BO Y T E

Cao Ding Y te Phu Tho

illllBDIHIIHl
KM.007837

KY THUAT XET NGHIEM

VISINH LAM SANG

NHA XUAT BAN Y HOC

 BO Y TE

KY THUAT XET NGHIEM

VISINH LAM SANG
(Sach dung Dao tao Cit nhan Ky thuat y hoc)
Ma so: DK.01.W.10

N H A X U A T BAN Y HOC
H A N O I - 2006
CHi DAO BIEN SOAN:

Vu khoa hoc Oao tao - Bo Y te

TAC GIA:

TS.BS. Pham Hung Van

THAM GIA TO CHlTC BAN THAO

ThS. Phi Van Tham va Ban thi/ ky HDTOSGK va TLDH

© Ban quyen thuoc Bo Y te (Vu khoa hoc va Dao tao)

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 LOI GIOI THIEU

ThUc hien Nghi dinh 43/2000/ND - CP ngay 30/8/2000 cua Chi'nh phu quy
dinh chi tiet va hudng dan trien khai luat Giao due, Bo Giao due va Dao tao, Bo Y
te da ban hanh chUdng trinh khung dao tao CCt nhan Ky thuat y hoc. Bo Y te to
chiic bien soan bo tai lieu day hoc cac mon hoc cd sd va chuyen mon theo chUdng
trinh mdi nham tting budc xay dUng bo sach chu&n trong cong tac dao tao Cil nhan
Ky thuat y hoc.
Sach ky thuat xet nghiem vi sinh lam sang dUdc bien soan theo chUdng
trinh khung da dUdc phe duyet. Nam 2005, cuon sach nay dUdc Hoi dong chuyen
mon tham dinh sach giao khoa va tai lieu day hoc cua Bo Y te th&m dinh. Bo Y
te thong nhat sii dung la tai lieu day hoc chinh thufc cua nganh trong giai doan
hien nay. Trong thdi gian tii 3 den 5 nam, sach c in dUdc chinh ly, bo sung va
cap nhat. Tai lieu dUdc bien soan gom 21 bai, moi bai co muc tieu hoc tap va cac
noi dung thiet yeu. Trong do, noi dung the hien dUdc cac yeu c&u: kien thiic cd
ban, chinh xac khoa hoc^cap nhat dUdc tien bo khoa hoc hien tai va thuc tien
Viet Nam. Sach dung de dao tao COf nhan Ky thuat y hoc dong thdi cung la tai
lieu tham khao tot cho sinh vien cac chuyen nganh khac va cac can bo y te quan
tam den cong tac xet nghiem.
Vu Khoa hoc Dao tao, Bo Y te xin chan thanh cam dn TS.BS. Pham Hung Van
cac giang vien TrUdng Dai hoc Y DUdc TPHCM da tich cUc tham gia bien soan cuon
sach nay. Cuon sach nay dUdc viet lan dau va dang dan tvfng budc hoan thien nen
noi dung bien soan khong tranh khoi nhufng thieu sot va can dUdc bo xung. Vu
Khoa hoc va Dao tao mong nhan dUdc nhieu y kien dong gop cua doc gia va dong
nghiep de cuon sach nay cang hoan thien hdn.

VU KHOA HOC VA DAO TAO

BO Y TE

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 LOI NOI OAU

Khac vdi thvt nghiem sinh hoa va huyet hoc tai benh vien la cac thCr nghiem
ma ngay hom nay chi co khau lay m&u tren benh nhan la phai lam b&ng tay con t&'t
ca cac cong doan con lai deu co the thtic hien dtfdc tren may til dong, xet nghiem vi
sinh lam sang la mot loai xet nghiem v ln con co da so cac budc thtfc hien phai lam
bang tay va khong chi the la mot xet nghiem doi hoi ngiidi lam xet nghiem phai c6
mot kien thvfc rat he thong va kha toan dien de co the quyet dinh dUdc cac bade di
tiep theo trong tien trinh lam xet nghiem cho den khi co ket qua chung cuoc. Ngay
ca khi co dUdc ket qua chung cuoc, viec danh gia va bien luan ket qua nhu the nao
de hiiu dung nhat cho lam sang cung doi hoi ngiidi lam x6t nghiem phai co nhiing
hieu biet toi thieu ve khia canh vi sinh trong lam sang. Chinh vi nhiing ly do vay
nen vi sinh lam sang la mot xet nghiem cho den ngay hom nay co the noi la cham
phat tren nhat so v6i cac xet nghiem khac tai cac phong thi nghiem lam sang tai
cac benh vien cua chung ta. Trong linh viic giang day cung vay, do sU thieu thon
cac phUdng tien va thiet bi nen cac kien thiic cung nhu thiic hanh vi sinh lam sang
dUdc giang day cho cac sinh vien cung con rat ngheo nan, chap va va thieu he
thong; va cung chinh nhii vay da anh hif6ng them den chat liidng kem coi cua cac
xet nghiem vi sinh lam sang tai cac benh vien hien nay.

Vdi 27 nam kinh nghiem trong giang day cung nhu lam viec trong linh vUc vi
sinh lam sang, tac gia da co" gang tong ket va he thong cac kien thtic c&n thiet nhat
<? w
va cd ban nhat de giup cac sinh vien cung nhtf cac nha vi sinh lam sang co the nam
dude khi xay dung, thUc hanh cac xet nghiem vi sinh lam sang cung nhu khi bien
luan cac ket qua ma cac xet nghiem vi sinh lam sang mang lai. Cac kien thiic nay
dude trinh bay trong nhieu phan, trong do phan dau tien la cac ky thuat lay va lam
xet nghiem vi sinh lam sang cac benh pham khac nhau difdc trinh bay trong cuon
sach nay, cac phan con lai se lien quan den viec dinh danh cac vi khu&n gay benh
thudng phan lap dUdc tii cac benh pham khac nhau va cac ky thuat khang sinh do
thudng qui dUdc thUc hien tai cac phong thi nghiem benh vien dang diidc viet va se
dude tac gia dua ra trong mot thdi gian ngan sap tdi.

Tac gia luon cho rang mot nha vi sinh lam sang diing trUdc mot benh pham
can phai lam xet nghiem vi sinh lam sang giong nhu mot bac si dieu tri diing tnidc
mot benh nhan can phai dUdc chan doan va dieu tri. Bac si can phai chan doan
dung benh ma benh nhan dang mac phm de dua ra dUdc dieu tri dung va thich hdp
cung giong nhu nha vi sinh lam sang trUdc mot benh pham phai dua ra dUdc mot

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qui trinh xet nghiem thich hdp de thUc hien theo dung tien trinh va cuo'i cung co
mot ket qua xet nghiem vi sinh lam sang hutu dung dUdc cho lam sang. Trong cac
budc xet nghiem vi sinh lam sang phai thiic hien, budc lay benh ph&m va lam cac
benh pham la quyet dinh nhat cho sii thanh bai cua ket qua xet nghiem. Chinh vi
vay, tac gia hy vong r&ng cuon cac ky thuat lay va lam cac benh pham khac nhau
de lam xet nghiem vi sinh lam sang nay se dap ling dUOc phan nao yeu cau cua cac
nha vi sinh lam sang trong nUdc cung nhu cac sinh vien khoa ky thuat y khoa de
xay dung dUdc cd sd cua cac xet nghiem vi sinh lam sang cung nhu cac phong thi
nghiem vi sinh lam sang tai cac benh vien cua chung ta hien nay.
Di nhien sach cung khong the nao tranh dUdc cac sd sot ve kien thiic, ve ngiJ
phap, va ca ve in an. Tac gia chan thanh Udc mong dUdc nhan sU dong gop cua qui
doc gia.

TS. BS. PHAM HUNG VAN

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 MUC
■ LUC
■

Mau mau va cay mau 7

Cac m&u mu, chat dich va cay mu, chat dich 18
Mliu phan va cay phan 25
M lu nUdc tieu va cay nUdc tieu 34
Dich nao tuy 43
M lu quet hau, hong va cay mau quet hau hong 53
Benh pham tai, mfit, mui, xoang va cay benh pham tai m&t mui xoang 59
Ddm, dich hut ddm qua mui, dich hut riia phe quan qua noi soi va cay ddm,
benh pham chtia ddm 67
Cay dinh liidng mau ddm '8
M lu benh pham sinh due va cay mau lay tti d\idng sinh due 84
Cay ky khi va cay m lu benh pham ky khi 92
Khao sat trUc tiep
Nhuom GRAM 105
Nhuom METHYLEN BLUE kiem 109
Nhuom khang ACID ^^
Thii nghiem LAL phat hien ENDOTOXIN (lim u lu s amebocytes lysate) 116

Kiem tra vi sinh khong khi 121

Thu nghiem huyet thanh hoc 124
Thu nghiem hoa mien dich hoc 126
Danh gia mot thu nghiem dung de phat hien mot tac nhan gay benh 131
Ti’ ang bi cd ban cho mot phong thi nghiem vi sinh lam sang 138
Tai lieu tham khao 143
Phu luc A: Bang tra ciiu cac sd do va qui trinh lam xet nghiem
vi sinh lam sang cac benh pham khac nhau 144
I hu luc B: Bang tra ciiu cac hinh anh minh hoa 144
Phu luc C. Bang tom tat cac vat lieu lay mau va moi trUdng phan lap
tUdng Ung cho cac benh pham khac nhau. 145

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 BA11. MAU MAU VA CAY MAU

MUC TIEU

1. Giup diidc lam sang cho cac chi dinh cay mau thich hdp nhat vi biet diidc
chi dinh cay mau va thdi diem tot nhat de thiic hien cay mau.
2. Thiic hien dung cach xet nghiem cay mau vi biet diidc cach thiic hien cay
mau nhu the nao, bao gom the tich mau nen lay de cay bao nhieu, nen cay
mau may Ian1 ?...
3. Liia chon diidc phiidng tien thich hap nhat cho cay mau trong cac benh vien
hien nay nhd biet diidc phiidng tien (moi tritdng) phu hcfp nhat de cay mau.
4. Thiic hien tot qui trinh theo doi cay mau va cho cac ket qua chinh xac - kip
thdi den lam sang vi biet diidc qui trinh theo doi cay mau.
5. Bien luan diidc mot cach chinh xac ket qua cay mau vi biet diidc cac vi
khuan thiidng gap trong cay mau la cac vi khuan nao? Lam the nao de co
the nhan biet diidc mot cay mau diidng tinh that sii vdi mot cay mau diicfng
tinh do ngoai nhiem.

1. CHiOjNH CAY MAU

- Phai chi dinh cay mau trUdc cac trUdng hdp nhiem trung co the co du
khuan huyet tam thdi (transient bacteremia) hay nhiem trung huyet
(septicemia)...
- Do vay, nen chi dinh cay mau trade cac benh nhan co mot trong cac trieu
chiing nha sot, dn lanh, lanh run, tieng thdi tim (cardiac murmur) nghi
ngd viem noi tam mac, co xucit huyet d da hay niern mac, xucit huyet
dang sao (splinder) tren mong tay, choang.

2. THOI DIEM CAY MAU

- Phai cay mau trudc khi benh nhan dung khang sinh he thong. Trong
benh vien, bac si phai cho cay mau trade khi bat dau cho benh nhan dung
khang sinh.
- Tuy nhien trong cac trUdng hdp benh nhan dang dieu tri khang sinh nhung
cac trieu chang cua du khuan huyet hay nhiem trung huyet van khong
thuyen giam thi bac si cung nen cho chi dinh cay mau de phat hien tac nhan
vi khuan gay nhiem trung.

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 - Thdi diem tot nhat de cay mau la khi benh nhan bi dn lanh hay dang
lanh run trUdc khi sot, hay luc benh nhan dang len can sot...
- Co the cay mau 2 Ian trong vong 1 gia dau va thiic hien cay mau tai 2 vi tri
lay mau khac nhau tren cd the (vi du: lan dau lay mau tay phai thi lan sau
lay mau 6 tay trai).

3. CACH LAY MAU BE CAY

- Lay mau tinh mach bang phUdng phap vo trung (sat trung da bang con 70%,
chd kho roi m6i choc kim lay mau).
- The tlch mau diidc lay de cay nen chiem 1/10 hay toi da la 1/5 the tich mdi
triidng cay mau. Thong thudng lay 3-10ml mau de cay vao moi trUdng cay
mau co the tich 50ml. Doi v6i triidng hdp benh nhan la tre em, lay khoang 2-
3ml mau de cay la vifa.

4. MOI TRUONG CAY MAU

- BHI, TSB hay Columbia broth, cho vi khu^n hieu khi.

- De cay ky khi, them vao moi triidng cac chat khif nhii Thioglycollat,
L-cystein.
- De khang dong, tot nhat la dung Sodium Polyanethol Sulfonat (SPS), neu
khong co thi dung citrat hay heparin.

5. THEO DOI CAY MAU

- Chai cay mau dUdc u trong tu am 35°C hay 37°C va theo doi moi ngav trong
5-7 ngay xem co dau hieu co vi khuan moc hay khong trong moi trUdng cay
mau long, do la: (1) Co hat dong tren mat hong cau, (2) Due deu hav co
mang, (3) Tan huyet, (4) Dong huyet tUdng, (5) Co gas, (6) Co hat trang
trong ldp hong c&u hay mat ldp hong cau.
- Neu la chai cay mau co 2 pha thi trUdc khi u va moi ngay sau khi quan sat
mat thach cua pha dac xem co khom vi khuan moc hay khong? Neu khong
co khom vi khuan moc tren pha dac, trang pha long len pha dac. Co mot so
vi khuan rat de bi ly giai sau khi moc trong moi trUdng long nhii S.
pneumoniae, hay kho moc thanh khom tren pha dac nhu Streptococci, do do
nen lam mot cay truyen mu len mat thach (thach mau hay thach nau. sau
do u trong tu am C 0 2 hay binh nen) sau khi u chai cay mau qua dem hoac
sau 24 gid.

Bat eu' luc nao phat hien co dau hieu vi khuan moc (tren pha dac thay co
khom vi khuan moc. hay tren pha long thay co mot trong cac dau hieu vi
 khuan moc nha ke tren) hay nghi ngd vi khu&n moc, tien hanh cay phan lap
ngay, tot nh&'t la tren thach bo dudng nhat la thach nau co bo sung XV
(CAXV = Chocolate Agar Isovitex), neu khong co thi cay tren thach mau
(BA) hay thach nau (CA), dong thdi lam mot phet nhuom gram khao sat
true tiep. Neu ket qua nhuom gram thay co vi khuan, co the lam khang sinh
do trtfc tiep tti chai cay mau. Neu tren pha dac co vi khuan moc thi tien
hanh dinh danh ya lam khang sinh do ngay.
- Sau 5 ngay theo doi chai cay mau, phai cay truyen mu mot lan nUa de chac
ch^n khong co vi khuan moc trong chai cay mau trade khi tra ldi cay mau
am tinh.

6. CAC VI KHUAN HAY VI NAM THUONG LA TAC NHAN NHI§M KHUAN HUYET
HAY NHllM NAM HUYET

- Gram (-) - Gram (+) va nam men

+ Escherichia Coli + Staphylococcus aureus
+ Klebsiella spp + S. epidermidis
+ Enterobacter spp + S. pyogenes (nhom A)
+ Proteus spp + S. agalactiae (nhom B)
+ Samonella typhi + Streptococci tieu huyet a
+ Samonella (ngoai S. typhi) (viridans)

+ Pseudomonas aeruginosa + Streptococcus pneumoniae

+ Neisseria meningitidis + E. faecalis (nhom D)

+ Haemophilus influenzae + Listeria monocytogenes

+ Bacteroides fragilis + Clostridium perfringens

+ Brucella spp + Peptococcus spp

+ Pseudomonas pseudomallei + Peptostreptococcus spp

+ Candida albicans
A va cac nam men khac

7. VAN DE VI KHUAN NGOAI NHllM

Cac vi khu^n ngoai nhiem co the

- TO da:

+ Staphylococcus epidermidis,
+ Propionobacterium acnes,
+ Diphtheroides.

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 - Ti* moi triidng xung quanh:
+ Clostridium spp.
+ Acinetobacter spp.
+ Bacillus spp.
- Loai trif dUdc ngoai nhilm khi ket qua cay mau cho thay vi khuan:
+ Cung phan lap dUdc tti hai chai cay mau cay tir mot benh nhan.
+ Cung phan lap dUdc tif mot benh pham khac cung tren benh nhan do.
+ Vi khuan moc nhanh (trong vong 48 gid).
+ Cac dong vi khuan co cung type sinh hoc va de khang nhu nhau vdi
khang sinh dUdc cay tii hai chai cay mau khac nhau tren cung mot benh
nhan.

8. TRA LOI KET QUA CAY MAU DUONG TINH

Tat ca cac vi khuan moc dUdc trong chai cay mau deu phai tra ldi cho bac si
dieu tri. Tham khao vdi bac si lam sang de rut kinh nghiem trong cac trUdng hdp
ngoai nhiem. Cac vi khuan nghi la ngoai nhiem, khong can thiet phai lam khang
sinh do va thong bao cho bac si biet s\i nghi ngd nay. Phai thifdng xuyen thong bao
cho bac si lam sang biet cac ket qua tam thdi bat cii khi nao thay co dau hieu vi
khuan moc bang cac phieu tra ldi ket qua tam thdi.

9. CAY MAU - CAC CAU HOI THUONG GAP

9.1. Tren thirc te, ty le cay mau dirong tinh tai cac benh vien rat thap, co phai la
do chi djnh cay mau qua rong rai khong?

- That sU ty le cay mau dUdng tinh thap tai cac benh vien khong phai do chi
dinh cay mau qua rong rai ma lai la do lam sang it bao gid nghi den viec cho
chi dinh cay mau sdm ngay tu: dau. Lam sang chi cho cay mau sau khi da
that bai dieu tri khang sinh va chinh chi dinh cay mau qua hep nay da lam
cho ty le cay mau dUdng tinh tai cac benh vien rat thap.
- Cay mau la mot xet nghiem vi sinh lam sang can phai thUc hien trUdc cac
trUdng hdp benh nhan co chan doan lam sang nghi ngd co du khuan huyet
tam thdi (nhiem trung ho hap cap...), nhiem trung huyet, hay co nguv cd
nhiem trung huyet (thudng han, nhiem trung tieu, nhilm trung gan mat...).
Cay mau cung can phai thuc hien tren cac benh nhan co cac dau hieu nhiem
trung mach mau hay tim mach (viem van tim cap tinh hay ban cap. viem
noi mac mach mau...). Noi chung chi dinh cay mau rat rong rai, co the gin

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 nhu mot chi dinh lam cong thiic bach c£u trUdc cac benh nhan co dau c
nhiem trung.

9.2. Thdi diem tot nhat de cay mau la khi nao va nen cay mau may lan?

- Tot nhat la cay mau trUdc khi cho benh nhan dung khang sinh he tl
Tuy nhien v ln phai chi dinh cay mau trong cac trUdng hdp benh nha
dung khang sinh ma van con cac dau hieu nhiem trung tien trien.
- Thdi diem cay mau tot nhat la luc benh nhan dang len cdn sot hay luc
nhan ret run. Co the thuc hien hai lan cay mau cach nhau 1 gid tai 2 1
lay mau khac nhau (lan d&u lay mau tay phai, lan sau lay mau tay
chang han...).

9.3. Nen lay the tich mau la bao nhieu de cay?

- The tich mau dUdc cay la 1/10 hay toi da la 1/5 the tich moi trUdng cay n
- NgUdi ldn, moi lan cay, lay 5-10ml mau cho vao chai 50ml moi trUdng
con co the tvf 1ml den 3ml cho chai 30 den 50ml moi trUdng.

9.4. Phtfdng tien nao hien nay la thich hdp nhat cho cac benh vien de cay ma

- PhUdng tien cay mau thich hdp nhat cho dieu kien cua cac phong thi ngl
lam sang hien nay la chai cay mau 2 pha do cong ty Nam Khoa san j
Chai cay mau nay co du cac liu diem cua mot moi trUdng cay mau ly tu
cua phUdng phap cay mau tai giUdng thuan tien, cua phUdng phap the*
de phat hien vi khuan rat tien ldi.

9.5. Qui trinh theo doi cay mau dufdc thuc hien nhu the nao? Bao lau sau thi
sang se co ket qua cay mau?

- De theo doi cay mau, can phai u chai cay mau trong tu am 35-37°C liei
trong 5-7 ngay. Sau ngay dau, phai cay mu len hop thach CA de phat
difdc cac vi khu&n moc nhanh va kho thanh khom tren mat thach ngb
cua chai cay mau 2 pha, cac ngay sau trang pha long len pha dac de ci
khuan moc trong pha dac len mat nghieng. Bat cut luc nao phat hien
vi khuan moc trong pha long hay tren pha dac thi phai tien hanh
danh va khang sinh do. Ngay theo doi cuoi cung, phai cay mu mot lan
len thach CA de dam bao khong co vi khuan moc triidc khi tra ldi ket
cay mau am tinh.

- Mot qui trinh cay mau va theo doi cay mau de tra ldi ket qua cho lam
toi da la 7 ngay. Nhieu trUdng hdp vi khuan moc nhanh, ta co the co ket
 trong vong 48 gid. Vi cay mau c£n phai co thdi gian nhli v&y nen bat ciif luc
nao chung ta phat hien co vi khuan moc tren chai cay mau thi phai tra ldi
ket qua tam thdi cho lam sang trUdc khi cho ket qua chung cuoc.

9.6. Lam the nao nhan biet dirdc mot chai cay mau dirong tinh la do ngoai nhiem

- Mot ket qua cay mau dudng tinh do ngoai nhilm co the nhan biet dUdc khi:
(1) Cac vi khuan phan lap dUdc la cac vi khuan hoai sinh nhu Bacillus
subtilis, Bacillus cereus...\ (2) Vi khuan cay dUdc tren hai chai ca'y mau tren
cung mot benh nhan trong cung mot thdi diem hay trong hai thdi diem cach
nhau 30’ den 1 gid lai co ket qua khac nhau.

- Mot ket qua cay mau chac chan la dUdng tinh that sU khi: (1) Vi khua’n ca'y
diidc la vi khuan chac chan gay benh, khong the hoai sinh nhu: S. typhi, S.
pneumoniae, N. meningitidis...-, (2) Vi khuan moc ra't nhanh, chi 24 gid sau
khi cay; (3) Vi khuan cay dUdc tren hai chai ca'y mau tren cung mot benh
nhan cho ket qua giong nhau; (4) Vi khuan tii cay mau giong het vi khua’n
phan lap dUdc tii mot nhilm trung khac la nguon go'c gay du khuan huyet
hay nhiem trung huyet.

10. CAU HOI ON TAP

a. Hay cho biet khi nao mot benh nhan can phai diidc ca'y mau?
1 b. Cay mau cho benh nhan vao thdi diem nao la tot nhat? Can phai cay bao
nhieu lan?
c. The tich mau diidc cay la bao nhieu va ty le mau diidc ca’y so vdi moi
triidng cay mau nen la bao nhieu thi tot?
d. Moi trUdng cay mau tot nhat la gi ? Khang dong bang gi la tot nhat va
tai sao?
e. Chai moi triidng cay mau thich hdp nhat cho cac benh vien hien nay tai
Viet Nam la loai nao? Phan tich cac liu khuyet diem cua chai ca'y mau nay?
f. Hay cho biet cach theo doi mot chai ca'y mau 2 phase trong phong thi
nghiem? Da'u hieu nao cho biet chai ca'y mau diidng tinh (co vi khuan
moc)? Thdi gian theo doi chai cay mau cho den khi tra ldi diidc ket qua
cuo'i cung la bao lau?
g. Hay cho biet cac vi khuan thudng gap trong ca'y mau dudng tinh?
h. Lam the nao co the nhan biet diidc mot ca'y mau la dudng tinh that stf
hay do ngoai nhiem?

12
PHU• LUC
• 1:

San pham lien quan: Chai cay mau HAI PHA

Mota

- Chai cay mau HAI PHA (BIPHASIC) la san pham tii de tai nghien ciiu cap
bo cua TS. BS. Pham Hung Van. San pham nay chi dung mot lan
(disposable), khong dung lai diidc. Cau tao (xem hinh diidi) gom cac thanh
phan sau:
+ Chai bang nhtfa polystyren.
+ Nut cao su de choc kim cay mau qua, lam bang cao su trung tinh.
+ Nap van bang nhiia mau do de me) nap chai cay mau cay truyen khi da co
dau hieu vi khuan moc trong pha long hay tren be mat pha dac.
+ Nap che bang nhUa mau xanh la de bao ve phan nut cao su.
+ Phase dac la mat moi triidng BHI agar (Difco, Biorad hay Merck).
+ Phase long la BHI long (Difco, Biorad hay Merck) co SPS (Sigma).
+ Vach ngan pha dac va pha long.
- Chai cay mau diidc boc trong mang co, co ghi lo va ngay san xuat. Khi chiia
dung, bao quan trong toi, nhiet do phong thi nghiem va co han dung 2 nam
ke tut ngay san xuat. Neu bao quan trong tu lanh, SPS se bi ket tua lai,
nhiing ket tua nay se tan sau khi u trong tu am vai gid.

Cach cay mau

- Xe rach mang co, rut nap che ra khoi nap van (khong van nap).
- Sat trung mat nut cao su cua nap van bang con 70% roi chd kho.
- Choc kim cay qua nut cao su, bdm 3-10ml mau vao pha long.
- Day nap che lai. Lac deu de tron, trang qua mat moi tnidng pha dac. U 35-37°C.

Cac uu diem cua chai cay mau hai pha

- Tranh ngoai nhilm nhd:

+ Khong can md nut chai khi cay mau.
+ Cay truyen ngay trong chai cay mau kin tii pha long qua pha dac.
- Moi triidng cay mau ly tiidng vi:

13
+ Che tii moi triidng BHI cua Difco, Biorad hay Merck.
+ Co SPS vdi cac cong dung.
Khang dong, nhd do vi khuan khong bi bay trong cuc mau dong.
tJc che thuc bao.
tic che bo the.
\Jc che tac dong cua cac khang sinh Aminoglycosides.
Tach rdi pha dac khoi pha long de:
+ Neu trong mau co chat tic che vi khuan (khang sinh), chat lie che tir mau
hoa vao pha long se se khong tham qua pha dac. Nhd vay vi khuan khong
moc trong pha long van co the moc trong pha dac.
+ Lam dUdc khang sinh do trUc tiep tu: chai cay mau dudng tinh.
Tiet kiem thdi gian, co the lam khang sinh do trUc tiep t\l chai cay mau
dUdng tinh bang cach dung pha long lam huyen dich vi khuan trai len mat
thach Mueller Hinton de lam khang sinh do, doc ket qua sau 6-8 gid u. 95%
cac trUdng hdp nay co ket qua phu hdp vdi phUdng phap chuan.

Nut cao su

Nut cao su

Phase dac

nhi/a

Phase long

Hinh 1.1: Cau tao chai cay mau hai pha

PHU LUC 2:

Mot so diem can can nhac de Ilia chon chai cay mau 2 pha
hien dang co mat tren thj trUdng Viet Nam

Chai ca'y mau 2 pha san xuat trong nude

- Cong ty san xuat dat ISO 9001: 2000, WHO-GMP/GLP (UKASS)

- Khang dong bang SPS cua Sigma (My).
- Moi trUdng BHI cua Merck (Dtic), hay Biorad (My).
- The tich moi trUdng 50ml tUdng thich de cay mau cho ca ngUdi ldn (5ml
mau) hay tre con (l-3m l).
- Chai bang nhila trong suot, rat de phat hien khom vi khuan moc
- Tach rdi pha dac ra khoi pha long nen khom vi khuan se de moc va de tach rdi.
- D l thao tac trong thi nghiem vi nhe, mieng chai rong d§ cho vong cay vao
ben trong de lay dich ca'y cung nhu khom vi khuan.
- Cac vi khuan kho moc nhu H. influenzae hay S. pneumoniae van moc dUdc
de dang.

Chai cay mau 2 pha cua An Do (Hi-Media)

Khong chac khang dong bang SPS cua Sigma (My).

Moi trUdng BHI cua HiMedia (An Do), kem xa ve chat ludng so vdi BHI cua
Merck hay Biorad.
The tich moi trUdng < 20ml chi tUdng thich de ca'y mau cho tre con (l-2ml).
Muon dung cay mau cho ngtidi ldn thi phai dung chai co the tich moi trUdng
50ml vdi gia thanh cao hdn.
Chai bang thuy tinh day, kho phat hien khom vi khuan moc tren pha dac va
pha long. •
- Khong tach rdi pha dac ra khoi pha long nen khom vi khuan se rat kho moc
tren pha dac va de bi bong khoi pha dac khi thao tac. Ngoai ra neu benh
nhan da dung khang sinh thi khang sinh se tham vao pha dac va tic che vi
khuan moc tren pha dac.
Rat kho thao tac trong thi.nghiem vi nang, mieng chai hep kho cho vong ca'y
vao ben trong de lay dich cay cung nhti khom vi khuan.

15
Thii nghiem cho thay vi khuan H. influenzae va S. pneumoniae rS't kho
trong chai cay mau nay.

Hinh 1.2: Chai cay mau 2 pha co vi khuan moc (trai)

va chai cay mau 2 pha chi/a cay benh pham (phai)

Hinh 1.3: Chai cay mau 2 pha nude ngoai dung moi tri/dng Hi - Media cua An Do
 Sd do 1: Qui trinh vi sinh lam sang cay mau
vdi chai cay mau hai pha
Neu khong co
Neu co
dau hieu VK moc
D1 L5c tron, trang len pha dac dau hieu VK moc
Qui trinh B
Qui trinh A
■ s r i/ r s Thuc hien cay mu
Lay vai giot
Tren pha dac khong VK 1
moc. Lay vai giot moi trUdng
U tiep 37 C ^m m i
qua dem
II moi trifdnq
Neu tren pha dac co
m khom vi khuan
~ '/ tz
CAXV hay
I BA. U
1
Dinh danh, khdng sinh do
37-C/CO, tA±Jt±dt±±dt±J
Tiep tuc den ngay 7 Nhuom gram
Neu khong co VK | Neu co vi khuan i_am KSO

Chd KQ cay tren

CAXV hay BA

Co VK moc Khong co VK moc

D2 t
Quan sat chai cay mau moi ngay, cho
den N5. Sau khi quan s a t , lie tron -
n f khang sinh do
Dinh danh,
' hfhfc)hltdt±ft±dhi Qui trinh B KQ sd bo:
trang len pha dac. Sau do tiep tuc u
KSD trUc tiep

D3-D5
Qui trinh A Truoc khi tra ket qua chung
cuoc, thuc hien cS’y mu Ket qua chung cuoc
--I nhUqui^inhB
n
X TJ >
X - £.
CZ ua chung cuoc sau 5 ngay:
O Q Cay mau (-) neu khong co VK moc
<1 x r r , » > <2: Cay mau (+) vdi KQ djnh danh va KSD
rn>l
-7 ! O Z 0
 BAI 2. CAC MAU MU VA CHAT D|CH
VA CAY MU, CHAT D|CH

MUC TIEU

1. HtC&ng dan diidc lam sang hay co the thiic hien lay dung cac benh pham
cho cac mau mu va chat dich vi biet diidc cac loai benh pham va cac cach
lay benh pham thich hdp trong cac triidng hdp lam xet nghiem vi sinh cac
mau mu va chat dich.
2. Thiic hien diidc cac qui trinh nay mot cach chinh xac trong khi lam xet
nghiem cac mau mu va chat dich do biet diidc qui trinh thiic hien vi sinh
lam sang bao gom khao sat triic tiep, nuoi cay phan lap cac vi khuan gay
benh hien dien trong cac mau mu va chat dich.
3. Lila chon diidc phiidng tien thich hdp nhat cho cay mu va cac chat dich
trong cac benh vien hien nay do cd diidc phiidng tien thich hdp nhat de
thiic hien diidc xet nghiem vi sinh lam sang cac mau mu va chat dich.

1. CHiO|NH

Tat ca cac triidng hdp co mu, chat dich nhti:

- Mu ap xe.
- Vet thudng nhiem trung, bao gom cac vet loet, cat, Id, mo hau phlu, loet do
nam lau.
- Cac mach lUdn.
- Cac mach dan tii xoang hay hach bach huyet.
- Cac dich tiet nhu dich mang phoi, khdp, mang bung.
- Cac mau nao mu xUdng khi giai phau.

2. CAC LOAI BENH PHAM VA CACH LAY

- Mu ap xe, dich mang phoi, mang bung, khdp: lay bang phUdng phap vo
trung nhu khi lam tieu phlu, sau khi sat trung vung da ben ngoai va chd
kho, choc kim hut lay mu hay chat dich. Cho mu hay chat dich vao lo lay
benh pham vo trung (nap van chat) hay tube Eppendorf biopure (tinh sach
sinh hoc), hay de nguyen ong kim hut mu, roi gui ngay den phong thi

18
 nghiem de yeu c&u c&y ngay. Co the tam mu vao tam bong roi cho vao moi
trUdng chuyen chd Stuart -Amies (dung cap tube dua tam bong vo
trung I tube dua Stuart -Amies), hay co the cay ngay tai gitfdng benh vdi chai
2 mat thach cay cac dich khong tap nhiem (xem gidi thieu chai 2 mat thach
cay DNT va cac dich khong tap nhiem) roi chuyen ve phong thi nghiem.
- Cac vet thiictng nhiem trung: lau sach vung da lanh chung quanh vdi con
70%. Lau sach mu tren vet thudng bang gac vo trung tham nUdc muoi sinh
ly vo trung. Dung tam bong vo trung lay mau de quet lay mu, chat dap nat,
hay mo (ngay dudi ldp mu da chui sach); hay lay mau cho vao lo lay benh
pham vo trung, hay tube Eppendorf biopure roi gui ngay den phong thi
nghiem de yeu cau cay ngay. Neu chUa cd the gtii ngay, cho tam bong da
quet mu vao moi trUdng chuyen chd Stuart -Amies (dung cap tube dua tam
bong vo trung I tube dua Stuart -Amies).
- Cac nao mu hay mo khi gidi phau: cung dUdc lay bang quet tam bong hay
trUc tiep cho mau vao lo lay benh pham vo trung, hay tube Eppendorf
biopure roi giii ngay den phong thi nghiem. Neu chUa cd the giii ngay, cho
vao moi trUdng chuyen chd Stuart -Amies (dung cap tube dua tam bong vo
trung I tube dua Stuart -Amies).
- Cac mach liicfn hay mach dan: dung tam bong manh vo trung luon vao mach
lUdn; hay pipet Pasteur nhiCa hut lay mu cho vao lo lay benh pham vo trung,
hay tube Eppendorf biopure roi gtfi ngay den phong thi nghiem. Neu chUa cd
the gtii ngay, cho vao moi trUdng chuyen chd Stuart -Amies (dung cap tube
dua tam bong vo trung I tube dua Stuart -Amies).

3. KHAO SAT DAI THE

- Mau: do, vang, xanh...

- Mui: thoi, tanh, hang...
- Ti'nh chat: dac, long, nhay, cd mau...

4. KHAO SAT VI THE

Nhom gram. Neu ket qua nhuom gram thay cd vi khuan thuan khiet, cd the
lam khang sinh do trUc tiep m iu benh pham. Nhuom khang acid (neu cd yeu cau).

5. NUOI CAY

- Cay ngay vao cac hop thach phan lap:

- Toi thieu la BA hay BA cd nalidixic acid (BANg) va MC hay EMB.
- Neu cd dieu kien, cay them MSA hay DNA agar, BA cd gentamicin.
- Neu nghi nam, cay them thach Sabouraud.

19
 - Cac hop BA phai dUdc u 35-37°C trong tu &'m C 0 2hay binh nen. Cac trtfdng
hdp khac, u khi trUdng binh thudng.

- Quan sat hop thach lien tuc trong 3 ngay, mot khi c6 khom vi khu&n moc,
tien hanh dinh danh va lam khang sinh do ngay.

- Cay du phong vao mot orig Thioglycollat hay BHI, u dong thdi vdi cac hop
thach phan lap. Neu tren hop thach phan lap khong co vi khuilin moc ma
orig BHI hay Thioglycollat due thi cay phan lap tii cac ong moi tnldng nay.

6. CAC VI KHUAN GAY BENH CO TH^ PHAN LAP Ol/QC

- ThUdng gap
+ Streptococcus pyogenes,
+ Staphylococcus aureus,
- It gap hdn
+ Cac trUc khu&n Enterobacteriaceae,
+ Pseudomonas va cac trUc khuan gram (-) khong len men,
+ Streptococci (cac loai khac),
+ Clostridium perfringens,
+ Bacteroides va cac vi khuan ky khi khac.
- Rat hiem gap
+ Bacillus anthracis,
+ M. tuberculosis,
+ M. ulcerans,
+ Pasteurella multocida.

7. CAY MU, CHAT DjCH - CAC CAU HOI THUONG GAP

7.1. Tai sao co nhieu trudng hop cay mu do nhiem trung vet thuong ho cho ket
qua khong phu hop vdi hieu qua dieu trj khang sinh trong lam sang?

- Thuc te dung nhu vay, ly do la khi lay mu lam xet nghiem, thudng chung ta
hay lay mu tren be mat vet thudng nhilm trung va nhu vay co khi vi khuan
phan lap dUdc khong phai chinh la vi khuan gay benh ma chi la cac vi
khuan tam tru trong mu, do mu cung la moi trUdng tot cho nhieu loai vi
khuan tang trUdng.

20
 - Chinh vi vay, lay mu de tim vi khuan gay benh trong cac triidng hdp vet
thiidng hd, can phai lau sach mu va chi quet lay chat dich tren be mat ldp
mo cua vet thudng, day mdi chinh la benh pham co nhieu kha nang chtia vi
khua’n gay nhiem trung vet thiidng.

7.2. Tai sao co trudng hop cay mu hay chat djch ket qua am tinh, tham chi co khi
khao sat true tiep qua phe't nhuom gram van thay co hien dien vi khuan?

