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Bi 3 QUAN ST HNH THI VI SINH VT
PHNG PHP LM TIU BN
I. Quan st hnh thi vi sinh vt:1. Quan st i th: Vi khun: quan st
khun lc trn a petri (hnh dng. kch thc, mu sc, b mt, mp khu
lc).
Mc: quan st mt trn v di khun lc, mu sc, sc t do nm mc tit ra mi
trng thclng t.
2. Quan st vi th: Quan st hnh thi, cu to, t bo VSV, ch c th thc
hin i vi sgip ca knh hin
vi c phng i i tng nghin cu ln hang trm ln (knh hin vi quang hc)
v lnhng vn ln (knh hin vi in t).
3.
Phng php lm tiu bn:Mc tiu lm tiu bn: Quan st t bo sng ca VSV.
Tbo cnh v nhum mu, c im hnh thi, m s
lng VSV, kim tra thun khit ca ging, phn loi vi khun.Lm tiu bn t
bo sng:
Tiu bn git p (nm mc, vi khun): Mc ch: Xc nh hnh thi cc tbo VSV,
xc nh kch thc v s sp xp ca chng. Phng php: Chn nm ng ngy tui (cn
non) 3-5 ngy. Chun b lampe sch. Nh 1 git lactofenol, xanh cotn,
blue metyl ln lambe. Vi vi khun: To dch huyn ph (nc thi). Ly lambe
sch p ln(trnh bt kh). Quan st vt knh 10 (i th), a0 (chi tit). Tiu
bn git treo: (vi khun, nm men). Mc ch: Quan st s sinh sn ca VSV, s
hnh thnh v s ny mm ca bo t, pht hin khnng d
ng v mi quan h ca cc t bo VSV vi cc kch thch ha hc.
Phng php: dng que cy hnh trn ly VSV t ln l knh mng (lamelle),
quay ngc lknh xung pha di v t l knh ln 1 phin knh lm. Git huyn ph
ny phi treo l lnkhng c tip xc vi cc mp v y ca ch lm hnh trn trn
phin knh.
Cn s dng cc phin knh v trng cc dch huyn ph VSV th c chun b vi
mitrng lng.
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Phng php ct thch (nm mc): Sau 3-5 ngy cy mc trn a petri, dng dao
m ct ngang khun lc v ct 3ng cn li
to thnh hnh ch nht, 2 ngy. Quan st vt knh 10, thy khun ty v cc
cung sinh bot xut hin ni mp ct ca khun lc.
Tuy nhin vic lm tiu bn bng t bo sng hn ch vic bt mu ca VSV, tn
ti cc VSVkhng mong mun trong khng kh, d b nhim E.coli trong khng
kh.Lm tiu bn cnh (cc t bo cht):
Nhum gram: 1884, nh bc hc an Mch Cristian Gram xut phng php nhum
cbit. y l phng php nhum phn loi vi khun. Theo khnng bt mu vi cc
thucnhum tm kt tinh v iod, tt c cc vi khun c chia lm 2 nhm ln:
Nhm1: gic phc cht to thnh gia tm tinh khit v iod, khi x l bng
cn, vi khunnhum gram +.
Nhm 2: vi khun khng c khnng gic phc cht v b mt mu khi x l bng
cn, vkhun nhum gram -.
Phng php: Bc 1: lm vt bi (tri trng) vi mc ch cnh VSV trn tiu bn.
Vi khun trong mi trng lng: dng que cy u trn ly 1 git vi khun t ln
lamelle sac Vi khun trong mi trng c: ly 1 git nc v trng t ln
lamelle. Ly vi khun trn u trong mt git nc (tri mng trn lamelle).
Cnh vt bi: kh t nhin hoc cnh nh trn ngn la n cn Mc ch cnh: gn t bo
VSV ln phin knh, trnh b ra tri v sau.
=>Vt bi dbt mu vi thuc nhum
Bc 2: Nhum tiu bn bng Crytasl Violet (Getian violet) 30 giy vi
khun c ngmmu.
Bc 3: Ra nc, nhum lugol 1 giy (ra nc hoc khng). Bc 4: Ra nc, ty
cn 30 giy, ra nc. Bc 5: Nhum Fushin 30 giy, ra nc, kh. Bc 6: Nh du,
quan st vt knh 100. Tin hnh lm vi vi khunBacilus Lm tiu bn vi vi
khunBacilus (khng nhum nm men, mc,). Tri trng bng que cy u trn
trong nc ct. Sau khi tri trng ta h tiu bn trn ngn la n cnnhm lm kh
vt bi, trnh tra tri,
tiu dit cc vi khun tn ti trong khng kh, ditE.coli.
u tin ngi ta nh thuc tm tinh th (Crystal Violet) th G+ khng c lp
v nhy bo vnn c mu tm ,G- th c nn khng bt mu thuc tmGram +: c khong
40 lp peptydoglycan, nn khi bt mu phm nhum th bm rt cht vbn vng.
Gram -: c 1 hoc 2 lp peptydoglycan, do s lp vch t bo t nn d b ty
khinhum mu.
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Ra b phm nhum tha Ph Lugol ln pht vi trng Ra bng alcol 95 (lu :
do vch t bo gram - t nn d dng b ty trng, mt mu phm
nhum tm)
Ph Safranin ln pht vi khun (do VK gram + c vch qu dy, thm tm
Crystal Violenn ko thm na).
Ra nc loi b phm tha Thm kh lamel. Nh du quan st vt knh. Kt qu:
gram + : mu tm ; gram -: mu hng . Lu : khi tin hnh thao tc nhum
gram nn tun thng theo cc bc trn, nu nhum
khng ng gram m v gram dng c th cho cng mt mu.
Hnh 1.1. Cc bc tin hnh nhum Gram v v d minh ho kt qu.
Yu cu: Ging vi khun nhum Gram l ging ng ngy tui (18-24 gi). Nui
cy mi trng thc
hp.
Nhum theo ng trnh t v thi gian. Sau khi quan st vt knh du, lau
sch knh bng xylem.4. Bn lun kt qu th nghim:
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Sau khi nhum gram ta squan sat c vi khun di knh hin vi v phn bit
c gram v gram dng.
Quan st vi knh hin vi phng i ca vt knh l 40 ln. y cha tnh phng
ica thknh (thng l 10X) vy tnh ra th phng i ca mu l 40x10 = 400
ln.
ngha nhum gram: phn bit gram m, gram dng. ngha thc t:ng dng
trong y hc xc nh mu c b nhim trng qua vic nhum gram
t xc nh c bin php iu tr.
Khi tri trng khng lm qu dy rt kh quan st v khi ra tri s khng ra
ht mu, ly qutha sgy kh khn trong vic quan st.
Khi lm tiu bn trn ngn la n cn khng c h qu nng t bo c th b teo
hoc cht bo.
Khng mu tm lu gram m s khng b kh mu. Ra ty khng qu mnh gram dng
s b ra tri.
KhunBacillus
II. Quan st nm mcAspergillus oryzae:1. Cc c im sinh hc cn quan
st: c im ca si nm: mu sc c vch ngn hay khng c vch ngn. Hnh dng, cu
to v cch sp xp ca bo t. S ny chi ca nm mc. Hnh dng slng bo t trong
ti bo t.
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2. Cch quan st: Lm tiu bn nhum n nm mc cy trong mi trng dinh dng
vi xanh metyl Loeffler. Quan st s ny chi ca nm mc.
NmAspergillusi oryzae: vi cc bo t nang
http://upload.wikimedia.org/wikipedia/commons/2/2f/Aspergillus_fumigatus_01.jpg
