Anaerobic Culture Systems in Microbiology

1. Anaerobic Culture Systems
Dr. Avizit Sarker
MBBS (DMC), MD (Microbiology), BCS (Health)
Department of Microbiology
Dhaka Medical College

2. Introduction
• An anaerobic bacteria culture system is a method of producing an
environment free from O2 either by displacement of O2 by inert gases or
by absorption of O2 by chemical or biological technique for isolation,
identification and preservation of anaerobic bacteria from a clinical
specimen.

3. Classification of bacteria according to oxygen
requirement
• Obligate Aerobes : They need oxygen for survival & growth.
e.g. : Mycobacterium tuberculosis, Nocardia asteroids etc.
• Facultative Anaerobes : They make ATP by aerobic respiration if oxygen is
present, but are capable of switching to fermentation if oxygen is absent.
e.g. : E. coli, Salmonella, Streptococcus, Staphylococcus etc.

4. • Obligate Anaerobes : They can grow only under conditions of high
reducing intensity. Oxygen is toxic for them.
➢ Strict obligate anaerobes : They can’t grow when oxygen level is > 0.5%.
Usually lack of SOD & Catalase.
e.g. : Cl. hemolyticum, Cl. novy, Cl. tetani, Treponema denticola.
➢ Moderately obligate anaerobes : They can grow in presence of ≤3%
oxygen. They have small amount of SOD & Catalase.
e.g. : Bacteroid fragilis, Prevotella species, Fusobacterium species,
Cl. perfringens. etc.
Most anaerobic infection caused by moderately obligate anaerobes.

5. • Aero tolerant Anaerobes : Don’t utilize oxygen. Produce ATP by
fermentation. Protect themselves from ROS by SOD & Peroxidase
(Don’t have Catalase).
e.g. : Cl. histolyticum, Streptococcus mutans etc.
• Microaerophiles : Require lower amount (5-10%) of oxygen for
survival. Higher oxygen tension is toxic to them. They can’t ferment.
Most of them are Capnophilic (8-10% CO2).
e.g. : H. pylori, Campylobacter species etc.

7. Classification of anaerobic bacteria

9. Sites of anaerobic infection

10. When to suspect anaerobic infection
• Foul smelling discharge.
• Necrotic gangrenous tissue and abscess formation.
• Free gas in tissue.
• Black discoloration of exudates.
• Sulphur granules in discharge.
• Bacteremia or Endocarditis with no growth on aerobic blood cultures.

11. Suitable specimen
• Abscess contents
• Blood
• Deep aspirate wound
• Transtracheal aspirates
• Bronchoscopy aspirates
• Amniotic fluid
• Endometrium lochia
• Urine Catheterized/Suprapubic aspirates
• CSF
• Normal sterile tissue

12. Unsuitable sample
• Specimens from the sites where anaerobic bacteria are normal flora
• Gingival, Throat or Nasopharyngeal swab
• Vagina & Cervical swab
• Gastric contents, small bowel contents, feces, rectal swabs, colostomy
stomata
• Materials adjacent to skin & mucus membrane
• Voided urine
• Specimens not submitted in anaerobic transport media & when transit
time exceeding 20 minutes.

13. Sample collection
Needle aspiration is preferable than swab culture :
• Better survival of pathogen
• Greater quantity
• Less contamination
• Ulcer : debridement & collect from base & edge.
• If no pus or fluid comes on aspiration inject sterile saline
subcutaneously and resample.
• The last and least way is to use deep swab and rapidly to transfer to
anaerobic transport media.

14. Sample processing & transport
• Pus : Aspirated into a syringe & inject into an Anaerobic
transport vial.
• Pieces of infected tissue : Should be transported in a loosely
capped container with an anaerobic atmosphere bag.
• Small tissue biopsy : In a semisolid anaerobic transport media.

