Anaerobic bacteria

1. Non sporing anaerobes
Dr Dipankar Pattnaik

2. Things to be covered
•Introduction
•Types of anaerobes
•Anaerobic organism & their classification
•Human infections by anaerobes
•Methods of diagnosis
•Anaerobic Culture techniques
•Pitfalls in anaerobic bacteriology
•Summary
•References

3. Introduction
•Anaerobic bacteriology has always been a time consuming &
expensive process. Because culture & identification of anaerobes is
typically slow.
•Major problems is that most infections involving anaerobes are
mixed.
•Aside from time factor, how much data are useful to the
clinician? Is the clinician interested in accurate speciation or will
general identification, with susceptibility data.
•Rapid diagnostic procedures may be employed for presumptive or
definitive identification.

4. Types of anaerobes
•Obligate anaerobic bacteria- Are those bacteria that can grow in the
absence of free oxygen, but fails to multiply in the presence of oxygen
on the surface of nutritionally adequate solid media incubated in room
air or in a CO2 incubator (containing 5-10% CO2). eg.- Bacteroides
fragilis, Clostridium perfringens, C. novyi, Porphyromonas,
Fusobacterium.
•Aerotolerant anaerobes- Anaerobic bacteria that will show limited or
scanty growth on agar in room air or in a 5-10% CO2 incubator, but
show good growth under anaerobic conditions. eg.- C. carnis, C.
histolyticum, C. tertium etc.
•Microaerophilic bacteria- Organism require O2 as a terminal electron
acceptor, yet these do not grow on the surface of solid media in an
aerobic incubator (21% O2) & grow minimally if at all under anaerobic
condition eg.- Campylobacter. jejuni (grow in 5% O2).

5. Anaerobic bacteria- Classification
A)Gram-negative bacilli (curved, spirals & spirochete forms)-
Bacteroides, Borrelia, Butyrivibrio, Capnocytophaga,
Campylobacter, Fusobacterium, Leptotrichia, Porphyromonas,
Prevotella, Treponema etc.
B)Gram-positive cocci- Anaerococcus, Coprococcus, Micromonas,
Peptococcus, Peptostreptococcus, Streptococcus, Gemella etc.
C)Nonsporing Gram-positive bacilli- Actinomyces, Arcanobacterium,
Bifidobacterium, Eubacterium, Lactobacillus, Methanobacterium,
Mobiluncus, Propionibacterium etc.
D)Gram-negative cocci- Acidaminococcus, Anaeroglobus, Veillonella
etc.

6. it is essential to isolate and identify anaerobic bacteria because
1)These are associated with high morbidity & mortality.
2)Treatment varies with bacterial species involved.
•Currently >3/4th of anaerobes isolated from different clinical
specimens are Bacteroides fragilis group, Prevotella,
Porphyromonas, Fusobacterium, anaerobic cocci, and the
anaerobic gram-positive, non-spore forming rods.
•Most of them are resistant to penicillin and its analogues; they
are resistant to many cephalosporins including third gen.,
tetracyclines, aminoglycosides also emergence of resistance to
newer quinolones, and clindamycins.

7. •Anaerobes causes infections involving virtually every organ &
anatomic region of the body.
•Most deep seated abscesses and necrotizing lesions are
polymicrobial, and may include obligate aerobes, facultative
anaerobes, or microaerophiles
•Within past few decades, however, endogenous anaerobic
infections have become far more common. As;
Laboratory recovery of anaerobes is improved
Compromised host immune response due to immunosuppressive
drugs.