- Cay mu ket qua am tinh co the la do: (1) Khang sinh da lam sach dUdc vi
khuan va mu chi chiia xac vi khuan, xac bach cau; (2) Mu gtii den phong thi
nghiem khong dUdc cay ngay, dac biet cac trUdng hdp mu diidc lay trUc tiep
ma khong cho vao moi trUdng chuyen chd; (3) Vi khuan co qua it trong mau
mu (nhu mu ket mac hay giac mac mat), trong trUdng hdp nay co the lam
tang sinh mu trong BHI khoang 2-3 gid roi mdi cay.
- Ngoai ra, cay mu co the am tinh trong cac trUdng hdp nhiem trung ky khi
ma trong khi do chung ta chi cay mu theo qui trinh hieu khi. Cac mau mu
chung ta co the nghi ngd co nhiem trung ky khi la cac mau co mui thoi, co
gas (co bot khi), mu ap xe... (xem them d phan qui trinh cay ky khi).

7.3. Khi nao co the lam khang sinh do true tiep mau mu?

- Da so" cac mau mu deu co the lam khang sinh do trUc tiep, nhUng tot nhat la
cac mau mu khong bi tap nhiem (nhti mu ap xe). Co the quan sat qua mot
phet nhuom gram de co the biet dUdc mau khong bi tap nhiem nhd chi thay
mot hinh thai vi khuan thuan khiet.
- Tuy lam khang sinh do trUc tiep nhUng cung khong the bo qua khau dinh
danh va khang sinh do bSng qui trinh thudng qui vi qui trinh nay mdi co the
cho dUdc ket qua chinh xac va d&y du.

7.4. Hay cho biet phifdng tien thich hop nhat de lay, chuyen chd cac quet mu,
cac chat djch va cac mu abcess?

- Khong nen dung cac phiidng tien tu che trong benh vien nhii: cac que tam
bong tu quan cho vao ong nghiem nhet gon roi hap hay say khii trung; hay
cac lo kieu chai peni hay tube thuy tinh nhet gon roi hap say vo trung de lay
cac benh pham mu va cac chat dich. Ly do la cac phtidng tien nay rat kho
thao tac khi lay benh pham tren benh nhan, hay khi lay benh pham de lam
xet nghiem trong phong thi nghiem. Ngoai ra cac phiidng tien nay cung
khong an toan khi thao tac hay chuyen chd vi nguy cd ri benh pham len
mieng tube hay chai. Nguy cd ngoai nhiem benh pham cung rat cao khi
dung cac phUdng tien nay.
- Sau day la cac phUdng tien thich hdp nhat de lay va chuyen chd cac benh
phaim mu va chat dich:

21
 Hinh 2.1: Tube dua tSm bong vo trung di/dc thiet ke rat thuan ldi de lay cac quet benh phar

Hinh 2.2: (A) Cap tube dua tSm bong vo trung/moi trifdng chuyen chd Stuart - Amies:
(B) CSp tube dua tSm bong manh vo trung/moi truting chuyen chd Stuart-Amies

(1) Tam bong vo trung lay m lu dUdc thiet ke vdi phan n ip tube g in s in tam
bong do vay khi rut nap tube ra khoi ong, tam bong se dUdc rut ra theo va tay ngUdi
thao tac cung se khong cham vao mieng tube, sau khi lay quet mu xong tam bong
co the dUdc cho vao lai trong tube hay cho vao tube dua chtia moi trtfdng chuyen
chd Stuart -Am ies di kern.
 (2) Cap tube dua tam bong vo trung/tube dua moi trUdng chuyen chd Stuart -
Amies dUdc thiet ke de t&m bong cua tube dua tam bong vo trung sau khi lay mau
xong cho vCfa van vao tube dua moi triidng chuyen chd Stuart -Amies vdi thao tac
rat de dang vk thu&n tien.
(3) Lo vo trung l&y m lu rat thich hdp de lay cac chat dich hay mu abcess nhd
nip v&n khi md ra tay ngUdi thao tac se khong cham mieng lo (tay cham mieng lo
se rat d l co nguy cd ngoai nhiem), sau khi l&y benh pham xong van chat nap, lo se
rdit kin khong bi ro ri khi chuyen chd m&u.
(4) Tam bong manh vo trung lay mau rat thich hdp de lay cac quet mu tii cac
ong hep nhu mach lUdn, ong catheter... va cung tUdng thich de co the cho vao tube
dua moi triidng chuyen chd Stuart - Amies.

8. CAU HOI ON TAP

a. Cho biet cac chi dinh lam sang de cay mu va cac chat dich.
b. Hay cho biet cac loai benh pham mu va chat dich, va cach lay cac loai
benh pham nay.
c. Hay cho biet phUdng tien lay va chuyen chd cac benh pham mu va cac
chat dich thich hdp nhat cho cac benh vien hien nay va phan tich cac Uu
khuyet diem cua cac phUdng tien nay.
d. Hay cho biet qui trinh xet nghiem vi sinh lam sang thiic hien tai phong
thi nghiem doi vdi cac benh pham mu va chat dich.
e. Hay cho biet cac vi khuan co the gap dtidc trong cay mu va cac chat dich.

23
Sd do 2: Qui trinh vi sinh lam sang JT
cac m lu mu va chat dich 0
Ca'y ngay tai giudng
vao chai 2 mat thach
vdi cac bp khdng tap

,
u H ,
nhi£m (xem qui trinh a _ .
phan chai 2 mat thach
, ,
mu-choc hut mu
Quet mu, choc hut mu abces ^ ^ abces (MT chuy«n cho
(khong MT c h i^e n chd) khdng tap nhi&ri) s,uart j" Am« )

--------------Co th4 chd den cu6 ngay

Lam xet nghiem ngay

Chd KQ
”
Neu khong co VK,
hay da nhiem
1
Cay phan ldp tren BA hay
phan lap J BANg, MC/EMB
KSTT: nhuom GRAM

Neu cd KQ sd bo
VK thuan KSTT
khiet
U 37°C/CQ2 (BA hay BANg)

Chon khdm tach rdi

Cay tang sinh
len BA
KQ sd bo: KSD tri/c tiep

U 37°C/ti/ sang den chieu

Oinh danh
tdt±±±Jhft±±±(

KQ chung cuoc

LM UUM u Mh < 0 -
i •i (7
 BAI 3. MAU PHAN VA CAY PHAN

M U C T liU

1. Hiidng dan diidc lam sang hay thiic hien hay lay dung mau phan lam xet
nghiem vi sinh lam sang phat hien cac tac nhan vi khuan gay benh nhd
biet diidc cac cach lay va chuyen chd mau phan.
2. Thiic hien diidc cac qui trinh vi sinh lam sang mau phan mot cach chinh
xac va thich hdp nhd biet diidc qui trinh thiic hien.
3. Liia chon diidc phiidng tien thich hdp nhat cho cay phan trong cac benh
vien hien nay nhd biet diidc phiidng tien thich hdp nhat de thiic hien diidc
xet nghiem vi sinh lam sang mau phan.

1. CHI DjNH CAY PHAN

- Chi dinh cay phan khi benh nhan bi tieu chay hay bi cac roi loan tieu hoa
nghi do bi nhiem trung tieu hoa.
- Diing trU6c cac benh nhan co mot trong cac trieu chving sau day, nen cho chi dinh
cay phan: tieu chay, ly vdi phan co mu, nhay hay mau, bi ccfn dau bung.

2. THOI DIEM LAY PHAN

- Nen lay vao giai doan sdm, cang sdm cang tot cua benh.
- Lay phan khao sat trUdc khi benh nhan dung khang sinh.

3. CACH LAY MAU PHAN

- Co the lay phan tuoi, tot nhat la vung nh&y mau, cho vao lo sach; rong
mieng; nap van chat co gan mai cheo (dung lo lay mau phan khong F2M),
g&i den phong thi nghiem. Phan tifdi phai dtidc cay trong vong khong qua 2
gid sau khi lay mau.
- Co the dung tam bong nhung vao phan, vung nhay mau, cho vao moi trUdng
chuyen chd Cary -Blair (dung cap tube dua tam bong vo trung I tube dua
Cary -Blair) roi gvfi den phong thi nghiem. Moi triidng chuyen chd Cary -
Blair co the giii m lu phan trong hdn 48 gid. Tuy nhien, phai tien hanh cay
cang sdm cang tot. Trong trtfdng hdp nghi benh nhan bi tho ta (do vi khuan
ta), co the cho tam bong lay phan vao ong moi trUdng pepton kiem de vtia
tang sinh. vvfa chuyen chd den phong thi nghiem.

25
 - Co the lay miku phan bang tam bong quet hau mon roi cho vao moi tnfdng
chuyen chd (dung cap tube dua tam bong vo trung I tube dua Cary -Blair) de
giii ngay den phong thi nghiem.
- Co the khong dung moi trUdng chuyen chci neu tam bong l&'y phan (dung
tam bong vo trung lay mau) dUdc nuoi cay trong vong 30 phut sau khi
lay mau.

4. KHAO SAT DAI THE

Quan sat mau phan va ghi nhan:

- Long hay dac? Co nhay, co mau khong?
- Mau: trang, vang, nau den? Co giun san khong?

5. KHAO SAT VI TH^

Chi khao sat vi the cac trUdng hdp sau:

- Lam phet soi tifdi trong trUdng hdp nghi ta.
- Lam phet nhuom gram trong trtidng hdp nghi ta va nghi Campylobacter jejuni.

6. NUOI CAY

- Co the cay phong phu vao cac moi trUdng

+ GN broth de phong phu ca Salmonella lan Shigella.
+ Pepton kiem de phong phu Vibrio.
-i- Campy-thio de phong phu C. jejuni.
- Cay ngay phan, hay cay tti moi trUdng phong phu da cay phan vao cac hop
thach phan lap:
+ Toi thieu la MC (hay EMB) va SS (hay XLD hay HE).
+ Neu co yeu cau tim Vibrio, cay them len MEA hay TCBS.
+ Neu co yeu cau tim C. jejuni thi cay them Campy -CAP.
+ Neu nghi nam, cay them thach Sabouraud.
- Cac hop thach phan lap phai dUdc u 35-37°C trong tu am. Rieng hop Campy-
CAP thi u vi hieu khi trong nhiet do 42°C.
- Quan sat hop thach sau khi u qua dem, chon khom vi khu£in nghi ngd de
tien hanh dinh danh va lam khang sinh do.

- Doi vdi cac khom vi khu&n nghi ngd Salmonella hay Shigella thi co the lam
phan ting tu vdi khang huyet thanh dac hieu de dinh nhom hay dinh type.

26
7. CAC VI KHUAN LA TAC NHAN GAY BENH TiM THAY TRONG MAU PHAN

- Cac vi khu&n chac chan gay benh: Salmonella, Shigella, cac E. coli gay benh
(ETEC, EPEC, EIEC, VETEC), S. aureus (co enterotoxin), V. cholerae va cac
Vibrio khac, Campylobacter jejuni va cac Campylobacter khac, Yersinia
enterocolitica va cac Yersinia khac, Clostridium difficile (co toxin).
- Cac vi khuan co the gay benh: Plesiomonas, Aeromonas.
- Cac vi khuan khac co the gay benh neu chiem da so do bi loan khuan.

8. CAY PHAN - CAC CAU HOI THUONG GAP

8.1. Phuang tien nao thich hop de lay va chuyen chd mau phan?

- Neu lay mau phan tUdi thi tot nhat la dung lo sach co mai cheo gan tren nap
de lay mau. Cung co the dung tam bong vo trung de quet lay mau phan.
Mau phan tUdi phai diidc gtii den phong thi nghiem de lam xet nghiem ngay
vi neu cham tre thi se lam giam cd hoi phat hien cac vi khu&n gay benh.
- Trong dieu kien hien nay, tot nhat la dung cap tube dua tam bong vo
trung/ tube dua Cary -Blair de lay mau phan. Ly do la vdi phUdng tien
nay, phong thi nghiem co the de don mau lai de cay vao cuo'i ngay hay vao
thdi diem thich hdp nhat trong ngay ma khong can phai cay ngay moi khi
. nhan diidc mau.
- Tranh dung cac lo penicillin riia sach, tube nap gon vdi tam bong tu quan
hay cac lo nhUa, hu thuy tinh de lay mau phan vi nhu vay se khong an toan
trong khau gui m lu hay trong thao tac xet nghiem.

8.2. Tai sao da so' cac trudng hop tieu chay xet nghiem phan thudng am tinh?

- Co the benh bi tieu chay khong phai do vi khuan ma do cac nguyen nhan
khac nhu do virus, do tham thau, do dau bao tii, hay do cac roi loan tieu
hoa khac. Mot tac nhan virus thudng gay tieu chay la Rota virus va phong
thi nghiem vi sinh lam sang co the phat hien tac nhan nay neu co phUdng
tien sinh hoc phan tu (PCR, hay chi can ly trich ARN toan phan roi dien di
de phat hien 11 vach ARN dac hieu cua virus) hay mien dich (tu latex hay
dien di mien dich doi liiu de phat hien khang nguyen dac hieu cua virus
trong phan).
- Co the benh nhan bi tieu chay do cac tac nhan vi khuan ngoai kha nang
phat hien cua phong thi nghiem do thieu phUdng tien, hay do phong thi
nghiem khong co kinh nghiem de phat hien nhu E. coli (EPEC, ETEC,
EIEC, EHEC, VETEC), S. aureus tiet enterotoxin, ma muon phat hien

27
 chinh xac phai co them phiidng tien mien dich hay sinh hoc phan ta; hay
Campylobacter jejuni, Clostridium difficille, Y. enterocolitica, ma muon phat
hien phai co phUdng tien nuoi cay thich hdp; Vibrio cholerae, Aeromonas,
Plesiomonas ma muon phat hien thi phai co kinh nghiem hay phai c6 quan
tam trUdc.
- Cung co the do khong dung dung phUdng tien de lay va chuyen chd benh
pham, keo theo hau qua la vi khuan gay benh khong con nhieu trong benh
phfi’m, hay do phong thi nghiem cham tre lam xet nghiem cac m lu phan
tUdi khong diidc lay vao moi trUdng chuyen chd. Ngo&i ra mot nguyen do
niia co the la do phong thi nghiem khong co phiidng tien vi sinh toi thieu de
phan lap vi khu&n gay benh trong m lu phan cung nhu dinh danh dUdc vi
khualn gay benh co trong mau phan.
- Cuo'i cung, co the do benh nhan da dung khang sinh triidc khi lay m lu nen
khong the cay dUdc vi khuan gay benh co trong phan.

8.3. Vdi qui trinh xet nghiem vi sinh lam sang m lu phan nhu da trinh bay, co phai
chung ta chi phat hien duoc mot so chur khong phai tat ca cac vi khuan gay benh?

- Qui trinh da trinh bay cho phep chung ta co the phat hien hau het cac vi
khuan gay benh ke ca C. jejuni va Vibrio spp. Chi co C. difficille, S. aureus
sinh Enterotoxin va Y. enterocolitica la khong the phat hien diidc vi din
phai co them phUdng tien nuoi cay thich hdp, tuy nhien day la 2 tac nhan
khong thUdng gap.
- Vdi C. jejuni, vi day la vi khuan cung kha thiidng gap, do vay cac benh vien
cung nen quan tam de trang bi cho phong thi nghiem phUdng tien nuoi cay
phat hien dUdc tac nhan nay (xem phan ky thuat phan lap C. jejuni).
- Vdi cac vi khu&n E. coli gay benh, de phat hien dUdc EPEC, phong thi
nghiem phai trang bi them bo khang huyet thanh phat hien (co the mua tii
Biorad). Vdi cac E. coli khac nhu ETEC, EIEC, EHEC, VETEC thi neu
muon phat hien chinh xac can phai co them cac phUdng tien mien dich hay
sinh hoc phan tii (PCR).
- Vdi S. aureus sinh enterotoxin, de phat hien chinh xac thi can phai co them
bo thuo'c thu ELISA phat hien doc to' hay phUdng tien sinh hoc phan tu phat
hien enterotoxin gen b&ng ky thuat PCR.
- Vdi cac phong thi nghiem khong the trang bi them cac phiidng tien phat
hien chinh xac E. coli, neu khong phat hien dUdc cac tac nhan vi khuan gay
benh khac ma tren hop thach phan lajD vi khuan E. coli lai chiem da so' thi
nen lam khang sinh do vi khuan nay de tra ldi ket qua cho lam sang.
- Cac vi khu^n khong gay benh dudng ruot nhu Proteus, Klebsiella.
Pseudomonas... co the la tac nhan gay tieu chay trong cac triidng hdp loan
khuan do vay van nen lam khang sinh do va tra ldi ket qua cho lam sang
neu chiem da so tren hop thach phan lap va khong co cac vi khuan gay
benh khac.
9. CAU HOI ON TAP

a. Cho biet cac chi dinh de c&y phan.

b. Hay cho biet cach lay m&u phan nhtf the nao?
c. Hay cho biet phtfdng tien de lay va chuyen chd m lu phan thich hdp nhat
cho cac benh vien hien nay, va phan tich cac Uu khuyet diem cua cac
phUdng tien nay.
Hay cho biet qui trinh xet nghiem vi sinh lam sang thtfc hien tai phong
thi nghiem doi vdi benh pham phan.
Hay cho biet cac vi khuan gay benh co the gap dUdc trong cay phan.

r
LO\6trCjng
Dtfngmfiu
____ Turf.__
____________ $6._____
_____ Gtflh
4 A
1. j J

Hinh 3.1: Lo lay phan ti/di co trang bi mai

Hinh 3.2: Lo lay phan khao sat ky sinh trung
cheo gan tren nap rat thuan tien cho
co tui F2M s in sang de dung bao quan phan
benh nhan lay phan va nap v3n chat
khao sat ky sinh trung. Vdi Ip nay chung ta
tranh nguy cd ri phan ra mieng lo
co the lifu m lu hdn 1 tua"

Hinh 3.3: C3p tube dua tSm bong vo trung/moi trudng chuyen chd Cary - Bair
co the giup lull mau phan trong 48 gid

29
 Sd do 3: Qui trinh vi sinh lam sang
-1 khao sat m lu phan
Phan ti/oi" Cay ngay -

Co the chd
den chieu
Cary-
Biair
Mdi tn/dng phan ldp

—► MT tartg sinh 1. MC/EMB

GN broth (pepton 2. SS/HE/XLD
kiem cho Vibrio,
DO 3. (Neu cd yeu c i u 'V T l'
hay Campy-thio
TCBS, MEA cho Vibrio>
cho C jejuni, neu U 37°C/6-8h
Campy-BAP cho C. U 37*C/(jua (06
co yeu cau) jejuni) vdi Campy-BAP thi uvi
' 1 ' hieu kh. 42*C va t*> tuc
qui trinh cho C. jefuni )

D1 Chon khom VK nghi ngd

- Khdng I6n men lactose
- Khom E.cok
- Khom JU the
Cay tang sinh len BA/NA

K han^ sinh <3o

Dinh type, group ▼
Dinh danh

0000000000

KQ so bo: type va group

VK gay benh

D2
KQ chung cuoc

tJ U ttijU U U U M

30
PHU• LUC
• 1:

Ky thuat phan lap Campylobacter jejuni

Lay mau

Mau co the lay tUdi gtii den phong thi nghiem de lam xet nghiem ngay. Neu
de cham tre hdn 2 gid thi mau phai lay vao moi trUdng chuyen chd/tang sinh
Campy -thio.

Phan lap Campylobacter jejuni

Co the cay phan lap ngay, hay co the giuf tang sinh m lu trong Campy - thio d
tu am 42°C trong 4 - 6 gid roi mdi cay phan lap. Co hai cach:

- Phan lap trUc tiep: cay benh ph&m b ln g phUdng phap 3 chieu tren moi
trUdng Campy - BAP, roi u hop thach trong mot binh u ky khi (Genbox-
anaer cua bio -Merieux) dung bao sinh vi hieu khi (Microanaer cua bio -
Merieux). Giut binh trong tu am 42°C trong 24 den 48 gid.

- Phan lap qua loc: dung mot loc cellulose acetat 0,65^m vo trung dat tren
mat hop thach Campy - BAP nhung khong co khang sinh. Dung pipet
Pasteur vo trung hut benh pham hay dung dich tang sinh benh ph&m, nho
9
5 - 1 5 giot len tam cua giay loc. U ngUa hop thach trong tu am 37°C hay
42°C trong 1 gid. Sau do lay td giay loc ra khoi mat thach, tranh khong cho
*> o . ' 9 x
benh pham nho trUc tiep len m at thach. U hop thach nay trong dieu kien vi
^lieu khi nhu tren.

Bjnh danh Campylobacter jejuni

- Sau khi u, quan sat mat thach. C. jejuni cho khuan lac kha tieu bieu: mau
xam nhat hay xam vang nhat, hdi nhay, doi khi co duoi theo dudng cay
{hinh 10). Kich thudc kha ldn: 1,5 - 2 mm.

- Nhuom gram, vi khuan an mau gram [-], hdi nho, hinh cong, co the thay
dang dac trUng hinh canh chim hai au. Doi khi co hinh dau nga (hinh 3.1).

- Soi tUdi qua kinh hien vi dao pha hay nen den, thay vi khuan di dong nhu
ten ban.

- ThU nghiem oxidase [+], catalase [+]

Hoi du dUdc cac yeu to tren'la co the dinh danh Campylobacter jejuni.

31
PHU LUC 2:
• •

Moi tri/dng can thiet de phan lap C. jejuni

Dung djch khang sinh 100x

- Cong thiic
Vancomycin lOOmg

Trimethoprim 50mg
Polymyxin B 25000UI
Amphotericin B 20mg

Cephalothin 150mg

Nude cat 100ml

- Vo trung qua loc milipore. Phan phoi vao cac chai, moi chai 2ml de che 200
ml moi trUdng. GiCi cac chai trong tu dong -70°C

Moi trudng chuyen cho/tang sinh Campy -thio

- Cong thiic
Thioglycolat media 30gr
Bacto agar l,6gr
Nude cat 1000ml
- Dun tan moi trUdng va agar, de nguoi, chinh pH den 7,2.
- Phan vao cac chai Duran loai 250ml nap van chat, moi chai cho vao chinh xac
200ml moi trUdng. Hap khu trung 121°C/15 phut. De nguoi,'them vao moi
chai 2ml dung dich khang sinh 100X d tren. Co the de nguyen chai trong tu
lanh 4°C, hay phan phoi vo trung 5ml Campy -thio vao cac tube 15 x 125mm
vo trung nap van chat roi giif trong tu lanh 4°C (khong qua 2 thang).

Moi trudng phan lap Campy -BAP

- Cong thiic
Brucella agar base 43gr
Nude cat 1000ml
- Dun tan moi trUdng va agar, de nguoi, chinh pH den 7,2.
- Phan vao cac chai Duran 250ml n ip van chat, moi chai 200ml moi trUdng.
Khu trung b&ng hap ifdt 121°C/15 phut. De nguoi den 45°C, them vao moi
chai 10ml mau cvfu da mat fibrin va 2ml dung dich khang sinh 100X d tren.
Lac nhe de tron deu, tranh bot. Do vao hop petri vo trung loai 90 mm, moi
hop do 18 - 20 ml moi trUdng. Chd dac lai hoan toan.
Gifi cac hop thach trong tu lanh (khong qua 2 thang).

L.

t§ §

Hinh 3.1: Hinh anh Gram vi khuan Campylobacter jejuni

Mb

Hinh 3.2: Hinh anh khom vi khuan Campylobacter jejuni Iren thach Campy - BAP

33
 BAI 4. MAU Nl^fC TIEU VA CAY NlfOTC TIEU

MUC TIEU

1. Thiic hien diidc hay hiidng dan lam sang thiic hien diidc lay dung mau
niidc tieu, tranh ngoai nhiem, de lam xet nghiem vi sinh lam sang phat
hien cac tac nhan vi khuan gay benh.
2. Thiic hien diidc qui trinh xet nghiem vi sinh lam sang mau niidc tieu,
dac biet qui trinh cay dinh liidng mau niidc tieu.
3. Liia chon diidc phiidng tien thich hdp nhat cho cay niidc tieu trong cac
benh vien hien nay nhd biet diidc phiidng tien thich hdp nhat de thiic
hien diidc xet nghiem vi sinh lam sang niidc tieu.

1. CHI OjNH CAY NUdC TIEU

- Cac triidng hdp bac si lam sang nghi ngd nhiem trung tieu (NTT) cap tinh,
man ti'nh, co trieu chiing hay khong co trieu chiing.
- Nen cho chi dinh cay nUdc tieu doi vdi cac benh nhan co mot trong cac trieu
chiing nghi ngd benh nhan bi (1) nhiem trung bang quang nhii dai ra mu, dai
kho, dai ra mau, dai dau, dau tiic vung tren xitdng mu hay bung dadi,
hay (2) nhiem trung than nhii dau liing, tile ca n g vung g o c son g - siidn.

2. THOI DIEM CAY NUdC TIEU

- Tot nhat la buoi sang, lay trUdc khi benh nhan dung khang sinh.
- Trong dem, benh nhan co nhin tieu cho den khi lay m lu.

3. CACH LAY BENH PHAM

- Nude tieu nen dUdc lay vao cac lo vo trung nap day chat (lo vo trung lay
mau). Tot nhat la cac lo mieng rong, hay cac ong nghiem ly tam nap chat.
Co the lay nUdc tieu va cay ngay tai gitidng vao chai ca'y 2 mat thach cay va
dinh liidng niidc tieu {xem hiidng dan va qui trinh sii dung chai 2 mat thach
cay va, dinh liidng niidc tieu).

Nude tieu sau khi lay xong phai dUdc giii den phong thi nghiem khao sat
ngay. Neu cham tre, co the giii trong tu lanh 4°C, nhung khong qua 4 gid.

34
 - Nude tieu phai dUdc lay bang phUdng phap vo trung, tranh toi da sU nhiem
ban tii cd quan sinh due ngoai. Sau day cac phUdng phap lay nUdc tieu tu:
benh nhan.

3.1. Doi vdi phu nurhay tre gai ldn, benh nhan tii lam hay dieu dudng giup

- Rua tay ky bang xa phong, nUdc roi lau kho bang khan sach.

- Cdi quan, vach am moi, rijfa sach roi tham xa phong trong ra ngoai bang
bong hay gac vo trung.
- Rtia sach xa phong roi tha'm kho bang gac vo trung. Suot qua trinh v ln
dung tay vach am moi khong cho dung vao phan ben trong.
- Tieu bo phan dau, lay nUdc tieu phan con lai.

3.2. Doi vdi dan dng hay tre trai ldn, benh nhan tu lam hay dieu dudng giup

- Riia tay sach nhu tren.

- Keo phan da qui dau tut ra sau.
- Rvfa sach cung nhu tren.
- Tieu bo phan dau, lay phan nUdc tieu con lai.

3.3. Doi vdi tre nho, kho lay hon vi benh nhan khdng biet hop tac

- TrUdc khi lay nUdc tieu nen cho benh nhan uong nhieu nUdc.

- Cho benh nhan ngoi tren dui me, riia sach bo phan sinh due ngoai roi xi be
tieu, hting lay nUdc tieu cang nhieu cang tot.

3.4. Cd the lay blng cac phirong phap khac

- Choc qua da tren xUdng mu.

- Bang ong thong tieu hay qua noi soi bang quang.

4. KHAO SAT TRl/C TIEP

Tron deu nUdc tieu roi nho 1 giot trUc tiep tren lam. Chd kho tu nhien roi
nhuom gram. Quan sat dudi vat kinh dau.
- Neu co l't nhat 1 te bao vi khuan va/hay bach cau hien dien tren mot quang
trUdng, co the nghi ngd benh nhan bi nhiem trung tieu. Co the lam khang
sinh do trUc tiep tif mau ntidc tieu nay.
- Neu toan phet nhuom khong hay phat hien dUdc rat it te bao vi khuan hay
bach cau. benh nhan chac chan khong bi nhiem trung tieu.

35
5. CAY NlTCJC TIEU

- Dung vong ca'y dinh lUdng 0,01 (lOpl) hay 0,001 (lfil), l&'y d ly mot vong c&'y,
cung co the dung micropipet vdi dlu tip vo trung l&'y 1^1 nUdc tieu, trai toan bo
len be mat hop thach nuoi cay (thtidng la BA hay BANg v& MC hay EMB).
- U BA hay BANg trong binh nen, MC hay EMB u thudng, tl't ca trong tu am
35-37°C, de qua dem.
- Doc ket qua b ln g cach dem so' khom trung de biet so' lvfdng vi khuln trong
mam cay ban dau, sau do suy ra toan bo so liidng vi khuln so'ng (Colony
Forming Unit=CFU) trong 1 ml nUdc tieu. Neu co:
+ < 10000 CFU/ml nUdc tieu, khong co nhiem trung tieu.
+ > 100000 CFU/ml nUdc tieu, ch ic ch in co nhilm trung tieu, dinh danh vi
khuan va lam khang sinh do.
+ 10000 < 100000 CFU/ml nUdc tieu, nghi ngd nhiem trung tieu. Trong
trUdng hdp nay, neu benh nhan co dau hieu lam sang chac chan nhiem
trung tieu hay bi nhiem trung tieu tai phat nhieu lln , co the dinh danh
vi khuln phan lap dUdc va lam khang sinh do.

6. KHAO SAT SINH HOA, TE BAO VA CAN LANG

- Thuc hien sau khi ca'y.

- Doi vdi sinh hoa, dung giay nhung.
- Quay ly tam lay can de khao sat te bao va can ling.

7. CAC VI KHUAN THUONG LA TAC NHAN GAY NHliM TRUNG TIEU

- Enterobacteriaceae (Escherichia coli, Klebsiella spp, Proteus spp...)

- Enterococcus spp.

- Pseudomonas aeruginosa, va cac trUc k h u ln khong len men khac.

- Staphylococcus aureus.
- S. epidermidis.
- S. saprophyticus.
- Candida albicans.
- M. tuberculosis.

36
8. C A Y Nl/CJC TI^U - C A C C A U H 6 l THUCJNG G A P

8.1. Lam the nao de lay dung nude tieu lam xet nghiem vi sinh lam sang?

- Lay dung nUdc tieu de lam x6t nghiem vi sinh lam sang la phai lay nUdc
tieu giuta dong, lay bang phUdng phap vo trung, trong do quan trong nhat
la phai rvLa sach bo phan sinh due ngoai trUdc khi hutng lay nUdc tieu giuta
dong.

- Dung cu dung nUdc tieu de lam xet nghiem vi sinh lam sang cung phai thich
hdp, nghla la phai dung cac lo vo trung va co the hutng lay nUdc tieu d§ dang
cung nhu khong chay tham ra ngoai trong qua trinh chuyen chd den phong
thi nghiem. Thich hdp nhat la cac lo dung tich khoang 50-100ml, vo trung,
mieng rong, co nap van chat va co nhan de ghi ten benh nhan cung nhu cac
thong tin toi thieu khac ve benh pham.
- Tren thuc te rat ft khi lam sang quan tam stf dung dung dung cu lay nUdc
tieu, rat nhieu ndi chi dung cac lo thuy tinh kieu chai dung thuoc chich rtfa
sach, nhet gon hay cac tube thuy tinh nhet gon roi say hay hap de vo trung.
Cac vat lieu nhu vay khong chi rat kho hiing nUdc tieu giuta dong ma con bi
tham ra mieng lo hay tube gay nguy cd ngoai nhiem cho mau va con co the
nguy hiem cho ngtidi xet nghiem.
- Ngoai ra cung co nhieu ndi lam sang khong quan tam hu6ng dan hay giup
benh nhan lay dung nUdc tieu giuta dong va lay vo trung, tham chi co khi
benh nhan lai lay nUdc tieu trong bo de dUa cho lam sang. Chinh vi vay m lu
nUdc tieu giii den phong thi nghiem co the bi ngoai nhiem va gay sai lech
ket qua.

8.2. Quay ly tam nude tieu va lam phet nhuom gram can lang co gia trj gi khdng?

- Hien nay van co mot so phong thi nghiem lam phet nhuom gram can ling
ntidc tieu sau ly tam va tra ldi cho lam sang cac hinh anh gram vi khuan ho
quan sat dUdc. That sU ket qua nay khong co y nghla gi trong viec cho biet
benh nhan co hay khong co bi nhiem trung tieu va bi nhiem trung do vi
khuan gram nao vi da so' cac can lang nUdc tieu du khong bi nhilm trung
tieu v ln co chufa mot it vi khuan do ket qua cua sU tap nhilm khi nUdc tieu
di qua dUdng sinh due ngoai.
- Tuy nhien nhuom gram can l&ng nticic tieu sau ly tam lai rat co gia tri de
phat hien vi khuan lau (N . gonorrhoeae) la cac song cau gram [-] hinh hat ca
phe noi te bao trong cac te bao niem mac dUdng tieu hay dtidng sinh due.

37
8.3. Neu chua the cay ngay, co the luu mau nude tieu trong tu lanh 4°C diwc
khong? Va luu duoc trong bao lau?

- Tot nhat la nUdc tieu phai dUdc chuyen den phong thi nghiem ngay sau khi
lay va phai dUdc cay ngay vi neu de cham tr i cac vi khu^n tap nhiem se
tang sinh trong nUdc tieu va lam sai lech ket qua cay dinh ltfdng. Chinh vi
v&y, trong cac phien trUc, hay khi khSng co ky thuat vien cSy dinh lUdng
nUdc tieu, tot nhat phong thi nghiem dung chai cay nUdc tieu hai mat thach
vi vdi phUdng tien nay bat cd ai cung thuc hien dUdc ky thuat ctfy dinh
liidng nUdc tieu.

- Co the givi nUdc tieu trong tu lanh 4°C, nhung khong dUdc qua 4 gid. Tuy
nhien co nhieu mau nUdc tieu bi due do stf hinh thanh cac tinh the khi de
lanh, do vay cac mau nay khong the khao sat can lang dUdc.

8.4. Phat hien nitrit trong nude tieu co chinh xac khong de xac djnh nhiem
trung tieu?

- Phat hien nitrit de xac dinh nhiem trung tieu dtia tren nguyen tac la neu co
nhi§m trung tieu, vi khuan trong nUdc tieu se khii dUdc nitrat trong nUdc
tieu thanh nitrit. Tuy nhien khong phai tat ca cac vi khuan deu co kha nang
nay, do vay v ln co nhieu trUdng hdp n h ilm trung tieu nhung khi thu nitrit
van am tinh.

- Ngoai phUdng phap tim nitrit, cung co the tim sU hien dien cua bach c&u
trong nUdc tieu de xac dinh nhiem trung tieu vi mot khi co nhiem trung tieu
thi co phan Cfng viem va co phan ilng viem la co hien dien bach cku. NgUdi
ta phat hien bach cau bang cach phat hien men myeloperoxidase hay mot so
men dac hieu cua bach c&u co trong nUdc tieu khi co bach c&u.

- Ca hai phUdng phap tren v^n la cac phUdng phap gian tiep phat hien nhilm
trung tieu, khong the cho biet vi khuan gay benh la vi khuan gi va cung
khong the cho biet khang sinh do. Chinh vi vay, phUdng phap vi sinh cay
dinh liidng phat hien nhiem trung tieu la phUdng phap chuan nhat va hijtu
dung cho lam sang nhat.

38
PHU
• LUC
•

San pham lien quan: Chai cay djnh lUdng nude tieu hai mat thach
BANg va MC
N ip ngoai
Muc dich sur dung

Chai c§fy hai mat thach B A N g

va MC da dUdc nghien cufu san xuat
ra de thuc hien dUdc ky thuat c&y Nap cao su
dinh lUdng nUdc tieu mot cac de dang
thay phUdng phap cay dinh lUdng N ip trong
kinh dien phai dung micropipett vo
trung 1^1 hay phai dung vong cay Thach MC
dinh liidng.
Than chai
polystyren
Dong goi
Thach mau
Chai cay hai mat thach BANg - BANg
MC la chai b&ng polystyren co do hai
mat thach BANg va MC tren hai mat
hong cua chai. Chai co nap van chat
vdi mat nap bfing cao su de dang
dam kim bdm mam cay vao. Chai con
co mot n&p ngoai boc bao ve ldp n ip Hinh 4.1; Cau tao chai cay nifdc tieu 2 mat
trong. Tat ca dUdc boc kin b in g thach BANg va MC
mang co.

Cach sur dung

- Xe rach mang co, rut nap ngoai khoi nap trong, van nap trong de md chai.
- Do nUdc tieu can cay vao chai, day chat nap trong. Lac nhe de trarg deu
nUdc tieu len hai mat thach. Mcl n&p trong de do bo tat ca nUdc tieu trong
chai ra ngoai. Van nap trong trd lai nhung giU. nap long. Day nap ngoai len.
u chai d vi tri th&ng trong binh nen roi de trong tu am 35-37°C. Quan sat
khom vi khuan moc tren hai mat thach moi ngay trong 2 ngay. Doc ket qua
dinh lUdng nhu hinh dudi.
- Ket qua dinh danh sd bo co the doc nhu sau:
- Neu chi co vi khuan moc tren mat thach BANg ma khong moc dUdc tren mat
thach MC thi vi khuan phan lap dUdc co the la cau khuan gram [+] nhu
ntreptococcus faecalis hay Staphylococcus aureus hay S. saprophyticus.

39
 - Neu vi khuan moc tren MC ma khong moc tren BANg thi do la cac true
khuan gram [-] de moc.
- Tat ca vi khuan phan lap dtidc deu phai nhuom gram, dinh danh vk l&m
khang sinh do.

