Rabu, 15 September 2010

Pewarnaan Malachite Green

Pewarnaan malachite green adalah pewarnaan yang biasanya digunakan untuk melihat bakteri batang pembentuk spora.

Untuk membuat larutan malachite green dapat digunakan bahan kimia dari Malachite Green Oxalate dari Sigma Aldrich atau dari Fisher Scientific.

Untuk membuatnya , larutkan 5 gram Malachite Green Oxalate dalam 100 ml Aquadest.



Untuk konfirmasi spora, panaskan sample 80 derajat C selama 10 menit. yakinkan bahwa media sudah mencapai suhu tersebut sebelum inokulasi. Pindahkan dan dinginkan dalan kotak es. Kemudian inkubasi pada temperatur optimum microbe selama 5 - 7 hari. Spora akan tumbuh sedangkan bakteria vegetative akan mati.

Selain itu, terdapat beberapa tekhnik yang dapat digunakan untuk manganalisa bakteri batang pembentuk spora.
( masih bahasa asli, takut salah translatenya )

1. POPPING TEST.
Immerse suspect culture in an acidic oxidizer (e.g. 0.1% KMnO4 in 0.3 N HNO3). The spore cortex ruptures and the cellular milieu spills out for staining with basic dyes. Mount a dried film of the suspected spores on as a dried film on a slide; expose to the reagent for 10 - 20 minutes; observe under oil immersion without staining or with staining.

2. Negative Staining.
Use 7% (w/v) nigrosin and observe for highly refractile elliptical or spherical bodies.

3. Dorner Spore Stain.
Heat fix the suspension on a slide and cover with thin filter paper cut to fit; saturate with carbolfuchsin and steam for 10 min.; remove paper and decolorize with acid-alcohol for 1 min. and rinse with tap water; blot dry; place a drop of nigrosin on the smear and cover with another slide; slowly draw the two apart to form a thin film; cover with a cover slip or dry; observe. The endospore is red. (This is also called "Flemming's Stain").

4. Alternate Dorner Spore Stain.
In a test tube mix an aqueous suspension of the culture with an equal volume of carbolfucshin; heat in a boiling water bath for 10 min.; Mix 1 loopful of 7% nigrosin with 1 loopful of the stained suspension on a glass slide; mix and spread until a thin film forms and dries; examine.






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