CHNG 1

CC PHNG PHP XC NH VI SINH VT TRONG THC PHM

CHNG 1

MT S PHNG PHP XC NH

VI SINH VT

I. 1. HA CHT V MI TRNG PHN TCH VI SINH VT

-Trong phn tch, kim nghim vi sinh vt, mi trng cn cho vic tng sinh, phn lp, phn bit, nhn

ging, cy chuyn, bo qun, nh danh vi sinh vt

- Mi trng cn cha y cc thnh phn v

ngun carbon, m, khong v vi lng v c cc

iu kin l ha thch hp

PHN LOI MI TRNG NUI CY VSV

* Theo ngun gc

- T nhin
- Tng hp - Bn tng hp * Theo trng thi vt l - Lng (Brothe)

- Rn
- Bn lng (bn rn)

PHN LOI MI TRNG NUI CY VSV

* Theo mc ch

- Mi trng tin tng sinh

- Mi trng tng sinh - Mi trng chn lc - Mi trng phn bit - Mi trng th nghim sinh ha

PHNG PHP PHA CH MI TRNG

Mi trng t pha ch Chn b nguyn liu: Cn, ong thnh phn Pha ch: Ha tan, b sung agar (mi trng rn) Lc iu chnh pH Phn phi vo dng c Hp kh trng (1210C/30p)

 Mi trng ng kh: c s dng ph bin trong cc phng kim nghim vi sinh Cn lng mi trng B sung nc (theo t l), ha tan Phn phi vo dng c cha Hp kh trng (1210C/15)
Note: Thng thng mi trng c pha ch c gi tr pH thch hp, khng cn phi hiu chnh pH sau khi c pha ch .

Vi khun gram dng trn mi trng chn

lc CAN (Colistan Nalidixic Acid Agar )

S. epidermidis v S. aureus trn mi trng chn

lc phn bit Mannitol Salt Agar (MSA)

Mi trng kim tra kh nng sinh Indol

Mi trng kim tra kh nng ln men Dextrose (Glucose)

I.2. K THUT LY MU, VN CHUYN MU V BO QUN MU THC PHM

K HOCH LY MU THC PHM

Mt k hoch ly mu chi tit cn c cc yu t sau: n,

c, m, M
- n: s mu th - c: s mu c php nm trong khong ln cn gii hn (m c M) - m: khong chp nhn (mt vi sinh <m)

- M: khong khng chp nhn (mt vi sinh >M;

thng thng 10 x m M

V d: Tiu chun v tng s vsv trong sp trng c

thanh trng Pasteur: n=5, c=2, m= 5.104, M= 106

CFU/25g

- K hoch 2 thuc tnh - Ch nu ra m - Nu tt c cc mu m hoc c mu trong n mu th >m: Chp nhn l hng

- Nu >c mu trong s n mu th >m: t chi l

hng

- K hoch 3 thuc tnh - t ra c m v M - Ch tiu m thng phn nh ngng trn ca GMP - Ch tiu M nh du gii hn trn (nguy him/khng chp nhn. - Nu tt c cc mu m hoc c mu trong n mu th nm trong khong >m v M: Chp nhn l hng - Nu >c mu trong n mu th nm trong khong >m v M Hoc c mt mu >M: T chi l hng

- La chn k hoch ly mu - K hoch 2 thuc tnh: i tng vsv quan tm khng c php c trong thc phm; - K hoch 3 thuc tnh: Nu cho thc phm mt s lng nht nh vsv trong mt n v th tch.

