tCvn T i u c h u n V i t N a m

tcvn 6187-2 : 1996

ISO 9308-2: 1990 (E)


Cht lng nc - Xc nh - Pht hin v m vi khun
coliform, vi khun coliform chu nhit v
Escherichia coli gi nh

Phn 2. phng php nhiu ng
( s c xc sut cao nht )

Water quality - Detection and enumeration of organisms thermotolerant
coliform organisms and presumptive Escherichia coli
Part 2: Multiple tube (most probable number) method



















H Ni - 1996
TCVN 6187-2:1996
2








Li ni u

TCVN 6187-2: 1996 hon ton tng ng vi ISO 9308-2: 1990 (E)
TCVN 6187-2: 1996 do Tiu ban k thut nc tinh lc TCVN/TC/F9/SC1 thuc Ban k
thut tiu chun TCVN/TC/F9 ung bin son, Tng cc Tiu chun- o lng - Cht
lng ngh. B Khoa hc, Cng ngh v Mi trng ban hnh.
TCVN 6187-2:1996
3
Ti u chun Vit Nam TCVN 6187-2:1996


Cht lng nc - Xc nh - Pht hin v m vi khun coliform,
vi khun coliform chu nhit v Escherichia coli gi nh

Phn 2. phng php nhiu ng ( s c xc sut cao nht )

Water quality - Detection and enumeration of organisms thermotolerant
coliform organisms and presumptive Escherichia coli

Part 2: Multiple tube (most probable number) method

1. Phm vi p dng
Tiu chun ny quy nh phngphp pht hin v m s lng vi khun coliform,
coliform chu nhit v escherichia coli gi nh c trong nc bng cch nui cy trong mt
mi trng lng mt h gm nhiu ng nghim v tnh ton "s c xc xut cao nht" ca
chng c trong mu th.
Phng php ny c th p dng cho mi loi nc, k c cc loi nc c cha mt lng
ng k vt cht l lng. Vic la chn cc php th dng pht hin v xc nhn cc vi
khun nhm coliform, k c escherichia coli, c th c xem nh l b phn ca mt dy
lin tip. Quy m ca vic xc nhn vi mt mu th ring bit no , mt phn tu thuc
vo bn cht ca mu nc v phn khc vo nhng l do kim tra.
trong thc t, vic xc nh E.coli gi nh trong nc c nu iu 3.3 ca tiu chun
ny, thng thng l cung cp mt chng c ca s nhim phn.
2. Tiu chun trch dn
ISO 3696:1987 Nc dng cho phng th nghim phn tch. Yu cu k thut v phng
php th.
ISO 5667 -1: 1980 Cht lng nc - Ly mu. Phn 1: Hng dn xy dng cc phng
n ly mu.
TCVN 5992: 1995 (ISO 5667 -2 Cht lng nc - Ly mu. Hng dn cc k thut ly
mu.
TCVN 6187-2:1996
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TCVN 5993: 1995 (ISO 5667 -3 Cht lng nc - Ly mu. Hng dn bo qun v x l
mu.
ISO 6887:1983 Vi sinh hc - Hng dn chung v chun b cc dch pha long kim tra
vi sinh vt.
ISO 8199:1983 Cht lng nc -Hng dn chung v m s lng vi sinh vt bng cch
nui cy.
3. nh ngha
