GIAOTRINHTHUCHANH 1 3zd2o

V SINH C NHN V NGUYN TC LM VIC
1. Khi lm vic trong phng th nghim vi sinh hc phi tuyt i v sinh, tn trng tnh
ngn np v trt t ca phng th nghim.
2. Mi c nhn, mi nhm lm th nghim vi dng c ring, khng c mn dng
c ca cc nhm khc. Nu thiu phi hi cn b PTN hoc GVHD, khng c t ng s
dng dng c ca ngi khc.
3. Phi mc o bluose.
4. Khng ni chuyn, khng n ung, khng ht thuc l, khng i li ln xn trong
PTN.
5. Khng cc vt phm, canh trng vi sinh vt hoc mi trng nui cy vi sinh vt
gy bn ra bn gh, sch v hoc qun o. Trng hp gy bn phi v sinh ngay.
6. Sau khi lm xong, phi v sinh ni lm vic ca mnh, v sinh cc dng c, my mc
th nghim. Ra tay sch v ly cn st khun trc khi ra khi PTN.
7. Khi tin hnh th nghim phi c s ghi chp cc cng vic th nghim. Trong s ghi
chp cc iu kin lin quan n bi th nghim. Phi ghi chp y , cn thn, r rng.
H V TN SINH VIN MSSV

...
Gio trnh Thc hnh Vi sinh vt k thut Mi trng
BI 1: CC QUY TC PHNG TH NGHIM V

K THUT PHA CH MI TRNG
1. CC QUY TC PHNG TH NGHIM

1.1. Tit trng b mt v mi trng lm vic
Phng th nghim vi sinh phi c v sinh trc v sau khi s dng.
Dng c thit b c kh trng hay v sinh sch s v sp sp ngn np.
B mt lm vic phi c tit trng bng cn 700.
1.2. My mc, dng c trong phng th nghim:
Dng c bng thu tinh hoc kim loi:
- Cc dng c ng mi trng dinh dng phi c kh khun tuyt i trc khi
s dng. Sau khi s dng xong phi ra sch v kh khun li, sy kh, bao gi v vo
ni quy nh.
- Cc my mc, thit b cn c chnh xc cao (cn phn tch, bung m) cng
cn phi ch v sinh trong v sau khi tin hnh th nghim
C hai phng php tit trng: nhit kh hoc nhit m.
*Phng php tit trng bng nhit kh: Sy trong t sy nhit 170 -1800C
*Phng php tit trng bng nhit m: c thc hin trong thit b tit trng p
sut 1atm, 1210C trong thi gian 20-30 pht.
Dng c bng plastic chu nhit: Tit trng bng nhit m.
Mi trng nui cy vi sinh vt: Thng c tit trng trong thit b tit trng
1210C, 20-30 pht.
i vi nhng thnh phn ca mi trng dinh dng nhy cm vi nhit cao
khng th s dng phng php tit trng bng nhit trong thit b tit trng. Trong nhng
trng hp cn s dng mng lc vi khun tit trng.
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2. K THUT PHA CH MI TRNG

Mi trng dinh dng l hn hp gm cc cht dinh dng v cc cht c nhim v
duy tr th oxi ho kh, p sut thm thu ca t bo v s n nh pH ca mi trng.
C cc cht dinh dng cn thit; C pH thch hp; C nht nht nh; Khng
cha cc yu t c hi; Hon ton v trng.
Lm mi trng thc hin vic phn lp, nhn ging, gi ging vi sinh vt, ng
thi nui cy v nghin cu cc c im sinh hc ca chng.
2.1. Nguyn tc
Da trn c s nhu cu v cc cht dinh dng v kh nng ng ho cc cht dinh
dng ca tng loi sinh vt.
m bo s cn bng v p sut thm thu gia mi trng v t bo vi sinh vt.
m bo cc iu kin ho l cn thit cho cc hot ng trao i cht ca vi sinh
vt.
2.2. Pha mi trng
Cn, ong tht chnh xc tng thnh phn mi trng v pha ch theo ng trnh t
hng dn trong ti liu.
+ Mi trng lng: Cn, ong cc cht ri cho ho tan vo nc.
+ Mi trng c: Cn agar v ho cht cho vo bnh tam gic hay becher ri ho
tan trong nc, un trn bp hay hp trong autoclave.
2.3. Chun b mi trng
a. Thch nghing
Cho vo ng nghim vi mt lng mi trng c 1/3 n 1/4 ng nghim.
ng thch v tr nghing thch hp cho n lc thch c li hon ton.
V tr u trn ca mt nghing khng vt qu 2/3 chiu cao ng nghim.
Khng thch chm vo nt bng, khi lm nghing tt, mt thch nghing bng
phng, khng b t, chiu di va phi, khng ngn qu, di qu.
b. Thch ng
Cho vo ng nghim vi mt lng mi trng chim 1/2 n 2/3 ng nghim.
Thch ngui s ng li v ta s c ng thch ng.
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i vi bnh cu hay bnh tam gic, lng mi trng chim 1/2 - 1/3 bnh.
c. Lm thch hp petri
un thch chy lng, ngui n 50-600C.
Tay phi cm bnh (hoc ng) mi trng chy lng, gi hi nghing, dng tay
tri xoay v rt nt bng ra, v khun ming bnh (hoc ming ng trn n cn).
Dng tay tri h m np hp va phi v nhanh tay rt vo hp mt lng mi
trng thch hp.
Nhanh chng y np hp li, xoay trn hp t t trn bn thch dn thnh mt
lp u trn y hp. Khng xoay mnh lm thch bn tung ln.
H m np hp v yn cho n lc thch c li hon ton.
y np li, lt ngc hp cho y ln trn.
Hp thch tt, mt thch phi u, bng phng, lp thch khng dy qu hoc
mng qu (thng khong 2mm).
Hinh 1: Mt s dang mi trng trong ng nghi m va ia petri
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S mi trng cha dng n phi bo qun ch mt, nhit t 0 - 50C, khng

mi trng b kh, b tc dng ca nh sng.
D mi trng mi chun b hay bo qun lu, trc khi em dng u phi kim
tra s v khun. Mun vy, ta mi trng trong t m c nhit t 37 - 380C trong 2
3 ngy ly ra quan st v loi b nhng ng (hoc bnh) mi trng c vi sinh vt pht trin
(b c hoc c khun lc).
2.4. iu chnh pH ca mi trng:
iu chnh pH ca mi trng dng HCl 10% hay NaCl 10%.
Ngoi ra c mt s ho cht khc nh: H3PO4, H2SO4, KOH, NaHCO3, Na2CO3...
Lp nhn mi trng va thc hin:
Tn mi trng: ....................................................
Kh trng: ngy ......... thng ........... nm ............
3. DNG C, HA CHT V MI TRNG
3.1. Dng c
Dng c S lng n v
n cn 6 Ci
a petri 60 Ci
ng nghi m 60 Cai
Pipet 1ml 6 Ci
Pipet 10ml 6 Ci
Becher 100ml 12 Ci
3.2. Ha cht v mi trng

Agar 30g
Mi trng tng hp PCA (plate count agar) 23.5g
Cn 960 2 lt
Nc ct 5 lt
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4. THC HNH
Pha cn 700
mi trng thch ng nghim v a petri
Chun b mi trng PCA (plate count agar)
Vit bo co
5. CHUN B
Mu nc: sinh hot v nc thi
Mu t
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HNG DN VIT BAO CAO BA I 1

1. S liu bao cao
NG NGHIM NG NGHIM NG NGHIM
. .. ..
AT
KHNG
IA PETRI IA PETRI IA PETRI

. .. ..
AT
KHNG
IA PETRI (PCA) IA PETRI (PCA) IA PETRI (PCA)

. .. ..
AT
KHNG
Nhn xt kt qu:
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2. Cu hi n bi:
2.1. Ti sao phi v trng? Cc phng php v trng v tit trng?
2.2. Ti sao li m np a petri khi chun b mi trng thch? Ti sao phi iu chnh
pH?
2.3. C my dng mi trng thch ng nghim? Nu cc c trng cho tng loi mi
trng?
3. Yu cu bi hc
- Nm vng ni dung bi hc
- Lp bo co kt qu v nhn xt
- Tr li cu hi
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BI 2: NH LNG TNG VI KHUN HIU KH

