C TNH MT S MI TRNG PHN LP V CHN ON VI KHUN GY

BNH

Trong phng th nghim y t, phn lp v nh danh vi khun gy bnh cn

phi s dng n nhiu loi mi trng nui cy v ho cht thch hp cho mt s
th nghim, c bit l th nghim sinh ho. Bn cnh , vic nh gi cht lng
pha ch mi trng ng quy cch cng l mt trong nhng khu quan trng trong
cng tc xt nghim vi khun.
Cho n nay, c nhiu loi mi trng khc nhau c dng cho phn lp v
nh danh vi khun, v thng c xp thnh 3 loi ch yu l: mi trng nui cy
c bn, mi trng tng sinh vi khun (c giu cht dinh dng) v mi trng nui
cy chn lc. Mi loi mi trng s c s dng vi nhng mc ch khc nhau
trong nui cy, phn lp v nh danh vi sinh vt c trong mu xt nghim ca bnh
nhn.
1. MT S MI TRNG NUI CY THNG THNG
1.1. Mi trng Chocolate agar
Mi trng thch chocolate l mi trng giu dinh dng v thng c
dng cho vi khun Haemophilus, S.pneumoniae v cc loi vi khun kh pht trin
khc. y l mi trng thch mu cu, khi thm mu cu vo mi trng c bn
trong iu kin nhit gii phng t bo hng cu v nicotinamid - adenin
dinucleotid (NAD). Mi trng a c bo qun 40C. a mi trng cy
bnh phm c m nhit 350C trong kh trng c 5% CO2, thi gian 18 24
gi, quan st s pht trin ca vi khun.
1.2. Mi trng Simmons citrate
Mi trng thch Cimmons citrate c s dng cho cc trc khun ng
rut gram m khc nhau. Ging nh nguyn l s dng mi trng acetat ca vi
khun, phn bit cc loi vi khun ngi ta da vo s khc bit do kh nng s
dng citrate nh l ngun carbon duy nht ca chng phn lp. Mi trng c pH
khong 6,9. ng mi trng sau khi cy huyn dch vi khun ln mt nghing, t
m 35oC, quan st s thay i trong 4 ngy.
1.3. Mi trng Deoxychocolate citrate

Thch Deoxychocolate citrate l mi trng chn lc, mi trng khc nhau

dng cho phn lp trc tip cc cn nguyn gy bnh ng rut t phn v bnh
phm nc tiu hoc gin tip t canh thang giu dinh dng nh selenite-F. Cc
thnh phn chn lc c trong mi trng nh citrate natri v deoxycholate natri
phi hp vi nhau c ch cc trc khun ng rut khc m khng phi l cn
nguyn gy bnh nguy him.
Thnh phn khc nhau y l lactose, cn nguyn gy bnh ng rut
khng ln men ng s xut hin cc khun lc khng mu. Nu ln men ng
lactose, xut hin nhng khun lc chuyn t mu hng sang mu , l kt qu
ca s thay i pH trong qu trnh ln men ng lactose.
Cy khun lc ln mi trng a, m 35 0C/48 gi. Cht nui cy m
nhiu nh mu phn, nc tiu, hoc cc cht khc ca c th th c th cy trc
tip vo mi trng, ngc li nu cht nui cy t th nn lm thm mt bc l cy
vo mi trng tng sinh trc ri mi cy vo mi trng chn lc.
1.4. Mi trng Kligler Iron Agar (KIA)
Mi trng thch Kligler Iron Agar c s dng nui cy trc tip cho
trc khun gram m ln men ng glucose, y l c im c bn trong phn loi
bc u ca cc trc khun gram m. Mi trng ny cng ng thi th tnh ln
men ng lactose, sinh hi trong qu trnh ln men carbohydrat v sinh H2S. Tt c
nhng c im c s dng cho cc trc khun gram m thuc h vi khun
ng rut.
Mi trng KIA gm glucose v lactose (c th ln men carbohydrat),
phenol (ch th pH), pepton (ngun carbon/nitrogen) v mui st cng vi thiosulfate
natri. C 3 hnh thi ln men carbohydrat c th xy ra l:
-

Acid (mu vng) phn gc v kim (mu ) phn nghing ca mi trng, khi
cho thy vi sinh vt ln men ng glucose nhng khng ln men ng lactose.