i vi mc ch ca tiu chun ny, p dng cc nh ngha sau:
3.1 Vi khun coliform: L cc sinh vt c kh nng sinh trng hiu kh nhit hoc
35
0
C 0.5
0
C hoc 37
0
C 0.5
0
C trong mt mi trng nui cy c lactoza th lng, km
theo vic to thnh axit v sinh kh trong vng 48 h.
3.2 Cc vi khun coliform chu nhit: L cc vi khun coliform nh m t mc 3.1, c
cng c tnh ln men trong vng 24 h, nhit 44
0
C 0.25
0
C hoc 44,5
0
C 0.25
0
C.
3.3 Escherichia coli gi nh: L cc vi khun coliform chu nhit nh m t mc 3.2
m cng sinh indol t tryptophan trong vng 24 h, hoc 44
0
C 0.25
0
C hoc 44,5
0
C
0.25
0
C.
4. Nguyn tc
Cy cc phn mu th, c pha long hoc khng pha long vo mt dy ng nghim
cha mt mi trng nui cy chn lc dng lng c lactoza.
Kim tra cc ng th sau 24 h v 48 h nui nhit hoc 35
0
C hoc 37
0
C; cy chuyn
tip t mi ng c biu hin c km theo sinh kh vo mt mi trng khng nh chn lc
hn v khi mun tm E.coli gi nh cy vo mt mi trng m qua c th quan st thy
s to thnh indol.
Nui cc mi trng khng nh ny cho ti 48 h nhit hoc 35
0
C hoc 37
0
C pht
hin cc vi khun coliform, v 44
0
C trong khong 24 h pht hin cc loi coliform chu
nhit v E.coli gi nh.
Bng cc bng thng k, tnh ton s xc xut cao nht ca cc dng coliform,
coliform chu nhit v E.coli gi nh c th c mt trong 100 ml mu th, t s cc ng th
kt qu xc nhn dng tnh.
TCVN 6187-2:1996
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5. Dch pha long,mi trng nui cy v thuc th
5.1 Cc dng nguyn liu chnh
Hy s dng cc thnh phn c cht lng ng nht v cc ho cht thuc loi phn tch
pha ch cc mi trng nui cy v thuc th v tun theo cc ch dn cho trong ph
lc B. i vi cc thng tin v vic bo qun, xem trong ISO 8199. Mt khc, s dng cc
mi trng hon chnh dng kh v theo st cc ch dn ca nh sn xut.
pha ch cc m trng, s dng nc ct bng dng c thu tinh hoc nc loi ion,
khng cha cc cht c th c ch sinh trng ca vi khun trong cc iu kin th v ph
hp vi ISO 3696.
5.2 Dch pha long
to nn cc pha long ca mu th, s dng mt trong cc dch pha long c kin
ngh ph luc B. Chun b dch pha long theo ch dn cho trong ph lc B.
5.3 Mi trng phn lp
S dng mt trong cc mi trng nui cy di y, cc hng dn v chun b mi
trng a ra trong ph lc A.
5.3.1 Canh thang lactoza
5.3.2 Canh thang macConkey
5.3.3 Mi tr
2)
ng glutamat-lactoza-format ci tin
2)
5.3.4 Canh thang lauryl- tryptoza (lactoza)
5.4 Mi trng khng nh
S dng mt hoc mt s cc mi trng sau:
5.4.1 Cc mi trng kim tra tnh sinh kh;
5.4.1.1 Cc thang lc sng-lactoza (mt)
5.4.1.2 Mi trng EC
5.4.2 mi trng kim tra tnh sinh indol:

