I.

HNH NH NHUM GRAM

Shigella c 4 nhm:
+ Shigella dysenteriae

+ Shigella flexneri

+ Shigella boydii

+ Shigella sonnei

II. BNG TNH CHT SINH HA

Loi

Glucose
khng
sinh hi

Manit- Lactos Ureas Indol

ol
-e
-e
e

metyl

VP

Citrate

Di
ng

S.Dysenteri
-ae
S.Flexneri
S.Boydii
S.Sonnei

+
+
+

+
+
+

+
chm

+
+
+

Ch thch: *: S.Dysenteriae type 2 dng tnh, cn li m tnh.

Hnh nh:
Shigella ln men glucose khng sinh hi, ln men
Manitol ( tr Shigella dysenteriae khng ln men
Manitol), hu ht khng ln men Lactose.

Ln men
glucose
Ln men
Mantiol
Khng
ln men
lactose
Khng
sinh H2S

 Khng sinh H2S

Urease m tnh

Phn ng indol thay i

Phn ng Metyl dng tnh

 Phn ng VP m tnh

Phn ng citrate m tnh

III. MT S HNH NH KHUN LC

TRONG TNG MI TRNG:

Shigella boydii trn mi trng Hektoen Enteric Agar.

Shigella sonnei trn mi trng Hektoen Enteric Agar, 48 gi

37C

Shigella flexneri trn mi trng SS Agar.

Shigella flexneri trn mi trng XLD Agar.

\
Shigella sonnei trn mi trng XLD Agar, 72 gi nhit 37C

Shigella sonnei trn mi trng XLD agar, 48 gi 37C

Shigella sonnei trn mi trng Chocolate Agar, 48 gi 37C

Shigella sonnei trn mi trng Macconkey, 72 gi 37C

Shigella sonnei trn mi trng Macconkey, 48 gi 37C

Shigella sonnei trn mi trng Macconkey, 24 gi 37C

Shigella boydii mc trn mi trng Blood agar plate (PAP).

IV. QUI TRNH NH DANH:

4.1 Nguyn tc
Shigella l trc khun Gram (-) kch thc khong 0,4x2 mcm hiu kh
hoc k kh, khng di ng, khng to bo t, khng ln men lactose,
saccharose; ln men dextrose nhng khng sinh hi, khng sinh
ihyrosunfua. Pht hin shigella theo nguyn tc tng sinh mi trng
ph hp, phn lp nhn dng cc c trng v khng nh bng nhum
Gram v cc phn ng sinh ha.

4.2 Trang thit b

a) phng php truyn thng:
-Dng c thit yu : n cn , que cy, a petri
-Giai on tng sinh:
Bnh phm
Canh trytone soya
-Giai on tng sinh chn lc:
Canh tng sinh GN
-Giai on phn lp:
Mi trng HE

 XLD
MAC
Deoxycholate Citrate Agar
-Giai on khng nh bng phng php sinh ha v huyt thanh:
Mi trng khng chn lc (nh TSA)
Simmon citrate
Arginie decarboxylase
Lysine decarboxylase
Urease
Malonate
MR
VP
Salicine
Xylose
Cellobiose
Andonitol
Dulcitol
Inositol
-Khng nh bng th nghim khng huyt thanh:
Nc mui sinh l
Huyt thanh
b) Phng php hin i:
Phng php PCR:
B kch PCR

 Dung dch m PCR

Mi
Nc
..
-Phng php LA( latex agglutiuation)
B kch Wellolex
B kch Bactigen

4.3 i tng thc hin:

Cc i tng thc hin gm c:
- C nhn xt nghim.
- K thut vin xt nghim.
- Ging vin b mn vi sinh.

4.4 An ton sinh hc:

Trong qu trnh nh danh vi khun Shigella cn phi m bo an
ton sinh .
- Phng xt nghim phi m bo an ton sinh hc cp 2. Ch
cc yu cu sau:
+ C s vt cht:
T ng qun o thng v dng c nhn, ch n ung v ngh
ngi phi b tr bn ngoi phng xt nghim.
Bn ra tay c vi nc gn ca ra vo.
C phng tin cu ha, x l s c in.
Vi ra mt khn cp trong khu vc xt nghim.
Hp ng thuc v dng c s cu ban u c trang b thch
hp v sn sng cho s dng.
C h thng in y v n nh, tip t ton b h thng.
Nn c my pht in d phng h tr cc trang thit b thit yu nh
t m, my lnh,
Nn lp t h thng n chiu sng khn cp trong tng hp c
s c mt in nghin cu vin c th ri khi phng xt nghim mt
cch an ton.
Nn c phng tm c vi hoa sen trong khu vc phng xt
nghim x dng trong trng hp khn cp.
+ Thit b:
T an ton sinh hc cp 2
Ni hp t (autoclave)
T m