Xc nh hay nh lng vi sinh vt l xc nh s lng c th tn ti trong mt n
v vt phm hoc canh trng. nh lng, cn phi thao tc ng quy cch mt s vn
nh: ly mu, pha long mu
1. PHNG PHP LY MU
nh gi chnh xc s lng vi sinh vt, yu cu u tin l mu phi c ly v
bo qu ng k thut.
Ty theo yu cu nghin cu, i tng nghin cu v cc ch tiu sinh hc cn phn
tch m khi lng mu cng nh quy cch ly mu (s lng, v tr, thi gian) khc
nhau.Tuy nhin, vic ly mu cn tun theo nhng yu cu sau:
- Dng c ly mu v cha mu phi v trng: m bo thun khit, trnh tp
nhim m bo chnh xc ca kt qu phn tch.
- Dng c ly mu v cha mu phi sch, khng c hi vi vi sinh vt, trnh lm
cht hoc c ch vi sinh vt trong vn chuyn v bo qun.
- Cc yu cu v khi lng v tnh c th ca mu:
+ khi lng theo yu cu phn tich
+ ng ni quy nh v vo thi im ph hp vi yu cu nghin cu
+ Mu trung bnh v in hnh
- Bo qun: mu ly xong phn tch ngay. Nhit thng khng mu qu 30 pht.
Nu ly mu xa, cn c thit b phn tch hin trng hoc bo qun nhit thp (0-50C),
thi gian bo qun khng qu 24 gi.
- Cc yu cu khc: mu cn c dn nhn v ghi r
+ M s ca mu
+ Loi v mc ch ly mu
+ Tn v chc nng ca ngi ly mu
+Yu cu phn tch
2. PHA LONG MU
2.1. Chun b mu
Yu cu ca vic ly mu:
- Ly mu c tnh cht i din.
- Lng mu ly va phi, phn tch cc c tnh l, ho, sinh hc.
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- Dng c ly mu, cha mu phi v trng.

- Ly mu xong phi phn tch ngay v khng c qu 24h.
- Ghi nht k ca mu v ni thu mu.
2.2. Pha long mu :
- Chun b mt bnh cha mu c n phn tic h
- Mt s ng nghim cha 9ml nc ct v trng
- Mt s pipet v trng.
- Pha long mu cn phi thc hin trong t cy hoc phng v trng
a. Mu trng thi lng :
Pha long mu theo dy thp phn 1/10 (10-1), 1/100 (10-2), 1/1000 (10-3), 10-n
Hnh 2.1: Phng php pha long mu nc theo dy thp phn
b. Nu mu nghin cu trng thi c (nh t, lng thc, thc phm)

Mi trng t nhn chung rt thun li cho nhiu nhm vi sinh vt pht trin. H vi
sinh vt t rt a dng gm vi khun, nm, x khun S lng vi sinh vt ph thuc vo
hm lng cht hu c, tnh cht c l cng nh m ca t, s lng c th thng rt
ln.
Cc bc tin hnh:
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- Ly mu t: mu t mang phn tch c tnh i din nn s lng mu ty theo

yu cu nghin cu khc nhau. Mu t ly t 10-20g cho mi v tr, ly 4 mu ca 4 gc
vung v 1 mu tm, su nht nh v mu phi c trn u trc khi s dng.
- Chun b dng c:
Mt binh tam giac 250 ml cha 90ml nc ct v trng
Mt s ng nghim cha 9ml nc ct v trng
Mt s pipet v trng.
- Pha long mu: cn 10 g t (hoc mu) cho vo binh tam giac cha 90ml nc c t
v trung. Lc 10 pht, lng 30 giy ri tip tc pha long mu nh trng thi lng.
Tu theo s c on s lng vi sinh vt trong mu m pha long nhiu hay t.
Hnh 2.2: Phng php pha long mu c theo dy thp phn
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3. K THUT TRI A
Hnh 2.3: Minh ha k thut tri a

Dng pipet v khun ly 0,1 ml dch huyn ph cho vo mi a thch (tng
ng vi 2 git dch).
S t bo cy trn b mt thch phi c dn u v khong 25-300 CFU (coliform
unit: n vi khu n la c).
S dng que cy gt bng thu tinh dn u cc t bo trn b mt thch, que gt
thu tinh c v khun bng cch nhng vo trong cn, t chy phn cn cn li trn que
cy trn ngn la n cn. Lm ngui que cy bng cch nh n vo b mt thch, ri dn
u lng cht lng cha t bo trn .
Lp cht lng trn thch cng mng, s hp thu xy ra cng nhanh.
t cc a thch va cy vo t m ci nhit 300C v trong 48 gi.
Kt qu:
S khun lc (CFU) x pha long
CFU/g = -------------------------------------------------
Th tch mu (ml)
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4. THC HNH
4.1. Dng c
Tn S lng n v
n cn 6 Ci
a petri 60 Ci
Pipet 1ml 6 Ci
Pipet 10ml 6 Ci
Becher 100ml 12 Ci
Que trang 6 Ci
ng nghim 30 Ci
Mi trng Plate count agar (PCA) tng hp 23,5g hay mi trng thnh phn
gm: Trypton, 5.0g; Yeast extract, 2.5g; Glucose, 1.0g; Agar,15g.
Cn chnh xc khi lng mi trng cho vo becher 1,5 lt v thm vo 1 lt
nc ct. un nng hn hp n khi tan u ri cho vo erlen 1,5 lt em hp kh trng.
Lu , trong khi un mi trng cn phi khuy thng xuyn mi trng khng b
lng v kht.
Sau , hp kh trng 1210C, 1atm v thi gian 30 pht.
Chun b:
Cn 960 2 lt
Nc ct 5 lt
NaCl tinh khit 10g
4.3. Thc hnh
- Pha nc mui sinh l 0,9%
- Pha long mu (mu nc v t)
- a.
- Tri a
c kt qu v vit bo co
12

1. S liu bao cao
1 2 3
Nng pha long .. .
AT
S khu n la c
KHNG
Nhn xt kt qu:
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2. Cu hi n bi:
2.1. Ti sao phi pha long mu? Cc bc pha long mu?
2.2. Vi sinh vt hiu kh c u trong mi trng? ngha ca vic xc nh tng vi
khun hiu kh?
2.3. Ti sao phi kh trng hay st trng ton b: mi trng, dng c, tay trong qu
trnh thao tc? ngha vic kh trng v st trng?
3. Yu cu bi hc
- Tr li cu hi
13
BI 3: XC NH TNG VI KHUN K KH
1. K THUT MI TRNG VI KHUN K KH

Phn lp cc vi sinh vt k kh trn mi trng c a petri.
Dng mi trng c trong ng nghim em chng cch thu loi b khng kh
trong mi trng.
ngui mi trng cn 45 - 500C.
Ht 0,1 ml mu vo ng mi trng, y nt li, lc trn quanh trc ng nghim.
Rt nhanh mi trng ng nghim vo np di ca a petri v y tht nhanh
np trn li, sao cho gia mt np v mi trng khng cn khng kh.
Dng parafin hn kn phn tip xc gia 2 np ca a petri.
Nui trong bnh ym kh: sau khi cy vi khun xong, cho vo bnh ym kh. Thoa
mt lp parafin lng ln ming bnh, t n cn hay n cy ln v t vo bnh, y np
li, hoc dng my ht chn khng. Cho vo t m, nhit v thi gian thch hp.
Sau khi vi sinh vt pht trin, chn cc khun lc ring r trong khi mi trng,
dng que cy ct c khi mi trng ri cy vo mi trng lng thch hp.
Ngoi ra, ngi ta cn s dng phng php xc nh tng vi khun k kh bng ng
nghim y nt cao su nhng mi trng nui cy phi b sung y glucose, trypticase,
yeast v mt s cc mui khong khc cho vi khun pht trin, c bit phi b sung cc tc
nhn kh nh cystein v Na2S vo mi trng.
Thao tc thc hin:
Mi trng sau khi hp kh trng ngui n nhit 40-460C. B sung vi git
Na2S + NaHCO3 kh ht O2 trong mi trng.
Cn 1g mu t v pha long mu bng nc ct v trng theo dy thp phn lin
tip, tin hnh pha long n nng thch hp sao cho khun lc mc trn a khong 30 -
300. Khng mu trong nc pha long lu hn 30 pht.
Ht 1ml mu pha long vo ng nghim v trng.
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Rt mi trng nhit 40-460C vo ng nghim c cha mu. Rt mi trng

nh nhng trnh khng to bt kh v khng mi trng trn ra ngoi.
y ng nghim bng nt cao su v trng. Ch cho thch ng cng li v em
nhit phng.
Sau 3 ngy m cc khun lc ring r quan st c.
2. THC HNH
2.1. Dng c
Tn S lng n v
n cn 6 Ci
ng nghim 60 Ci
Pipet 1ml 6 Ci
Pipet 10ml 6 Ci
Becher 100ml 12 Ci
Thnh phn mi trng
NH4NO3 2g
KH2PO4 1g
NaCl 10g
MgSO4.7H20 3,3g
Glucose 1g
Pepton 5g
Cao men 0,5g
Agar 12g
Sau khi kh trng 1210C trong 15 pht, mi trng c pH 7.0 7.2.