8. Anaerobic infection
•Abscess of any organ
•Actinomycosis
•Aspiration pneumonia
•Complication of appendicitis or cholecystitis
•Dental & periodontal infection
•Endocarditis
•Meningitis, usually following brain abscess
•Otitis media, sinusitis
•Necrotizing pneumonia
•Osteomyelitis,
•Peritonitis,

9. Table. shows incidence of
anaerobes in various infections
S. N.Type of infection Incidence (%)
1.Lung abscess, necrotizing pneumonia 62-93
2.Bacteremia 6-10
3.Brain abscess 60-89
4.Chronic sinusitis 52
5.Thoracic empyema 76
6.Intra abdominal/pelvic abscess 60-100
7.Perirectal abscess 75
8.Gas gangrene 85-95
9.Post appendectomy 40
Finegold SM et al. 2000

10. CLINICAL ISOLATION PROFILE
•Gram negative bacilli followed by gram
positive cocci .

11. ANAEROBIC GRAM NEGATIVE BACILLI
•Bacteroides ( most common )
•Prevotella
•Porphyromonas
•Fusobacterium
•Bilophila
All non motile

12. 1. Bacteroides group
•B. fragilis (most common)
•B.thetaiotaomicron

13. •Gram negative bacilli
with rounded ends
•1.5-9 µm vs 0.5-0.8
µm
•Nonmotile
•Broth culture :
pleomorphic with
vacuoles
•Capsules

14. Colony on CDC anaerobic blood agar
•Non hemolytic
•Semi opaque
•Grey colony with concentric whorls inside

15. Key features…of this species
•Growth enhanced by bile
•Disc technique:
(R) : Penicillin
Kanamycin,
Colistin
Vancomycin
(S): Rifampin

16. Presumpto Quadrant Plates 1,2,3

17. Quadrant plate & Anaerobic plate

18. Bacteroides fragilis

19. Regarding biochemicals…
•All are sachharolytic
• Fermentation patterns along with indole
help to differentiate among species (para
dimethyl aminocinamaldehyde
INDOLERHAMNOSE
FERMENTATION
B.fragilis
--
B.thetaiotaomicron
++

20. B .thetaiotamicron

21. B. ureolyticus
•DISC TECHNIQUE
•Sensitive :
Penicillin
Rifanpin
Kanamycin
•UREASE

22. Sub-species Rhamnose Trehalose Mannitol Indole
Ovatus + + + +
Thetaiotamicron + +/ - - +
Distasonis +/ - + - -
Vulgatus + - - -
Fragilis - - - -
SOME DIFFERENTIAL CHARACTERISTICS OF SUB-SPECIES OF B. FRAGILIS

23. Pigmented anaerobic bacilli are no
longer classified in the genus
Bacteroides
Prevotella-porphyromonas group
2
nd
most common group
Next to bacteroides

24. Based on fermentation of
carbohydrates
SACCHAROLYTIC
•PREVOTELLA
•P.intermedia
•P. nigrescens
•P. melaninognica
ASACCHAROLYTIC
•PORPHYROMONAS
•P. asaccharolytica

25. Porphyromonas
•Tan to buff colonies : brown-black pigment
•Brick-red fluorescence (UV)
•Inhibited by bile
•Disc technique :
(R) : Kanamycin
(S): Penicillin
Rifampin
•Failure to grow in
Kanamycin-Vancomycin BA
as Vancomycin inhibits
growth.

26. Definitive identification of
Porphyromonas to species level
is difficult.
Rapid ID 32 A system or Rapid ANA II
determines enzyme activities
Within 4 hrs

27. Prevotella
•Brown to black colonies on BA
•Brick –red fluoescence (Long wave UV)
•Produce indole
•Ferments glucose etc.

28. COCCO BACILLI

29. P. intermedia resembles
P. nigerscens which can be sort
out by using egg-yolk agar
Lipase produced by P. intermedia

30. Prevotella- porphyromonas group
•2
nd
most common group( anaerobic bacteria )
•Normal microflora of oropharynx,GIT,GU syst.
•Lesions like oro-facial origin & anaerobic
pluropulmonary infections they out number
the Bacteroides group .
•Like B . fragilis group they produce
β- lactamases .

