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Enterobacteriaceae Curs

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L9 Enterobacteriaceae Lecture prepared by Dr. M.Watts
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Lecture prepared by Dr. M.Watts

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� students will become familiar with

� a range of tests designed to identify


� a range of diseases caused by these


� Know where they fit into the classification


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Medically important bacteria:So far.. Gram +

� Gram-positive cocci

� Micrococaceae� Streptococcacaea

� Gram-positive rods� Aerobic� Bacillus spp.� Lactobacillus sp. � Corynebacterium spp� Listeria spp.� Erysipelothrix rhusiopathiae� Nocardia sp. � Streptomyces spp.

� Anaerobic � Actinomyces sp.� Clostridium spp

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Now: Gram Negative


� Cocci (only a few)

� Aerobic

� Neisseria

� Moraxella

� Anaerobic

� Veillonella sp.

� Rods (several hundred)

� Aerobic

� Anaerobic

� Facultative/fermentative

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GNRs: Facultative/fermentative

� Enterobacteriaceae

� Fermenting glucose and lactose

� Citrobacter sp.

� Enterobacter sp.

� Escherichia coli

� Klebsiella pneumoniae

� Fermenting glucose but NOTlactose

� Proteus sp.

� Salmonella enteriditis

� Salmonella typhi

� Shigella sp.

� Serratia marcescens

� Yersinia enterocolitica

� Yersinia pestis

� Others (non-enteric) misc GNBs� Pseudomonas spp.� Aeromonas sp. � Plesiomonas shigelloides� Vibrio cholerae� Vibrio parahaemolyticus� Vibrio vulnificus

sugar fermented is hugely important:

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What are they?

Where do you find them?Are they medically important?Which are most commonly encountered?How to recognise and identify

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What are they?

� Gram negative rods grouped together on strong phenotypic grounds

� Most confirmed by molecular methods

� Currently, they are classed into 31 genera and over 100 species.

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Where do you find them?

� Primarily the bowel

� 1012 per gram of faeces

� large range of animals, both warm and cold blooded

� Also on some plants and in the soil….

� wherever you find animal faeces

� Prefer moist environments

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Are they medically important? YES!

� Cause a range of infections

� Enteric: Salmonella,

Shigella, E. coli.

� UTI: E. coli.

� Nosocomial:

� Septicaemia,

� Pneumonia

� Wound infection

� Have the opportunity

� reasonably resistant to antibiotics

�have the necessary virulence attributes

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E. coliClinical aspects:

� normal flora of GIT � produces vitamin K in the large intestine

� most commonly isolated pathogen in hospitalized patients� UTI, neonatal meningitis,

gastroenteritis, wound infections, pneumonia, septicaemia.

� LPS (endotoxin) is released when the cell dies� Treating infections with antibiotics may

place the patient in severe shock

� some strains have acquired additional genetic information from plasmids, transposons, and phages which allows them to be pathogenic

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E.coli: a highly variable organism

� 5 classes cause diarrhoeal diseases

� ETEC (Enterotoxigenic)

� EPEC (Enteropathogenic)

� EIEC (Enteroinvasive)

� EHEC 0157


� EaggEC

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EHEC 0157(Enterohaemorrhagic)

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How can variants of the same organism be so different?

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Salmonella nomenclature controversial

� original taxonomy based on clinical considerations,

� e.g., Salmonella typhi, Salmonella cholerae-suis, Salmonella abortus-ovis,

� Now known that all Salmonella serovars form a single species (Salmonella enterica) composed of seven subgroups

� Subgroup 1 contains most common serotypes� typhi

� cholerae-suis

� paratyphi

� gallinarium

� pullorum

� Subgroup 2 : salamae

� Subgroup3a: arizonae

� Subgroup3b: diarizonae

� Subgroup 4: houtenae

� Subgroup 5: bongori

� Subgroup 6: indica

Eg Salmonella enterica serovars (e.g.,

Enteritidis, Typhi, Typhimurium)

Ie Salmonella ser. Typhimurium (not


Some books:

S. Typhimurium

S. Typhi

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Why the problem?

� three kinds of major antigens

� Somatic (O) or Cell Wall Antigens67 used for serological identification.

� Surface (capsular) Antigens Vi antigen well known

� Flagellar (H) AntigensAntiflagellar antibodies can immobilize bacteria with corresponding H antigens.

So many surface antigens

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Clinical Aspects:

� two distinct diseases

� enteric fever (typhoid),

� bacterial invasion of the bloodstream (S. typhi)

� acute gastroenteritis

� foodborn Salmonella: (S.typhimurium, S.enteritidis)

� chicken and eggs reservoir.