Cch pha hn hp Na2S + NaHCO3: Cn 0,75g Na2S v 0,3g NaHCO3 pha trong
30ml nc ct.
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Mi trng sau khi hp kh trng ngui n nhit 40-460C. B sung vi

git Na2S + NaHCO3 kh ht O2 trong mi trng.
- Cn 960 2 lt
- Nc ct 5 lt
- NaCl tinh khit 10g
2.3. Thc hnh
- Pha long mu (mu nc v t)
- ng nghim
c kt qu v vit bo co
3. CHUN B
mi trng PGA
16

1. S liu bao cao
1 2 3
Nng pha long .. .
AT
S khu n la c
KHNG
Nhn xt kt qu:
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2. Cu hi n bi:
2.1. Ti sao phi s dng dung dch Na2S + NaHCO3 ?
2.2. Vi sinh vt k kh c u trong mi trng? ngha ca vic xc nh tng vi
khun k kh?
2.3. Phn bit s khc nhau gia vi khun k kh v hiu kh?
2.4. ngha vic xc nh tng vi khun k kh trong mi trng?
3. Yu cu bi hc
- Tr li cu hi
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BI 4: XC NH TNG VI NM
1. NGUYN TC
Nm men v nm mc u thuc nhm a mt, nhit thch hp cho s pht trin
trong khong 20 280C, mt s t l a nng v a lnh. Vi nm c kh nng sinh trng
trong vng pH t 2-9, nhng pH thch hp nht nm trong khong 4 6,5. Hu ht vi nm
u thuc nhm hiu kh bt buc, mt s c th pht trin trong iu kin k kh.
Mt vi nm trong mu c xc nh di dng tng vi nm bng k thut pha
long, tri v m khun lc trn mi trng thch hp.
2. THC HNH
2.1. Dng c
Tn S lng n v
n cn 6 Ci
a petri 60 Ci
Pipet 1ml 6 Ci
Pipet 10ml 6 Ci
Becher 100ml 12 Ci

Thnh phn mi trng:
Khoai ty 300g
Glucose 20g
Agar 15g
Pha ch mi trng: B sung khong 500ml nc my vo khoai ty un si k v

lc ly dch lc, sau b sung ng v agar vo hp kh trng. S dng acid lactic chnh
pH n 5,5 c ch s pht trin ca vi khun. Kh trng 1210C trong 15 pht.
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Chun b:
- Cn 700 2 lt
- Cn 960 2 lt
- Nc mui sinh l 1 lt
- Nc ct 5 lt
2.3. Thc hnh
- mi trng
- Tri a
c kt quv vit bo co
3. CHUN B
Chun b mi trng Lauryl Sulphate broth (LSB)
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1. S liu bao cao
Nng pha long 1 2 3
.. .
AT
S khu n la c
KHNG
Nhn xt kt qu:
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2. Cu hi n bi:
2.1. Vi n m la gi?
2.2. Phng phap xac inh
t ng vi n m?
2.3. Phn bi t khu n la c vi khu n va vi n m?
3. Yu cu bi hc
- Tr li cu hi
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BI 5: NH LNG VI SINH VT
V PHNG PHP MPN
1. CC PHNG PHP NH LNG VI SINH VT

1.1. m trn lam pht knh
Tiu bn vi sinh v t c nhum mu (nhum n).
Hnh 5.1: Din tch hnh vung pht knh

S : Di chuyn vt knh trong hnh vung pht knh, m s VSV/1VT
a. Pht knh:
- Dng lam sch v bt ch m (bt lng kim), k mt hnh vung S = 400 mm2,
mt trn lam. Mc ch khng cho vi sinh v t lan ra khi din tch m.
- Ht 0.02-0.2 ml dung dch pha long , nh vo gia hnh vung, mt trn lam.
- t que cy, ngui, dng cnh vng cy u que dn u git mu ra xung
quanh, va chm vo cnh hnh vung.
- C nh tiu bn, nhum n bng thuc nhum blue methylen.
- Nh mt git du soi, xem vt knh x100.
b. Cch m
- m s t bo VSV/ 1VT (VT: vi trng).
- m ht tt c t 30 50 VT/ hnh vung pht knh. Theo s hnh v.
c. Cng thc tnh
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Trong :
X = S VK m c trong mt vi trng
400 = din tch hnh vung pht knh, bng 400mm2
0.0004 = din tch ca vi trng, R2 = 0.0004 mm2
S vi trng c c trong S: 4cm2
V = th tch git VK pht knh

H = h s pha long mu
1.2. m trong bung m:
a. Cu to bung m.
- Bung m l mt phin knh trong, dy, hnh ch nht.
- Gia l phn lm phng, c k mt hoc hai khung m gm 400 nh. Bn ngoi
c ghi tn loi bung m, v ghi cc thng s k thut nh hnh trn.
Hnh 5.2: Cu to bung m
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b. Cu to khung m.
Hnh 5.3 Cu to khung m
Cu to mt khung m c:
- Trng hp1: 1 khung c 16 ln, 1 ln c 25 nh. Vy 1 khung c 400 nh.
- Trng hp 2: 1 khung c 25 ln, 1 ln c 16 nh. Vy 1 khung c 400 nh.
- Din tch 1 nh l 1/400 mm2.
Vy V nh = 0.1 mm x 1/400 mm2 = 1/4000mm3.
c/ Cch tin hnh.
- t lammen sch ph ln khung m.
- Dng ng nh git ht dung dch nm men pha long, b i vi git u, bm
nh vo rnh bung m, dung dch thm vo k bung m v lammen.
- Dung dch chy trn t t vo cc rnh, lan ta lp y khp lammen, hi
tha mt t. Nu b bt kt li trong lammen th phi lm li.
- t bung m ln bn kp ca knh hin vi, dng kp c nh bung m.
- Thao tc knh hin vi, dng vt knh x10 iu chnh s b trc, sau dng vt
knh x40 m.
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- m s t bo trong 5 ln (4 cnh, 1 gia), m ln lt tng nh

trong 1 ln. Trong tt c cc nh cn m s t bo nm hn trong trc, sau m
s t bo nm cnh pha trn v cnh bn phi .
- m tt c 80 nh c trong 5 ln (T/h: 1 ln c 16 nh).
- Hoc m 125 nh c trong 5 ln (T/h: 1 ln c 25 nh).
d. Cng thc tnh.
Trong : a = s t bo trung bnh c trong 1 nh (V = 1/ 4.000 mm3)

4.000 = s qui i t 1/4.000 mm3 thnh 1 mm3.
1.000 = s qui i t 1 mm3 thnh 1ml (1ml = 1.000 mm3)
H = h s pha long (v d: H = 10-2)
e. Ch :
- Phng phap nay chi s du ng quan sat nhng vi sinh v t co kic h thc ln nh
bao t cua n m men va n m m c.
- Ch m c tng s t bo, khng th bit t bo no cn sng hay cht.
- Nn nhu m mau (methylene blue) d quan sat va m chin h xac.
- Nng dch huyn ph pha long sao cho mt trong mi nh khng qu 10
t bo.
- S dng xong phi ra bung m ngay v lau kh.
- t chnh xc cao, th s t bo trung bnh m c trong 1 nh: 2.5 < a
<10.
1.3. Phng php trai ia
Tng t thao tc trai ia , s lng t bo vi sinh vt c xc nh qua khun lc.
Khun lc l cc c th hay t bo vi sinh vt c nui cy trong cc iu kin mi trng
thch hp, sau phn chia to thnh nhiu c th hay qun th vi sinh vt m mt thng
nhn thy c.
24
1.4. Phng php MPN (Most Probable Number)

Phng php MPN (phng php c s 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 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 3 pha long bc 10 lin tip, tng cng 3 x 3 = 9 ng nghim.
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 nh 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/100ml hay s MPN/1g mu. chnh xc ca tr s MPN ph
thuc vo s lng ng nghim lp li trong mi pha long.
C th l xc nh s lng Coliforms trong thc phm, nc ung, nc sinh hot
hoc nc thi.
Thao tc:
25
Bc 1: Ly mu v pha long mu n 3 nng lin tip thch hp.

