1. Isolation and identification of pyogenic cocci
Experiment Three
Isolation and identification of pyogenic cocci

2. Objective of Experiment
To master basic principle 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. PROCEDURE Smears Morphological characteristics (Gram-stain)
(Microscopic examination)
Colonial characteristics
Hemolytic reaction
Pigmentation
Specimens
morphology observation
colonial
Isolation culture
(blood agar plate)
stained and observe
typical colonies
Direct identification
Pure culture
Antibiotic susceptibility test

4. Identified method for Staphylococci
Gram-stain
Isolation and culture
Pure culture
Direct identification

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

7. Direct identification
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. Isolation and identification of bacteria
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 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. acid-fast stain of Nocardia asteroides
Gram stain of
Nocardia asteroides
acid-fast stain of Nocardia asteroides

32. flagella