Acid (mu vng) phn gc v acid (mu vng) phn nghing ca mi trng,
khi cho thy vi sinh vt ln men c ng glucose v ng lactose.

Kim (mu ) phn gc v kim (mu ) phn nghing mi trng, khi

cho thy vi sinh vt khng th ln men ng glucose v ng lactose. Cc
sn phm sinh ra khng phi l sn phm acid.
Nu sinh hi, c hi phn gc ca ng mi trng, mi trng b chia ct

hoc y mi trng ln khi y ng. Nu thy mu en phn gc ng, vi sinh vt

 sinh ra hydrogen sulfid gas t thiosulfid. Phn ng c c sau khi nui cy t 18

24 gi, nu mi trng c c qu sm, vi sinh vt ch c th ln men ng
glucose. Nu c qua mun, cc cht gy men lactose c th dng ht lactose v
bt u d ho pepton, phn nghing ca mi trng tr li mu .

1.5. Mi trng MacConkey Agar

Thch MacConkey l mi trng chn lc cho cc vi khun thuc h vi khun
ng rut v cc trc khun gram m khc c trong mt qun th hn tp v phn
bit chng c ln men lactose hay khng ln men lactose. Mui mt v tinh th mu
tm c ch hu ht cc vi sinh vt gram dng nhng cho php cc trc khun gram m
mc c. ng lactose nh mt ngun cung cp carbohydrat duy nht. Trc khun
gram m, nu ln men lactose s sinh ra khun lc mu hng hoc . Ngc li, nu
khng ln men lactose th khun lc khng c mu hoc khun lc c mu trong.
Sau khi cy vi khun ln a mi phn lp, t vo t m 35 0C, thi gian t
18 24 gi. Vi khun c th ln men lactose mnh hoc yu, sau 24 gi nui cy sinh
ra nhng khun lc c mu hoc xut hin mu hng nh sau 24 48 gi. Khng
nui cy ko di qu 48 gi, nh th c th lm o ln kt qu.
Thch MacConkey Sorbitol, c cha cc thnh phn ging nh thch
MacConkey thng thng khc nhng c thm D-sorbitol. Mi trng ny c dng
cho phn lp E.coli O157:H7.
1.6. Mi trng Mueller-Hinton Agar
Mi trng thch Mueller-Hinton Agar l mi trng trong, dng cho th
nghim tnh nhy cm ca vi sinh vt vi khng sinh. Mi trng cng thng c
dng th nghim s thu phn tinh bt. Thch Mueller-Hinton c cha hn dch
ng vt, casamino acid v tinh bt gip cho hu ht cc vi sinh vt pht trin. Hn
na, c th cho thm mu cu vo cng thc c bn thc hin th nghim tnh
nhy cm ca chng Streptococci. Khi un nng mu cu to thnh chocolate vi
thch Mueller-Hinton th c th th nghim vi cc vi sinh vt kh mc, nh
Heamophilus v Neisseria.
1.7. Mi trng dinh dng (Nutrient Agar)

Mi trng thch dinh dng c s dng phn bit gia mt s t loi

Neisseria d mc vi cc loi Neisseria khc, nh N.gonorrhoeae v N.meningitidis.
S t loi Neisseriae d mc th pht trin c trn mi trng thch dinh dng,
ngc li c nhiu loi khng pht trin c. Thch dinh dng cng thng c
dng lm mi trng lu gi chng. Thch dinh dng c cha cc cht dinh dng
ng vt v c bit l hm lng protein thp. Chng phn lp no pht trin c
trn mi trng ny th cng c ngha l chng rt d mc v khng i hi bt c
thnh phn c bit no.