K HOCH THU MU THC PHM

Mu:

- Phi mang tnh i din (nhiu thi im, vi tr)

- /v mu tp bit: s dng que bng v trng

qut 1 din tch b mt nht nh or ct lt 2-3mm)

- Trnh s tp nhim vi sinh vt ngoi lai (cha mu trong cc bnh bng nha c np v trng) - Ghi ch mu: thi gian, nhit , ni ly mu, phng tin vn chuyn,

Phng php ly mu tht

Ni ly mu S lng mu cn ly1

Thi im ly mu

Qui m nh

Qui m va

Qui m ln

Ch tiu VSV kim tra

CSGM tru b

Sau khi khm a thn tht i hoc trc khi c thu thp pht.

tht, trc khi tiu th/s ch 7lm lnh, mu 1 - 3 4 - 6 12 trong vng 30

- VKHK TS - Enterobacteriaceae

- Salmonella

CSGM ln

Sau khi khm tht, trc khi a thn tht i tiu th/s ch hoc trc khi lm lnh, mu c thu thp trong vng 30 pht.

1-3 4-6

712

- VKHK TS - Enterobacteriaceae - Salmonella

CSGM Sau khi khm tht, trc khi cu, d, a thn tht i tiu th/s ch ch, nga hoc trc khi lm lnh, mu c thu thp trong vng 30 pht.

1- 3 4 - 6

712

- VKHK TS - Enterobacteriaceae - Salmonella

Ni ly mu CSGM gia cm2

S lng mu cn ly1 Thi im ly mu Qui m nh Qui m va Qui m ln

Ch tiu VSV kim tra - VKHK TS - Enterobacteriaceae - Salmonella

Sau khi a thn tht i lm lnh t nht 1,5 gi (c trong kho lm lnh chng hoc sau khi treo thn tht li trn dy) hoc trc khi a i tiu th/s ch.

1-3

46

7 - 12

- Campylobacter

C s pha lc/s ch

Sau khi lc xng, bt u lm lnh hoc ng lnh tip theo, thu thp mu l cc 4ming tht pha lc, tht s ch 1 - 3 6 hoc tht xay trc khi bao gi chn khng hoc bao gi kn.

- VKHK TS - E.coli 7 - 12

- Salmonella

- VSV khc khi yu cu

C s bo qun (Kho lnh)

Trong kho lnh, ti thi im Cho 1 kho lnh ly - VKHK a lnh bo qun mu. Mu ly l tht 5 n v mu ti 5 - Enterobacteriaceae lnh hoc lnh ng ti 5 v tr. - Salmonella im: 4 gc v 1 gia. Gp li l 1 mu.

Cc sn phm lnh ng:

- Lau cn pha ngoi PE

- t vo khay trng men v trng

- a vo bung v trng r ng t nhin (T0 phng); 2-50C (18h); 450C (15 pht)

Mu hp: - Kim tra hp c b phng, bin dng, h - Lau chi bng cn bn ngoi

- a vo phng v trng kim

Cc sp bao b, nylon, thy tinh

- Kim tra kn ca bao b

- Lau cn pha ngoi

- a vo phng v trng kim

Trong qu trnh vn chuyn: tch (0-40C)

VN CHUYN V BO QUN MU THC PHM

- Mu phi c bo qun -200C cho n khi phn

- Mu phi xt nghim trong vng 36h (6h)

- Vi sinh vt c th b tn thng nn trong nhiu trng hp cn giai on tng sinh.

CHUN B MU THC PHM

- Gii ng mu trc khi phn tch . k v trng (2-50C trong 18h, 450C trong 15 pht kt hp lc bnh cha mu tng tc gii ng v ng nht nhit trong mu)

- ng nht mu
. k v trng, mu lng (lc k), mu rn (stomacher)

- Cn mu
. k v trng, sai s cho php 0,1g . nh tnh: 10g; nh lng: 25g

Stomacher

SP c hon ton:
10g(25g) mu nghin nt trong ci s v trng Erlen c cha sn bi thy tinh v 90ml (225ml) nc ct v trng hoc nc mui sinh l lc u, lng cn ht phn dch cy mu, pha long 10-1 (10-2)

Cc sp va c va lng:
Cn 100g mu (c ci ln nc) ci nh v trng, nghin cn 10g (dch nghin) Erlen (90ml NMSL + bi thy tinh) lc u, lng cn ht phn dch cy mu.