2)
C sn dng thng phm kh "mi trng gutamat ci tin c mui khng". Thng tin ny c a ra vi tnh
tin li cho ngi s dng tiu chun ny v khng to nn mt s xc nhn ca tiu chun v sn phm ny.
TCVN 6187-2:1996
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Nc trypton.
5.4.3 Mi trng ng n, kim tra c tnh sinh kh v sinh indol:
Canh thang tryptoza-mannitol-lauryl c tryptophan.
5.5 Thuc th
5.5.1 Thuc th kovac th indol
5.5.2 Thuc th oxidaza dng cho php th oxidaza.
6. Thit b
Cc thit b thng thng ca phng th nghim vi sinh gm c:
6.1 T sy hi nng kh trng kh v mt ni hp p lc.
Ngoi cc thit b v trng sn, thit b thu tinh v cc trang b khc cn c kh trng
theo ch dn nu trong ISO 8199.
6.2 T m hoc b iu nhit, c th iu chnh c nhit hoc 35
0
C 0.5
0
C, hoc
37
0
C 0.5
0
C.
6.3 T m hoc b iu nhit, c th iu chnh c nhit hoc 44
0
C 0.25
0
C, hoc
44.5
0
C 0.25
0
C.
6.4 pH mt.
7. Ly mu
Lymu v chuyn v phng th nghim theo ISO 8199, ISO 5667, TCVN 5992:1995 (ISO
5667-2) v TCVN 5993:1995 (ISO 5667-3).
8. Cch tin hnh
8.1 Chun b mu th v cc mi trng cy
chun b mu th, tin hnh pha long mu v cyvo mi trng phn lp cc phn
mu th, theo nh cc ch dn cho ISO 8199. i vi cc phn mu th c th tch ln
hn 5 ml, s dng cc ng mi trng phn lp nng kp.
8.2 Nui cc ng th
TCVN 6187-2:1996
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Nui cc ng mi trng cy mu trong 48 h nhit 35
0
C 0.5
0
C, hoc 37
0
C
0.5
0
C.
8.3 Kim tra cc ng th
Kim tra cc ng mu ly sau 18 24 h nui mv coi l c phn ng dng tnh i vi
cc ng c biu hin c do vi khun sinh trng v sinh kh bn trong cc ng ln ngc
(ng Durham), cng vi vic to thnh axit nu trong mi trng phn lp c cha mt ch
th pH.
Nui tip trong t ng nhng ng nghim cha c bt k du hiu no hoc mi bin i
ni trn v kim tra li chng sau 48 h tm phn ng dng tnh.
8.4 Cc php th khng nh
iu quan trng cn lu l cc phn ng dng tnh trong cc ng mi trng phn lp th
ch l kt qu gi nh v coliform. Do cn phi tin hnh cc php th xc nhn, tt hn
l trn cc chng cy thun khit.
8.4.1 Cy truyn, nui m v kim tra
Cy truyn t mi ng mi trng phn lp c kt qu dng tnh vo mt hoc mt s ng
mi trng khng nh (5.4) tm tnh sinh kh v sinh indol.
Ch thch: - Nu mi trng c ch ti thiu nht (canh thanglactoza) c dng phn lp, th
nn cy truyn tip vo c hai trong s cc mi trng khng nh chn lc hn (canh thang lc
sng- lactoza (mt) hoc canh thang EC) khng nh.
8.4.1.1 Vi khun coliform
khng nh s c mt ca vi khun coliforrm chu nhit, nui mt ng mi trng EC
khc (5.4.1) nhit 44
0
C trong 24 h v kim tra tnh sinh kh.
khng nh s c mt ca E.coli gi nh cy vo ng trypton (5.4.2) to indol 44
0
C
trong vng 24h. Sau thm 0.2 ml 0.3 ml thuc th kovac (5.5.1) vo ng nc trypton