1.8. Mi trng Oxydative-Fermentative (OF)

Mi trng thch Oxydative-Fermentative hoc OF l mi trng lun c
dng xc nh cch s dng carbonhydrat ca vi sinh vt l kiu oxy ho, ln men
hoc tr. y l mi trng rt quan trng nh danh cc trc khun gram m
khng ln men ng glucose nh: Pseudomonas, Acinetobacter spp .
Mi trng c cha mt lng nh pepton ngn cn s to thnh sn phm
kim, v cc sn phm ny c th trung ho mt lng nh sn phm acid qua qu
trnh oxy ho. Lng ln carbonhydrat trong mi trng lm tng s lng acid m
n c th to thnh. Mi trng cng c lng nh thch to cho mi trng khng
rn cng khng lng, to iu kin cho php acid to thnh trn b mt v khuch tn
qua mi trng. Mu xanh bromthymol nh ch th mu pH pht hin acid.
1.9. Mi trng th nghim tnh di ng
Mc ch ca mi trng th tnh di ng l xc nh mt vi khun c th di
ng c hay khng. y l th nghim c bit dng cho nh danh cc thnh vin
ca h vi khun ng rut, thng cho 2 loi Shigella v Klebsiella lun khng di
ng, v cc loi Yersinia chc chn di ng nhit phng nhng khng di ng
350C.
Cc vi khun khng di ng l do khng c lng roi, ch pht trin trong
ng cy v xung quanh mi trng trong. Cc vi sinh vt di ng, s dng lng
roi di chuyn ra ngoi ca ng cy v xut hin m khi trong mi trng.
Lng thch trong mi trng thp lm cho mi trng khng lng, khng cng
cho php pht hin di ng tt nht.

Dng u que cy chc thng vo mi trng, thn trong di chuyn u que

cy trn 1 ng, khng chc que cy xung tn y tupe. mi trng nui cy
350C. Bn cht lng roi l protein, do vy khng to thnh tt nhit cao, mt s
nh sinh vt hc thch nhit t 18 200C.
1. 10. Mi trng thch Salmonella- Shigella (SS Agar)
Thch Salmonella- Shigella thng dng chn lc Salmonella v mt vi
chng Shigella t mu xt nghim phn. Nhng vi sinh vt ny c phn bit trn
mi trng thch SS bng c im khun lc trn mi trng. Thnh phn mi
trng SS c cha mui mt, natri citrate v xanh brillian, c ch cc trc khun
gram dng v rt nhiu trc khun ln men ng lactose thng thng khc tm
thy trong phn. Nu mt vi sinh vt pht trin trn mi trng v ln men lactose,
th n s sinh ra acid v thay i thnh mu - hng. Nu thiosulfate natri tham gia
vo nh mt ngun sulfur cho qu trnh sinh ra hydrogen sulfid. Nu hydrogen sulfid
hin din trong mi trng th s to thnh cht kt ta mu en gia khun lc.
Khun lc Shigella xut hin khng mu trn mi trng SS, v nhng vi sinh
vt ny khng ln men ng lactose hoc sinh hydrogen sulfid. Khun lc
Salmonella khng mu v c im en gia khun lc, v vi khun ny to ln
hydrogen sulfid nhng khng ln men ng lactose. Khun lc chuyn t mu hng
n mu cho thy sinh vt ln men ng lactose, nu c mu en gia th
cng sinh ra hydrogen sulfid. Nu Proteus pht trin trn mi trng ny th kh nng
to thnh nhng m s b hn ch.
1. 11. Mi trng Thiosulfate Citrate Bile Salts Sucrose (TCBS)
Mi trng thch Thiosulfate Citrate Bile Salts Sucrose l mi trng chn lc
dng phn lp cc loi Vibrio t mu xt nghim phn c ln vi vi khun ch
khc. Cc loi Vibrio c phn bit bng c im do khun lc sinh ra trn mi
trng. Cc kiu khun lc khc nhau c sinh ra t cc loi Vibrio khc nhau, th
hin qua s ln men ca sucrose. V d: V.cholerae v V. alginolyticus sinh ra cc
khun lc mu vng, v chng ln men ng sucrose, ngc li V. parahaemolyticus
v V.vulniticus lun sinh ra khun lc mu xanh do thiu ln men sucrose. Vi sinh vt
Vibrio khng sinh ra hydrogen sulfid.
Khi dng thch TCBS, chng ta nn dng cht nui cy c, nhng loi Vibrio
d cht t ngt v mi trng ny c ngn chn c iu . Mu xt nghim

