By: Dr Muhammad Noman Naseem

M.Phil. Scholar (Pathology)nomannaseem97@yahoo.com

Isolation and Identification of

Salmonella & E.coli

Salmonella

Sample Collection • Both clinical tissue and environmental sample should

be collected.• Clinical tissues samples include liver, spleen, heart,

ovary, yolk sack, synovia, eye and brain.• Caeca and caeca tonsils may be cultured.• Cloaca swabs. • Eggs

• Environmental sample can be taken by Drag swab Floor litter(dry areas) Dust Cage housing Feed

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• To determine Salmonella infection in 1- day old chicks , 4- methods have been used.

Take 25, 1-day old chicks (in a group of 5) and pool internal organs, yolk sac and intestinal section from each group in three separate sterile plastic bags and add selective enrichment broth.

Culture chick meconium. 5g of meconium collected from chicks.

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Culture the paper that line the boxes in which chicks are transported, or surface of chick paper may be swabbed with DSSM.

50-100 chicks that have been held for 48-72 hours with only clean water available for consumption. This promotes the wide spread of infection among box mates and increase the likelihood of detection by culturing the paper line.

Material Required for Isolation

• Selective Enrichment Broth Tetrathionate enrichment broth.(Tetrathionate

reductase enzyme)

Selenite enrichment broth.

Rappaport Vassiliadis enrichment broth.

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• Selective MediaBrilliant green agar

MacConkey agar

XLD4

ProcedureSample(clinical or environmental)

Inoculate on selective enrichment broth

37c 24 hours

Inoculate on selective media

37c 24-48 hours

Check results

RESULTSOn MacConkey agar• Salmonella gives colourless colonies

• E.coli gives pink colour colonies

PrincipalComposition of macConkey,s agar

• Lactose, bile salts, indicator system(neutral red).

• Lactose fermenter bacteria have lactase enzyme.

• Due to lactose fermentation ,PH decrease, indicator which is colorless at 6.8 PH, become pink

at acidic PH.

• Lactose positive are E.coli, Enterobactor, Klebsiella

• Lactose Negative are Salmonella, Proteus, Yersinia, Pseudomonas, Shigella.

• Staphylococcus and Streptococcus no growth.

RESULTS

On Brilliant green agar(BGA).• Salmonella colonies are red.

• E. coli colourless colonies.

Principal• Composition ; lactose, sucrose, phenol red,

brilliant green dye.

• Phenol red is indicator.

• Brilliant green inhibit G +ve bacteria and most of G –ve bacilli other than Salmonella spp.

• Phenol red turns the medium yellow with formation of acid, if lactose and sucrose are fermented.

RESULTS

On XLD4• Colonies are red with a black center due to

H2S production.

Principal• Normal PH of this medium 7.4, normally

appear red due to indicator phenol red.

• As PH decrease due to sugar fermentation, phenol red change to yellow.

• Salmonella fermented xylose to produce acid result in PH decrease.

• After exhausting the xylose supply, Salmonella colonies decarboxylate lysine.

• Result in increase in PH (alkaline) lead to red color colonies.

CONTINUE• Salmonella metabolize thiosulfate to produce

H2S which leads to formation of colonies with black center.

• E.coli can not decarboxylate lysine, so no reversion of PH, yellow colonies.

• Shieglla form red color colnies because not ferment xylose.

• Pseudomonas gives pink color colonies.

Motility Determination

Hanging Drop slide

Soft Agar Stabbing (Tube Method). SIM

SIM medium• Sulfide indole motility (SIM) medium is a

semisolid agar used to determine the• hydrogen sulfide (H2S) production,• indole formation,• and motility.• SIM medium is used to differentiate members

of the family Enterobacteriaceae.• SIM Medium, is rich in tryptophan. Organisms

possessing the enzyme tryptophanase degrade tryptophan to indole.

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• Indole is detected upon the addition of KovacsReagent following incubation of the inoculated

medium.

• Indole combines with p-dimethylamino-benzaldehyde and produces red colour.

• A negative indole test produces no color change upon the addition of Kovacs Reagent..

Result InterpretationTest Organisms Results

Salmonella Indole –veMotility +veH2S +ve

Escherichia coli Indole +ve+ve or –ve for motility-ve for H2S

Identification

TSI slant ( 1% lactose, 1% sucrose, 0.1% glucose)• Salmonella produce alkaline red slants and

acid yellow butt with gas bubbles and blackening.

• S.gallinarum not form gas in TSI.

• S.pullorum show gas production in TSI.

TRIPLE SUGAR IRONINGREDIENTS FUNCTION RESULT/INTERPRETATION

Phenol red a pH indicator:below 6.8 it is yellowabove 8.2., it is red

Phenol red turns yellow in an acid environment. indicating whether the acids of fermentation have been produced. Failure to turn the butt yellow indicates that no fermentation has occurred, and that the bacterium is an obligate aerobe.