( cy mu): Vic xut hin mt mu sau khi lc nh chng t s c mt ca indol._
Ch thch
2. Vic s dng canh thang lauryl-trypo-mannit c tryptophan cho php s hnh thnh indol v sinh
kh do E.coli gi nh c th hin trong mt ng n;
TCVN 6187-2:1996
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3. Vic pht hin E.coli gi nh c xem nh l chng c m bo v s nhim phn. tuy nhin
cc php th su hn khng nh E.coli c th phi c tin hnh nu xt thy cn thit (xem
8.5);
4. Khi cy truyn t cc khun lc trn mng lc vo cc ng mi trng khng nh, tt hn cng
nn cy ln mt a mi trng thch dinh dng th oxidaza.
8.5 Php th oxidaza
Mt vi chng vi khun tm thy trong nc cng c th ph hp vi nh ngha v vi khun
coliform hu ht cc mt, nhng chng ch c kh nng sinh kh t lactoza, nhit
di 37
0
C m thi. Do chng cho cc kt qu m tnh trong php th khng nh chun
i vi vi khun coliform v s hin din ca chng trong nc th thng thng khng
c coi l ng k. Cc chng aeromonas, xut hin mt cch t nhin trong nc, gy
nhiu cho vic xc nh ch nhit 37
0
C v thp hn, vic khng nh bng php th
oxidaza ch cn n khi xc nh coliform.
8.5.1 Tin hnh php th oxidaza vi cc ging cy thun khit t cc vi khun ln men
lactoza, mc trn mi trng thch dinh dng, nh sau:
- nh 2 hoc 3 git thuc th oxidaza (5.5.2) mi c chun b ln mt mig giy lc trong
mt a Petri;
- bng que cy thu tinh, tm bng hoc que cy c dy bch kim (khng phi loi Ni-
crom), dn mng mt t mu cy ln trn giy lc c chun b sn (xem ch thch 4).
- quan st thy xut hin mt mu xanh-tm sm trong vng 10 giy l phn ng dng
tnh.
Ch thch - Trong mi trng hp s dng thuc th oxidaza cn thc hin cc php th kim tra
vi mu cy mt loi vi khun cho phn ng dng tnh v mt loi cho phn ng m tnh (E.coli)
bit trc.
9. Biu th kt qu
T s ng mi trng phn lp v cc php th khng nh cho cc kt qu dng tnh,
tnh ton, bng cch tham kho cc bng tra thng k trong ISO 8199, s xc xut cao
nht ca vi khun coliform chu nhit v E.coli gi nh c trong 100 ml mu th.
10 Bo co kt qu
Bo co kt qu gm nhng thng tin sau:
TCVN 6187-2:1996
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a) tham kho tiu chun ny.
b) mi chi tit cn thit nhn bit y v mu th.
c) k thut v mi trng phn lp dng.
d) cc mi trng v cc php th khng nh dng.
e) thi gian, nhit v cc iu kin nui cy.
f) cc kt qu trnh by ph hp vi iu 9.
g) bt k mt thng tin no khc lin quan ti phng php.
TCVN 6187-2:1996
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Ph lc A
(Tham kho)
Thng tin su hn v vi sinh hc lin quan ti kim tra nc i vi
nhm vi khun coliform
i vi cc mc ch kim tra nc hng ngy, nhm vi khun coliform c th c mt
bng cc thut ng vi sinh chung, khng mang tnh phn loi hc, nh sau:
