CCH PHA CH MT S MI TRNG NUI CY VI SINH VT TRONG DY THC HNH SINH HC PGS.

TS Bin Vn Minh Trng HSP i hc Hu

Cng nh ng - thc vt, sinh trng cc t bo vi sinh vt (VSV) cn cc cht dinh dng thch hp. Trong nui cy nhn to ngi ta chun b cc loi thc n cung cp cho tng nhm VSV khc nhau. Dng thc n ny c gi l mi trng nui cy. Mi trng nui cy thng c gi theo tn ngi ch to cng thc, v d mi trng Hansen, Sabouraud hoc theo ngun cht dinh dng chnh c trong mi trng, v d mi trng thch - tht - pepton Mt mi trng c s dng nui cy VSV phi c cc cht dinh dng cn thit, c pH thch hp, c nht nht nh, khng cha cc yu t c hi v phi tuyt i v trng. Lm mi trng nhm thc hin vic phn lp, nhn ging, gi ging VSV, ng thi nui cy v nghin cu cc c im sinh hc ca chng. 1. Nguyn tc pha ch mi trng - Pha ch mi trng phi da trn c s nhu cu v cc cht dinh dng v kh nng ng ho cc cht dinh dng ca tng loi VSV. - Cn phi iu chnh t l v nng cc cht trong thnh phn mi trng m bo s cn bng v p sut thm thu gia mi trng v t bo VSV . - m bo cc iu kin l - ho cn thit cho cc hot ng trao i cht ca VSV - Mi trng phi v trng tuyt i khi nui cy ch pht trin mt loi VSV mong mun. 2. Phn loi mi trng - Da vo mc ch s dng, ngi ta chia mi trng thnh cc loi: Mi trng c s, mi trng lm giu, mi trng chn lc v mi trng gim bit. a. Mi trng c s (minimum medium): l mi trng c cc cht cn thit cho a s cc VSV pht trin c. Mi trng ny thng c dng nui cy cc VSV d pht trin. V d, mi trng thch tht peptone dng phn lp, nui cy vi khun. b. Mi trng lm giu (enrichment medium): trn mi trng c s cho thm mt s cht dinh dng c bit thch hp vi vic nui cy mt s nhm VSV. Cc cht b sung thm c th l mu, huyt thanh, cao nm men, m ng vt hoc nc chit thc vt. V d, nui cy vi khun Bordetella pertussis ngi ta dng mi trng c s Difco 0048 ( kh trng mi trng 1100C trong 30 pht) c b sung mu th v trng ( lc qua nn lc), sao cho nng cui l 15%. c. Mi trng chn lc (Selective medium): l mi trng, ngoi cc cht dinh dng c s cn c mt s cht c hiu kch thch VSV pht trin hoc mt s cht km hm c ch s pht trin ca cc VSV khc. Mi trng ny thng c dng nui cy, phn lp mt loi VSV nht nh. V d, mi trng Hansen dng phn lp, nui cy nm men. d. Mi trng gim bit (differential medium) dng trong vic gim nh cc loi VSV khc nhau xc nh v tr phn loi ca chng. Chng hn, khi xc nh kh nng sinh protease th b sung casein hay gelatin vo mi trng, kh nng sinh amylase thm tinh bt tan vo mi trng, kh nng sinh lipase thm du n v ch th mu, kh nng sinh H2S thm Pb acetate... - Da vo trng thi vt l, ngi ta chia mi trng lm 4 loi: + Mi trng lng hay dch th (liquid medium): Trong thnh phn ch c cc cht dinh dng v nuc, c dng nui tng sinh, th nghim cc c tnh sinh l, sinh ha hoc dng gi ging v bo qun VSV khng tng trng tt trn mi trng rn. + Mi trng c hay cng (solid medium): l mi trng dch th c b sung 2% thch (agar - agar) dng phn lp VSV hiu kh. + Mi trng bn c (semisolid medium): l mi trng dch th cha 0,35-0,75% thch (dng kim tra kh nng di ng) v mi trng xp l mi trng c s dng cc nguyn liu lm xp nh cm, cm, mt ca, than bn(dng nui cy cc VSV hiu kh dng si).

3. Cc bc pha ch mt mi trng nui cy Bc mt: Chun b dng c: pipet, ng ong, ng nghim, cn, bnh cu. Bc hai: i vi mi trng lng: cn, ong cc thnh phn ca mi trng ri cho vo bnh cu; i vi mi trng c thm 2% thch (cn thch ngm vo nc trc 5-10 pht, vt thch, vt kh). Bc ba: un si nh la v khuy u cho tan ht ri lc qua vi mn khi cn nng (500C) hoc lm trong bng cch dng lng trng 1 qu trng g vi 1 lit mi trng. nh trng si bt ri cho vo mi trng, khuy u, un si 5 pht, lng ri lc qua vi mn. Bc bn: o pH ca mi trng bng my o pH. Chnh pH n gi tr thch hp bng dung dch NaOH 10% hoc HCl 10% . Bc nm: Dng phu thy tinh, rt mi Lm nt bng theo trnh t a, b, c. trng vo cc dng c (1/4 chiu cao ng nghim ch thch nghing, 1/2 chiu cao ng nghim - thch ng, 1/3 chiu cao a petri mi trng thch a). Lm nt bng (dng bng khng thm nc) nt ming ng nghim (nt khng c qu cht hoc qu lng, thng di 3-4cm). Bao giy chng thm trn mi u ng nghim hay bnh nn ng mi trng (hnh trn). Bc su: Hp 1100C mi trng trong 30 pht, em ra ch thch a, hay lt nghing cho mi trng ng li ta c mi trng thch nghing (xem hnh di). Bc by: Nhn nh kt qu.

