anaerobic bacteria

Isolation and identification of anaerobes from clinicalspecimens depend on:•Proper collection of specimens•Prompt transport to the laboratory•To perform special techniques for processing and culture

SPECIMEN COLLECTION!Avoid oxygen contact and contamination withnormal flora.• Aspirated material from deeper tissues,abscess…etc (with syringe)• Surgical drainage material (abscess)• Tissue biopsy samples• Samples collected with anaerobic swabsystemsCollection of specimens with swabs should bediscouraged

TRANSPORTATIONSpecimens should be transported to the laboratoryas soon as possible (within 10-30 mns)• Anaerobic transport systems• Anaerobe swab systems• Anaerobic liquid medium for tissue samples• Sterile screw cap tubes for large volumes of purulent material.Specimens should not be transported in syringe

DIRECT EXAMINATION :Macroscopic examination :• The foul odor andpurulence of thespecimen• Presence of blood,necrotic material andsulphure granules

DIRECT EXAMINATION :Microscopic examination :• Smear preparation• Fixation with methanol• Gram staining• Observation of PMNL’sand bacteria withdifferent morphotypes(mixed bacteria)* isimportant

CULTIVATIONInoculation of specimen :• Specimens for anaerobic culture must beinoculated onto aerobic medium along with theanaerobic media.• Inoculation of anaerobic liquid media such asthioglycolate medium is also very important forthe possibility of anaerobic incubation systemfails.

CULTIVATION :Anaerobic Media:The media must be,* supplemented with hemin and vit K ,* freshly prepared and properly stored.Solid media ;-Non-selective media• Anaerobic blood agar• Schaedler agarLiquid media ;Thioglycolate mediumChopped meat broth• Brucella agar• Columbia agar-Selective media• Phenyl ethyl alcohol agar• Bacteroides bile esculin agar

CULTIVATIONAnaerobic atmosphere*Anaerobic jar- GasPak systems• Commonly used.• The GasPak envelope is cut, placed in the jar and 10 ml of wateris put inside the kit.• The water and the chemical agent of GasPak reacts with eachother by the help of palladium catalyst placed in the jar,oxygen is removed, water and carbondioxide is generated• The atmosphere in the jar consist of 80-90 % N 2 , 5-10 % H 2 , 5-10 % CO 2 .• Rezasurin or methylen blue indicators are used to monitoranaerobic system.*Anaerobic chambers

Examination of growth:Incubation period:• At 37 C at least for 48 hrs.• Some of the anaerobes may require longer incubation upto 3 weeks.Evaluation:• The growth on anaerobic medium is compared with thaton the aerob culture plate.• Selective media are also helpful for isolation andidentification of certain species.

Identification:• Conventional methods• Commercial identification systems• Research:• Gas-liquid chromotography• MALDITOF (matrix assisted laser desorptionionisation-time of flight)• Molecular methods

Laboratory practice:On the front bench:• Pigmented anaerobic bacteria• Lecithinase activity on Egg Yolk Agar• Double zone of beta hemolysis• Thioglycolate medium• Chopped meat broth• Anaerobic jar-GasPak

Laboratory practice:On the front bench• Clostridium tetani, gram positive bacilli withterminal spore• Clostridium botulinum, gram positive bacilli withsubterminal spore• Clostridium perfringens, gram positive bacilliwith spores usually not seen• Fusobacterium nucleatum, fusiform gramnegative bacilli• Actinomyces meyeri, filamentous gram positivebacilli

Pigmented anaerobic bacteria Prevotella bivia

Laboratory practice:On each bench,there will be ready made and fixed slides forGram staining• Clostridium tetani, gram positive bacilli withterminal spore• Clostridium botulinum, gram positive bacilli withsubterminal spore• Clostridium perfringens, gram positive bacilliwith spores usually not seen• Fusobacterium nucleatum, fusiform gramnegative bacilli

Lecithinase (+)

Double zone of beta hemolysis

Thioglycolate mediumChopped meat broth

Anaerobic jarGasPak system

Clostridium tetani, gram positive bacilli with terminal spore

Clostridium botulinum, gram positive bacilli withsubterminal spore

Clostridium perfringens, gram positive bacilli withspores usually not seen

Fusobacterium nucleatum, fusiform gram negative bacilli

Actinomyces meyeriFilamentous gram positive bacilli

Gram stain• Crystal violet (1 min)• Wash with water• Lugol (1 min)• Wash with water• Alcohol (30 sec)• Wash with water• Fuchsin (30 sec)• Wash with water