Cc vi khun coliform l cc vi khun hnh que (dng thng), gram m, khng c bo t,
oxidaza m tnh; chng c kh nng sinh trng hiu kh v k kh khng bt buc trong s
c mt ca mui mt (hoc cc yu t tc ng b mt khc c c tnh c ch sinh trng
tngt). Chngcng c th ln men lactoza v (mannit) km theo to thnh axit, kh v
andehyt trong vng 48 h, khi nui cy nhit 35
0
C n 37
0
C.
Cc vi khun coliform chu nhit l cc vi khun coliform cho thy c cng kh nng ln
men v cc c tnh sinh ho khi nui cy nhit 44
0
C n 44.5
0
C. E.coli gi nh l
cc vi khun coliform chu nhit, chng cng c kh nng sinh indol t tryptophan.
E.coli c th c xem l E.coli gi nh, chng cng c mt kt qu dngtnh trong php
th metyl v c th loi cacboxyl ca axit glutamic, nhng chng khng c kh nng to
ra metyl axetyl cacbonol, s dng xitrat nh ngun cacbon duy nht, hoc sinh trng trong
canh thang kali xyanua (KCN).
TCVN 6187-2:1996
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Ph lc B
(Than kho)
Cc mi trng nui cy, thuc th v nc pha long Mi trng
phn lp
- Canh thang lactoza
Mi trng nng kp:
Pepton 10 g
Lactoza 10 g
Cao tht b 6 g
Nc ct 1000 ml
Ho tan cc thnh phn trn trong nc si. Nu cn, chnh pH sao cho khi kh trng pH =
6.9 0.2.
Pha ch mi trng nng n bng cch pha long mi trng nng kp vi mt th
tch nc ct tng ng.
- Canh thang MacConkey
Mi trng nng kp:
Mui mt 10 g
Pepton 40 g
Lactoza 20 g
Natri clorua 10 g
Bromo cresol (hoc Cresol)
ta dung dch 1% (th tch/ th tch)
pha long trong etanol 2 ml
Nc ct 1000 ml
TCVN 6187-2:1996
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Ho tan pepton, natri clorua v mui mt trong nc bng cchun nng v gi 4
0
C qua
m. em lc khi dung dch vn cn lnh, b sung lactoza v ho tan. Chnh pH n 7,4
0.2 v b sung bromocresol ta.
Mi trng nng n:
Chun b mi trng nng n bng cch pha long mi trng nng kp vi mt th
tch nc ct tng ng, hoc c th lm ring bng cch gim mt na lng cc cht
trong thnh phn.
Phn phi mi trng nng n thnh cc lng 5 ml/mt ng v mi trng nng
kp thnh tng lng 10 ml v 50 ml. Mi ng nghim hoc l thu tinh c dng cn c
mang mt ng ln men ln ngc (durham). Hp p lc 150
0
C trong 10 pht.
- Mi trng format-lactoza-glutamat ci tin
3)
Mi trng nng kp:
Lactoza 20 g
L (+) Mui natri glutamat 12.7 g
L (+) Arginin monohidro clorua 0.048 g
L (-) Aspartic axit 0.04 g
L (-) Xystin 0.04 g Natri format 0.5 g
Dikali hidro photphat 1.8 g
Amoni clorua 5 g
Magie sunfat (MgSO
4
.7H
2
O) 0.02 g
Canxi clorua(CaCl
2
.2H
2
O) 0.02 g
Vy st(III) Xitrat 0.02 g
Thiamin(aneurin hidroclorua) 0.002 g
Axit nicotic 0.002 g
Axit Pantotenic 0.002 g
Bromocressol ta
{ dung dch 1%(khi lng/khi lng)
trong cn etanol} 2 ml
Nc ct 1000 ml