Glucose if only glucose is fermented, only a small amount of acid is produced

If only glucose is fermented, only enough acid is produced to turn the butt yellow. The slant will remain red.

lactosesucrose

if the culture can ferment either lactose (lac+) and/or sucrose (suc+), a large amount of acid is produced

a large amount of acid turns both butt and slant yellow, thus indicating the ability of the culture to ferment either lactose or sucrose

FeSO4(ferrous sulfate)

A source of iron and sulfur

A few bacteria are capable of reducing the sulfateto H2S (hydrogen sulfide). The iron combines with the H2S to form ferrous sulfide, a black compound. This will turn the butt black. Thus, a black butt indicates H2S production.

TRIPLE SUGAR IRON AGAR: INTERPRETATION OF RESULTS

SCORING THE SLANTS: Examine the slant and butt, and record data using the following criteria

SLANT COLOR Code letter: Interpretation

RED Rdoes not ferment either lactose or sucrose

YELLOW Yferments lactose and/or sucrose

TRIPLE SUGAR IRON AGAR: INTERPRETATION OF RESULTS

SCORING THE BUTT COLOR AND CONDITION

BUTT COLOR/CONDITION Code Letter Interpretation

RED Rno fermentation, the bacterium is an

obligate aerobe

YELLOW Y

some fermentation has occurred, acid

has been produced, it is a facultative

anaerobe.

GAS FORMED YG

Seen as cracks in the agar, bubbles, or

the entire slant may be pushed up of the

tube.

BLACK "+" H2S has been produced

BACTERIUM SLANT BUTT H2S

Shigella R Y

Salmonella typhimurium R Y(G) +

Salmonella pullorum R Y(G) +

Salmonella gallinarum R Y +

Salmonella typhi R Y +

Aerobacter aerogenes Y Y(G)

Escherichia coli Y Y(G)

Citrobacter freundii Y Y (G) +

Proteus vulgaris Y Y(G) +

Klebsiella pneumoniae Y R or Y

Pseudomonas aeruginosa R R

TSI agar slant results: (from left) preinoculated (as control), P. aeruginosa, E. coli, Salmonella Typhimurium, Shigella flexneri

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LYSINE IRON SLANT (LI)• Decarboxylate lysine

• Deep purple slant.

• Alkaline or neutral butt with blackening due

H2S production.

Principal• The indicator in LIA is bromocresol purple.

• An alkaline reaction is seen by the presence of a purple color, and an acidic reaction is indicated by the appearance of a yellow color.

• Sodium thiosulfate is incorporated into this medium as the source of hydrogen sulfide, and ferric ammonium citrate as the indicator, which turns the butt black in the presence of free hydrogen sulfide gas.

Serological Typing

• 1st of serological typing is to serogroup the isolate based on their somatic O-group antigen using commercially available polyvalent antisera.

• Than check using individual single factor O-group antisera.

• Serotyping based on flagella anti-gen can also be done.

E.coli

Sample Collection

• Only internal organ(liver) and blood, not feaces or intestine.

• When fibro purulent lesion swab sample of exudate should be collected from pericardial sac, air sac & joints.

• When PM changes are obvious, bone marrow may be useful because they are less likely than other tissues to contain intestinal E.coli.

Preferred Culture MediaSelective media• MacConkey,s agar

• Tergitol 7 agar

• Tryptose blood agar with 5% bovine blood can be used a primary culture medium to support growth of E.coli & other bacterial pathogens.

• Blood sample should be diluted (1:10) in brain heart infusion broth than inoculate on culture media.

Isolation • Samples should be inoculated and incubated

on selective media for 18-24 hours at 37c for isolation of E.coli.

• On macConkey agar, E.coli produce 1-2 mm diameter pink colonies.

• On Tergitol 7 agar, produce 1-2 mm diameter yellow colonies with yellow zone.

Principle In Tergitol 7 agar

• Tri-phenyltetrazolium chloride (TTC) is added.

• This is not reduce by E.coli.

• Other bacteria reduce it to form formazan(a red color product).

Identification

• Suspected colonies inoculated on triple sugar iron slants.

• On TSI produce acid and gas not H2S.

• On SIM medium

• E.coli is +ve for indole reaction.

• +ve or -ve for motility.

• -ve for H2S

Pathogenicity• To check pathogenicity of E.coli

• Inoculate 0.1 ml broth culture in young chicks (less than 3-weeks of age).

• Pathogenic isolates should produce death or show characteristics lesion of colibacillosis with in 3-days.

• Now a days, PCR is used.

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• Pathogenic serotypes O78, O2, O1, O35 and O36.

• O78:K80 and O2:K1 are mostly isolated from clinical cases of colibacillosis.

DifferenceCHARACTER SLAMONELLA E.coli

macConkey,s agar Colorless colonies Pink color colonies

Brilliant Green agar Colonies are red Colorless colonies

XLD4 Red colonies with black center

yellow colonies

Tergitol 7 agar Red colonies yellow colonies

TSI slant Red slant, yellow butt,H2S +

Gas +ve or _ve

Yellow slant and buttGas +ve and H2S -ve

SIM medium Indole –veMotility +ve

H2S +ve

Indole +ve+ve or –ve for motility

-ve for H2S