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

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

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.

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: http://www.bd.com/ds/productCenter

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

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

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)

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.

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)

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

Anaerobic bag or pouch 출처 : http://www.medixcorp.com/catalog http://www.sanilaboshop.es/GasPak-EZ-Anaerobe-Pouch-System

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

Anaerobic chamber Catalyst 출처: http://kp.caeonline.com

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

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

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 +++

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

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)

Clues from primary culture plates Agar pitting -Bacteroides ureolyticus group 출처: http://www.microbes-edu.org Black or tan pigmentation -Porphyromonas spp. Pigmenting Prevotella spp.

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)

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

Clues from primary culture plates Molar tooth - Actinomyces spp. Pink to red colony - Actinomyces odontolyticus 출처: http://www.jcdr.net

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

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)

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

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

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

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

Anaerobic gram negative bacilli

Anaerobic gram negative bacilli

Pigment production gram negative bacilli

Pigment production gram negative bacilli

Non-pigmented Prevotella spp.

Spore-forming gram positive bacilli

Non-Spore forming gram positive bacilli

Non-Spore forming gram positive bacilli

Anaerobic gram positive cocci

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