Tin li hn c, nn chun b mi trng ny vi lng l 10 lit hoc nhiu hn. Nu nh
khng phn phi ngay vo cc ng nghim, th lactoza v thiamin ngoi, khng pha,
vag b sung chng ngay trc khi phn phi. cho tin hn, mt vi thnh phn c b

3)
C bn sn dng kh "mi trng glutamat iu chnh khong". Cng c th cn iu chnh PH
TCVN 6187-2:1996
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sung vo mi trng dng cc dung dch ring r v cc dung dch na s c chun b
nh sau:
Dung dch 1:
L (+) Arginin monohidro clorua 0.4 g
L (-) Aspartic axit 0.48 g
Nc ct 50 ml
un nng ho tan hon ton.
Dung dch 2:
L (-) Xystin 0.4 g
Natri hidroxit (5mol/l) 10 g
Nc ct 90 ml
un nng ho tan.
3) C bn sn dng kh ''mi trng glutamat iu chnh khong". Cng c th cn
iu chnh pH.
Dung dch 3:
Axit nicotic 0.02 g
Axit Pantotenic 0.02 g
Nc ct 5 ml
Ho tan m khng un nng.
Dung dch 4:
Vy st(III) Xitrat 0.2 g
Nc ct 10 ml
un nng ho tan.
Dung dch 5:
TCVN 6187-2:1996
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Canxi clorua(CaCl
2
.2H
2
O) 2 g
Nc ct 100 ml
Axit clohtdric m c 0.1 ml
Ho tan khng cn un nng, v kh trng 121
0
C trong 20 pht. Bo qun lm dung
dch gc.
Dung dch 6: Kh trng dung dch thiamin 0.1% trong nc ct.
Tt nht, nn pha ch dung dch ny bng cch: Thm lng cha trong mt ng ampul
(100 ml), mt cch v trng, vo 99 ml vo nc ct v trng. Bo qun dung dch 4
0
C v
loi b sau 6 tun l.
chun b 10 lit mi trng nng kp, ho tan cc lng tng ng ca L (+) mui
natri glutamat, natri focmat, dikali hidro photphat, amoni clorua v magie sunphat trong 9 lit
nc ct nng. Sau b sung ton b cc dung dch 1, 2, 4, v 4 ml ca dung dch 5.
Chnh pH ti 6.8 hoc cao hn nu thy cn thit, sao cho pH cui cng, sau khi hon
chnh v kh trng mi trng, l 6.7. Nu dng cng loi thit b v phng php kh
trng, th s c cng mt s thay i v pH lun xy ra trong qu trnh kh trng. C th
cn n mt vi php th s b to c pH chnh xc trc khi kh trng.
Sau khi chnh pH, thm 20 ml dung dch bromo cresol ta 1% etanol vikhong 810 ml nc
ct, b sung cho n th tch cui cng l 10 lit. Nu lng mi trng ny khng cn s
dng ngay, cho vo cc chai dung tch 500 ml v em hp p lc 115
0
C trong 10 pht.
Khi dng n, b sung lng cn thit cho dung dch lactoza v thiamin (dung dch 6),
cho tan u v sau phn phi thnh tng lng 10 ml v 50 ml. Mi ng nghim v l
c s dng cn mang mt ng ln men ln ngc (ng durham). iu quan trng l cn
bo m sau khi hp v trc khi s dng, ng Durham ny cha y mi trng. Nu
khng s to nn mt kt qu dng tnh gi mo v tnh sinh kh. Kh trng nhit
115
0
C trong 10 pht hoc t vo mt ni hi nc 100
0
C trong 3 ngy, mi ngy 30
pht.
Mi trng nng n:
Chun b mi trng nng n bng cch pha long mi trng nng kp vi mt th
tch mc ct tng ng v phn phi thnh tng lng 5ml/ng nghim c cha ng ln
men ln ngc (Durham). Kh trng nhit 115
0
C trong 10 pht, hoc trong lung hi
nc si 100
0
C trong 3 ngy lin tip, mi ngy 30 pht.
TCVN 6187-2:1996
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Ch thch 6 - Vic b sung 0.1% casein thu phn axit khng c vitamin c th cho cc kt qu
nhanh hn.
- Canh thang lauryl tryptoza (lactoza)
Mi trng nng kp:
Tryptoza 40 g
Lactoza 10 g
Natri clorua 10 g
Dikali hidrophotphat 5.5 g
Kali dihidrophotphat 5.5 g
Natri lauryl sunphat, tinh khit cao 0.2 g
Nc ct 1000 ml
Cho tryptoza, natri clorua, lactoza v mui photphat vo nc v un nng ho tan. B
sung natri lauryl sunphat v trn nh
nhng trnh si bt. Chnh pH n 6.8 0.2. Chun b mi trng nng n bng
cch pha long mi trng nng kp vi mt th tch nc ct tng ng.
Phn phi mi trng nng n thnh tng lng 5 ml, v mi trng nng kp thnh
tng lng 10 ml v 50 ml. Mi ng nghim hi\oc l nh c s dng cn phi c mt
ng ln men ln ngc. Hp p lc 115
0
C trong 10 pht.
Cc mi trng khng nh
- Canh thang lactoza - lc sng (mt)
4)
( kim tra tnh sinh kh)
4) Mi trng ny khng phi lun lun cho cc kt qu lp li c, v nn kim tra cc
c tnh ca n trc khi dng.
Pepton 20 g
Lactoza 10 g