� implicated in more than 50,000 cases of bacterial food poisoning in the United States every year

� S. typhi only carried by humans

� Role of carriers

� About 5% of patients clinically cured from typhoid remain carriers for months or even years.

� Antibiotics are usually ineffective on Salmonellacarriage because the site of carriage (gall bladder) may not allow penetration by the antibiotic.

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Outbreak of salmonella

� http://video.google.com/videoplay?docid=623


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Shigella� Severe diarrhea accompanied by fever

and also invasive

� reservoir human only� small inoculum (10 to 200 organisms) is

sufficient to cause infection.

� Shiga toxin A:B, enters cell disrupts protein synthesis

� Outbreaks in daycare, nursing homes

� spread� Four “ f’s “ faeces food flies fingers

� four species � Serotype A- S. dysenteriae� Serotype B- S. flexneri� Serotype C- S. boydii (rare)� Serotype D- S. sonnei (most


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� Klebsiella pneumoniae

� Common cause of nosocomial pneumonia and UTI(second only to E. coli)

� Produces a heat-stable enterotoxin

� contain resistance plasmids (R-plasmids)

� can be transferred to other enteric bacteria (not necessarily of the same species)

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P. mirabilis

• Proteus: urinary


• Can form stones

• caused by infection of the urine with urea-

splitting bacteria.


Staghorn calculus

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Yersinia pestis

•Yersinia pestis rodent pathogen,

•The flea draws viable Y. pestis organisms into its intestinal tract.

•These organisms multiply in the flea.

•humans an accidental host

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Los Angeles Times

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� NF gut can all cause opportunistic infections


� E. tarda known to cause gastroenteritis and wound infections

� CITROBACTER� C. freundii is suspected to cause diarrhea

� C. diversus has been linked to a few cases of meningitis in newborns.

� ENTEROBACTER� E. aerogenes and E. cloacae are sometimes associated with UT & RT infections


� Serratia marcescens is considered a harmful human pathogen

� known to cause urinary tract infections, wound infections, and pneumonia.

� also have many antibiotic resistance properties

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Uses biochemistry of bacteria

Think about what they do and where they live

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12 most common genera

� Often collectively called “coliforms”.

� Escherichia coli: type species


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Which tests are most useful?

� Diagnostic microbiology

� Most useful tests for quick ID

� Empirical:

� Ist stage tests

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E.coli, a vibrio & a pseudomonad

Which is which??

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OF test: most are fermenters

Which is fermenter? Which is oxider?


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Have GNR fermentative :

???? how to differentiate Enterobacteriacea

from ‘the others’

� Oxidase neg: enterobacteriacea

� Oxidase pos: = ‘the


� Vibrios

� Aeromonads

� Pseudomonads (aerobic)

� Will do these next lecture

The OXIDASE test

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Nitrate reduction

� +ve for Enterobacteriaceae

� Detects whether bacteria uses nitrate (NO3)as electron acceptor (ie)

� Nitrate reduced to nitrite (or other compounds) via nitrate reductase

� NO3 ----> NO2 ----> NH3 or N2

� Grow organism in nitrate broth: test for end products� reagents: a-naphthylamine and sulfanilic acid

� Formation of red color after addition of the

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Nitrate reduction

Medium undefined: contains large amounts of nitrate (KNO3). Which are +ve?

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Another example: Following incubation and addition of nitrate reagents:

3 4

Which are positive??

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Now which are positive? (Following addition of Zn dust)

What about 1?

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differentiating pathogens from non-pathogens: ability to ferment lactose


0 - 1%


0 – 1%






Citrobacter freundii (50%)

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Commonly used lactose containing selective and differential media

� MacConkey





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MacConkey Agarselective and


contains� lactose, � bile salts, � neutral red (and crystal violet).

� selective media � Gram-positive organisms are inhibited by the bile salts and the

crystal violet.

� Differential� When bacteria ferment lactose and produce enough acid products

to reduce the pH below 6.8, the neutral red turns from colorless to red.

� Thus, MacConkey is a differential media on which lactose fermenting colonies appear red (or pink). Nonlactose fermentersare colorless.

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Reactions on Mac

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Have a guess?

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� selective and differential medium designed for the isolation of Gram-negative enteric pathogens from clinical specimens.

� contains xylose, lysine, sodium desoxycholate, sodium thiosulfate and ferric ammonium citrate.

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� Differentiation of Shigella and Salmonella from nonpathogenic bacteria is accomplished by three reactions:

� 1) xylose fermentation, � 2) lysine decarboxylation, and

� 3) hydrogen sulfide production.

� Xylose: enterics (except Shigella) ferment xylose rapidly.

� Salmonella rapidly exhaust xylose and decarboxylate lysine, and revert to alkaline conditions (simulates the Shigella reaction).

