Materials Required:

1. Clean glass slides

2. Inoculating loop
3. Bunsen burner
4. Bibulous paper
5. Microscope
6. Lens paper and lens cleaner
7. Immersion oil
8. istilled !ater
". 18 to 24 #our cultures o$ organisms

Reagents:

1. %rimar& 'tain ( Cr&stal )iolet
2. Mordant ( *rams Iodine
3. ecolouri+er ( ,t#&l -lco#ol
4. 'econdar& 'tain ( 'a$ranin


Procedure:

Part 1: Preparation of the glass microscopic slide

*rease or oil $ree slides are essential $or t#e preparation o$ microbial smears. *rease or oil
$rom t#e $ingers on t#e slides is remo.ed b& !as#ing t#e slides !it# soap and !ater. /ipe t#e
slides !it# spirit or alco#ol. -$ter cleaning0 dr& t#e slides and place t#em on laborator&
to!els until read& $or use.

Part 2: Labeling of the slides

ra!ing a circle on t#e underside o$ t#e slide using a glass!are(mar1ing pen ma& be #elp$ul
to clearl& designate t#e area in !#ic# &ou !ill prepare t#e smear. 2ou ma& also label t#e
slide !it# t#e initials o$ t#e name o$ t#e organism on t#e edge o$ t#e slide. Care s#ould be
ta1en t#at t#e label s#ould not be in contact !it# t#e staining reagents.

Part 3: Preparation of the smear

Bacterial suspensions in broth: /it# a sterile cooled loop0 place a loop$ul o$ t#e
brot# culture on t#e slide. 'pread b& means o$ circular motion o$ t#e inoculating loop
to about one centimeter in diameter. ,3cessi.e spreading ma& result in disruption o$
cellular arrangement. - satis$actor& smear !ill allo! e3amination o$ t#e t&pical
cellular arrangement and isolated cells.
Bacterial plate cultures: /it# a sterile cooled loop0 place a drop o$ sterile !ater or
saline solution on t#e slide. 'terili+e and cool t#e loop again and pic1 up a .er& small
sample o$ a bacterial colon& and gentl& stir into t#e drop o$ !ater4saline on t#e slide
to create an emulsion.
Swab Samples: 5oll t#e s!ab o.er t#e cleaned sur$ace o$ a glass slide.

Please note: It is .er& important to pre.ent preparing t#ic10 dense smears !#ic# contain an
e3cess o$ t#e bacterial sample. - .er& t#ic1 smear diminis#es t#e amount o$ lig#t t#at can
pass t#roug#0 t#us ma1ing it di$$icult to .isuali+e t#e morp#olog& o$ single cells. 'mears
t&picall& re6uire onl& a small amount o$ bacterial culture. -n e$$ecti.e smear appears as a
t#in !#itis# la&er or $ilm a$ter #eat($i3ing.

Part : !eat "i#ing

7eat $i3ing 1ills t#e bacteria in t#e smear0 $irml& ad#eres t#e smear to t#e slide0 and allo!s
t#e sample to more readil& ta1e up stains.

-llo! t#e smear to air dr&.
-$ter t#e smear #as air(dried0 #old t#e slide at one end and pass t#e entire slide
t#roug# t#e $lame o$ a Bunsen burner t!o to t#ree times !it# t#e smear(side up.

8o! t#e smear is read& to be stained.

%lease 8ote9 :a1e care to pre.ent o.er#eating t#e slide because proteins in t#e specimen can
coagulate causing cellular morp#olog& to appear distorted.

Part $: %ram Stain Procedure


1. %lace slide !it# #eat $i3ed smear on staining tra&.

2. *entl& $lood smear !it# cr&stal .iolet and let stand $or 1 minute.

3. :ilt t#e slide slig#tl& and gentl& rinse !it# tap !ater or distilled !ater using a !as#
bottle.

4. *entl& $lood t#e smear !it# *ram;s iodine and let stand $or 1 minute.

5. :ilt t#e slide slig#tl& and gentl& rinse !it# tap !ater or distilled !ater using a !as#
bottle. :#e smear !ill appear as a purple circle on t#e slide.

6. ecolori+e using "5< et#&l alco#ol or acetone. :ilt t#e slide slig#tl& and appl& t#e
alco#ol drop b& drop $or 5 to 1= seconds until t#e alco#ol runs almost clear. Be
care$ul not to o.er(decolori+e.

7. Immediatel& rinse !it# !ater.

8. *entl& $lood !it# sa$ranin to counter(stain and let stand $or 45 seconds.

". :ilt t#e slide slig#tl& and gentl& rinse !it# tap !ater or distilled !ater using a !as#
bottle.

1=. Blot dr& t#e slide !it# bibulous paper.

11. )ie! t#e smear using a lig#t(microscope under oil(immersion.

