dr. shivam seminar staining mehtods

7/24/2019 Dr. Shivam Seminar Staining Mehtods

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STAINING

METHODS


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Signifcance o staining

Dyes give colour to microbes.

Iincrease visibility.

For recognition; accentuate specifc morphologicaleatures.

Preservation or uture study.


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Dyes

ACIDIC D!S

"egative charged groups#inds to positively charged cellstructures

Acid ushin$ rose bengal and eosin

#ASIC D!S

Positively chargedgroups#inds to negativelycharged cellstructures%ethylene blue$ #asicuchsin$Crystal voilet.


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Staining methods or bacteria

Simple staining

highlight entire microorganism.

&ne reagent

%ethylene blue or basic uchsin.

Di'erential staining

(rouping o bacteria.

%ultiple reagents

(ram staining and Acidast staining


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Structural) special staining*

%ultiple reagents are used .

+sed to identiy and study the structure o bacteria

Alberts staining ,

-olutin granule !ndoSpore staining

Flagellar staining

Capsular Staining"egativestaining/


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Staining or parasites

Iron0haemato1ylin stain

2richrome stain

%odifed acid0ast stain

lishman

and giemsa staining


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Staining o ungus

Calco3our staining ,

For tissues , PAS Periodic acid0 schi' /

(%S (rocott0gomori methanamine0silver /

Staining or viruses0

Inclusion bodies Immunopero1idasestaining


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(4A% S2AI"I"(

5667

8A"S C84IS2IA" (4A%

DA"IS8 #AC2!4I&9&(IS2 56:05


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2ypes

(ram positive (ram

negative

!1ceptions

5. Chlamydia 0e1ist e1clusively =ithin hostcells

>. %ycoplasma$ +reaplasm09ac? cell =all

. Spirochaetes0Insu@cient dimension tobe resolved by lightmicroscopy.


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hen stained =ith basic dye and iodine

Decolourisation =ith acetone or alcohol

4esist0 gram positive Do not resist0gram

negative


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Cell =all theory

#asic dye B Iodine

Dye iodine comple1

Attach to bacterial cell =all.

5. (ram positive0 thic? and dense

peptidoglycan layer =hich is lesspermeable to dye0iodine comple1.

>. Iodine binds =ith peptidoglycan anddecreases its permeability.

. Also 2eichoic acid combines =ith basic dye.


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(ram positive

5. (ram positive0 thic? and densepeptidoglycan layer =hich is lesspermeable to dye0iodine comple1.

>. Iodine binds =ith peptidoglycan anddecreases its permeability.

. Also 2eichoic acid combines =ithbasic dye.

7. Protoplasmic theory0 (ram positivehave acidic protoplasm =hich helpsin retainin basic d e.


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(ram negative

9ipid theory0 (ram negative have more lipids intheir cell =all =hich are easily dissolved by

decolouriser.thereby$ increasing cell =allpermeability

%agnesium ribonucleate theory0 (ram

negative has more magnesium ribonuclease =hichhelps in orming a dye0iodine comple1. 2his dye0iodine magnesium ribonuclease comple1 is noteasily removable by decolouriser.


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Ideal conditions &+"( C+92+4!

28I" S%!A4

F4!S8 4!A(!"2

C&"24&9 C+92+4!


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StepsPrepare a smear.

Fi1ing either physical and chemical.

Application o P4I%A4 S2AI" -iolet dye/.

Application o %&4DA"2 Iodine solution/.

Application o D!C&9&+4I!4.

Application o C&+"2!4S2AI" Pin?)4ed dye/.


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GRAM

POSITIVE

BACTERIA

(VIOLET)


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GRAM

NEGATIVE

BACTERIA

(RED)


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BothGrampositive

+

Gram

e!ative


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ORIGINAL MET"OD O#GRAM ($%%&)

PRIMAR'STAIN

IODINESOLTION

DECOLORIER CONTERSTAIN

REAGENT ANILINE *GENTIANVIOLET

IODINE+POTASSIM IODIDE+ATER

ABSOLTEALCO"OL

BISMARC,BRON

TIME

&- se. $ mi ti/ .o/o0r.aeses to

.ome

&- se.