� Lactose and sucrose, added in excess, prevent lactose fermenters from similarly reverting.

� The production of hydrogen sulfide under alkaline conditions results in the formation of colonies with black centers, whereas, under acidic conditions, this black precipitation is inhibited

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E. coli on XLD agar: lactose

and sucrose fermented,

lysine and H2S negative.

E. coli on XLD agar: lactose and sucrose

fermented, lysine and H2S negative.

Shigella on XLD agar:

non-fermentive, no H2S

Salmonella on XLD agar: lysine positive,

xylose fermented, positive H2S

(colonies with black centers)

Proteus on XLD agar:

swarming inhibited, xylose only

fermented, lysine negative.

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Reactions on XLD

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EMB: � Peptones 10.0;

� di-potassium hydrogen phosphate 2.0;

� lactose 5.0;

� sucrose 5.0;

� eosin Y;

� methylene blue 0.07; agar-agar 13.5.

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KIA formula

� Meat extract...........................................................................3,00

� Yeast extract..........................................................................3,00

� Peptone.................................................................................20,00

� Lactose..................................................................................10,00

� Sodium chloride..................................................................... 5,00

� Dextrose................................................................................ 1,00

� Ammonium Ferrous citrate......................................................0,50

� Sodium thiosulfate................................................................... 0,50

� Phenol red.............................................................................0,03

� Agar.......................................................................................15,00

� Final pH 7,4 ± 0,2

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� 1. Fermentation of glucose, lactose

� 2. Production of gas during fermentation

� 3. Production of H2S from the sulfur source

� Phenol red is used as PH indicator: yellow in acid, red in alkaline

KIA:allows for determination of:

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KIA interpretation



erroneously inoculated a Gram + or some other weird thingA/K

nonfermenters such as Pseudomonas and othersK/K

Salmonella, Edwardsiella, ShigellaK/A

E. coli, Yersinia, Aeromonas, VibrioA/A



Why include


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KIA interpretation

� If the butt of tube is yellow (A): glucose fermenter

� If the slant of tube is yellow (A): lactose fermenter

� If the slant is red (K): non-lactose fermenter

� Black ppt: H2S prodn

� Gas: gaps in agar

� A/A, K/A and K/K

� Could you get A/K??????

� (If the butt of tube is red (K): non-glucose fermenter)

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Carbohydrate fermentation

� Single CHO

� Test Result

� 1.Control Negative

� 2. S. aureus ?

� 3. P. vulgaris ??

� 4. P.aeruginosa

� 5. E. coli ??

+ve Acid prodn: color change from red to yellow.

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Amino acid decarboxylation

� Decarboxylation only takes place in an acid environment

� & needs to be anaerobic

� Net reaction is alkaline

� How can you be sure decarboxylation has occurred?

� 2 tubes inoculated:

� one without amino acid but

both with glucose (for……….?)

� sterile mineral oil overlay

� Tube without aa is control

(goes yellow)

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Typical reactions: which set is +ve?

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Motility test

� A non-motile organism will

have a clearly defined edge

as it grows on the stab line

� Motile organisms will be

turbid throughout the tube

or have fuzzy, diffuse

growth at the edges.

� Some organisms are so

motile that the entire tube

becomes very turbid


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Indole-methyl red-VP-citrate

IMViC tests (4 tests)

� used to differentiate Enterobacter and Klebsiella from E.coli

� three sets of media are inoculated:

� indole test (tryptone broth)

� MR-VP broth,

� Simmons citrate medium

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The Indole Test

� tests the ability of organism to split indole from tryptophan (ie have tryptophanase)

� Indole reagents:

� Kovac's reagent: P-dimethylaminobenzaldehydein alcohol

� Positive reaction: formation of red color at the interface of the broth and reagent

tryptone broth



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� All enterics oxidize glucose for energy

� end products vary depending on bacterial enzymes

� MR and VP tests are used to determine what end products result

� MR-VP media buffered-dextrose peptone broth

� tests are read from a single inoculated tube of MR-VP broth.

� After 24-48 hours of incubation the MR-VP broth is split into two tubes.

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Methyl red:

� It tests the ability of organism to produce and maintain strong, mixed acid from buffered-dextrose peptone broth

� E. coli is one of the bacteria that produces acids, causing the pH to drop below 4.4.

� When the pH indicator methyl red is added to this acidic broth it will be cherry red (a positive MR test)

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Vogues Proskauer

� Klebsiella and Enterobacterproduce more neutral products from glucose (acetoin)

� pH rises above 6.2.

� The reagents: alpha-naptholand potassium hydroxide.

� If acetoin is present reagents turn a pink-burgundy color (a positive VP test).

� color may take 20 to 30 minutes to develop.