Bc 2: Mi pha long ht 3 ln, mi ln 1 ml cy vo 3 ng nghim mi trng
Lauryl Sulphate broth (LSB). Ht 3 nng lin tip, mi nng 3 ng nghim.
Bc 3: m t 35 370C, thi gian 24 - 48 gi.
Bc 4: ng dng tnh l c bt kh trong ng durham.
Bc 5: Tra bng Mac Crady lot 3 ng nghim 3 nng lin tip.
Bng MPN/100ml tnh theo lng mu l 10 ml, 1ml v 0,1ml tng ng MPN/ml
ca mu pha long 10-1, 10-2 v 10-3 dng phn tch.
Kt qu tra bng MPN/ml = kt qu tra bng x f/10
( f pha long thp nht 10n)
Lu : Cc ng nghim c kh nng hin din ca coliform l dng tnh c hai mi
trng LSB v BGBL.
2. THC HNH
2.1. Dng c
Tn S lng n v
n cn 6 Ci
ng nghim 100 Ci
a petri 20 Ci
Pipet 1ml 6 Ci
Pipet 10ml 6 Ci
Becher 100ml 12 Ci
Mi trng LSB va BGBL
Cn chnh xc khi lng mi trng tng hp hay thnh phn ca tng mi trng.
un nng cho mi trng ng nht ri phn phi vo cc ng th nghim c ng durham.
Lu : lng mi trng trong ng nghim phi cao hn ng durham. Cn loi b
cc ng durham c bt kh sau khi hp kh trng.
Hp kh trng mi trng 1210C, 1atm, 15 pht.
26
Chun b:
Cn 960 2 lt
Nc ct 5 lt
2.3. Thc hnh
Mu nc: nc thi v nc sinh hot
Mu t
nh lng trc tip s lng t bo nm men bng bung m hng cu.
nh lng E.coli v Coliforms bng phng php MPN.
c kt qu
Vit bo co
3. CHUN B
Violet Red Bile Agar (VRBA)
27

1. S liu bao cao
1.1. K t qua quan sat trn bu ng m h ng c u:
Nng pha long 1 2 3
.. . ..
S t bao
1.2. K t qua m b ng phng phap MPN

Nng pha long 1 2 3
.. .
AT
Tra bang
.
KHNG
Nhn xt kt qu:
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28
2. Cu hi n bi:
2.1. Nh c i m cua phng phap i m t bao vi sinh v t b ng bu ng m h ng c u la
gi?
2.2. Thao tac s du ng kin h hi n vi quan sat va m t bao vi sinh v t?
2.3. Ti sao phai s du ng mi trng BGBL sau khi xac inh
b ng mi trng LSB?
3. Yu cu bi hc
- Tr li cu hi
29
BI 6: XC NH E.COLI V COLIFORM
1. TNG QUAN
Vi khun Coli (Total coliform) tn ti trong t nhin, trong phn ngi, phn gia sc,
trong nc, trong nc thi hay trong cc vt phm c ngun gc khc nhau. Theo c tnh
nui cy, ngi ta chia chng lm hai nhm:
- Fecal Coli (Escherichia coli - E.coli) c ngun gc t phn (ngi, ng vt, c..)
- Non fecal Coli (Bacterium coli) c ngun gc khc (t, cy c, nc thi)
Vi khun E.coli thuc nhm vi trng ng rut Enterobacteriaceae, chng hin
din nhiu i trng nn cn gi l vi khun i trng. Vi khun E.coli nhim vo t,
nc t phn ca ng vt. S c mt ca chng chng t nc b nhim phn hoc nc
thi sinh hot. Chng s gy bnh khi gp iu kin thun li cho s pht trin ca chng.
- Hnh dng: Vi khun thuc loi trc khun gram m, di ng bng tim mao quanh
t bo, khng to bo t, loi c c lc th c capsul, loi khng c c lc khng c
capsul. Kch thc trung bnh (0,5 x 1-3) hai u trn. Mt s dng c lng bm (pili).
- c im nui cy v sinh ha: L loi hiu kh hay hiu kh ty nghi. Nhit
thch hp 370C nhng c th mc trn 400C, pH 7,4.
- Trn mi trng VRBA to khun lc mu tm.
- E.coli gy bnh truyn nhim tiu chy hay nhim trng huyt tr s sinh Ngoi
ra, nc thi nhim E.coli cn c th nhim cc vi sinh vt khc c trong ng rut nh vi
khun t (Vibrio choleras), vi khun l (Shigella),
2.1. Dng c
Tn S lng n v
n cn 6 Ci
a petri 60 Ci
Pipet 1ml 6 Ci
Pipet 10ml 6 Ci
Becher 100ml 12 Ci
Mi trng VRB
30
Cn mi trng tng hp hay thnh phn sau thm nc nc ct thch hp ri un

nng cho ha tan cc cht vi nhau. mi trng thch a m khng cn phi hp kh
trng.
Chun b:
Cn 700 1 lt
Cn 960 2 lt
Nc ct 5 lt
Nc mui sinh l 1 lt
3. THC HNH
Mu nc va mu t:
- Pha loan g mu theo day n ng th p phn
- Cho n n ng thich h p, l y m t l ng mu va phai cho vao ia petri co cha mi
trng VRB.
- Trai ia va em u nhi t thic h h p, sau 24 gi o c k t qua.
c kt qu:
Sau 24 gi cc khun lc c trng l E.coli v Coliform c mu ta, ng knh
0.5mm hoc hn, i khi c bao quanh bi mt vng hi ca mt kt ta.
Vit bo co
4. CHUN B
Mi trng NA
31

1. S liu bao cao
1 2 3
..
.
AT
S khu n la c
KHNG
Nhn xt kt qu:
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2. Cu hi n bi:
2.1. Coliform l g? E.coli l g? Ti sao phi nhn bit Coliform v E.coli?
2.2. Mi trng v iu kin nui cy Coliform? Cc phng php xc nh E.coli
hc, u im v nhc im ca mi phng php?
2.3. So sanh mu phn tich theo tiu chu n Vi t Nam (TCVN)?
3. Yu cu bi hc
- Tr li cu hi
32
BI 7: CC PHNG PHP PHN LP,

NUI CY V BO QUN VI SINH VT
1. KHI NIM
Phn lp vi sinh vt l qu trnh tch ring cc loi vi sinh vt t qun th ban u v
a v dng thun khit.
y l mt khu c ngha rt quan trng trong vic nghin cu v ng dng vi sinh
vt.
Vi sinh vt dng thun khit l ging vi sinh vt c to ra t 1 t bo ban u.
Nguyn tc:
- Tch ri cc t bo vi sinh vt; Nui cy cc t bo trn trong mi trng dinh
dng c trng cho khun lc ring r, cch bit nhau.
- Sau khi phn lp vi ln, t khm thun cy truyn sang ng mi trng thch
nghing hay ng nghim canh gia tng s lng vi sinh vt.
2. PHNG PHP PHN LP VI SINH VT

2.1. Mi trng thch a
2.1.1. K thut hp ria
- Dng que cy vng thao tc v trng thu ging.
- Ria cc ng trn a petri cha mi trng thch hp (ria ch T v ria bn gc).
Sau mi ng ria, t kh trng u que cy v lm ngui trc khi thc hin ng ria
tip theo.
- Bao gi a petri, nhit v thi gian thch hp trong t m.
33
Hnh 7.1: K thut hp ria
2.1.2. K thut hp tri

