1. Experiment 6 Isolation & identification of pathogenic cocci

2. The common procedures of the Isolation & Identification of pathogenic cocci
Morphological characteristics
Smears
(Microscopic examination)
(Gram-stain)
Colonial characteristics
colonial
Specimens
morphology observation
Hemolytic reaction
Isolation culture
Pigmentation
(blood agar plate)
stained and observe
typical colonies
Direct identification
Pure culture
Antibiotic susceptibility test

3. Materials Specimen: Pus swab (#1 and #2), and throat swab
Culture : blood agar plate (BAP)
Equipments: alcohol lamp, inoculating loop, slide,
marking pencil
Reagent:
gram stain reagent: Crystal violet
Iodine solution
Alcohol 95%
Safranin solution

4. Procedures test2：Smears test1：Isolation culture
Inoculate the swabs to blood agar by four-area streak technique separately.
Incubate them overnight at 37℃.
test2：Smears
Make Gram stain of swabs, and examine them under oil-immension objective.

5. Four-area streak dilution technique
area 1 rolled with pus swab
area 2-4 streaked with inoculating loop
1/10
1/5
1/4

6. Gram stain Crystal violet for 1min, then wash Prepare a smear
Pass the slide over flame
Stain
Crystal violet for 1min, then wash
Iodine for 1min, then wash
95% alcohol for 15sec, then wash
Safranin for 1min, then wash
Dry with bibulous paper
Observe it under oil-immension objective
Record the results.

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

8. Gram stain
BAP
swab #1
G+ cocci,irregular
Yellow, hemalytic
swab #2
white, non-hemalytic

9. Coagulase Test Procedures 1) Divide the slide into two equal parts
2) place two loops of NS on the left side and on the right side respectively
3) Emulsify the colonies in them respectively
4) Gently shake the slide and observe the result
5) Repeat the above steps for another bacterium’s colonies