Mau difdng tinh M lu am tinh Mau nghi ngd

(nhilm trung tieu) (khong nhilm trung tieu) (co the nhilm trung tieu)
%
Hinh 4.2: Danh gia cac mCrc do vi khuan moc tren chai cay nifdc tieu
hai mat thach BANg va MC

Hinh 4.3: Lo nap do vo trung lay nude tieu

giup benh nhan de dang lay nude tieu va tranh
nguy cd nude tieu bi ri ra ngoai trong qua trinh
chuyen chd den phong thi nghiem hay khi thao
tac vdi benh pham

Hinh 4.4: Chai 2 mat thach BANg va MC dung

cay dinh lifdng nirdc tieu, rat thich hdp trong cac
phien true khi phong thi nghiem khong co nhan
vien true chuyen khoa vi sinh

40
9. CAU HOI ON TAP

a. Cho biet cac chi dinh de c&y nUdc tieu.

b. Hay cho biet cac cach l§ty mau niidc tieu nhu the nao?
c. Hay cho biet phUOng tien lay va chuyen cho mSu nUdc tieu thich hop
nhat cho cac benh vien hien nay va phan tich cac liu khuyet diem cua cac
phUOng tien nay.

d. Hay cho biet qui trinh xet nghiem vi sinh lam sang thiic hien tai phong
thi nghiem doi vdi benh pham nUdc tieu.
e. Hay cho biet cac vi khuan gay benh co the gap dUOc trong cay niidc tieu.

41
C6 NTT. djnh danh VK v i KSD
 BAI 5. BICH NAO TUY
■

MUC TIEU

1. Lay, chuyen chd dich nao tuy va thay diidc tinh cap thiet phai giii gap dich
nao tuy den phong thi nghiem de cay ngay de tang cd hoi bat diidc vi khuan
gay benh.
2. Thiic hien diidc day du cac biidc de khong bo sot cac cd hoi phat hien cac
tac nhan vi khuan gay benh nhd biet diidc qui trinh thiic hien vi sinh lam
sang mau dich nao tuy.
3. Phat hien diidc cac tac nhan thiidng gay viem mang nao mu du cac tac nhan
nay da so rat la kho moc vi biet dung cac phiidng tien can thiet va thich hdp
nhat de thiic hien diidc xet nghiem vi sinh lam sang dich nao tuy.

1. CHI DjNH CHOC DO D|CH NAO TUY (DNT) KHAO SAT VI SINH LAM SANG

- DNT nen diidc choc do de khao sat vi sinh lam sang triidc cac benh nhan co
cac trieu chiing nghi ngd viem mang nao.
\

- Khong nen choc do DNT triidc cac benh nhan co dau hieu gia tang ap liic noi
so. Dau hieu nay diidc kham pha qua soi day m&t thay gai thi bi phu ne.

2. THOI DIEM CHOC DO DNT

- Cang sdm cang tot, ngay sau khi co chan doan lam sang.
- TrUdc khi benh nhan dung khang sinh dieu tri he thong.

3. CACH LAY VA CHUYEN CHCJ

- DNT phai dUdc bac si chuyen khoa choc do, phiidng phap vo trung.
- 5 -10ml DNT diidc lay vao 2 lo vo trung nap chat (cd the dung tube n ip
vang vo trung hay tube Eppendorf biopure) roi gUi ngay den phong thi
nghiem, khong cham tre. Mot lo khao sat vi khuan hoc va mot lo khao sat
sinh hoa te bao.
- Cd the cay ngay tai giUdng vdi chai 2 mat thach cay DNT va cac dich khong
tap nhiem (xem hiidng dan va qui trinh sii dung chai 2 mat thach cay DNT
va cac dich khong tap nhiem).

43
4. QUAN SAT OAI THf:

Ghi nhan cac tinh chat dai the cua DNT, nhii la:

- Trong, keo
- Due, mu
- Vang, hay vang chanh
- Co mau, co sdi fibrin, co can

5. KHAO SAT VI KHUAN HOC

Khao sat vi khuan hoc theo cac budc sau

5.1. Sura soan benh pham

- Neu DNT due, khong can ly tam.

- Cac trUdng hdp khac, ly tam DNT 10 den 15 phut d toe do cao nhat cua
may ly tam ban. Lay phan can khao sat vi the va nuoi cay, ph&n nUdc
lam phan iing hoa mien dich phat hien khang nguyen hoa tan va lam
phan iing sinh hoa.

5.2. Khao sat vi the

- Lam phet soi tUdi

Nho mot giot can ly tam vao lam kinh, day lamel len. Doc dudi quang trUdng
x400, dong bdt hay ha bdt tu quang de thay r5 do tUdng phan. Tim:
+ Bach cau (da nhan hay lympho), hong cau
+ Vi khuan, nam men
+ Ddn bao (Naegleria fowleri)
Neu co nam men nghi Cryptococcus neoformans, quan sat dich nao tuy dudi
giot m tfc tau hay vdi n ig r o s in 20%
Lam phet nhuom
+ Gram
+ Khang acid (neu co yeu cau)
Ket qua phet nhuom va ket qua soi tUdi phai thong bao ngay cho bac si dieu tri.

5.3. Cay phan lap vi khuan gay benh

- Vi la mau benh pham khong bi ngoai nhiem nen cd the cay nhieu mam cay.

44
 - Co the chi can c&y vao thach nau co bo sung XV (CAXV), u d nhiet do 35-
37°C trong binh nen.
- Neu khong co dieu kien ma DNT la cua tre con, co the cay vao hop BA co
vach mot vach S. aureus th in g goc vdi dudng cay mam cay de kich thich H.
influenzae moc dUdc, u b nhiet do 35-37°C trong binh nen.
- Phan DNT con lai co the cay liiu dtf phong (back-up) vao dung dich BHI.
- Theo doi lien tiep trong toi da 3 ngay, bat ci'1 luc nao co sU hien dien cua
khom vi khuan, tien hanh dinh danh va khang sinh do. Neu co dieu kien,
dinh type hay group huyet thanh cua vi khuan.
- Vi viem mang nao mu (VMNM) la mot benh nguy hiem, de doa tinh mang,
nen mot khi co vi khuan moc thi phai tien hanh nhuom gram khom vi
khuan va ket hdp vdi tinh chat khom vi khuan moc tren moi trUdng phan
lap, phai tra ldi ket qua tam thdi cho bac si lam sang ngay.
- Neu cd yeu cau tim C. neoformans, cay DNT tren thach Sabouraud, tren
BHI, u d nhiet do 35-37°C. Cung theo doi lien tuc trong 3 ngay.
- Neu cd yeu c&u tim M. tuberculosis, cay DNT vao moi trUdng Lowenstein -
Jensen (nen cay 3 ong), u d nhiet do 35-37°C. Theo doi lien tuc trong 2 thang.

6. TIM KHANG NGUYEN HOA TAN CAC VI KHUAN GAY BENH THUONG GAP
TRONG DNT BANG CAC PHUONG PHAP HOA MI§N DjCH

Cd the phat hien khang nguyen hoa tan cac vi khuan gay benh cd trong DNT
bang mot trong cac phan iing hoa m iln dich nhtf:
- Tu latex vdi bo thuoc thvi MENINGITEX, PASTOREX..
- Dien di mien dich doi ltfu (CCIE)
- ELISA
Trong cac phifdng phap tren thi phUdng phap tu latex cho ket qua nhanh
nhat (khong qua 3 phut), ddn gian nhat (cd the thuc hien ngay tai giiidng benh), it
chiu anh hUdng cua cac thong so' ky thuat va rat nhay cam.

7. TAC NHAN VI KHUAN GAY VIEM MANG NAO

- Tre sd sinh (cho den 2 thang tuoi)

+ Escherichia coli,
+ Cac Enterobacteriaceae khac (Salmonella spp, Citrobacter spp),
+ Streptococcus agalactiae (nhom B),
+ Listeria monocytogenes,
- Cac lila tuoi khac

45
 + Haemophilus influenzae type B(a),
+ Neisseria meningitidis,
+ Streptococcus pneumoniae,
+ Mycobacterium tuberculosis,
+ Listeria monocytogenes^',
+ Cryptococcus neoformans(b>,
+ Staphylococci'c>.

Bang 5.1: Bien luan ket qua sinh hoa -te bao -vi the - dai the djch nao tuy de ti/d o co the
suy doan ra diidc tac nhan vi sinh vat gay viem mang nao my
Thong so VMN vi khuan VMN lao VMN vi nam VMN virus
Bach cau Da nhan Odn nhan Odn nhan Ddn nhan
Glucose Rat thap Thap Thap Binh thudng
5-20mg/100ml 20-40mg/100ml 20-40mg/100ml 65-70mg/100ml
Protein Tang Tang Tang Hdi tang trong giai
doan dau
Vi the Thudng phat hien Hiem khi thay Thudng phat hien am tinh
dUdc vi khuan dUdc AFB d i/d c vi nam
Dai the Due Vang chanh Due nhe Trong

la) La tac nhan gay VMNM thtfdng gap nhat d tuoi til/ 6 thang den 2 tuoi. Hiem d tuoi tren 5 tuoi.
,b) Gap d ngudi bi suy giam m iln djch (vi du AIDS)
tc) Gap d ngudi chiu p h iu thuat so nao hay sau dan luu

8. CAU HOI ON TAP

a. Cho biet cac chi dinh lam sang de cay dich nao tuy, va thdi diem tot nhat
de lay dich nao tuy.
b. Hay cho biet cac cach lay va chuyen chd dich nao tuy den phong thi
nghiem de lam xet nghiem vi sinh lam sang.
Hay cho biet phUdng tien lay va chuyen chd dich nao tuy thich hdp nhat
cho cac benh vien hien nay, va phan tich cac Uu khuyet diem cua cac
phifdng tien nay.

Hay cho biet qui trinh xet nghiem vi sinh lam sang thuc hien tai phong
thi nghiem doi vdi benh pham dich nao tuy.
e. Hay cho biet cac vi khuan gay benh co the gap dUdc trong cay dich nao tuv.
f. Hay cho biet cac phUdng phap hoa mien dich phat hien khang nguyen
hoa tan cac vi khuan gay benh viem m ang nao m u co trong dich nao tuy.
Phan tich cac Uu khuyet diem cua cac phUdng phap nay.

Bien luan ket qua sinh hoa te bao xet nghiem dich nao tuy.

46
 H J
• L 4-<-

J
Naegleria fowleri C.neoformans soi dudi C.neoformans
soi tUdi dudi KHV KHV dao pha soi tUdi dudi KHV

Hinh 5.1: mot so hinh

anh quan sat true tiep
phet soi tuoi hay phet
nhuom gram cac mau
dich nao tuy benh nhan
VMNM

Neisseria meningitidi qua Streptococus pneumoniae qua

phet gram djch nao tuy phet gram dich nao tuy

47
PHU LUC 1:

San pham lien quan:

CHAI HAI MAT THACH CAXV DE CAY DjCH NAO TUY HAY CAC CHAT DICH
KHONG TAP NHllM KHAC

N ip ngoai
Muc dich sur dung

Chai cay hai mat thach CAXV

da dUdc nghien ciiu san xuat ra de
thiic hien dUdc ky thuat cay tai
N ip cao su
giUdng hay cay trong phien trUc cac
mau dich nao tuy; hay cac dich
khong tap nhiem khac nhii dich N ip trong
mang phoi, mu abcess, dich mang
bung, dich mang tim, dich khdp... Thach CAXV

Dong goi Than chai

polystyren
Chai cay hai mat thach CAXV
la chai bang polystyren co do hai Thach CAXV
mat thach CAXV tren hai mat hong
cua chai. Chai co nap van chat vdi
mat nap bang cao su de dang dam
kim bdm mam cay vao. Chai con co
mot nap ngoai boc bao ve ldp nap
trong. Tat ca dUdc boc kin bang
mang co.

Cach sur dung Hinh 5.2: Cau true cua chai 2 mat thach CAXV
dung cay djch nao tuy va cac dich
Xe rach mang co, rut nap khong tap nhilm khac
ngoai khoi nap trong.
Md nap trong de cho benh
pham trang len hai mat
thach, sau do van chat lai
nap trong. Co the khong md nap trong ma dung bdm tiem hut lay benh
pham roi bdm benh pham qua nap cao su (sau khi sat trung bang con va de
kho) de trang len hai mat thach. De chai nam len mat ben (khoang vai phut
cho moi mat ben) de benh pham diidc trang deu len 2 mat thach. Sau do
dung ddng chai len, ndi long nap trong, day nap ngoai len, cho vao binh nen
va de vao tu am 35-37°C. Xeu la tu am C 0 2 thi khong can cho vao binh nen.
 - Quan sat khom vi khu^n moc tren hai mat thach moi ngay trong 2 ngay.

- Do mSt thach c£y benh ph^m la thach CAXV giau bo diidng va giau XV, do
v^y ta't ca cac vi khuan du kho moc nhii H. influenzae, N. meningitidis, S.
pneumoniae deu co the moc dUdc. Khi thay co sii hien dien cua bat ky khum
vi khu£n nao moc tren mat thach thi phai lam phet nhuom Gram de quan
sat hinh thai vi khusin va tien hanh dinh danh, khang sinh do theo hiidng
ma hinh anh Gram vi khuan gdi y.

PHU
• LUC
• 2:

MENINGITEX - phat hien nhanh khang nguyen hoa tan cac vi

khuan thudng gay viem mang nao mu

Nguyen tic thur nghiem

Thii nghiem thiic hien diia theo nguyen tac cua phan iing tu latex, gac khang
the dac hieu dUdc gan tren hat latex se bi tu lai khi gap cac khang nguyen tUdng
iing va hien tiidng tu nay se thay diidc bang mat thiidng.

Thanh phan thuoc thur

Mot bo thuoc thii gom 5 tube nap chat chiia thuoc thii la 1 ml huyen dich cac
hat latex da diidc gan khang the dac hieu Neisseria meningitidis group A (lo NMA),
Neisseria meningitidis group B (lo NMB), Neisseria meningitidis group C (lo NMC),
Haemophilus influenzae (lo Hib) va Streptococcus pneumoniae (1 lo PNE). Ngoai ra
con co lo chiia 200pl khang nguyen chiing (lo C [+]). Hop thuoc thii phai dUdc bao
quan tai nhiet do 4°C, co han dung 2 nam tii sau ngay san xuat.

Phirong phap thirc hien thur nghiem

Chuan bi mot lam soi kinh hien vi mdi, lau sach bang con, roi sau do lau kho.
Nhd khi cam lam phai cam tren canh lam chii khong cam tren mat lam. Dung but
aceton hay but md ke 5 o tren lam. Nho len moi o mot giot (khoang 10pl) dich nao
tuy (DNT). Lac manh cac tube thuoc thijf MENINGITEX de thanh huyen dich due
deu nhii suta. Sau do nho tren 6 dau tien mot giot (khoang lOfal) NMA, o thii 2 mot
giot (khoang lOpl) NMB, o thii 3 mot giot (khoang lOfil) NMC, o thii 4 mot giot
(khoang lOpl) Hib, o thii 5 mot giot (khoang 10p.l) PNE. Thay dau cone sau moi lan
lay va nho thuoc thu tren lam. Dung que tam tron deu giot thuoc thii vao giot dich
nao tuy, viia tron vila trai rong hon hdp ra cho gan day mat o, nhd thay que tam

49
sau khi tron hon hop trong moi o. Sau khi tron xong, giii canh lam giila hai ng6n
tro va cai, \kc nghieng tdi roi nghieng lui trong vong 3 phut, vtia l&c vtia quan sat
xem o nao tu lai de ghi nhan ket qua [+] khang nguyen hoa tan cua vi khu&n gay
benh co trong dich nao tuy tUOng iing vdi khang the dac hieu co trong giot thuoc
thii. Co hien tUdng tu trong o khi cac hat latex khong con d dang huyen dich due
deu nhu stia ma tu lai thanh hat ldn cdn tr&ng tren nen dung dich trong dfin di.
Hat ldn cdn tr&ng cang 16n khi phan ting tu cang manh, cang nho khi phan iing tu
cang yeu. Neu sau 3 phut phan ting tu chua thay ro, co the quan sat qua mot gUdng
lorn anh phan chieu cua cac o qua gUdng lom de phat hien hien ttidng tu.

Kiem tra thuoc thur

Cti moi vai thang kiem tra thuoc thii xem co con du nhay khong vdi chiing [+]
va vdi nUdc muoi sinh ly. ThUc hien tren 2 lam soi kinh hien vi, moi lam cung ke 5
o. ThUc hien nhu tren nhung tren mot lam, thay vi nho vao moi o 1 giot DNT, cho
vao moi o mot giot 10 pi chting [+] (tti lo C [+]) va tren lam kia cung thay vi nho v&o
moi o 1 giot DNT thi nho vao moi o mot giot 10 pi nUdc muoi sinh ly. Thuoc thii con
hieu luc khi ca 5 o nho chting [+] deu co hien tUdng tu so vdi ca 5 6 nho nUdc muoi
sinh ly deu khong co hien tUdng tu.

00010
Ooc ket qua: Hib
NMA NMB NMC Hib PNE

1 §1 @ 1
Chifng am (nUdc muoi SL) Chifng f+1

Hinh 5.3: Mo ta cach lam xet nghiem phat hien cac vi khuan gay benh co trong dich nao tuy
bang bot thuoc thif MENINGITEX

50
Hinh 5.4: Chai 2 mat thach CAXV cay dich nao tuy va cac dich khong tap nhilm khac, rat thi'ch
hdp trong cac phien true khi phong thi nghiem khong co nhan vien trUc chuyen khoa vi sinh

c D

Hinh 5.5: Cac phUOng tien thich hop de lay djch nao tuy co the la:
(A) Tube ly tam vo trung 15ml nap van (tra i), (B) tube vo trung 5ml, tube vo trung 1,8ml va
(D) tube nap bat kieu Eppendorf biopure

51
O'
to
 BAI 6. MAU QUET HAU, HUNG
VA CAY NlAU QUET HAU HUNG

MUC TIEU

1. Thiic hien diidc cach lay cung nhii hiidng ddn diidc lam sang lay diidc
benh pham nay nhd biet diidc cach quet hau hong.
2. Thiic hien diidc qui trinh vi sinh lam sang mdu quet hau hong de co the
phan lap diidc cac vi khuan dich md yeu cau cua lam sang muon tim.
3. Chon liia vd sit dung dung cac phiidng tien de khao sat diidc vi sinh lam
sang mdu quet hau hong nhdm vao cac vi khuan dich cdn tim theo yeu cau
cua lam sang nhd biet diidc phiidng tien cdn thiet vd thich hdp nhat de
thiic hien diidc xet nghiem vi sinh lam sang quet hau hong.

1.CHi DjNH

- Quet hau hay quet hong dtfdc chi dinh trUdc cac benh nhan c§n xac dinh co
phai bi nhilm trung vung hau, hong do vi khu^n hay khong. Nghla la nen
co chi dinh quet hau, hong triidc cac benh nhan co cac trieu chiing: dau, rat
vung hau, hong. Kham thay niem mac h&u hong sUng do, phu ne, viem
amidan, co mang mu hay mang gia, phu ne liidi, ludi do dau tay va siing
hach co.
- Ngoai ra quet hau, hong con co khi dUdc chi dinh de phat hien ngUdi lanh
mang vi khu&n nhu S. aureus, N. meningitidis, S. pyogenes (nhom A),
C. diphtheria.

2. THOI DIEM QUET HAU, HONG

Phai lay m iu triidc khi benh nhan dung khang sinh he thong.

3. CACH LAY MAU

- Cho benh nhan ngoi, dau hdi ngtia ra sau. Yeu cau benh nhan ha mieng ra,
co the v\ia ha mieng vila phat am A..A..A..
- Dung cay de liidi, de liidi cua benh nhan de thay ro hong.
- Lay tam bong vo trung (tam bong vo trung lay mdu) quet vao vung amidan,
vung thanh sau hong hay vung bi viem nhiem. Tranh quet vao liidi hay vom
khiku cai hay niem mac ma, mieng.

53
7.2. Neu ket qua cay quet hau hong co H. influenzae, S. pneumoniae, S. aureus
hay N. meningitidis thi day cd phai la cdc vi khuan gay viem hpng khong v i co
can tra ldi cho lam sang khong?

- Cac vi khuan tren deu co the la vi khuan thiidng tru d hJiu hong, khong phai
la tac nhan gay viem hong. Rieng H. influenzae, neu la type B thi c6 thi li
tac nhan gay viem hong.
- Khi ket qua cay la mot trong cac vi khuein tren thi c6 the tra ldi cho lam
sang neu co yeu c£u ro rang tim cac vi khuan tren, nhung phai ghi chu day
co the la vi khuan thudng tru khong gay benh viem h£u hong.

7.3. Xin cho biet gia tri cua cac thur nghiem hoa mien djch (sac ky mien djch hay tu
latex) phat hien trifc tiep va nhanh lien d u tieu huyet nhdm A trong quet hpng?

- Day la cac thii nghiem hoa m iln dich phat hien khang nguyen carbohydrat
C dac trUng cua lien cau tan huyet nhom A trong hau hong.
- Cac thii nghiem nay co do nhay va do dac hieu rat cao, khong thua gi nuoi
cay. Lai co the thiic hien rat de dang tai cac phong mach hay phong kham
va ket qua cung rat nhanh chi trong vong khong qua 10 phut sau khi lay
mau. Chinh vi vay day la t h i nghiem nen co tai cac phong mach hay phong
kham vi viem hong la mot benh canh rat thudng gap va yeu c lu tim lien chu
tan huyet nhom A cung la mot yeu chu rat din thiet de giup chan doan phan
biet vdi cac tac nhan viem hong khac cung nhii giup bac si cd ngay chi dinh
dieu tri chinh xac ngay tii dau. Hinh dtidi day mo ta nguyen tac hoat dong
cua thii nghiem.
A. Cho tam bong quet hong vao
tube chira dem ly trich khang
nguyen carbohydrate C dac hieu 1. Ldp gdn thuy tinh tam khang the ddn d in g dac hieu k h in g nguyen
lien cau tan huyet beta nhdm A. ^ ■ < ------6 carbohydrat C cu lien c4u lan huyet nhdm A . 2. V ach t^m k h in g th^ tbo M e
Khang nguyen di/Oc ly trich se hieu k h in g nguyen. 3. V a ch tam k h in g d i e hieu k h in g the chuflt 4. G iip thim
tham vao ldp gon thuy tinh cd tam • d i y . 5. Thanh nitro ce llu lo se de s i c ky 6. T i m bong quet hpng duoc ngim

khang the don dong gan mau dac tube chtfa d e m ly trich k h in g nguy£n. 7. K h in g nguyen carbohydrat C caa
“ hen cau tan huyet nhdm A da dirdc ly trich.
hieu khang nguyen
2 3 3
B . Khang nguyen di/oc ly trich se
bi khang the don dong gan mau i i
(cd the la cac hat vang the keo)
tom tat
8. CAU HOI ON TAP

Cho biet cac chi dinh lam sang de l&y mau quet hau hong khao sat vi
sinh lam sang.
Hay cho biet cac cach lay va chuyen chci mau quet hau hong den phong
thi nghiem de lam xet nghiem vi sinh lam sang.
Hay cho biet phUdng tien lay va chuyen chd mau quet hau hong thich
hdp nhat cho cac benh vien hien nay va phan tich cac tfu khuyet diem
cua cac phUdng tien nay.
Hay cho biet qui trinh xet nghiem vi sinh lam sang thiic hien tai phong
thi nghiem doi vdi benh pham quet hau hong de phat hien lien c^u tieu
huyet nhom A va de phat hien cac vi khuan dich khac theo yeu cau cua
lam sang.
Hay cho biet cac vi khuan gay benh can phai phan lap trong m lu quet
hau hong.

Quet tam bong len hach amidan

\
tranh dung vao li/di, nien mac ma
hay vom khau cai

Hach amidan

Hinh 6.2: Cach lay benh pham quet hau hong bang quet tSm bong len hach amidan,
tranh cham vao liidi, niem mac ma hay vom khau cai

Hinh 6.3: Hinh anh vi the vi khuan bach cau. (A) Phet nhuom
methylen blue kiem (B) Phet nhuom gram

57
 Sd do 6: Qui trinh vi sinh 13m sang khao sat
mau quet hau hong tim lien cau tan huyet beta

Benh pham quet hau hong, khong can

moi trudng chuyen trci
DO

Phan lap: BANg/BAGe

hay BA

D1

U 37 C/bin h nen/4-6 gid Qay tang sinh bang dudng Phat hien cac khom
zic-zac tren mat BA Streptococci tan huyet beta

prZ't, •\
I ?/ •)

D2
KQ chung cuoc: dinh danh
va khang sinh do

58
 BAI 7. BENH PHAM TAI, MAT, MUI, XQANG
VA CAY BENH PHAM TAI, MAT, MOl, XOANG

MUC TIEU

1. Hiidng dan diidc lam sang hay tham chi co th i thiic hien diidc tot biidc
lay vd chuyen chd mdu nhd biet diidc cach lay vd chuyen chd cac mdu
benh pham tai mat mui xoang.
2. Thiic hien diidc day du cac biidc trong gui trinh xet nghiem vi sinh lam
sang cac mdu benh pham lay tii tai, mat, mui xoang de khong bo sot cac
cd hoi phat hien cac tac nhan vi khuan gay benh.
3. Si} dung diidc phiidng tien cdn thiet vd thich hdp nhat de thiic hien diidc
xet nghiem vi sinh lam sang cac benh pham lay tii tai, mat, mui, xoang;
nhd vay co the phat hien diidc cac tac nhan gay benh cho dii la cac tac
nhan rat kho moc.

1. CHI DJNH

- Cac benh ph&m tai, m it, mui, xoang dUdc chi dinh lay trUdc cac trtidng hdp
viem cap tinh hay m an tinh cac vung tai, mat, mui, xoang.

- Cu the la viem tai ngoai cap tinh, viem tai gitia cap hay man tinh, viem
xoang cap hay man tinh, viem mui cap hay man tinh, viem ket mac cap hay
man tinh.

2. THOI DIEM LAY BENH PHAM

Tot nhat la trtidc khi benh nhan dung khang sinh he thong hay tai cho.

3. CACH LAY BENH PHAM

3.1. Trudng hop viem tai ngoai

- Neu chua vd mu, sat trung da b&ng con 70%, chd kho, sau do choc hut hay
rach lay mu cho vao tube vo trung (tube nap vang vo trung hay tube
Eppendorf biopure). Neu da vd mu, lau sach mu roi dung tam bong vo trung
ep nan mu tham vao tam bong (dung tam bong vo trung lay mdu). Cac m lu
mu lay khong co moi trtidng chuyen chd phai gtii ngay den phong thi
nghiem de yeu cau cay ngay.

59
 - Thich hdp nhat la dung tam bong tham hay lay mu cho vao moi trudng
chuyen chd (dung cap tube dua tam bong vo trung I tube dua Stuart Amies
hay cap tube dua tam bong manh vo trung* I tube dua Stuart -Amies) Ufy
hay tham mu. Mu lay ra, co the cho vao lo hay tube nSp chat vo trung hay
tham vao tam bong vo trung roi cho vao tube n&p chat vo trung, gui ngay
den phong thi nghiem. Trong trUdng hdp cham tre thi cho vao tube moi
trUdng chuyen chd Stuart -Amies roi giii den phong thi nghiem.

3.2. Trudng hdp viem tai giiifa

- Neu cap tinh va chua v3 mu, bac si chuyen khoa lay qua choc hut xuyen
mang nhi cho vao tube vo trung (tube nap vang vo trung hay tube Eppendorf
biopure). Neu da vd mu hay chay mu man tinh, lau sach mu vung tai ngoai
roi dung tam bong vo trung tham mu vao tam bong (dung tam bong vo trung
lay mdu, hay tot nhat la tam bong manh vo trung*). Cac mau mu lay khong
co moi trUdng chuyen chd phai giii ngay den phong thi nghiem de yeu cau
cay ngay...
- Thich hdp nhat la dung tam bong tham hay lay mu cho vao moi trUdng
chuyen chd (dung cap tube dua tam bong vo trung / tube dua Stuart Amies
hay cap tube dua tam bong manh vo triing* / tube dua Stuart -Amies) Ifiy
hay tham mu. Mu lay ra, co the cho vao lo hay tube nap chat vo trung hay
tham vao tam bong vo trung roi cho vao tube nap chat vo trung, gui ngay
den phong thi nghiem. Trong trUdng hdp cham tre thi cho vao tube moi
trUdng chuyen chd Stuart -Amies roi giii den phong thi nghiem.

3.3. Trudng hdp viem xoang

- Neu cap tinh thi lay quet mui sau hay quet mu khe giufa. De lay quet mui
sau, bac si chuyen khoa dung kep nong mui benh nhan roi lay quet mui sau
bang mot tam bong nho, cong mem (tam bong manh vo trung*), luon tU lo
mui qua vach mui trUdc va mui sau, dua sau vao trong cho den khi can lai la
hau. De lay quet mu khe giuta can phai lay qua noi soi tai mui hong. Quet
mui sau va quet mu khe giuta dUdc cho vao tube vo trung hay vao moi
trUdng chuyen chd Stuart -Amies roi giii den phong thi nghiem (dung cu
thich hdp nhat de lay mau nhu vay la cap tube dua tam bong manh vo
trung I tube dua Stuart -Amies)
- Neu man tinh, lay mau thii la mau sinh thiet hay dich riia xoang bang niidc
muoi sinh ly khong co chat sat trung. Manh sinh thiet hay dich riia diidc cho
vao lo vo trung (dung lo vo trung lay mdu) hay tube vo trung (tube
Eppendorf biopure) roi gui ngay den phong thi nghiem.
* 1 rUdng hdp vung phai lay mu qua hep nhii mu khe giuta, mu tai ngoai.
mu tai giUa

60
3.4. Trudng hdp viem mui

- Bac si chuyen khoa la'y quet mui sau hay quet mu khe giuta roi guti den
phong thi nghiem.
- Quet mui triidc chi diidc la'y de khao sat tinh trang mang vi khuan S.
pneumoniae, H. influenzae 6 cac benh nhan tre em bi nhilm trung ho hap
cap. Ngoai ra, quet mui trU6c con diidc la'y de dieu tra tinh trang ngiidi lanh
mang S. aureus.. Dung cu thich hdp nha't de lay quet mui triidc la dung cap
tube dua tam bong vo trung I tube dua Stuart - Amies.

£.5. Trudng hdp viem ket mac mat

- Benh pham la quet mu ket mac mat diidc bac si chuyen khoa la'y bang ky
thuat vo trung.
- Dung cu thich hdp nhat de la'y m lu la cap tube dua tam bong vo trung I tube
dua Stuart Amies hay cap tube dua tam bong manh vo trung * /tube dua
Stuart - Amies.

4. CAC VI KHUAN GAY BENH CO THi TIM THAY TRONG B|N H PhAm

4.1. Cac trudng hdp viem xoang, mui cap tinh va man tinh

- S. pneumoniae,
- H. influenzae,
- M. catarrhalis,
- Cac Streptococci tan huyet P,
- S. aureus,
. - Klebsiella va cac vi khuan thuoc ho Enterobacteriaceae,
- Vi khuan ky khi (Bacteroides va cac vi khuan khac).

4.2. Trudng hdp viem tai giura cap

- S. pneumoniae va cac Streptococci khac,

- M. catarrhalis,
- H. influenzae.

4.3. Trudng hdp viem tai giura man tinh

- P.aeruginosa,

- Proteus spp.,

61
 - Cac vi khu&n ky khi,
- Cac vi khuan khac: hiem gap.

4.4. Trudng hdp viem ket mac mat cap tinh hay man tinh

- Haemophilus spp.,
- Moraxella spp.,
- N. gonorrhoeae,
- S. aureus,
- S. pneumoniae,
- S. pyogenes,
- P. aeruginosa,
- Cac vi khu&n khac: hiem gap.

5. KHAO SAT TRUC TIEP

- Lam phet nhuom gram (sau khi tien hanh nuoi c&y neu benh ph&m chi 1&
mot que tam bong).
- Ghi nhan su hien dien cac vi khu&n trong mau va tra ldi sd bo cho bac si lam
sang.

6. TIEN HANH NUOI CAY

6.1. Cay phan lap tren cac hop thach phan lap* toi thieu la

- Thach mau (BA) thudng hay BA co nalidixic acid (BANg).

- Co the them BAGe (thach mau co gentamicin chon loc cac vi khuan
Pneumococci va Streptococci).
- Thach nau chon loc H. influenzae (CAHI).
- Thach chon loc trUc khugin Gram [-] d§ moc (EMB hay MC).
- Co the them thach nau chon loc Neisseria (CATM) khi co yeu cau.
- Co the them Sabouraud Dextrose Agar co hay khong co khang sinh de cay
chon loc vi nam.

Cac hop thach BA. BAGe, CAHI, CATM phai u trong binh nen, cac hop thach
khac u khi trUdng binh thudng. Tat ca de trong tu u 35-37°C va quan sat moi ngay
trong lien tiep 3 ngay.
6.2. Cac khom mpc tren hop thach phan lap deu phai tien hanh djnh danh va lam
khang sinh do

7. CAY BENH Ph A m TAI, MAT, MUI, XOANG - CAC CAU HOI THUONG GAP

7.1. Trudc benh nhan bj viem mui, lamf sang co khi lay benh pham b&ng tam bong
cho vao mui roi quet lay djch mui xanh hay vang chay ra tir mui benh nhan de
lam xet nghiem vi sinh; cach lay benh pham nay co gia trj khong?

- Vdi cach lay benh pham tren thi chung ta chi lay dUdc quet mui trudc va
nhu vay thi co the benh pham nay bi ngoai nhiem vdi vi khuan thudng tru
vung mui trudc nhu S. pneumoniae, H. influenzae, va S. aureus ma cac vi
khu^n nay cung rat co the la tac nhan gay viem mui hay viem xoang mot
khi lan tCf ndi thudng tru sang xoang hay mui sau. Chinh vi vay ket qua cay
cac benh pham nay khong the khang dinh diidc tac nhan vi khuan phan lap
diidc co phai la tac nhan gay benh hay khong.
- Vay thi benh pham nao la thich hdp nhat tren cac benh nhan bi viem mui?
Cung nhu trong viem xoang cap, benh ph&m thich hdp nhat phai la cac quet
mu lay tu: khe giOa cua mui hay la quet mui sau. Cac benh pham nay khong
the lay dUdc bang cac que tam bong thong thudng vi khong the tranh khoi bi
dung vao mui triidc khi lay mau. PhUdng tien thich hdp nh&'t de lay dUdc
quet mu khe giiia va mu quet mui sau phai la cac que tam bong manh lam
b in g sdi nhom. Vdi que tam bong nay, lam sang cd the de dang lay mau
quet mu khe giiia hay quet mui sau qua noi soi hay que kep nong lo mui,
tranh cham vao mui trudc.

7.2. Vi sao tai nhieu benh vien, ket qua vi sinh lam sang cac benh pham lay tirtai
mat mui xoang thudng cho ket qua am tinh hay khong phu hop vdi hieu qua dieu
trj khang sinh tren benh nhan?

- Co the ket qua am tinh that svf do benh nhan da dieu tri khang sinh trudc
lam cho cac vi khuan khong the phan lap dUdc do da bi tieu diet bdi khang
sinh hay da lan tron trong cac vi tri sau hdn, cung cd the tac nhan khong
phai vi khuan ma la cac tac nhan khac.

- Nhiing nguyen nhan khac lam ket qua am tinh la cd the lien quan den van
de ky thuat nhii lay va chuyen chd benh pham, quan trong nhat la benh
pham sau khi lay khong cho vao moi trUdng chuyen chd va khi den phong
thi nghiem lai khong dUdc cay ngay trong khi do da sd" cac tac nhan gay
benh dudng tai mat mui xoang lai la cac tac nhan vi khuan rat kho moc nhii
H. influenzae, S. pneumoniae, va M. catarrhalis. Ngoai ra mot nguyen nhan
khac rat thiidng gap tai cac benh vien chung ta hien nay la phong thi nghiem

63
 chi dung cac moi tnidng tii pha che thong thtidng nhii thach mau ngtfdi vi
thach Mac Conkey hay tham chi chi dung thach dinh diidng thudng (Nutrient
Agar) ma khong dung cac thach phan lap thich hdp nhi't cho cac benh phim
lay ttif tai m it mui xoang nhii thach mau ctTu va thach CAXV d i phan lip
dUdc cac vi khuan rat thiidng gap nhung kho moc neu tren.

- Nguyen nhan lam cho co sii khac biet giiia ket qua vi sinh vdi hieu qua dieu
tri khang sinh tren benh nhan co the la do vi k h u in phan lip dUdc khong
phai la vi khuan gay benh ma chi la cac vi khuan thudng tru vung mui
trudc, vung tai ngoai... va nguyen nhan thiidng l i vi lam sang li'y benh
phSm bi tap nhiem vdi cac vung nay do dung phUdng tien ltfy m lu khong
thich hdp nhu da de cap d phan tren; hay cung cd the vi k h u in phan lip
dUdc chi la vi khuan tam tru trong benh p h im vi du vi khu&n tam tru trong
mu tai ngoai, mu xoang hay mu tai giiia chay vao tai n goii... v i de giai
quyet dUdc nguyen do nay lam sang c in phai chui sach mu d cac vung tren
va chi lay mu mdi chay ra hay mdi nan ra.

7.3. Xin cho biet lam the' nao de co the lay benh pham tiTcac loet giac mac?

- Lay benh pham tCf cac loet giac mac doi hdi phai sii dung mot cay nao bing
kim loai de nao dUdc vet loet cho vao ong moi triidng BHI bo’ sung XV roi
mdi chuyen ve phong thi nghiem. Neu khong cd cay nao niy, lam sin g cd
the dung que tam bong manh vo trung quet lay mu tii vet loet cho v io ong
, BHI -XV.

- Tai phong thi nghiem, ong BHI -XV dUdc u trong tu am 35 - 37°C trong 6 gid
hay qua dem de hom sau ca'y phan lap tiep len cac moi triidng phan lap nhii
da hudng d in trong qui trinh. Ong BHI - XV v ln phai dUdc tiep tuc u trong
tu am va theo doi lien tuc trong 7 ngay de phat hien vi khuan hay vi nam
moc neu lan phan lap dau khong cd vi khuan moc.

