The Enterobacteriaceae: Isolation and Identification

Background: The Enterobacteriaceae is a remarkable group of medicallyimportantGram-negative bacillicontainingwithinitsranks organismsthatcausesuchdevastatingdiseasesas the plague(Yersiniapestis),typhoidfever (Salmonellatyphi) and Traveler’sdiarrhea(Escherichiacoli). Membersofthisgroupofbacteriaarealso amongthe most commonly isolated bacteria inthe clinicaland public healthlaboratories. Besides the numerous

diseasesassociated withtheseorganisms, thecoliformbacteriainthisgroupareusefultoolstodetectfecal contamination of food and water.

Sincethese organisms areverycommon and cause a wide range of diseases,itis imperative thatanystudentof microbiology be familiar with theisolation and identification of these bacteria. Table 1 outlines the basic characteristics of Enterobacteriaceae.

Table1-CharacteristicsoftheEnterobacteriaceae

GramStain / All are Gram negative rods
Oxidase Reaction / All are oxidase negative
Catalase Reaction / Allarecatalasepositive
Glucose Fermentation / Most ferment glucoseandproducegas
Nitrate Reduction / Most reducenitrate tonitrite
Oxygen Requirements / All are facultative
Media Requirements / All with grow on standard lab media such as TSA or NutrientAgar

Materials:

1)Reagents:

a.Kovacs’ reagent

2)Media:

a.2 EMB plates

b.2 MacConkey’s agar

c.2TripleSugarIron(TSI)stabs

d.2Motilitystabs

e.2 tubes of BHI broth (for Indole test)

f.1 Enterotube per group

3)Cultures ofEscherichia coli,Enterobactercloacae,Proteusvulgaris,Klebsiellapneumoniae, Salmonella typhimurium, Shigellasp.

Protocol:

1)Gramstain your assigned organisms. Pool the results forthe entire class. Put your results in theTable 2.

2)Streakyourassigned organisms ontoseparate EMB and MacConkey’s plates. Streak for isolated colonies.

3)Inoculate separate TSI slants with your assigned organisms. Carefullyfollow the instructor’sinstructionson how to inoculate these tubes.

4)Inoculate separate Motility stabs with your assigned organisms.

Day 2 Protocol

5)Observe the EMB and MacConkey’s plates for growth, colony morphology and color. Lactose fermenters will generally produce small dark to black colonies or dark colonies with pink centers on EMB agar. Lactose fermenters will produce pink colonies on MacConkey’s agar. E.coli may produce a metallic green-sheen on EMB agar. Record your results in Table 3 below.

Table2-GramStainResults

Table 3 – Growth Results on EMB/MacConkey’s

2)Interpret theTSI slants using theTable provided (Table 4) and record yourobservationsinTable5.TSI media contains three sugars(glucose,lactose and sucrose) andanironingredientfor detecting H2Sproduction - thus the name TripleSugarIron.IfH2S is produced the media will be blackened.Ifthesugar is fermented the mediumwillturnfromorange-redorpinktoyellow.If H2S is produced themediumwill be blackened.

Table 4 - Appearance ofReactedTSI Slants

Butt / Slant / Interpretation
Yellow / Yellow / Glucose andLactose Fermented
Yellow / Orange-redorpink / Onlyglucose fermented
Orange-redorpink / Orange-redorpink / No fermentation
Black / Black / H2S production

Table5–ResultsofReactionsonTSISlants

Organism / Butt/Slant / Interpretation
E.coli
Salmonella
Shigella
Klebsiella
Proteus
Enterobacter

3)ObservetheMotilitystab.Ifyou rotate the tube and notice a distinctvertical line ofmicrobes thatdisappearswhenyourotatepasta certainpoint,considerthisa non-motileresult.Ifitisdifficulttosee the initial inoculation line, due to adiffuseopaquecolor,considerthisa motileresult.

4)ToBHIbrothadd5-10dropsof Kovacs’reagentandshakegently.A positiveIndoleresult will be a redring immediatelyforming on the topof the broth.

Recordallofthe biochemical resultsinTable 6.

Table6-BiochemicalTestson members oftheEnterobacteriaceae

Organism / Motility / Indole
E. coli / ( ) yes
( ) no / ( ) yes
( ) no
Salmonella / ( ) yes
( ) no / ( ) yes
( ) no
Shigella / ( ) yes
( ) no / ( ) yes
( ) no
Klebsiella / ( ) yes
( ) no / ( ) yes
( ) no
Proteus / ( ) yes
( ) no / ( ) yes
( ) no
Enterobacter / ( ) yes
( ) no / ( ) yes
( ) no

NOTES:

Enterotubes

1)Using your EMB plates, inoculate anEnterotubeasinstructed.

2)Place theEnterotubesinto theincubator.

3)You will check themthe next lab period andscore theresults. Place your resultsinthescoresheetsonthenextpage or the sheets provided by the instructor. The instructor will explain how to score the tubes. Be sure to follow those instructions carefully.

Enterotube Score Sheets Organism

Enterotube Score Sheets Organism

Appendix1-Keyto the Enterobacteriaceae

I)Hydrogen Sulfide Production:

a)the organism produces hydrogen sulfide

1)the organism produces phenylalanine deaminase –Proteusvulgaris

2)the organism does not produce phenylalanine deaminase –Salmonellasp.

b)the organism does not produce hydorgen sulfide

1)the organism ferments lactose:

a)theorganism utilizes citrate as acarbonsource (positive citrate test)

1)theorganism is motile –Enterobacter cloacae

2)the organism is not motile –Klebsiellapneumoniae

b)theorganism does not utilize citrate as a carbon source (negativecitratetest) – E coli

2)the organism does not ferment lactose:

a)theorganism is motile –Serratiamarcesens(late fermenter –appearslactose negative at 24 hours, but maybecomepositive at 48 to72 hours)

b)the organism is not motile –Shigellasonnei

Table of results for the organisms used in class

Bacteria / Motility / LactoseFermentation / Indole Production / MethylRed / CitrateUtilization / Urea Hydrolysis / Phenylalaninedeaminase / Hydrogen sulfide production
Escherichiacoli / Motile / Fermentation / Yes / Yes / No / No / No / No
Enterobactercloacae / Motile / Fermentation / No / No / Yes / No / No / No
Proteus vulgaris / Motile / NoFermentation / Yes / Yes / No / Yes / Yes / Yes
Serratiamarcescens / Motile / NoFermentation / No / No / Yes / No / No / No
Klebsiellapneumoniae / Not Motile / Fermentation / No / No / Yes / Yes / No / No
Salmonella typhimurium / Motile / NoFermentation / No / Yes / Yes / No / No / Yes
Shigellasonnei / Not Motile / NoFermentation / No / Yes / No / No / No / No