Umar Aziz Bsc(hon.

)poultry science 17-21

Collection , Isolation
and submission of salmonella
Introduction
•Salmonella is gram negative bacteria, facultative anerobic and
facultative intracellular parasite.
• it is a genus of rod-shaped (bacillus) Gram-negative bacteria of the
family Enterobacteriaceae. The two species of Salmonella are
Salmonella enterica and Salmonella bongori. S. enterica is the type
species and is further divided into six subspecies that include over
2,600 serotypes. Salmonella was named after Daniel Elmer Salmon
(1850–1914), an American veterinary surgeon.
collection
• Salmonella can be collect from uncooked chicken, egg , turkey ,
ducks etc. and also from vegetables and unpasteurized milk ,Culture
of paper, and from litter by boot swab
•Example for collection of salmonella (eggs)
•Materials:
•We have used three type of media
1. Pre-enrichment
2. Selective-enrichment
3. Selective agar media
Collection
Egg
• Equipment:
1. Cotton or cotton swab
2. Glass apparatus
3. Burner
4. micropipette
Collection
1. Forceps
2. Saline water
3. Alcohol
4. Gloves
5. Inoculation loop
Description of media
Pre-enrichment media
• Buffered peptone water is a pre-enrich media use to inc. the recovery
of injured salmonella from food prior to selective media and isolation
Ingredients
• Proteose peptone 10.000
• Nacl 5.000
• Disodium phosphate ,anhydrous 3.500
• Monopotassium phosphate 1.500
• Final ph (at 25c) 7.2
Media
• Selective media
• Green agar
• XLD4
• Macconkey agar
• Primary isolation media
• Blood agar incubate in the air at 35 to 37 c for 18 to 24 hour
• CLED agar incubate in the air at 35 to 37 c for 18 to 24 hour
• XLD agar incubate in the air at 35 to 37 c for 18 to 24 hour
• DCA incubate in the air at 35 to 37 c for 18 to 24 hour
• Colony formation
• on blood agar the colony are moist and 1-2 mm in diameter
• on CLED it cannot fermentate with lactose but some serotype
s.arizonae, s.indiana may fermentate with lactose
• on XLD red colony appear and a black center
• on DCA all colonies are black center and colorless colony
Isolation example
• Salmonella can be selected by using Salmonella Shigella Agar
(SSA) selective media. Five-fold serial dilution of the sample was
carried out and inoculated using the pour plate method
• one mililiter (1ml) of serial dilution was inoculated on sterile Petri
dish, after that media was poured aseptically on the inoculated
plates. The plates should be incubated at 370C for 24 hours. After
incubation morphologically colonies Salmonella sp. are shown with
colorless colony with black center
Isolation result and discussion
• Soil sample was taken from three different locations in poultry
coop soil. The three retrieval locations have different characteristic.
The location of the first sample was a location with good intensity
of light, the second sample was a location with less intensity of
light and near to wellspring , and the last sample was the location
that didn’t expose with light. Physicochemical characteristics of
soil sample test result were presented in Table1 with three
replications. The picture of the soil sample from three locations
was presented in next slide
• The condition of the poultry coop is a factor which great influences
the poultry’s health. Exploration of Salmonella is an effort to
understand the condition of the poultry coop and preserve the
health of the poultry.
Identification methods and explain
• On diff. media
• There are two types of fermentation happen on it lactose and non-lactose
fermentation the color occurs respectively bright pink and transparent color colony
• By H2S
• it can react with triple sugar iron it can produce the h2s gas, and cause a blackish
color around the test tube
• By agglutination
• We can also identify salmonella by agglutination test with salmonella galinaraum
take a drop of serum and mix with antigen
• Blood cluster
• 85% of blood cluster are positive
Intrepretation of results
identification
• Biochemical test
• In this kind of test, we can identify the salmonella on the basis of ferment so the salmonella in
ferment with the maltose, dextrose, mannitol, sorbitol and clearly not fermentate with the
lactose and sucrose
• ureae test
• salmonella can not produce urease
• oxidase test
• salmonella is oxidase negative if positive appear to discard and take the negative it can
agglutinate with the polyvalent o and antiserum then again there are two results positive and
negative in negative case urease is added at 37c for an hour then on CLED plate then again
watch the negative and positive results discrete positive results now first positive of
agglutination and CLDE plate again for biochemical test after that check pure culture there will
be two results salmonella related and not related take related and done the agglutination tests
Differents results for identification
Salmonella is motile positive how ?
Biochemical and serological reactions
of Salmonella
Storage of salmonella
• Salmonella is not able to grow at low temperatures and samples should
be refrigerated if they cannot be sent for analysis immediately. The cells
survive well in frozen foods and other materials, but samples should be
kept frozen prior to testing. Typically, 25g food samples are cultured in
detection testing, but dried foods require a resuscitation stage for sub-
lethally damaged cells in non-selective pre-enrichment media, such as
buffered peptone water, before further culture. Furthermore, some
dried foods, notably herbs and spices and dried onions, contain
compounds that could inhibit Salmonella growth in enrichment
cultures. These compounds should be neutralised, either by the
addition of a suitable neutralising agent, or by additional dilution
Preservation

• Differences in the ability to withstand freeze-drying were

demonstrated among strains of Salmonella typhimurium. On the
average, the number of viable cells in freeze-dried cultures stored
at 5 C for 12 to 18 months was approximately one half as large as
that found 24 hr after freeze-drying. The viability in samples stored
at higher temperatures declined rapidly and was correlated with
the dryness of the sample. The virulence for mice of three strains
of S. typhimurium did not change appreciably when samples were
kept for 1 or 2 years as freeze-dried samples stored at 5 C, or as
agar cultures stored at 5 C or at room temperature
Submission
• Submit 1 isolate of each somatic group recovered from each
analytical unit, unless otherwise instructed. Submit cultures on BHI
agar slants in screw-cap tubes (13 × 100 mm or 16 × 125 mm) with
caps secured tightly. Label each tube with sample number,
subsample (analytical unit) number, and code, if applicable. Submit
a copy of the Collection Report, FD-464, or Import Sample Report,
FD-784 for each sample
Submission
• Place cultures in culture container with official FDA seal. Place
accompanying records inside shipping carton but not within
officially sealed culture container. Submit memo or cover letter for
each sample number to expedite reporting of results. Prepare
cultures for shipment according to requirements for shipment of
etiological agents . Label secondary shipping container according
to Send container by most rapid mail service available. Maintain
duplicate cultures of those submitted for serotyping only on those
samples under consideration for legal action.
Submission guidline
• Microbiology Field laboratories should follow the following guidance
in sending Salmonella isolates for serotyping:
• Name
• Adress
• Street no
• Dr name
• Fax no
• Cell no
• etc