&ifferences 'ncountered in a Real laborator(:

In an actual laborator& setting0 t#ere are certain important steps t#at are not necessaril&
applicable in a .irtual lab9

1. -l!a&s !ear glo.es0 and lab coat.
2. :ie &our #air properl& to pre.ent an& contamination $rom t#e culture &ou are !or1ing
!it#.
3. -$ter entering t#e lab0 ma1e sure t#at t#e microscope is !or1ing properl&. >culars
and ob?ecti.e lenses s#ould be cleaned be$ore and a$ter eac# use !it# lens paper.
4. -d?ust t#e illumination be$ore using t#e microscope.
5. %repare &our !or1 space @Laminar -ir Alo! CabinetB or lab benc# b& !iping do!n
t#e area !it# disin$ectant.
6. %roperl& ad?ust t#e $lame o$ t#e Bunsen burner. :#e proper $lame is a small blue coneC
it is not a large plume0 nor is it orange.
7. /ipe t#e glass slide !it# spirit and !a.e t#e slide o.er t#e Bunsen burner to remo.e
an& un!anted microorganisms in t#e slide.
8. Label one side o$ t#e glass slide !it#

1. 2our initials
2. :#e date
". /#ile $laming t#e inoculation loop be sure t#at eac# segment o$ metal glo!s
orange4red(#ot be$ore &ou mo.e t#e ne3t segment into t#e $lame.
1=. >nce &ou #a.e $lamed &our loop0 do not la& it do!n0 blo! on it0 touc# it !it# &our
$ingers0 or touc# it to an& sur$ace ot#er t#an &our inoculums. I$ &ou do touc# t#e tip to
anot#er sur$ace or blo! on it0 &ou !ill #a.e to re($lame t#e loop be$ore &ou proceed
to &our e3periment.
11. -llo! &our loop to cool be$ore &ou tr& to pic1 up &our organism. I$ &ou pic1 up
organism !it# a #ot inoculation loop0 &our cells !ill be 1illed and !ill a$$ect &our
results.
12. /#en remo.ing t#e caps $rom tubes0 al!a&s 1eep t#e caps in &our #and. 8e.er set
t#em on t#e table0 as t#e& could pic1 up contaminants.
13. -l!a&s #andle open tubes at an angle near to t#e $lame o$ t#e burnerC ne.er let t#em
point directl& up0 since airborne or ot#er en.ironmental organisms could $all into t#e
tube and cause contamination.
14. -s soon as &ou trans$er t#e organism into t#e slide0 $lame &our loop. 8e.er place a
contaminated tool on &our !or1benc#.
15. :r& to prepare a single cell la&er o$ organism @a t#in smearB. >t#er!ise t#e all cells
!ill appear as gram positi.e in t#ic1 area.
16. o not o.er!arm t#e cells. r& t#e slide t#oroug#l& prior to #eat $i3ing.
17. Dse &oung0 .igorous cultures rat#er t#an older cultures $or &our e3periment.
18. ecolorisation step s#ould not e3ceed t#e time limit.
1". /#ile !as#ing t#e slide a$ter staining0 do not let t#e !ater stream $all directl& on t#e
smear. :#is ma& disrupt t#e smear. Let t#e stream o$ !ater $lo! slo!l& along t#e
sur$ace0 suc# t#at onl& t#e stain is $looded and t#e smear is intact.
2=. -l!a&s pre$er to obser.e under 1=E $irst. :#is !ill gi.e &ou an idea o$ t#e location o$
a good area $or obser.ation. -$ter t#is &ou ma& pre$er to s!itc# o.er to 4=E.
21. o not e.er obser.e at a specimen at 1==E !it#out oil.
22. /#ile $ocusing t#e microscope0 glass slides s#ould be #andled care$ull& to a.oid t#e
c#ance o$ c#ipping or brea1ing.
23. -$ter t#e obser.ation0 !ipe t#e microscopic lens !it# an absorbent paper and co.er
t#e microscope properl&.
24. iscard all contaminated materials properl& and return &our supplies to t#e proper
storage locations0 and clean up &our !or1ing area.
25. -l!a&s disin$ect &our !or1 area !#en &ou are $inis#ed.
26. ,nsure proper #and !as#ing be$ore &ou lea.e $rom t#e laborator&.


)(pical %ram*negati+e bacteria:

1. Bordetella pertusis0 t#e causati.e agent o$ !#ooping coug#
2. Salmonella typhi0 t#e causati.e agent o$ t&p#oid
3. Vibrio cholera0 t#e causati.e agent o$ c#olera
4. Escherichia coli0 t#e normall& benign0 ubi6uitous0 gut(d!elling bacteria

)(pical %ram*positi+e bacteria:

1. Staphylococci suc# as Staphylococcus epidermidis and Staphylococcus aureus !#ic#
is a common cause o$ boils.
2. Streptococci suc# as t#e man& species o$ oral streptococci0 Streptococcus pyogenes
!#ic# causes man& a sore t#roat and scarlet $e.er and Streptococcus pneumoniae
!#ic# causes lobar pneumonia.
3. Clostridia suc# as Clostridium tetani0 t#e causati.e agent o$ tetanus @loc1?a!B.
4. Actinomyces suc# as Actinomyces odontolyticus !#ic# is $ound in mout#.
5. 'pecies o$ t#e genus Bacillus suc# as Bacillus subtilis !#ic# are common microbes
li.ing in soil.