+ S dng trang thit b bo h:

Mc o chong, hoc ng phc xt nghim trong sut qu trnh
lm trong phng xt nghim.
eo gng tay trong tt c qu trnh tip xc vi bnh phm, ha
cht c nguy c gy hi. Sau khi s dng, tho b gng tay v ra tay
ng cch.
eo khu trang thng hay khu trang c hiu qu lc cao trong
qu trnh lm.
+ An ton trong quy trnh xt nghim:
Thc hin ng thao tc v k thut. Cn thn trong lc tip xc
vi bnh phm v ha cht nguy him. Trnh lm v, ri vi hay c
kh nng phi nhim. Khi xy ra s c phi x l dung quy trnh.
Tt c cc thao tc cn c thc hin theo phng php gim ti
thiu vic to cc git hay dung kh.

4.5 Bnh phm:

Bnh phm phn:
bnh st thng hn nn cy phn tun l 3-4 khi cha dng
khng sinh, nn lp li nu m tnh. Trong ng c do ni c t ca vi
khun, cy phn ngay t tun u cng sm cng tt.
+ Cch ly:
Ly phn ti, tt nht l vng c nhiu nhy mu, cho vo l
sch: rng ming, np vn cht c gn mi cho (dng l ly mu phn
khng F2M) . Phn ti phi ccy trong vng khng qu 2 gi sau khi
ly mu.
Nu cn a ti phng xt nghim xa th cho phn vo dung dch bo
qun:
Glyxcrin
250 ml
Nacl
5g
Nc ct 750 ml
C th dng bng tm nhng vo phn, vng nhy mu, cho vo
mi trng chuyn ch Cary Blair ( dng cp tube a tm bng v
trng/ tube
a Cary Blair) ri gi n phng th nghim. Mi trng chuyn ch
Cary Blair c th gi mu phn trong hn 48 gi. Tuy nhin phi tin
hnh cy cng sm cng tt. Trong trng hp nghi ng bnh nhn b th
t (do vi khun t), c th cho tm bng ly phn vo ng mi trng
pepton kim va tng sinh, va chuyn ch n phng th nghim.
C th ly mu phn bng tm bng qut hu mn ri cho vo
mi trng chuyn ch (dng cp tube a tm bng v trng / tube a
Cary Blair) gi ngy n phng th nghim.

 C th khng dng mi trng chuyn ch nu tm bng ly

phn (dng tm bng v trng ly mu) c nui cy trong vng 30
pht sau khi ly mu.
+ Cy phn:
C th cy phong ph vo mi trng GN.
Cy ngay phn, hay cy t mi trng phong ph cy phn
vo hp thch phn lp : MC(hay EMB) v SS ( hay XLD hay HE).
Cc hp thch phn lp phi c 35-37C trong t m.
Sau 24 gi, chn khm khun nghi ng tin hang nh danh v
lm khng sinh .
+ Phn ng ngng kt xc nhnhm v type huyt thanh: sau khi
nui cy xc nh l Shigella th tin hnh xc nh nhm bng khng
huyt thanh a gi nhm. Nu mt trong nhm khng huyt thanh a gi
nhm ngng kt th tin hnh phn ng ngng kt vi cc khng huyt
thanh n
gi theo tng nhm.

4.6. Tin trnh thc hin

*Phng php truyn thng:
1. Cy bnh phm vo mi trng phn lp:
Tin hnh: Cy mu ln mi trng theo nguyn tc vch ba chiu.
Cc bc thc hin:
Bc 1: Nung u que cy vng, lm ngui trong khng kh
khong 15giy. Cho vng que cy v huyn dch c vi khun cn phn
lp hoc c th ly trc tip t mu.
Bc 2: Tay tri m np hp thch, t u que cy vo mt gc hp
thch chm nh loi bt t bo.
Bc 3: y nh u que cy lt trn b mt thch theo ng zch
zc.
Bc 4: Xoay a v tip tc vch cc ng zch zc tng t sao
choc ac ng khng trng ln nhau nhm to khong trng cho cc
khun lc mc ri nhau.
Bc 5: y np hp thch li.
Bc 6: Kh trng que cy trc khi cm vo gi.