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The Citrate Test: Citrate use

alkalinises the medium

� Simmon's medium � Typical Composition (g/liter)

Ammonium dihydrogen phosphate 1.0;

� di-potassium hydrogen phosphate 1.0;

� sodium chloride 5.0;

� sodium citrate 2.0; sole C and energy source

� magnesium sulfate 0.2;

� bromothymol blue 0.08;

� pH of 6.9: agar-agar 13.0.

� yellow at acidic pH's (around 6), and blue at more alkaline pH's (around 7.6).

� positive citrate: blue � Enterobacter and Klebsiella + E.coli -

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Why does citrate use alkalinise the medium??

Bacteria that use citrate also utilize the ammonium salt

as a nitrogen source and create ammonia as a result.

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IMViC Interpretation

� E.coli gives ++--

� Enterobacter and Klebsiella give the reverse:


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ONPG test

� Principle

� Lactose utilization requires 2 main enzymes, � permease and b-galactosidase

� Enzymes ONLY induced in the presence of the lactose substrate (inducer)� Need high lactose medium

� Regular LFs produce both permease enzyme and b-galactosidase

� True NLFs lack both of these enzymes

� Late LFs produce b-galactosidase but lack permease (egShigella sonnei)

� ONPG: (Ortho-nitrophenyl-b-D-galactopyranoside ) Artificial substrate

� turns yellow in the presence of beta-galactosidase.


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ONPG test: detects the presence of β-


� organism needs to be growing in/on any medium with lactose (to induce the production of galactosidase)

� Pipette 0.5ml of the saline into a sterile tube.

� Inoculate with the bacterium and add the ONPG disc in a sterile manner (forceps dipped in alcohol and flamed) to the tube.

� Incubate at 37C for 4 hours.

� INTERPRETATION:� any shade of yellow + for galactosidase


� Precautions� A heavy inoculum is necessary to obtain

a high concentration of enzyme.

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Urease test

� Some bacteria produce urease:

� Urease splits urea into ammonia and carbon dioxide

� Organisms that produce urease will turn hot pink due to ammonia prodn

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Urease +ve Proteus

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�characteristic red pigment.

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Summary: Recognition and Identification

� Is it a “coliform” or another type of GNB?

� Facultative, growth on Mac: then do oxidase (-)

� [OF (F) and NO3 reduction tests not usually needed]

� Which coliform is it?

� Is it a major pathogen (salmonella or shigella or E.coli 0157?)

� Requires a “battery” (10-20) of biochemical tests

� Usually commercially packaged

� Many of these are broadly discriminatory.

� A range of CHO’s added for more fine discrimination.

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Characteristics shared by all


� Gram negative rods

� oxidase negative

� All can ferment glucose

� Facultative anaerobes

� reduce nitrate to nitrite

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Then more tests to speciate

� Lactose fermentation

� CHO fermentation

� Decarboxylase reactions

� H2S production

� IMViC reactions:

� · I: indole

� · M: methyl red

� · V: Voges-Proskauer

� · C: citrate

� And others

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BUT………consider: Biochemical variability from tables

--57--Pr. mirabilis

-vvvvSalmonella spp.

9483-7795E. coli

-++++K. pneumoniae



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Probability and identification


989595E. coli

+++Result in your test


Could be either E.coli or Shigella, but E. coli more

probable. More tests in your panel = better


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API strips are therefore very useful!!!!!

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The use of codes and code books

� Do tests in “set” order

� group the results in sets of three.

� Assign 0 for a (-) and 1, 2 or 4 for a (+).

� - - - = 0, + - - =1, - + - = 2, + + - = 3

� - - + = 4, + - + = 5, - + + = 6, + + + =7

� ( note that if 1, 2, 3 then + + - =3 and - - + = 3)

� 514 is unique to those results.





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API profile result sheet

Spot the deliberate mistake!!!!!!

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Mechanics of identification

� Do the 10 or 20 tests.

� + - - + + - + + + - - + + + - - + +

� Number?

� 137436

� Now compare your results with a table of expected

results for over 100 different organisms.

� This could be a table of 30 x 100!

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� Many systems now available.

� organism in diluent.

� aspirates into “black box”

� Distributes inoculum into wells with dehydrated substrates and incubates.

� Instrument reads results with spectro.

� Determines + or -, and compares with database.

� Prints out name of organism.

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Major ID tests


� After growth on Mac, do oxidase test.

� If oxidase negative, do biochemical tests.

� – Carbohydrate fermentation

� – KIA agar/TSI

� – IMViC

� – Urease

� – Decarboxylase reactions

� – ONPG

� – H2S

� – Motility

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Compose a flow charts from major tests

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Enterobacteriaceae flow chart

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End of lecture

� Review questions