1. Culture & Identification of Anaerobic bacteria 무산소 세균의 배양과 동정
서울아산병원 진단검사의학과
박숙자

2. Contents 1. Collection and Transport of Specimens
2. Culture for anaerobe
3. Incubation systems for anaerobe
4. Identification of anaerobic bacteria

3. 1. Collection and Transport of Clinical Specimens
for Anaerobic Culture
The best specimen for anaerobe culture is collected with needle and syringe after surface decontamination
Swab specimen should be discouraged or rejected.
Avoid extremes of heat or cold.
-hold the specimen at room temperature until
processing.

4. Anaerobic specimen collection device
1. Collection and Transport of Clinical Specimens
Anaerobic specimen collection device
Syringe
Vacutainer anaerobic
specimen collector
Transport swab for aerobe
and anaerobe
출처:

5. 2. Culture for Anaerobes Specimen preparation
Purulent specimens with anaerobic transport vial
- Vortex grossly
Large volumes of non purulent specimens
- Centrifuge and use the sediment
Bone or tissue
- Grind with 1ml of liquid medium(Thio)
Swab
-Wring out swabs in 0.5ml liquid medium

6. 2. Culture for Anaerobes Inoculation procedure
Aerobic and anaerobic media, liquid medium and slide for Gram stain
- Purulent specimens : 1drop
- Non-Purulent specimens : 2-3drops
- Liquid medium : 0.5~1.0ml specimen on the bottom
- 1drop of specimen spread on a glass slide
Swabs
- inoculate the nonselective plates first
Two swabs
- one for media, one for gram stain

7. 2. Culture Media for Anaerobes
medium
purpose
limitation
BBE
Rapid isolation, and presumtive identification of Bacteroides fragilis group and Bilophila wardsworthia.
Some strain of
F. mortiforum,
K. pneumoniae, yeast may grow. B. vulgatus may not form black colony
PEA
Prevent swarming of Clostridium and Proteus spp.
A few organisms occasionally do not grow
BHI
Brucella
CDC
Columbia
TSA
Schaedler
General all-purpose
Aerobes will grow on medium.(gram stain and aerotolerance test)
KVLB or LKV
Rapid isolation and selection of Bacteroides species and Prevotella spp.
Earlier detection of pigment producing.
Yeast and other kanamycin-resistant organisms may grow. (gram stain and aerotolerance test)

8. 2. Culture Media for Anaerobes
medium
purpose
limitation
THIO
(with vitamin K1, hemin, marble chip or sodium bicarbonate)
.Chopped meat carbohydrate
.Chopped meat-glucose
Enrichment broth media ;
-backup source of culture if anaerobic environment fails, for small numbers
Some anaerobes may be inhibited by growth of rapidly growing facultative anaerobes.
THIO
(with vitamin K1, hemin, marble chip or sodium bicarbonate)
.Chopped meat carbohydrate
.Chopped meat-glucose
Enrichment broth media
-backup source of culture if anaerobic environment fails, for small numbers
Inhibited by rapidly growing facultative anaerobes.
CCFA
Recovery and presumtive identification of
C. difficile
Additional identification methods should be used.
EYA
Clostridium spp.
- lecithinase or lipase may be useful for identification of anaerobic isolated.
-Lipase is observed under oblique light.
-compared with portion of EYA acting as negative control.

9. 3. Incubation systems for anaerobe
Immediately place inoculated plates into anaerobic environment
Incubated plates should not remain in the holding jar no longer than 1hr at room temperature
Incubation condition
35~37℃ (daily monitoring incubator)
24~48hrs
Gas mixture
80~90% N2, 5% H2, 5~10% CO2
Catalyst
Palladium-coated alumina pellets
Daily change
Reduce oxygen to water
Rejuvenate each time (160℃ 1~2h)

10. Methylene blue indicator
3. Incubation systems for anaerobe
Anaerobic indicator
Chemical indicator
Biological indicator
-F. nucleatum ATCC 25586,
-Porphyromonas levii ATCC 29147,
-Clostridium novyi ATCC 25758
Methylene blue indicator
BD Dry indicator
Mitsubishi indicator

11. Anaerobic bag or pouch 출처 : http://www.medixcorp.com/catalog

12. Absorbs atmospheric oxygen
Anaerobic jars
Gas Pack
Add water 10ml, produce H₂ and CO₂
Anaerobe Pack
Absorbs atmospheric oxygen

