microbiology project - latest in staining

8/17/2019 Microbiology Project - Latest in Staining

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Microbiology- TheLatest on Staining

A Presentation By

Caitlyn Davis andShyamalee Ramaraj


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Introduction

With the development of the microscope came the need todistinguish microscopic organisms from their environment. Bythemselves, they ere nearly invisi!le, containing transparentcytoplasm"s#, therefore they ere una!le to !e seen even ith anaided eye. Staining resolved this pro!lem !y enhancing the

visuali$ation of cells !y increasing contrast of the images of specimenseen under the microscope, as ell as pave ay for the study ofmicro!e morphology, arrangement, structure, si$e, meta!olism, and!ehavior. %hrough a variety of staining methods, various strains ofmicro!es have !een identified and utili$ed in the research fields ofa&uatic, food, and medical micro!iology. 'any micro!iological

advancements today can !e attri!uted to proper applications ofstaining.


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Introduction (cont.)

%his presentation aims to outline the significance of theapplication or procedures of staining methods andshocase past and present staining techni&ues inmicro!iology.

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Objectives

%he o!jectives of this presentation include the folloing*

%o revie !asic staining procedures discussed in the la!oratory

%o introduce ne methods employed today in individual cells of

tissues, viruses, and isolated culture samples of eu+aryotes and!acteria

%o understand the purposes and importance of simple, differential,and speciali$ed staining and its implications in areas such asa&uatic, medical, and food micro!iology as ell as epidemiology

and etiology %o help one to comprehend the value of staining and instill

interest and appreciation for the field of micro!iology and itsdevelopments.


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Outline/Overview o !resentation

%his presentation ill !e delivered in the folloing manner !eyondthis point*

. -ideo -irus staining

/. 0uic+ content revie of staining methods

. Purpose of Staining

/. Staining methods summary ta!le

1. 2noledge inventory

3. 4ther stains "not discussed in la!oratory#

5. -ideo 6luorescence staining

1. 7volution of Staining procedures in 'icro!iology 8 9ames to

Remem!er

3. :atest innovations in staining "main focus#

5. %a+e home lesson"s#


6/19

Short -ideo; 9egative stains and -iruses


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Staining, as mentioned previously, is important in the

identification of !acteria cells, eu+aryotic cells, and theirconstituent components ithin them "i.e., organelles#. %heapplication of certain light and dye com!inations can evenallo for the identification of viruses ithin host cells.

'orphology, si$e, and arrangement of targeted cells can

!e determined through staining procedures.Stains ma+e specimens visi!le. 'any sample cells on

smears are not visi!le to the na+ed eye and seemespecially transparent even under the microscope. %headdition of dyes allo for the coloring of inside the cells or

for the negatively;charged micro!e to attract or repel thestain, there!y filling the organism ith color, orsurrounding it and increasing contrast.


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Such properties of stains in the la!oratory also

ma+e them useful and effective in fields of medicaland food micro!iology for the classification,separation, and elimination of

-arious stains indicate the presence of different

organisms, thus certain types of stains target cellsthat they are specific to. 2noing andunderstanding the functions of specific stains canallo one to isely and correctly utili$e them on aparticular type>aggregation of cells ithout

destroying its>their structure in order to study thespecimen.


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Method o

Staining

Se"arates

s"eci#en into

Live/dead

s"eci#en$

!ri#ary Stain

(negative or

basic)

Mordant

and/or

decolor-i%er

&ounterstain 'otes (si#"le

s"ecial or

dierentialstaining)

Simple Staining 4rganism vs. non;

organism "organismvs. surroundings#

Dead specimen 'ethylene !lue,

safranin, orcrystal violet

"!asic stains#

9one. 9one. Simple staining,

!asic stain"positively

charged stain is

used#, allos for

contrast !eteenmicro!e and

surrounding

space.

?ram;Staining ?ram;Positive vs.?ram;9egative

Dead specimen Crystal violet"!asic stain#

?ram iodine"mordant#,

acid alcohol

"decolori$er#

Safranin Differentialstaining, "8# has

thic+er

pepdidoglycansu!layer than ";#.

AcidfastStaining

Acidfast"myco!acteria# vs.

non;acidfast

Dead specimen Car!olfuchsin"!asic stain#,

detergent

%ergitol is addedto primary stain

in heatless

method.

9o mordant.Acid alcohol

removes color

from non;acidfast

micro!es.

