Method of Anaerobiasis and Anaerobic culture

1. Santosh KumarYadav
M.Sc. Clinical Microbiology
(IOM,TUTH)
Method of Anaerobiasis and
Anaerobic Culture

2. Introduction

3. Introduction
Greek word ( Anaerobiasis)
An = negative
Aer = air
Biosis = way of life
► Literally : life in absence of environmental O2.
► Method of producing the 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.
Importance :-
- Processing of anaerobic specimens.
- Incubation & cultivation of anaerobes.
- Biochemical & Susceptibility tests of anaerobes.

4. Why O2 is toxic to anaerobes?
☺ Enzymes superoxide dismutase & catalase are absent in
anaerobes, which convert the lethal H2O2, Superoxide anion
formed in their cells due to the presence of oxygen.

7. Specimen selection & collection for Anaerobic
culture
Specimens usually are collected from warm, moist
environment that is low in O2
Specimen collected by needle & syringes are better for
anaerobic bacteriology than swab.They are less likely to be
contaminated by endogenous anaerobes. Following
aspiration of specimen , any air present in syringe & needle
should be rapidly expelled.
• It must be Rapid transported to Laboratory & processed
rapidly with minimum exposure to O2

9. Unacceptable Specimens for Anaerobic
Bacteriology
Throat swabs, nasopharyngeal swabs; sputum obtained by
nasotracheal or endotracheal suction; bronchial washings or other
specimens obtained via a bronchoscope; expectorated sputum
Gingival swabs or any other intraoral surface swabs
Faeces; rectal swabs; gastric contents (Except for Clostridium
difficile, Clostridium botulinum )
Voided or catheterized urine
Vaginal, cervical, or urethral swabs; female genital tract
specimens collected via the vagina; swabs of a vaginal discharge
Surface swabs from decubitus ulcers, perirectal abscesses, foot
ulcers, exposed wounds and other sinus tracts.

10. Transport of specimens
Different kinds of anaerobic transport systems
are available-
1. Rubber- stoppered collection vial
containing an agar & indicator system:
Vial is gassed out with oxygen free CO2 or
nitrogen. Specimen is injected through
the rubber stopper after all air is expelled
from the syringe and needle .

11. 2. For only a swab specimen
A special collection device with an oxygen
free atmosphere is required.
When re-inserting the swab, care must be
taken not to enter any air.

12. 3.Anaerobic bags or pouches
 Tissue can be placed in a small amount of liquid
(saline) to keep it from drying out and placed in an
anaerobic pouch ,O2 removal system is activated &
the bags is sealed
 These bags are useful as transport device eg. for
biopsy specimen
 All specimen should be held at R.T.
 Refrigeration can oxygenate the specimen .
Fig. 3Fig. 3

13. 4. Others
Any specimen suspected anaerobic bacterial infection
must be sent to the laboratory into anaerobic transport
medium or under anaerobic conditions such as Cooked
meat broth, Cary & Blair medium.
The aspirated material into syringe for anaerobic culture
may be simply sent to the lab by bending the needle or
probing the needle into rubber cork to prevent the
specimen from O2 contact.

14. Processing clinical specimens
1. Macroscopic examination of the specimen
2. Preparation of Gram stain smear for microscopic
examination
3. Inoculation of appropriate plated & tubed media
4. Anaerobic incubation of inoculated media

15. Macroscopic examination of specimen
Characteristics to note during macroscopic examination of a specimen
►Foul odor- many anaerobes, especially fusobacterium & porphyromonas spp have foul
smelling metabolic end product,
► Fluoresce when expose to UV light/wood lamp(366 nm) ?
pigmented species of porphyromonas & prevotella produce substances that fluoresce
under long wave UV light , brick –red fluoresce is presumptive evidence of these
organisms .
► Is the necrotic tissue or exudate black?
such discoloration may be due to the pigment produced by pigmented species of
porphyromonas & prevotella.
► Does the specimen contain sulphur granules ?
such granules are associated with actinomycosis, caused by Actinomyces, Propionibacterium
propionicus.

16. Direct microscopic examination
Gram stain
Direct smears for Gram stain must be prepared
Smear Should be Methanol fixed
To enhance the red color use Basic fuchsin as counterstain or safranin
for longer time (3 to 5 min.) is recommended.
Stain reveals the various type & relative number of microorganisms ,
presence of multiple distinct morphologic forms.

18. Inoculation into appropriate culture media
The choice of media is an extremely important aspect of successful
anaerobic bacteriology.
Anaerobe microorganisms are among the most difficult microbes to isolate
and grow because they need an environment that's both free of O2 &
reduced. The reduced environment is physiologically necessary for
anaerobes to produce energy for growth.
Anaerobes have special nutritional requirements for Vit k, hemin & yeast
extract.
The ideal media for use in the culture of anaerobes are those that have
never been exposed to O2 . Such media include freshly prepared media &
those stored under anaerobic condition.
Plates exposed to air for longer periods may contain toxic substances,
produced as a result of the reduction of the molecular O2, such media may
also have redox potentials above that required for anaerobes.