- Dng pipetman v u tp v trng, thao tc v trng chuyn 0,1 ml dch cha
ging vi sinh vt ln b mt mi trng trong a petri.
- Nhng u thanh gt (que tri) thu tinh vo cn 960, h qua ngn la kh trng.
u thanh gt ngui trong khng gian v trng ca ngn la.
Hnh 7.2: Cch dn vi sinh vt trn b mt mi trng bng que gt Drigalxki.
- M a petri, t nh nhng thanh gt ln b mt thch ca a petri. Dng u

thanh gt xoay, tri u dch ging ln b mt thch. Trong khi tri, thc hin xoay a mt
34
vi ln, mi ln khong 1/2 chu vi a to iu kin cho thanh gt tri dch ging u khp
b mt mi trng.
- Rt thanh gt khi a, y a, gi v nhit v thi gian thch hp trong t
m.
2.1.3. K thut hp
- Dng pipetman v u tp v trng, thao tc v trng chuyn 1ml dch cha ging
vi sinh vt ln b mt mi trng trong a petri.
- khong 15 - 20 ml mi trng un chy v ngui n 45 - 550C vo a
petri cy mu.
- Xoay nh ia petri cng chiu v ngc chiu kim ng h vi ln dung dch
ging c trn u trong mi trng cy.
- y np a petri, ng t nhin.
2.2. Phng php cy truyn
2.2.1. Cy truyn t ng nghim ny sang ng nghim khc
- Tay tri cm 2 ng nghim: 1 ng ging v 1 ng mi trng
- Tay phi cm que cy v kh trng bng n cn cho n khi nng dy cy
- Dng ngn t v ngn p t kp nt bng vo lng bn tay xoay nh, ko nt ng
ging ra
- Kh trng khng kh ming 2 ng nghim
- i khi que cy va ngui, kho lo a que cy tip xc vi khun lc trong ng
ging.
- Rt que cy ra, khng que cy chm vo thnh ng nghim v a ng vo mi
trng thc hin cc thao tc cy truyn.
- Kh trng li phn khng kh ni ming 2 ng nghim ri y nt bng
- Kh trng li que cy sau khi s dng xong
Ch : Nu ng ging l canh trng lng c vi sinh vt th dng pipet ht canh
trng thay que cy.
35
2.2.2. Phng php cy trn thch nghing

Phng php ny dng cy truyn cc vi sinh vt hiu kh.
+ Ho ging u que cy vo git nc y ng nghim.
+ Nh nhng lt que cy trn mt thch theo cc kiu: Hnh ch chi; Hnh vng
xon; Hnh vch ngang song song
Hnh 7.3: Cc kiu cy chuyn trong ng nghim
2.2.3. Phng php cy trn thch ng

Phng php ny dng cy cc vi sinh vt k kh.
- S dng que cy hnh kim
- Sau khi ly ging vi sinh vt,
dng que cy ny m su vo phn khi
thch hnh tr, m st y ng nghim v
m thnh 3 ng: 1 ng chnh gia, 2
ng st thnh ng tu yu cu.
- ng cy phi thng, nh nhng
khng gy nt, v mi trng
Hnh 7.4: K thut cy trn thch ng
36
3. CC PHNG PHP NUI CY V BO QUN VI SINH VT

3.1. Cc phng php nui cy
Kt qu ca vic nui cy: Sau khi nui thi gian v nhit thch hp cho mi
loi vi sinh vt:
- Trong mi trng lng: Vi khun pht trin s lm c mi trng.
- Trong mi trng c thch nghing: Nm men, vi khun pht trin s to nn
vt ni trn mt thch trong hay trng c. Nm mc s to nn nhng si mnh t vt cy.
- Trong mi trng thch ng: Cc vi khun k kh pht trin y ca ct mi
trng.
3.2. Cc phng php bo qun ging vi sinh vt
Phng php cy truyn nh k ln mi trng mi: Phng php ny p dng
bo qun tt c cc loi vi sinh vt.
Phng php gi ging trn mi trng thch c lp du khong: i vi vi sinh vt
k kh.
i vi vi sinh vt hiu kh:
+ Cung cp thng xuyn v y O2.
+ Lp mi trng nui cy c dy va phi
+ Cc bnh cha mi trng c lc thng xuyn trong qu trnh nui cung cp
thm oxi cho vi sinh vt.
+ Nu nui cy trong mi trng c khi lng ln phi tin hnh sc kh thng
xuyn hay nh k.
i vi vi sinh vt k kh, hn ch s tip xc vi oxi bng cch:
+ ln b mt mi trng parafin, du vaselin.
+ Cy trch su vo mi trng c.
+ Nui cy trong bnh ht chn khng.
+ Nui trong ng nghim c bit sau khi rt ht khng kh v hn kn li. un si
mi trng mt thi gian loi ht O2, ngui 450C. Dng ng ht cy vi sinh vt vo
y ng nghim. Lm ngui tht nhanh ri vazlin ln b mt hn ch s tip xc vi
O2 .
37
Phng php gi ging trn t, ct: Dng bo qun cc chng to bo t tim

sinh (hoc bo t v tnh) vi thi gian bo qun t mt n nhiu nm.
Trn ht: Dng bo qun cc chng c dng hnh si sinh bo t hoc khng. Thi
gian bo qun c th ti 1 nm.
Phng php ng kh: lm cho t bo mt nc trng thi t do. ng thi lm
gim, thm ch lm ngng hn qu trnh phn chia ca vi sinh vt. Thi gian bo qun ln
ti vi chc nm.

4.1. Dng c
Tn S lng n v
n cn 6 Ci
a petri 60 Ci
ng nghim 60 Ci
Pipet 1ml 6 Ci
Pipet 10ml 6 Ci
Becher 100ml 12 Ci
Mi trng NA 2lt
Cn 960 2 lt
Nc ct 5 lt
5. THC HNH
Mu vi khun nui cy t cac bai trc c em i phn l p.
c kt qu:
Vit bo co
6. CHUN B
Thu c nhu m Gram
C n 960 va 700
38

1. S liu bao cao
1 2 3
..
.
AT
S khu n la c
KHNG
Nhn xt kt qu:
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2. Cu hi n bi:
2.1. Ti sao phai ria theo hinh zic z c?
2.2. Mc ich cua vi c phn l p va bao quan gi ng vi sinh v t?
3. Yu cu bi hc
- Tr li cu hi
39
BI 8: QUAN ST HNH THI VI SINH VT

BNG KNH HIN VI
1. GII THIU CC LOI KNH HIN VI.
1.1. Knh hin vi nn en.
Nguyn tc: nh sng thng chiu t di ln, qua ra ca t quang nn en, chiu
ht vo xung quanh tiu bn, nhng tia sng ny b tiu bn lm khc x ri a vo vt
knh. Tiu bn c soi sng rc trn nn en, ging nh trong phng ti c mt tia
sng mnh chiu vo, gip thy r tng ht bi trong khng kh b tia sng chiu vo.
Chc nng: quan st hnh thi v c tnh ca mt s vi khun m knh hin vi
thng kh quan st.
1.2. Knh hin vi i pha.
Nguyn tc: nh sng thng b i pha v bin dao ng bi cu trc c bit
ca t quang knh, vt knh v th knh.
Chc nng: dng quan st r nt cc cu trc nh nh tin mao, cc lp mng.
1.3. Knh hin vi hunh quang.
Nguyn tc: chm tia t ngoi chiu vo tiu bn nhum mu bng cc
cht hunh quang. Trong t bo, cc cu trc khc nhau s pht quang vi mu sc khc
nhau.
Chc nng: quan st v phn bit cu trc t bo vi sinh vt.
1.4. Knh hin vi in t.
Nguyn tc: chm tia in t bc sng rt ngn, nng sut phn li rt ln nn
phn gii cao, gip phn bit 2 im rt gn nhau.
Chc nng: dng quan st virus, cu trc phn t ca t bo.
1.5. Knh hin vi quang hc
1.5.1. Nguyn tc
Dng nh sng c bc sng t 500nm 560 nm trong chm nh sng thng
to nng sut phn ly ln gip phn bit 2 im cch nhau khong 0.2 m tr ln. H thng
phng to ca knh hin vi quang hc gm hai b phn: Vt knh (quay v pha vt quan st)
40
v th knh (quay v pha mt nhn). Mi b phn ny l mt h thng thu knh hi t

phc tp, mu vt cn quan st AB c t trc vt knh mt khong cch ln hn tiu
c ca vt knh mt cht. nh tht o ngc AB ca vt s thu c bn kia
vt knh, nm trong khong tiu c ca th knh. Th knh hot ng nh mt knh lp.
Qua th knh, ngi ta s thy nh o AB c phng to ln ca nh tht AB.
Hnh 9.1: Nguyn tc quang hc ca knh hin vi.

1.5.2. Chc nng
Quan st t bo vi sinh vt, k sinh trng, t bo ng vt, thc vt.
1.5.3. Cu to
Hnh 9.2: Cc b phn KHV quang hc dng gng.
41
Hnh 9.3: Cc b phn KHV quang hc- dng n.