Bc 7: em nhit 37oC trong vng 18-24gi.

2. Chn khun lc nghi ng l Shigella
Quan st khun lc:
Trong mi trng cy SS: Khm khun khng mu, nh, trong,
bin u, v Shigella khng ln men trong ng lactose hoc
khng sinh H2S.
Trong mi trng cy McConKey: Khm khun nh, trong, khng
mu, bin u.
Vi cc mi trng khc nh DC (desoxycholate xitrat), DCLS
(DC c them ng lactose v saccharose), EMB, Endo, Istrati,
Hinton, DLC. Khng mu, trn, bng, nhn. Ring Sh.Sonnei c
khun lc nhn.
Sau khi chn c khm vi khun c c im nghi ng l
Shigella tin hnh cc th nghim sinh ha v huyt thanh hc.
3. Xc nh tnh cht sinh vt ha hc:
Nguyn tc nh danh
a) Da vo c im ca h Enterobacteriaceae

Ln men glucose

Bin i nitrat

Phn ng oxidase (-)

b) Da vo hnh thi hc v sinh l hc

Trc khun gram (-), khng di ng

Ln men glucose khng sinh hi (tr S. flexner type 6)

Khng ln men lactose, tr S. sonnei ln men chm ( ngy)

Khng s dng nitrat, khng sinh H2S

 c) Da vo tnh cht khng nguyn

Phn ng
Mannitol

Ornithine
decarboxylase

S. dysenteriae (A)

S. flexner (B)

S. boydii (C)

S. sonnei (D)

Nhm

Quy trnh tin hnh:

Giai on 1: Xc nh Shigella spp
Cc th nghim:
Trn mi trng nui cy (MC, XLD), chn khm khun lc trong,
b u v khng mu; y l du hiu ca nhm vi khun lactose
(-).
C th thc hin phn ng oxydase xc nh chnh xc khm vi
khun thuc tc Enterobacteriaceae.
Bc 1: Phn ng Oxydase:
Cc vi khun k kh khng c oxydase, cn cc vi khun hiu kh
tuyt i th lun c. Tuy nhin cc vi khun hiu k kh tu tin
th thng khng c oxydase.
pht hin tnh cht ny ta s nh mt git dung dch
imetylparaphenylen diamin ln mt ming giy lc ri ly mt
khun lc ca vi khun ln ming giy m ny. Nu vi khun c
oxydase th ming giy c mu hng tm, cn khng c oxydase th
ming giy khng i mu. Enterobacteriaceae cho phn ng m
tnh.
Bc 2: phn ng KIA
K.I.A l mi trng dng kho st s ln men glucose, lactose, kh
nng sinh H2S.

K.I.A l mi trng c, c iu ch theo th nghing su v c

cht ch th mu l Red Phenol.
a) phn ng oxi ha gc glucose v lactose.
Kt qu: Shigella spp ch ln men glucose, khng ln men lactose.
Phn su thch b acid ha c mu vng, phn nghing b kim ha
c mu .
Gii thch:
Ban u, vi khun ln men glucose, acid ha hai phn nghing v
su, sau v phn nghing vi khun mc nhanh kim ha s bin
dng pepton v thi ra NH3.
b) phn ng sinh H2S
Vi khun thy phn Sodium thiosulfate to thnh kh H2S, kh ny
phn ng
vi st to st sulfate ha tr 3 mu en. Shigella spp khng c
phn ng ny.
Bc 3: phn ng PAD (Phenyl Alanine Deaminase)
Phn ng ny nhm phn bit proteus spp vi shigella spp da trn
nguyn tc men Deaminase kh amin ca amino acid
phenylalanine thnh mt keto acid, cht ny kt hp vi ion st
trong thuc th Ferric chloride 10% cho ra mu xanh l cy.
Men ny c sn sinh bi proteus spp nhng shigella spp th
khng.
Bc 4: phn ng urease
Phn ng ny c thc hin trong mi trng Urea c
(Christensen Urea agar - thch nghing) hoc lng.
Cy khm khun thun khit t mi trng KIA, 37oC trong 1824h.
Dng tnh: mi trng c mu hng . m tnh: khng i
mu .