'ethylene !lue or

Differentialstaining,

separates !acteria

into those thatcontain mycolic

acid in their outer

cell alls vs. onesthat do not.

7ndospore

Staining

7ndospore vs.

vegetative cells

'eta!olically

inactive

'alachite green

"!asic stain#

9o mordant.

Water may

act as adecolori$er.

Safranin Differential

staining,

separatesorganisms

containingendospores from

vegetative cells.

9egativeStaining

'icro!e "delicate#vs. surroundings

:ive specimin 7osin or nigrosin"negative or

acidic stains#.

9one. 9one. Special staining,

6lagellar

staining

'otile micro!es vs.

surroundings

:ive specimen Car!olfuchsin or

pararosaliline"!asic stains#.

Potassium

alum ortannic acid.

9one. Special staining,


10/19

0ui$ time@

. " s+ittle# What color is methylene !lue

/. "/ s+ittles# s car!olfuchsin a mordant What is amordant

1. "1 s+ittles# What +ind of stain "!asic or acidic# is usedto identify viruses under a %7' in the previous video


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Some stains that e have notyet discussed include*

. Bismar+ Bron "mucins#

/. Carmine "glycogen#

1. Coomassie !lue "proteins#

3. DAP "fluorescent#

5. 7thidium !romide "apoptosis#

. ematoEylin "nucleus#

F. oechst stains "fluorescent, D9A#

G. odine "starch#

H. ndia in+I. 9eutral Red "nucleus#

. 9ile red "lipids#

/. 9ile !lue "nucleus#

1. 4smium tetroEide "lipids#

3. Rhodamine "proteins#

5. Ammonium moly!date

. Jranyl acetate

F. Jranyl formate

G. Phosphotungstic acid

H. 4smium ferricyanide

/I. Auroglucothionate

/. ?iemsa "pathogens, specifically viruses#

//. Acridine orange "D9A#

/1. Cresyl violet "histology#

/3. 'ethyl green "fluorescent, chromatin#

/5. SKBR ?reen 6luorescent stain"s#


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*ideo - +luorescence staining in #edicine


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II;F5I

Anton van:eeuenhoe+ uses

spice saffron tostain muscle tissue

G1I;G3I

Discovery of6luorescein "G1G,

G35#

G3;

GI

Discovery,synthesis, andutili$ation ofcarmine stain

"G53#

GI;GFH

6irst 6luoresceindye used in a

la!oratorysetting "GF#

Staining ithmetal

compounds Silver staining

"GF1#

GGI;GHI

Development ofAcid;fast

staining "GG/,pu!lishing in

GG1#

?ram stainingdeveloped andused "GG1#

Liehl;9eelsenmodification for

Acid;6aststaining "GG1#

6irst vieing ofvirus after

applying stain"GGF#

WeigertMsmodification of?ram Staining

"GGF#

GH;HI

Discovery of

hematoEylin andeosin stains

"GH#

?iemsa stain"HI3#

NensenMs

modification for?ram staining

"HIH#

H;H/5

2inyonMs Acid;6ast methodmodification

"H5#

7ndosporestaining method

development"H//#

2opeloff andBeermanMs ?ram

Stainmodification

"H//#

Cytochemiicalstaining ofperoEidase

"H/3#

H3I;HI

Jtili$ation of6luorochrome

Preston and'orrellMs

modification of?ram Staining

"H5#

Advancements inStaining ProceduresAt;A;?lance


14/19

GII GI G/I G1I G3I

• ,iscovery o +luorescein ( 01)

6luorescein is a fluorophore commonly used in microscopy, in a

type of dye laser as the gain medium, in forensics and serologyto detect latent !lood stains, and in dye tracing. n cellular

!iology, the isothiocyanate derivative of fluorescein is oftenused to la!el and trac+ cells in fluorescence microscopyapplications "for eEample, flo cytometry#. Additional !iologicallyactive molecules "such as anti!odies# may also !e attached to

fluorescein, alloing !iologists to target the fluorophore tospecific proteins or structures ithin cells.


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• +irst +luorescein dye used in a laboratory setting (2)With the development of the synthetic dye industry, it as not long !efore Adolf von Bayer

synthesi$ed the first fluorescent dye, fluorescein.

• ,evelo"#ent o 3cid-ast staining (4 "ublishing in )Paul 7rlich developed acid;fast staining in GG/ hile or+ing ith 'yco!acterium tu!erculosis.Liehl and 9eelson later improved the techni&ue !y adding heat to drive car!ol fuchsin into the cellall.