Sp lng hon ton:

- S dng trc tip mu hoc pha long ty theo mc nhim bn. - Lc k bnh cha mu, ht 10ml mu cho vo Erlen c sn 90ml nc ct hay nc mui sinh l tit trng ( pha long 10-1)

- Dung dch pha long: Tt hn nn dng nc pepton 0,1%; nc mui sinh l (0,85%), trong buffer nu cn.

I. 3. TH NGHIM SINH HA

Trong k thut phn tch vi sinh vt - nh danh vi sinh vt: da vo

- Cc c im v kiu hnh
- Cc p/ sinh ha c thc hin bi cc chng vi sinh vt - Cc p/ sinh ha c th thc hin theo 3 cch: - Truyn thng - B KIT - Thit b t ng

TRUYN THNG

Th nghim kh nng ln men

Xc nh kh nng s dng mt ngun carbon nht nh bi vsv tng trng theo con ng ln men Sp (ru, acid hu c, CO2) to thnh lm gim pH mi trng thay i mu mi trng
CO2 to ra c by bng ng durham lm ni ng.

Th nghim kh nng ln men

+ + -

Mi trng s dng: Phenol Red broth base: (pH 7,4) vng (pH 6,8)
Dng nh danh cc vsv thuc h Enterobacteriacea (vk ng rut): E.coli, Klebsiella, Staphylococcus (+); Corynebacterium (-)

Th nghim kh nng oxy ha ln men

(TN Hugh Leifson) Xc nh vi sinh vt d dng cn cung cp ngun carbon di dng cht hu c, thng l carbonhydrate. Vsv s dng CBH ny lm ngun nng lng theo con ng ln men hay h hp

Qu trnh ln men thng to mi trng c tnh acid cao hn qu trnh h hp.

Mi trng Hugh- Leifson c ch th pH l bromothymol blue (pH = 6.0: vng; pH= 7,6: xanh dng)

Th nghim Hugh Leifson

- 2 ng nghim cha mt hugh-Leifson; 1 ng c ph paraffin lng v trng trn b mt mi trng.

- Cy m su vsv, 24-48h trong cng k Sau p/: - Ln men: c 2 ng b oxh u t trn b mt v phn su ca mt - H hp: ng k kh b oxh u t trn b mt xung phn su ca mt; ng hiu kh ch b acid ha phn y

Th nghim Hugh-Leifson

Th nghim Bile Esculin

Phn bit vi sinh vt da trn kh nng thy phn lin kt glucoside trong esculin thnh esculentin v glucose Esculentin p/ vi mui st to phc hp mu nu hay en MT: Aesculin Medium hay Edwards Sau p/: ha nu hay en (+); ko i mu (-)

Th nghim Bile Escusin

Th nghim kh nng bin dng citrate

Citrate c s dng lm ngun carbon duy nht ca vsv.

Sp bin dng citrate thay i tu vo pH mi trng pH kim: acetate v formate pH trung tnh: acetate v CO2 pH acid: acetoin v lactate Mt Simmons citrate cha cht ch th mu Bromothymol blue pH trung tnh c mu xanh lc, pH kim c mu xanh dng (pH>7,6). Sau p/: mt c mu xanh lc (-); xanh dng (+)

Mi trng Simmons citrate agar c cht ch th mu bromothymol blue.

Sau phn ng: Xanh lc (-); xanh dng (+)

Th nghim kh nng bin dng malonate

- Mi trng manolate broth c cha bromothymol bue

Sau p/: Xanh lc (-); xanh dng (+)

Th nghim catalase
Phn bit vi sinh vt hiu kh v vi sinh vt k kh VSV hiu kh c enzyme catalase c kh nng phn gii H2O2 H2O v O2

Th nghim:
H2O2 30% (gi lnh) nh 1 git sinh khi vsv ln 1 git H2O2

Staphylococcus Streptococcus

Si bt (+): Bacillus, Micrococcus,

Khng xut hin bt kh (-): Clostridium,

Th nghim catalase

Th nghim decarboxylase
Phn loi v nh danh cc loi vi khun ng rut da trn loi enzyme decarboxylase xc tc phn ng phn gii 1 a.a c trng CO2 tng pH mi trng C 3 loi enzyme: ODC, LDC v ADC (ADH) Mi trng Decarboxylase basal medium cha ch th bromocrezol purple v 1 loi a.a (ornithin, lysin, arginin) Sau p/: Vng (-), tm (+)