Ngi u tin ngh n s dng mi trng c trong nghin cu vi sinh vt l Robert Koch vo nm 1881.

Thch l sn phm ch to t Rau cu ch vng (Gracilaria verucosa) l cht lm cng mi trng.

Cc loi mi trng nui cy vi sinh vt cn pha ch ti cc phng th nghim sinh hc.

4. Thnh phn mt s mi trng v cch pha ch a. Mi trng thch tht peptone (phn lp, nui cy vi khun) * Cng thc: Nc tht b 1000ml; peptone 10g; NaCl 5g; glucose 20g; thch 20g. Lm nuc tht b: Lc ht gn m ca tht, xay hoc bm nh, cn 500g ri ngm tht vo 1000ml nc ct, t lnh qua m. un t t cho ti si, vt bt, khuy u, duy tr si m trong 1gi, thnh thong khuy u. Lc qua giy lc loi m, b tht, ri thm nc ct cho 1000ml. Nu ct gi dnh th ng chai hp 1100C trong 30 pht. * Pha ch: + Chun b dng c: bnh cu (hoc ni nhm), ng ong, giy lc, dao, my xay tht, cn. Cn ong nc tht, peptone, mui ri cho vo ni nhm un nng cho tan. iu chnh pH n 7,2 7,4. Cho thch ( ngm nc trc 10 pht, ra sch) vo, un si, lc qua bng gc khi mi trng cn nng ri phn phi vo cc ng nghim, bnh cu hay bnh nn. Dng nt bng nt cc ng nghim, bao giy bt u ming ca chng. Hp 1100C trong 30 pht; ly cc ng nghim ra v t nm nghing cho thch ng. + Yu cu: thch phi tan u, mu vng nht hoc hi sm, khng qu cng hoc qu mm.

b. Mi trng Hansen (phn lp, nui cy nm men) Glucose 50g; peptone 10g; KH2PO4 3g; MgSO4.7H2O 3g; thch 20g; nc ct 1000ml; pH= 6,0. c. Mi trng Czapek (phn lp v nui cy nm mc) Saccharose 30g; NaNO3 2g; K2HPO4.3H2O 1g; KCl 0,5g; MgSO4.7H2O 0,5g; FeSO4.7H2O 0,01g; thch 20g; nc ct 1000ml; pH =7,0. d. Mi trng Gause 1 (phn lp v nui cy x khun) Tinh bt tan 20g; KH2PO4 0,5g; MgSO4.7H2O 0,5g; KNO3 1g; NaCl 0,5g; FeSO4 0,01g; thch 20g; nc ct 1000 ml; pH = 7,2 - 7,4 . Cch pha: Ha tan cc ho cht trong thnh phn mi trng vo nc, cn tinh bt ho tan trong nc m. Khuy cho tan u ri trn vo nhau, thm nc cho 1000ml. e. Mi trng nc chit khoai ty v gi u xanh (nui cy vi khun, nm men, nm mc) Khoai ty 100g, gi u xanh 100g, ng knh 50g; thch 20g; nc ct 1000 ml. un si khoai ty ( gt v, thi lt) cng gi u xanh vi nc ct trong bnh cu 10 pht, sau em lc ly dch trong, cho hn hp ng knh v thch vo bnh cha dch trong, un si, khuy u cho ho tan, lc qua bng gc khi mi trng cn nng ri phn phi vo cc ng nghim, bnh cu hay bnh nn. Dng bng nt cc ng nghim li, bao giy bt u ming ng, hp 1100C trong 30 pht; ly cc ng nghim ra v t nm nghing cho thch ng. Cu hi: 1) Trnh by nguyn tc c bn ca vic pha ch mi trng nui cy vi sinh vt? 2) Nu cc bc pha ch mt loi mi trng nui cy vi sinh vt? Nguyn nhn mi trng b h hng ? Phng php bo qun mi trng? 3) Trnh by cc loi mi trng nui cy VSV cn pha ch ti cc phng th nghim ? ng dng? 5. Nhng im cn lu : - Thch (agar - agar) l cht rn b sung lm cng mi trng, khng phi l cht dinh dng i vi VSV. Thch nng chy 1000C v ng li 400C. Nu cn cy VSV trc khi mi trng ng c, ngi ta mi trng ngui n 45 500C ri mi cy vo. - Vi cc bnh mi trng ng cn un cch thy mi trng lng ng u. Khi ly bnh ra khi ni phi dng khn sch thm kh bnh (trnh nc trn thnh bnh nh vo cc a mi trng gy nhim). C th t bnh vo ni hp un n khi ha tan, khng nn un bnh trn bp in. - Trong qu trnh thch ng khng c lm rung bn hay rung ng thch. Sau khi thch ng, ng ngui hn mi gi cc ng thch nghing bng giy v trng, t vo t m 30-370C kim tra v trng v lm kh mt thch. Sau vi ngy ly ra quan st. Nhng ng thch nghing v trng (khng cha cc khun lc VSV) c dng nui cy VSV. - Rt nhiu loi ng d b phn gii trong qu trnh kh trng pH kim (c bit khi c mt ca mui phosphate v peptone) lm cho mu mi trng chuyn thnh mu nu v cc sn phm to thnh c th c ch s sinh trng ca VSV. trnh tnh trng ngi ta kh trng mi trng pH acid nh hoc kh trng ring bit i vi ng. Ti liu tham kho

[1] V Th Minh c, Thc tp vi sinh vt hc, NXB i hc Quc gia H Ni nm 2001. [2] John William Henry Eyre, 2009. A Laboratory Guide for Medical, Dental, and Technical
Students, http://www.gutenberg.org/.