University of the Philippines, Baguio city College of Science, Department of Biology Biology 120: Microiology
!"ercise #$
B%C&!'$%( !)D*SP*'! %)D C%PSU(!S Davi+, risette- .aros, /amee - 'elucio, Mie Department of Biology, College of Science, University of the Philippines, Baguio City
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Endospore is a differentiated cell formed ithin the cells of certain gram positive !acteria that is e"tremely e"tre mely resistant to heat as ell as to other harmful agents, (#adigan, $%%&)' t is an adaptive feature in micro!es that help them survive in a nutrient deficient environment' Unlie any other cells, endospore are hard to stain using simple gram stains since since they do not a!sor! a!sor! them them ell' ell' So, it is seen unstain unstained ed and stand stand out under the microscope at as strongly infractile structure, (#adigan, $%%&)' Differential staining are essenti essential al in determ determini ining ng and visual visuali*i i*ing ng diffe differen rentt organi organism' sm' Simila Similarr to other other stain stain techni+ues such as the acidfast staining and -ram staining, a special method should !e used to stain endospore' .his is Scaeffer /ulton method, hich uses malachite green as a stain for such cell coat' t demonstrated the endospre forming ithin the vegetative cells and mae free spores easy to identify (0ussey et al', $%%)' 2n the other hand, capsule is a dense, elldefined polysaccharide or protein layer, non as the glycocaly", closely surrounding the cell all, (#adigan, $%%&)' -lycocaly" is a general term for any netor of polysaccharide or protein coating on the outside of the cell' n micro!es, there are to types of protein coating produced' .his includes the capsul capsulee and slime slime layer layer hich hich vary accordi according ng to their their organi organi*at *ation ion or distri distri!uti !ution' on' Capsule Capsule is closely associated associated ith cells hereas hereas slime slime layer is diffused' diffused' Compared Compared to Endospore, this structure can not retain the color of the staining agent' /or this reason, to methods are used to test !oth gram negative and gram positive !acteria' .hese methods are negative capsule staining and positive p ositive capsule stain' .his study aims to provide a !etter understanding on the appropriate methods used to distinguished to cell coats capsule and endospore (Scaeffer/ulton method and 3egative
staining, respectively)' 4lso, this e"periment aims to practiced !iology students in differential staining' .hrough this, the students ill not only !e a!le to familiari*e ith the techni+ues !ut also ill let them visuali*e the to protein coats hich are usually unstained and unseen in most !acterial specimens that are gramstained' M%&!'$%(S %)D M!&*DS %3!n+ospore Staining: Scaeffer4.ulton Metho+
Each mem!er of the group o!tained a /i" smear of B. Subtilis' .he smear as flood slide ith malachite green and as gently steamed at lo flame for a!out 5 minutes' t as counterstained ith 56 safranin for a minute' .hen, it as o!served under 2 for the evidence or appearance of endospore, green in color, and vegetative cells, red in color' B3 Bacterial Capsule Staining: )egative Staining
4 small drop of nigrosin as placed on the edge of a clean slide' .hen, a loopful of E. Coli as mi" ith the nigrosin solution' Using a ne clean slide, the mi"ture as runacross the idth of the slide' .he smear as airdried and covered ith safranin for a!out 7% secs' Safranin as removed using ater' .he smear as e"amined under 2 for the presence of capsule' '!SU(&S %)D D$SCUSS$*)
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#arise, 0', 8 9ayait*, 4' ($%%, :anuary 1)' Endospore Stain Protocol' ;etrieved 3ovem!er 11, $%1<, from http=>>'micro!eli!rary'org>component>resource>la!oratorytest>711$ #adigan, #'? #artino, :'? Dunlap, P? Clar, D'? $% %&' Broc Biology of #icroorganism' 1$ ed' Pearson Education, nc' South 4sia Pte @td'