ti l tt nht, v vi sinh vt ny nhy cm vi iu kin kh, nh sng v pH acid.

Nu x l chm th nn s dng mi trng Cary-Blair vn chuyn bnh phm tt
hn l mi trng vn chuyn c m glycerol. a mi trng nui cy nn nhit
350C, thi gian t 18 24 gi, di nht l 48 gi. Khng ly khun lc mi trng
TCBS lm th nghim oxydase.
1.12. Mi trng Esculin Agar
Mi trng thch esculin l mi trng phn bit kh nng thy phn esculin
ca vi khun. Nhng sn phm thu phn t phn ng esculin vi mui st c trong
mi trng gy ra kt ta cc thnh phn iron, v sn phm ca s kt ta lm
i mu ca mi trng t mu xm ti mu en. Cy vi khun ln phn nghing ca
mi trng, 350C trong iu kin hiu kh v quan st s pht trin, mi trng c
mu en cho thy c s thy phn esculin.

1.13. Mi trng Methyl Red-Voges-Proskauer (MR-VP)

Mi trng canh thang MRVP dng cho cc th nghim methyl v VogesProskauer.

Quy

trnh

ny

dng

phn

bit

cc

thnh

vin

thuc

Enterobacteriaceae (h vi khun ng rut), v d: E.coli, methyl dng v

Voges-Proskauer m; Enterobacter aerogene v Klebsiella pneumoniae cho cc phn
ng ngc li. Cc thnh vin thuc h vi khun ng rut c chia thnh 2 nhm
da trn cch chuyn ho ng glucose. Mt nhm sn sinh ra mt lng ln hn
hp acid (lactic, formic, succinic, acetic). Khi cho thuc th methyl vo mt trong
cc vi khun thuc nhm ny, mu xut hin do pH c tnh acid. Nhm th 2 sn
sinh ch yu cc sn phm trung tnh, acetoin hoc acetyl-methylcarbinol.
Th nghim methyl, canh khun phi c m 48 gi. Ly vo mi ng
nghim 0,5 n 1 ml canh khun, 350C, thi gian 18 24 gi l c th thc hin
c th nghim. Trong qu trnh th nghim, lc ng nghim canh khun hp th
oxy v tng thm phn ng.
1.14. Mi trng Blood Agar
Mi trng thch mu (Blood Agar) l mi trng thng xuyn dng nui
cy cc vi khun khng kh mc lm. Mi trng c pha ch hoc thch triptic soy