4)
Mi trng ny khng phi lun lun cho cc kt qu lp li c v nn kim tra cc c tnh c ch ca n
trc khi dng.
TCVN 6187-2:1996
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Mt b (kh) 20 g
Lc sng (dung dch 0.1% khi
lng dung dch nc) 13 ml
Nc ct 1000 ml
Ho tan pepton trong 500 ml nc ct. B sung 20 g mt b kh ho tan trong 200 ml
nc ct. Dung dch ny cn c ph khong 7.0 n 7.5. Dng nc ct a th tch
dung dch ln 975 ml. Thm lactoza v chnh pH n7.4. Thm dung dich lc sng v a
th tch chng ln n 1000 ml bng nc ct.
Phn phi vo cc ng nghim c ng ln men ln ngc (ng Durham), mi ng 5 ml, v
hp 115
0
C trong 10 pht.
- Mi trng EC ( kim ta tnh sinh kh)
Tryptoza hoc tryticaza 20 g
Lactoza 5 g
Hn hp mui mt s 1.5 g
Dikali hidropho
2
HPO
4
) 4 g
Kali dihidro photphat (KH
2
PO
4
) 1.5 g
Natri clorua (NaCl) 5 g
Nc ct 1000 ml
pH phi l 6.9 sau khi kh trng. Trc khi kh trng, phn phi vo cc ng nghim
vi cc lng ngp trn ng ln men (Durham) ln ngc, t nht ngp phn no sau
khi kh trng.
- Nc trypton ( th phn ng indol)
Mt vi loi pepton cho cc kt qu tt vi cc php th 35
0
C n 37
0
C th li khng tho
mn vi php th indol 44
0
C. Ngi ta thy tryptol tho mn iu v kin ngh dng
n.
Trypton 20 g
TCVN 6187-2:1996
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Natri clorua 5 g
Nc ct 1000 ml
Ho tan cc thnh phn trn trong nc v chnh pH n 7.5. Phn phi vo tng lng 5
ml v hp 115
0
C trong 10 pht.
Ch thch 7 - Vic b sung 0.1% (khi lng m/m) L hoc DL tryptophan ca th ci thin tnh
nng ca mi trng.
- Canh thang Lauryl -trypto-mannit c tryptophan
(Mi trng ng n kim tra c hai c tnh: sinh kh v sinh indol)
Tryptoza 20 g
Natri clorua 5 g
Mannitol 5 g
Dikali hidropho
2
HPO
4
) 2.75 g
Kali dihidro photphat (KH
2
PO
4
) 2.75 g
Natri lauryl sunphat 0.1 g
L (-) Tryptophan 0.2 g
Nc ct 1000 ml
Cho tryptoza, natri clorua, mannitol, cc mui phot pht v tryptophan vo nc v hm
nng nh ho tan. B sung natri lauryl sunphat v trn nh nhng trnh si bt.
Chnh pH ti 6.8 0.2. Phn phi vo cc ng nghim c cha ng ln men ln ngc
(Durham) trong mi ng 5 ml. Hp 115
0
C trong 10 pht.
Cc thuc th
Thuc th kovac v tnh sinh indol:
P- dimethylamino 5 g
Cn amylic 75 ml
Axit clohidric ( = 1.18 g/l) 25 ml
TCVN 6187-2:1996
18
Ho tan andehyt ni trn trong cn amylic. Cn thn b sung axit c vo. Trnh nh sng
v bo qun 4
0
C.
Ch thch 8 - Thuc th ny cn phi c mu vng nht n nu nht; mt vi mu cn
amylic khng tho mn c v cho mu ti vi andehyt.
- Thuc th oxidaza
Tetramethyl -p -phenylen diamin hidro clorua 0.1 g
Nc ct 10 ml
Thuc th ny khng gi c v do cn phi c pha ch mi lng nh cho mi
ln s dng nu cn n n.
Dch pha long
- Nc pepton (0.1%)
Pepton 1.0 g
Nc ct 1000 g
Ho tan pepton trong khong 950 ml nc. Chnh pH bng dung dch NaOH hoc HCl (1
mol/l), sao cho sau khi kh trng pH l 7.0 0.1. Thm nc ct cho 1000 ml, phn phi
thnh cc lng ph hp v kh trng 121
0
C 1
0
C trong 15 pht.
- Dung dch pepton mui:
Pepton 1.0 g
Natri clorua (NaCl) 8.5 g
Nc ct 1000 ml
Ho tan cc thnh phn trn trong khong 950 ml nc bng nc un si. Chnh pH bng
dung dch NaOH hoc HCl (1 mol/l), sao cho sau khi kh trng pH l 7.0 0.1. Thm nc
ct cho 1000 ml, phn phi thnh cc lng ph hp v hp 121
0
C 1
0
C trong 15
pht.
- Dung dch Ringer- nng mt phn t
Natri clorua (NaCl) 2.25 g
TCVN 6187-2:1996
19
Kali clorua (KCl) 0.105 g
Canxi clorua, khan 0.12 g
Natri hidro cacbonat 0.05 g
Nc ct 1000 ml
Ho tan cc thnh phn trn v phn phi thnh cc lng ph hp. Kh trng bng cch
hp p lc 121
0
C 1
0
C trong 15 pht. pH cui cng phi l 7.0 0.1.
- Dung dch m photphat
Kali hidro photphat (KH
2
PO
4
) 42.5 mg
Magie clorua (MgCl
2
) 190 mg
Nc ct 1000 ml
Cch pha ch:
a) dung dch photphat:
Ho tan 34 g mui photphat trong 500 ml nc ct. Chnh pH ti 7.2 0.5 bng dung dch
NaOH (1 mol/l) v thm nc ct n 1000 ml
b) dung dch magie clorua:
Ho tan 38 g magie clorua trong 1000 ml nc ct.
Dung dch cui cng.
s dng, thm 1.25 ml dung dch photphat a) v 5.0 ml dung dch magie clorua b) vo
1000 ml ncct. Phn phi thnh cc lng ph hpv kh trng bng cch hp p lc
121
0
C 1
0
C trong 15 pht. pH cui cng phi l 7.0 0.1.
Mi trng thch dinh dng:
Cao tht 1.0 g
Pepton 1.0 g
Natri clorua 5 g
TCVN 6187-2:1996
20
Thch 15 g
Cho cc thnh phn trn vo nc v un nng ho tan. Chnh pH ti khong 8.2 bng
NaOH (1 mol/l), v un si trong 10 pht. Lm trongbng cch lc v chnh pH ti 7.2 n
7.4. Phn phi vo cc l dung tch 100 ml v hp 121
0
C 1
0
C trong 15 pht.