- Trong trUdng hdp nghi tac nhan gay benh la nam, thi cac nao hay quet mu
vet loet phai dUdc ca'y ngay tren moi trudng Sabouraud Agar ben canh viec
tang sinh trong ong BHI - XV.

8. CAU HOI ON TAP

a. Cho biet cac chi dinh lam sang de lay cac benh pham tii tai, m it, mui,
xoang lam xet nghiem vi sinh lam sang.
b. Hay cho biet cac cac loai benh pham lay tii tai, mat, mui, xoang v i c ic
cach lay va chuyen chd cac loai benh pham n iy den phong thi nghiem de
lam xet nghiem vi sinh lam sing.

64
 c. Hay cho biet phiidng tien l&y va chuyen chd thich hdp nhat cho cac benh
vien hien nay de l&y cac benh ph^m tii tai, mat, mui, xoang va phan tich
cac liu khuyet diem cua cac phUdng tien nay.
/
d. Hay cho biet qui trinh xet nghiem vi sinh lam sang thuc hien tai phong
thi nghiem doi vdi benh pham l&y tii tai, mat, mui, xoang.
e. Hay cho biet cac vi khu^n gay benh can phai phan lap trong cac mau
benh pham lay tii tai, m it, mui, xoang.

Hinh 7.1: Tam bong manh vo trung rat tich hop de lay cac quet mu tCr cac khe hep nhii mu khe
giura, mu chay ra tii tai giura, quet mu nieu dao ngay trong long nieu dao; hay lay mu loet giac
mac la cac vet loet nho khong the dung tam bong binh thiidng de quet. Tam bong manh nay
diidc thiet ke de co the cho vCia van vao tube dua moi triidng chuyen chd Stuart - Amies

Hinh 7.2: Moi triidng tang sinh BHI-XV chCra trong cac tube 5ml rat can thiet de tang sinh cac
quet mu loet giac mac von dT chCra rat it cac vi khuan gay benh

65
 TT
Cac benh pham la mu tai Ly tam lay cSn tCr cac benh
hay mu xoang, co the lay pham Id djch nJa xoang ^ '
trifc tiep hay cho vao moi
trifdng chuyen chd

Lam mot phe t

nhuom gram
QO Ca'y phan lap tren:
- BAGe (chon loc PNE, STR)* U 37°C qua (Jem
- CAHI (chon loc HIN, BRA) BAGe (binh nen)
- BANg (chon loc Gr(+] cocci)* CAHI (binh n£n)
- MC/EMB (chon loc Gr[-] rod) BANg (binh nen)
Ket qua sd bo: KSTT
* Neu khong co BANg va BAGe, ■MC/EMB (binh thudng)
thay b ln g BA

D1 Chon khom vi khuan dich

tren cac hop thach phan lap
.chon loc

Cay tdng sinh bang difdng zic-zac len

mat thach BA hay mat thach thich hdp.
U 37°C/CO; trong 4-6 gid

Dinh danh sd bo

Gat vi khuan tang sinh tCr

di/dng cay zic-zac

Khano sinh do

tt± ± ± i_ m i “ f N

D2 r

Ket qua chung cuoc

Sd do 7: Qui trinh vi sinh lam sang m lu mu tai,
mu xoang, djch rCfa xoang

66
 BAI 8. DOMI, DjCH HUT DOfM QUA MUI, D|CH HUT RUTA
PHE QUAN QUA N0I SOI VA CAY DOM,
BENH PHAM CHUTA DOM
m

MUC TIEU

1. Hiidng ddn diidc lam sang cho chi dinh dung, cung nhii hiidng dan diidc
lam sang hay benh nhan lay dung cac loai benh pham nay nhd biet diidc
cac chi dinh, phiidng phap vd thdi diem de lay mdu ddm vd cac benh
pham cd ddm khao sat vi sinh hoc.
2. Danh gid diidc mdu ddm vd cac benh pham co ddm nhd vay cd the loai
bo, khong tien hanh nuoi cay cac mdu khong cd gid tri, tranh diidc cac
ket qua xet nghiem mdu ddm khong hUu dung cho lam sang.
3. Trien khai sit dung diidc cac phiidng tien thich hdp nhat vd day du nhat
de cd the phan lap diidc dung cac vi khuan gay benh cd trong mdu ddm
vd cac benh pham co ddm, du la cac vi khuan kho moc nhat, nhd biet
diidc qui trinh xet nghiem vi sinh lam sang mdu ddm vd cac benh pham
cd ddm

1. CHiOjNH

- Cac trUdng hdp viem nhiem dtfdng ho hap dudi nhii viem phoi, viem phe
quan cap, cdn cap cua viem phe quan man.
- Nen cho chi dinh lay mau trong cac trUdng hdp benh nhan co mot trong cac
trieu chiing sau: ho co mau hay ho nhieu, dau ngiic, kho thd, co dau hieu dac
phoi nhii co ran am va rft; giam tieng ri rao phe nang; go due khi kham
phoi; phim phoi co tham nhiem; co nang, co mu.

2. THOI DIEM LAY MAU

- Cang d giai doan sdm cua benh cang tot. Nghia la tien hanh lay mau ngay
sau khi co chan doan lam sang.

- Nen lay mau trUdC khi benh nhan dung khang sinh he thong.

67
3. CACH LAY MAU

3.1. Ddm

- Triidc het cho benh nhan sue mieng sach, khong sue mieng bang niidc sue
mieng co chat sat trung.
- Hufdng d^n benh nhan hit that sau vao roi hay co khac ddm ra. Co the giup
benh nhan khac ddm bang each vo nhe vao liing. Benh nhan khac ddm vao
lo vo trung, rong mieng, nap chat (dung lo vo trung lay mdu). Tranh lan
nUdc bot.

3.2. Djch hut ddm tren khi quan qua dudng mui (Naso-Tracheal-Aspirate)

- Triidng hdp benh nhan khong the khac dUdc ddm, nhu d tre em.

- Dung dung cu dac biet goi la bo hut khi quan (tracheal suction set), la mot
ong nghiem gan vdi mot nap van tren do co 2 voi, mot noi vdi mot ong thong
mem, mot noi vdi may bdm chan khong dang hoat dong va tren voi nay c6
mot van hong dang met. TrUdc het ngUdi me hay than nhan benh nhi am
ngiia be vao long, giii dau hdi ngiia ra sau. Dua ong thong qua mui benh nhi
cho den khi dau ong cham vao phan tren khi quan, luc do benh nhi se c6
phan xa ho. Ngay luc benh nhi ho, dung tay bit chat van hong lai, nhd vay
ddm dUdc hut vao ong thong. Sau do rut ong thong khoi mui benh nhi, roi
cho dau ong thong vao mot lo chiia 5ml nUdc muoi sinh ly vo trung. Nude
muoi sinh ly se riia dam dinh d thanh ong thong vao ong nghiem. Tat may
bdm, thao nap co voi khoi ong nghiem roi day chat ong nghiem bang mot
nap van khac co trong bo dung cu. Gui mau den ngay phong thi nghiem.
- Neu khong co dung cu nay, co the dung ong chich 60ml, noi mot dau vdi ong
thong mem de lay ddm theo cach nhu tren, nhung thay vi dung bdm chan
khong, hut ddm bang tay vdi ong chich tren.

3.3. Djch hut phe quan qua noi soi (BW = Broncho-Washing)

Do bac si chuyen khoa lay khi dang noi soi, cho vao tube vo trung nap chat roi
giii ngay den phong thi nghiem.

3.4. Cac benh pham khac

Nhii dich hut xuyen khi quan, choc hut phoi, do cac bac si chuyen khoa lay va
giii ngay den phong thi nghiem de khao sat.

4. DANH GlA MOT m Au CO GlA TRj DE KHAO SAT VI KHUAN HOC

- Vi ddm cung nhii dich hut ddm tren khi quan qua diidng mui (NTA) hay
dich rUa phe quan qua noi soi (BW) co the bi ngoai nhiem bdi cac vi khuan
thiidng tru vung hong, do do can phai danh gia trudc khi tien hanh nuoi cay.

68
 - Tot nha't cac m lu sau khi nhan phai dUdc tien hanh khao sat ngay, khong
cham tre. Neu vi mot ly do gi do chtia the khao sat ngay dtidc, co the giul
mku trong tu lanh, nhung khong qua 2 gid.

4.1. Khao sdt dai the mau ddm

Ghi nhan tinh chat dai the cua mSu ddm, cac tinh chat sau:
- Co nhieu nUdc hot khong?
- Co mu (purulent) khong, thudng mau xanh hay vang due?
- Co mu nhay (muco-purulent) khong?
- Co nhay (mucoid) khong?
Mau co lan nhieu nUdc hot la mau khong thich hdp de ca'y.

4.2. Khao sat vi the

- Dung mot que tre, go, vong ca'y, hay pipet Pasteur la'y mot it ddm tii vung
nhay mu, trai deu thanh mot phet 2 x 3 cm tren mot ta'm lam. De kho tii
nhien, sau do gan nhe tren ltia. ThUc hien phet nhuom gram.
- Khao sat dudi kinh hien vi, trtidc het dudi quang trtidng xlOO (vat kinh
xlO). Tim vung nhay nhdt roi ghi nhan so' liidng te bao v&y (squamous cells)
la cac te bao CO goc canh va te bao bach cau hay te bao mu (leukocyt hay
purulent cells) la cac te bao tron nho hdn va an dam mau toan te bao.

Bang 8.1: Thang diem Barlett dung danh gia mau ddm

Tinh chat dai /vi the Diem

Mau ddm
1 0 - 2 5 bach cau +1
> 25 bach cau +2
Nhay, mu, mu nhay +1
10 - 25 te bao vay -1
> 25 te bao vay -2
M lu ddm hut qua mui (NTA=nasal tracheal suction ), noi soi phe quan
10 - 25. bach cau +1
> 25 bach cau +2
Te bao tru +1
10 - 25 te bao vay -1
> 25 te bdo vay -2

69
 - Mau khong thich hdp la m lu co so ltfdng te bao v&y trong mot quang triidng
xlOO qua 25 te bao. Mau tin cky nhat la mSu co nhieu bach c&u hay tf> bko
m u , qua 25 te bao trong mot quang trtidng xlOO. Noi chung, m iu tin cfiy la
mSu co nhieu te bao bach c iu (> 25), it te bao vay (= 10), ty le te bao bach
cau/vay tot nhat la > 2,5.
- De de dang va khach quan, dung thang diem Barlett danh gia m lu ddm,
trong do ket hdp ca quan sat dai the lan vi the de cho diem.
Thang diem de danh gia la cong tat ca cac diem lai roi danh gia nhu sau:
< 0 khong tin cay de cay.
1-2 tin cay vtia.

> 3 rat dang tin cay.

Ddn gian hdn, co the danh gia dua vao so' ltidng te bao vay va te bao bach dlu
tren mot quang trUdng xlOO. Mau hoan toan tin cay de ca'y khi co > 25 bach c4u va
< 10 te bao vay. Mau hoan toan khong tin cay khi co < 25 bach clu va >10 te bao
vay, day la mau chi la nUdc bot hay dich tiet vung hau hong. Mau tin cay vtia khi co
khi co > 25 bach cau va > 10 te bao vay, day la mau bi ngoai nhilm nUdc bot hay
dich tiet vung hau hong.
Sau khi danh gia, chuyen sang vat kinh dau (xlOO), quan sat vung nhay nhdt
va quanh te bao bach cau de ghi nhan sU hien dien cac vi khuan theo nhu bang 8.2.

Bang 8.2: C ac ti'nh ch a t vi the cua cac loai vi khuan dich co th e quan sat
dUdc trong phet nhuom vi the

Tinh chat Nghi VK dich Tinh chat Nghi VK dich

Cau khuan gram [+] xep Staphylococci Cau khuan gram [+] xep doi Pneumococci
thanh chum
Cau khuan gram [+] xep Streptococci Cau khuan gram [-] xep doi M. catarrhalis
chuoi
Trife khuan gram [-] nho Haemophilus True khuan gram [-] I6n_______ True khuan gram [-] de moc

5. CAY PHAN LAP VI KHUAN GAY BENH

- Tat ca cac mau ddm sau khi danh gia neu tin cay thi tien hanh nuoi ca'y
ngay. Cac mau khong tin cay thi khong tien hanh nuoi cay ma yeu cau lam
sang lay mau lai. Rieng cac m lu tin cay vtia, co the yeu cau la'y mau lai hay
cung co the tien hanh nuoi ca'y nhung phai co lay mam ca'y la vung ddm mu,
tranh lay nhdt hay nUdc bot de nuoi ca'y.
- Moi mot mau ddm phai diidc tien hanh nuoi cay 3 chieu tren cac moi trUdng
phan lap du kha nang de cay ra dtidc cac vi khuan gay benh du kho moc.
Trinh bay dudi day la cac moi trUdng phan lap can phai dUdc sti dung de cay
ddm va cac benh pham co ddm

70
5.1. Phan lap c iu khuan Gram [+]
- Thach mdu ciCu (BA) phan lltp khong chon loc (ca vi khuan gram [+] lln vi
khu&n gram [-] deu moc dUdc), hay thach mdu co nalidixic acid (BANg) de
phan l^p chon loc c£u khu^n gram [+].

- Hay co the them thach mdu co gentamicin (BAGe) de phan lap chon loc S.
pneumoniae.

5.2. Phan lap H. influenzae va M. catarrhalis

- Thach nau mau ngUa (CAXV) phan lap khong chon loc H. influenzae va M.
catarrhalis.
- Hay co the dung thach nau mdu ngiia bo sung Bacitracin (CAHI) de phan
lap chon loc cac vi khu&n nay.

5.3. Phan lap true khuan dudng ruot va khong dudng ruot

- Thach Mac Conkey (MC) dung phan lap chon loc trUc khu&n dudng ruot hay
khong dUdng ruot.

- Hay co the dung cac hop thach phan lap khac co cung muc dich.
- Co mot so' trtic khu&n khong dtidng ruot khong moc tren MC, nhung trong
cac trtidng hdp nay, vi khuan co the phan lap dtidc tti thach mau.
Cac hop thach mau hay thach nau dUdc u trong tu am 35-37°C va giti trong
binh nen hay trong tu C 0 2. Thach MC hay loai ttidng dUdng thi khong c&n u C 0 2.
Sau khi u qua dem, quan sat cac hop thach phan lap de tim cac khom vi khu&n gay
benh. Tien hanh dinh danh va khang sinh do de co the tra ldi ket qua den lam sang
vao ngay hom sau. Cac trUdng hdp khong phat hien dUdc vi khuan gay benh thi co
the tra ldi ngay cho lam sang.

6. CAC VI KHUAN GAY BENH CO T H i TIM THAY TRONG MAU DOM, NTA VA BW

Thtidng khi mau tin cay, vi khuan phan lap dUdc tren mat cac hop thach phan
lap rat phu hdp vdi ket qua khao sat trtic tiep qua phet nhuom gram. Sau day la
cac vi khuan la tac nhan gay benh co the phan lap tti mau.

6.1. Th u d n g gap trong n h iim trung cong dong

- H. influenzae
- S. pneumoniae
- M. catarrhalis
- S. aureus
- K. pneumoniae

71
6.2. Th u d n g gap trong nhiem trung benh vien

- S. aureus
- P.aeruginosa
- K. pneumoniae
- Cac trUc khu^n gram [-] d i moc khac
Cac Enterobacteriaceae khac

7. C A Y DOM V A BENH PHAM C H lfA DOM - C A C C A U HOI T H U O N G G A P

7.1. De lay ddm lam xet nghiem vi sinh lam sang tim cac vi khuan ga y nhiem
trung ho hap dudi, thdi diem tot nhat co phai la va o luc sang sdm khong?

- Lay ddm vao sang sdm la lay ddm de khao sat tim vi khu^n gay benh lao v&
trong xet nghiem nay benh nhan lay ddm vao 3 ng&y lien tiep de giii den
phong xet nghiem lao moi ngay. Tai phong thi nghiem, m lu ddm dUdc ti£n
hanh nhuom khang acid (acid fast staining) va co the dUdc lam thu&n nhat
de cay vao moi trUdng cay lao.

- De lam xet nghiem vi sinh lam sang phat hien cac vi khuan gay nhiem trung
ho hap dudi, phai tien hanh lay ddm ngay sau khi co ch£tn doan va trudc khi
cho benh nhan dung khang sinh. Lam sang phai hudng dan benh nhan cach
lay ddm vi neu khong, rat nhieu benh nhan khong biet cach khac ddm ma chi
khac ra nUdc bot va nhu vay thi khong co gia tri de lam xet nghiem.

7.2. Tai sao co nhieu khi ket qua xet nghiem vi sinh lam sang khong phu hdp vdi
hieu qua dieu trj khang sinh tren benh nhan? V i du cay ra vi khuan P. aeruginosa
khang rat nhieu khang sinh trong khi do chi dieu trj benh nhan bang Augm entin
hay cephalosporin the' he 2 benh nhan van cai thien dUdc lam sang?

- Ddm la mot benh pham rat co gia tri de tti do co the phat hien dUdc tac nhan
vi khan gay nhilm trung cap dtidng ho hap dudi, nhung cung co the la benh
pham khong co gia tri gi neu nhu chung ta khong lay dUdc ddm that sti ma
benh pham gtii den phong thi nghiem chi la nUdc bot hay la benh ph§m ddm
nhUng lai tap nhiem qua nhieu nUdc bot vung hau hong.

- TrUdng hdp ke tren thudng la do benh pham gtii den phong thi nghiem chi la
nUdc bot hay tap nhilm nhieu nUdc bot ma phong thi nghiem khong danh
gia va v ln tien hanh nuoi ca'y. Chinh vi vay vi khu&n phan lap dUdc khong
phai la vi khuan gay benh ma chi la vi khu&n thudng tru hay tam tru tren
vung hau hong ma thoi.

72
 - Cung c6 the co mot ly do ntia la phong thi nghiem khong dung moi trtidng
CA mau ngtia (CAXV) va BA mau ctiu, chinh vi vay khong co kha nang
phan lftp dUdc S. pneumoniae, H. influenzae va M. catarrhalis la cac vi
khu^n ra't thiidng gap (>50% tac nhan gay nhilm trung ho ha'p dtidi la cac
tac nhan nay) nhUng lai rat kho moc va kho nhan dien tren cac moi trUdng
thong thtidng khac cua nhieu phong thi nghiem hien nay nhti BA mau ngtidi
va MC. Chinh vi chi dung cac moi trtidng nay nen phong thi nghiem chi
phan lap dUdc cac tac nhan vi khu^n d i moc, do la trtic khuan dudng ruot
hay khong dudng ruot ma cac vi khuEtn nay co khi chi la vi khuan tap nhiem
tti h£u hong.
i
7.3. Nhieu khi tren hop thach phan lap chi thay lien cau tan huyet alpha, nhung khong
co dac diem cua cac khuan lac S. pneumoniae. Trudng hop nay chung ta co tien hanh
tiep drnh danh va lam khang sinh do de tra ldi ket qua cho lam sang khong?

- Gan nhti 90% cac mau ddm la co lien cau tan huyet alpha khong phai S.
pneumoniae moc tren cac hop thach phan lap. Da so" cac trUdng hdp nay la
tap nhiem vi khuan lien cau tti vung hau hong.
- Tuy nhien trong trUdng hdp mau co gia tri va tren thach mau hay thach nau
chi thay toan la lien cau tan huyet alpha khong phai S. pneumoniae thi
chung ta phai tien hanh dinh danh va lam khang sinh do de tra ldi cho lam
sang vi day rat cd the la vi khuan gay benh cd nguon goc tti vi khuan thudng
tru vung hau hong nhung lan xuong dtidng ho hap dudi de gay benh.

7.4. Xin cho biet cach nhan dang cac khom S. pneumoniae, H. influenzae va
M. catarrhalis?

- Khom vi khuan S. pneumoniae moc tren thach mau ctiu cho tan huyet alpha
(tren mau ngUdi lai cho tan huyet beta nen rat d i nham vdi lien cau tan
huyet beta), tren thach nau cho mot vong mau vang ttidi quanh khom (bieu
hien tan huyet alpha tren thach nau). Khom vi khuan thudng det, khong loi,
va d gitia khom thtidng bi lom xuong do vi khuan bi tti tieu.
- Tren thach mau, khom vi khuan H. influenzae chi co the moc thanh cac
khom li ti ve tinh quanh cac khom vi khuan khac nhti Staphylococci,
Streptococci hay Bacillus. Tren thach nau (CAXV hay CAHI), H. influenzae
moc thanh cac khom due nhe, hdi det, dudng kinh #lm m rat de nhan dien.
- Khom vi khuan M. catarrhalis moc tren thach mau hay thach nau thtidng
hdi loi, co mau trang hay mau vang, dudng kinh <lm m . Dac diem la khom
bd nhu bot khoai tay khua'y, co nghla la dung vong cay cat la'y mot goc khom
rat d§ dang. Thti nghiem oxidase tren khom cho ket qua [+].

73
 Hinh 8.1: Mot so vat dung hien nay van con dung tai nhieu benh vien de lay mau benh pham
trong do co mau ddm (A,B,C,D), cac dung cu nay rat kho de lay mau vi mieng mieng Ip hay tube
rat nho, ngoai ra cung khong an toan vi benh pham co the dinh vao nut gon va ri len mieng lo
hay tube. Vdi Ip nhua chuyen dung nap vSn, dUdc khif trung bang gamma thi khi lay m lu nhif
ddm co the danh gia ngay la mau co gia tri vi chi chira ddm (G) hay it gia trj vi lin ni/dc bot (F).
hay hoan toan khong gia trj vi chi la nUdc bot khong phai ddm (E). Ngoai ra phUdng tien nay
cung rat an toan khi chuyen chd mau vi khong bi ri benh pham len nap.

Hinh 8.2: Cac dung cu dung de lay mau ddm qua ong thong m u i : (A) Bo lay ddm qua
ong thong mui (NTS set) ban s in tren thi tri/dng. (B) Neu khong cd bo NTS nay, cd the dung
xilanh 20 hay 50 ml g in vdi ong thong mem de thay the

74
Hinh 8.3: Ket qua danh gia m lu ddm qua phet nhuom gram quan sat di/di quang tri/dng x100:
(A) m lu khong tin cay vi co >25 te bao bieu mo va khong co te bao bach cau, (B) mau bi tap
nhilm ni/6c bot vi co > 25 te bao bieu mo va > 25 bach cau (C,D) m lu hoan toan tin cay vi co
<10 te bao bieu mo va > 25 bach cau. Ket qua quan sat m lu ddm tin cay qua phet nhuom gram
6 quang tri/dng x 1000: (E) song cau gram [+] nghi S.pneumoniae, (F) song cau gram
[-] nghi M.catarrhalis, (G.H) trUc khuan gram [-] nho va ngan nghi H.influenzae

75
8. CAU HOI O N T A P

a. Cho biet cac chi dinh lam sang va thdi diem tot nhat de lflTy ddm va cac
benh pham chtia ddm de lam x6t nghiem vi sinh lam s&ng.
1

b. Hay cho biet cac lay va chuyen chci ddm va cac loai benh pham c6 ddm
nay den phong thi nghiem de lam xet nghiem vi sinh lam sing.
c. Hay cho biet cach danh gia mot mSu ddm nhti the nao la co gia tri de nuoi ca'y.
d. Hay cho biet qui trinh x6t nghiem vi sinh lam sang thtic hien tai phong
thi nghiem do'i vdi dcJm va cac benh phjim chtia ddm.
e. Hay cho biet phUdng tien thich hdp nha't de lam x6t nghiem vi sinh lam
sang mSu ddm va cac benh pham co ddm.

f. Hay cho biet cac vi khu^n gay benh c£n phai phan lap trong ddm va cac
benh phgLm chtia ddm.

76
 Rifa ddm tin nhieu ni/dc bot vdi Ly tim l£y can ddm ti/ cac benh pham
5 ml ni/dc mudi sinh ty v6 trung la dich hut ddm tren thanh quan hay
qua dich hut ndi soi khf quan

Khdng d n rifa
ddm n6u khdng ty
1 lin ni/dc bot

"► Larp mpT phet nfrw.g gram

DO o
Quan sat phet ddm qua
"kinh hien vi vat kinh —
x 10 danh gia m lu
b ln g thang diem Bartlejt
M lu tin cay (diem
Bartlett > 2) M lu chi/a du tin cay Mau khong tin ca
(diem Bartlett = 1) (diem Bartlett > 0)

Yeu cau lay lai m lu .

khdng tien hanh nudi ca’y
Cay phan lap tren: U 37°C qua dem
- BAGe (chon loc PNE, STR)*
- BAGe (binh nen)
- CAHI (chon loc Hin, BRA)*
- CAHI (binh nen)
- BANg (chon loc Gr [+] cocci)'
- BANg (binh nen)
- MC/EMB (chon loc Gr[-] nod)
- MC/EMB (binh thudng)

* Neu khong co BANg va

BANe thay bang BA Dinh danh sd bo

D1 \
Chon khom vi khuan f e r n t-.V'X' I
dich tren cac hop thach
phan lap chon loc //,
/ y
Cay tang sinh bang Gat vi khuan tang sinh
di/dng zic-zac len mat ti/ di/dng cay zic-zac
thach BA hay mat thach
thich hop. U 370C/CO2
trong 4-6 g id "

Dinh danh
tt± ± ± ± j r t t t t N
aiVMMIV i M M H a n B M

D2 r

Ket qua chung cuoc

hut ddm tren thanh - khi quan/dich rifa khi quin
qua noi soi
IzMJsWUWWWV

/ 1
 BAI 9. CAY DINH LUDNG NlAU DDM
■ ■

MUC TIEU

1. Hiidng ddn lam sang cho dung chi dinh cay dinh liidng ddm md khong
phai qua han che hay qua rong khong cdn thiet vi biet diicfc cac chi dinh
lam sang de cho lam xet nghiem cay dinh liidng mdu ddm.
2. Thiic hien diidc qui trinh cay dinh liidng mdu ddm trong phong thi
nghiem vi sinh lam sang cua minh.
3. Trien khai thiic hien diidc xet nghiem cay dinh liidng mdu ddm tai benh
vien khi co yeu cau cua lam sang vi biet vd chuan bi diidc cac phiidng
tien cdn thiet vd thich hdp nhat de thiic hien diidc xet nghiem nay.

1. CHiOjNH

Hien nay co mot xu hudng mdi trong cay ddm, do la cay dinh lUdng. Diia vao
ket qua cay dinh lUdng co the phan biet tac nhan nao la tac nhan that sU gay benh
co trong m&u ddm, tac nhan nao la ngoai nhiem vung h&u hong. PhUdng phap dky
dinh lUdng mau ddm dUdc chi dinh thong thudng nhat la tren cac viem phoi nang
hay tren cac viem phoi benh vien. Tren viem phoi cong dong thi khong nhat thiet
phai ap dung phUdng phap nay.

2. THOI DIEM LAY MAU VA CACH LAY MAU

TUdng tu phUdng phap cay khong dinh lUdr _•

3. DANH GlA MAU DOM

Tru6c khi tien hanh nuoi cay, mau ddm cung dUdc danh gia dai the va sau do
lam mot phet nhuom gram de danh gia vi the nhu phUdng phap cay ddm khong
dinh ltfdng.

4. PHUONG PHAP CAY 0|NH LUONG

Trudc het lam tan ddm trong dung dich nU6c muoi sinh ly vo trung vtia pha
them NALC (SPUTAPREP-QT kit cua Nam Khoa) va sau do pha loang ddm. Tien
hanh nha sau:
- Trong mot tube 15ml vo trung n ip xanh (loai Falcon) da chtia 10ml nUdc
muoi sinh ly vo trung (NS), cho them vao 50mg NALC (N-Acetyl-L-Cystein),
ttic la toan bo bot NALC chtia trong mot tube Eppendorf. L ie nhe cho tan
hoan toan. Dung dich NS co NALC nay chi dung trong ngay va du cho 2 - 3
mau. Luon luon bao quan tube dung dich NALC da pha trong tu lanh 4°C.

7S
 - La'y mot the tich ddm can nuoi ca'y cho vao mot tube vo trung n ip van chat
(hay lo vo trung n ip do). Them vao mot the tich nhu vay dung dich NS co
NALC vtia mdi pha. L ie nhe cho den khi ddm tan trong dung dich nay. Nhu
vay chung ta da co ddm da pha loang 1/2. Sau khi ddm tan hoan toan, pha
loang tiep m lu ddm nay thanh 1/10 trong nUdc muo'i sinh ly vo trung. Nhu
vay chung ta da co ddm pha loang 1/20.
- Tien hanh cay dinh liidng mau ddm da pha loang 1/2 tren cac hop thach
mau ctiu (BA), thach nau mau ngiia co bacitracin (CAHI) va thach MC b in g
khuyen ca'y dinh liidng 10(^1 (0,01ml) va lfil (0,001ml) hay dung micropipett
de hut 10fj.l va Ipl ca'y len mat thach. Vdi mau ddm pha loang 1/20, dung
khuyen ca'y l(j.l hay dung micropipet de hut lpl ca'y len mat thach va cung
tren 3 loai hop thach nhu tren. PhUdng phap cay dinh liidng tren mat thach
dtidc thiic hien nhti ca'y dinh lUdng nUdc tieu. Sau khi ca'y, cac hop thach BA
va CAHI dUdc u trong khi trtidng C 0 2 con cac hop thach MC dUdc u trong
khi trUdng binh thUdng, nhiet do u la 35-37°C, thdi gian u qua dem hay toi
da 24 gid. Doc ket qua va dinh liidng cac loai vi khuan moc tren cac hop
thach nhu trong bang 4 trinh bay sau:

Bang 9.1: Cach doc ket qua dinh liidng cac loai vi khuan trong m iu ddm di/a tren
so liidng cac loai khom vi khuan moc tren cac hop thach

Hop thach M lu ddm pha loang Vong cay djnh lUdng The tich ddm di/cJc cay So vi khuan
dupe dinh li/dng
1 1/2 10^1 5nl 2N x 102/ml
2 1/2 V 0,5|il 2N x 103/ml

79
 N la so khom cua mot loai vi khuin dem dUdc tren cac hop thach. De c6 con so
dinh lUdng cua mot loai vi khuan thi chung ta nen la'y so' trung binh cua cac ket
qua. Vi du: ting vdi cac khom nghi ngd K. pneumoniae, ket qua tren hop thach MCI
la 200 khom, liidng vi khuin K. pneumoniae trong 1ml ddm la 40000 CFU/ml
(2x200xl02); tren hop thach MC2 la 15, lUdng vi khuin/m l ddm la 30000 CFU/ml
(2 x l5 x l0 3); va tren hop thach MC3 la 3, liidng vi khuin la 60000 CFU/ml (2x3xl03).
Nhu vay, ket qua cuo'i cung lUdng vi khuin K. pneumoniae trong m iu ddm se dUdc
tinh la" (40000 + 30000 + 60000)/3 = 43333 CFU/ml. Neu cac hop thach 1 va 2 c6 vi
khuan qua nhieu (>200 khom 2), chung ta chi nen dem so khom tren hop thach 3.

5. BIEN LU A N K E T Q U A

Cac vi khuin co liidng > 1000 CFU/ml thi phai dinh danh va lam khang sinh
do. Co nghla la tien hanh dinh danh va lam khang sinh do ba't cti khom vi khuin
nao moc dtidc tren hop thach 2 va 3 hay co so' lUdng > 5 khom tren hop thach 1. Tra
ldi lam sang ca ket qua dinh liidng la bao nhieu vi kh uin da dinh danh va lam
khang sinh do de lam sang co the tuy nghi sti dung.

6. CAY OjNH LUONG MAU DOM - CAC CAU HOI THUONG GAP

6.1. Tro n g trudng hop viem phoi cong dong, co can thiet phai ca'y dinh lupng
mau ddm?

- Viem phoi cong dong neu la nhe hay trung binh thi khong nha't thiet phai
cay dinh liidng vi cac vi khuin gay benh thudng la H. influenzae, M.
catarrhalis va S. pneumoniae. Ckc vi khuin nay du deu co the la vi khuin
thudng tru vung hau hong nhung kho co the moc dUdc tren moi trUdng nuoi
cay neu chung d dang thudng tru vi so' liidng ra't it. Chinh vi vay chung ta
chi can danh gia mau ddm qua phet nhuom gram, neu thay tin cay thi tien
hanh nuoi ca'y binh thudng chti khdng can ca'y dinh liidng.
- Doi vdi viem phoi cong dong nang, cac viem phoi benh vien thi rat ckn thiet
phai ca'y dinh liidng mau ddm vi tac nhan vi khuan gay benh thudng la cac
trUc khuin gram [-] de moc hay la tu cau ma day la cac vi khuin co the la
cac vi khuan tam tru rat de moc tren cac hop thach phan lap du so liidng vi
khuan hien dien it trong mau.

6.2. Khi tien hanh ca'y djnh lupng, co can danh gia mau ddm trudc qua phet
nhuom gram khong?

- Cay dinh lUdng nhim muc dich la chi bat cac vi khuin co so' liidng ldn trong
mau ddm vi day mdi chinh la tac nhan gay benh that sU. Do vay ve nguyen
tac khong nha't thiet phai danh gia mau ddm trU6c khi ca'y dinh liidng.
- Tuy nhien vi qui trinh ca'y dinh liidng ngdai viec phai dung bo thuoc thti lam
tan ddm, chung ta con phai sti dung so' liidng hop thach phan lap nhieu gap
3 lan phiidng phap ca'y khdng dinh liidng, dd vay ra't can thiet phai danh gia

80
 mSu ddm de chi tiep tuc qui trinh ca'y dinh liidng khi m&u ddm dUdc danh
gia la tin cay hay tin c&y vtia, nhd vay chung ta co the tiet kiem vi khong
phai tien hanh ca'y dinh liidng m lu khong tin cay.

6.3. Co khi nao trong ket qua cay dinh luung ddm cho ket qua so luung vi khuan
gay benh tren 1.000 hay 10000, ma ket qua danh gia ddm qua phet nhuom gram
quan sat 6 quang trudng x100 lai khong tin cay khong?

- Tren thuc te co phong thi nghiem cay dinh liidng ddm cho ket qua nhu tren.
Tuy nhien vdi nghien ctiu ma chung toi da tien hanh tai phong thi nghiem
cua minh thi 100% cac m iu khong tin cay deu cho ket qua dinh liidng vdi so
liidng vi khuan (iing vdi moi loai) <1000 CFU/ml.
- Ket qua nay cang khang dinh la khong nen cay dinh liidng khi phet nhuom
gram mau ddm quan sat dudi kinh hien vi vdi quang trUdng xlOO cho thay
cd >10 te bao bieu mo va <25 te bao bach cau.
- Do vay, cd le phong thi nghiem nay da quan niem sai ve cach tinh so' liidng
vi khuan, thay vi la so lUdng cua tiing loai vi khuan, ho lai tinh so lUdng cua
tat ca cac loai vi khuan hien dien trong mau ddm.

6.4. Cay djnh luong ddm co kha thi khdng vdi dieu kien hien nay tai cac phong
thi nghiem vi sinh lam sang benh vien?

- TrUdc day, kho cd the cay dinh liidng ddm vi doi hoi phai cd thuoc thti lam
tan ddm phai dat mua nUdc ngoai va gia thanh kha dat. Hien nay bo thuoc
thii lam tan ddm de cay dinh liidng ddm da dtidc cong ty Nam Khoa (dat ISO
9001:2000) nghien ctiu thanh cong va san xuat trong nUdc vdi san pham
mang ten SPUTAPREP -QT. San pham nay rat tiet kiem va tien dung nhd
dUdc chia nho de cd the thuc hien tren toi thieu 2 mau ddm trong ngay.
- Neu dUdc trang bi SPUTAPREP -QT; va cd them cac loai moi trUdng phan
lap la MC, BA mau ctiu, CA giau XV (cac moi trtidng nay cung da dUdc cong
ty Nam Khoa san xuat dudi dang che san hay san sang de che); cac phong
thi nghiem vi sinh lam sang benh vien hoan toan cd the cay dinh liidng dUdc
m lu ddm ma khdng gap bat cti kho khan hay trd ngai gi.

6.5. Khi nao thi nen chi dinh cay dinh lueng ddm?

- That ra ca'y dinh liidng ddm cd the chi dinh cho ta't ca cac trtidng hdp ca'y
ddm. Tuy nhien nhu da ndi d tren, cay dinh liidng ddm ton kem gap 3 lan
hdn cay khong dinh lUdng. Do vay, chung ta khong can thiet cho cay dinh
lUdng doi vdi cac trudng hdp viem phoi cong dong nhe va vtia.
- Rat can thiet phai cho cay dinh liidng ddm cac trtidng hdp viem phoi nang,
cac trUdng hdp viem phoi benh vien, cac triidng hdp viem phoi nam tai cac
phong cap ctiu va phong cham soc tang cUdng (ICU).

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 - Cung nen cho cay dinh lUdng khi khi quan sat dai the cac m&u ddm l&y dUdc
thay cd lan nUdc hot.
- Mot nguyen tac ma chung toi cho la rat c£n thiet phai tuan theo de tiet kiem
chi phi cho benh nhan va benh vien, do la khdng ctfy dinh liidng khi quan
sat vi the m lu ddm qua phet nhuom gram cho th&y lit m lu ho&n toan khdng
tin cay.

7. CAU HOI ON TAP

a. Cho biet cac chi dinh lam sang de cay dinh liidng m lu ddm.
b. Cd c&n danh gia mau ddm trong cay dinh liidng khdng?
c. Neu cu the cach cay dinh lUdng mau ddm nhu the nao va ve sd do thiic hien.
d. Bien luan mot ket qua cay dinh lUdng ddm.

