 30 pht, ly ra

lng. Lc b kt ta, pha long t 6 8o Brix, thm 2% agar, un v khuy u cho

n khi thch tan ht. Cho mi trng thch vo cc dng c cha, hp tit trng 121oC /
15 pht.
- M7 (mi trng BGBL 2% Broth dng pha sn)
Peptic digest of animal tissue
1%
Lactose
1%
Oxygall
2%
Brilliant green
0.0133g/L
Dng 40g hn hp pha sn pha thnh 1000ml mi trng.
- M8 (mi trng Plate Count Agar ATCC medium 1048): 1000ml
Agar
150 g
Yeast extract
2.5 g
Trypton
5g
Glucose
1.0g
- M9 (mi trng LST - Lauryl Sufate Broth/Lauryl Trypton Broth): 1000ml
Trypton
20 g
Lactose
5g
NaCl
5g
2.75 g
K2HPO4
2.75 g
KH2PO4
Sodium lauryl sufate
0.1 g
pH 6.8 0.2 Nc 1000ml
- M10 (Mi trng VRBL- Crystal Violet neutral Red Bile Lactose agar):
Pepton
7g
Neutral Red
0,03g
Yeast Extract
3g
Crystal Violet
0,002g
Lactose
10g
Agar
15g
NaCl
5g
Nc n
1000 ml
Mui mt
1,5g
Ha tan cc thnh phn trn trong nc, yn vi pht, t ln bp un si v
khuy u, si 2-5 pht. Lm ngui n 45oC s dng.
Ch :
- Khng si qu lu
- Khng c tit trng mi trng
- Pha ch xong s dng ngay trong vng 3 gi

TI LIU THAM KHO

1. Phng php phn tch vi sinh vt trong nc, thc phm v m phm Trn Linh
Thc Nh xut bn Gio dc, 2003
2. Thc tp Vi sinh vt hc thc phm Nguyn c Lng, Nguyn Chc, L Vn
Vit Mn i hc Bch Khoa TP.HCM
3. Th nghim Vi sinh vt hc thc phm L Vn Vit Mn, Li Mai Hng Nh
xut bn i hc quc gia TP.HCM, 2006
4. Handbook of Microbiologycal Media for the Examination of Food Ronald M.
Atlat CRC Press, Taylor &Francis Group, 2006
5. Laboratory Manual of Food Microbiology for Ethiopian health and Nutrition
Research Institute Dr. Ciira Kiiyukia Unido project, 2003
6. Food Microbiology Laboratory Manual Professor Nagendra Shah School of
Molecular Sciences Victoria University, 2004