*enerall& cocci are *ram(positi.e but t#ere are e3ceptions. :#e most signi$icant $rom a
clinical point o$ .ie! is t#e gonococcus0 Neisseria gonorrhoea !#ic# t&picall& appears as a
*ram(negati.e diplococcus loo1ing .er& muc# li1e a pair o$ 1idne& bean.
,b-ecti+es9

1. :o di$$erentiate bet!een t#e t!o ma?or categories o$ bacteria9 *ram positi.e and *ram
negati.e.
2. :o understand #o! t#e *ram stain reaction a$$ects *ram positi.e and *ram negati.e bacteria
based on t#e bioc#emical and structural di$$erences o$ t#eir cell !alls.

Clic1 to .ie! t#e -nimation

Principle:

Staining is an au3iliar& tec#ni6ue used in
microscopic tec#ni6ues used to en#ance t#e clarit&
o$ t#e microscopic image.'tains and d&es are !idel&
used in t#e scienti$ic $ield to #ig#lig#t t#e structure
o$ t#e biological specimens0 cells0 tissues etc.

:#e most !idel& used staining procedure in
microbiolog& is t#e *ram stain0 disco.ered b& t#e anis# scientist and p#&sician !ans
.hristian /oachim %ram in 1884. *ram staining is a di$$erential staining tec#ni6ue t#at
di$$erentiates bacteria into t!o groups9 gram(positi.es and gram(negati.es. :#e procedure is
based on t#e abilit& o$ microorganisms to retain color o$ t#e stains used during t#e gram
stain reaction. *ram(negati.e bacteria are decolori+ed b& t#e alco#ol0 losing t#e color o$
t#e primar& stain0 purple. *ram(positi.e bacteria are not decolori+ed b& alco#ol and !ill
remain as purple. -$ter decolori+ation step0 a counterstain is used to impart a pin1 color to
t#e decolori+ed gram(negati.e organisms.


"ig: !ans .hristian /oachim %ram

0mportance of a %ram Stain:

:#e *ram stain is a .er& important preliminar& step in t#e initial c#aracteri+ation and
classi$ication o$ bacteria. It is also a 1e& procedure in t#e identi$ication o$ bacteria based on
staining c#aracteristics0 enabling t#e bacteria to be e3amined using a lig#t microscope. :#e
bacteria present in an unstained smear are in.isible !#en .ie!ed using a lig#t microscope.
>nce stained0 t#e morp#olog& and arrangement o$ t#e bacteria ma& be obser.ed as !ell.
Aurt#ermore0 it is also an important step in t#e screening o$ in$ectious agents in clinical
specimens suc# as direct smears $rom a patient.

:#e *ram stain procedure enables bacteria to retain color o$ t#e stains0 based on t#e
di$$erences in t#e c#emical and p#&sical properties o$ t#e cell !all.

11 %ram positi+e bacteria9 'tain dar1 purple due to retaining t#e primar& d&e called
Cr&stal )iolet in t#e cell !all.
,3ample9 Staphylococcus aureus

"ig: %ram positi+e bacteria

2. %ram negati+e bacteria9 'tain red or pin1 due to retaining t#e counter staining d&e
called 'a$ranin.
,3ample9 Escherichia coli


"ig: %ram negati+e bacteria

Bacterial Morpholog(:

Bacteria are .er& small unicellular microorganisms ubi6uitous in nature. :#e& are
micrometers @1Fm G 1=
(6
mB in si+e. :#e& #a.e cell !alls composed o$ peptidogl&can and
reproduce b& binar& $ission. Bacteria .ar& in t#eir morp#ological $eatures.

)he most common morphologies are:

.occus 2pleural: .occi3:

'p#erical bacteriaC ma& occur in pairs @diplococciB0 in groups o$ $our @tetracocciB0 in grape(
li1e clusters @Staphylococci)0 in c#ains @StreptococciB or in cubical arrangements o$
eig#t or more @sarcinaeB.
Aor e3ample9 Staphylococcus aureus, Streptococcus pyogenes

Bacillus 2pleural: Bacilli3:

5od(s#aped bacteriaC generall& occur singl&0 but ma& occasionall& be $ound in pairs @diplo(
bacilliB or c#ains @streptobacilliB.
Aor e3ample9 Bacillus cereus, Clostridium tetani

Spirillum 2pleural: Spirilla3:

'piral(s#aped bacteria

Aor e3ample9 Spirillum, Vibrio, Spirochete species.

Some bacteria ha+e other shapes such as:

.occobacilli: ,longated sp#erical or o.oid $orm.
"ilamentous: Bacilli t#at occur in long c#ains or t#reads.
"usiform: Bacilli !it# tapered ends.