c bn c lm giu thm bng cch thm 5 10% mu cu, mu th hoc mu

ngi ph v si fibrin. S kt hp ca mu khng ch cung cp cht dinh dng
cho vi khun pht trin m cn cho php pht hin c tnh tan mu ca vi khun.
1.15. Mi trng Mannitol Sald Agar
Mi trng thch Mannitol l mi trng nui cy chn lc c bn dng
tng sinh v nh danh Staphylococci t mu xt nghim c ln vi vi khun ch khc.
m mui cao (7,5%) c tc dng c ch hu ht vi khun gram m v gram
dng tr Staphylococcus spp. Vi khun Staphylococcus aureus c kh nng ln men
ng mannitol, sinh ra sn phm acid lm pH ca mi trng gim, phenol t mu
chuyn sang mu vng, khun lc in hnh ca Staphylococcus aureus xut hin
mu vng. Nhng loi Staphylococcus v Micrococcus khc thng khng ln men
ng mannitol, do sinh ra khun lc mu hi .
Sau khi cy vi khun ln a mi trng, m 35 0C trong kh trng khng c
CO2, thi gian 24 48 gi. Mt s chng Staphylococcus aureus c th ln men
ng mannitol chm, do vy khng nn loi b mi trng nui cy trong vng
48 gi. Cc chng ngh n l S.aureus cn c lm thm cc th nghim nh
coagulase hoc bng mt thng quy chun khc.

1.16. Mi trng Selenite F

Canh thang Selenite F l mi trng tng sinh cho s t thnh vin ca
Salmonella v mt vi chng Shigella t phn v mu xt nghim cha s lng ln vi
sinh vt hp. Mi trng c Selenite natri c ch rt nhiu trc khun gram m
v cu khun ng rut (Enterococci) nhng cho php tng sinh Salmonella v mt
vi loi Shigella. Selenite hiu qu nht l trung ho pH, trong qu trnh pht trin ca
vi khun lm gim Selenite, cc sn phm kim ho c sinh ra, cho nn
ng lactose cng c trong mi trng ny. Cc cht ln men ng lactose sinh ra
acid, v cc sn phm kim c trung ho v tr li mi trng trung ho pH.
1.17. Mi trng Thayer-Martin, Modified (MTM)
Thch Modified Thayer-Martin l mi trng giu dinh dng dng tng sinh
Neisseria gonorrhoeae v N. meningitidis t mu xt nghim c cc vi khun ch hp.
Mi trng thch Modified Thayer-Martin giu dinh dng, do vy nhiu loi Neisseria

kh mc cng c th pht trin c. Mi trng c cc yu t pht trin nh

hemoglobin, vitamin, cocarboxylase, diphosphopyridin nucleotid v glutamin.
Cng thc ca Modified cha nhiu thch ngn chn s lan to ca
proteus. Thch Modified Thayer-Martin c cha mt s khng th, c ch vi khun ch
thng thng v ngn chn s pht trin ca hu ht cc vi khun nh vancomycin
c ch cu khun gram dng pht trin, colistin c ch trc khun gram m v
nistatin ngn chn s pht trin ca nm. a mi trng MTM sau khi cy vi
khun, c m trong kh trng c CO2 hoc bnh nn trong vi ngy.
1.18. Mi trng Peptone-Yeast Extract-Glucose (PYG)
Canh thang Peptone-Yeast Extract-Glucose l mi trng c dng cho nui
cy vi khun. C th dng thng quy ghi sc k lng pht hin cc sn phm
chuyn ho cui cng trong canh cy PYG phn lp vi khun k kh. Canh thang PYG
c cha mt s cht dinh dng v thnh phn b sung kch thch vi khun k kh
pht trin. Thnh phn dinh dng gm vitamin K (cho sinh sc t Prevotella v
Porphyromonas), cao men, hemin v glucose. Cht Resazurin p ng nh cht ch
th vi khun k kh. Khi mi trng xut hin mu hng ngha l c hin din ca oxy.
1.19. Mi trng Lowenstein-Jensen (LJ)
Mi trng Lowenstein-Jensen c dng nui cy Mycobacterium spp.
Trong mi trng hu ht c cha thnh phn c th c ch s pht trin ca
Mycobacteria. Bt khoai ty, trng v glycerol c trong mi trng LJ c tc dng
gip kh c mi trng ny, ng thi cung cp cht dinh dng cho vi khun ny
pht trin. Mi trng LJ m bo tt trong 1 thng nu vn cht np, ni kh ro
v nhit t 4 60C. Mi trng LJ cng phi c gi ni ti, v xanh malachite
rt nhy vi nh sng. Bnh phm c nui cy ln mi trng, m trong kh
trng CO2 (5 10%), t 6 10 tun. Mt iu quan trng l phi ni lng np cho
thay i khng kh bn trong.
2. CC TH NGHIM TRN MI TRNG THNG THNG
Mi trng nui cy c th c chun b t nhng thnh phn ring hoc
chun b t mi trng di dng bt kh c thng mi ho. Ngy nay, mi
trng dng bt kh c thng mi ho tr ln ph bin ti cc phng th

