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

2. Objective of Experiment
To master primary principle and method of isolation and identification pyogenic cocci from clinical specimens .
To diagnose clinical disease and guide us to use medicines .

3. Morphologic observation
1.Staphylococci
G+, arranged in grape like irregular clusters
2.Streptococci
G+, arranged in chain
3.Pneumococci
G+, Lancet-shaped cell.
The Capsule stain: the capsule appears colorless in contrast to the blue of the cell
4.Meningococci
G-, kidney-shaped ,arranged in pairs
5.Gonococci

4. Streptococcus
Staphylococcus

5. Staphylococcus
Streptococcus

6. Diplococcus

7. N. Gonorrhoeae
N gonorrhoeae infections have a high prevalence and low mortality
Acquired by sexual contact and usually affect
Mucous membranes of the urethra in men
Endocervix in women
Infection may disseminate to a variety of tissues
One of the most common STIs in the world

8. Morphological characteristics (Gram-stain) (Microscopic examination)
PROCEDURE
Isolation culture
Smears
(blood agar plates)
Morphological characteristics
(Gram-stain)
(Microscopic examination)
Colonial characteristics
Hemolytic reaction
Pigmentation
Specimens
morphology observation
colonial
Gram staining
observation with the microscope
Pure culture
typical colonies
Antibiotic susceptibility test
Direct identification

9. Identified method for STAPHYLOCOCCI
Gram-stain
Isolation and culture
Pure culture
Direct identification
The antibiotic susceptibility test

10. Gram-stain Morphological characteristics
-Typical staphylococcus cells are spherical,arranging in irregular clusters,which just like as clusters of grape.
The cell is about 1 μm in diameter.
Typical cells are Gram-positive,nonmotile and do not form spore.

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

12. Isolation and culture Cultural
Specimens planted on blood agar plates give rise to typical colonies in 18 hours at 370C
Hemolysis and pigment production may not occur until several days later and are optimal at room temperature.

13. Isolation and culture colonial characteristics
Colonies of staphylococci on solid meida are round,smooth,raised,and glistening.S.aureus forms gray to deep golden yellow colonies.
S.epidermidis colonies are gray to white on primary isolation.
Various degrees of hemolysis are produced by S.auerus and occasionally by other species.

15. Pure culture MATERIALS: PROCEDURE . Agar slope culture
Colonies on agar plate
PROCEDURE
With the flame-sterilized wire inoculating loop, transfer a small amount of bacteria from the colony on agar plate. Then streak on the agar slope.
Sterile the mouth of tubes, replug the test tubes and flame the loop.
Label and incubate at 37℃ for hours .

16. Directed identification
The Coagulase Test
The Dnase Test
The mannitol fermentation test.
The phage typing test
Animal Experiment

17. The Coagulase Test
Possession of the enzyme coagulase which coagulase plasma is an almost exclusive property of Staph.aureus.There are two ways of performing this test
The Slide Coagulase Test
The Tube coagulase Test

18. The Coagulase Test
Coagulase is an enzyme converting fibrinogen into fibrin promoting blood clotting. It has been speculated that this enzyme might be a virulence factor with the coagulated blood around the bacteria protecting them from the immune system.
However, coagulase-negative strains are often as pathogenic as coagulase-positive strains.

19. The Slide Coagulase Test

20. The Tube coagulase Test
positive negative

21. 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.

22. 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.

23. 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.

24. Animal Experiment Isolation and identification of bacteria Vomit
excrement
remaindered food
observation
filter
Meat soup media
6--8W cat
food poisoning
injection

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

26. The antibiotic susceptibility test
PROCEDURE
Streaking the S.aureus 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 ar 370C hours.
Observe the results the plates are examined for the present of zones of inhibition of bacterial growth around the filter paper.The sensitivity of the organism is indicated by the diameter of the zone of growth inhibition.

28. Hiss’ stain Dissection of white mice Materials
Animals:mice died from infection
Instruments for autopsy:sterile tweezers and scissors,anatomic board
Pins,iodine,ethanol,cotton absorbent gauze balls,3%lysol ,and so on

29. How to make a smear
Open the abdomen cavity from pubis and expose the abdominal viscera .
Check the intestine
Put tissue fluid on the slide

30. Examine under the oil immersion lens
Hiss capsule staining
1%crystal violet in alcohol
Smear
Dry
2mins
Purple black cells of bacteria and colorless capsule
Wash with 20%Cuso4
Examine under the oil immersion lens
Blot dry with bibulous paper

31. Pneumococcus