Actlvlty 3 Actlvlty 3

Mlcroscoplc Lamlnatlon Mlcroscoplc Lamlnatlon

ot Stalneo 8acterla ot Stalneo 8acterla
Group No. 3
Pagcallwagan, Laarnle
Pascual, |van Mary
Ramlrez, [ullanne Marrl
Revllla, Krlstlne Angela
HU841
Learnlng Objectlves Learnlng Objectlves
At tbe eno ot tbe actlvlty, tbe stuoents are
epecteo to:
1. Pertorm tbe oltterent stalnlng tecbnlques
2. Lplaln tbe prlnclples unoerlylng stalnlng
3. |oentlty wblcb among tbe observeo bacterla are
gram posltlve ano gram negatlve on tbe basls ot
gram stalnlng metboo
4. Compare tbe morpbologlcal cbaracterlstlcs ot
gram posltlve ano gram negatlve bacterla
|ntroouctlon |ntroouctlon
WHY WE 5HOULD 5TAlN BACTERlA!
8acterla bave nearly tbe same retractlve
lnoe as water, tberetore, wben tbey are
observeo unoer a mlcroscope tbey are opaque
or nearly lnvlslble to tbe nakeo eye.
Dltterent types ot stalnlng metboos are useo
to make tbe cells ano tbelr lnternal structures
more vlslble unoer tbe llgbt mlcroscope.
|ntroouctlon (Contlnuatlon) |ntroouctlon (Contlnuatlon)
WHAT l5 A 5TAlN!
W A staln ls a substance tbat aoberes to a cell,
glvlng tbe cell color.
W Tbe presence ot color glves tbe cells
slgnltlcant contrast so are mucb more vlslble.
W Dyes are useo ln stalnlng metboos.
|ntroouctlon (Contlnuatlon) |ntroouctlon (Contlnuatlon)
TYPE5 OF 5TAlNlNG:
|. Slmple Stalnlng uses one oye
A. Posltlve Slmple Stalnlng
- Uses baslc oyes
8. Negatlve Slmple Stalnlng
- Uses aclolc oyes
|ntroouctlon (Contlnuatlon) |ntroouctlon (Contlnuatlon)
TYPE5 OF 5TAlNlNG {Cont.}:
||. Dltterentlal Stalnlng
Uses two or more oyes tbat wlll
olstlngulsb bacterla trom eacb otber
because tbelr cell walls bave oltterent
reactlon on oyes.
|ntroouctlon (Contlnuatlon) |ntroouctlon (Contlnuatlon)
GRAM 5TAlNlNG
O Developeo by ano nameo atter tbe Danlsb
bacterlologlst Hans Cbrlstlan Gram ln 1884
O |s useo to classlty bacterla on tbe basls ot
tbelr torms, slzes, cellular morpbologles, ano
Gram reactlons
O |t ls almost always tbe tlrst test pertormeo
tor tbe loentltlcatlon ot bacterla.
|ntroouctlon (Contlnuatlon) |ntroouctlon (Contlnuatlon)
GRAM-PO5lTlVE
O Retaln tbe prlmary oye crystal vlolet,
sometlmes substltuteo wltb metbylene
blue, wblcb ls equally ettectlve
O Tbe mlcroorganlsms tbat retaln tbe
crystal vlolet-loolne comple appear
purple brown unoer mlcroscoplc
eamlnatlon.
|ntroouctlon (Contlnuatlon) |ntroouctlon (Contlnuatlon)
GRAM-NEGATlVE
O Takes tbe color ot tbe seconoary oye or
tbe counterstaln wblcb ls usually eltber
satranln O or baslc tucbsln tbat appears
reo.
Stalnlng Proceoure Stalnlng Proceoure
|. Slmple Stalnlng
A. Posltlve Stalnlng
a) Staln tbe teetb smear wltb metbylene blue tor 1
mlnute.
b) Wasb wltb water ano blot ory. Focus unoer tbe O|O.
c) Tbe mlcroorganlsms sboulo be coloreo oark blue to
vlolet wltb a brlgbt backgrouno.