"ig: &ifferent bacterial morpholog(

%ram Stain Mechanism:

%ram Positi+e .ell 4all:

*ram(positi.e bacteria #a.e a t#ic1 mes#(li1e cell !all !#ic# is made up o$ peptidogl&can
@5=("=< o$ cell !allB0 !#ic# stains purple. %eptidogl&can is mainl& a pol&sacc#aride
composed o$ t!o subunits called 8(acet&l glucosamine and 8(acet&l muramic acid. -s
ad?acent la&ers o$ peptidogl&can are $ormed0 t#e& are cross lin1ed b& s#ort c#ains o$ peptides
b& means o$ a transpeptidase en+&me0 resulting in t#e s#ape and rigidit& o$ t#e cell !all. :#e
t#ic1 peptidogl&can la&er o$ *ram(positi.e organisms allo!s t#ese organisms to retain t#e
cr&stal .iolet(iodine comple3 and stains t#e cells as purple.
Lipoteic#oic acid @L:-B is anot#er ma?or constituent o$ t#e cell !all o$ *ram(positi.e
bacteria !#ic# is embedded in t#e peptidogl&can la&er. It consists o$ teic#oic acids !#ic#
are long c#ains o$ ribitol p#osp#ate anc#ored to t#e lipid bila&er .ia a gl&ceride. It acts as
regulator o$ autol&tic !all en+&mes @muramidases9 Bacterial en+&mes located in t#e cell !all
t#at cause disintegration o$ t#e cell $ollo!ing in?ur& or deat#.B



Medical 5ele.ance o$ *ram %ositi.e Cell /all9

L:- also #as antigenic properties t#at stimulate speci$ic immune responses !#en it is
released $rom t#e cell !all a$ter cell deat#. Cell deat# is mailnl& due to l&sis induced b&
l&so+&mal acti.ities0 cationic peptides $rom leucoc&tes0 or beta(lactam antibiotics.

%ram 5egati+e .ell 4all:

*ram(negati.e bacteria #a.e a t#inner la&er o$ peptidogl&can @1=< o$ t#e cell !allB and lose
t#e cr&stal .iolet(iodine comple3 during decolori+ation !it# t#e alco#ol rinse0 but retain t#e
counter stain 'a$ranin0 t#us appearing reddis# or pin1. :#e& also #a.e an additional outer
membrane !#ic# contains lipids0 !#ic# is separated $rom t#e cell !all b& means o$
periplasmic space.


Medical 5ele.ance o$ *ram 8egati.e Cell /all9

:#e cell !all o$ *ram(negati.e bacteria is o$ten a .irulence $actor t#at enables pat#ogenic
bacteria to cause disease. :#e .irulence o$ *ram(negati.e bacteria is o$ten associated !it#
certain components o$ t#e cell !all0 in particular0 t#e lipopol&sacc#aride @ ot#er!ise 1no!n
as L%' or endoto3inB. In #umans0 L%' elicits an innate immune response c#aracteri+ed b&
c&to1ine production and acti.ation o$ immune s&stem. In$lammation occurs as a result o$
c&to1ine production0 !#ic# can also produce #ost to3icit&.

Stain Reaction:
:#e $our basic steps o$ t#e *ram 'tain are9


13 6pplication of the primar( stain .r(stal 7iolet 2.73 to a heat*fi#ed smear of
bacterial culture1
C) dissociates in a6ueous solutions into C)H and Cl I ions. :#ese t!o ions t#en penetrate
t#roug# t#e cell !all and cell membrane o$ bot# *ram(positi.e and *ram(negati.e cells. :#e
C)H ions later interacts !it# negati.el& c#arged bacterial components and stains t#e bacterial
cells purple.


23 6ddition of %ram8s 0odine1
Iodine @I I or I3 IB acts as a mordant and as a trapping agent. - mordant is a substance t#at
increases t#e a$$init& o$ t#e cell !all $or a stain b& binding to t#e primar& stain0 t#us $orming
an insoluble comple3 !#ic# gets trapped in t#e cell !all. In t#e *ram stain reaction0 t#e
cr&stal .iolet and iodine $orm an insoluble comple3 @C)(IB !#ic# ser.es to turn t#e smear a
dar1 purple color. -t t#is stage0 all cells !ill turn purple.

33 &ecolori9ation with :$; eth(l alcohol.
-lco#ol or acetone dissol.es t#e lipid outer membrane o$ *ram negati.e bacteria0 t#us
lea.ing t#e peptidogl&can la&er e3posed and increases t#e porosit& o$ t#e cell !all. :#e C)(I
comple3 is t#en !as#ed a!a& $rom t#e t#in peptidogl&can la&er0 lea.ing *ram negati.e
bacteria colorless.

>n t#e ot#er #and0 alco#ol #as a de#&drating e$$ect on t#e cell !alls o$ *ram positi.e
bacteria !#ic# causes t#e pores o$ t#e cell !all to s#rin1. :#e C)(I comple3 gets tig#tl&
bound into t#e multi(la&ered0 #ig#l& cross(lin1ed *ram positi.e cell !all t#us staining t#e
cells purple.

:#e decolori+ation step must be per$ormed care$ull&0 ot#er!ise o.er(decolori+ation ma&
occur. :#is step is critical and must be timed correctl& ot#er!ise t#e cr&stal .iolet stain !ill
be remo.ed $rom t#e *ram(positi.e cells. I$ t#e decolori+ing agent is applied on t#e cell $or
too long time 0 t#e *ram(positi.e organisms to appear *ram(negati.e. Dnder(decolori+ation
occurs !#en t#e alco#ol is not le$t on long enoug# to !as# out t#e C)(I comple3 $rom t#e
*ram(negati.e cells0 resulting in *ram(negati.e bacteria to appear *ram(positi.e.