PHU• LUC
•

San pham lien quan: SPUTAPREP-QT - Bo thuoc thur lam tan

ddm de cay djnh lUdng ddm

Nguyen tac hoat dong

Bo thuoc thti hoat dong diia tren nguyen t ic dung N -Acetyl-L-Cystein

(NALC) de lam tan ddm va cac nhay nhdt trong ddm, nhd vay lam dong nhat dUdc
mau de cd the tien hanh cay dinh liidng, tach chiet dUdc nucleic acid cua virus hay
vi khuan gay benh cd trong ddm de lam thti nghiem PCR...

Thanh phan thuoc thur

SPUTAPREP-QT danh cho 10 mau ddm gom cac thanh phan sau:
1. N-Acetyl-L-Cystein (NALC): bot nguyen chat chtia trong 5 tube Eppendorf
nhd, mdi tube chtia NALC du dung cho xti ly 2 mau ddm.
2. Dung dich nUdc muoi sinh ly vd trung: chtia trong tube 15ml nap van chat,
mdi tube chtia 10ml nUdc muoi sinh ly vd trung.

Bo thuoc thti SPUTAPREP -QT chtia dung hay ngay sau khi dung nen dUdc
giti trong tu lanh 4°C cho den ngay het han ghi tren hop (2 nam sau ngay san xuat).

Phifdng phap lam tan ddm vdi SPUTEPREP -QT

1. Trong mot tube 15ml vd trung nap xanh (loai Falcon) da chtia 10ml nUdc muoi
sinh ly vd trung (NS), cho them vao 50mg NALC (N-Acetyl-L-Cystein). ttic la
toan bo bot NALC chtia trong mot tube Eppendorf. Lac nhe cho tan hoan
toan. Dung dich NS cd NALC nay chi dung trong ngay va du cho 2 - 3 m lu.
Luon luon bao quan tube dung dich NALC da pha trong tu lanh 4°C.
 2. Ltfy mot the tich ddm c ln nuoi c£y cho vao mot tube vo trung nap van chat
(hay lo vo trung n ip do). Them vao mot the tich nhu vay dung dich NS co
NALC viia mdi pha. L ie nhe cho den khi dam tan trong dung dich nay. Nhu
vay chung ta da co ddm da tan va dUdc pha loang 1/2. Ddm da tan nay san
sang cho qui trinh cay dinh liidng hay qui trinh tach chiet nucleic acid cho
PCR hay RT -PCR de phat hien cac tac nhan vi khuan hay virus gay benh co
trong mau ddm.

Hinh 9.1: Mot so hinh anh cay dinh li/dng m lu ddm tren mat thach phan lap:
(A). Thach nau vdi khom vi khuan nghi ngd M.catarrhlis, (B) Thach nau vdi nhieu khom vi khuan
nghi ngd S.pneumoniae co tap nhilm vi khuan hau hpng khac, (C,D) Thach mau vdi nhieu khom vi
khuan nghi ngd Streptococus feacalis, (E) Thach MC vdi cac khom vi khuan nghi ngd Acinetobacter

83
 BA110. MAU BENH PHAM SINH DUC
VA CAY MAU LAY TUT DlTDTNG SINH DUC

MUC TIEU

1. Thiic hien lay dung benh pham diidng sinh due vi biet diidc cach lay cac
loai benh pham diidng sinh due tren cac doi tiidng benh nhan khac
nhau.
2. Thiic hien diidc day du cac biidc vd phan tich diidc cac ket qua de khong
bo sot cac cd hoi phat hien cac tac nhan vi khuan gay benh vi biet diidc
qui trinh thiic hien vi sinh lam sang cac mdu lay tii diidng sinh due.
3. Phat hien diidc cac tac nhan thiidng gay nhiem trung lay truyen bang
diicfng sinh due du la kho moc vi biet diidc phiidng tien cdn thiet vd thich
hdp nhdt de thiic hien ditdc xet nghiem vi sinh lam sang cac mdu lay til
diicfng sinh due.

1. CHiOjNH

Khi nghi ngd nhiem trung ben h lay tru yen b a n g diidng s in h d u e (dan 6ng,
dan ba) hay khi bi huyet trang nghi do nhiem trung (dan ba).

2. THOIBIEM

- Cang sdm cang tot, ngay trong giai doan dau cua benh.
- TrU6c khi dung khang sinh.

3. CACH LAY BENH PHAM

3.1. 0 dan ong

- Tut da qui dau ra sau, dung con 70% lau sach da qui dau, chd kho.

- Benh nhan vuot nhe dudng vat doc theo ong dai de ra cho dUdc m6t giot mu
va tham giot mu len mot que tam bong vo trung, phan con lai quet tren mot
lam ki'nh. Que tam bong dtidc cho vao moi trUdng chuyen chd Stuart -Amies
(thich hdp nhat la dung cap tube dua tam bong vo trung!tube dua Stuart
Amies). Gtii ngay den phong thi nghiem de khao sat cang s6m cang tot.

84
 - Neu benh nhan khong vuo't dtidc mu, co the dung que gon manh luon vao
ong dai sau khoang 3-4 cm roi vtia xoay nhe vtia rut ra roi cho ngay vao moi
trUdng chuyen chd Stuart -Amies (dung cap tube dua tam bong manh vo
trung /tube dua Stuart Am ies).
- Neu chi c ln ca'y phan lap N. gonorrhoeae thi tot nhat la cay tren thach phan
lap CATM ngay sau khi lay benh pham, cach lay la dung vong cay nhua hay
vong c&y kim loai hoac que gon manh (dung tube dua tam bong manh vo
trung) luon vao ong dai sau khoang 3-4 cm roi vtia xoay nhe vtia rut ra.
Ngay sau do vach tren hop thach CATM roi gtii den phong thi nghiem de u
va theo doi.
- Cac loai benh pham khac co the lay de khao sat la quet hau mon, quet hong,
quet mu khi lay noi soi trUc trang d ngUdi dong tinh luyen ai.

3.2. d phu nur

- Benh nhan dUdc nam tren ban kham phu khoa, rtia sach bo phan sinh due
ngoai bang nUdc, tham kho, sau cho cho mo vit vao. M o vit dUdc vo trung
bang ha'p Udt hay say kho chti khong phai bang hoa cha't.

- Dung tam bong quet lay huyet trang d thanh sau cua am dao de khao sat T.
vaginalis va na'm (dung tam bong vo trung lay mdu, hay tam bong dai vd
trung lay mdu). Tam bong nay dUdc cho vao mot lo co dung dich bao quan
F2M (lo co F2M de lay mdu) roi gtii ngay den phong thi nghiem de soi tUdi.
Neu muon ca'y vi sinh hay nam thi lay quet huyet trang bang que tam bong
roi cho vao moi trtidng chuyen chci Stuart -Amies (dung cap tube dua tam
bong manh vo trung / tube dua Stuart Am ies).
- Doi vdi ngUdi bi nghi nhiem lau, lay benh pham tti ldng cd tti cung bang cach
dung gon vo trung lau sach huyet trang roi dung mot tam bong manh dua
vao long cd tti cung xoay nhe trong 10 giay trudc khi rut ra. Lay mau bang 2
que tam bong, mot que quet len mot lam kinh, que con lai cho vao moi
trUdng chuyen chd Stuart -Amies roi gtii den phong thi nghiem de khao sat
ngay (dung cap tube dua tam bong manh vo trung I tube dua Stuart A m ies).

3.3. d tre con

- Co the lay mu mat neu nghi bi viem mu ket mac mat do lau.
- Cung co the la'y mu tti am dao cua be gai (trtidng hdp bi hiep dam).

3.4. Cac trudng hop dac biet

- Tim Chlamydia trachomatis bang ky thuat nhuom khang the huynh quang
trtic tiep: lay benh pham tti long cd tti cung nhti trong trtidng hdp tim vi
khuan lau. Quet tam bong nay len mot lam lo chuyen dung, de kho tu nhien

85
 roi nho len mot giot aceton hay methanol, chd kho. Boc lam b ln g mot td gi&y
nhom roi gtii ngay den phong thi nghiem. Triidng hdp chtia gtii den phong
thi nghiem dUdc, co the bao quan lam trong tu lanh 4°C trong toi da khong
qua 1 tuan.

- Cac vet loet: lau sach b ln g gon vo trung roi nan ch&'t dich vao mot tam bong
vo trung, phan con lai quet tren mot lam kinh. Que tam bong diidc cho v&o
mot tube n ip chat vo trung hay vao moi trUdng chuyen chd Stuart -Amies
(dung cap tube dua tam bong vo triing/tube dua Stuart Am ies), gtii ngay
den phong thi nghiem de khao sat cang sdm cang tot.

4. KHAO SAT TRlJC TIEP

4.1. Benh pham lay d dan ong hay tre con

- Lam phet nhuom gram, khao sat dtidi kinh hien vi do phong dai ldn nh&'t
(vat kinh dau): neu chi co dudi 4-10 bach cau trong mot quang tnidng va
khong co song cau gram [-] noi te bao thi rat nhieu kha nang benh nhan bi
viem nieu dao khong phai do lau. Neu co tren 10 bach cau trong mot quang
trUdng va/hay phat hien co song cau gram [-] noi te bao, co the chac tren
98% benh nhan bi lau.

- Khong lam phet nhuom gram quet hong hay quet hau mon. Tuy nhien neu
la quet mu lay tii noi soi trUc trang thi rat co gia tri de khao sat gram.

4.2. Benh pham lay d phu niir

- Soi tUdi rat can thiet de phat hien Trichomonas vaginalis va nam men.

- Lam phet nhuom gram cac quet benh pham tren lam kinh va quan sat dtidi
kinh hien vi, vat kinh dau. Vdi quet am dao: (1) Neu cd It hdn 5 bach cau
trong mot quang trUdng va cd trUc kh uin gram [+] (thudng la
Lactobacillus), thi day la mot quet am dao binh thudng. (2) Neu cd It hdn 5
bach c lu trong mot quang trUdng, khong cd cac trUc khuan gram [+]
(Lactobacillus), cd nhieu trtic khuan gram [-] nhd quanh te bao kem cac vi
khuan khac nhu cau trUc kh uin gram n [-], trUc kh uin gram [-], trUc khuin
gram [-] cong, thi cd nhieu kha nang benh nhan bi viem am dao do
Gardnerella vaginalis. (3) Neu cd tren 10 bach cau trong mot quang trtidng,
cd kha nang benh nhan bi nhiem C. trachomatis. Vdi quet co tti cung: co phat
hien cac song cau gram [-] noi te bao. Tuy nhien ket qua gram khong the ch ic
benh nhan bi lau vi do dac hieu chi dat 50-90%, do nhay chi 50-70%.

86
4.3. Trudng hop benh pham la nude tieu

La'y m §u niidc tieu bang phUdng phap vo trung nhu da de cap d phan cay nUdc
tieu nhung khong phai la'y giuta dong ma tot nha't la lay dau dong hay cuo'i dong.
Nude tieu giii den phong thi nghiem phai dUdc tien hanh xet nghiem ngay bang
each ly tam d to'c do cao nha't cua may ly tam ban, sau do chi lay can de viia lam
mot phet nhuom gram, viia cay tren mdi trUdng phan lap. Neu quan sat nhuom
gram cd song c&u gram [-] dac trUng va noi te bao , cd the tra ldi ket qua sd be cho
bac si la nghi ngd lau ciu.

4.4. Trudng hop loet ha cam

Soi tUdi dudi kinh hien vi nen den hay dao pha tim xoln khuan giang mai.

4.5. Trudng hop tim C. trachomatis

Nhuom khang the huynh quang roi doc dudi kinh hien vi huynh quang.

5. NUOI CAY, PHAN LAP

- Tren thach nau Thayer Martin (CATM) de cay phan lap vi khuan N.
gonorrhoeae (vi khuan lau), them thach BANg (cd the thay bang BA neu
khdng cd BANg) va MC cho cac vi khuan de moc khac.
- Cd the cay them tren thach nau neu nghi cac nhiem trung vi khuan kho moc
khac.
- Cd the cay them thach Sabouraud cd hay khdng cd khang sinh neu cd yeu
cau tim nam men.
- Cac hop BA va CA phai dtidc u 35-37°C trong binh nen. Cac trUdng hdp
khac, u khi trtidng binh thudng. Theo doi lien tuc trong 3 ngay. TrUdng hdp
cay vi khuan N. gonorrhoeae, chi md binh sau 48 gid u.
- Neu cd vi khuan moc, tien hanh dinh danh va lam khang sinh do.

6. CAC VI KHUAN GAY BENH CO T H I TIM THAY TRONG DUONG SINH DUC

6.1. Thudng gap

- N. gonorrhoeae,
- Treponema pallidum (soi ttidi).

6.2. ft gap

- H. ducreyi,

- Gardnerella vaginalis (nhom gram),

87
 - Candida albicans (nhuom gram),

- Trichomonas vaginalis (soi tUdi).

6.3. Rat hiem gap

- Chlamydia trachomatis.

7. CAY MAU BENH PHAM LAY TLf Ol/ONG SINH DUC - CAC CAU HOI THUONG GAP

7.1. Mot ngirdi dan ong thudng hay cam thay ngura rat & dudng tieu nhung lai
khong bj ra mu tai nieu dao ma chi hay ra chat nhay trang trong vao sang sdm;
nhu vay hp co bj lau khdng va lam the nao tim duoc tac nhan gay benh trong
trudng hop nay?

- Trieu chiing tren co the la lau nhung benh nhan da dtidc dieu tri khong triet
de trudc do hay la ban tinh (hoac vd) cua ho khong dtidc dieu tri triet de.
Tuy nhien cung co the ho nhiem tac nhan gay benh khac khong phai l&u
nhu Trichomonas vaginalis, Enterococcus faecalis, hay Streptococci nhom B.
Do vay, trudc het phai hoi ky benh sti cua benh nhan xem trade day co trieu
chting cua lau khong, cd dieu tri khdng? dieu tri nhu the nao? cd dieu tri ca
cho ban tinh khong?...
- Trong trUdng hdp nay cach lay benh pham tot nhat la dung mot tam bong
manh hay mot vong cay nhtia nhd luon vao nieu dao qua da^i dudng vat de
ngoay nhe va lay chat nhay ra lay len hop thach CATM va hop thach CA.
Cac hop thach nay phai dUdc chuyen den phong thi nghiem de u ngay trong
tu am va binh nen hay tu am C 0 2. Ngoai ra cung phai lay them benh pham
vdi cach nhu tren de soi tUdi va lam mot phet nhuom gram de khao sat trUc
tiep ntia.

7.2. Mot so' phong thi nghiem hay lam xet nghiem khao sat true tiep can ling
nude tieu de tim vi khuan lau. Xet nghiem nay cd tin cay khdng?

- Neu muon tim vi khuan lau trong nUdc tieu thi nha't thiet phai khao sat can
lang nUdc tieu vdi nUdc tieu lay dau ddng hay cud'i dong vao sang sdm. Tuy
nhien neu chi diia vao phet nhuom gram thi kho cd the ket luan chinh xac
lau du cd the thay dUdc hinh anh dien hinh song cau gram [-] noi te bao, ma
phai nuoi ca'y can lang tren moi trUdng CATM.

- Tuy nhien vi khuan lau trong nUdc tieu cd the kho nuoi ca'y thanh cong. do
vay hien nay mot ky thuat chinh xac va nhay cam nha't la ngudi ta tien
hanh thti nghiem PCR phat hien N. gonorrhoeae dong thdi vdi
C. trachomatis trong can lang nUdc tieu.

88
7.3. Hifn nay co thur nghiem xet nghiem nhanh tim N. gonorrhoeae va
C. trachomatis. Thur nghiem nay hoat dong tren nguyen tac nao va co dang tin
cay khong?

- Cac thti nghiem nhii vky da so' deu dua tren nguyen tAc thii nghiem sac ky mien
dich (xem trang 45) phat hien khang nguyen vi sinh vat co trong benh pham.
- Vi la thii nghiem phat hien khang nguyen nen gia tri hdn la thii nghiem
phat hien khang the. Tuy nhien do nhay cam va do dac hieu cao hay khong
thi lai rat thay doi tuy theo hang san xuat. Do v&y triidc khi ap dung chung
ta phai nghien ctiu so sanh vdi phUdng phap nuoi c&y (doi vdi vi khu&n N.
gonorrhoeae), hay phUdng phap mien dich huynh quang trUc tiep (doi vdi C.
trachomatis), hay neu cd dieu kien thi so vdi phUdng phap PCR. Tuy nhien
doii vdi cac hang san xuat ma san ph&m nay da dUdc FDA danh gia chap nhan
lUu hanh thi chung ta cd the sti dung ngay ma khong can phai nghien ctiu.

7.4. Ket qua nhuom Grarn mot phet am dao hay co tur cung thay co song cau
Gram [-], noi te bao. Co the nghi ngd benh nhan nhiem N. gonorrhoeae khdng?

- Neu la mot quet mu nieu dao d dan ong va ket qua nhu v&y thi cd the khang
dinh ch&n doan benh nhan bi lau. NhUng trong trudng hdp benh ph&m la
quet am dao hay co’ tti cung cua phu nti thi chi cd the nghi ngd chti khdng
the chSc chan, can phai nuoi cay.
- De nuoi cay, can phai cay ngay quet nay len hop thach Thayer Martin
(CATM) cd cac khang sinh tic che vi khuan va nam va u ngay trong binh nen
hay tu u C 0 2 5%. Sau khi phan lap, cac khom nghi ngd phai dUdc dinh danh
xac dinh N. gonorrhoeae bang phUdng phap len men dudng nhanh (3 loai
dudng: glucdse, lactdse, maltose, va sucrose). Vi khuan N. gonorrhoeae chi
len men dtidng glucose, khdng len men cac dudng khac.

7.5. Xin cho biet gia trj cua thfr nghiem miin djch9huynh quang phat hien
C. trachomatis trong cac benh pham la cac mau quet co tur cung

- Day la thti nghiem phat hien trUc tiep vi khuan C. trachomatis b4ng ky
thuat mien dich huynh quang trUc tiep dung khang the dac hieu
C. trachomatis gan huynh quang. Neu lam dung thed chi dan cua nha san
xuat thi thti nghiem rat dang tin cay.
- Mot kho khan khi thtic hien thti nghiem nay la nen dung dung cu lay m&u
thich hdp do la nhiing ban chai quet lay co tti cung ma hien nay neu phai
dat mua tai Viet Nam thi gia thanh kha dSt.

8. CAU HOI ON TAP

a. Cho biet cac cach lay benh pham tti dtidng sinh due d cac doi tUdng khac nhau.
b. Hay cho biet cac phUdng phap khao sat trUc tiep va cac cach danh gia
ket qua khao sat trtic tiep tren cac benh ph&m lay tti dudng sinh due.

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 c. Hay mo ta qui trinh nuoi cay va phfin lap cac vi khu&n gay benh tti cac
benh pham lay tti dtidng sinh due vk tti dd cho biet cac phUdng tidn c&n
thiet de thuc hien dUdc qui trinh nay.

d. Hay cho biet cac vi khu&n gay benh co the tim th&y tti dudng sinh due.

Hinh 10.1: Phet nhuom gram mot

m iu mu nieu dao cua mot ngudi dan
ong nghi bj lau. Quan sat d quang
khuan x1000. Co nhieu te bao bach
cau da nhan co cac song cau gram [-]
noi te bao. Vdi ket qua nay cd the
chan doan gan nhu 100% benh nhan
bj lau. Tuy nhien cung can phai tien
hanh nuoi cay de lam khang sinh do

Hinh 10.3: Hinh anh vi the mot phet te bao

Hinh 10.2: Phet nhuom gram mot
mau can lang ni/dc tieu cua mot
ngudi dan ong nghi bj lau cho thay
cd mot sd te bao bach cau da nhan
cd cac song cau gram [-] noi te bao.
Ket qua nay cd the giup chan doan
benh nhan bj lau, tuy nhien can xac
dinh bang nuoi cay
am dao benh nhan nhiem Gardnerella
vaginalis. (A) phet soi ti/di chi thay true
khuan cong bam day len te bao chat te bao
bieu mo am dao. (B) phet nhuom gram cung
cho thay trUc khuan G.vaginalis bam day len
te bao bieu mo am dao. (C) Cung phet
nhuom gram quet am dao, ngoai G.vaginalis
(g), chung ta cdn thay cac vi khuan khac nhu
Bacteroides (b) - Mobiimcus (c) -
Peptostreptococci (p)

90
 Sd do 10: Qui trinh vi sinh lam sang cac mau
benh pham lay tCr dudng sinh due

DO
KSTT: GRAM
Cay phan lap ngay
-C ATM
- BANg* va MC/EMB
* Neu khong c6 BANg, thay b ln g BA
KQ so bo: KSTT
U 37°C/C02 (CATM/BA)

Tren CATM co khom Chpn cac khom VK Cay tang sinh len
nghi N. gonorrhoeae khac tren BANg va BA/NA
D1 MC/EMB

U 37°C/ti/ sang|d6n ch iiu

Dinh danh (RNIS) KSD (CAIS) Djnh danh KSD j Djnh danh KSD
Theo qui trinh N.gonorrhoeae Theo qui trinh VK khdng phai N gonorrhoeae Theo qui trinh VK kh6ng phai N.gonorrhoeae

LUJJUULJULJLr IJUUUUULiJLJLr''

KQ chung
cuoc R N IS
 BA111. CAY KY KHI VA CAY MAU BENH PHAM KY KHI
■ a •

MUC TIEU

1. Chi dinh lam sdng diidc de cay ky khi vd cdc triidng hdp ndo khong nen
cay ky khi vd co the giup diidc lam sdng cho dung chi dinh cay ky khi vd
tranh cdc chi dinh khong cdn thiet.
2. Thiic hien diidc khau lay benh pham cay ky khi mot cdch chinh xdc vd
hodn hdo nhd vay tang diidc cd hoi phan lap diidc vi khuan ky khi gdy
benh tii cdc benh pham vi biet diidc cdc phiidng tien vd cdc cdch lay vd
chuyen chd cdc mdu benh pham d ecay ky khi.
3. Thiic hien diidc cdy ky khi nhd do co the trien khai diidc xet nghiem cdy
ky khi tai phong thi nghiem lam sdng tai cdc benh vien vi biet diidc cdc
phiidng tien cdn thiet vd thich hdp nhdt de co the thiic hien diidc xet
nghiem ndy.
4. Thiic hien diidc day du qui trinh cdy vd theo doi ky khi, dong thdi tao diidc
moi lien lac vdi lam sdng trong qua trinh lam xet nghiem cdy ky khi.

1. CHiDINH

1.1. Lam sang nghT den nhiem trung ky. khi khi

- Dich ri: hoi thdi, co hdi, co mau den; huynh quang do, hat sulfur.
- Cac nhiem trung gan niem mac.
- Vet thudng co hoai tu, mo dap nat, mang gia.
- Viem noi tam mac cay mau hieu khi am nghiem.
- Nhiem trung d cac benh ac tinh, benh co pha huy mo, benh co hu hai mach mau.
- Nhiem trung khi dang dung khang sinh Aminoglycosides.
- Nhilm trung huyet co viem tinh mach thuyen tkc, co vang da, sau nao thai,
sau giai phau vung bung.

1.2. Cac benh pham nen chi djnh cay ky khi

Tat ca cac benh pham lay tu: cac ndi khdng bi ngoai nhiem vi khu&n thudng
tru ky khi, ky khi tuy nghi va hieu khi:
- Vet thudng va ap xe, hut mu.

92
 - Mo (sinh thi£t, tti thiet, ph&u thu&t).
- Cac dich cd the (DNT, mang phd’i, mang bung, mang tim, dich khdp).
- Mau, tuy xUdng.
- Dich hut khi quan qua da.
- Dich choc hut phoi (cung nhu cac dich choc hut tti cac ndi khac cua cd the).
- Cac hat luu huynh lay tti benh nhan nghi nhiem Actinomyces.
- Nude tieu (choc hut qua da tren xUdng mu, lo md nieu quan, tham tich, nieu
quan, phau thuat md bang quang hay md than).
- Nieu due (chi lay tti cac ndi khong cd vi khuan ky khi thudng tru).
- Ruot non (blind loop va cac hoi chting kem hap thu tUdng tu).
- Phan hay benh pham tti da day ruot (de phat hien doc to' va cay phat hien
cac vi khuan nghi ngd gay benh nhu Clostridium difficile, C. perfringens va
C. botulinum).

1.3. Cac benh pham khong nen chi djnh cay ky. khi

Do khong the phan biet dUdc tac nhan gay benh va vi khuan thudng tru nhU:
- Quet hdng hay mui hau, quet nUdu rang.
- Ddm, dich hut noi soi phe quan khdng lay bang ong thong cd hai nong va cd
bao ve.
- Cac dich da day va ruot non (trti trtidng hdp blind lddp va cac hoi chting
ttidng tu), phan (trti trtidng hdp tim doc to' va phat hien cac clostridia gay
benh), quet hau mon, mach lUdn tti ruot gia ra da va thong noi ruot gia da
day (colostomy stomata).
- Cac benh pham lay tti be mat cac vet loet do nim lau, cac quet tti cac be mat
khac, dudng dan tti xoang (sinus tract) va cac hoai tti (eschar).
- Benh pham lay gan da va niem mac b&ng cac phUdng phap khdng thich hdp,
de bi ngdai nhiem.
- Nude tieu (vdided urin), quet am dao hay co’ tti cung.

2. CAC PHlfONG PHAP LAY BENH PHAM DE CAY KY KHI

- Tot nhat la benh pham sau khi lay phai dUdc chd ngay vao trong mdi trtidng
chuyen chd chuyen dung nhu Carrypdrt (dung cap tube dua tam bdng vd
trung/tube dua Carryport).

- Benh ph&m cd the dUdc lay b&ng tam bdng vd trung dua sau vad trdng mdi
trtidng chuyen chd. Hay sau khi cho vao mdi trtidng chuyen chd thi dung
tam bdng vd trung d&y sau vao ben trong mdi trUdng.
 - Cac benh pham khac nha DNT, chat dich, sau khi hut v&o ong kim, dfiy chat
nap kim lai va gtii nguyen den PTN.

3. QUI TRlNH VI SINH LAM SANG KHAO SAT MAU KY KHl

* N G A Y 1:

3.1. Mau ducsc lay vao moi trudng chuyen chd ky khi

Do la Carryport (Nam Khoa); trUdng hdp dich hay mu, co the hut bang ong
chich insulin khong cho bot khi lin vao roi mang thang den PTN.

3.2. Phong thi nghiem phai tien hanh khao sat ngay theo cac budc sau

Cay vao cac moi trUdng phan lap ky khi va /hay hieu khi:
- Cay hieu khi: BA hay BANg, EMB hay MC. BA u trong binh nen, MC hay
EMB d khi trUdng binh thudng. Theo doi theo qui trinh cac mau benh ph&m
cay hieu khi.
- Cay ky khi: BA ky khi (BAYK) va BAYK co phenyl Ethyl Alcohol (BAYK-
PEA) hay BAYK co nalidixic acid (BANg). U trong binh hay tui ky khi (vi
du: Genbox cua bio -Merieux) va khi trUdng ky khi dUdc tao ra bang phUdng
phap hoa hoc (vi du: bao Genbox anaer cua bio -Merieux khong can dung
xuc tac hay bao Gaspak cua BD vdi xuc tac Pallidium -aluminium). Binh u
ky khi dUdc giti trong tu am 37°C trong 48 gid.
- Lam phet nhuom gram benh pham, ghi nhan sU hien dien cua cac vi khuan
trong mau trudc khi co ket qua cay. Tra ldi ket qua sd bo cho bac si dieu tri
qua dien thoai hay qua phieu tra ldi ket qua tam thdi.
* NGAY3: (vi phai u binh ky khi tren 48 gid)
- Md binh ky khi hay tui ky khi. Quan sat cac mat thach, ghi nhan cac loai
khom va hinh thai cac khom. Lam phet nhuom gram khao sat dudi kinh
hien vi.
- ThUc hien cay lai thuan khiet cac khom trong moi trUdng BAYK/u ky khi,
dong thdi lam thti nghiem ldai trti vi khuan phan lap dtidc la YK tuy nghi,
HK tuyet doi bang cach cay them tren mat thach BA /u binh nen.
- Tra ldi them ket qua sd bd chd lam sang.
* N G A Y 4 H A Y 5:

- Quan sat BA /u binh nen. Neu khdng co vi khuan moc thi vi khuan phan lap
dUdc la YK tuyet doi.

- Md binh ky khi. Lay cac khdm vi trung tren thach BAYK da xac dinh la ky
khi tuyet dci. Lam phet nhupm gram quan sat hinh dang vi khuan qua kinh
hien vi.

94
 - Tien h&nh qui trinh dinh danh vdi API 20A va lam khang sinh do vdi ATB -
ANA, u ky khi 24-36 gid. Neu khong co API 20A thi co the tien hanh dinh
danh ky khi theo qui trinh kinh dien vdi mot so moi trUdng va phan iing
sinh hoa dac biet.
- Tra ldi ket qua chung cuoc cho lam sang sau khi doc ket qua dinh danh va
khang sinh do.

4. CAC PHUONG PHAP TAO KHf TRUONG KHONG CO 0 2 (OXY PHAN Tlf) DE
CAY KY KHf

4.1. Phuong phap dung bao sinh khi COz va khur khi 0 2 trong binh u hay trong
bao u

PhUdng phap nay dung mot tui kin chiia mot bao hoa chat ma khi tui kin nay
bi xe ra de bao hoa chat tiep xuc vdi khong khi thi se xay ra phan ting hoa hoc khti
0 2 trong khong khi va sinh ra khi C 0 2 vdi ty le 5% trong binh u hay tui u tUdng ting
vdi kha nang khti 0 2 cua bao hoa chat. Hang bio -Merieux co dua ra thi trUdng san
pham co cong dung nhu tren va co ten la Genbox -anaer dung cho binh u cho
khoang 10 hop thach phan lap va Genbag -anaer danh cho tui u cho khoang 2 hop
thach phan lap. Cac san pham nay rat de dang sti dung vi khong can phai cho nUdc
vao tui hay la phai dung chat xuc tac. Trong dan dung chung ta co the thay san
ph&m co cong dung khti 0 2 (nhUng khong sinh C 0 2) trong cac tui banh trung thu
nh&m muc dich giti banh trung thu d dieu kien ky khi nhd vay ma bao quan dUdc
banh khong bi nam moic trong thdi gian dai.

4.2. PhUOng phap dung bao sinh khi C 0 2 va H2de H2 khti 0 2 trong binh u nhd chat
xuc tac

Hang BD (My) dua ra san pham co ten la Gaspak hoat dong theo nguyen tac
nay. Gaspak la mot tui kin chtia hai vien hoa chat ma khi mot goc tren cua tui kin
nay bi xe ra va cho vao do 10ml ntidc thi nUdc se tham dan vao hai vien hoa chat de
co mot vien sinh ra khi C 0 2 va mot vien sinh khi H2. Khi H2 sinh ra se nhd chat xuc
tac (la cac vien da pallidium -aluminium gan tren nap binh, dUdc goi la xuc tac
lanh; hay la vong day bach kim nung nong nhd dien trd cung gan tren nap binh,
dUdc goi la xuc tac nong) phan ting vdi 0 2 trong khong khi binh u thanh H20 bam
vao thanh binh. Mot luu y cua phtidng phap nay khi dung cac vien da xuc tac
pallidium -aluminium la phai say kho cac vien da nay 180°C trong vong 1 gid ngay
trudc khi sti dung hay sau khi sti dung va sau do giti trong cac binh kin (dessicator)
co silicagel hut am vi cac vien da nay chi xuc tac phai ting ket hdp 0 2 vdi H2 khi
chung d dang khan nUdc. Mot bat tien ntia la cac tui Gaspak khong the chuyen chd
dUdc bang dUdng hang khong vi nguy cd gay chay no nen ngUdi sti dung phai dat
hang trUdc kha lau mdi co.

95
4.3. PhUdng phap dung binh khi C 0 2 va binh khi H2 phoi tron lai de thay khong
khi trong binh u, sau do chat xuc tac se ket hop H2vdi 0 2 de khur hoan toan 0 2
trong binh u

PhUdng phap nay re tien nhat nhung muon ap dung thi phai trang bi cho d&y
du cac binh cung cap H2 va 0 2, may tron khi de tron H2 va C 0 2 tvf hai binh cap khi
va bdm chan khong noi vdi binh u. Sau khi cho cac hop thach da cay benh pham v&o
trong binh u, phai thuc hien qui trinh thay khong khi trong hinh u bang hon hdp
khi nhu sau: (1) Day chat binh u roi noi voi cap khi va voi hut chan khong v&o binh
qua nSp binh, dong khoa voi cap khi va md khoa voi hut roi bat may hut chan
khong de hut khong khi ra khoi binh cho den ap luc dat <10 2 thi khoa voi. (2) Md
khoa voi cap khi de cung cap hon hdp khi vao binh u cho den khi ap ltic khi trong
binh trd lai binh thudng thi dong voi cap khi. (3) Tiep tuc lai qui trinh nay them viki
lan ntia de dam bao khong khi trong binh da dUdc thay hoan toan bang hon hdp
khi. Hon hdp khi nay van co the con 0 2 vi kho co the co dtidc cac binh cap khi C 0 2
va binh cap khi H2 khong bi tap nhiem 0 2, tuy nhien sau vai gid khi 0 2 trong hon
hdp khi trong binh se bi khti hoan toan nhd cac xuc tac nong hay xuc tac lanh gan d
nap binh. Cung nhu phUdng phap dung bao Gaspak, neu chung ta dung xuc tac
lanh thi phai khti nude xuc tac lanh bang cach say 180°C trong hdn mot gid de dam
bao xuc tac hoat dong tot.
Dong ho do ap luc

Khoa dong md Khoa dong md

Vdi ndi vdi may Vdi ndi vdi may hut

tron khi chan khdng

Xuc tac nong

haylanh

Ong chi thi

ky khi

Hinh 11.1: Cau true mot binh u ky khi dung phtfdng phap thay khi

96
 Hi£n nay co may tron khi thuc hien viec hut chan khong cua binh va cho khi
vao binh mot cach ho&n toan tu dong, vl du may Anoxomat mark II cua Ha Lan
(Hinh 11.2) va nhd \ky ngticJi lam x6t nghiem khong can phai thtic hien cac dong
tac md, dong van xa va hut khi ntia.

5. CHI THj KY KHf

Chi thi ky khi la mot phUdng tien giup chung ta co the kiem tra dUdc la trong
binh ky khi that sU da hoan toan het 0 2 chua. Cac chi thi ky khi thudng hoat dong
dua tren nguyen tac khti mau cua mot cha't mau khi chat mau nay d dang khti va
co mau khi d dang oxid hoa. Cac cha't mau co dac tinh nhu vay la Methylen blue va
Resazurin va day cung la hai cha't thudng dUdc cho vao moi trUdng Thioglycolat hay
moi trtidng chuyen chd benh pham de cay ky khi cung vdi muc dich cho biet cac moi
trUdng nay co cl tinh trang khti 0 2 trong phan sau dudi be mat cua moi trUdng hay
khong de san sang sti dung. Neu 6 phan sau cua ong moi trUdng co mau thi ngUdi
ta phai dun cach thuy chung de dudi het 0 2 (khi do phan sau cua moi trUdng se mat
mau) va de nguoi roi mdi sti dung.
Chung ta co the tu pha chi thi mau de kiem tra tinh trang ky khi cua binh u,
nhUng ddn gian nhat la chung ta co the dung mot ong moi trtidng chuyen chd ky
khi hay mot ong moi trUdng thioglycollat de lam chi thi ky khf. Cach sti dung la
trudc het phai dun cach thuy cac ong moi trUdng nay de dudi het 0 2 khoi moi
trtidng va sau do de nguoi, luc nay phan mat ong moi trUdng se co mau trd lai. Ndi
long nap ong moi trUdng va cho vao binh u ky khi trUdc khi day nap. Binh u ky khi
dUdc coi la ky khi hoan toan khi be mat cac ong moi trUdng nay mat mau hoan toan
sau khi tien hanh u ky khf khoang 1-2 gid.

Hinh 11.2: May axonomat Mark II

cua Ha Lan de tao khi trudng ky khi

6. CAY MAU BENH PHAM KY KHI - CAC CAU HOI THl/ONG GAP

6.1. Mot so bac sT lam sang muon tim tac nhan vi khuan ky khi gay viem phoi. Yeu
cau nay co hop ly khong va phong thi nghiem co nen dap umg yeu cau nay khong?

- Thuc te co mot so bac si co yeu cau nay, tuy nhien nhti da trinh bay la cay
ky khi khong nen thuc hien tren cac benh pham ddm hay dich rtia khi quan

97
 vi day la cac mau thti t§p nhiem, khong the phan biet dtfdc vi kh&n ky khi
gay benh vdi vi khuan ky khi tap nhilm. Do vky, neu muon tim tac nhan vi
khuan ky khi gay viem phoi thi phai co phUdng tien My dUdc benh ph&m \k
ddm lay qua noi soi bang ong thong co hai nong va co bao ve.
- Tuy nhien cung can phai biet rSng tac nhan gay viem phoi cong dong riit
hiem khi la vi khuan ky khi, ma thudng la S. pneumoniae, H. influenzae, vk
M. catarrhalis.... Tac nhan gay viem phoi benh vien cung vay, thudng la S.
aureus, cac trUc khuan gram [-] dudng ruot hay khong len men. Do vay theo
chung toi nghi yeu cau tim vi khu&n ky khi gay viem phoi la khong hdp ly
llm . Chi nen nghi den tac nhan vi khuikn ky khi trong cac trUdng hdp viem
phoi tac nghen u (do hit di vat hay do ddm qua dac va nhieu bit lay khi quan
trong cac trUdng hdp COPD nang), cac trUdng hdp viem phoi co kem ap xe
phoi. Trong cac trtidng hdp nay benh pham tot nhat la choc hut phoi qua
thanh ngUc de hut lay mu ap xe hay cac chat dich vung nhu mo phoi bi ton
thudng.

