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 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,P H decrease, indicator which is colorless at 6.8 P H, become pink at acidic P H. 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 P H of this medium 7.4, normally appear red due to indicator phenol red. As P H decrease due to sugar fermentation, phenol red change to yellow. Salmonella fermented xylose to produce acid result in P H 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 (H 2 S) 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 COLORCode letter:Interpretation REDR does not ferment either lactose or sucrose YELLOWY ferments lactose and/or sucrose

23. TRIPLE SUGAR IRON AGAR: INTERPRETATION OF RESULTS SCORING THE BUTT COLOR AND CONDITION BUTT COLOR/CONDITIONCode LetterInterpretation REDR no fermentation, the bacterium is an obligate aerobe YELLOWY some fermentation has occurred, acid has been produced, it is a facultative anaerobe. GAS FORMEDYG Seen as cracks in the agar, bubbles, or the entire slant may be pushed up of the tube. BLACK"+""+"H 2 S has been produced

24. BACTERIUM SLANT BUTTH2S ShigellaRY Salmonella typhimuriumRY(G)+ Salmonella pullorumRY(G)+ Salmonella gallinarumRY+ Salmonella typhiRY+ Aerobacter aerogenesYY(G) Escherichia coliYY(G) Citrobacter freundiiYY (G)+ Proteus vulgarisYY(G) + Klebsiella pneumoniaeY R or Y Pseudomonas aeruginosaRR

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 1 st 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 coloniesPink color colonies Brilliant Green agar Colonies are redColorless colonies XLD4 Red colonies with black center yellow colonies Tergitol 7 agar Red coloniesyellow 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