Th nghim decarboxylase

Th nghim coagulase nh danh Staphylococcus, loi ny c kh nng tit enzyme coagulase lm kt t cc thnh phn huyt tng to khi ng huyt tng Th nghim c thc hin vi huyt tng th hay ngi dng ng kh Tin hnh trn phin knh/ng nghim

Sau p/ (khong 4 pht)

Xut hin m ngng kt: + Ko c p/ ngng kt: -

Th nghim Coagulase

Th nghim urease
Xc nh kh nng to enzyme urease ca 1 s vsv, c bit l nhm Proteus

Enzyme ny xc tc p/ phn gii ure thnh NH3 v CO2 tng pH mi trng

Mi trng lng Rustigian Stuarts Urea broth hay mt c Christensen Urea cha ch th phenol Sau p/: khng i mu (mu vng cam): -; tm:+

Th nghim urease

++

++++

Th nghim genlatinase nh gi kh nng tit enzyme genlatinase phn gii genlatine thnh polypeptide v a.a Mi trng nui cy vsv c b sung gelnatine hoc nui cy vsv trn mt gelatine c b sung 5-10ml trichloacetic acid TCA l kt ta gelatine.

Sau p/:
ng nghim thch nghing b tan chy: + Khng thay i trng thi: * Chng vsv: Aeromonas hydrophyla (+); E.coli (-)

Th nghim gelatinase
+

Th nghim kh nng sinh H2S

Xc nh kh nng phn gii cc a.a cha lu hunh (cystein, cystin, methionin) H2S nh enzyme desulfuahydrase H2S to kt ta mu en vi ch th sulfide (Fe II, Fe III, amonium sulfate st ) cha trong mi trn. Mi trng th nghim: KIA, TSI, SIM, PIA, BSA Sau nui cy: Xut hin mu en (+), ko xut hin mu (-) VSV: E.coli (+), Klebsiella (-), Proteus mirabilis (-)

Th nghiem kha nang sinh H2S

Th nghim kh nng sinh indol

Xc nh kh nng chuyn ha cc sp trung gian ca qu trnh oxy ha thnh indol.

Indol c xc nh nh p/ vi thuc pdimethylaminobanzaldehide to phc quinon co mu MT th nghim: nc trypton, MIU, SIM; thuc th l Kovacs v Erlich Sau p/: xut hin lp mu trn b mt mt (+), mu vng (-) VSV: E.coli (+), Klebsiella (-), Serratia marcescen (-), Proteus rettgeri (+)

Th nghim kh nng sinh idol

Th nghim kh nng sinh idol

Th nghim KIA, TSI

Mi trng KIA, TSI s dng ng thi th nghim kh nng s dng cc ngun carbon khc nhau (glucose v lactose) v kh nng sinh H2S. KIA cha 1%lactose, 0,1% glucose, cht ch th Phenol red; TSI c thm 1% succrose
Trn mt KIA: - glucose (+): B mt c mu , phn su mu vng - glucose, lactose: c b mt v phn su u c mu vng - H2S: c mu en trong thch v c vt nt - Th nghim HsS nh Na2SO3 trong tp mi trng v

Th nghim KIA, TSI

Th nghim nitrase (kh nitrate)

nh gi kh nng s dng enzyme nitratase kh nitrate thnh nitrite v cc sp khc

Nitrit to thnh s c nhn bit nh phn ng vi sulphanilamide v N-napthylenediamide hydrochloride pH acid cho phc cht mu hng Mt nitrate broth, sodium nitrate Sau p/: khi thm thuc th vo v acid ha mi trng

Mu hng: +
Khng xut hin mu hng: -

Th nghim nitratase

Th nghim oxidase

Nhm xc nh s hin din ca h enzyme oxidase vsv

Hot tnh oxidase c pht hin nh thuc th pphenylenediamin, nu c s hin din ca enzyme, thuc th b oxy ha indolphenol c mu xanh dng

Mi trng th nghim: nutrient aga; ly 1 t sinh khi t trn 1 tm giy lc c tm thuc th pphenylenediamin Sau th nghim: Xanh dng (+); khng i mu (-)

Th nghim oxidase

Th nghim ONPG
Xc nh hot tnh enzyme -galactosidase tham gia vo qu trnh ln men lactose vsv.