6.2. Co nhieu trtidng hdp benh nhan co dau hieu nhiem trung huyet ro rang
nhung cay mau vin bj am tinh. Co phai nhu vay la benh nhan co the bj nhiem
trung huyet vi khuan ky khi khdng?

- Co nhieu trUdng hdp nhu vay, nhUng khong phai tat ca deu la do nhiem
trung vi khuan ky khi ma con co the la do benh nhan bi nhiem trung tai cho
nao do ma vi khuan khong vao mau va trieu chting benh ly giong nhiem
trung huyet la do phan ting toan than cua cd the doi vdi tinh trang nhiem
trung nay.
- De co the cay mau ky khi, co the cay mau trong moi trtidng dac biet, do la
cac chai moi trUdng thioglycolat. Cac chai cay mau nay dUdc che tti moi
trUdng thioglycolat long la moi trUdng du bd dudng va co du chat khti de duy
tri tinh trang ky khi trong moi trUdng.

6.3. Tai sao chung ta khong siir dung phUdng phap cay khom vi khuan nay tren
ong thach nghieng sau de co the phan biet dtidc mot khom vi khuan moc tren
mat thach phan lap u ky khi la khom vi khuan ky. khi hay ky. khi tuy nghi?

- Ong thach nghieng sau dUdc che tti moi trUdng thach thioglycolat. dUdc lam
dac trong ong sau khi khti trung de co mot phan sau va mot phan nghieng
nhu ong moi trUdng KIA hay TSI. NgUdi ta cay vao ong thach nghieng nhti
cay vao ong thach KIA hay TSI, ttic la sau khi lay mam cay la khom vi
khuan len que hay vong cay, dam sau vao phan sau va sau do cay zic -zac
tren phan mat nghieng. U ong moi trtidng trong dieu kien hieu khi qua dem
hay 24 gid. Quan sat vi khuan moc trong ong, neu chi moc d phan sau ma
khong moc dUdc tren mat nghieng thi do la vi khu&n ky khi. Neu \i khuan
moc dUdc tren ca mat nghieng lan dudi phan sau thi do la vi khujin ky khi
tuy nghi.

98
 - Tuy nhien vi da so Streptococci khong the moc dUdc tren mat thach
thioglycolat du chung co la ky khi tuy nghi, do vay thii nghiem nay co the
khong phan biet dUdc Streptococci ky khi vdi Streptococci ky khi tuy nghi.
Day chinh la mot ly do giai thich tai sao co mot vai cong trinh nghien ctiu vi
khuan ky khi lai cho ket qua khang sinh do co ty le de khang qua cao khang
sinh Metronidazol vi co the trong cac cong trinh nay cac tac gia da cho cac
chung Streptococci ky khi tuy nghi thanh Streptococci ky khi.

6.4. Doi vdi phong thi nghiem khong co phUdng tien cay ky khi, lam the nao co
the nhan biet duoc co tac nhan vi khuan ky khi trong benh pham?

- Du khong co phUdng tien cay ky khi nhung phong thi nghiem van co the
nhan biet dUdc sU hien dien cua vi khuan ky khi bang cach song song vdi qui
trinh cay hieu khi (cay khong ky khi), luon cay mau vao mot ong moi trUdng
thit bam (choped meat) hay ong moi trUdng thioglycolat. Neu mau cay hieu
khi khong co vi khuan moc ma trong cac ong moi trUdng tren lai nhieu vi
khuan thi rat co kha nang co vi khuan ky khi, luc do chung ta se tien hanh
nhuom gram de tra ldi cho lam sang ket qua nhuom gram va cac nhan xet
cua minh.

- De dam bao cac ong thioglycolat va ong thit bam hoan toan khti het 0 2 trong
phan sau, trudc khi cho benh pham vao moi trUdng ngUdi ta phai dun soi cac
ong moi trUdng nay trong 10 phut de dudi het 0 2 ra khoi moi trUdng va sau
do de nguoi roi mdi cay benh pham vao.

7. CAU HOI ON TAP

a. Cho biet lam sang nen cho chi dinh cay ky khi trticfc cac trUdng hdp nao?

Cac benh ph&m nao khong nen cho lam xet nghiem tim vi khuan ky khi
gay benh, phan tich ly do tai sao?
c. Cho biet cac cach lay va chuyen chd benh phiam de lam xet nghiem tim vi
khuan ky khi va phan tich ly do tai sao?
d. Cho biet qui trinh cay va theo doi cay ky khi dUdc thuc hien trong may
ngay vdi cac bu6c tien hanh nhu the nao cho tting ngay?

99
 Benh pham cay ky khi phai di/oc C iy phan lap trfen m6i trudng
giOf trong moi tn/dng chuyen chd BAYK+PEA/BAYKNg.
chuyen dung la CARRYBAC

DO

U ky khf trong binh ky khf v6i bao

Genbox - anaer. Be trong tu 5m
37°C qua 2 dem

D1

Chon khom vi k h u in , c5y

zic - zac tren hai mat thach BAYK

Mot u ky khf
Mot u binh nen 37°C trong 24h
37°C qua dem

D2

Chon vi khuan chi moc ky khf,

khong moc trong binh nen

Thi/c hien dinh danh v6i API

20A va khang sinh do vdi ATB -
ANA. U ky khi/370C/24 gid Sd do 11: Qui trinh vi sinh lam
sang cac mlu khao sat ky khi

D3 Doc ket qua chung cuoc: Djnh danh va khang sinh do

100
 BA112. KHAO SAT TRUC TIER ■

MUC TIEU

Su! dung cd hieu qua phiidng tien khao sat trite tiep trong xet nghiem vi
sinh lam sdng cdc mdu benh pham khac nhau nhd biet diidc cdc loai
khao sat trite tiep cd the thiic hien diidc tai cdc phong thi nghiem lam
sdng vd cong dung cua tiing loai.
2. Phuc trinh ditdc cdc ket qua khao sat trite tiep vd dong thdi tham van
ditdc cho lam sdng cdc y nghla hvtu dung lam sdng md ket qua khao sat
trite tiep nhd biet ditdc vai tro vd y nghla cua khao sat trite tiep trong xet
nghiem vi sinh lam sdng.
Tranh ditdc cdc triidng hdp phai lam khao sat trite tiep mot cdch vd ich ■
tham chi cdn cho cdc thong tin sai lac den lam sdng vd khdng bo sot cdc
tritdng hdp cdn thiet phai lam khao sat trite tiep vi cdc y nghla hCtu dung
md ket qua khao sat trite tiep mang lai cho lam sdng vd biet ditdc cdc
tritdng hdp benh pham ndo cdn phai lam khao sat trite tiep, cdc tritdng
hdp benh pham ndo khdng that sit cdn khao sat trite tiep.

1.CAC PHUONG PHAP KHAO SAT TRl/C TIEP

1.1. Soi tUdi

- Qua ki'nh hien vi thudng

+ Soi titdi khdng cdn nen, la phUdng phap soi ttidi qua kinh hien vi dong bdt
tu quang, vdi benh pham dUdc dat trong mot giot nUdc muoi sinh ly tren
mot lam kinh, treo hay ep dtidi mot lamel. PhUdng phap nay dung de xem
sU di dong cua vi khuan.

+ Soi titdi cdn nen, la phtidng phap soi ttidi qua kinh hien vi dong bdt tu
quang vdi benh pham dtidc dat trong mot giot dung dich mau lam nen
nhu dung dich mUc tau; nigrosin; m ethylen blue, tren mot lam ki'nh, ep
dudi mot lam el. PhUdng phap nay dung xem nang vi khuan, hay tim
nam men co trong benh pham nhu Cryptococcus neoformans trong dich
nao tuy.

- Qua kinh hien vi nen den hay dao pha

+ Qua kinh hien vi nen den, muc dich thong thudng nhat la xem hinh dang
va sU di dong cua vi khu&n co trong mot benh pham dat trong mot giot
nUdc muoi sinh ly tren mot lam kinh ep dudi mot lamel. PhUdng phap
nav dUdc dung de tim xoan khuan giang mai. vi khuan leptospira, hay
khao sat su di dong vi khuan.

101
 + Qua kinh hien vi dao phase, muc dich thong thudng nh&'t 1& xem nang vi
khuan nhu la S. pneumoniae, hay tim nam men co trong benh ph&m nhu
Cryptococcus neoformans trong dich nao tuy.

1.2. Nhuom

- Nhuom gram, la phUdng phap nhuom thong thudng nhat trong cac phong
thi nghiem vi sinh. PhUdng phap nhuom gram cho phep xac dinh dUdc
hinh dang, cach sap xep va phan biet vi khu&n la thuoc loai gram [+] hay
gram [-].

- Nhuom ddn M ethylen blue kiem, la phUdng phap hay dUdc dung de nhuom
khao sat co sU hien dien cua vi khuan Corynebacteria hay khong vi phUdng
phap nay cho phep nhuom vi khuan va cac hat bien sac co trong vi khuan.

- Nhuom khdng acid, la phUdng phap hay dUdc dung de nhuom va phat hien
cac vi khuan khang acid nhu cac Mycobacteria.

- Phiidng phap nhuom huynh quang, la phUdng phap nhuom vi khuan b&ng
pham mau huynh quang va chi ap dung cho mot so trUdng hdp nhu nhuom
huynh quang rhodamin phet ddm tim vi khuan lao.

- PhUdng phap nhuom khdng the dac hieu danh dau men hay danh dau
huynh quang, la cac phUdng phap phat hien trUc tiep vi sinh vat muon tim
co trong benh pham nhd khang the dac hieu khang nguyen vi sinh vat dUdc
danh dau bang men (phat hien qua quan sat bang kinh hien vi thudng) hay
bang huynh quang (phat hien qua quan sat bang kinh hien vi huynh quang)

- Cdc phiidng phap nhuom khac , nhu nhuom nang, flagella, sp ore... chi dUdc
dung trong cac trUdng hdp dac biet.

2. VAI TRO VA Y NGHIA CUA KHAO SAT TRUC TIEP

2.1. Cho ket qua rat sdm, gan nhu chung cuoc

Co nhuing ket qua khao sat trUc tiep giup bac s! lam sang va phong thi
nghiem nghi ngay den tac nhan gay benh vdi sU chinh xac gan nhu 9 9 % , vi du:

- Ket qua khao sat trUc tiep dich nao tuy thay co song cau gram [-]; nghi ngay
den tac nhan. meningitidis, thay song cau gram [+] hinh mui giao; nghi
ngay den S. pneum oniae , hay thay trUc khuan gram [-] nho; nghi ngay den
H. influenzae...

- Ket qua soi tUdi mu nieu dao tii dan ong thay co song c&u gram [-]; nghi
ngay den tac nhan N. gonorrhoeae...
- Ket qua soi tUdi dich nao tuy thay co nam men co nang; nghi ngay den tac
nhan nam men C. neoformans...

102
 - Ket qua khao sat trUc tiep phet quet cd tti cung phat hien C. trachomatis
bang phUdng phap nhuom khang the huynh quang dac hieu C. trachomatis
dudng tinh la du de ket luan benh nhan bi nhiem vi khuan nay.

Cac ket qua nhu tren rat co gia tri giup cho bac si dieu tri chon dUdc khang
sinh dieu tri ban d&u va giup phong thi nghiem biet dtfdc hudng phan lap; dinh
danh; khang sinh do trong xet nghiem cay va phan lap tiep theo.

2.2. Cho ket qua sdm va goi y

Rat nhieu ket qua khao sat trUc tiep, neu biet tan dung, bac si lam sang se co
hudng dieu tri ban d&u cung nhu phong thi nghiem co hudng phan lap va dinh
danh, vf du:

- Khao sat trUc tiep nUdc tieu, thay co 1 vi khuan/quang trUdng xlOO, co the
nghi ngay la benh nhan bi nhiem trung tieu va co the chon lua khang sinh
dieu tri budc dau tuy theo hinh anh gram cua vi khuan hien dien trong mau.

- Khao sat phet nhuom gram mau mu, hay abcess, hinh anh vi khuan thay
trong benh pham qua phet nhuom gram gdi y dUdc tac nhan vi khu&n gay
benh, nhd do bac si lam sang se co hudng dieu tri ban dau cung nhu phong
thi nghiem co htidng phan lap va dinh d anh....

2.3. Cho ket qua danh gia m lu co tin cay de nuoi cay, phan lap hay khong va cho
ket qua gcfi y

- Lam mot phet gram mau ddm, quan sat d quang trUdng xlOO, co the danh
gia mau tin cay hay khong de co the tiep tuc thuc hien qua trinh nuoi cay
phan lap

- Cung qua phet nhuom gram mau ddm, neu mau tin cay, co the tiep tuc qua
quang trUdng xlOOO de quan sat hinh anh gram cac vi khu&n hien dien va
ket qua nay se rat co gia tri gdi y cho phong thi nghiem hudng phan lap vi
khuan gay benh va bac si se co the co hudng dung khang sinh nao trong
dieu tri ban dau.

2.4. Co nhung trtidng hop mau khong can phai lam khao sat true tiep

- M au quyet hong, neu khong co yeu cau tim vi khuan bach hau thi khong can
thiet phai lam khao sat trtic tiep vi khong co sU khac biet gitia mau khong
benh vdi mau benh.

- M au phan, neu khong co yeu cau tim Campylobacter, V. cholerae thi khong
can thiet phai lam- khao sat trUc tiep.

3. CAU HOI ON TAP

a. Cho biet cac phUdng phap khao sat trUc tiep va cong dung cua tting
phUdng phap nay?

103
 b. Cho biet vai tro va y nghla cua cac ket qua khao sat tnlc tiep doi vdi lam
sang? Neu cac vi du cu the.

c. Cho biet cac trUdng hdp nao khong nhat thiet phai lam khao sat trUc tiep
va cac trUdng hdp nao bat buoc phai lam khao sat trUc tiep trudc khi tien
hanh nuoi cay phan lap? Tai sao?

Hinh 12.1: Soi tifdi nhieu khi rat co gia tri trong xet nghiem vi sinh lam sang: (A) Mot phet soi tUdi
djch nao tuy trong giot muc tau cho hinh anh nhu vay rat co gia tri de chan doan viem mang nao do
Cryptococcus neoformans. (B) Phet soi tUdi qua kinh hien vi nen den giot moi trudng EMJH cay mau
benh nhan cho ket nhu tren giup ket luan mau cay co vi khuan Leptospira. (C) Phan ifng phong
nang (quellung reaction) dung dinh type S.pneumoniae co the doc ket qua qua phet soi tuoi trong
giot nigrosin nhutren. (D) Soi tUdi mot giot moi trUdng cua chai cay mau qua kinh hien vi dao pha vdi
hinh anh nhutren giup sdm nhan biet chai cay mau [+] trUc khuan

104
 BA113. NHUdM
m GRAM

MUC TIEU

1. Nhan dinh diidc mot ket qua nhuom gram tot hay co van de ve mat ky
thuat nhd biet ro nguyen tac cua phiidng phap nhuom gram.
2. Thiic hien diidc tot ky thuat nhuom gram cdc mdu benh pham khac nhau
nhd nam viing diidc cdc biidc thiic hanh trong ky thuat nhuom gram.
3. Tranh diidc cdc yeu to ky thuat lam sai lech ket qua nhuom gram vd co
the bien luan diidc mot ket qua nhuom gram co van de ve mat ky thuat
nhd biet diidc cdc yeu toky thuat anh hiidng len ket qua nhuom gram.

1. NGUYEN TAC

Do sU khac biet ve cau true vach te bao nen trong qua trinh nhuom gram, vi
khuan gram [+] se giii dUdc phtic hdp tim Gentians -iod khong bi tay mau bdi
alcohol, trong khi vi khuan gram [-] khong giti dUdc phtic hdp mau nay, do vay ket
qua sau khi nhuom la vi khuan gram [+] van giti dUdc mau tim cua gentian, con vi
khuan gram [-] an mau hong cua pham mau safranin hay fuchsin.

2. BO THUOC NHUOM GRAM

De nhuom gram, can phai co bo thuoc nhuom gram gom cac lo thuoc thti sau:
Lo thuoc nhuom gram la dung dich tim gentian
• Lo lugol la dung dich iod
• Lo alcohol 95%
• Lo safranin chtia dung dich safranin
Bo thuoc nhuom gram dUdc chtia trong cac chai sam mau, nap van chat, giti
trong toi d nhiet do phong.

3. PHUONG PHAP THL/C HIEN

- TrUdc het lam mot phet rat mong benh pham hay vi khuan tren lam kinh,
de kho tu nhien, roi sau do gan tren lam bang cach hd nhanh qua ngon ltia
den con hay den bunsen 3 lan. Luu y la chi hd nhe tren ngon ltia de lam chi
am len vtia phai chti khong lam lam bi qua nong vi nhu vay se lam bien the
hinh dang vi khuan.

- Dat lam tren gia nhuom, cho vai giot thuoc nhuom gram (crystal violet = tim
gentian) phu trum len phet vi khuan, de yen trong 1 phut. Sau do cam lam len
va rtia sach mau thtia dinh tren lam dudi mot voi ntidc may chay rat nhe.

105
 - Dat lam trd lai tren gia nhuom, cho tiep vai giot lugol (iod) phu trum tiep
len phet vi khuan, de yen trong 1 phut. Sau do dim lam len vk rUa sach
lugol thifa dinh tren lam dudi mot voi nUdc may chay rSit nhe.
- Tay mau bang cach nho len goc tren cua lam nhuom dung dich alcohol 95%
cho den khi giot alcohol rdi khoi lam sach mau. Sau do c&m lam len va riia
sach alcohol khoi lam dudi mot voi nUdc may chay rat nhe.
- De lai lam tren gia nhuom roi phu vai giot safranin len phet nhuom, de yen
1 phut. Sau do cim lam len va rtia sach safranin thCfa dinh tren lam dudi
mot voi nUdc may chay rat nhe.
- Tham kho lam b&ng cach ep lam giuta 2 td giay tham. De kho lame hoan
toan trong khong khi. Sau do nho mot giot dau soi kinh len phet nhuom va
quan sat qua kinh hien vi, trudc het d vat kinh x 10 de tim vung phet benh
pham, sau do xoay sang vat kinh xlOO (vat kinh d&u) de quan sat hinh thai
va cach an mau cua vi khuan.

4. DOC KET QUA

Quan sat phet nhuom gram qua kinh hien vi, dudi vat kinh d&u, chung ta se
thay vi khuan gram [+] an mau tim, con vi khuan gram [-] an mau hong.
Khi tra ldi mot ket qua nhuom gram, phai tra ldi cac chi tiet sau:
- Hinh dang vi khuan.
- Cach sap xep cac vi khuan.
- Cach an mau cua vi khuan, tiic la vi khuan gram [+] hay gram [-].

5. CAC SAI LAM CO THE GAP TRONG PHUONG PHAP NHUOM GRAM

- Co nhulng sai lam trong phUdng phap nhuom gram lam cho vi khu&n gram
[+] nhuom thanh mau hong cua vi khuan gram n [-], do la u:

+ Phet vi khuan d liia cay qua gia tren 24 gid, luc do cau true vach vi khuan
khong con ben chat nhu cau true vach vi khuan gram [+] liia cay tre, do
vay mat kha nang ngan can sU t&y mau cua alcohol.
+ Dung dich lugol khong con tot nufa do da pha qua lau va da mat di iod, co
the nhan biet trUdng hdp nay khi dung dich lugol khong con sam mau nuta.

+ Tay mau bang alcohol qua lau da lam cho vi khuan gram [+] cuo’i cung
cung bi tay mau, hay dung Ion chai tay mau alcohol acid cua phUdng
phap nhuom khang acid.

- Co nhutng sai lam trong phUdng phap nhuom gram lam cho vi khu&n gram [-]
nhuom thanh mau tim cua vi khuan gram n [+], do la:

+ Phet vi khuan qua day lam cho alcohol khong the tay mau toan bo vi
khuan trong phet nhuom.
+ Tay alcohol trong thdi gian qua ngan hay dung dich alcohol bi pha qua
loang lam cho mau gram khong dUdc tay khoi te bao vi khuan.

106
 - Ngoai ra con co mot sai lam ky thuat n€fa ma chung ta thudng gap, do la sau
khi phu gram, chung ta khong riia sach mau thifa tren lam do vay khi
chung ta phu lugol tren phet nhuon thi mau gram thiia se bi tua lai bam
tren be mat phet nhuom. Ket qua khi nhin qua ki'nh hien vi chung ta se
thay rat nhieu tinh the mau gram bam tren phet nhuom lam lam tUdng
hinh anh vi nam. .

6. CAU HOI ON TAP

a. Cho biet nguyen tac cua phUdng phap nhuom gram.

b. PhUOng phap tien hanh nhuom gram nhu the nao? Ket qua an mau cua
vi khuan sau moi giai doan nhuom nhu the nao?

c. Cho biet cach tra ldi ket qua cua mot phet nhuom gram vi khuan.

d. Cho biet cac sai lam thudng gap trong phUdng phap nhuom Gram va hau
qua cua cac sai lam nay.

1a. Phu thuoc nhuom 2a. Phu dung djch

Gram trong 1’ bang nude may lugol trong 1’

3a. Tay mau bang alcohol 3b. Riia sach alcohol

niidc sach cho den khi giot alcohol roi bang nude may
khoi lam het mau

4a. Phu thuoc nhuom 4b. Riia sach safranin 3b. Tham kho lam giua
safranin trong 1’ bang nude may hai to giay tham

Hinh 13.1: Hinh ve mo ta cac giai doan tien hanh cua phUdng phap nhuom gram

107
Hinh 13.2: Mot bo thuoc nhuom gram day du Hinh 13.3: Ket qua nhuom gram la gram [+]
an mau tim, gram [-] an mau hong

108
 BA114. NHUDMMETHYLEN BLUE KIEM
m

MUC Tl£U

1. Sit dung hieu qua phiidng phap nhuom nhuom methylen blue kiem, dac
biet trong phat hien vi khuan Corynebacterium diphthriae trong cdc
mau quet hau hong nhd biet ditdc nguyen tac vd muc dich cua phitdng
phap nhuom ndy.

2. Thiic hien diidc tot ky thuat nhuom ddn ndy nhd nam vitng ditdc cdc
biidc thiic hanh trong ky thuat nhuom methylen blue kiem.

1. NGUYEN TAC

Methylene blue khi dUdc lam kiem hoa thi se nhuom dtidc cac hat bien sac
cua cac trUc khuan Corynebacteria, dac biet la trUc khuan bach hau. Do vay nhuom
methylene blue kiem ngoai muc dich dung de nhuom ddn, nhuom nen sau khi
nhuom khang acid, con co muc dich chinh la nhuom cac phet benh pham quet hau
hong de phat hien Corynebacterium diphtheriae.

2. BO THUOC NHUOM METHYLEN BLUE KIEM

De nhuom methylen blue kiem, can co chai thuoc nhuom methylen blue kiem
chiia trong chai sam mau, nap van chat, giii trong toi 6 nhiet do phong.

3. PHUONG PHAP THUC HIEN

- TrUdc het lam mot phet mong benh pham hay vi khuan tren lam kinh, de
kho tu nhien, roi sau do gan tren lam bang cach hd nhanh qua ngon liia den
con hay den bunsen 3 lan. Luu y la chi hd nhe tren ngon liia de lam chi am
len viia phai chut khong lam lam bi qua nong vi nhu vay se lam bien the
hinh dang vi khuan.

- Dat lam tren gia nhuom, cho vai giot thuoc nhuom methylene blue kiem phu
trum len phet vi khuan, de yen trong 1 phut. Sau do cam lam len va riia
sach mau thufa dinh tren lam dudi mot voi nU6c may chay rat nhe.
- Tham kho lam bang cach ep lam giiia 2 td giay tham. De kho lam hoan toan
trong khong khi. Sau do nho mot giot dau soi kinh len phet nhuom va quan
sat qua kinh hien vi, trudc het d vat kinh xlO de tim vung phet benh pham,
sau do xoay sang vat kinh xlOO (vat kinh dau) de quan sat hinh thai va cach
an mau cua vi khuan.

109
4. DOC KET QUA

Te bao vi khuan bat mau xanh bien nhat, cac hat bien sac bat mau tim den
(xem hinh 21A).

Hinh 14: Mot chai nhiia sam mau chtia ben

trong thuoc nhuom alkalin methylen blue

110
 BA115. NHUOM KHANG ACID
■

MUC Tieu

1. Tranh cdc sai sot ky thuat neu co trong khi thiic hanh nhuom khdng
acid nhd biet diidc cdc nguyen tac cua phiidng phap nhuom khdng acid,
biet diidc trong phiidng phap nhuom khdng acid, khau ndo la quyet dinh
diidc tinh chinh xdc cua ket qua.
2. Thiic hien diidc tot ky thuat nhuom khdng acid ndy nhd nam vitng diidc
cdc biidc thiic hanh trong ky thuat nhuom ndy.
3. Bien luan diidc ket qua phiidng phap nhuom khdng acid dac biet khi
phet nhuom co sd sot xdy ra nhd biet diidc cdc sd sot ky thuat cd the xdy
ra trong khi thiic hanh phiidng phap nhuom khdng acid.

1. NGUYEN TAC

Do te bao vi khuan khang acid co ldp vo sap bao boc nen khi da nhuom dUdc
phtic hdp mau carbolfuchsin, phtic hdp nay se khong bi t&y mau bdi dung dich tay
mau manh (acid, alcohol acid). Tuy nhien de phtic hdp mau nay co the tham xuyen
qua ldp vo sap cua vi khuan, co hai cach: (1) Dun nong phet nhuom vdi dung dich
mau carbolfuchin, nhd do ma carbolfuchsin tham qua ldp vo sap cua vi khuan de
nhuom mau vi khuan; day la phUdng phap nhuom nong Ziehl Neelsen. (2) PhUdng
phap thuf hai la phUdng phap nhuom lanh con goi la phUdng phap Kinyoun, trong
phUdng phap nay ngUdi ta dung dung dich carbolfuchsin dam dac, nhd vay khi phu
dung dich mau dam dac nay len phet nhuom vdi thdi gian lau, carbolfuchsin van co
the tham qua ldp vo sap de nhuom mau vi khuan.

2. BO THUOC NHUOM KHANG ACID

De nhuom khang acid, can phai co bo thuoc nhuom khang acid gom cac lo
thuoc thii sau:
- Lo thuoc nhuom carbolfuchsin la dung dich basic fuchsin vdi phenol, dUdc
pha dam dac de van co the dung cho phUdng phap nhuom lanh.
- Lo alcohol -acid vdi alcohol 70% va acid chlohydric 3%.
- Lo methylen blue chiia dung dich mau methylen blue.
Bo thuoc nhuom khang acid dUdc chtia trong cac chai sam mau, nap van chat,
giti trong toi d nhiet do phong.

3. PHUONG PHAP THUC HIEN

PhUdng phap trinh bay sau day la phUdng phap nhuom lanh, vdi cac budc nhti sau:

111
 - TrUdc het lam mot phet mong benh pham hay vi khuan tren lam kinh, de
kho tu nhien, roi sau do gan tren lam bang cach hd nhanh qua ngon lua ddn
con hay den bunsen 3 lan.

- Dat lam tren gia nhuom, cho vai giot thuoc nhuom carbolfuchsin phu trum
len phet vi khuan, de yen trong 5 phut. Sau do c im lam len va n ia sach
mau thiia dinh tren lam dudi mot voi nUdc may chay ra't nhe.

- Tay mau bang cach nho tren goc tren cua lam nhuom dung dich alcohol acid
cho den khi giot alcohol rdi khoi lam sach mau. Sau do riia sach alcool acid
khoi lam dudi mot voi nUdc may chay ra't nhe.

- De lai lam tren gia nhuom roi phu vai giot methylen blue len phet nhuom,
de yen 1 phut. Sau do cam lam len va riia sach methylen blue thtia dinh
tren lam dudi mot voi nUdc m ay chay rat nhe.

- Tham kho lam bang cach ep lam givia 2 td giay tham . De kho lam hoan toan
trong khong khi. Sau do nho mot giot dau soi kinh len phet nhuom va quan
sat qua kinh hien vi, trUdc het d vat kinh xlO de tim vung phet benh pham,
sau do xoay sang vat kinh xlOO (vat kinh dau) de quan sat tim trUc khuan
khang acid.

4. DOC KET QUA

Quan sat phet nhuom khang acid qua kinh hien vi, dudi vat kinh dau, trtic
khuan khang acid an mau do canh sen, con vi khuan thudng cung nhu cac nen khac
nhu te bao bieu mo hay bach cau an mau xanh methylen blue.

Khi tra ldi mot ket qua nhuom khang acid, khong dUdc ket luan la dUdng tinh
M. tuberculosis ma chi tra ldi co hien dien trUc khuan khang acid.

Rieng doi vdi mau ddm, can quan sat 3 dong, moi dong quan sat khoang 100
quang trUdng (QT) dau, ghi nhan ket qua theo bang dtidi day de tra ldi tren phieu
tra ldi ket qua:

Bang 15: Cach ghi ket qua quan sat phet nhuom khang acid mot m lu ddm

So true khuan khang acid/so quang trudng Cach ghi ket qua
1-2/300 QT (3 dong) +/-
1-9/100 QT (1 dong) 1+, so trung binh /100 QT
1-9/10 QT 2+, so trung binh /10 QT
1-9/1 QT 3+, so trung binh /1QT
>9/1 QT 4+, >9/QT

112
5. CAC SAI LAM KY THUAT CO THf: GAP TRONG PHUONG PHAP NHUOM
KHANG ACID

Phet nhuom qua day se lam alcohol acid khong the tay het mau
carbolfuchsin, vi v&y nhiing trUc khuan khong khang acid van an mau do canh sen.
Tuy nhien co the phan biet dtidc day khong phai la vi khuan khang acid khi quan
sat mau vung nh&y hay te bao bieu mo hay bach cau chung quanh v&n con mau do
chiing to mau khong bi tay het. Ngoai ra, true khuan khong co hinh dang tieu bieu
cua true khuan khang acid: map, khong manh khanh nhu trUc khuan khang acid
thuc sil.

Mau carbolfuchsin bi can, se dong len phet nhuom lam alcohol acid khong the
tiy het mau va do vay vi khuan thtidng van nhuom mau do canh sen nhu tren. De
tranh tinh trang nay, phai thtidng xuyen loc mau qua giay loc moi khi kiem tra nho
mau carbolfuchsin len mot lam trong thay cd can sau khi do’ bo carbolfuchsin khoi lam.

Thdi gian phu mau carbolfuchin qua ngan khong du 5 phut, hay dung
carbolfuchsin pha cho phUdng phap nhuom nong de nhuom lanh, do vay tnic khu&n
khang acid khong b&t dUdc mau carbolfuchsin.

De tranh cac sai lam tren, nen nhuom kiem tra chat lUdng trudc moi lo thuoc
nhuom mdi pha va dinh ky moi thang mot l&n doi vdi lo thuoc nhuom dang sti dung.

6. CAU HOI ON TAP

a. Cho biet nguyen tac cua phUdng phap nhuom khang acid.

b. Cd m ay phtidng phap tien hanh nhuom khang acid? PhUdng phap nhuom
lanh tien hanh nhu the nao? Ket qua an mau cua vi khu&n sau moi giai
doan nhuom?

c. Cho biet each tra ldi ket qua cua mot phet nhuom khang acid mot m lu ddm.

d. Cho biet cac sai lam thtidng gap trong phUdng phap nhuom khang acid
va hau qua cua cac sai lam nay.

* c 6n o ty hah khoa

Hinh 15.1: Mot bo thuoc nhuom khang

acid hoan chinh bao gom mot chai carbol
fuchsin, mot chai alcohol acid va mot chai
methylen blue

113
 Hinh 15.2: Mot so hinh anh quan sat qua kinh hien vi thay co true khuan khang acid.
(A) Phet nhuom khang acid lam tC/ khom vi khuan M.tuberculosis, (B) Mau ddm tap trung
va thuan nhat; (C) M iu ddm thay nhieu M.avium\ (D) Mau ddm khong tap trung

PHU LUC

San pham lien quan SPUTAPREP Bo thuoc thu thuan nhat, loai trif
tap nhiem va tap trung ddm

Nguyen tac hoat dong

Nhd tac dung cua dung dich kiem cd chiia N -Acetyl-L-Cystein (NALC) ma cac
chat nhay cd trong ddm bi lam tan hoan toan va dong thdi vi khu§n tap nhiem
trong ddm cung bi pH kiem ly giai. Sau do, mau ddm da thuan nhat va loai trii tap
nhiem nay dUdc trung hoa lai bang dem phosphat, va cuoi cung can ddm diidc tap
trung bang ly tam. Can ddm nay se tap trung rat nhieu Mycobacteria de san sang
cho nuoi cay, khao sat triic tiep, hay lam ly trich ADN cho thii nghiem PCR.

Trinh bay

SPUTAPREP gom cac thanh phan sau:

- N-Acetyl-L-Cystein (NALC) bot nguyen chat chiia trong 5 tube Eppendorf
nho, moi tube chiia NALC du dung cho xii ly 2 m lu ddm.
- Dung dich MUCOPREP cd sodium citrat va NaOH, 50ml chiia trong lo n&p
van chat. LUdng dung dich nay du dung cho xii ly 10 mau ddm.

114
 - Dung dich dem phosphat dam dac 10 lan, 5ml, chtia trong tube 5ml, n&p
van chat.
Bo thuoc thii SPUTAPREP chtia dung hay ngay sau khi dung nen dUdc giti
trong tu lanh 4°C cho den ngay het han ghi tren hop (2 nam sau ngay san xuat).

Qui trinh thuan nhat, loai trti ngoai nhiem va tap trung mau ddm vdi SPUTAPREP

- TrUdc het, pha loang dem phosphat 10X vdi 45ml nUdc cat vo trung de co
50ml dem phosphat IX. Giti dem phosphat IX nay trong mot lo sach va trti
trong tu lanh 4°C.
- De xti ly mau ddm, dau tien chu&n bi mot tube ly tam 15ml nap van. Cho
vao tube 10ml MUCOPREP va toan bo bot NALC tti 1 tube Eppendorf. Lac
nhe cho tan NALC trong MUCOPREP, chia 5ml dung dich nay vao mot tube
15ml khac, nhu vay la cd 2 tube, moi tube cd 5ml de xti ly cho hai mau ddm.
Dung dich NALC chi dung trong ngay, khong lUu lai cho ngay sau.
- Cho toi da 5ml ddm vao tube 15ml chua 5ml NALC tren. Lac deu, hay vortex
trong vong vai phut cho tan hoan toan ddm. Sau do cho them 5ml dem
phosphat mot lan. Lac nhe de tron deu.
- Ly tam vdi mot may ly tam ban toe do toi da (4.000 RPM) trong 15 phut. Do
bo phan nUdc ndi, chtia khoang 0j5ml can. Can dUdc dung lam phet nhuom
khang acid, cay vao moi trUdng cay lao (Lownstein Jensen hay Bactec), hay
de ly trich ADN cho PCR lao.

C 6n g ty TNHH u n s u it - thuong m »l - dich vu NAM KHOA

P TAnMUng Q ? TP Hd Chi
Pt*0»w 77*581# 771632® Fa* 84 « 7718329

Hinh 15.3: Hai bo thuoc thir thuan nhat, loai tri/tap nhilm va tap trung ddm thudng dung
va cac thanh phan chinh ben trong. Bo SPUTAPREP-LJ danh cho xii ly mau ddm cay
tren moi trudng Loweinstein Jensen hay lam PCR. Bo SPUTAPREP - OGW
danh cho xir ly mau ddm cay trdn moi trudng Ogawa

115
 BA116. THUT NGHIEM LAL PHAT HllN ENDOTOXIN
■ m

(LIMULUS AMEBOCYTES LYSATE)

MUC TIE U

1. Hieu diidc nguyen tac cua thu! nghiem LAL phat hien endotoxin va cdc chi
dinh dp dung de co the diia vao thanh mot phiidng tien chan doan vi sinh
lam sdng giup chan doan phat hien nhanh nhiem trung trong mot sd
triidng hdp cdn thiet.
2. Thiic hien diidc xet nghiem LAL vdi ket qua chinh xdc khong bi dm tinh
hay diidng tinh gid nhd biet diidc cdc doi hoi dac biet ve vat lieu khi tien
hanh lay, chiia benh pham, cung nhii khi tien hanh lam xet nghiem.
3. Lam diidc thit nghiem LAL dinh tinh vd dinh liidng mot cdch chinh xdc,
khong sd sot.

1. N G U Y E N T A C

Endotoxin la lipopolysaccharid co nguon goc tti vach te bao cua vi khuan

gram [-], co mot tinh chat rat dac trUng la ly giai te bao amebocyt tuan hoan trong
mau con sam (horseshoes = Limulus polyphemus). Dich ly giai amebocytes se bi due
di cung nhu bi dong lai thanh gel do do nhdt bi gia tang va do due cung nhu do dong
ciing se tuy thuoc vao liidng endotoxin cd trong m lu thii. Cd che cua hien tUdng nay
that ra chua ro, cd the giong nhu hien ttidng dong mau b ngtidi trong do co hai qua
trinh xay ra: (1) Endotoxin vdi sil hien dien cua ion calci kich hoat mot he thong
men tien dong giong trypsin. (2) He thong men bi kich hoat nay se lam bien doi
chat gay dong (coagulogen) bang mot qua trinh ly giai protein cd gidi han de san
sinh ra cac protein cd the dong dac dUdc.
De doc dUdc ket qua cua thti nghiem LAL, cd the cd hai phUdng phap:
- PhUdng phap phat hien dung dich thii nghiem bi tang do nhdt, ket qua la do
due tang len va bi dong dac lai (doc bang mat thudng, hay doc bang may do
do due).
- PhUdng phap sinh mau dua tren nguyen tac protein bi ly giai se lam mot
protein khac (cd ma so S -2423) trong dung dich sinh mau sinh ra mau, do
dam nhat cua mau se tuy thuoc vao ham liidng endotoxin thii nghiem va
chinh nhd vay cd the dinh lUdng dtidc endotoxin.

