PrincipleSalmonellaisagramnegativemesophilicbacteriumthatcangrowatrefrigerationtemperature(4to

10)0C,withrapidgrowthbetween25and430C,althoughitisusuallysensitivetoabove550C.

Salmonellaisamajorzoonoticpathogenandthecauseofnumerousoutbreaksworldwideeachyear.

EggshellscanbecomecontaminatedwithSalmonellaeitherbecauseofaninfectionoftheoviductorby
environmentalcontaminationduetothesheddingofthebacteriabyinfectedanimals.

Materials:
1) Media-We have used three types of media.These are
I.
pre-enrichment
II.
Selective enrichment
III.
Selective agar media
2) Sample-We have used hens egg
3) Equipment i.
Cotton and Cotton swab
ii.
Glass apparatus- Testtubes, Pipette, Petri-dish, Conical flask Beaker
iii.
Bunsen burner
iv.
Balance meter
v.
Glassware marker pen
vi.
Inoculation loop
vii.
Micropipette
viii.
Hand gloves
ix.
Forceps
x.
Saline water
xi.
Alcohol

Description of media:
1) Pre-enrichment media:-BufferedPeptoneWaterisapreenrichmentmediumusedforincreasingthe
recoveryofinjuredSalmonellaspeciesfromfoodspriortoselectiveenrichmentandisolation

2) Selective enrichment media:-The objective of selective enrichment media is to

select salmonellae feomall oter organisms present in the pre-enriched sample.The
ideal selective enrichment should repress competing organisms and allow the
salmonellae to multiply without restriction.Traditionally, the following families of
selective enrichments are used:(1)tetrathionate media, (2) selenite-based media
and (3) Rappaport media.
Beside selective agents to suppress the growth of non-salmonella strains, the
selective enrichments media may also contain agents which will indicate the
presence of salmonella spp. Additional to selective agents, a low P H and elevated
incubation temperature(approximately 420C) of the medium will further inhibit the
growth of interfering background flora.
RAPPAPORTVASSILIADISSALMONELLAENRICHMENTBROTH
Formula/Liter
SoyPeptone.........................................................................4.50g
SodiumChloride.....................................................................8.0g
PotassiumPhosphate,monobasic.......................................0.60g
PotassiumPhosphate,dibasic.............................................0.40g
MagnesiumChloride,anhydrous*......................................13.58g
MalachiteGreen.................................................................0.036g
FinalpH:5.20.2at25C

SoyPeptoneisthecarbonandnitrogensourcesforgeneralgrowthrequirementsinRappaportVassiliadis
SalmonellaEnrichmentBroth.MagnesiumChlorideraisestheosmoticpressureinthemedium,andPotassium
Phosphateactsasabuffer.MalachiteGreenisinhibitorytoorganismsotherthanSalmonellaspp.ThelowpHof
themedium,combinedwiththepresenceofMalachiteGreenandMagnesiumChloride,selectforthehighly
resistantSalmonellaspp
SELENITECYSTINEBROTH
Formula/Liter
EnzymaticDigestofCasein...................................................2.5g
EnzymaticDigestofAnimalTissue.......................................2.5g
Lactose.....................................................................................4g
SodiumPhosphate..................................................................10g
SodiumSelenite........................................................................4g
LCystine..............................................................................0.01g
FinalpH:7.00.2at25C

EnzymaticDigestofCaseinandEnzymaticDigestofAnimalTissueareusedasnitrogenandvitaminsourcesin
SeleniteCystineBroth.LactoseisthecarbohydrateandDisodiumPhosphateisthebuffer.SodiumSeleniteisthe
selectiveagentagainstGrampositivebacteriaandmostentericGramnegativebacilli.LCystineisareducingagent
Selectiveplatingagar:

XLD Agar (Xylose Lysine Desoxycholate Agar) is a moderately selective and

differential medium for the isolation and differentiation of gram-negative enteric
pathogens (Salmonella and Shigella) from clinical specimens.

XLD Agar is both a selective and differential medium. It contains yeast extract as a
source of nutrients and vitamins. It utilizes sodium desoxycholate as the selective
agent and, therefore, is inhibitory to gram-positive micro-organisms. Xylose is
incorporated into the medium since it is fermented by practically all enterics except
for the shigellae and this property enables the differentiation of Shigella species.
Lysine is included to enable the Salmonella group to be differentiated from the non
pathogens since without lysine, salmonellae rapidly would ferment the xylose and
be indistinguishable from nonpathogenic species. After the salmonellae exhaust the
supply of xylose, the lysine is attacked via the enzyme lysine decarboxylase, with
reversion to an alkaline pH which mimics the Shigella reaction. To prevent similar
reversion by lysine positive coliforms, lactose and sucrose are added to produce
acid in excess. 1 To add to the differentiating ability of the formulation, an H2S
indicator system, consisting of sodium thiosulfate and ferric ammonium citrate, is

included for the visualization of the hydrogen sulfide produced, resulting in the
formation of colonies with black centers. The non pathogenic H2S- producers do not
decarboxylate lysine; therefore, the acid reaction produced by them prevents the
blackening of the colonies which takes place only at neutral or alcaline pH. 1

Procedure:Sample collection:-At first hens eggs were collected from retailer shop with a
view to isolation of salmonellae spp. on eggshell
Microbiological Analysis:i.
ii.
iii.
iv.
v.
vi.
vii.

viii.

At first we autoclaved all necessary instrument which are autoclavable.

At first we washed out our hand with alcohol to minimize further
contamination of eggshell and other instruments
With forceps a cotton swab was taken and immersed it into saline
water in a beaker
With this wet cotton swab rubbed gently on an eggshell
After rubbing, that cotton swab was immersed into BufferedPeptoneWater
whichisapreenrichmentmedium
Preenrichmentmediumcontainingthatcottonswabwasincubatedfor(18to24)hat
370C
Afterincubation,1mlbrothfrompreenrichmentmediumwastransferredtoaselective
enrichedmediumforenhanchingthegrowthofsalmonellaespp.andincubatedat41.50C
for(24to48)h
ApetriplatewithXLD Agar (Xylose Lysine Desoxycholate Agar) was

labelled on one corner of the plate.

ix.

x.

Withsterileinoculationloop,aloopfulbrothfromincubatedenrichmentmediumwas
transferredtothatpetriplatewithXLD Agar (Xylose Lysine Desoxycholate

Agar) and streaked on that plate according to Four Quadrent

Streaking Method.
After streaking, this petri-plate incubated at inverted position for
24 h at 370C.

Result:Organisms that ferment xylose will acidify the medium and produce yellow

colonies. Organisms able to remove the carboxyl group from (decarboxylate) L-lysine
will release alkaline products and produce red colonies. Organisms able to reduce sulfur
will produce a black precipitate in the growth due to the reaction of ferric ammonium
citrate with H2S.
After overnight incubation I have observed my petri-plate and seen that two single colonies
at 3rd quadrent position of the plate produced a black precipitate in the growth due to the
reaction of ferric ammonium citrate with H2S.