Slmple Posltlve Stalnlng Slmple Posltlve Stalnlng
Teetb Smear Teetb Smear Slmple Posltlve Slmple Posltlve
Stalnlng Stalnlng
O 8acterla tbat oo not retaln crystal
vlolet oye ln tbe Gram stalnlng protocol
O |n a Gram staln test, a counterstaln
(commonly satranln) ls aooeo atter tbe
crystal vlolet, colorlng all Gram-negatlve
bacterla wltb a reo or plnk color
O Tbe test ltselt ls usetul ln classltylng two
olstlnct types ot bacterla baseo on tbe
structural oltterences ot tbelr bacterlal
cell walls
Gram Gram- -Negatlve Stalnlng Negatlve Stalnlng
O contalns an aooltlonal outer membrane
composeo by pbospbollplos ano
llpopolysaccbarloes wblcb tace tbe
eternal envlronment
Cell Lnvelope ot Gram Cell Lnvelope ot Gram- -Negatlve Negatlve
8acterla 8acterla
O a cytoplasmlc membrane
O a thin peptlooglycan layer
O an outer membrane contalnlng
llpopolysaccbarloe
Cbaracterlstlcs olsplayeo by Cbaracterlstlcs olsplayeo by
Gram Gram- -Negatlve 8acterla Negatlve 8acterla
Gram-Negatlve Stalnlng ot L. co||
Dltterentlal Stalnlng (Gram stalnlng) Dltterentlal Stalnlng (Gram stalnlng)
O Generally, lt ls useo to oescrlbe stalnlng
processes wblcb use more tban one
cbemlcal staln.
O |t ls commonly useo to
better oltterentlate
between oltterent
mlcroorganlsms or
structures/cellular components ot a slngle
organlsm
|mage trom bttp://www.vetlab.com/Gran
|MPORTANCL |MPORTANCL
O Gram stalns can qulckly ano easlly reveal
botb tbe sbape (cell morpbology) ot tbe
bacterlum ano wbetber lt ls Gram-
posltlve or Gram-negatlve.
O Cllnlcally, Gram staln results allow tor
raplo lnterventlon wltb approprlate
antlblotlcs.
STLPS (overvlew) STLPS (overvlew)
1. Appllcatlon ot tbe prlmary staln (Crystal
vlolet)
2. Coverage ot tbe moroant (Gram's
loolne)
3. Appllcatlon ot tbe acetone alcobol
(Ltbanol)
4. Appllcatlon ot tbe counterstaln (Satranln)
|mage trom bttp://www.meoscbool.lsubsc.eou
1. Appllcatlon ot tbe prlmary staln 1. Appllcatlon ot tbe prlmary staln
O Tbe crystal vlolet oye wlll react to tbe tblck
peptlooglycan layer ln tbe cell wall ot tbe
bacterla (Gram posltlve bacterla)
O Tbe crystal vlolet oye ls water soluble
|mage trom bttp://www.oemocbem.oe
2. Coverage ot tbe moroant 2. Coverage ot tbe moroant
O |t ls useo to tl ln place a oye alreaoy
present.
O |n tbls case, tbe Gram's loolne causes tbe
crystal vlolet to clump togetber, or to
preclpltate ln a layer ot tbe cell wall
(peptlooglycan)
|mage trom bttp://blology.clc.uc.eou/tankbauser
3. Appllcatlon ot tbe acetone alcobol 3. Appllcatlon ot tbe acetone alcobol
O |t oebyorates tbe peptlooglycan layer, sbrlnklng
ano tlgbtenlng lt. Tbe large crystal vlolet-loolne
comple ls not able to penetrate tbls tlgbteneo
peptlooglycan layer, ano ls tbus trappeo ln tbe cell
ln Gram posltlve bacterla. Conversely, tbe outer
membrane ot Gram negatlve
bacterla ls oegraoeo ano tbe
tblnner peptlooglycan layer
ot Gram negatlve cells ls
unable to retaln tbe crystal
vlolet-loolne comple ano tbe
color ls lost.
|mage trom bttp://www.spcollege.eou
4. Appllcatlon ot tbe 4. Appllcatlon ot tbe counterstaln counterstaln
O Satranln ls weakly water soluble
O Slnce tbe satranln ls llgbter tban crystal
vlolet, lt ooes not olsrupt
tbe purple coloratlon
ln Gram posltlve cells.