3 .ounterstain with Safranin
:#e decolori+ed *ram negati.e cells can be rendered .isible !it# a suitable counterstain0
!#ic# is usuall& positi.el& c#arged sa$ranin0 !#ic# stains t#em pin1. %in1 colour !#ic#
ad#eres to t#e *ram positi.e bacteria is mas1ed b& t#e purple o$ t#e cr&stal .iolet @Basic
fuschin is sometimes used instead o$ sa$ranin in rare situationsB.



"ig: .olour changes that occur at each step in the staining process
,b-ecti+e:

:o stud& and gain e3pertise on di$$erential and c&tological staining tec#ni6ues.

)heor(:

i$$erential staining is a tec#ni6ue t#at #elps to c#aracteri+e t#e microorganisms depending
on t#e di$$erence in t#e p#&sical and c#emical nature o$ t#e microorganism. :#e di$$erential
and c&tological staining tec#ni6ues discussed in t#is c#apter #elp to di$$erentiate bet!een
acid $ast and non acid $ast cells and to .isuali+e t#e intracellular constituents o$ t#e microbial
cells including endospores0 capsules0 metac#romatic granules0 and $lagella. In t#is tec#ni6ue
t#e di$$erential stains applied on t#e bacterial smear re.eals t#e di$$erent t&pes o$ cells at one
point0 re.eals one part o$ t#e cell !it# one color and ot#er parts a di$$erent color.

&ifferential Staining
*ram stain tec#ni6ue is a di$$erential staining tec#ni6ue0 !#ic# separates bacteria into t!o
groups @discussed in earlier c#aptersB0 *ram(positi.e bacteria and *ram(negati.e bacteria.
-not#er di$$erential staining tec#ni6ue is acid($ast tec#ni6ue !#ic# di$$erentiates species o$
M&cobacterium $rom ot#er bacteria. M&cobacterium species due to its special cell !all resist
t#e e$$ect o$ t#e decolori+er acid(alco#ol and retain t#e color o$ t#e primar& stain
carbol$uc#sin stain and stains t#e acid $ast cells in brig#t red color. >t#er bacteria lose t#e
stain and ta1e on t#e subse6uent color o$ t#e counter stain met#&lene blue stain and stain t#e
cell as blue. ,ndospore staining is a special staining tec#ni6ue0 to obser.e bacterial spores0
!#ere t#e spores ta1e t#e color o$ t#e primar& stain Malac#ite green0 !#ile t#e counterstain0
sa$ranin0 gi.e color to t#e non(spore $orming bacteria. 'peci$ic stains suc# as nigrosine0
Indian in1 etc #elp to .isuali+e t#e bacterial stains !#ic# cannot be stained b& usual staining
met#ods. :#e metac#romatic granules0 t#e c#aracteristic $eature o$ Corn&bacterium
dip#t#eriae0 can be di$$erentiated $rom t#e bacterial cells !it# t#e #elp o$ -lbert staining
tec#ni6ues. Alagella are t#e t#in delicate structure $or bacterial motilit&. :#e t#in structures o$
t#e bacterial $lagella ma1e it di$$icult to obser.e under brig#t $ield microscope. 'pecial stains
and mordents suc# as Lei$son;s stain are re6uired $or staining t#e bacterial $lagella.


'ndospore Stain:

Schaeffer*"ulton Method

,ndospore staining is used to .isuali+e speciali+ed cell structures. :#e endospore stain is used
to determine t#e #ig#l& resistant spores o$ certain microorganisms !it#in t#eir .egetati.e
cells. :#e multiple t#ic1 coats o$ t#e spore made t#e endospore resistant to stain !it# most
d&es. In 'c#ae$$er(Aulton met#od0 t#e primar& stain0 Malac#ite *reen0 is added o.er t#e #eat
$i3ed bacterial smear and #eated o.er a steam bat# $or $e! minutes. :#is !ill so$ten t#e #ard
outer co.erings o$ t#e spore and t#e primar& stain gets stic1 to t#e spore. /#en ta1en $rom
t#e steam bat# $ollo!ed b& $urt#er cooling #ardens t#e outer la&er o$ t#e spore. uring t#is
stage bot# t#e spore and .egetati.e cells appear as green in color. But later t#e t#ic1 outer
la&er ma1es t#e spore resistant to t#e action o$ decolori+ing agent @!aterB0 but #o!e.er0 !ater
can easil& decolori+e t#e .egetati.e cells. /#en counterstained !it# 'a$ranin0 .egetati.e
cells are easil& stained !it# 'a$ranin0 and t#e cells appear in red or pin1 color.

&orner Method

orner met#od is an alternati.e met#od $or staining t#e endospores. In t#is staining process0
2( 3ml brot# culture o$ microorganism and e6ual .olume o$ Basic $uc#sin is #eated in a
!ater bat# at 1==
o
C $or 1= minutes. :#e e3tensi.e boiling so$tens t#e structure o$ t#e
bacterial spores and t#e basic $usc#in get into t#e spores. -$ter t#e boiling process0 t#e
microbial culture(basic $usc#in stain is allo!ed to cool $or sometime !#ic# #ardens t#e outer
co.ering o$ t#e spore. In order to gi.e a color to t#e bac1ground and to di$$erentiate bet!een
t#e .egetati.e cells and endospores0 a t#in $ilm o$ loop$ul o$ t#e microbial culture(basic
$usc#in stain and 2 nigrosin on a clean glass slide. 'ince nigrosin is negati.e c#arged0 t#e
bacterial cells cannot easil& ta1en up b& t#e cells. -$ter staining t#e .egetati.e cells appear
become colorless0 t#e endospores stains as red !#ic# can be present as terminal or sub
terminal. :#e bac1ground is stained as dar1 b& t#e 8egrosin stain.