Mi trng th nghim: ONPG broth cha onitrophenyl D galactopyranoside, sp to thnh onitrophenol c mu vng VSV: Serratia marcescens (+), Proteus rettgeri (-)
Sau p/: mu vng (+); ko i mu (-)

Th nghim ONPG

Th nghim MR (Methyl Red) Phn bit vsv da vo s khc bit trong qu trnh to v duy tr cc sp c tnh acid t s ln men glucose. VSV ln men glucose to v duy tr cc sp c tnh acid lm i mu thuc th. Nu sp acid tip tc cc sp trung tnh: khng i mu thuc th Cht ch th mu pH mt l methyl red Sau th nghim: mt chuyn mu (+), mt khng i mu (-) VSV: Proteus rettgeri (+), Serratia marcescens (-)

Th nghim MR (Methyl Red)

_ + + +

Th nghim VP (Voges-Proskauer)

Th nghim VP (Vosges- Proskauer)

Phn bit cc loi vk trong h ng rut Enterobacteriaceae, da vo s oxh acetoin c to ra t 2,3 butadiol thnh diacetyl. Diacetyl c xc nh da vo p/ kt hp vi guanidine ca pepton to phc mu

MTTN: MR-VP
Thuc th: Barritt, Koblentz cha -naphton v KOH

Sau p/: mu (+), ko thay i mu (-)

VSV: E. coli (-), Enterobacter cloacea (+)

Th nghim cAMP
Cyclic adenosine monophosphate (cAMP, cyclic AMP or 3'-5'-cyclic adenosine monophosphate)

- Phn bit cc nhm Streptococcus. - P/ CAMP thc hin gia nhn t CAMP ca Streptococcus nhm B tit ra v -hemolysin do Staphylococcus aureus lm tng hot tnh ca hemolysin gy ph v hng cu (tan huyt) Th nghim ng thi chng staphylococcus aureus v chng th nghim, 2 ng cy vung gc cch nhau 2mm
-

Th nghim CAMP

- Mttn: Tryptose Blood Agar Base -

Sau p/: c vng tan huyt (+), khng c vng tan huyt (-) VSV: Strep. agalactiar (-), Strep nhm B (+)

Th nghim tnh di ng

KHNG TRUYN THNG

1. Phng php pht quang sinh hc ATP trong gim st v sinh

2. Phng php Elisa (Enzyme-linked ImmunoSorbent Assay) 3. Phng php lai phn t (Hybridization) 4. Phng php PCR (Polymerase Chain Reaction)

5. Mt s phng php th nhanh khc

- K thut phn tch v tng mt - K thut mng petri (petrifilm)

- K thut Redigel
- K thut dn in, tr khng (conductance/impedance) - KT o vi lng calorie (microcalorimetry) - KT o mc phng x

1. Phng php pht quang sinh hc ATP trong gim st v sinh - Phn t ATP c mt trong tt c cc t bo sng, s pht hin ATP nhn bit cc cht sng tn ti. - ATP c pht hin nh lng nh sng pht ra thng qua s kt hp vi 1 enzyme luciferase nh mt my o nh sng - u im: nhy (1pg ATP = 10-12g), nhanh (vi pht) - Nhc im: t tin, ha cht n nh s pht sng, cng knh ( c s ci tin mang i c)

C ch phn ng
Luciferase + Luciferin + ATP Luciferase-Luciferin AMP + PPi Luciferase-Luciferin AMP + O2 Oxyluciferin + AMP + CO2 + hv (pht quang)

Dng c qut b mt

My o sng

Dng c qut b mt Clean - Trace

2. Phng php Elisa (Enzyme-linked ImmunoSorbent Assay)

PP elisa - Nguyn tc: S dng khng th n dng ph ln ngoi nhng a ging. Nu c khng nguyn mc tiu trong mu, khng nguyn ny s c gi li trn b mt ging. Nu b sung khng th th cp c gn enzyme nh horseradish peroxidase.