• 5ra# staining develo"ed and used ()n GG1;3, Dr. ?ram discovered a method for staining certain !acterial cells purple>!lue hile

staining eu+aryotic cells, and many other !acterial cells pin+. 7ssentially, Dr. ?ram found that

!acteria fell into to distinct categories hen stained se&uentially ith crystal violet folloed inse&uence !y a !ath in an iodine solution, a ash ith a destaining agent and a counter;stain.

• +irst viewing o virus ater a""lying stain (2)n GGF, Buist N.B. visualised one of the largest, -accinia virus, !y optical microscopy after

staining it. -accinia as not +non to !e a virus at. that time

G5I GI GFI GGI GHI


16/19

HII HI H/I H1I H3I

• ?iemsa stain "HI3#?iemsa stain, named after ?erman chemist and !acteriologist ?ustav ?iemsa,

is used in cytogenetics and for the histopathological diagnosis of malaria and otherparasites. ?iemsa stain is used in ?iemsa !anding, commonly called ?; !anding,to stain chromosomes and often used to create a +aryogram "chromosome map#.

• 7ndospore staining method development "H//#n H//, Dorner pu!lished a method for staining endospores. t employed a

lengthy heating step !ut resulted in differential staining of endospores and vegetativecells in the same sample. 7ndospores and free spores appeared green or !lue incontrast to red dye ta+en up !y the vegetative cells.

• Jtili$ation of 6luorochrome "H3I;H5I#

A fluorophore "or fluorochrome, similarly to a chromophore# is a fluorescentchemical compound that can re;emit light upon light eEcitation. 6luorophorestypically contain several com!ined aromatic groups, or plane or cyclic moleculesith several O !onds. 6luorophores are sometimes used alone, as a tracer in fluids,as a dye for staining of certain structures, as a su!strate of en$ymes, or as apro!e or indicator "hen its fluorescence is affected !y environmental aspects suchas polarity or ions#.

%ried;and;tested staining techni&ues such as ?ram and Acid;fast staining


17/19

%ried;and;tested staining techni&ues such as ?ram and Acid;fast staininghave seen decades of use in the science of micro!iology. At the sametime though, perpetual research in staining methods; !oth old and novel;for staining continue to !e developed in pursuance of identifying,isolating and studying ne and old micro!es ali+e this includescomponents relating to samples of multicellular organisms.

Some recent methods>modifications in staining, include*

• Research specific stain using periodic acid;Schiff, ColemanQs 6eulgen

solution, 'ayerQs hematoEylin, and methylene !lue in order to reducedistortions in a HHF pu!lished study containing elico!acter pylori,pathogenic ? !acteria

• Contrast;enhancing dye composed of osmium tetroEide stain alongith eEcess thiocar!ohydra$ide "4%4# for lipid;containing mem!ranes"H#

•

Dou!le immunoen$ymatic staining methods involving al+alinephosphates and peroEidases to shade developing en$ymes "/I/#• 9e staining techni&ue for fungal;infected plant tissues using cotton

!lue and saphranin to identify em!edded fungal structures in delicatetissues "/I1#

• 7lectrophoresis staining to promote active anti!ody !inding in tissues

to reduce time of staining "/I3#


18/19

&onclusion

Staining is a very important procedure in the realm ofla!oratory micro!iology. t allos one to differentiatevarious types of cells, !oth pro+aryotic and eu+aryotic from

each other hen "a# specific dye"s# is applied. t hasevolved from the simple histological staining practices ofAnton -an :eeuenhoe+Qs time to the various differentialand special staining techni&ues of today. While not toomany entirely ne stains are in eEistence today, t isevident that a com!ination of staining techni&ues oreEisting stains yield ne methods testing for the presenceor proper visi!ility of target compounds.


19/19

7Etra slide

While the same staining techni&ues are repeatedlyemployed in the science of micro!iology to meet currentdemands, such as ?ram staining and Acid;6ast staining,as they are relia!le and accurate methods for studyingmicroscopic specimen, ne methods for staining have

!een and are !eing developed to target specific micro!esand histological samples and their constituents. %heevolution of the differential staining proceduresmentioned a!ove reflects the rapidly increasing need toaccommodate and eEpand the isolation, identification,and study of microorganisms and their meta!olism,

structures, and arrangement ithin their surroundings,as ell as the !ehavior of cells of multicellularorganisms.

microbiology project - latest in staining

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