This presentation is made by concerning three books. The data used in this is mainly revolve about poultry point of view. REFERENCE Isolation and identification of avian pathogen(AAAP)

1. By: Dr Muhammad Noman Naseem
M.Phil. Scholar (Pathology)
nomannaseem97@yahoo.com
Isolation and
Identification of
Salmonella & E.coli

2. Salmonella

3. 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

4. Continue………..
• 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.

5. Continue………..
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.

6. Material Required for Isolation
• Selective Enrichment Broth
Tetrathionate enrichment broth.(Tetrathionate
reductase enzyme)
Selenite enrichment broth.
Rappaport Vassiliadis enrichment broth.

7. Continue……
• Selective Media
Brilliant green agar
MacConkey agar
XLD4

8. Procedure
Sample(clinical or environmental)
Inoculate on selective enrichment broth
37c 24 hours
Inoculate on selective media
37c 24-48 hours
Check results

9. RESULTS
On MacConkey agar
• Salmonella gives colourless colonies
• E.coli gives pink colour colonies

10. Principal
Composition 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.

11. RESULTS
 On Brilliant green agar(BGA).
• Salmonella colonies are red.
• E. coli colourless colonies.

12. 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.

13. RESULTS
On XLD4
• Colonies are red with a black center due to
H2S production.

14. 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.

15. 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.

16. Motility Determination
Hanging Drop slide
Soft Agar Stabbing (Tube Method). SIM

17. 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.

18. Continue……
• Indole is detected upon the addition of Kovacs
Reagent 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..

19. Result Interpretation
Test Organisms Results
Salmonella Indole –ve
Motility +ve
H2S +ve
Escherichia coli Indole +ve
+ve or –ve for motility
-ve for H2S

20. 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.

21. TRIPLE SUGAR IRON
INGREDIENTS FUNCTION RESULT/INTERPRETATION
Phenol red a pH indicator:
below 6.8 it is yellow
above 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.
lactose
sucrose
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 sulfate
to 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.

22. 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 R
does not ferment either
lactose or sucrose
YELLOW Y
ferments lactose and/or
sucrose

23. TRIPLE SUGAR IRON AGAR: INTERPRETATION OF RESULTS
SCORING THE BUTT COLOR AND CONDITION
BUTT COLOR/CONDITION Code Letter Interpretation
RED R
no 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

24. 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

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

26. Continue…….
LYSINE IRON SLANT (LI)
• Decarboxylate lysine
• Deep purple slant.
• Alkaline or neutral butt with blackening due
H2S production.

27. 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.

28. 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.

29. E.coli

30. 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.

31. Preferred Culture Media
Selective 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.

32. 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.

33. 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).

34. 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

35. 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.

36. Continue…..
• Pathogenic serotypes O78, O2, O1, O35 and
O36.
• O78:K80 and O2:K1 are mostly isolated from
clinical cases of colibacillosis.

37. Difference
CHARACTER 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 butt
Gas +ve and H2S -ve
SIM medium Indole –ve
Motility +ve
H2S +ve
Indole +ve
+ve or –ve for motility
-ve for H2S