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opelo' E #eermans %odifcation 5>/

PRIMAR'STAIN

IODINESOLTION


REAGENT MET"'LVIOLET

STAIN ($1)+

SODIM

BICARBONATE

(-1)

IODINE+

SODIM"'DRO2IDE

(&1)

ACETONE

( $33 1)

BASIC#SC"IN

(343-1)

TIME - MINS 5 MINS 5 * 6 SECS 63 SECS


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AD-A"2A(!*

5/ sodium bicarbonate , strengthens gram0positive staining /

>/ sod. 8ydro1ide 0 more al?aline sol. strongergram positive staining/

/ Acetone 0 astest and most specifc.

7/4ecommended or anaerobic organisms.


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DISAD-A"2A(!*

5/ %ethyl -iolet E Sodium #icarbonateprecipitate =ithin e= days

cannot be ?ept.

>/Acetone 0 evaporate astly

short period o e1posure

di@cult to control0manyslides

P4!S2&" E %&44!99S


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P4!S2&" E %&44!99S%&DIFICA2I&" 5/

PRIMAR'STAIN

IODINESOLTION

DECOLORIER

CONTERSTAIN

REAGENT CR'STAL

VIOLET

+MET"'LATED SPIRIT

+

AMMONIMO2ALATE $1

IODINE

+POTASSIMIODIDE

+DISTILLEDATER

ACETONE

+LI7OR IODI#ORTIS(IODINE +POT IODIDE+

MET"'LATEDSPIRIT +DISTILLEDATER)

(346-1io8ie)

IE"L *

NEELSEN9SCARBOL#SC"IN

+DISTILLEDATER

(-1)

63 SECONDS 63 63 SECONDS 63 SECONDS


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Advantage*

5/(ram positive staining can bestrengthened by addition o ammoniumo1alate.

>/Addition o small conc. o iodine to

acetone slo=s its rate o decolouriHation.

Disadvantage*

5/ Iodine0Acetone produces an irritantaerosol =hen e1pelled.


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%&DIFI!D P4!S2&" E %&44!99S%!28&D 5/

PRIMAR'STAIN

IODINESOLTION

DECOLORIER

CONTERSTAIN

REAGENT CR'STAL

VIOLET+ABSOLTEALCO"OL+DISTILLED

ATER

IODINE

+POTASSIMIODIDE+DISTILLEDATER

EA,

IODINEACETONESOLTION(3436- 1)

IE"L:

NEELSEN9SCARBOL#SC"IN+DISTILLEDATER

TIME 63 SECONDS 63 SECONDS $3 SECONDS 63SECONDS


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Advantage * 4educed conc. o Iodine inAcetone to one0tenth .:J/.


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K!"S!"S %&DIFICA2I&"

PRIMAR'STAIN

IODINESOLTION



+DISTILLEDATER

(34-1)

IODINE+

POTASSIM IODIDE

+DISTILLEDATER

ABSOLTEALCO"OL

($33 1ET"ANOL)

NETRALRED

+$ 1 ACETICACID

+DISTILLEDATER

(34$1)

TIME 63 SECONDS 63SECONDS

NTIL COLORCEASES TOCOME

$ * 5MINTES


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For e1amination o smears or (onocococci

%eningococci.

!1cellent results =ith reedom rom deposit.

hen organisms are scanty LSA"DIF&4DSC&+"2!4S2AI"M used.

%alachite green$Pyronine$D/


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!I(!42S %&DIFICA2I&"

PRIMAR'STAIN

IODINESOLTION


REAGENT SATRATEDALCO"OLIC

SOLN4 O#GENTIANVIOLET

+- 1P"ENOL

IODINE+

POTASSIMIODIDE

+DISTILLEDATER

ANILINE+

2'LOL

CARMINICACID

+POTASSIMALM

+DISTILLEDATER

TIME 5 * 6MINTES

$ MINTE NTIL STAINCEASES TOCOME OT

$3 MINTES


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Advantage*4ecommended or

staining sections otissueaniline0 Nylol asdecolouriHer/

Disadvantage* Primary stain mi1ture0tends toprecipitate


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7IC, GRAM9S MET"OD #OR SINGLESLIDES