6cid "ast Stain:

<iehl 5eelsen Method

:#e Jie#l 8eelsen Met#od is used $or staining t#e M&cobacterium in clinical specimens. :#e
t#ic1 outer !a3& co.ering @m&colic acidB o$ t#e M&cobacterium cell !alls act as a barrier
and does not allo! all t#e stains to enter into t#e cell. In order to .isuali+e t#ese cells #ig#er
concentrations o$ t#e staining solution is needed and once t#is stain enters t#e cell0 it is too
di$$icult to remo.e t#e stain using a decolori+er. /#en t#e clinical specimen is stained !it#
basic d&es suc# as carbol$uc#sin @primar& stainB !it# t#e continuous application o$ #eat0
so$tens t#e !a3& lipid outer co.ering o$ t#e cell !all and t#e stain readil& enters t#e cell and
stains t#e cell c&toplasm. /#en decolori+ing agents suc# as acid(alco#ol is added o.er t#e
primar& stain0 some bacterial cells cannot be easil& decolori+ed and suc# bacterial cells are
called as acid $ast bacteria. :#e bacteria !it# #ig# concentration o$ lipid are easil&
decolori+ed b& t#e decolori+ing agent and are said to be non(acid $ast bacteria. Ainall&0 t#e
addition o$ t#e counter stain0 Met#&lene blue0 d&es t#e colorless non acid $ast cells as blue
t#us di$$erentiating t#em $rom t#e pin1 acid $ast bacteria !#ic# are una$$ected b& t#e
Met#&lene blue.

.apsule Stain:

Capsules are t#e gelatinous outer la&er o$ t#e bacterial cells and t#ese structures cannot retain
t#e color o$ t#e staining agents. :#e capsules can be .isuali+ed b& means o$ t!o met#ods.

Positi+e .apsule Staining

'ince capsule is !ater soluble in nature0 it is too di$$icult to stain t#e capsule !it# normal
staining met#ods. :#e positi.e capsule staining met#od @-nt#on& Met#odB uses t!o reagents
to stain t#e capsular material. :#e primar& stain Cr&stal .iolet is applied o.er a non #eat $i3ed
bacterial smear so t#at bot# t#e bacterial cells and capsular material ta1e up t#e color o$ t#e
primar& stain. :#e ionic nature o$ t#e bacterial cell binds t#e cr&stal .iolet stain more strongl&
!#ile t#e non(ionic nature o$ t#e capsule get ad#ere !it# t#e cr&stal .iolet stain. /#en t#e
decolori+ing agent copper sul$ate is added o.er t#e bacterial smear0 t#e loosel& ad#ered
cr&stal .iolet stain is !as#ed o$$ $rom t#e capsular material !it#out remo.ing t#e tig#tl&
bound cr&stal .iolet $rom t#e cell !all. :#e capsular material absorbs t#e lig#t blue color o$
t#e copper sul$ate in contrast to t#e purple bacterial cell.

5egati+e .apsule Staining

-not#er simple met#od to .isuali+e t#e bacterial capsules is b& using negati.e staining
:ec#ni6ue. uring staining t#e non #eat $i3ed bacterial smear !it# t#e acidic stains suc# as
8igrosin !ill not penetrate t#e bacterial cells @since bot# acidic stain and bacterial sur$ace #as
negati.e c#argeB. Instead t#e acidic stain deposits around t#e bacterial cells and create a dar1
bac1 ground and t#e bacteria appear as unstained !it# a clear area around t#em0 capsule.
5ote9 I$ &ou #eat $i3 t#e bacterial smear $or capsule staining0 t#e cells !ill s#rin1 creating a
#allo! +one around t#e bacterial cell and !ill be mista1en $or t#e capsule.

Metachromatic %ranule Staining: 6lbert8s Staining

-lbert;s staining is speciall& demonstrates t#e presence or absence o$ t#e metac#romatic
granules0 a c#aracteristic $eature o$ Corn&bacterium dip#t#eriae. uring gram staining i$ a
smear appears as purple rods !it# straig#t or slig#tl& cur.ed !it# clubs at t#e end0 !it# a
c#aracteristic ) s#ape t#en it is suspected as Corn&bacterium dip#t#eriae. :#e $urt#er
con$irmation can be done b& -lbert;s staining tec#ni6ue. :#is tec#ni6ues emplo& t!o stains
-lbert 'tain -@ combination o$ :oluidine blue0 Malac#ite green0 *lacial acetic acid0 alco#ol
and distilled !aterB and -lbert 'tain B@Iodine0 potassium iodide and distilled !aterB. -$ter
t#e staining process t#e metac#romatic granules appear as bluis# blac1 !#ere as t#e cell
appears as green color.