16. • Preservation time : 72 hours at 20-25°C (within anaerobic transport
system).
• Specimens must be transported to the lab within 20 minutes if transport
system not used.
• Never refrigerate the sample for anaerobic culture.

17. Direct examination
• Foul odor
• Purulent appearance
• Necrotic tissue
• Gas or Sulphur granules

18. Actinomycosis - sulfur granule

19. Gram stain & Microscopy
• Background & cellular characteristic
• Gram reaction
• Size, shape & arrangement
• Number of bacteria
• Presence of spores, their shape & position

20. Culture
Use anaerobic media containing reducing substances :
• Glucose 1%
• Sucrose
• Ascorbic acid 0.1%
• Thioglycolate 0.1%
• Cysteine 0.05%
• Cooked meat

21. Non selective anaerobic media
• Robertson’s cooked meat broth
• Trypticase soya agar
• Brain heart infusion agar
• Thioglycolate agar
• Brucella agar
• Peptone-Yeast extract glucose broth

22. Selective anaerobic media
• Anaerobe Phenylethyl Alcohol blood agar
• Anaerobe Kanamycin-Vancomycin blood agar. e.g. Bacteroides,
Prevotella, Fusobacterium & Veillonella.
• Bacteroides Bile-esculin agar. e.g. Bacteroides fragilis group.
• Cycloserine-Cefoxitin-Fructose agar. e.g. Cl. difficile.

23. Robertson's cooked meat Broth
Composition :
• Cooked meat medium
• Peptic digest of animal tissue
• Dextrose
• Sodium Chloride
• Yeast extract
• Iron filling
• Hemin
• Vitamin K
• Reducing substances : Hemin, Glutathione, Cysteine, Sulfhydryl
compounds.
• Sterilization : Autoclave 121°C for 30 min

25. Trypticase soya agar

26. Thioglycolate broth
• Composition :
✓ Peptic digest of casein
✓ Dextrose
✓ Yeast extract
✓ Sodium Chloride
✓ 0.1% thioglycolate
✓ L-cystine
✓ Resazurin
• Reducing agent : Thioglycolate.
• Sterilization : Autoclave 1210C
for 15 min.

27. Anaerobic methods
▪ Removal of oxygen :
✓ Anaerobic jar technique
▪ Absorption of oxygen by chemicals :
✓ Pyrogallic acid + NaOH
✓ Mixture of Chromium & H2SO4
✓ Gas Pak
✓ Pellets of Na Borohydride
✓ Citric acid
✓ Na Bicarbonate
▪ By displacement of oxygen & replacement by other gas :
✓ McIntosh field’s jar

28. Anaerobic methods
• Anaerobic jar technique by evacuation-replacement method
• Anaerobic jar technique by disposable gas generator (Gas Pak)
• Anaerobic glove boxes
• Anaerobic disposable plastic bags
• Candle jar

29. Evacuation-replacement method
(McIntosh field’s jar)

30. • Principle :
➢ Evacuation & replacement method.
➢ Air is removed from sealed jar → Outlet.
➢ Air is replaced with 85% Nitrogen, 10% Hydrogen & 5% CO2.
➢ The residual oxygen left behind is converted to water using Spongy
palladium or platinum catalyst.

31. Gas Pak

33. Anaerobic glove box

34. Anaerobic glove box
• Made of flexible clear plastic.
• Technologist uses gloves that form air tight seals around the arms to
handle items inside the chamber.
• Anaerobic environment is maintained inside the chamber.

35. • Consists of a clear, plastic bag (1-3 petri dishes). With a gas
generator inside.

36. Candle Jar
• The burning candle consumes
oxygen & replaces carbon dioxide
until it extinguishes itself.
• Suitable for growing
microaerophiles & capnophiles.

38. Questions
• What do you mean by anaerobic bacterial culture system.
• Classification of bacteria according to oxygen requirement with
example.
• Name some selective and non selective anaerobic culture
media.
• Short note : 1. McIntosh field’s jar
2. Gas Pak