2. C H iO jN H A p dung

Thii nghiem LAL dtidc chi dinh de phat hien va dinh ltidng endotoxin trong
cac mau dich truyen hay thuoc tiem trong san xuat dtidc pham vi cd do nhay cao
hdn thii nghiem phat hien chi nhiet to" thuc hien tren tho.

116
 Thti nghiem LAL cung co the ap dung tai cac phong thi nghiem vi sinh lam
sang de phat hien mot cach gian tiep nhilm trung vi khuan gram [-] trong cac dich
khong tap nhiem cua cd the nhU: dich nao tuy, dich mang phoi, dich mat, dich o
bung. PhUdng phap cd ldi diem la cd ket qua nhanh chi vai gid sau khi giii mau di
lam xet nghiem. Tuy nhien phUdng phap nay khong the thay the nuoi cay vi khong
cho biet vi khuan gay nhiem trung la vi khuan gi va khong the cho biet dUdc ket
qua khang sinh do.
De co the cd ket qua khong bi dudng tinh gia vi mau bi tap nhiem endotoxin
trong lay va chiia m lu, cac vat lieu dung de lay va chiia mau phai that sU tinh sach
va khong cd endotoxin. Tot nhat la dung cac vat lieu bang nhUa polystyren vo trung
bang gamma de chiia mau (lo vo trung nSp do cua cong ty Nam Khoa) va lay mau
bang ong chich thuy tinh vo trung (da xti ly nhu hudng dan d muc tiep theo). De ket
qua khong bi am tinh gia thi cac vat lieu de lay va chtia m lu cung khdng cd dac
tinh hap phu endotoxin, nghia la chung ta phai tranh dung cac vat lieu bang nhUa
polypropylen (nhUa PP).

3. CHUAN BI V A T LIEU

3.1. Vat lieu th u y tinh

Tot nhat la nen dung cac vat lieu thuy tinh mdi. Vdi cac vat lieu thuy tinh
mdi, can boc giay nhom roi hap khti trung 121°C trong 1 gid, sau do say kho 175°C
trong 3 gid.
Vdi cac vat lieu thuy tinh da dung roi, can phai ngam trong dung dich 1% tay
kiem, vi du dung dich 1% E-TOXA-CLEAN® cua Sigma (Catalog No. 210-3), thdi
gian ngam tot nhat la qua dem. Khi ngam cd the dung choi de cha sach. Sau khi
ngam riia 8-10 lan dudi vdi nUdc nong dang chay, sau do rtia 5 lan dudi vdi nUdc cat
hay nUdc khti ion. Cuo'i cung trang lai mot lan vdi nUdc da khti endotoxin. Say kho
dung cu trong tu say, sau do boc giay nhom roi khti trung giong nhu tren.

3.2. Vat lieu nhtia

Chi dung cac vat lieu nhtia vo trung, khong nhiem endotoxin de pha cac thuoc
thti. Chi dung mot lan, khong dung lai. 0 day chung toi khuyen dung cac pipet
Pasteur nhua vd trung, cac d lu type nhtia vd trung dung trong cac phong thi
nghiem thao tac vd trung. Rieng vat lieu dung pha gam chuan endotoxin hay pha
benh pham thi can phai bang polystyren chti khdng the bang polypropylen vi
polypropylen hap phu dtidc endotoxin. O day chung toi khuyen dung cac pipet vd
trung cd chia do, bang polystyren, vo trung (loai dong gdi rieng le, dung mot lan).

4. TIE N HANH TH Lf NGHIEM

7,

0 day trinh bay phUdng phap thti nghiem dung thuoc thti E -toxat cua Sigma.
PhUdng phap nay cd the dung vdi so' ltidng m lu nho.

117
4.1. Pha gam endotoxin chuan

Dung micropipet vdi tip vo trung va khong nhiem endotoxin hut mot the tich
nU6c cat khong nhiem endotoxin (Sigma, catalog No 210-7) cho vao chai chiia
endotoxin dong kho (Sigma, catalog No 210-SE) de dat nong do endotoxin 4000 EU/ml
(n.do goc). Vortex de tron deu trong vong it nhat 2 phut. Sau do vortex them 3 lftn
trong vong 30 phut (moi lan 30 giay, nghi 10 phut). Sau khi da pha. dung dich
endotoxin chuan nay dUdc giut trong tu lanh, ben hdn 2 tu&n neu khdng bi tap
nhilm. Tranh giii trong tu dong. Mdi lan dung thi phai vortex tron deu nhu tren.
Pha gam endotoxin chuan trong tube polystyren vd trung, khdng nhiem
endotoxin va cd nap day. Neu khdng cd tube polystyren thi phai dung cac tube thuy
tinh da khvi trung va khu endotoxin. Pha nhu trinh bay trong bang 16.1.
Cti mdi do pha loang, can phai vortex khoang 30-60 giay trudc khi pha mot do
pha loang ke. Bat cii mot do pha loang nao neu de lau qua 30 phut thi phai vortex
trd lai nhu tren trudc khi dung. Endotoxin vdi nong do tii 400 EU/ml trd len thi cd
the ben den hdn 1 tuan neu giii trong tu lanh (khdng giut dong da).

Bang 16.1: Cach pha dung dich endotoxin chuan

Ong so The tich dung dich endotoxin dUdc lay Nude khdng Nong do
endotoxin endotoxin
1 0,2 ml n.do goc 1,8 ml 400 EU
2 0,2 ml tUong 1 1,8 ml 40 EU
2 0,2 ml tir ong 2 1,8 ml 4 EU
2 0,3 ml tUong 3 2,1 ml 0,5 EU
2 1 ml tUong 4 1 ml 0,25 EU
2 1 ml tUong 5 1 ml 0,125 EU
2 1 ml ti/ ong 6 1 ml 0,06 EU
2 1 ml ti/ ong 7 1 ml 0,03 EU
3 1 ml ti/ ong 8 1 ml 0,015 EU

4.2.Tien hanh th u nghiem

TrUdc het mdi m lu thii nghiem phai dUdc chinh pH dat tii 6-8 (thich hdp nhat
la 6,8 den 7.,). Chinh pH vdi dung dich HC1 0,1M khdng lln endotoxin va dung giay
thii pH chii khdng dung pH ke.

Vd nhe chai chiia E -TOXATE (Sigma, catalog No 210-20) xuong mat ban de E -
TOXATE khdng dinh tren nap cao su. Dung micropipet vdi tip vd trung va khdng
nhilm endotoxin hut 2 ml nUdc cat khdng nhilm endotoxin cho vao chai. Khong
dUdc l^c manh, chi lan tron chai de hoa tan. Ngay sau khi hoa tan, cho lo vao trong
da bao. Phan phoi vao cac tube thuy tinh vd trung da khii endotoxin va cd day
mieng tube bang giay nhdm. moi tube 0,1 ml. Cac tube E -TOXATE nay phai luon
giii trong da bao. Dung dich E -TOXATE da pha xong nen dUdc dung ngay trong
ngay. Cac tube dung dich E -TOXATE chua dung thi phai giii trong tu dong va khi

118
do do nhay se giam di ra't it. Tranh ra dong nhieu lan vi nhu vay thi do nhay se
giam di ra't nhanh.
PhUOng phap dinh lUOng: do la trUdng hdp kiem tra chat lUdng phat hien
endotoxin trong thuoc tiem, dich truyen; moi mku thii nghiem rieng le phai dung 6
tube E -TOXATE danh so' tti 1 den 6 va cho vao tube mau thti, chting [-], chting tic
che (l/C), cung nhu cac nong do endotoxin chuan theo htidng d in trinh bay trong
bang 7.
PhUdng phap dinh tinh: do la trUdng hdp phat hien endotoxin trong cac benh
pham nhu mau, huyet thanh, dich nao tuy, dich mat... de tim bang chting sdm phat
nhien cd vi khuan gram [-] trong mau. Trong muc dich nay, cac mau thti phai lay vo
trung bang cac dung cu khdng chtia endotoxin va khdng cd tinh cha't ha'p phu
endotoxin. Moi mau thti nghiem rieng le phai dung 4 tube E -TOXATE danh so' tti 1
den 4 va cho vao tube mau thti, chting [-], chting [+], chting tic che (L/C) theo hudng
dan trinh bay bang 16.2.

Sau do, day mieng tube lai bang giay nhdm. U ngay cac tube trong may cach
thuy 37°C, khdng dUdc dao dong ong nghiem trong suo't qua trinh u vi nhu vay du
thti nghiem [+] thi dung dich trong tube cung khdng the dong lai dUdc ma chi tang
do nhdt ma thdi.

Bang 16.2: Cach chuan bi cac tube thti nghiem trong phi/ong phap djnh lUdng

Tube De phat hien Mau thur Nude khong Endotoxin chuan

endotoxin
1 Endotoxin 0 ml 0,1 ml dung dich 0,03 EU /ml
0,03 EU/ml \
2 Endotoxin 0,06 EU/ml 0 ml 0,1 ml dung dich 0,06 EU /ml
3 Endotoxin 0,125 EU/ml 0 ml 0,1 ml dung dich 0,125 EU /ml
4 Chufng [-] 0 ml 0,1 ml 0,0 ml
5 Chifng UC 0,1 ml 0,0 ml 0,01ml dung djch 4EU/ml
6 Endotoxin 0,1 ml 0,0 ml ■ 0,0 ml
mau thif

Bang 16.3: Cach chuan bi cac tube thif nghiem trong phUdng phap djnh tinh
Tube De phat hien Mau thur v Ni/dc khong endotoxin Endotoxin chuan
1 ChCfng [+] 0 ml 0,1 ml dung djch 4 EU /ml
2 Chifng [-] 0 ml 0,1 ml 0,0 ml
3 Chifng uC 0,1 ml 0,0 ml 0,01ml dung djch 4 EU /ml
4 Endotoxin 0,1 ml 0,0 ml 0,0 ml
mau thif \

5. DOC KET QUA

Sau khi u 37°C trong mot gid, can than lay tting tube ra khoi may cach thuy,
nghieng nhe tti tti den mot goc 180°, vtia nghieng vtia quan sat gel neu da hinh

119
thanh trong ong nghiem. Ket qua dUdc doc la [+] khi dung dich trong ong hoan to&n
thanh gel ciing cho phep nghieng hoan toan ong nghiem ma gel khong vd ra. Neu
khong co gel cting thi ket qua phai doc la k [-]. Luu y la dong tac nghieng ong thu
nghiem de doc ket qua chi lam mot l&n vi neu lam lai thi gel se bi v3 ngay.
Doi vdi phUOng phap dinh lUOng, ket qua dinh lUdng bien lu&n nhu vi du
trinh bay trong bang 16.4.
Doi vdi phUOng phap dinh tinh, ket qua m&u thii co endotoxin khi chiing [+]
cho ket qua [+], chiing [-] cho ket qua [-], khong co tic che (ket qua chiing iic che [+])
va m lu thii cho ket qua [+]. M lu thii khong co endotoxin khi chiing [+] cho ket qua
[+], chiing [-] cho ket qua [-], khong co iic che (chiing iic che [+]) va mliu thii cho k£t
qua [-]. Ket qua khong doc dUdc khi co lie che (tiic la ket qua chiing iic che la [-]).
M&u co ket qua [+] la mau co nhiem vi khu&n gram [-] vi endotoxin la thanh ph£n
lipopolysaccharid cua vach vi khuan gram [-].

Bang 16.4: Cach bien luan ket qua trong phi/dng phap dinh li/ong

Tube Ket qua Tube Ket qua

1 [-1 1 [-1
2 M 2 M
3 M 3 M
4 (-1 4 [-1
5 M 5 H
6 M 6 [-1
Mau co chifa Mau co chCra chat
endotoxin > 0,06 IU/ml Crc che thif nghiem

6. CAU HOI O N T A P

a. Cho biet nguyen t&c cua thti nghiem LAL.

b. Cho biet cac ap dung cua thu nghiem LAL, dac biet la trong xet nghiem
vi sinh lam sang.
c. De co the co ket qua chinh xac, hay cho biet cac vat lieu dung de lay va
chiia benh pham phai dap iing dUdc cac dieu kien gi? Va muon dap iing
cac dieu kien nay thi phai dung cac vat lieu gi?
d. Cac vat lieu nhua va thuy tinh dung trong thi nghiem LAL phai dap iing
dUdc cac dieu kien gi? Va muon dap iing c.ac dieu kien nay thi phai dung
cac vat lieu gi?
e. Hay trinh bay cach lam mot thi nghiem LAL va cach doc - phan tich ket qua.

120
 BA117. KIEM TRA VI SINH KH0NG KHI

MUC TIE U

1. Diia diidc viec kiem tra vi sinh khdng khi vdo thanh mot trong cdc qui
trinh giam sat nhiem trung tai benh vien vi biet ro muc dich vd chi dinh
cua viec kiem tra khdng khi.
2. Liia chon diidc phiidng phap kiem tra vi sinh khdng khi ndo tai benh
vien cua minh cho phu hdp vdi dieu kien tai chinh cung nhii phiidng tien
tai phong thi nghiem nhd biet diidc nhieu phiidng phap kiem tra vi sinh
khdng khi khac nhau.

3. Ap dung diidc phiidng phap lang bui cua Koch dung trong kiem tra vi
sinh khdng khi tai benh vien cua minh vi day la phiidng phap phu hdp
nhat hien nay.
4. Diia tren ket qua kiem tra vi sinh khdng khi, danh gid chat liidng khdng
khi phong mo vd danh gid chat liidng khdng khi ndi chung, tuy thuoc
vdo cdc chi dinh thiic hien kiem tra khdng khi tai khoa phong ndo.

1. MUC DICH

Dung kiem tra chat lUdng khong khi phong mo cua cac benh vien. Cung co the
dung kiem tra chat liidng khong khi tai cac benh phong trong cac chUdng trinh
kiem soat nhiem trung tai benh vien.

2. C AC P H U O N G P H A P KIEM T R A VI SINH K H O N G KHI

Cd nhieu phUdng phap:

- PhUdng phap hut bui qua mang loc bang gelatin (dung mang loc cua hang
Sartorius), sau do dat mang loc nay len mat mdi trUdng, u trong dieu kien
thich hdp (37°C/khi trUdng thudng hay khi trUdng C 0 2 neu la thach mau/24
gid). Sau do doc ket qua dem so liidng khom vi khuan moc de tinh ra so'
liidng vi sinh trong the thich khdng khi dUdc hut qua mang loc.
- PhUdng phap hut bui trUc tiep len mat hop thach mdi trUdng dinh dudng dat
trong may hut (dung may MAS100 cua Merck), hop thach sau do dUdc lay ra
khoi may va u trong dieu kien thich hdp (37°C/khi trUdng thudng hay khi
trUdng C 0 2 neu la thach mau/24 gid). Doc ket qua dem so' liidng khom vi
khuan moc de tinh ra so' liidng vi sinh trong the thich khong khi dUdc hut
qua may.

121
 - PhUdng phap lang bui cua Koch dua tren nguyen tic de bui lang tren mat cac
hop thach dinh dudng trong mot thdi gian nha't dinh roi sau do u cac hop
thach trong dieu kien thich hdp (37°C/khi trudng thUdng hay khi trudng C 0 2
neu la thach mau /24 gid). Doc ket qua dem so' ltfdng khom vi khultn moc de
tinh ra so' liidng vi sinh trong the thich khdng khi qua mot cong thiic cho sin.

3. PHL/ONG P H AP L A N G BUI C U A K O C H KI^M T R A VI SINH K H O N G KHf

3.1. PhUdng phap thuc hien

Day la phUdng phap dUdc nhieu benh vien sti dung de kiem tra vi sinh khdng
khi phong md. ThUdng thi mdi phong md se dUdc kiem tra nam cum, mot cum d
giiia va bon cum bon goc. Moi cum dat 3 hop thach dinh dudng loai dudng kinh
90mm. Ba loai hop thach dinh dudng do la: mot hop thach mau (BA) cho toan bo vi
khuan hieu khi, mot hop thach Mac - Conkey (MC) cho trUc khu&n gram [-] de moc
va mot hop thach Sabouraud (SA) cho vi nam. Tat ca cac hop thach se dUdc md nap
lan lUdt sau khi dUdc dat day du tai 5 vi tri cua phong md va de yen trong phong
trong 10 phut; sau do dUdc day nap lai cung lan lUdt, hop nao md nap trudc thi dUdc
day nap trUdc. Tat ca dUdc dem ve phong thi nghiem va u d cac dieu kien thich hdp
(37°C/khi trUdng thudng cho cac hop thach MC va khi trUdng C 0 2 cho cac hop thach
BA, rieng hop thach SA thi u d nhiet do phong thi nghiem). Thdi gian u la 24 gid,
rieng hop thach SA thi u 48 gid mdi doc ket qua. LUu y la khi thuc hien dat cac hop
thach kiem tra vi sinh khdng khi trong phong md thi cac nhan vien thuc hien viec
nay phai riia tay va mac do giong het phau thuat vien de tranh dua vi trung tap
nhiem vao phong md. Thdi diem thuc hien kiem tra tot nha't la vao buoi sang sau
khi van hanh he thong den cUc tim va cac may loc khdng khi khti trung phong md
va trudc khi thuc hien trUdng hdp md dau tien.

3.2. Phirong phap tinh toan ket qua

Dem so' khom vi khuan moc tren mdi hop thach. Tong so' vi khuan /m3 kh6ng
khi tai mdi cum dUdc xac dinh theo cong thiic sau:

v _ A x 100x100
A— *
Sxk

Vdi: X la tong so' vi khuan/m3 khdng khi tai cum khao sat (CFU/m3); A la tong
so' khom vi khuan moc tren ba loai moi trUdng; S la dien tich ctia hop petri chiia
mdi trUdng, dUdc xac dinh b in g cong thiic S = TtR2 (R tinh bang cm) = 3 .1 4 1 6 x 9 2;k
la he sd va phu thuoc vao thdi gian dat hop thach (la 1 neu dat 5 phut, la 2 neu dat
10 phut, la 3 neu dat 15 phut).

Vi du neu tren BA chung ta dem cd 32 khom. tren MC dem cd 2 khom. tren

SA dem co 10 khom; vay thi A se la 32 + 2 + 12 = 46. Neu thdi gian dat hop thach la
10. va hop thach co dudng kinh 90mm (9cm) thi X se la:

122
 4 6 x 100x100
X = -------------------------- = 3615CFU/m
63,62 x 2

So' liidng vi sinh cd trong lm 3 khdng khi trong phong diidc tinh bang so trung
binh cong cua so liidng vi sinh cd trong lm 3 cua 5 cum khao sat. Can cii vao ket qua
so' vi sinh cd trong lm 3 khdng khi cua phong, cd the danh gia chat liidng khdng khi
dua theo tieu chu^n cua V. Omelanskii (Nga) sau day:

Bang 17: Tieu chuan danh gia chat liidng khdng khi phong md theo V. Omelanski (Nga)

So ltfdng vi sinh/m3 khong khi Chat ltfdng khdng khi

<312 (<5 khuan lac 5 13 dTa thach) Tot
312 - 1250 (5 - 20 khuan lac/3 dTa thach) Kha
1250 -> 1562 (20 - 25 khuan lac/3 dTa thach) Vtfa
> 1562 (>25 khuan lac/3 dTa thach) Kem

4. TIEU CH U AN VI SINH K H O N G KH(

Cho den hien nay chung ta chua cd tieu chuan qui dinh chat lUdng khdng khi
diia theo liidng vi sinh vat trong lm 1khdng khi. Sau day la vai tieu chuan dung de
tham khao:
- Theo Preobran (Phap), khdng khi sach khi cd so' liidng vi khu&n
< 1000CFU/m3
- Theo Ginokova (Nga), khdng khi sach khi chi cd 5 - 10 khom moc tren mat
thach de trong 10 phut; vCfa khi cd 20 - 25 khom; xau khi cd >25 khom.
- Theo Romanovic, trong cd s6 san xuat thiic pham, khdng khi rat tot khi chi
cd < 20 khom moc tren mat thach de trong 10 phut va khdng cd khum nafn
moc; tot khi cd 20 - 50 khom va 2 khom nam moc; viia khi cd 50 - 70 khom
va 5 khom nam moc; xau khi cd >70 khom vi khuan va >5 khom nam moc.

5. CAU HOI O N T A P

a. Cho biet cac phUdng phap cd the dung de kiem tra khdng khi.
b. Trinh bay phUdng phap l&ng bui de kiem tra khdng khi phong mo va tieu
chuan de danh gia chat liidng khdng khi phong mo.
c. Cho biet cac tieu chu&n hien nay dung de danh gia chat liidng khdng khi.

123
 BA118. THUT NGHIEM HUYET THANH HOC
■ a

MUC TIE U

1. Biet diiac muc dich cua cdc thii nghiem huyet thanh hoc, nha do phan tich
diiac y nghla hiiu dung lam sdng cua cdc thit nghiem huyet thanh hoc.
2. Biet diiac cdc cdch bien luan ket qua cdc xet nghiem huyet thanh hoc,
nhd do co the them cdc binh luan di kem ket qua xet nghiem de lam sdng
phan tich vd sii dung dung ket qua xet nghiem trong chan doan phat
hien tac nhan gdy benh.

1. MUC DICH C U A THLT NGHIEM H U Y E T TH A N H H OC

Thii nghiem huyet thanh hoc co muc dich tim khang the dac hieu mot loai
khang nguyen cua vi sinh vat gay benh co trong huyet thanh benh nhan, nhd do cd
the gian tiep xac dinh dUdc tac nhan vi sinh vat gay benh. Vi du: thti nghiem
Widal chan doan benh thudng han, thii nghiem tim HBsAg de xac dinh benh nhan
cd khang the dac hieu viem gan B.

2. BIEN LU A N K E T Q U A M O T T H lf NGHIEM H U Y E T TH A N H H O C

Vi muc dich la tim khang the dac hieu khang nguyen vi sinh vat nen thii
nghiem huyet thanh hoc khdng phai la thii nghiem trUc tiep phat hien tac nhan vi
sinh vat gay benh. Do vay, bien luan thii nghiem huyet thanh hoc phai dua vao gia
tri dinh liidng cua ket qua thti nghiem.
Ket qua dinh lUdng cua mot thti nghiem huyet thanh hoc thudng dUdc hien
thi bang hieu gia khang the dac hieu, tiic la do pha loang cao nhat ctia huyet thanh
benh nhan ma thii nghiem van cdn dudng tinh (vi du trong thti nghiem Widal, ket
qua cho biet TO la 1/100, cd nghla la huyet thanh benh nhan pha loang 1/100 van
cdn tu dUdc vdi khang nguyen 0 cua S. typhi thti nghiem). Hien nay, vdi mot so bo
thti nghiem ELISA, ket qua dinh liidng thudng dUdc hien thi b ln g gia tri OD neu
thuc hien thti nghiem tren mot do pha loang huyet thanh benh nhan.
Cd nhieu cach bien luan ket qua ctia thti nghiem huyet thanh hoc dua vao gia
tri dinh liidng de nhd do cd the phat hien dUdc tac nhan vi sinh vat gay benh:

2.1. Dua va o huyet thanh kep

- Thti nghiem tren huyet thanh lay 2 lan cach nhau 7-14 ngay.
- Ket qua [+] khi hieu gia huyet thanh lan 2 cao > 4 lan hieu gia huyet thanh
lay lan 1. Doi vdi cac ket qua dinh lUdng hien thi bang gia tri OD thi cac nha
san xuat thudng dua ra mot cong thiic tinh toan de cho biet nhu the nao la
cd sU gia tang cd y nghla gia tri dinh liidng ctia huyet thanh lan hai so vdi
huyet thanh lan mot.

124
 - Phiidng phap huyet thanh kep thudng it htifu dung cho lam sang do mat thdi
gian tinh, nhung rat cd y nghla de chan doan xac dinh.

2.2. Dira vao hieu gia khang the dac hieu thuoc ldp IgM

- Cd the chi can thii nghiem tren huyet thanh lay mot lan va xac dinh khang
the dac hieu la thuoc ldp IgM, la ldp globulin mien dich chi xuat hien trong
giai doan sdm cua benh, ton tai khi cd hien dien tac nhan vi sinh vat gay
benh trong cd the va bien ma't nhanh sau khi het benh - khdng cdn hien dien
vi sinh vat gay benh trong cd the.
- Ket qua dUdc coi la [+], ttic la cd sii hien dien tac nhan vi sinh vat gay benh
trong cd the khi hieu gia khang the hay gia tri OD cua ket qua thu nghiem
cao hdn hieu gia hay gia tri OD cat, ttic la hieu gia hay gia tri OD trung
binh cua ngUdi binh thudng.
- Xet nghiem phat hien khang the dac hieu thuoc ldp IgM rat hutu dung cho
lam sang do khdng mat thdi gian tinh va rat cd y nghla de chan doan phat
hien tac nhan vi sinh vat gay benh ma khdng can phai thti nghiem tren
huyet thanh lay lan hai. Tuy nhien, chi cd cac thti nghiem mien dich nhu
ELISA, IFA (mi§n dich huynh quang), RIA (m iln dich phong xa) mdi cd the
xac dinh dUdc khang the dac hieu la thuoc ldp nao.

2.3. Dua vao hieu gia khang the dac hieu so vdi gia trj cat

- Phai xac dinh hieu gia hay gia tri OD cat ctia quan the bang mot dieu tra
huyet thanh dich te hoc.
- So sanh hieu gia hay gia tri OD ctia huyet thanh thti nghiem vdi hieu gia
hay gia tri OD cat cua quan the; neu hieu gia huyet thanh thti nghiem cao
> 4 lan hieu gia cat, hay OD huyet thanh thti nghiem cao hdn k x OD gia tri
cat (k la tri so do nha san xuat bo thti nghiem qui dinh) thi cd the cho ket
qua [+] ttic la cho chan doan xac dinh tac nhan vi sinh vat gay benh.
- Ket qua nay kha hutu dung cho lam sang do khdng mat thdi gian tinh, tuy
nhien chi cd y nghla vtia phai de chan doan xac dinh.

3. CAU HOI ON TAP

a. Cho biet muc dich ctia thti nghiem huyet thanh hoc.
b. Thti nghiem huyet thanh hoc tren huyet thanh kep la nhu the nao? Tai
sao phai lam thti nghiem huyet thanh hoc tren huyet thanh kep? Lam
the nao de bien luan ket qua?
c. Thti nghiem huyet thanh hoc tim khang the thuoc ldp IgM dac hieu
khang nguyen vi sinh vat gay benh cd y nghla nhu the nao trong chan
doan phat hien tac nhan gay benh?
d. Cd khi nao chung ta chi can diia vao ket qua ctia thti nghiem huyet
thanh hoc tren huyet thanh benh nhan chi lay cd mot lan ma van cd the
chan doan dUdc tac nhan gay benh, khong can phai xac dinh khang the
dac hieu tim dUdc la thuoc ldp IgM?

125
 BA119. THUTNGHIEM HOA NIllN DICH HOC
■ m m

MUC TIEU

1. Biet diidc muc dich cua cdc thii nghiem hod mien dich hoc, nhd do phan
tich diidc y nghla hiiu dung lam sdng cua cdc thii nghiem hod mien dich
hoc.
2. Biet diidc cdc cdch bien luan ket qua cdc xet nghiem hod mien dich hoc,
nhd do co the them cdc binh luan di kem ket qua xet nghiem de lam sdng
phan tich vd sii dung dung ket qua xet nghiem trong chan doan phdt
hien tdc nhan gdy benh.

1. MUC DICH THIT NGHIEM HOA MI§N D|CH HOC

Muc dich cac thu nghiem hoa mien dich hoc la phat hien khang nguyen cua
tac nhan vi sinh vat trong benh pham, nhd do co the chan doan xac dinh dUdc mot
cach tUOng doi trUc tiep tac nhan vi sinh vat gay benh. Vi du thu nghiem phat hien
khang nguyen hoa tan cac vi khuan thudng gay viem mang nao mu bang bo thuoc
thii tu latex co ten Meningitex la mot thu nghiem hoa mien dich; thti nghiem phat
hien khang nguyen be mat virus viem gan B (tim HBsAg) trong huyet thanh de
phat hien tinh trang nhiem viem gan sieu vi B la thti nghiem hoa mien dich hoc
mac du benh phaim de thti la huyet thanh benh nhan; hay thti nghiem tu tren kinh
vi khuan Shigella spp. phan lap dUdc vdi khang huyet thanh dac hieu de dinh
nhom A. B, C, va D cung la thti nghiem hoa mien dich hoc; hay thti nghiem mien
dich huynh quang trUc tiep phat hien Chlamydia trachomatis trong phet co tti cung
la thti nghiem hoa mien dich.

2. CAC LOAI THI/ NGHIEM HOA M liN DjCH

Tuy thuoc vao khang nguyen dUdc phat hien, co hai loai thti nghiem hoa m iln
dich. do la:

2.1. Phat hien khang nguyen hoa tan

Thudng, cac thti nghiem nhu tu latex, ELISA, CCIE (Counter Current
Immuno-Electrophoresis = dien di mien dich doi lUu), la cac thti nghiem hay dUdc
dung de phat hien khang nguyen hoa tan co trong benh pham.
Doi vdi tu latex, thudng cac nha nghien ctiu hay san xuat gan khang the dac
hieu khang nguyen len tren cac hat latex bang polystyren, nhd vay khi gap khang
nguyen dac hieu thi cac hat latex se tu lai va ket qua se thay dUdc rat de dang bang
mat thudng. Doi vdi thti nghiem ELISA, cac nha nghien ciiu hay san xuat thudng
phu khang the dac hieu khang nguyen tren cac gieng ELISA bang polystyren nhd

126
vay se tom bat dUdc khang nguyen trong benh pham (goi la ky thuat ELISA tom
bat khang nguyen). Doi vdi thii nghiem CCIE, cac nha nghien ctiu hay san xuat
thudng cho khang the dac hieu khang nguyen vao mot gieng va benh pham vao
mot gieng doi dien tren thach dien di, sau do dien di de khang the di chuyen ve
phia cUc am hudng ve gieng chtia benh pham va khang nguyen trong benh pham
se di chuyen ve phia cUc dudng hudng ve gieng chuta khang the; nhd vay sU gap gd
khang nguyen - khang the se hinh thanh mdt vach ket ttia thay dUdc tren thach
giiia hai gieng.
Thti nghiem phat hien khang nguyen hoa tan cac vi khuan thudng gay viem
mang nao mu nhu N. meningitidis, H. influenzae, vk S. pneumoniae bang phan iing
tu latex vdi thuoc thti MENINGITEX; hay thti nghiem phat hien Rotavirus trong
phan bang ky thuat CCIE; hoac thti nghiem phat hien HBsAg bang ky thuat
ELISA tom bat khang nguyen la nhiing minh hoa ba phUdng phap noi tren dung
phat hien khang nguyen hoa tan.

2.2. Phat hien khang nguyen hiiru hinh

ThUdng, cac thti nghiem nhu mien dich huynh quang trUc tiep (Direct-
Immuno-Fluorescent), hay nhuom khang the gan men peroxidase (Immuno-
Peroxidase-Staining), la cac thti nghiem hay dUdc dung nhat.
Trong thti nghiem mien dich huynh quang, benh pham dUdc phet tren lam
kinh hien vi, sau khi de khd dUdc co" dinh bang aceton. Nho khang the dac hieu
khang nguyen vi sinh vat muon tim va da gan huynh quang (thUdng la FITC =
Fluorescent Thio Cyanat) len phet benh pham nay, sau do soi dudi kinh hien vi
huynh quang. Neu trong benh pham cd vi sinh vat muon tim thi vi sinh vat nay se
bi bam khang the dac hieu gan huynh quang, do vay se bi phat huynh quang va
ngudi lam xet nghiem se de dang thay dUdc hinh anh cac vi sinh vat bi phat huynh
quang nay tren met nen toi khong phat huynh quang.
Thti nghiem nhuom khang the gin men cung cd nhiing budc thuc hien giong
nhu nhuom khang the huynh quang, nhung khdng can phai quan sat phet benh
pham qua kinh huynh quang ma chi can qua kinh hien vi quang hoc thudng. Neu
phet benh pham cd chtia khang nguyen vi sinh vat muon tim thi se bi khdng the gan
men bam vao va se bi sinh mau ket ttia do tac dong ctia men tren dai chat sinh mau.
Thti nghiem nhuom mien dich huynh quang trUc tiep phat hien vi khuan
Chlamydia trachomatis trong cdc quet cd tti cung, phat hien cac vi khuan khdng
dien hinh trong mdu ddm la nhiing thti nghiem hien nay thudng dUdc dung de chan
doan phat hien cac tac nhan gay benh ndi tren vi phUdng phap nuoi cay khd cd the
thuc hien dUdc tai cac phdng thi nghiem lam sang.

3. BIEN LUAN KET QUA MOT THLf NGHIEM HOA MI§N DjCH HOC

Vi muc dich la phat hien cd hay khdng khang nguyen vi sinh vat gay benh
trong benh pham nen thti nghiem hoa mien dich hoc thudng chi can la thti nghiem
dinh tinh, xac dinh cd (dUdng) hay khdng cd (am), ma khong can gia tri dinh liidng.
Vi vay khi tra ldi ket qua thti nghiem hoa mien dich, ngUdi lam xet nghiem khdng

127
can thiet phai tra ldi con so' dinh ltfdng. Mac du hien nay vdi nhieu thu nghiem hoa
mien dich dtfa tren phan ting ELISA, ket qua phai doc bang may quang pho vdi gia
tri OD, nhung gia tri OD nay chi can cho ngtfdi lim xet nghiem de so vdi OD nen
nhd do biet dtfdc ket qua mau benh ph&m la [+] hay [-]. Khi tra ldi ket qua cho lam
sang, khong c&n thiet phai dua ket qua gia tri OD cua thti nghiem vi neu lam v&y
co doi khi lam roi tri lam sang hay co khi lam sang hieu nham tri so OD cang ldn
thi nhiem trung cang nang so vdi ket qua co tri so' OD nho hdn.

4. CAU HOI ON TAP

a. Cho biet muc dich ctia thti nghiem hoa mi§n dich hoc.
b. Cac thti nghiem nao thtfdng dtfdc dung de phat hien khang nguyen hoa
tan va khang nguyen hutu hinh cac vi sinh vat gay benh co trong benh
pham? Noi qua nguyen tac ctia cac thti nghiem nay va cho cac vi du
minh hoa.
c. Hay cho biet co khi nao thti nghiem hoa mien dich cho chung ta ket qua
co gia tri dinh ltfdng khong? Neu co, ngtfdi lam xet nghiem co can thiet
phai tra ldi cho lam sang ket qua thti nghiem hoa mien dich hoc vdi ket
qua dinh ltfdng di kem theo hay khong? Tai sao can? Tai sao khong din?

Hinh 19: Quan sat qua kinh hien vi huynh quang phet quet co tti cung nhuom khang the huynh
quang d3c hieu C.trachomatis (trai) va phet ddm nhuom khang the huynh quang d§c hieu
Legionella pneumophila (phai)

128
PHU LUC

San pham lien quan: TYPHELISAM - Bo thuoc thur LISA phat hien
va djnh luong khang the IgM dac hieu Salmonella typhi de chan
doan phat hien sdm thuong han

Nguyen tac hoat dong

Bo thuoc thii hoat dong bang nguyen tac cua phan iing ELISA gian tiep phat
hien khang the: khang nguyen S. typhi da g in trong cac gieng nhua polystyren tom
bat cac khang the dac hieu co trong huyet thanh ngUdi va sau do phat hien khang
the dac hieu bam tren khang nguyen va xac dinh khang the nay thuoc ldp IgM
bang cong hdp khang IgM ngtidi danh dau men peroxidase. San pham nay la ket
qua nghien ctiu cua CN. Pham Thai Binh dudi sU hudng dan cua TS. BS. Pham
Hung Van vdi sti tai trd cua chUdng trinh vUdn Udm cua Thanh Doan va cua cong ty
Nam Khoa.

Trinh bay

Co cac thanh phan sau day, tat ca giti d 4°C

- R l: ban nhua polystyren lam ELISA, vdi 12 thanh rdi, moi thanh cd 8 gieng,
cac gieng nay da dUdc phu khang nguyen trich tti S. typhi.
- R2: dung dich riia PBS -T, 100ml x 4.
- R3: dung dich pha loang PBS -T-BSA, 120ml x 1.
- R4: cong hdp la khang the khang IgM ngUdi g in men peroxidase (HRPO), 12ml.
- R5a: cd chat TMB, 0,65ml x 1 tube
- R5b: peroxid UPR, 0,65ml x 1
- R5c: dung dich dem cd chat, 12ml x 1.
- R6: dung dich ngting phan ting, 12ml x 1
- R7a: huyet thanh chting (+), 0,1ml x 1
- R7b: huyet thanh chting (-), 0,1ml x 1
- R7c: huyet thanh chuan, 0,1ml x 1

Tien hanh phan umg

- Pha loang huyet thanh chting [+], chting [-], chuan va cac huyet thanh thu
nghiem 1/160 trong cac tube eppendorf (10pl huyet thanh cho vao 1590pl
R2). Cho vao cac gieng, moi gieng lOOpl cac huyet thanh da pha loang nay.
U 37°C trong 30 phut.

129
 - Riia gieng 4 lan vdi R2, ngam 1 phut d lan rtia cuoi. LUu y phai giu sach sau
moi lan do bo dung dich riia.
- Cho vao cac gieng lOOpl cong hdp, gai nhe day gieng de tron deu. U 37°C
trong 30 phut.
- Riia gieng 4 lan vdi R2, ngam 1 phut 6 lan riia cuoi. Luu y phai giu sach sau
moi lan do bo dung dich riia.
- Pha 1ml cd chat trong mot tube Eppendorf bang cach hut 0.9ml R5c cho v&o
tube roi them va 50pl R5a va 50pl R5c. Lac tron that deu. Dung dich n&y chi
pha ngay sau khi hoan tat budc 4.
- Cho vao cac gieng, moi gieng lOOpl cd chat vtia chuan bi xong. Giti trong toi
trong 15 phut. Doc ket qua bang may doc ELISA d budc song 450nm.