1.5.4. Cch s dng.
Trc khi s dng phi kim tra tt c cc b phn ca knh, dng khn mm
lau cc b phn ca knh (Ch : ch lau bn ngoi, khng tho ri cc b phn ca knh),
knh ngay ngn va vi tm ngi, sau tin hnh tun t cc bc tip theo soi tiu
bn.
Bc 1: Ly nh sng.
Bc 2: t tiu bn vo mm knh.
Bc 3: Quan st.
42
Hnh 9.4: Hnh v v hnh chp knh hin vi cc vi nm.
Staphylococcus spp. Streptococcus spp.
43
Bacillus spp. Lactobacillus subtilis.
E.coli : KHV X100 KHV IN T

Hnh 9.5: Hnh chp knh hin vi cc vi khun.
2. CC PHNG PHP NHUM MU

2.1. PHNG PHP LM TIU BN TM THI
2.1.1. Cch lm tiu bn git p
- Dng que cy hoc ng ht ly ging vi sinh vt lm vt bi.
- t l knh ln git canh trng tht nh nhng trnh khng to thnh bt kh.
Mun vy mt mp l knh tip xc vi phin knh ri t t h l knh xung.
- a tiu bn ln quan st trn knh hin vi.
2.1.2. Cch lm tiu bn git treo
Loi tiu bn ny dng theo di s sinh sn, s hnh thnh bo t, kh nng di
ng v phn ng ca t bo vi sinh vt vi cc loi kch thch
44
- Dng phin knh c bit c phn lm hnh trn gia.

- Bi vaseline quanh phn lm ca phin knh.
- Cho 1 git canh trng ln gia l knh.
- Thn trng xoay ngc l knh cho git canh trng xung pha di ri t ln
phn lm ca phin knh.
2.1.3. Cch lm tiu bn nhum mu
a. Nguyn tc
Phng php ny s dng thuc nhum khng hoc t c vi vi sinh vt v c
pha long nng m bo cho t bo vi sinh vt vn sng v hot ng sau khi nhum
mu.
b. Cch nhum
+ Nh 1 git thuc nhum xanh methylen 0,001% ln phin knh.
+ Nh 1 git canh trng vi sinh vt vi thuc nhum.
+ y l knh ln git dch.
+ Quan st tiu bn vt knh (x 10) ri (x 40)
2.2. PHNG PHP LM TIU BN C NH
Nhum t bo vi khun dng phn bit hnh dng t bo vi khun (cu,
trc, xon) hay phn bit cc thnh phn ring bit trn mt t bo vi khun.
C 2 phng php nhum c bn:
- Nhum n: Dng mt loi thuc nhum quan st hnh dng t bo.
- Nhum kp: Dng 2 thuc nhum phn bit vi khun G+ v G-, phn bit t bo
dinh dng v bo t, hay phn bit vi khun lao vi cc vi khun khc...
2.2.1. Nguyn tc chung lm tiu bn nhum
- Pht knh tiu bn
Lau nh tiu bn sch bng giy mm, h qua n cn.
Dng bt ch m hoc bt lng ghi tn mu, v v vng trn khong 15mm, mt
di lame knh nh du vt khun pha trn lame.
t nng que cy (trc v sau khi thao tc), m nt bng, h nhanh ming ng
nghim.
45
Trng hp 1: mu nui cy trong canh dinh dng, a u que cy vo ming

ng nghim (vn gi gn ngn la), nhng vo dung dch canh cy, ly 1 vng que cy.
Ly que cy ra, h nhanh ming ng nghim v nt bng, y nt bng li. Pht canh khun
trn vng que cy vo mt trn lam, gia vng trn, dn u ra xung quanh.
Trng hp 2: mu nui cy trong thch dinh dng, nh git dung dch NaCl
9 ln gia vng trn (mt trn lam). Thao tc ging trng hp 1, nhng dng cnh
vng trn ca u que cy t nh ln khun lc vi khun, ri t vo git NaCl 9 trn
lam, dn mng v u.
- C nh mu
Mc ch git cht vi khun v lm cho vi khun bm cht vo lame. C nh mu
bng cch kh t nhin.
Ch :
Nu c nh khng tt vi khun s tri i trong qu trnh nhum.
Khi c nh mu, ch h nhanh ch khng t trn ngn la.
Khng c chm vo thnh khi a u que cy vo v ra khi ng nghim vi
khun.
46
Hnh 9.6: S th t pht knh.
2.3. CC PHNG PHP NHUM.

2.3.1. Phng php nhum gram (Christian Gram):
a. Nguyn tc:
Do s khc bit v cu trc vch t bo nn trong qu trnh nhum Gram, vi
khun Gram [+] s gi c phc hp tm Gentian-iode khng b ty mu bi alcool,
trong khi vi khun Gram [-] khng gi c phc hp mu ny, do vy kt qu sau khi
nhum l vi khun Gram [+] vn gi c mu tm ca gentian, cn vi khun Gram [-] n
mu hng ca phm mu safranin hay fushin.
b. Thao tc:
- t tiu bn pht knh v c nh mu ln thanh thy tinh ch U, trn thau nha.
- t ming giy lc ln vng pht knh.
47
- Nh dung dch Crystal violet thm t ht giy lc. t 1 2 pht (nu vi khun
ly t canh lng 2 pht, ly t thch dinh dng 1 pht). Ra nc, thm kh.
- Ty cn 960 t 15 30 giy (t canh lng ty 15 giy, t thch dinh dng ty 30
giy). Ra nc, thm kh. Ty cn bng cch nghing tiu bn, cho cn chy t t
mp trn phin knh. Quan st mp di cho n khi git cn va mt mu tm.
- C inh
mau tim b ng iodine la dung dich
lugol.
- t ming giy lc ln vt khun, nh dung dch Fuschin kim long (hoc Safranin
O), 1 pht. Ra nc, thm kh.
- Quan st bng vt knh du, phng i 1.000 ln.
Vi khun Gr+ bt mu tm Crystal violet
Vi khun Gr- bt mu hng Fuschin (Safranin O)
Hnh 9.7: S th t nhum Gram

c. c kt qu:
- Quan st pht nhum Gram qua knh hin vi, di vt knh du, chng ta s thy vi
khun Gram [+] n mu tm, cn vi khun Gram [-] n mu hng.
- Khi tr li mt kt qu nhum Gram, phi tr li cc chi tit sau:
+ Hnh dng vi khun.
+ Cch sp xp cc vi khun.
+ Cch n mu ca vi khun, tc l vi khun Gram [+] hay Gram [-].
48
2.3.2. Phng php nhum Ziehl Neelsen:

Dng phn bit vi khun Lao vi cc vi khun khc
Do t bo vi khun khng acid c lp v sp bao bc nn khi nhum c phc
hp mu carbolfuchsin, phc hp ny s khng b ty mu bi dung dch ty mu mnh
(acid, alcool acid). Tuy nhin phc hp mu ny c th thm xuyn qua lp v sp ca vi
khun, c hai cch:
- un nng pht nhum vi dung dch mu carbolfuchin, nh carbolfuchsin thm
qua lp v sp ca vi khun nhum mu vi khun; y l phng php nhum nng
Ziehl Neelsen.
- Phng php th hai l phng php nhum lnh cn gi l phng php Kinyoun,
trong phng php ny ngi ta dng dung dch carbolfuchsin m c, nh vy khi ph
dung dch mu m c ny ln pht nhum vi thi gian lu, carbolfuchsin vn c th thm
qua lp v sp nhum mu vi khun.
2.3.3. Nhum bo t
Mt s vi khun nh Bacillus hay Clostridium c kh nng to thnh bo t. Bo t
c th tn ti trong mi trng thiu cht dinh dng, chu c nhit v mt s cc ha
cht m th sinh dng khng th.
3. THC HNH
Quan st hnh thi vi sinh vt c phn l p va nui c y t cac bai trc.
V li hnh quan st
Vit bo co
49

1. S liu bao cao
1.1. K t qua quan sat hnh thi vi nm trn knh hin vi:
Mu vi sinh vt 1 2 3
.. . ..
Hnh dng
1.2. K t qua quan st hnh thi vi khun

Mu 1 2 3
.. .
Hnh dng
Bt mu ( )
Nhn xt kt qu:
...............................................................................................................................................
...............................................................................................................................................
...............................................................................................................................................
...............................................................................................................................................
...............................................................................................................................................
...............................................................................................................................................
...............................................................................................................................................
...............................................................................................................................................
...............................................................................................................................................
...............................................................................................................................................
2. Cu hi n bi:
2.1. Trnh by thao tc s dng knh hin vi quang hc? nu s khc bit khi thao tc
quan st vi nm v vi khun, ti sao?
50
2.2. Ti sao phi nhum Gram khi quan st vi khun? Phn loi vi khun bt mu
nhum Gram?
2.3. Trnh by thao tc nhum Gram?
3. Yu cu bi hc
- Tr li cu hi
51
PH LC
A. HA CHT.
1. Cn Ether T l v th tch 3:1.

