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BACTERIAL IDENTIFICATION2 Basis:1) Genotypic criteria
Molecular testing-study the genes
Detection of :
Gene
Part of gene
Nucleic acid sequence
Polymerase chain reaction (most common
test)- Involve amplication of nucleic acid
sequence !ighly specic " sensitive
#tili$ed for:
Micro%es &'c cannot gro& in vitro
eg Mycobacterium leprae are pathogens
*iruses (acellular: can+t gro& outside
host cell not unless cm is made up ofactual living cell)
esearch purposes
2) Phenotypic criteria
,nalysis of gene products eadily availa%le
Physiological activities
Meta%olic activities
Basis:o Microscopic morphology " staining
characteristicso olony morphology
o .nvironmental requirements for gro&th
(ta/ing note of incu%ation)o esistance'suscepti%ility to
antimicro%ial
agentso Nutritional requirements " meta%olic
capa%ilities (0iochemical tests: performedfor studying
%iochemical prole)
Biochemical Tet
Most common approach of %acterial ID
Identication %y com!ination o" tet
o .sta%lished %iochemical
characteristic'prole of un/no&nisolates
General Procedure:
1 2election of inoculation of tests (%asisof test 3 initial gram
stain --gram stain
result after cultivation)4 Incu%ation for su%stance utili$ation5
Detection of meta%olic activity
(su%strate utili$ation)6 ,nalysis of meta%olic proles: ta/e
note of all results of the %iochemicaltests to compare to
esta%lished%iochemical characteristic'proles
2 Types:
i# Eta!lihin$ En%ymatic Capa!ilitie2 types:a) &in$le En%yme
Tet
Detects (in)capa%ility of %acteria to
produce a specic en$yme Principle:
&'B&TRATE (Rea$ent)
PROD'CT& (Relt)
#sually performed in organismsgro&n in culture #sually
rapid
an+t identify %acteria alone: Need
su%sequent ID tests
Examples: Catalae Tet
Catalase4!474---------8 4!47 9 74(5) (Production of%u%%les)
(*)esult:
- apid appearance of%u%%les'e;ervescence-
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o Non-utili$er: organism &'c
does not have the capa%ilityto utili$e !7
Principle:
o 2u%strate (!7) utili$ation
produces acid productso Products are detected %y p!
indicators ommonly used p! indicators:
o 0romcresol purple: Purple -8Cello&
o ,cid Buchsin: Pale Cello& -8
Pin/o Phenol ed: ed -8 Cello&
o Neutral ed: ed --8 Pin/-
red2) Amino Aci- De$eneration
Detects a%ility of %acteria to
produce en$ymes that:o
Decar%oAylaseo Deaminate
o Dehydroly$e ,mino acids
used:o
Phenylalanineo ?ryptophan
o >ysine
o 7rnithine
o ,rginine
Decarboxylase
.n$yme that attac/ the
car%oAyl group Borms amine" 74
- detected after
decar%oAylation
Reaction examples:
Lysine decarboxylase! >ysine ---------------------------8
adaverine
Ornit"ine decarboxylase2! 7rnithine -----------------------8
Putrescine#r$inine dihydrolase
%! ,rginine --------------------------8
itrullineCitrulline decarboxylase
&! itrulline -------------------------87rnithine
Deaminase
.n$yme that attac/'remove
the amine group Produces aci-ic pro-ct
Reaction examples:
P"enylalanine deaminase! Phenylalanine -------------8
Phenylpyruvicacid
Tryptop"an deaminase2! ?ryptophan ----------8 Indole-
pyruvicacid
4Triple &$ar Iron A$ar (T&IA) om%ination of !7
fermentation " ,mino ,cidDegeneration
Components:
Protein sources: (amino
acids)o 0eef eAtract
o Peptone
o Ceast eAtract
o Proteose peptone
!7: (triple sugar)
o Glucose (1)
o 2ucrose (1=)
o >actose (1=)
p! indicator: Phenol ed
2ulfur source: 2odium
thiosulfate Iron salts (either of the t&o):
o Berrous sulfate
o Berric ammonium citrate
Principles:
Detects fermenting capa%ility
o Glucose@ 2ucrose "'or
>actose
o 4-eaction ham%er1# Aero!ic lant
.Aposed to
atmospheric 74 0ecomes more
al/aline(redcolor) due to theo,i-ati.e-ecar!o,ylationo" the
aminoaci-
,llo&s detection of
lar$e quantities of
acid (only then &illit change in color)
2# Anaero!ic -eep Not eAposed to
atmospheric 74 Maintain usual p!
of the agaro Minimal
decar%oAylation of proteins
,llo& detection of
mall quantities of
certain amino
Deamination
reaction
Decar!o,yla
tion reaction
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acid products tochange in colorfrom red to yello&
Possible Results:
Glcoe "ermenter5
Nonlactoe 6 Noncroe"ermenter 78A
o ,l/aline slant',cidic
deepo edslant'Cello&deep
2mall quantity of acid
products
Glcoe "ermenter5
Lactoe 68or &croe"ermenter A8A
o ,cidic slant',cidic
deepo Cello& slant'Cello&
deep >arge quantities of acid
Non$lcoe9 Nonlactoe9Noncroe "ermenter 787
o ,l/aline slant',l/aline
deepo edslant'eddeep
4A87 is not possi%le (you maynot have sta%%ed the ?2I,
,lso detects production of !42
gaso !42 gas reacts &' iron salts to
produce the %lac/ precipitate(Berrous sulde)
!42 (9) : %lac/ ppt !42 (-) : &'o %lac/ ppt
Detects the a%ility of gas
production during fermentation Gas (9) : crac/s' Epulling
a&ayF Gas (-) : no crac/s' Epulling
a&ayFmore fermentation of sugar more gas production
:) &in$le &!trate 'tili%ation ,%ility of micro%e to
gro& in the
presence of a singlenutrient'car%on source
Examples: Citrate Tet
o itrate as sole source of
!7o p! indicator: 0romthymol
%lueo Principle:
itrate converted to N!5 N!5 converted to N!67!
0romthymol %lueproduces a green color
Lactoe Broth
o >actose as sole source of
!7o Indicator: Durham
fermentation tu%eo Principle:
Bermentation of lactose
Gas production is
o%served 0u%%le formation in DB
ii# Eta!lihin$ Inhi!itoryPro;le
,%ility to gro& in one'more inhi%itory
agents 0ile salts
Nal (most common)
.thanol
,nti%iotics
Example: Nal ?olerance ?est
o 1st0roth: &' H Nal
o 4nd0roth: &'o H Nal
Tolerates 'aCl:gro&th in %oth
%roths (ntolerant to 'aCl: gro&th only in
4nd%roth)Obli$ate #naerobes: gro&th only in1st%roth
