Culturing microorganisms

8/18/2019 Culturing microorganisms

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Culture and identification of

infectious agents, Lecture 25

Dr. Alvin Fox


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Key Terms

Key TermsIsolation (culture)

• Agar plate

plate/colonies

• Liquid media

Identification & taxonomy• amily

• !enus

• "pecies

•

Type• "train

After culture

• #ioc$emical (p$ysiological) tests• !enetic tests

% olymerase c$ain reaction (')

% "equencing

% *A%*A $omology/arrays% estriction en+ymes (digests)

• '$emical

% fatty acid/protein profiling

• Immunological

irect detection (i,e, -it$out culture)

• '

• Antigen detection

• "taining (e,g, !ram stain)• "erolo anti.od detection


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Taxonomy

0 Defines common traits among strains for

a bacterial species

0 sually genetic

0 Allo!s development of diagnostic "its


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#pecies versus strains

$ selecting discriminating featuresselecting discriminating features


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ClassificationClassification

0 #train% one single isolate or line#train% one single isolate or line

0 Type% sub$set of speciesType% sub$set of species

0 #pecies% related strains#pecies% related strains

0 &enus% related species&enus% related species

0 Family% related generaFamily% related genera


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Streptococcus pyogenes

#ot$ terms define t$e species name

!enus part "pecies part

"o Streptococcus pyogenes and Streptococus agalactiae

-ould .e t-o species in t$e same genus

In a report

4n first usage5 Streptococcus pyogenes

4n second usage5 S. pyogenes


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Identification of infectious agentsIdentification of infectious agents

in the diagnostic laboratoryin the diagnostic laboratory

0 Aids treatmentAids treatment

0 'elps antibiotic selection'elps antibiotic selection0 &eneral (ospital laboratory&eneral (ospital laboratory

7 p(ysiological testsp(ysiological tests

0 )ore fully e*uipped laboratories)ore fully e*uipped laboratories 7 &enetic tests&enetic tests


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"teps in isolation and identification"teps in isolation and identification

0 #tep +%#tep +% #trea"ing culture plates#trea"ing culture plates

7 colonies on incubation e.g 2- (rcolonies on incubation e.g 2- (r 7 si/e, texture, color, (emolysissi/e, texture, color, (emolysis 7 oxygen re*uirementoxygen re*uirement

$ .l d l l


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CDC0Dr. 1ames Feeley

"$eep .lood agar plate culture

Bacillus anthracisBacillus cereus,


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1:

)ixed colonies


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Isolation and identificationIsolation and identification

0 #tep 2% Colonies &ram stained#tep 2% Colonies &ram stained

7 cells observed microscopicallycells observed microscopically


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&ram negative&ram negative &ram positive&ram positive

'eat0Dry'eat0Dry

Crystal violet stainCrystal violet stain

odineodine FixFix

#afranin stain#afranin stain

Alco(olAlco(ol dede%stainstain


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!ram stain morp$ology!ram stain morp$ology

0 #(ape#(ape

7 cocci roundcocci round

7 bacilli rodsbacilli rods

7 spiral or curved e.g. spiroc(etesspiral or curved e.g. spiroc(etes

0 #ingle or multiple cells#ingle or multiple cells

7 clusters e.g. stap(ylococciclusters e.g. stap(ylococci

7 c(ains e.g. streptococcic(ains e.g. streptococci

0 &ram positive or negative&ram positive or negative


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#tep 3%#tep 3%

solated bacteria are speciatedsolated bacteria are speciated

0 &enerally using p(ysiological tests&enerally using p(ysiological tests


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Clinical )icrobiologyClinical )icrobiology

Laboratory 4enc(Laboratory 4enc(


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#tep -%#tep -%

Antibiotic susceptibility testingAntibiotic susceptibility testing


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oo

gro!t(gro!t(

Antibiotic susceptibility testingAntibiotic susceptibility testing

#usceptible#usceptible ot susceptibleot susceptible

4acterial4acterial

la!nla!n

&ro!t(&ro!t(

Antibiotic dis" Antibiotic dis"


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)olecular)olecular

differentiationdifferentiation0 &enomics&enomics

0&ene c(aracteri/ation&ene c(aracteri/ation 7 6C7 6C7

7 #e*uencing#e*uencing 7 7estriction digestion7estriction digestion

0 'ybridi/ation probes, arrays'ybridi/ation probes, arrays

0 8 guanine$cytosine8 guanine$cytosine


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+9# r7A #e*uencing+9# r7A #e*uencing

0 Differentiates bacterial speciesDifferentiates bacterial species

0 Development of clinical tests basedDevelopment of clinical tests based

on se*uence e.g. 6C7on se*uence e.g. 6C7


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2:

7eal$time 6C7

Cycle one

Cycle t!o

Cycle 3:

223:3:

ds DAds DA

DyeDye


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DA$DA (ybridi/ationDA$DA (ybridi/ation

+::8 'omology

;;

'eat'eat

:8 'omology

#train +#train +

#train 2#train 2


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6rofiles6rofiles0 Long c(ainLong c(ain fatty acidsfatty acids$ structural e.g. cell membrane$ structural e.g. cell membrane

0 #(ort c(ain#(ort c(ain

$ metabolic$ metabolic

$ volatiles$ volatiles$ Fatty acids0alco(ols$ Fatty acids0alco(ols


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0 6rotein profiling% defining a species by6rotein profiling% defining a species byc(aracteristic lo! molecular !eig(tc(aracteristic lo! molecular !eig(t

proteinsproteins

0 6roteomics% defining6roteomics% defining allall proteinsproteins

expressed by a species under specificexpressed by a species under specific

gro!t( conditionsgro!t( conditions


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4acterial DA se*uences can be4acterial DA se*uences can be

amplified directly from (uman bodyamplified directly from (uman bodyfluidsfluids


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Laboratory diagnosis !it(outLaboratory diagnosis !it(out

cultureculturen general, !(en speed is of essence


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)icroscopy)icroscopy0 spinal fluids meningitisspinal fluids meningitis0 sputum tuberculosissputum tuberculosis

0 sensitivity poorsensitivity poor


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"treptococcal Agglutination Test

Latex beadsLatex beads

#treptococcal#treptococcalantigenic extractantigenic extract

AntibodyAntibody


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#erologic identification#erologic identification

0 antibody response to t(e infecting agent

0 several !ee"s after an infection (as occurred


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;pidemics;pidemics

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0 associated !it( particular strains0types

0 state or federal laboratory system

Documents

Culturing microorganisms