ong 750 ml cn 960 + 250 ml ether, ta c dung dch cn ether t l 3:1. Dung
dch ny dng ty sch vt knh du, hoc cc dng c dnh du soi.
2. Cn acid (dng nhum Ziehl Neelsen )

Cch pha: ong 97 ml cn 960 + 3 ml acid HCl hoc H2SO4 m c.
3. Dung dch sunfo cromic: K2Cr2O7 + H2SO4

Cng thc:
K2Cr2O7 60g
H2SO4 m c 66ml
Nc 1000ml
Cch pha:
Cn 60g K2Cr2O7, ho vo 500ml nc. Thm t t 66ml H2SO4 m c, cui cng
li thm 500ml nc na. Bicromate kali s tc dng vi acid sulfuric v lm sinh ra acid
cromic. Cht ny c tc dng oxi ha mnh do c th ty sch cc vt bn trn dng c
thy tinh.
Thi gian ngm dng c thy tinh t 1 2 ngy, x tht k bng nc sch, ra
bng savon bt, ri ra li bng nc sch.
Dung dch ny c th dng nhiu ln cho n khi dung dch t mu cam bin
thnh mu lc en th b i.
Lu : Dung dch ny rt c. Do khi ngm phi y kn, lc ra nn eo khu
trang, mang gng cao su v knh bo him.
52
4. Cn 700:
Dng st khun tay, bn , gh, t cy,
Cch pha: cn 700 t cn 960. V khng i hi chnh xc, nn c th p dng cng
thc:
V1C1 = V2C2
5. Nc mui sinh l 9 v khun:

Cn chnh xc 9g NaCl tinh khit cho vo cc c cha 991ml nc ct (va
1000ml ), dng a thy tinh khuy u. em hp v khun t0 = 1210C, 1atm, 15 20
pht.
B. THUC NHUM.
1. Crystal violet (C25H30N3Cl . 9H2O = 570,11 ):
Cng thc:
a) Crystal violet 0,4g
Cn 960 10ml
b) Phenol 1g
Nc ct 100ml
Cch pha: Trn hai dung dch a v b li vi nhau, khuy cho ha tan u ri em lc.
Ch : dung dch thuc nhum ny lun bo qun trong chai mu, trnh nh sng.
2. Lugol:
Cng thc:
KI 2g
Iod tinh th 1g
Nc ct 300ml
Cch pha: Ha 2g KI vo 5ml nc ct, sau thm 1g iod. Ch cho iod tan ht mi
thm nc va 300ml.
53
3. Fuchsine kim ( C19H18N3Cl = 323,82 ):

Cng thc:
a) Fuchsine kim 0,3g
Cn 960 10ml
b) Phenol 5g
Nc ct 35ml
Cch pha:
Trn dung dch a v b vi nhau, khuy cho tan u em lc.
Bo qun trong chai mu.
Trc khi dng pha long 5 ln (dch pha long khng gi c lu cn dch c c
th gi c trong nhiu thng ).
4. Safranin O ( C20H19N4Cl = 350,80 ):

Cng thc:
Safranin O ( dung dch 2,5% trong cn 960 ) 25ml
Nc ct 75ml
5. Methylene blue ( C37H27N2S2 = 799,80 ):

Cng thc:
a) Methylen blue 3g
Cn 96% 30ml
b) Dung dch KOH 0.01% 1000ml
Cch pha: Trn hai dung dch a v b li vi nhau khuy ho tan u em lc.
54
MI TRNG
I. MI TRNG NUI CY NM MC V NM MEN.

1. Mi trng Sabouraud: ( nui cy nm mc v nm men)
Cng thc:
Pepton 20g
Mantose hay glucose 40g
Agar 18g
Nc ct va 1000ml
( pH: 5,5 6,0 kh khun trong ni p sut t0 = 1210C (1atm)/ 15- 20 pht).
2. Mi trng PGA
Thnh phn mi trng PGA:
Khoai ty 300g
Glucose 20g
Agar 15g
B sung khong 500ml nc my vo khoai ty un si k v lc ly dch lc, sau
b sung ng v agar vo hp kh trng. S dng acid lactic chnh pH n 5,5 c ch
s pht trin ca vi khun. Kh trng 1210C trong 15 pht
Mi trng PGA sau khi hp kh trng ngui n nhit 40-460C v a
ngui.
II. MI TRNG NUI CY I VI VI KHUN.

1. Mi trng Nutrient Broth (NB):
Cng thc:
Cao tht 5g
Pepton bt 10g
NaCl 5g
Nc ct 1000ml ( pH: 7,4 7,6 ).
55
Cn 13g bt mi trng NB ha vo 1000ml nc ct, khuy u. em hp kh

trng 1210C (1am)/ 15 20 pht.
2. Mi trng Nutrient Agar (NA):
Thnh phn mi trng nh mi trng NB nhng c b sung 18g agar trong
1000ml mi trng.
Cn 13g bt mi trng NB + 18g agar ha vo 1000ml nc ct, ri khuy u.
Kh khun trong ni p sut t0 = 1210C (1atm)/ 15 20 pht.
3. Mi trng thch bn lng di ng:
Cng thc:
Nutrient broth 13g
Agar 5g
Nc ct 1000ml pH: 7,2
Pha ch: em cc thnh phn ca mi trng trn ha tan trong nc ct, ri un
cch thy cho agar ha tan u. Phn vo trong cc ng nghim 16 x 120 mm, mi ng
khong 5ml. em hp kh khun 1210C (1atm)/ 15 20 pht. Ly ra ng cho mi
trng ng li.
4. Mi trng Plate Count Agar (PCA):

Thanh ph n:
Trypton 5g
Yeast extract 2.5g
Glucose 1g
Agar 15g
Nc ct 1000ml
Pha ch: em cc thnh phn ca mi trng trn ha tan trong nc ct, ri un
cch thy cho agar ha tan u. em hp kh khun 1210C (1atm)/ 15 20 pht. Phn
ph i vao cac ia petri, mi ia d y khong 10 - 20mm, ngu i cho mi trng ng li.
56
5. Mi trng Brilliant Green Bile Lactose Broth (BGBL)

Peptone 10g
Lactose 10g
M t bo 20g
Brilliant green 0,0133g
Nc c t 1000 ml
Hoa tan peptone va lactose vao trong 500ml nc c t, cho m t bo vao trong khoang
200ml va brilliant green vao trong khoang 100ml nc c t. Tr n u 3 dung dich
va thm
nc u 1000 ml, chinh pH khoang 7,4. Rot vao ng nghi m co ng Durham 10ml mi
trng BGBL. H p 1210C, 1atm trong 15 phut.
6. Mi trng Lauryl Sulphate Broth (LSB)

Tryptose 20g
Lactose 5g
Sodium chloride 5g
KH2PO4 2,75g
K2HPO4 2,75g
Lauryl sulphate 0,1g
Nc c t 1000ml
Hoa tan cac ch t vao 1000 ml, chin h pH khoang 7,4. Rot vao ng nghi m co ng
Durham 10ml mi trng LSB. H p 1210C, 1atm trong 15 phut.
7. Mi trng Violet Red Bile Agar (VRB ho c VRBA)
Cao n m men 3g
Peptone ho c gelysate 7g
NaCl 5g
Mu i m t 1,5g
Lactose 10g
Neutral red 0,03g
57
Crystal violet 0,002g

Agar 15g
Nc c t 1000ml
Hoa tan cac thanh ph n trong nc, i u chinh pH 7,4. un si trong 2 phut, khng
h p kh trung. S du ng vao mi trng ia
8. Mi trng Potato Glucose Agar (PCA):
- Thnh phn mi trng:
Khoai ty 300g
Glucose 20g
Agar 15g
Pha ch mi trng: B sung khong 500ml nc my vo khoai ty un si k v

lc ly dch lc, sau b sung ng v agar vo hp kh trng. S dng acid lactic chnh
pH n 5,5 c ch s pht trin ca vi khun. Kh trng 1210C trong 15 pht.
58
TI LIU THAM KHO