13. Anaerobic chamber
Catalyst
출처:

14. Anoxomat Mark II • Fast anaerobes and micro-aerophilic condition
• Low gas consumption
• Fully automatic self control
• Flexible
Catalyst

15. Anoxomat Mark II
1. Select recipe
Failed
Passed
2. Self control

16. 4. Identification of anaerobic bacteria
Aerotolerance test
Aerotolerance test must be performed
Chocolate agar
48hr, CO₂ incubation
BAP
Brucella
Type of bacteria -
++++
Anaerobic bacteria
+/-
Facultatively anaerobic bacteria
> Anaerobic bacteria
Aerobic bacteria
+++

17. Microscopic Examination
Clostridium perfringens
- Large gram (+) rods
with boxcar-shaped cell
& no spore
Pigmenting Prevotella group
and Porphyromonas group
- Gram(-)coccobacillary form 출처

18. Microscopic Examination
Fusobacterium nucleatum
-Gram(-)bacilli with tapered ends
Bacteroides spp.
-Pleomorphic pale staining
gram(-)bacilli
Veillonella spp.
- Very small gram(-)cocci
출처: 눈으로보는미생물검사
(이장호선생님)

19. Clues from primary culture plates
Agar pitting
-Bacteroides ureolyticus group
출처:
Black or tan pigmentation
-Porphyromonas spp.
Pigmenting Prevotella spp.

20. Clues from primary culture plates
Fried egg
- F. necrophorum or F. varium
출처: www. JCM riken.jp
Double zone of beta hemolysis
- C. perfringens
출처: 눈으로보는미생물검사
(이장호선생님)

21. Clues from primary culture plates
Greening of medium
- Fusobacterium spp.
출처: 눈으로 보는 미생물검사
(이장호선생님)
Medusa-head
- C. septicum

22. Clues from primary culture plates
Molar tooth
- Actinomyces spp.
Pink to red colony
- Actinomyces odontolyticus
출처:

23. Clues from primary culture plates
Swarming growth
- C. septicum
C. sordellii
C. tatani
Large with irregular margin
- Clostridium spp.

24. Commercial kit for the identification of anaerobes
Overnight biochemical systems
- API 20A(bioMerieux, Inc.)
- AN Microplate (Biolog)
2. Rapid enzymatic systems
-Rapid ID 32A (bioMerieux, Inc.)
-RapID ANA II (Remel, INC)
-Anaerobe panel(Bechman-coulter, Inc)
-Anaerobe ANI Card (bioMerieux, Inc.)
-Crystal ANR ID(BD Biosciences)

25. Additional tests for anaerobic bacteria
Catalase test
Urease test
Pigment production
Esculine test with BBE agar
SPS disk test
Spot indole test
Special-potency antimicrobial agent disks
Single rapid enzymatic test

26. Special-potency antimicrobial agent disks
Organism
Resposne to
Kanamycin 1,000μg
Vancomycin 5μg
Colistin 10μg
Gram-Positive organisms V S R
Bacteroides fragilis group
Bacteroides ureolyticus group
Fusobacterium spp.
Porphyromonas spp.
Prevotella spp.
Gram-negative cocci

27. Special-potency antimicrobial agent disks
C. difficile
B. fragilis CL CL

28. Single rapid enzymatic test
L-Proline-aminopeptidase
- Presumptively identify
Clostridium spp.
Proline (-)
Proline (+)
마지막 남은 슬라이드에서는 제가 검사실에서 경험했던 쉽게 동정되지 않았던 몇 가지 사례를 말씀 드리겠습니다.
CDSA(BD Biosciences)
CDIF(bioMerieux. Inc

29. Anaerobic gram negative bacilli

30. Anaerobic gram negative bacilli

31. Pigment production gram negative bacilli

32. Pigment production gram negative bacilli

33. Non-pigmented Prevotella spp.

34. Spore-forming gram positive bacilli

35. Non-Spore forming gram positive bacilli

36. Non-Spore forming gram positive bacilli

37. Anaerobic gram positive cocci

38. 감사합니다. **참고문헌: Clinical Microbiology Procedures Handbook
(Third Edition)
감사합니다.