nghim vi khun lm sng. Mt s im quan trng kim sot cht lng ca mi

trng nh sau:
-

Khng dng mi trng ht hn s dng;

Hp mi trng cn c vn cht bng np trnh m;

Gi mi trng vo ni ti, thong kh, mt;

Khng s dng mi trng dng bt kh b ng mu hoc vn cc. Thng

xuyn o mi trng d tr theo nguyn tc: ngoi vo, trong ra.

Khi chun b mi trng cn phi tun th nghim ngt ch dn ca nh

sn xut.

Khng mi trng c pha ch ngoi nh sng v nhit phng.

Kim tra tit trng v pH ca mi trng chun b.

Bng 1. Cc th nghim trn mt s mi trng thng dng

Mi trng

T m

Thch mu
(Blood Agar)

Sinh vt kim tra

S. aureus

Thch MacConkey
vi crystal violet

Methyl red/VogesProskauer

Pht trin v tan mu

hon ton

24h, CO2
S.pneumoniae

Thch Chocolate

Kt qu mong i

24h, CO2

24h

Pht trin v tan mu

khng hon ton

H.influenzae

Pht trin

E.coli

Khun lc c mu

P.mirabilis

Khun lc khng mu

E.faecalis

Khng pht trin

E.coli

(+)/(-)

K.pneumoniae

(-)/(+)

48h

E.coli ATCC 25922

Mueller-Hinton

ln ca vng v
khun

24h
Agar

P.aeruginosa ATCC

ln ca vng v

27853

khun

Peptone water
(indole)

Thch Simmons
citrate

K.pneumoniae

(-)

E.coli

Khng pht trin

Pht trin v sinh sc t

K.pneumoniae

24h

(TCBS)

Thayer Martin Agar

(+)

48h

Thch Thiosulfate
citrate bile salt

E.coli
24h

sanh nc bin

Vibrio spp.( Khng

Khun lc mu vng

dnh)
N.meningitidis

Pht trin

24h,

N.gonorrhoeae

Pht trin

CO2

Staphylococci

Khng pht trin

E.coli

Khng pht trin

Bng 2. Gim st cht lng ca cc th nghim thng dng

Quy trnh

Vi sinh vt

Phn ng mong i

S. aureus

Phn ng si bt

Streptococcus spp.

Khng si bt

S. aureus

To thnh vn cc sau 4 g

E. coli

Vng mu trn b mt

E. aerogenes

Vng mu vng trn b mt

E. coli

Lp tc c mu

E.. aerogenes

Khng i mu

P.aeruginosa

Mu tm trong vng 20 giy

E. coli

E. aerogenes

Catalase

Coagulase

Indole

Methyl red

Oxidase

Voges

Khng mu trong vng 20

giy
Mu

Proskauer
a

khng

Bacitracin

khng

Optochin

E. coli

sinh Streptococcus group A +

Khng i mu
C vng c ch

E. faecalis

C vng c ch

sinh S. pneumoniae

C vng c ch

S. viridans

Khng c vng c ch

P.aeruginosa

Mu tm trong vng 30 giy

E. coli

Khng i mu

a Oxidase

Lu : cc th nghim kim tra cht lng phi c thc hin cho mi ln

pha
dung dch.

mi