1. Trn Linh Thc (2005), Phng php phn tch vi sinh vt trong nc, thc
phm v m phm, NXB Gio dc.
2. Nguyn Vn Minh, Dng Nht Linh, Gio trnh THC TP VI SINH C S,
Trng i hc M Thnh ph H Ch Minh, Khoa Cng ngh sinh hc.
3. Nguyn Th Sn, Trn L Minh, Ti liu hng dn th nghim VI HA SINH
NG DNG TRONG CNG NGH MI TRNG, NXB Bch Khoa H Ni.
4. S tay phn tich t, nc, phn bon, cy tr ng.
59
BNG TRA MPN
BNG TRA MPN DNG CHO LOT 3 NG NGHIM 3 NNG
PHA LONG LIN TIP
S lng ng dng tnh S S lng ng dng tnh S

S ml mu s dng MPN/100ml S ml mu s dng MPN/100ml
10 1 0,1 10 1 0,1
0 0 0 - 2 0 0 9
0 0 1 3 2 0 1 14
0 0 2 6 2 0 2 20
0 0 3 9 2 0 3 26
0 1 0 3 2 1 0 15
0 1 1 6 2 1 1 20
0 1 2 9 2 1 2 27
0 1 3 12 2 1 3 34
0 2 0 6 2 2 0 21
0 2 1 9 2 2 1 28
0 2 2 12 2 2 2 35
0 2 3 16 2 2 3 42
0 3 0 9 2 3 0 29
0 3 1 13 2 3 1 36
0 3 2 16 2 3 2 44
0 3 3 19 2 3 3 53
1 0 0 4 3 0 0 23
1 0 1 7 3 0 1 39
1 0 2 11 3 0 2 64
1 0 3 15 3 0 3 95
1 1 0 7 3 1 0 43
1 1 1 11 3 1 1 75
1 1 2 15 3 1 2 120
1 1 3 19 3 1 3 160
1 2 0 11 3 2 0 93
1 2 1 15 3 2 1 150
1 2 2 20 3 2 2 210
1 2 3 24 3 2 3 290
1 3 0 16 3 3 0 240
1 3 1 20 3 3 1 460
1 3 2 24 3 3 2 1100
1 3 3 29 3 3 3 -
BNG TRA MPN DNG CHO LOT 5 NG NGHIM 3 NNG

PHA LONG LIN TIP
S lng ng dng tnh S S lng ng dng tnh S

S ml mu s dng MPN/100ml S ml mu s dng MPN/100ml
10 1 0,1 10 1 0,1
0 0 0 0 4 0 2 20
0 0 1 2 4 0 3 25
0 0 2 4 4 1 0 17
0 1 3 2 4 1 1 20
0 1 0 4 4 1 2 25
0 1 1 6 4 2 0 20
0 2 2 4 4 2 1 25
0 2 3 6 4 2 2 30
0 3 0 6 4 3 0 25
1 0 1 2 4 3 1 35
1 0 2 4 4 3 2 40
1 0 3 6 4 4 0 35
1 0 0 8 4 4 1 40
1 1 1 4 4 4 2 45
1 1 2 6 4 5 0 40
1 1 3 8 4 5 1 50
1 2 0 6 4 5 2 55
1 2 1 8 5 0 0 25
1 2 2 10 5 0 1 30
1 3 0 8 5 0 2 45
1 3 1 10 5 0 3 60
1 4 0 11 5 0 4 75
2 0 0 5 5 1 0 35
2 0 1 7 5 1 1 45
2 0 2 9 5 1 2 65
2 0 3 12 5 1 3 85
2 1 0 7 5 1 4 115
2 1 1 9 5 2 0 50
2 1 2 12 5 2 1 70
2 2 0 9 5 2 2 95
2 2 1 12 3 2 3 120
2 2 2 14 5 2 4 150
2 3 0 12 5 2 5 175
2 3 1 14 5 3 0 80
2 4 0 15 5 3 1 110
3 0 0 8 5 3 2 140
3 0 1 11 5 3 3 175
3 0 2 13 5 3 4 200
3 1 0 11 5 3 5 250
3 1 1 14 5 4 0 130
3 1 2 17 5 4 1 170
3 1 3 20 5 4 2 225
3 2 0 14 5 4 3 275
3 2 1 17 5 4 4 350
3 2 2 20 5 4 5 425
3 3 0 17 5 5 0 250
3 3 1 20 5 5 1 350
3 4 0 20 5 5 2 550
3 4 1 25 5 5 3 900
3 5 0 25 5 5 4 1600
4 0 0 13 5 5 5 1800
4 0 1 17 - - - -
HNG DN BO CO TH NGHIM
I. CCH LY MU
II. CC CH TIU PHN TCH
III. PHNG PHP PHN TCH
IV. KT QU
NHT K LM VIC
NGY MI S KT QU GHI CH
TRNG LNG
GIO VIN HNG DN
VU NGC DUNG

GIAOTRINHTHUCHANH 1 3zd2o

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V SINH C NHN V NGUYN TC LM VIC

H V TN SINH VIN MSSV

BI 1: CC QUY TC PHNG TH NGHIM V

1. CC QUY TC PHNG TH NGHIM

2. K THUT PHA CH MI TRNG

Hinh 1: Mt s dang mi trng trong ng nghi m va ia petri

S mi trng cha dng n phi bo qun ch mt, nhit t 0 - 50C, khng

3.2. Ha cht v mi trng

HNG DN VIT BAO CAO BA I 1

IA PETRI IA PETRI IA PETRI

IA PETRI (PCA) IA PETRI (PCA) IA PETRI (PCA)

BI 2: NH LNG TNG VI KHUN HIU KH

- Dng c ly mu, cha mu phi v trng.

Hnh 2.1: Phng php pha long mu nc theo dy thp phn

b. Nu mu nghin cu trng thi c (nh t, lng thc, thc phm)

- Ly mu t: mu t mang phn tch c tnh i din nn s lng mu ty theo

Hnh 2.2: Phng php pha long mu c theo dy thp phn

Hnh 2.3: Minh ha k thut tri a

HNG DN VIT BAO CAO BA I 2

1. K THUT MI TRNG VI KHUN K KH

Rt mi trng nhit 40-460C vo ng nghim c cha mu. Rt mi trng

Sau khi kh trng 1210C trong 15 pht, mi trng c pH 7.0 7.2.

Mi trng sau khi hp kh trng ngui n nhit 40-460C. B sung vi

HNG DN VIT BAO CAO BA I 2

2.2. Ha cht v mi trng

Pha ch mi trng: B sung khong 500ml nc my vo khoai ty un si k v

HNG DN VIT BAO CAO BA I 4

1. CC PHNG PHP NH LNG VI SINH VT

Hnh 5.1: Din tch hnh vung pht knh

V = th tch git VK pht knh

Hnh 5.2: Cu to bung m

Hnh 5.3 Cu to khung m

- m s t bo trong 5 ln (4 cnh, 1 gia), m ln lt tng nh

Trong : a = s t bo trung bnh c trong 1 nh (V = 1/ 4.000 mm3)

1.4. Phng php MPN (Most Probable Number)

Bc 1: Ly mu v pha long mu n 3 nng lin tip thch hp.

HNG DN VIT BAO CAO BA I 5

1.2. K t qua m b ng phng phap MPN

Cn mi trng tng hp hay thnh phn sau thm nc nc ct thch hp ri un

HNG DN VIT BAO CAO BA I 6

BI 7: CC PHNG PHP PHN LP,

2. PHNG PHP PHN LP VI SINH VT

Hnh 7.1: K thut hp ria

2.1.2. K thut hp tri

Hnh 7.2: Cch dn vi sinh vt trn b mt mi trng bng que gt Drigalxki.

- M a petri, t nh nhng thanh gt ln b mt thch ca a petri. Dng u

2.2.2. Phng php cy trn thch nghing

Hnh 7.3: Cc kiu cy chuyn trong ng nghim

2.2.3. Phng php cy trn thch ng

Hnh 7.4: K thut cy trn thch ng

3. CC PHNG PHP NUI CY V BO QUN VI SINH VT

Phng php gi ging trn t, ct: Dng bo qun cc chng to bo t tim

4. DNG C, HA CHT V MI TRNG

HNG DN VIT BAO CAO BA I 7

BI 8: QUAN ST HNH THI VI SINH VT

v th knh (quay v pha mt nhn). Mi b phn ny l mt h thng thu knh hi t

Hnh 9.1: Nguyn tc quang hc ca knh hin vi.

Hnh 9.2: Cc b phn KHV quang hc dng gng.

Hnh 9.3: Cc b phn KHV quang hc- dng n.

Hnh 9.4: Hnh v v hnh chp knh hin vi cc vi nm.

Staphylococcus spp. Streptococcus spp.

Bacillus spp. Lactobacillus subtilis.

E.coli : KHV X100 KHV IN T