19. PRAS Anaerobic media
 Commersial media that prepared, packaged ,
shipped and stored under anaerobic condition
i.e. not exposed to O2 until they are inoculated
Called Prereduced anaerobically sterilized
(PRAS) media.
 Reducing agents eg pallidium chloride added to
media before autoclaving to attempt Prereduce
stage .
Only PRAS media meet the requirements of all
clinical anaerobes for isolation and growth & best
support the growth of anaerobes
• ready & easy to use – save time & trouble Fig. PRAS Media

20. Other media recommended for recovery of
Anaerobes

21. Methods of Anaerobiosis
1.By incorporating reducing agents in the media -
Reducing agents -
 0.5 - 1% glucose
 0.1% thioglycollate
 0.1 % sodium mercaptoacetate
 0.1% cysteine
 0.1% ascorbic acid
 Cooked meat pieces
Two mostly used liquid culture media are
(a) Thioglycollate broth –
 Nutrient broth + 1 % Thioglycollate
 Used for anaerobic culture for blood.
 Indicator methylene blue or resazurin .

22. (b) Robertson’s cooked meat media –
 Nutrient broth + piece of fat free minced meat of beef
heart + glucose
 unsaturated fatty acids in meat utilise O2 for
autooxidation , catalysed by haematin in meat.
 Glutathione & cysteine also use to remove
O2.
 Media water bath 80 C for ½ hour to remove
O2 .
 Complete anaerobiosis surface of medium
covered with 1 cm layer with sterile liquid
paraffin or melted vasper.

23. 2. By displacement of oxygen
i. Cultivation in vacuum
ii. Displacement of oxygen by inert
gases like H2 or N2 .
Widely tried method Candle jar
method.
Drawback :
repeated evacuation and re- filling.
O2 cannot remove completely

24. 3. Anaerobic jars
 Tightely sealed chamber that maintain anaerobic atmosphere used for incubation &
storage of anaerobes.
principle:- Replacement & combustion of atmospheric oxygen
H2 + O2 --- catalyst platinum or palladium -→ H2O
• Indicator : reduced methylene blue Colorless anaerobically and regains blue color onIndicator : reduced methylene blue Colorless anaerobically and regains blue color on
exposure to oxygenexposure to oxygen
• Useful for for small laboratories
• The major disadvantage of the anaerobic jar system is plates have to be removed
from the jar to be examined.

25. McIntosh and Filde's anaerobic jar
Method of use : The culture media are placed in side the jar , stacked up one on the
other, 3
/4
th
of the air inside is pumped out and replaced by a 10%CO2+90%H2 . The
catalyst (Palladium) acts and the oxygen is used up in forming water.. The jar is
incubated at temperature 37⁰C
Other method utilizes a hydrogen generating package combined with
catalyst to remove moelcule of O2 (Gas pak jar). The gas-pak jar system is
most commonly used anaerobic jar.
 Contains disposable packet of albuminium foil contains pellets of sodium
borohydride, Cobalt chloridel , citric acid & NaHCo3 .
Generate H2 & CO2 when H2O(10ML) is added.
H2 + O2 ---Catalyst pallidium ------→ H2O
while molecular O2 converted to H2o the atmosphere become anaerobic .
Methylene blue used as indicator.

26. Anaerobic Gas pak jars

27. Anaerobic Gas pak jars

28. Commercial anaerobic jars

29. 4. Anaerobic chamber
 The ideal anaerobic incubation system provide
O2 free environment for
inoculation,incubation,identification &
susceptibility tests.
• Use of gloves or sleeves forming airtight seals
around the arms to handle items inside the
chamber
• Inside 37⁰C incubator, enclosed heated block
for loop sterilization.
• Anaerobic air circulaed by fan to maintain
homogeneity.
• All anaerobic chambers contains the followings:
-Catalyst usually palladium coated alumina pelletes
- Desiccant silica gel - absorb H2O when H2 combine free O2
- H2 gas (5 to 10%)
- CO2 gas ( to 10%)
- N2 gas (80 to 90%)
- Indicator usually methylene blue
Fig : Anaerobic
chamber

30. 5.Absorption of O2 by chemicals
A. pyrogallic acids :
Buchner (1888) first introduced alkaline pyrogallol for
anaerobiosis which absorb O2.
B. Mixture of powder chromium & H2SO4 :
Two chemical + O2 - chromous sulphate

31. 6.Others methods
1. Use of semisolid agar ( 0.05 to 0.2%) : prevent convention current
of air
2. By biological methods : Microorganisms have been used to achieve
anaerobiosis.
► By keeping aerobe & anaerobe in separate petridish & sealed together.
►Slow & ineffective

32. Identification
Colony morphology
Pigmentation
Gram staining
Fluorescence
Catalase test
Spot indole test
Urese test
Motility test
SPS disc test
Nitrate disc test
Bile tolerent test
Lecithinase, lipase and proteolytic activities, etc

35. THE END