"lagella Staining

Bacteria are motile b& means o$ $lagella. :#e $lagella are too t#in to be seen in ordinar&
stains0 special stains and tec#ni6ues needed to .isuali+e t#e $lagellum enoug# stain to obtain a
.isible t#ic1ness. 'peciali+ed stains are usuall& $ound in microbiolog& laboratories to detect
t#e presence or absence o$ $lagella to indicate t#e nature o$ t#at bacterium @motile4non(
motileB.


Liefson Stain

/#en a bacterial culture is stained !it# Lie$son stain0 t#e tannic acid component o$ t#e stain
produce a colloidal precipitate !#ic# can be ta1en up b& t#e bacterial $lagella !ill become
colori+ed !#ic# can be easil& .isuali+ed using microscop&. :#e concentration o$ t#e tannic
acid and d&e is important in staining t#e bacterial $lagella !#ile t#e alco#ol concentration in
t#e Lie$sons stains #elps in maintaining t#e solubilit& o$ t#e components. >n microscopic
obser.ation0 t#e bacterial cells and $lagella !ill stain red and t#e $lagellar arrangement can be
.isuali+ed easil&. :#e age o$ t#e bacterial culture0 condition o$ staining solutions0
concentrations o$ t#e staining solution etc can also a$$ect t#e staining reaction.
Materials Required:

'ndospore Staining

Schaeffer*"ulton Method
1. Clean glass slide in bo3
2. Inoculation loop
3. :est organism
4. -bsorbent paper
5. Boiling !ater in tripod stand
6. /ater in !as# bottle
7. Bibulous paper
8. :!ee+ers
". Microscope
1=. %rimar& 'tain I Malac#ite green
11. Counter stain ( 'a$ranin

Staining Steps:

1. -septicall& trans$er t#e bacterium !it# an inoculating loop to a clean glass slide and
prepare a t#in smear o$ t#e bacterium.
2. -ir dr& and #eat($i3 t#e bacterial smear.
3. Co.er t#e bacterial smear !it# a piece o$ absorbent paper cut to $it t#e smear and
slide.
4. %lace t#e slide o.er a container o$ boiling !ater.
5. 'aturate t#e absorbent paper !it# malac#ite green stain solution and steam $or 5
minutes. Keep t#e paper moist b& adding more stain as re6uired.
6. 5emo.e t#e absorbent paper using $orceps0 allo! t#e slide to cool0 and rinse t#e slide
!it# !ater $or 3= seconds.
7. /as# t#e slide !it# !ater.
8. Counter stain !it# sa$ranin $or 3= seconds.
". /as# t#e slide !it# !ater and blot dr& t#e slide.
1=. ,3amine t#e slide under t#e oil immersion lens $or t#e presence o$ endospores.

'#pected Result:

>n microscopic obser.ation endospores appear in green color and t#e .egetati.es cells as
pin1.


&orner Method

1. :est organism
2. *lass slides in t#e bo3
3. Inoculation loop
4. Basic $uc#sin
5. 8egrosin
6. Microscope
7. /ater bat#

Staining Steps:

1. Mi3 an a6ueous suspension o$ bacteria !it# an e6ual .olume o$ basic $uc#sin in a test
tube.
2. Keep t#e test tube in a boiling !ater bat# $or 1= minutes.
3. -dd a loop$ul o$ t#e boiled basic $uc#sin(organism suspension to one side o$ t#e glass
slide.
4. 7< nigrosin stain is added o.er t#e boiled basic $uc#sin(organism suspension.
5. Ma1e a t#in smear o$ t#e culture using a second glass slide ma1ing a 45 degree angle
!it# t#e $irst slide.
6. -ir dries t#e smear $or some time.
7. ,3amine t#e slide under t#e oil immersion lens $or t#e presence o$ endospores.

'#pected Result:

>n microscopic obser.ation0 endospores !ill appeared as red0 and t#e .egetati.e cells as
colourless0 in a dar1 bac1ground.

6cid "ast Staining

<iehl 5eelsen Method

1. Clean glass slide in t#e bo3
2. Inoculation loop
3. :est organism
4. -bsorbent paper
5. Bea1er !it# !ater in tripod stand
6. :!ee+ers
7. Bibulous paper
8. Microscope
". %rimar& stain ( Carbol $usc#in
1=. ecolori+er ( 25< sulp#uric acid
11. Counter stain ( Met#&lene blue


Staining Steps9

1. -septicall& trans$er t#e bacterium !it# an inoculating loop to a clean glass slide and
prepare a t#in smear o$ t#e bacterium.
2. 7eat( $i3es t#e bacterial smear.
3. Co.er t#e bacterial smear !it# a piece o$ absorbent paper cut to $it t#e smear and
slide.
4. %lace t#e slide o.er a container o$ boiling !ater.
5. 'aturate t#e paper !it# carbol $usc#in steam $or 5 minutes. Keep t#e paper moist b&
adding more stain as re6uired.
6. 5emo.e t#e absorbent paper using t!ee+ers0 t#e e3cess stain is !as#ed !it# !ater
and allo! t#e slide to cool $or some time.
7. ecolori+e t#e slide !it# 25 < sul$uric acid.
8. 5inse t#e slide !it# !ater.
". Counter stain t#e cells !it# Met#&lene blue stain $or 45 seconds.
1=. /as# t#e slide !it# !ater and blot dr& t#e slide.
11. ,3amine t#e slide under t#e oil immersion lens to obser.e t#e acid $ast or non acid
$ast(cells

'#pected Result:

>n microscopic obser.ation0 t#e acid $ast bacterium !ill appear as pin1 coloured cells.

