THC HNH VI SINH I CNG

BO CO THC HNH BI 4: PHNG PHP NHUM MU VI SINH VT

I. NGUYN TC Nhum mu l qu trnh lm cho t bo hoc cc thnh phn ca t bo vi sinh vt c mu di tc dng ca cc loi thuc nhum. 1. MC CH Gip vic quan st hnh dng t bo, cc thnh phn cu trc t bo ca vi sinh vt c d dng. Gip phn bit cc chng vi sinh vt vi nhau do vic n mu khc nhau i vi cc loi thuc nhum to iu kin cho vic phn loi, nh dng vi sinh vt ( nhum mu Gram ). 2. PHN LOI
Nhum mu vi sinh vt Nhum c nh Nhum khng c nh

Nhum n

Nhum kp

Nhum Gram

Nhum Ziehl - Neelsen Nhum bo t

Nhum tin mao

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THC HNH VI SINH I CNG

bi ny s tm hiu qu trnh nhum khng c nh v phng php nhum Gram thuc phng php nhum c nh. II. DNG C, MI TRNG V HA CHT Cc dung dch nhum: Dung dch xanh methylen Dung dch tm kt tinh ( Crystal violet ) Dung dch lugol Dung dch ty mu Dung dch nhum b sung ( Safranin O hoc Fuchsin )

III. TIN HNH TH NGHIM 1. NHUM MU VI SINH VT KHNG C NH Nh 1 git thuc nhum xanh methylen 0.001% ln phin knh. Nh 1 git canh trng vi sinh vt hoc ha mt t sinh khi vi sinh vt vo git thuc nhum. y l knh ln git knh Quan st tiu bn vt knh (x10) ri (x40). 2. NHUM MU VI SINH VT C NH ( s dng phng php NHUM GRAM ).

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THC HNH VI SINH I CNG

Quy trnh nhum Gram:
Vt

Ph tm kt tinh ( Crystal violet ) 1pht Ra nc

15s Ph lugol

1pht Ra nc

15s Ty cn

15s Ra nc

15s Ph safranin hoc Fuchsin 1pht Ra nc

15s nhum

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THC HNH VI SINH I CNG

Kt Qu: Th t 1 2 3 4 Thuc th Crystal Violet 1 Ra nc 15 Lugol 1 Ra nc 15 Ty mu 5 - 15 Ra nc 15 Saranin 1 Ra nc 15 Kt qu Gram ( + ) Tm Tm Tm Tm Tm Gram ( - ) Tm Tm Khng mu Hng Hng

Xem tiu bn vt knh x100 IV. BO CO THC TP Sau khi nhum, vi khun Gram dng c mu xanh en hay tm, Gram m c mu vng hay ta. Gii thch nguyn nhn? Qu trnh nhum tri qua cc loi thuc nhum sau: Nhum bng dung dch tm kt tinh vi khun bt mu tm. Nhum tip bng dung dch lugol mu tm ca dung dch tm kt tinh gm cht hn trn vi khun. Khi ph ln dung mi hu c Etanol, Axetol th vi khun Gram m b ty, lipit ca lp mng ngoi b tan lm tng tnh thm ca mng dn n s ra tri phc cht tm tinh th - Iot v lm cho vi khun mt mu. Khi nhum b sung chng s bt mu vi thuc nhum (mu vng vi Safranin, ta vi Fuchsin). i vi nhm gram dng, dung mi hu c Etanol, Axetol lm cho cc l trong Peptidoglican co li do phc cht tm tinh th - Iot b gi li trong t bo. Nu khng nhum b sung thuc nhum Safranin hoc Fuchsin, vi khun Gram m c mu g? Ti sao? Nu khng nhum b sung thuc nhum Safranin hoc Fuchsin, vi khun Gram m s khng c mu, i vi vi khun Gram m, hn hp kh mu ng vai tr l cht lm tan mng ngoi ca thnh t bo, c bn cht l lipide. Lp peptidoglycan mng khng th gi li phc hp tm tinh th-iot v t bo Gram m b kh mu.

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THC HNH VI SINH I CNG

V hnh th trng knh hin vi c vi sinh vt trong trng hp nhum vi sinh vt khng c nh v nhum vi sinh vt c nh.

Nhum vi sinh vt c nh ( Nhum Gram Gram m)

Nhum vi sinh vt khng c nh

Nu t nht 5 nguyn nhn khi nhum vi khun Gram ( + ) cho kt qu vi khun Gram ( - ) v ngc li. 5 nguyn nhn khi nhum vi khun Gram dng cho kt qu vi khun Gram m ( vi khun Gram dng c mu hng ): Thi gian ty mu qu lu lm cho phc cht tm kt tinh iot b ra tri. La cy qu gi, v t bo gi thng mt kh nng gi mu thuc nhum tm kt tinh khi ty bng ethanol. Dung dch lugol b hng nn khng to c phc vi dung dch tm kt tinh, kt qu s b ty mu khi dng dung dch ty mu. Thi gian bt mu thuc nhum tm kt tinh cha lu. Thi gian dung dch lugol to phc vi dung dch tm kt tinh cha . 5 nguyn nhn khi nhum vi khun Gram m cho kt qu vi khun Gram dng ( vi khun Gram m c mu tm ): C nh tiu bn khi cn t , cc hp cht protein c trong mi trng hoc trong mu th lm tiu bn kh ty mu.

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THC HNH VI SINH I CNG

Thi gian ty mu qu nhanh khin mu tm ca dung dch tm kt tinh cha thot ra ht. Thi gian bt mu thuc nhum tm kt tinh qu lu. Thi gian dung dch lugol tao phc vi dung dch tm kt tinh qu lu. Qu trnh c nh mu cha thi gian cng vi qu trnh ra bng nc qu lu hoc ra trc tip vo mu khin cho vi khun gram m b ra tri.

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