PRIMAR'STAIN

IODINESOLTION

DECOLORIER

CONTERSTAIN

REAGENT CR'STALORMET"'LVIOLET

($1)

IODINESOLTION

ACETONE BASIC#SC"IN

(34-1)

TIME - SECONDS - SECONDS 5 SECONDS - SECONDS


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GRAM MET"OD #OR MLTIPLESLIDES

PRIMAR'STAIN

IODINESOLTION



LGOL9SIODINE $1

IODINE *ACETONE(s/o; a.ti!8e.o/o0ri


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STAINING O# ACID #ASTBACILLI

E"RLIC"=S MET"OD4

IE"L:NEELSEN MET"OD4


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E"RLIC"9S MET"OD

Ori!ia/ metho8($%%5)4

se8 Ai/ie:Getia Vio/etstai4

Stro! Nitri. A.i8 as8e.o/o0ri


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DISADVANTAGE>

&rdinary aniline dye sol. do not readilypenetrate

tubercle bacilli.

rest structures and cells ,

counterstain


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I!890"!!9S!"S %!28&D

(iven in 566:/.

Po=erul staining solution used containing

Phenol.

Application o heatDo not boil/.

Any strong acid as DecolouriHer.

Counter stain.


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principal


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ACID #ASTBACILLI

PRIMAR'STAIN

RED


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T'PES

N Metho8 A (si!/e smear9s)4

N Metho8 B (m0/tip/e

smear9s)4


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MET"ODA

PRIMAR' STAIN DECOLORIER CONTERSTAIN

REAGENT

BASIC #C"SIN ( $1)

+

P"ENOL +ALCO"OL

+D

+

"eat(8o ot ?oi/)

CONCENTRATEDSLP"RICACID

(531)

+

D

LOE##LER9SMET"'LENEBLE

OR

MALAC"ITEGREEN

TIME - mi $3 mi $-:53 se.


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MET"ODB

(MLTIPLESLIDES)

PRIMAR' STAIN DECOLORIER CONTERSTAIN

REAGENT

N Car?o/@0.hsi

+"eat(Do ot?oi/)

A.i8:A/.oho/

531 s0/ph0ri.a.i8

A!ai531"5SO&

Di/0teMa/a.hite

Gree

TIME mi

6 mi

- mi

- mi

53:63 se.

I%P&42A"2 P&I"2S


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I%P&42A"2 P&I"2S Alcohol as secondary decolouriHation*

5/DecolouriHation is completed moreOuic?ly.

>/Identifcation o 2ubercle bacillisome

acid0ast bacilli are decolouriHed by alcohol/.

/ept or > min.

Acid0Alcohol as decolouriHer*

5/ J8C9 in /!1pensive$less corrosive$convienient to ma?e.

MODI#ICATIONS O# N


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MODI#ICATIONS O# NMET"OD

A. Conc. & 8>S7-aries /

BACTERIA PRIMAR'STAIN

DECOLORIER

CONTERSTAIN

LEPROS'BACILLI

SAME -1 "5SO& SAME

NOCARDIA SAME $1"5SO& SAME

SPORESSAME

345-1:34-1"5SO&

SAME


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#. Cold %ethod inyouns %ethod/

8eating is not reOuired. Phenol concentration in carbol uchsin is

increased.

Duration o carbol uchsin staining is more.

C. %alachite green can be used ascounterstain.

D. Acid0alcohol can be used as decolouriser.


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Alberts Staining

2o demonstrate the metachromatic granules oCorynebacterium diphtheriae

Stain* Alberts stain 2oludine blue$%alachite

green$ (lacial aceticacid$

Alcohol Q Alberts iodine.


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Steps %a?e flm$ dry$ f1.

Cover slide =ith Alberts stain or 0:min.

ash in =ater.

Cover slide =ith Alberts iodine or 5min. ash and blot dry.

4!S+92S*

(ranules000000000#9+IS80#9AC

&rganisms00000009I(82 (4!!"


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principle


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ALBERT9SSTAINING


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thas

dr. shivam seminar staining mehtods

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