Isolation and identification of pyogenic cocci

1. Experiment Three Isolation and identification of pyogenic cocci

2. Objective of Experiment • To masterbasicprinciple and method,which use to isolate and identify pyogenic cocci from clinical specimens. • To diagnose clinical disease and guide doctors to select proper medicines .

3. Colonial characteristics colonial Hemolytic reaction morphology observation Pigmentation PROCEDURE Smears Morphological characteristics (Gram-stain) (Microscopic examination) Specimens Isolation culture (blood agar plate) stained and observe typical colonies Directidentification Pure culture Antibiotic susceptibility test

4. Identified method for Staphylococci • Gram-stain • Isolation and culture • Pure culture • Directidentification

5. Staphylococci are Gram-positive cocci, typically arranged in clumps or Grape-like clusters

7. Directidentification • The mannitol fermentation test. • The Coagulase Test • The Dnase Test • The phage typing test • Animal Experiment

8. The mannitol fermentation test. • Inoculate the bacteria into a mannitol micro-tube,incubate at 370C for 18h.S.aureus will ferment mannitol to produce acid,which causes the medium to turn yellow.

9. Test of Microbiological experiment • While a patient is infected by pathogenic enterobacteria (Salmonella),how to diagnose with microbiological methods in lab? (simple procedure) • While a patient is infected by pyogenic cocci,how to diagnose with microbiological methods in lab?(simple procedure) • While a patient is infected by influenza virus,how to diagnose with microbiological methods in lab?(simple procedure)

10. The Coagulase Test • Coagulase is an enzyme converting fibrinogen into fibrin promoting blood clotting. • It might be a virulence factor with the coagulated blood around the bacteria protecting them from the immune system. • Coagulase-negative strains are often as pathogenic as coagulase-positive strains.

11. The Slide Coagulase Test

12. The Tube coagulase Test Left tube coagulase positive Right tube coagulase negative

13. The Dnase Test • Inoculate Dnase agar plates with a loop so that the growth is in plaques about 1 cm in diameter.Incubate at 370C overnight.Flood the plate with 1 N hydrochloric acid.Clearing around the colonies indicates Dnase activity.The hydrochloric acid reacts with unchanged deoxyribonucleic acid to give a cloudy precipitate.A few other bacteria,e.g. Serratia,may give a positive reaction.

14. The phage typing test • This test is used to trace the infective agent in epidemiology if necessary. • It is usually not done for routine clinical purpose.

15. Animal Experiment vomit excrement remaindered food Meat soup media Isolation and identification of bacteria filter injection 6—8w cat observation Food poisoning

16. The antibiotic susceptibility test • This test is helpful for the treatment of S.aureus infection. • materials • Staphaureus（isolated from the pus of a patient）. • Several kinds of filter paper （each contains different kinds of antibiotics） • Nutrient agar plate

17. The antibiotic susceptibility test • Streaking the staphaureus on agar plate • (thoroughly covered the plate) • Put 4 kinds of paper contained different antibiotics • on the plate (each paper are far away about 2 cm) • Incubate at 370C for 18-24 hours. • Observe the results the plates • The sensitivity of the organism is indicated by the diameter • of the zone of growth inhibition.

19. Laboratory Diagnosis of Pathogenic Enterobacterial Infection

20. Dimidiation of the enterobacteria according to the fermentation of lactose • Lactase fermenters:saprophytic and commensal Escherichia Klebsiella Citrobater Enterobacter • Non lactase fermenters: pathogens Salmonella Shigella some Citrobacter Proteus Serratia

22. Procedure Colonial characteristic observation Specimens isolation Gram Staining (SS/EMB plate) Serological identification TSI Biochemical reaction

23. Specimens • Different specimens should be taken depending on the kind and the process of the disease. blood bone marrow Urine stool

24. Isolation • Culture medium: S.S agar • Method: streak plate • Result: Pathogenetic colonies: middle size, red Suspect colonies: colorless, small, opaque

25. Biochemical reactions of Salmonella, etc Species bottom slope H2S motility E.coli AG AG - + Salmonella A - +/- + Other Salmonella AG - +/- + Shigella A - +/- - bottom: ferment dextrose slope: lactose A:acid AG: acid and gas

26. Antigens of salmonella • O antigen polysaccharide of LPS somatic antigen used to divide Salmonella to 42 groups A-Z groups are pathogenic stable to heat (remains activity at 100 ℃ ) H antigen flagella antigen divide Salmonella tow phase: special no special Vi antigen related to the virulence of Salmonella sensitive to heat ( lose activity at 60 ℃)

28. Salmonella

29. Serological Identification of Salmonella Select the specimen React with A-Z polyvalent antiserum Agglutination reaction (＋) (－) React with several individual types of anti-O and anti-H antiserum React with anti-Vi antiserum Identify its group and phage

30. Gram Stain

31. Gram stain of Nocardia asteroides acid-fast stain of Nocardia asteroides

32. flagella