lab 14- Ecoli & Kleb 22. enterobacteriace pptx

Enterobacteriace E col and Kleb lab

1. Lab-14 (see Lab manual) Enterobacteriaceae E.coli & Klebsiella

2. Enterobacteriaceae Large group of Gram negative rods. Natural habitat is the intestinal tract of human and animals. Some enteric organism eg . Escherichia coli and Proteus are part of the normal flora, and incidentally cause disease. Some strains are opportunistic pathogens Some strains are true pathogens ( Salmonella, Shigella,Yersinia , some strain E. coli )

3. Characters of Enterobacteriaceae All Enterobacteriaciae Gram-negative rods , arranged singly Facultative anaerobic , ferment glucose with acid production Reduce nitrates into nitrites Oxidase negative Motile except Shigella and Klebsiella Non-capsulated except Klebsiella Grow on bile containing media ( MacConkey agar)

4. Enterobacteriaceae spp. Escherichia coli ( E.coli ) Klebsiella Proteus Salmonella Shigella

5. Classification of Enterobacteriaceae according to lactose fermentation (growth on MacConkey Agar) Enterobacteriaceae Lactose Fermenters Lactose Non- Fermenters Escherichia coli Klebsiella spp Enterobacter spp Citrobacter spp Salmonella spp Schigella spp Proteus spp Yersinia spp Pink colonies Colorless colonies Acid Neutral red No acid

6. Lactose fermentation - Some ferment lactose rapidly - Some does it slowly - The others do not ferment lactose at all

7. Growth on MacConkey agar Uninoculated plate Lactose non fermenters Salmonella, Shigella , Proteus Lactose fermenters E. coli, Citrobacter Klebsiella , Enterobacter Colorless colonies Pink colonies

8. Escherichia coli Lactose fermenter Motile Non capsulated Non- sporulated

9. Klebsiella Lactose fermenter Polysaccharide capsule Non motile

10. Laboratory diagnosis Specimens : urine, pus, stool. Staining method : Gram stain (G- ve bacilli). Culture : 1- MacConkey’s agar 2- Eosin- Methylene Blue (EMB) agar 3- Blood agar Biochemical test: IMViC tests Motility test The API 20E System Vitek test

11. Cultivation on Mac Agar E. coli Klebsiella

12. E. coli on Mac. agar

13. Klebsiella pneumoniae on blood agar

14. Klebsiella and E. coli on EMB agar

15. Metabolic reactions & Biochemical Tests The IMVC tests were developed in order to distinguish of E. coli and Klebsiella from related species that are Lactose fermenters I ndole M ethyl red V oges-Proskauer C itrate

16. Principle: This test demonstrates the ability of certain bacteria to decompose the amino acid tryptophan to indol , which accumulates in the medium. Results E coli + ve red colour Klebsiella - ve Indol production test

17. Methyl Red test (MR) Principle: To detect acid production during fermentation of glucose as shown by changing the colour of methyl red indicator Results E coli + ve (red colour ) Klebsiella - ve (yellow colour )

18. Principle: Many bacteria ferment glucose with production of acetoin or its reduction products, these can be detected by oxidation under alkaline conditions. Results: E coli - ve (no pink-red colour ) Klebsiella + ve (pink-red colour ) Voges-Proskauer (VP) test

19. To detects the ability of an organism to use citrate as carbon & energy source for growth and ammonium salt as the source of nitrogen. Principle: Degradation of citrate cause alkalinization of the medium , then change in the pH , the indicator become blue. Citrate test on Simmon’s citrate agar Results: E. coli - ve (No colour change ) Klebsiella + ve ( colour change from green to blue )

20. The API 20E System

21. Motility Test + ve - ve + ve

22. Vitek identification : Automated system for bacteria identification & antibiotic susceptibility testing. * Panels are incubated & inhibition of bacterial growth by the antibiotic is measured using turbidimetry , spectrophotometry , fluorescence * Obtain the result in a period of fewer than 18 hrs.

23. Vitek identification cards

24. Differentiation between E. Coli and Klebsiella Klebsiella E. coli Test IMViC - + I = Indole - + M = Methyl red + - V = Voges - Proskaur + - C = Citrate - + Motility