3. Lai phn t (Hybridization) Nguyn tc: da vo s bin tnh tch mch ca phn t DNA ti nhit nng chy Tm thnh 2 mch n (pp o c) -Nu gim nhit t ngt: khng c s ti bt cp tr li - Nu ta gim nhit t t th c s ti bt cp ca cc mch n DNA: cc phn t lai

- c im ca lai phn t c hiu tuyt i: s ti bt cp ch xy ra gia hai trnh t hon ton b sung Cc trnh t b sung c th l DNA, RNA dn n hnh thnh cc phn t lai DNA-DNA, DNA-RNA, RNA-RNA

ng dng lai phn t pht hin vi sinh vt trong thc phm? - nh lng vi sinh vt v c t - S dng mu d (probes), trn th trng c cc b kit (clostridium botulinum Genetrak; Escherichia coli Gene-trak; staphylococcus aureus AccuProbe; )

Southern blot method

Southern blot method

Dot & slot bot method

Dot & slot bot method

4. PHNG PHP PCR (Polymerase chain reaction)

- Phng php PCR dng tng hp DNA da trn khun DNA ban u, khuych i, nhn s lng bn sao ca khun ny thnh hng triu bn sao nh enzyme polymerase v mt cp mi (prime) c hiu cho on DNA ny (Karl Mullis v cng s, 1985)

- Phng php PCR cho php tng hp rt nhanh v chnh xc tng on DNA ring bit. y l phng php hin i v thun tin cho vic xc nh s c mt ca mt gen ca mt i tng vi sinh vt vi chnh xc cao.

- Hin nay pp PCR c s dng pht hin, to t bin gen, chn on bnh, pht hin mm bnh c trong thc phm - Phn ng PCR gm nhiu chu k lp li ni tip. Mi chu k gm 3 bc: Bin tnh: Mch DNA tch thnh mch n (94950C, 30s) Lai: Cc mi bt cp vi mch khun (40-700C, 30-60s) Ko di: Enzyme polymerase hot ng, tng hp DNA (720C, 30s vi pht) - Chu k 3 bc lp li nhiu ln, sau 30-40 chu k s to ra 106 bn sao

Sau phn ng PCR, DNA c nhum bi ethidium bromide, quan st qua in di sn phm PCR trong gel agarose v quan sat di tia UV

My in di ng

Bn in di

My in di ngang

My lun nhit (My PCR realtime)

Cc thnh phn ca phn ng PCR - DNA polymerase: cn thit tng hp DNA

- DNA mch khun: trnh t DNA mc tiu c trng cho vi sinh vt mc tiu hoc l gen quy nh vic tng hp mt loi c t ca vi sinh vt ny
- Mi: l nhng on DNA ngn c kh nng bt cp b sung vi mt u ca mch khun - Cc loi nucleotid A, T, X, G; nc, dung dch m, ion Mg2+

Quy trnh thc hin:

- Tng sinh: vi sinh vt c nui cy tng sinh trong mi trng chn lc (TSB) 10-20 gi - Thu dch nui cy, tch chit DNA vi sinh vt - Thc hin phn ng PCR

- in di trn gel agarose 1,5%, c kt qu

trn n UV

LOCAD-PTS reader [left] and swabbing unit [right]

IN DI (GEL ELECTROPHORESIS)

IN DI (GEL ELECTROPHORESIS)

in di ngang in di ng

IN DI (GEL ELECTROPHORESIS)

IN DI (GEL ELECTROPHORESIS)

IN DI (GEL ELECTROPHORESIS)