Bin lun: shigella spp khng c men urease nn cho phn ng m

tnh
Bc 5: th nghim kh nng di ng
Nguyn l: nhng vi khun c lng, chng c th c pht hin
bng cch nui cy vo mi trng thch mm khi chc que cy c
vi khun thnh mt vch thng gia ng
Cy m su vi sinh vt vo mi trng thch mm (0,5% agar).
Vi sinh vt di ng s lm mi trng c,
pht trin lan ra khi vt cy.
Vi sinh vt khng di ng s pht trin quanh
ng cy, mi trng khng b c.

Giai on 2: Xc nh cc loi Shigellla.

Shigella

Ch sinh acid

Mannitol (+)

Mannitol (-)

SH.dysenteriae

Lactose (-)
)

Lactose (+)
)

Indole (-)

Indole (+)

Sh.boydii
)

Sh.flexner
)

S nh danh cc loi Shigella

4. Th nghim vi khng huyt thanh

Thc hin t nghim khng huyt thanh bng huyt thanh a gi A, B,
C, D t cc dng c nui cy trn mi trng thch khng chn lc.
a) Phng php ngng kt trn phin knh:
Cc bc tin hnh:
-Nh mt git khng huyt thanh ln phin knh.
-Dng que cy ly 2-3 khm vi khun cho vo git huyt thanh.
-nh cho khm vi khun tan u.

Sh.sonnei
)

-Quan st,nu c ngng kt xy ra th phn ng dng tnh

Nhm huyt thanh

Tn loi vi khun Shigella

A1A2

Sh.dysenteriae

Sh.flexneri

C1C2C3

Sh.boydii

Sh.sonnei

Lm phn ng ngng kt trn phin knh vi cc khng huyt thanh

khng O ca cc nhm A, B, C, D. Tin hnh cc i chiu m tnh trn
nc mui sinh l.
Nu ngng kt vi khng huyt thanh nhm A th kt lun l S.
Dysenteriae
Nu ngng kt vi khng huyt thanh nhm B th kt lun l S.
flexneri.
Nu ngng kt vi khng huyt thanh nhm C th kt lun l S.
boyii.
Nu ngng kt vi khng huyt thanh nhm D th kt lun l S.
sonnei.
Lu :
Nu mt gc vi khun cho phn ng sinh ha c nghi ng l
Shigella nhng khng cho phn ng dng tnh vi bt c loi
huyt thanh ngng kt no k trn.Nguyn nhn l do mt s
Shigella to thnh khng nguyn b mt (khng nguyn K) c th
lm ch mt khng nguyn O v lm cho phn ng ngng kt
khng din ra. V th khi khng c biu hin ngng kt cn thc
hin vic un si dch vi khun trong khong 30 pht loi b
khng nguyn b mt. Sau tin hnh li th nghim ngng kt.
Nu mt vi khun c nghi ng l Shigella li cho phn ng
huyt thanh ngng m tnh vi huyt thanh ngng kt nhm
shigella sau khi huyn trc vi khun c un nng. Ta ly huyn
trc vi khun cha un lm phn ng huyt thanh ngng kt

Salmonella v c vi gc Samonella khng sinh H2S v khng di

ng.
Mt s chng shigella khng ngng kt vi cc khng huyt thanh
tng ng do c khng nguyn K che ph khng nguyn O. Nu
nghi ng c khng nguyn K th un huyn dch vi khun (ha
trong nc mun sinh l) 100oC trong 20 pht ri lm li phn
ng.

*Phng php PCR:

K thut PCR dng pht hin shigella c tin hnh trn cp mi SHIG
khuch i cho trnh t 320bp trn plasmid xm nhim c hiu cho
Shigella v cp mi 16S khuch i cho trnh t c kch thc 1007bp
nm trong vng bo tn ca 16S rRNA hin din trong mi loi vi khun.
So vi phng php nui cy truyn thng, phng php ny cho hiu
qu chnh xc v hay hn ch, n pht hin nhiu vi khun gy bnh
hn v tn rt t thi gian.Ring i vi Shigella th cho php pht hin
shigella mc 10CFU/25g mu sau 1214h tng sinh,cho kt qu sau
24h.
HT