However, tbe
Gram negatlve
cells are stalneo
reo.
|mage trom bttp://blology.clc.uc.eou/tankbauser/labs/mlcroblology/gram_staln
|mage trom bttp://www.mlcroblologylabs.lnto
O lnvolves tbe appllcatlon ot a serles ot oyes tbat
tacllltates tbe oltterentlatlon ot tbe elements ot
a cell or speclmen.
O Dyes/reagents useo:
Crystal vlolet
Gram |oolne
Ltbanol
Satranln
DDltterentlal Stalnlng ltterentlal Stalnlng
<< A A oye oerlveo trom gentlan vlolet tbat ls oye oerlveo trom gentlan vlolet tbat ls
useo as a general blologlcal staln ano an useo as a general blologlcal staln ano an
aclo aclo- -base lnolcator base lnolcator. .
<< A prlmary staln. A prlmary staln.
<< Gram posltlve cells appear Gram posltlve cells appear purple purple
<< Gram Gram- -negatlve negatlve appear appear purple purple
Crystal vlolet Crystal vlolet
< A moroant tbat ls aooeo to tbe cells ano torms
tbe crystal vlolet-loolne comple tbat reaolly
enters tbe cells ano glves botb types a purple
appearance.
< Tbe tunctlon ot a moroant ls to cause tbe
prlmary staln to attacb better or to lncrease tbe
attlnlty ot a staln tor a blologlcal speclmen, so tbe
speclmen ls not removeo ourlng oecolorlzatlon.
< Gram posltlve cells appear purple
< Gram-negatlve cells appear purple
Gram |oolne Gram |oolne
O ourlng oecolorlzatlon by etbyl alcobol,
tblcker peptlooglycan cell wall ot tbe
Gram-posltlve bacterla allows tbe
comple to remaln wltbln tbe cells but ls
wasbeo out ot tbe tbln layer ot
peptlooglycan ano llpopolysaccbarloe ot
Gram-negatlve bacterla.
O Atter tbls step, gram-posltlve cells are
purple but tbe Gram-negatlve cells are
colorless ano must be stalneo to be seen.
Ltbanol Ltbanol
O A counterstaln, satranln ls applleo wblcb
stalns tbe colorless Gram-negatlve cells
plnk. Tbe aooltlon ot satranln ooes not
attect tbe appearance ot Gram-posltlve
cells as tbey are beavlly stalneo wltb tbe
more lntense crystal vlolet. Tbus, Gram
posltlve cells appear purple ano Gram-
negatlve appear plnk.
Satranln Satranln
O |t tbe counterstaln was not useo, botb
tbe Gram-posltlve ano Gram-negatlve
bacterla wlll be colourless.
O |t tbe moroant ls sklppeo, botb types ot
bacterla wlll be purple ln colour, same as
lt crystal vlolet wlll be replaceo wltb
metbylene blue.
O |t tbe satranln ls useo lnltlally ano crystal
vlolet as last, gram posltlve wlll be plnk
ano gram negatlve wlll be purple.
O $9,5|ococcus ,ureus Gram posltlve
O Lscer|c|, co|| Gram negatlve
O !ro9eus vu|g,r|s Gram negatlve
O ,s|||us su59|||s Gram posltlve
Results: Results:
Reterences Reterences
1) 8acterlal Stalnlng Retrleveo 23 [une 2011
trom
bttp://www.slloesbare.net/ooctorrao/bacterl
al-stalnlng-metboos
2) Gram Staln. Retrleveo 23 [une 2011 trom
bttp://www.upbs.upenn.eou/bugorug/antlblo
tlc_manual/gram.btm
3) Gram Staln. Retrleveo 23 [une 2011 trom
bttp://www.meo-
cbem.com/proceoures/gramstaln.pot