.apsule Staining

Positi+e .apsule Staining

1. *lass slides in t#e bo3
2. Inoculation loop
3. :est organism
4. Cr&stal .iolet
5. 2=< copper sul$ate
6. Bibulous paper
7. Microscope

Staining Steps9

1. 2 drops o$ Cr&stal )iolet @primar& stainB is added to one side o$ t#e glass slide.
2. - loop$ul o$ bacterial culture is mi3ed !it# Cr&stal )iolet stain.
3. Ma1e a t#in smear o$ t#e culture using a second glass slide ma1ing a 45 degree angle
!it# t#e $irst slide.
4. ecolori+e and counter stain t#e bacterial smear !it# 2=< copper sul$ate.
5. -ir dries t#e smear $or some time.
6. ,3amine t#e slide under t#e oil immersion lens $or t#e presence o$ capsules o$
bacteria.

5egati+e .apsule Staining

1. *lass slides in t#e bo3
2. Inoculation loop
3. Bunsen burner
4. :est organism
5. 8egrosine
6. Microscope

Staining Steps:

1. -dd one drop o$ nigrosin onto t#e end o$ a clean slide.
2. - loop$ul o$ bacterial culture is added onto t#e drop o$ nigrosin and mi3 !ell.
3. Ma1e a t#in smear o$ t#e culture using a second glass slide ma1ing a 45 degree angle
!it# t#e $irst slide.
4. -ir dries t#e smear $or some time.
5. ,3amine t#e slide under t#e oil immersion lens $or t#e presence o$ capsules o$
bacteria.

'#pected Result:

>n microscopic obser.ation0 t#e capsulated bacterium can be obser.ed in a dar1 bac1ground.

Metachromatic %ranule Staining

6lbert8s Staining
1. *lass slides in t#e bo3
2. Inoculation loop
3. :est organism
4. -lbert stain -
5. -lbert stain B
6. /ater in t#e !as# bottle
7. Bibulous paper
8. Microscope.

Staining Steps:

1. -septicall& trans$er t#e bacterium !it# an inoculating loop to a clean glass slide and
prepare a t#in smear o$ t#e bacterium.
2. -ir dries t#e smear $or some time.
3. Co.er t#e air dried smear !it# -lbert 'tain -.
4. 5emo.e t#e e3cess stain b& !as#ing !it# !ater.
5. Co.er t#e bacterial smear !it# -lbert 'tain B. Let it stand $or 2 minutes.
6. /as# t#e slide !it# !ater and blot dr& t#e slide.
7. ,3amine t#e slide under t#e oil immersion lens $or obser.ing t#e metac#romatic
granules o$ bacteria.

'#pected Result:

:#e metac#romatic granules appear as bluis# blac1 and t#e bodies o$ t#e bacillus appear as
green.

"lagella Staining

Liefsons "lagella Stain
1. Clean glass slides in t#e bo3
2. Inoculation loop
3. :est organism
4. Lei$son;s stain
5. /ater
6. Microscope

Staining Steps:

1. Ma1e a rectangular border on t#e sur$ace o$ t#e glass slide !it# a grease pencil.
2. -septicall& trans$er t#e bacterium !it# an inoculating loop to a clean glass slide.
3. -llo! t#e smear to run do!n in an inclined slide.
4. Alood t#e slide !it# Lie$sonLs Alagella 'tain and allo! staining $or 1= I 15 minutes. -
$ine rust colored precipitate $orms t#roug#out t#e slide.
5. -ir dries t#e slide.
6. ,3amine t#e slide under t#e oil immersion lens to obser.e t#e t&pe o$ $lagella.

%ra( Method
1. 8utrient agar plate !it# test organism
2. 'calpel
3. :!ee+ers
4. *lass slides in t#e bo3
5. Basic $usc#in
6. Microscope


Staining Steps:

1. Cut a small portion o$ t#e agar !it# t#e test organism using a sterile scalpel.
2. 5aise t#e agar bloc1 !it# t#e aid o$ a $orceps.
3. %lace t#e agar bloc1 in a sterile glass slide0 !it# t#e bacterial culture $acing
do!n!ards. Keep it undisturbed $or some time.
4. :a1e t#e agar bloc1 and discard it in #a+ardous bo3.
5. -llo! t#e bacterial culture in t#e glass slide to air dr& $or some time.
6. Alood t#e bacterial culture !it# basic $uc#sin until a golden precipitate is obser.ed.
7. *entl& !as# !it# !ater.
8. ,3amine t#e slide under t#e oil immersion lens to obser.e t#e t&pe o$ $lagella.

'#pected Result:

>n microscopic obser.ation t#e $lagella present around t#e bacterial cells are obser.ed.