Doc ket qua

- Xac dinh gia tri OD450 cua diem cat bang cach lay gia tri OD450 cua chting
chuan do dUdc (OF) nhan vdi gia tri OD450 cua chting chuan (CF) ghi trong
tai lieu hudng d in kem theo bo thti nghiem.
- Xac dinh cac chi so khang the cua huyet thanh chting [+] chting [-] va cac
huyet thanh thti nghiem bang cach la'y gia tri ODvso do dUdc tren cac m lu
chia cho gia tri OD450 cua diem cat.
- Bien luan nhti sau:
+ Chi so' khang the cua huyet thanh chting [+] phai p > 1,05, cua huyet
thanh chting [-] phai p < 0,95.
+ Cac mau huyet thanh thti cd chi so' khang the > 1,05 la dudng tinh,
< 0,95 la am tinh.
+ Cac m lu huyet thanh thti co chi so khang the tti 0,95 den 1,05 thi co the
dudng tinh va cung cd the la am tinh. Can phai lam lai tren mau la'y sau
do mot tuan mdi quyet dinh dtidc.

130
 BAI 20. DANH GlA MOT THUT NGHIEM DUNG DE
PHAT HIEN MOT TAC NHAN GAY BENH
m m m

MUC TI§U

1. Dinh nghla diidc chuan vang de co the dung lam chuan danh gid mot xet
nghiem dang dung hay sap trien khai trong phong thi nghiem.
2. Tinh diicfc do nhay cam (Se) do dac hieu (Sp) cua mot xet nghiem de co
the tinh toan, phan tich vd biet diidc y nghla cua hai tri so ndy khi danh
gid mot xet nghiem dang dung hay sap trien khai dung.
3. Tinh diidc gid tri tien doan diidng (PPV), gid tri tien doan dm (NPV) de
co the giup cdc nhd lam sdng danh gid diidc sit hiiu dung lam sdng cua
ket qua xet nghiem vd dong thdi cho ditdc chi dinh xet nghiem co hieu
qua nhat trong chan doan benh.
4. Tinh diidc he so kappa de co the itng dung trong danh gid do phu hdp
cua hai thii nghiem khac nhau dung cho cung mot muc dich phdt hien
tdc nhan vi sinh vat gdy benh.
5. Tinh diidc he so bien thien (CV) de co the phan tich diidc do lap lai cua
mot xet nghiem dang dung hay sap trien khai trong phong thi nghiem.

1. CHUAN VANG LA Gi?

De danh gia mot thti nghiem dtidc nghien ctiu dung phat hien mot tac nhan
gay benh, ngUdi ta phai so sanh thti nghiem nay vdi chuan vang (gold standard).
Chuan vang la tieu chuan dung de xac dinh benh that sU va khong benh that sti.
Chugtn vang cd the chi can dua vao mot xet nghiem, mot dau chting lam sang, hay
phai dtia vao sti ket hdp nhieu dau hieu xet nghiem va dau chting lam sang; nhung
quan trong nhat la phai dUdc nhieu ngtidi thtia nhan tren y van.

2. DO NHAY CAM VA DO DAC HIEU CUA THlf NGHIEM

Doi vdi cac nha nghien ctiu san xuat bo thti nghiem hay doi vdi cac phong thi
nghiem ap dung bo thti nghiem, co hai thong so' can phai dUdc danh gia, do la do
nhay cam va do dac hieu cua thti nghiem.
• Do nhay cam (sensitivity = Se) cua thti nghiem la ty le phan tram cho
ket qua [+] trong so' nhting m lu benh that sti.
• Do dac hieu (specificity = Sp) cua thti nghiem la ty le phan tram cho ket
qua [-] trong so' nhting m lu khong benh that sti.

131
 Vi du: De nghien ctiu Se va Sp cua thii nghiem ELISA phat hien khang the
dac hieu chan doan lao phoi, nha nghien ciiu thii tren 100 m lu huyet thanh benh
nhan bi lao (xac dinh b&ng chu^n vang la nuoi day ddm [+] vi khufin M. tuberculosis
hay phet nhuom khang acid mau ddm [+] kem X quang phoi th&'y ton thudng tham
nhiem dinh phoi dien hinh lao); va 100 m lu huyet thanh benh nhan dtfdc xac dinh
bi viem phoi do tac nhan vi khuan khac va loai trti nhiem lao. Ket qua dUdc trinh
bay trong bang sau:

Bang 20.1: Vi du ket qua thur nghiem ELISA phat hien khang the dac hieu de chan doan lao.

Benh nhan lao phoi [+] Benh nhan lao phoi [-] Tong
ELISA [+] a = 75 b = 30 a+b = 105
ELISA [-] c = 25 d = 70 c+d = 95
Tong a+c = 1 0 0 b+d =1 00 a+b+c+d = 200

• Do nhay cam cua thti nghiem ELISA la:

a 75
Se = ----------- = ----------- =75%
a +c 75 + 25
• Do dac hieu cua thii nghiem ELISA la:
d 70
Sp = ----------- = ----------- =70%
b+d 30 + 70

3. GIA TRj TIEN DOAN DUONG VA GIA TRj TIEN DOAN AM CUA THLf NGHIEM

Doi vdi cac nha lam sang hay dieu tri thi gia sti dung cua mot thti nghiem la
phai tra ldi dtfdc hai cau hoi: (1) Neu thti nghiem co ket qua [+] thi ty le chinh xac
de ket luan dtfdc benh nhan bi benh la bao nhieu phan tram? Thong so" nay dtidc goi
la gia tri tien doan dtfdng (PPV = Positive predictive value). (2) Neu ket qua thti
ngiem la [-] thi ty le chinh xac de ket luan benh nhan khong bi benh la bao nhieu
phan tram? Thong so" nay dtfdc goi la gia tri tien doan am (NPV = Negative
predictive value). Neu dua vao ket qua trinh bay trong bang 12 d tren thi gia tri
tien doan dtfdng (PPV) va gia tri tien doan am (NPV) cua thti nghiem ELISA phat
hien khang the dac hieu M. tuberculosis de chan doan lao se la:

a 75
PPV= ----------- = ----------- =71%
a+b 75 + 30

d 70
NPV = ----------- = ----------- = 74o/0
c +d 25 + 70

Tuy nhien, neu chung ta tinh PPV va NPV cua thti nghiem ELISA dtfa tren
ket qua nghien ctiu tren mot so ltfdng mau am hay mau dudng biet trtfdc nhtf tren

132
thi se khong noi chidc x6t nghiem ELISA htiu dung nhtf the nao khi dem ra ap dung
tren ttfng thtic te. Lay vi du, neu chung ta dung thti nghiem ELISA nay de tam
soat lao tren mot khu vtfc dan ctf co ty le mac benh lao la 1/10000 thi neu ket qua
ELISA [+], do chinh xac de xac dinh nhiem lao tren ngtfdi dtfdc lam thu nghiem se
la bao nhieu u (ttic la PPV); va neu ket qua ELISA [-] thi do chinh xac de xac dinh
khong bi nhiem lao tren ngUdi dUdc lam thti nghiem se la bao nhieu (ttic la NPV)
se la bao nhieu? Sau day la cach tinh: Neu khu vuc dan cU nay co 1000000 ngUdi,
thi so' ngUdi m&c benh lao se la 100 ngUdi, so ngUdi khong mac lao se la 999900
ngtfdi. Vi do nhay cua thti nghiem la 75% nen tren tren 100 ngudi mac lao nay,
thti nghiem se cho ket qua [+] tren 75 ngUdi, vi do dac hieu cua thti nghiem la
70% nen tren 999900 ngUdi khong bi nhiem lao nay se co 699930 ngUdi cho ket
qua ELISA [-] va 299970 cho ket qua ELISA [+]. Cac so lieu dUdc trinh bay vao
bang dtfdi day:

Bang 20.2: Vi du ket qua thif nghiem ELISA tam soat lao, gia dinh thtfc hien di/dc tren tat ca
1000000 ngudi trong quan the co ty le mac lao la 1/10000.
Benh nhan lao phoi [+] Benh nhan lao phoi [-] Tong
ELISA [+] a = 75 b = 299970 a+b = 300045
ELISA [-] c = 25 d = 699930 c+d = 699955
Tong 100 999900 1000000

PPV va NPV cua thti nghiem ELISA phat hien lao ting dung tren cac doi
ttfdng nay la:

a 75
PPV = ----------- = — - =002%
a+b 300045 ~

d 699930
Npv= --------
c +d
* liiiii •«*»*
Tinh toan tren chting minh la neu ket qua ELISA [+], kha nang xac dinh bi
nhiem lao tren ngtfdi dtfdc thti nghiem chi la 0,02%, con neu ket qua ELISA [-] thi
kha nang xac dinh khong bi nhiem lao tren ngtfdi thti nghiem la den 9999%. Co
nghla la vdi do nhay 75% va do dac hieu 70% thi ELISA cho ket qua [-] co gia tri
hdn nhieu so vdi ELISA cho ket qua, [+] trong muc dich sti dung de tam soat lao
tren ngtfdi binh thtfdng d quan the dan ctf tren.
Nhtf vay, stf htiu dung cua mot x£t nghiem doi vdi bac si lam sang hay doi vdi
nha dieu tri tuy thuoc rat nhieu vao xac suat chan doan trtfdc khi cho xet nghiem.
Trong trtfdng hdp ELISA chan doan lao neu tren, neu bac si da nghi ngd benh nhan
bi lao (xac suat 50%) thi PPV va NPV se la 71% va 74% nhtf la trong thiet ke thi
nghiem tim Se va Sp vdi 50% mau nhiem lao va 50% mau khong nhiem lao, nhtfng
neu chi cho xet nghiem cau may thi PPV se rat thap chi 0,02% vi luc do xac suat
chan doan lao tien xet nghiem rat thap nhtf la trong trtfdng hdp cho xet nghiem
ELISA de tam soat lao dai tra d quan the dan ctf trong vi du tren.

133
 Tom lai cac nha lam sang hay dieu tri thtfdng hay dat cau hoi ve dp nh$y cam
va do dac hieu cua mot thti nghiem ma khong biet rin g hai thong so nfcy it c6 gia
tri htiu dung doi vdi ho. Thong so" that sti htiu dung doi vdi cac nha lam sang hay
dieu tri chinh la PPV va NPV. Hai gia tri nay chi bi anh htidng mot phan bdi do
nhay va do dac hieu cua thti nghi£m, trong khi do lai bi anh htidng kha ldn bdi xac
suat chan doan cua bac si trudc khi dat but yeu c&u x6t nghiem. Neu do nghi ngd
benh nhan bi mac benh can chan doan cang cao, thi ket qua x6t nghiem cang htiu
dung cho lam sang. Con neu chi cho xet nghiem cau may, thi sti htiu dung lam sang
cua ket qua xet nghiem cang tha'p.

4. SO SANH HAI THlf NGHIEM KHAC NHAU CHO CUNG MOT MUC 0(CH PHAT
HIEN TAC NHAN VI SINH VAT GAY BENH

De so sanh hai xet nghiem, cac thong so" sau day can dtidc phan tich, do la: do
tUdng dong va do khac biet hay con goi la do phu hdp va do khong phu hdp. Cach
tinh toan dtfa vao bang trinh bay sau day:

Bang 20.3: Vi du so sanh ket qua thti nghiem ELISA va thti nghiem tu latex (LAT) phat hien
khang the dSc hieu M. tuberculosis tren 100 mau huyet thanh lay thti nghiem.
Thiir nghiem ELISA Tong
+
T"
Thif nghiem + a = 65 c=10 a+c = 75
LAT - b = 10 d = 15 b+d = 25
Tong a+b = 75 c+d = 25 n = 100

Do phu hap quan sat diiac (agreement degree) la ty le % cac ket qua ma ca hai
thti nghiem deu cho ket qua giong nhau. Do khong phu hap quan sat diidc
(disagreement degree) la ty le % cac ket qua ma hai thti nghiem cho ket qua khac
nhau. Phan tich bang tren chung ta thay:
• Do phu hdp quan sat dtidc la:
a+d 65+15
----------------- = ------------------------- = 80%
a+b+c+d 65+10+10+15

• Do khong phu hdp quan sat dtfdc la:

c+b 10+10
------------------------ = ------------------------------------ = 20 %
a+b+c+d 65+10+10+15

Tuy la do phu hdp quan sat dtfdc, nhtfng khong the dtfa vao tri so" nay de danh
gia mtic do phu hdp cua hai xet nghiem. Cac nha dich te hoc lam sang da nghien
ctiu, dtfa ra mot thong so, goi la he so kappa, dtfa vao gia tri cua he so"kappa, phong
thi nghiem hay cac nha lam sang co the danh gia do phu hdp cua hai thti nghiem
dtfa theo bang sau:

134
 Bing 20.4: Bang danh gia mtic do phu hop cua hai thti nghiem di/a theo he so kappa
He so kappa Mure do phu hdp He sd kappa Mure do phu hop
0,0-0,2 Qua thap 0 ,6 - 0,8 Kha
0 ,2 -0 ,4 Thap 0 ,8 - 1,0 Cao
0,4-0,6 VC/a

Sau day la cach tim he so kappa:

• Phu hdp quan sat diidc, tinh nhu sau:

(a+d)
x 100

Phu hop ngau nhien, tinh nhu sau:

*\
(c+d) (b+d)
* < f ! l xioo -------- i. X - ----- L x 100

. Phu hop hoan toan, gia dinh ca hai phu hcfp hoan toan vdi nhau, tri so'
se la 100
• Phu hop thtfc tai, tmh nhu sau:

Phu hop quan sat - Phu hdp nglu nhien

• Phu hop tiem an, tmh nhu sau:

Phu hop hoan toan - Phu hop ngau nhien

Tii cac gia tri tren, he so' kappa dtfdc tinh theo cong thiic sau:

Phu hop thtfc tai

He so kappa =-
Phu hop tiem an

Trong vi du trinh bay 0 bang 20.4, sau khi co dtfdc cac gia tri cua phu hcfp
ngau nhien (62,5), phu hdp quan sat (100), phu hop thtfc tai (17,5) va phu hop tiem
an (37,5); he so' kappa se la:
17.5
Kappa =■ = 0,47
37.5

Vdi gia tri cua he so' kappa tinh dtfdc, chung ta co the ket luan ELISA va LAT
phat hien khang the dac hieu M. tuberculosis co do phu hdp d mtic do vtia.

Ngoai ra, chung ta con co the phan tich do phu hdp dtidng, ttic la ty le % ca
hai thti nghiem deu cho ket qua [+] trong tong so' cac ket qua giong nhau cua hai
xet nghiem; va do phu hdp am la ty le % ca hai thti nghiem deu cho ket qua

135
[-] trong tong so cac ket qua giong nhau cua ck hai x6t nghiem. Trong vi du tren,
chung ta thay:
• Do phu hdp dtfOng cua ELISA va LAT la:
a 65
= 81%
a+d 65+15

Do phu hdp am cua ELISA va LAT la:

d 15
= 19%
a+d 65+15

5. DO LAP LAI CUA XET NGHIEM

Do lap lai cua ket qua dinh ltfOng cua xet nghiem khi lam nhieu lan tren cung
mot mau benh pham dtfdc goi la do lap lai cua x6t nghiem. Thong so' noi len do lap
lai cua mot xet nghiem dtfdc goi la he so' bien thien (CV = Coefficient o f variation)
cua ket qua xet nghiem.

Tren cung mot m lu benh pham, neu chung ta chia ra de lam cung mot x6t
nghiem, va cac ket qua dinh ltfdng se la A „ A2, A3, A4...A„. Chung ta se tinh dtfdc
ket qua trung binh A va do lech chuan a nhtf sau:

A1+A2+A3+A4+...+An
A= ---------
N

I (A- A)2
a=
N

He so bien thien chinh la ty le phan tram cua do lech chuan so vdi so' trung
binh, dtfdc tinh theo cong thtfc sau:

cv= 100x —- — %

He so' bien thien con dtfdc goi la he so' phan tan, cho biet do lap lai cua mot
thti nghiem co tot hay khong. Thtidng mot thti nghiem dtfdc danh gia co do lap lai
tot khi CV < 10%.

6. CAU HOI ON TAP

a. Cho biet chuan vang la gi? y nghla cua chuan vang trong danh gia mot
xet nghiem.
b. Hay cho biet cach tinh do nhay cam va do dac hieu cua mot xet nghiem.
y nghla cua hai thong so' nay trong danh gia xet nghiem. Vdi mot xet

136
 nghiem dang sti dung, lam the nao de nang cao do nhay cam hay do d§c
hieu cua x6t nghiem do?
c. Hay cho biet cach tmh gia tri tien doan dtfOng va gia tri tien doan am
cua mot x6t nghiem. Doi v6i bac si dieu tri thi phai lam the nao de gia
tri tien doan dtfOng va gia tri tien doan am cua mot xet nghiem co gia tri
cao nghla la x6t nghiem se hiiu dung cao doi v6i lam sang?
d. Cho biet cach nao de co the tim dtfdc do phu hop cua hai x6t nghiem
dtfdc sti dung cho cung mot muc dich phat hien tac nhan vi sinh vat gay
benh?
e. Cho biet cach nao de co the danh gia do lap lai cua mot xet nghiem?

137
 BAI 21. TRANG Bj CO BAN CHO M0T
PHONG THI NGHIEM VI SINH LAM SANG
■

1. THIET Bj CO BAN

- Mot tu lanh 2 ngan 180L den 240L, dung de dtfng cac moi trtfdng, va cac
thuoc thit che san can phai ltfu trtf d 4°C.
- Mot tu am co dung tich 80L den 100L, dieu chinh dtfdc nhiet do ttf nhiet do
+5 hay +10 tren nhiet do phong den 100°C dung de lam tu u vi khuan.
- Mot tu cay vi sinh khi tinh co den neon va d6n UV, kich thtfdc 0,6 x 1,2m.
- Mot kinh hien vi quang hoc thtfdng 2 mat cua Olympus, Nikon, hay cua mot
hang co uy tin ttf Nhat/Au My.
- Mot may ly tam ban co rotor ttfOng thich cho cac tube thi nghiem hay tube
Falcon 15ml (co the dung may ly tam Trung Quoc).
- Mot may cach thuy co the dieu chinh nhiet do ttf 30°C den 100°C
- Mot may vi tinh co kem may in dung de cai dat phan mem quan ly xet
nghiem vi sinh lam sang (phan mem SCM cua Pham Thai Binh).
Mat nghieng trudc cua tram dupe thiet k£ c o
canh tr^n, g in ban 16 de c o th6 de dang m d ra
Tram duoc dong lau chui, vfi sinh toan tram v i ch o thi£t bi vao
bang ki'nh 5 ly

Hinh 21.1: Mo hinh mot tu cay vi sinh khi tinh. Kich thirdc chieu cao 0,8m, chieu sau 0.6m.
Rieng kich thi/dc chieu dai thi tuy thuoc vao ban de tu cay, trung binh la 1,2m.

138
2. DUNG CV CO BAN

- Kim c&y, vong cay, va vong c&y dinh liidng; m6i loai can 2 cai.
- Micropipet dieu chinh dtfdc the tich ttf l-10pl, 20-200pl, 50-1000pl. Moi loai
mot cay.
- Mot but ngoi thep viet len kinh.
- Hai kep mui nhon de gSp dia khang sinh.
- Mot thtfdc do dtfdng kinh vong vo khu&n.
- Hai hop dtfng lam.
- Hai den con.
- Dau soi kinh hien vi.
- Mot binh thuy tinh mieng rong co n§p, dung tich 3-5L lam binh nen.
- Mot gia nhuom va khay nhuom.
- Hai ca nhtfa 2L co n&p de dtfng vat lieu ban sau khi dung xong.
- Hai binh tia dung tich 0,5L de dtfng con rtia tay
- Gia nhtfa dtfng ong nghiem thuy tinh va nhtfa cac loai
- Dung cu huy kim.
- Binh dtfng con 20L.
- Mot thung dtfng rac y te.
- Mot thung dtfng rac thong thtfdng.
- Hai keo cSt giay hay nylon.

3. VAT LIEU TIEU HAO

3.1. Loai thong thudng

- Pipet Pasteur nhtfa vo trung.

- Cac hop dau cone dung cho micropipet, gom cac loai cone trang l-20pl, cone
vang 20-200jil, cone xanh 50-1000pl. Tat ca deu dtfdc vo trung.
- Lam va lamel soi kinh hien vi.
- Giay lau kinh hien vi, loai khong co bui giay.
- Bao nylon mau den dtfng rac y te.
- Bao nylon trong dtfng rac thong thtfdng.
- Bang keo giay.

139
 - But aceton.
- Tam bong vo trung lam khang sinh do.

3.3. Cac bo thuoc nhuom

- Bo thuoc nhuom gram.

- Bo thuoc nhuom khang acid.
- Bo thuoc nhuom Methylen Blue kiem.
- Bo thuoc nhuom Nigrosin.
- Bo thuoc nhuom Giemsa.

3.4. Cac vat lieu lay va chuyen chd benh pham

- Tam bong vo trung lay mau.

- Tam bong manh vo trung lay mku.
- Tam bong que dai vo trung lay mau quet co tti cung.
- Tube dua moi trtfdng Stuart -Am ies.
- Tube dua moi trtfdng Cary —Blair.
- Lo nap van chat lay mau tap nhiem.
- Lo nap van chat vo trung lay mau khong tap nhiem.
' - Lo nap van chat co mai cheo.
- Lo nap van chat co mai cheo va co ttii F2M.
- Tube 1,8ml nap van vo trung.
- Tube 5ml nap van vo trting.
- Tube 15ml nap van vo trting.
- Tube Eppendorf biopure.
- Tube lay mau khong khang dong.
- Cac loai tube lay mau khang dong EDTA, citrat, heparin.

3.4. Cac moi trudng phong phu, tdng sinh thong thudng va che sin

- GN broth.
- Pepton kiem.

- Loeffler (huyet ttidng dong).

- Chai cay mau 2 pha.

140
 - Chai 2 mSt thach CA c&y dich khong tap nhilm.
- Chai 2 mat thach BANg vk MC c&y niidc tieu.

- Ong moi trtfdng BHI -XV (tube 5ml n ip van).

3.5. Cac mdi trudng phan lap thong thudng va che san

- BA mau ctfu

- BANg*

- CAXV

- CAHI

- CATM*

- MC (hay EMB)

- SS (hay XLD, hay HE)

- MEA (hay TCBS)*

- BA co tellurite *

- BAYK**

- Campy-BAP***

3.6. Cac kit dinh danh

- IDS 14GNR, hay mot so" cac dia giay sinh hoa le, hay mot so' ong moi trtfdng
sinh hoa le

- SAU-latex

- Dia bacitracin

- Dia optochin

- Que X, V, XV

- Dia novobiocin

- Dia oxidase

- H20 2 3%

- Bile Esculin

- TSB co 6,5% NaCl

- RNIS dinh danh Neisseria

- Tube KIA hay TSI

141
3.7. Cac moi trudng lam khang sinh do

- MHA
- MHBA
- HTM*

M iy lanh trdn trtin

1. Tu cay vi sinh khi tTnh
2. Kinh hien vi 2 mat
3. Lavabo rCra tay va nhuom lam
4. May cach thuy
5. May ly tam ban
6. Tu aim 80-100L
7. Tu lanh 180-200L
8. May vi tinh
9. May in
10. Tu dung ho so
11. Ke da co ngSn tu ben di/di

Hinh 21.2: Mo hinh mot phong thi nghiem vi sinh

lam sang tai benh vien (kich thifdc 3-4m x 4-5m)

3.8. Cac phUdng tien lam khang sinh do

- Lo niidc muoi sinh ly vo trung (2-3ml)

- Cac orig do due chuan 0,5 McF
- Cac dia khang sinh lam khang sinh do
- Dia nitrocefin tim [3-lactamase

3.9. Cac vi khuan chuan va tham chieu de kiem tra chat luong

- S. aureus ATCC 25923

- E. coli ATCC 25922
- E. coli ATCC 35218
- P. aeruginosa ATCC 27853
- S. faecalis ATCC 29212
- S. pneumoniae ATCC 49619
- H. influenzae ATCC 49247
- H. influenzae ATCC 49278

142
 - N. meningitidis group B
- S. typhi, Shigella flexneri
- K pneumoniae, Proteus vulgaris
- N. gonorrhoeae, M. catarrhalis
- V. cholerae

3.10. Cac moi trudng luu chung tam thdi

- Ong thach NA nghieng trong tube nhtfa 5ml nap van chat
- Ong thach CAXV nghieng trong tube nhtfa 5ml nap van chat
- Cryotube chiia BHI co 20% glycerol

TAI LIEU THAM KHAO

■

1. Bailey & Scott’ s Diagnostic Microbiology. 11th Edition. Betty A.

Forbes, Daniel F. Sahm, Alice S. Weissfeld. Mosby. 2002
2. Bacterial Pathogenesis - A Molecular Approach. Abigail A.
Salyers and Dixie D. Whitt. ASM Press. 1994
3. Clinical Bacteriology. J. Keith Struthers, Roger P. Westran. Manson
Publishing. 2003
4. Color Atlas and Textbook of Diagnostic Microbiology. 5th Edition.
Elmer W. Koneman, Stephen D. Allen, 'William M. Janda, Paul C.
Schreckenberger, Washington C. Winn, Jr. Lippincott. 1997
5. Immunobiology - the immune system in health and disease. 6th
Edition. Charles A. Janeway, Jr., Paul Travers, Mark Walport, Mark J.
Shlomchik. Churchill Levingstone. 2005
6. Jawetz, Melnick, & Adelberg5 s Medical Microbiology. 23th Edition.
Geo. F Brooks, Janet S. Butel, Stephen A Morse. Me Graw Hill. 2004
7. Manual of Clinical Microbiology. 6th Edition. Patrick R. Murray,
Ellen Jo Baron, Michael A. Pfaller, Fred C. Tenover, Robert H. Yolken.
ASM Press. 1995
8. Microbial Life. Jerome J. Perry, James T. Staley, Stephen Lory.
Sinauer. 2002
9. Mims’ Pathogenesis of Infectious Disease. 4th Edition. Cedric Mims,
Nigel Dimmock, Anthony Nash, John Stephen. Academic Press. 1997.
10. Problem-Oriented Clinical Microbiology and In fe c tio n . Hilary
Humphreys, William L. Irving. Churchill Livingstone. 1996

1 13
P H U• L U• C A

Bang tra cum cac sd do va qui trinh lam xet nghiem

vi sinh lam sang cac benh pham khac nhau
SO doI Ndi dung Trang
1 Qui trinh vi sinh lam sang cay mau vdi chai cay mau hai pha 17
2 Qui trinh vi sinh lam sang cac mau mu va chat djch 24
3 Qui trinh vi sinh lam sang khao sat mau phan 30
4 Qui trinh vi sinh lam sang khao sat m lu ni/dc tieu 42
5 Qui trinh vi sinh lam sang mau djch nao tuy 52
6 Qui trinh vi sinh lam sang khao sat mau quet hau hpng tim lien cau tan huyet beta 58
7 Qui trinh vi sinh lam sang m lu mu tai, mu xoang, djch rCfa xoang 66
8 Qui trinh vi sinh lam sang mau ddm/djch hut ddm tren thanh - khi quan/dich afa
khi quan qua noi soi 77
9 Cach pha loang va cay djnh lupng mau ddm tren cac hop thach 79
10 Qui trinh vi sinh lam sang cac mau lay tC/di/dng sinh due 91
11 Qui trinh vi sinh lam sang cac m lu khao sat ky khi 100

P H U LUC B

Bang tra curu cac hinh anh minh hoa

Hinh Ndi dung Trang
1 Hinh ve minh hoa cau tao chai cay mau hai pha 14
2 Hinh anh chai cay mau 2 pha co vi khuan moc va chifa cay benh pham 16
3 Hinh anh chai cay mau An Do 16
4 Hinh anh tam bong vo trung lay benh pham 22
5 Hinh anh cap tam bong vo trung va tam bong manh vo trung kem vdi tube moi
trudng chuyen chd Stuart -Amies 22
6 Hinh anh lo lay mau phan ti/di dung khao sat vi khuan 29
7 Hinh anh Ip lay m lu phan co F2M de tim ky sinh trung dudng ruot 29
8 Hinh anh cap tam bong vo trung kem vdi tube moi trudng chuyen chd Cary -Blair 29
9 Hinh anh gram vi khuan C. jejuni 33
10 Hinh anh khom vi khuan C. jejuni moc tren moi trudng phan lap 33
11 Hinh ve minh hoa cau tao chai cay nUdc tieu 2 mat thach 39
12 Hinh anh cac mCfc do vi khuan moc tren mat thach cua chai cay nUdc tieu 40
13 Hinh anh chai n ip do vo trung lay benh pham 40
14 Hinh anh chai cay nUdc tieu hai mat thach 40
15 Cac hinh anh khao sat true tiep mau djch nao tuy nhin qua kinh hien vi 47
16 Hinh ve minh hoa cau tao chai 2 mat thach de cay djch nao tuy 48
17 Hinh ve minh hoa cach lam thir nghiem vdi bo thuoc thir MENINGITEX phat hien
khang nguyen hoa tan cac vi khuan thudng gay VMNM 50
18 Hinh anh chai 2 mat thach de cay djch nao tuy 51
19 Hinh anh cac loai vat lieu vo trung nen dUdc dung de lay djch nao tuy hay cac dich
khong tap nhiem khac 51
20 Hinh ve minh hoa cach quet hpng de lay mau 57

144
 21 Hinh anh gram vi khuan bach hau 57
22 Hinh ve minh hoa nguyen t ic cua thuf nghiem m iln dich sac ky phat hien vi khuan
Streptococci tan huyet beta nhom A ti/ quet hong 56
23 Hinh anh tam bong manh vo trung nen dung de lay cac quet tai -mui-hong 65
24 Hinh anh cac tube nap van chat chtfa 5ml BHI co bd sung XV 65
25 Hinh anh cac dung cu nen dung va khong nen dung de lay m lu ddm 74
26 Hinh anh cac dung cu co the dung de lay dam qua dudng mui 74
27 Hinh anh nhuom gram ma dtfa vao do co the danh gia do tin cay mau ddm 75
28 Hinh anh khom vi khuan moc tren cac mat thach cay dinh ltfdng m lu ddm 83
29 Hinh anh gram phet mu nieu dao co N. gonorrhoeae 90
30 Hinh anh gram phet can lin g ntfdc tieu co N. gonorrhoeae 90
31 Cac hinh anh khao sat trtfc tiep quet am dao hay cd tif cung co G. vaginalis 90
32 Hinh ve minh hoa phtfdng phap tao khi trtfdng ky khi bang phtfdng phap thay khi 96
33 Hinh anh thiet bi Axonomat Mark II de cay ky khi 97
34 Cac hinh anh khao sat trtfc tiep b ln g phtfdng phap soi ttfdi 104
35 Hinh ve minh hoa cac btfdc thtfc hien phtfdng phap nhuom gram 107
36 Hinh anh bo thuoc nhuom gram 108
37 Hinh anh ket qua quan sat mot phet nhuom gram 108
38 Hinh anh bo thuoc nhuom Methylen Blue kiem 100
39 Hinh anh bo thuoc nhuom khang acid 113
40 Hinh anh ket qua quan sat phet nhuom khang acid 114
41 Hinh anh bo thuoc thCf lam thuan nhat va loai ta/tap nhiem m lu ddm 115
42 Hinh anh ket qua cac phet nhuom khang the huynh quang 128
43 Hinh ve minh hoa cau tao mot buong cay vi sinh 138
44___ Hinh ve minh hoa cach bd tri cac thiet bi va dung cu can thiet cho mot phong thi
______ nghiem vi sinh lam sang tai cac benh v i e n _________________________ 142

PHU LUC C:

Bang tom tat cac vat lieu lay mau va moi trirdng phan lap
ti/dng urng cho cac benh pham khac nhau
Benh pham Vat lieu lay mau Moi trtfdng phan lap
Mau Chai cay mau (hinh 1.1, trang 14, hinh 1.2, CA mau ngtfa (tot nhat) ± MC
trang 16) BA mau ctfu ± MC
Mu Tam bong vo trung lay m lu (hinh 2.1, trang Co the cay vao ong Thioglycollat hay
22) ± Moi trtfdng chuyen chd Stuart Amies BHI giau XV (hinh 7.2 trang 64) de Itfu
(hinh 2.2, trang 22) hay m lu , phong trtfdng hdp tren cac mat
thach phan lap khong co vi khuan moc
Tam bong manh vo trung lay mau (hinh BA m iu ctfu + MC ± MSA
7.1, trang 65) ± moi trtfdng chuyen chd ± Sabouraud Agar, hay
Stuart Amies (hinh 2.2, trang 16) hay BANg -t MC ± MSA ± Sabouraud Agar,
Lo lay mau vo trung nap do -van chat (hinh hay
3.1, trang 29) hay tube Eppendorf biopure, Chai 2 mat thach CAXV cay dich khong
hay tube 1,8ml vo trung, hay tube 5ml vo tap nhiem (hinh 5.2, trang 48) nhtf cay
trung F2M (hinh 5.5, trang 51) hay Pipet mu ao xe trong phien trtfc khong co vi
Pasteur nhtfa vo trung, hay ong chich nhtfa sinh.
vo trung. Day la cac loai phtfdng tien de lay
cac m lu mu ap xe.

145
Phan Lo lay m lu phan khong co F2M (hinh 3.2 N6n c3y phong phii vao GN broth ±
trang 29) dung lay mau phan tuoi -cay Pepton kiem cho Vibrio ± Campy-thio
ngay,hay cho C. jejuni
Tam bong vo trung lay mau (hinh 2.1, trang MC (hay EMB) ♦ SS (hay XLD. hay HE)
22) dung lay phan tifdi hay quet hau mon - ± TCBS/MEA cho Vibrio ± Campy-BAP
cay ngay, hay cho C. jejuni
n £ u KHONG c A y NGAY THI DUNG TAM
BONG VO TRUNG l A y MAU + Moi trtfdng
chuyen chd Cary -Blair (hinh 3.3, trang 29)
Ntfdc tieu Lo lay mau vo trung n ip do -van chat (hinh BA ± MC hay EMB, hay
3.1, trang 29) BANg + MC hay EMB, hay
Chai 2 mat thach BANg va MC (hinh 4.4,
trang 40) cay nubc tieu trong ph*en true
Dich nao tuy Tube 1,8ml vo trung n ip vang (hinh 5.5 CAXV ± MC, hay
trang 51) Chai 2 mat thach CAXV cay dich khong
Tube Falcon 15ml vo trung n ip xanh (hinh tap nhiem (dung cay DNT trong ph*en tnX)
5.5 trang 51)
Tube 5ml vo trung n ip do (hinh 5.5 trang 51)
Tube Eppendorf biopure (hinh 5.5 trang 51)
Quet hau hong Tam bong vo trung lay mau (hinh 2.1, trang BA, hay BAGe, hay
22) ± Moi trtfdng chuyen chd Stuart Amies BA-tellurite ± Loeffler (neu muon phan
(hinh 2.2 trang 29) lap bach hau), hay
CAHI (neu muon phan lap H.
influenzae), hay CATM (neu muon
phan lap N. gonorrhoeae)
Tai, m it, Tam bong vo trung lay m lu (hinh 2.1, trang Trong trtfdng hdp quet mu m it, nen
mui, xoang 22) ± Moi trtfdng chuyen chd Stuart Amies tang sinh vao BHI giau XV (hinh 7.2
(hinh 2.2, trang 22) hay trang 64) trtfdc khi cay vao moi trtfdng
Tam bong manh vo trung lay mau (hinh 7.1 phan lap.
trang 64) ± Moi trtfdng chuyen chd Stuart BA hay BANg hay BAGe + MC + CAXV
Amies (hinh 2.2, trang 22) hay
Lo lay mau vo trung n ip do -van chat (hinh
3.1 trang 29) hay tube Eppendorf biopure
hay tube 1,8ml vo trung, hay tube 5ml vo
trung (hinh 5.5 trang 51)
Ddm va cac Lo lay mau vo trung n ip do -van chat (hinh BA hay BANg hay BAGe + MC ♦ CAXV
benh pham co 3.1 trang 29) hay
ddm Bo lay ddm qua ong thong mui (hinh 8.2
trang 74)
Sinh due Tam bong vo trung lay mau (hinh 2.1 trang CATM ± BA hay BANg ± MC (neu cd
22) ± moi trtfdng chuyen chd Stuart Amies yeu cau cay vi khuan khac ngoai vi
(hinh 2.2 trang 22) hay khuan lau).
Tam bong manh vo trung lay m iu (hinh 7.1
trang 64) ± moi trtfdng chuyen chd Stuart
Amies (hinh 2.2 trang 22) hay
Lo co F2M de lay mau (hinh 3.2 trang 29)
de soi ttfdi tim T. vaginalis
Cay yem khi Tam bong vo trung lay mau (hinh 2.1 trang BAYK de cay yem khi + cac mo< ta rin g
22) ± moi trtfdng chuyen chd yem khi cay hieu khi mau mu (BA + MC).
Carryport, hay
Chai cay mau yem khi
C)ng chich nhtfa, loai ong chich insulin vo
trung de hut lay mu

146
 NHA XUAT BAN Y HOC

KY THUAT
m XET NGHIEM VI SINH LAM SANG
■

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HOANG TRONG QUANG

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Site ban in: NGUYEN TIEN DUNG
Trinh bay bia: CHU HUNG
K tvitin h : BUI HUE CHI
In 1000 cuon, kho 19 x 27cm tai Xuong in Nha xuat ban Y hoc.
So dang ky ke hoach xuat ban: 23 - 2006/CXB/36 - 271/YH
In xong va nop luu chieu quy II nSm 2006.