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
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