Cultivation of Anaerobes

Dr Mostafa Mahmoud, MD, Ph D,
Associate Prof. of Microbiology & Immunology

Faculty of Medicine, Ain Shams University
Bacterial classification by O2 requirement:-
1. Obligate aerobes: require O2 for growth i.e.
O2 acts as electron acceptor e.g. Ps aeruginosa.
2. Obligate anaerobes: grow only in complete
absence of O2. O2 is toxic to bacteria e.g. Cl.
tetani. Inorganic compound acts as an electron
acceptor.
3. Facultative anaerobes: can grow under
aerobic and anaerobic conditions e.g. E. coli.
4. Microaerophilic: grow best at low O2 tension
e.g. H. pylori.
When to suspect Anaerobic infections:
 Foul smelling discharge.
 Necrotic gangrenous tissue and abscess
formation.
 Free gas in tissue.
 Black discoloration of exudates
(Bacteroides melaninogenicus).
 Sulphur granules in discharge
(Actinomyces spp.).
 Bacteraemia or endocarditis with NO
growth on aerobic blood cultures.
What are risk factors for anaerobic
infection?
 Poor blood supply and tissue necrosis:
- Trauma. - Foreign body.
- Malignancy. - Surgery.
 Diabetes mellitus.
 Splenectomy.
 Immuno-compromised patients.
Sites with normal anaerobic flora:
 Mouth
 Throat
 Vagina
 Cervix
 Skin folds
 Intestine
Caution: when sampling these sites not suitable for
anaerobic culture but can cause anaerobic
infections in nearby tissues after trauma.
Suitable specimens for Anaerobic culture?
 These include:-
 Abscesses, Bites,
 Blood, Cerebrospinal fluid (CSF),
 Exudative body fluids,
 Deep wounds,
 Sterile surgical samples
Dead tissues.
 Tissue samples and biopsies.
 Transtracheal aspirate.
 Endometrial swabs.
Unsuitable specimens:
1. Specimens from sites in which anaerobic
bacteria are normal flora (e.g., throat, rectal
swabs, urine, bronchial washes, cervico-vaginal
mucosal swabs, sputum, saliva).
2. Voided and catheterized urine.
3. Gastric contents (lavage), small bowel
contents, feces, colocutaneous fistula and
colostomy contents should not be cultured for
anaerobic bacteria.
4. Specimens not submitted in anaerobic
transport media.
5. Improperly labeled specimen.
6. Follow the rejection policy for unacceptable
specimen.
Sampling for anaerobic culture:
1- Aspirate the specimen using a NEEDLE AND
SYRINGE is the best and convenient way for
sampling following the usual procedure and
remove the air from syringe immediately.
2- If no pus or fluid comes on aspiration inject
sterile saline subcutaneously and resample.
3- The last and least way is to use deep swab and
rapidly to transfer to anaerobic transport
media.
Anaerobic Specimen transport
 Must be done immediately to the lab within 1 hr
and less than 2 hr.
 Never refrigerate samples for anaerobic culture.
 If delay can not be avoided use the anaerobic
transport kit especially for swabs and small
volume samples.
 The anaerobic transport system is commercially
available and consists of group of vials, tubes
and bags that remove O2 and maintain the
anaerobic atmosphere for up to 72 H at 20-25 oC.
Fluid transport (aerobic & anaerobic) Anaerobic transport kit
Blood culture bottles for anaerobic blood culture
Caution
- Avoid shock to the anaerobic bacteria by
exposing them to Oxygen or dryness of
sample.
- Avoid exposure to cold as anaerobic
microorganisms are sensitive to cold.
 Avoid swabs in sampling as Swab fibers
contain ambient air and introduce oxygen to
the sample.
Anaerobic culture methods:
1. Use of media containing reducing substances
(Robertson Cooked Meat broth or
Thioglycolate broth).
2. Culture away from O2 (Deep agar tubes).
3. Chemical exclusion of O2 (anaerobic Gas Pak
system).
4. Mechanical exclusion of O2 (anaerobic
incubator).
1- Use of media containing reducing
substances.
A- Robertson Cooked Meat broth
 Composition: 5gm of cooked meat particles +
nutrient broth.
 The reducing substances are haemin and
glutathione in meat particles
 Uses: for anaerobic cultivation
 Sterilisation: Autoclave at 121°C for 30 min
 Robertson Cooked Meat Broth
B- Thioglycolate broth:
- Media for anaerobes supplemented with
nutrients like hemin and vitamin K, 1% glucose,
0.1% thioglycollate, 0.1% ascorbic acid, 0.05%
cysteine or red hot iron filings .
- Sterilize by autoclaving at 121°C for 15 minutes.
- Cool to 25°C and store in a cool dark place
preferably below 25°C.
- Before use the medium must be boiled in water
bath to expel any dissolved oxygen and then
sealed with sterile liquid paraffin.
Thioglycolate broth
 Culture away from O2 (Deep agar tubes).
- Simple way to produce anaerobic condition

- The agar surface can be overlaid with oil to
maintain the anaerobic condition.
- Sterilization of the media can be carried out in
the autoclave at 121 oC for 30 minutes.
- Inoculation is by deep stabbing.
O2 content of culture tube
Growth in deep agar

3- Chemical exclusion of O2 (anaerobic
Gas Pak system).
 Uses H2 to convert air O2 to H2O in the
presence of a catalyst.
 The reaction formula is (2H2 + 0 2 2H20).
 The source of H2 is the Gas Packet
commercially supplied.
 The catalyst is palladium contained in the lid
of the jar.
 Anaerobic indicator strips included to monitor
the anaerobic condition.
 GasPak needing H2O  GasPak Not needing H2O
4- Mechanical exclusion of O2 (anaerobic
incubator).
Thank You

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Dr Mostafa Mahmoud, MD, Ph D,

Associate Prof. of Microbiology & Immunology

- Simple way to produce anaerobic condition

Growth in deep agar

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