31. Contd..
•Gram stain: short coccobacilli
0.6-1µm × 0.3-0.4 µm
•Brick-red fluorescence (UV)
•Brown-black pigment
•Inhibited by bile
•Disc test:
(S) Penicillin , Rifampin .
(R) Kanamycin

32. Major problem faced with these
pigmented groups
•Fastidious
•Slow growing (2 days - 3 weeks )
•Some times may even fail to produce pigment

33. Fusobacterium
•Normal habitat : upper respiratory tract
gastrointestinal tract
genitourinary tract

34. Clinical syndromes…
•Like Prevotella-Porphyromonas group a/w
anaerobic pleuropulmonary infections i.e.
aspiration pneumonia, lung abscess
necrotizing pneumonia, thoracic empyema.
•Brain abscess, chronic sinusitis, metastatic
osteomyelitis, septic arthritis, liver abscess,
intra abdominal infections.

35. Species..
•F. nucleatum (most common)
•F. necrophorum
•F. mortiferum
•F. varium

36. Fusobacterium nucleatum….
•Patients with neutropenia & mucositis
following chemotherapy at high risk.
•Direct M/S: characteristic spindle shaped cell
i.e. long(5-10µm) filamentous tapered ends.
Whereas most other species donot have
fusiform shape ; rather rounded ends.

38. Contd..
•Anaerobic BA : 1-2 mm in diameter with
characteristic internal flecking referred as:
crystalline internal structures (CIS)
speckled opalescence

39. •Biochemically : inert
•Electrophoresis (DNA) : 3 sub species
but clinical significance not known.

40. Fusobacterium necrophorum….
•Lemiere’s syndrome ( necrobacillosis)
•Postanginal septicemia
•Liver abscess

41. Lemiere’s syndrome ( necrobacillosis)
•Life threatening
•Should be suspected in young patients with
septic thrombophlebitis of internal jugular
veins following URTI.
•12-25 yr healthy people
•Oropharyngeal infections (tosillitis,peritonsilar
abscess, pharyngeal abscess) followed by
anaerobic septicemia & subsequent
metastatic complications (lung , joints)

42. Contd..
•Direct M/S : curved forms &
spherical areas with in cells.
•On LD egg yolk agar: iridescent sheen(lipase).
• Three biovars i.e.
A, B, C .
clinical significance
not known

44. Species Aesculin
hydrolysis
MaltLactSucGrowth
in bile
indol

Resistant to

Other
Rifampicin
F. morteferum + + + - + - +
F. varium - - - - + + +
F. nucleatum - - - - - + -
F.
necrophorum
- - - - - + - Lipolyti
DIFFERENTIAL CHARACTERISTICS OF SOME FUSOBACTERIUM SPECIES

45. Antibiotic susceptibility….
•Resistant to erythromycin , tetracyclin ,
aztreonam , co-trimoxazole &
aminoglycosides.
•However sensitive to : metronidazole ,
clindamycin
chloramphenicol
nearly all β- lactam agents

46. F. mortiferum & F. varium
• May produce β- lactamases
•Coccoid to filamentous with spherical swelling
near centre or one end . (2-10µm ×0.5-2µm)
•BA : 1-2 mm diameter
fried - egg appearance
•Resistant to rifampin
separates it from not only other fusobacterium
species but also from Bacteroides,Prevotella-
Porphyromonas group

47. They can be differrentiated by ….
Esculin
hydrolysis
Lactose
fermentation
F. mortiferum+ v
F. varium
- -

48. F .murtiferum

49. ACTINOMYCES

50. Morphology
•Gram positive bacilli (0.5-1mm)
•Nonmotile
•Nonsporing
•Non-acidfast
•Filamentous (break up into coccoid and bacillary
forms)
• Exhibit true branching

51. Actinomycosis
•Caused by A. israelii
•Chronic suppurative and granulomatous disease
•Three types : Cervicofacial (mc)
Thoracic
Abdominal

52. Characterised by :
•formation of sinus tracts from suppurative lesions
•presence of pus
• sulphur granules
•colonies

53. Lab. Diagnosis
Samples :
•Pus
Bronchial secretions
Sputum etc
• Sulphur granules

54. Lab. Diagnosis contd..
Microscopy
•Sulphur granules are stained with Gram and ZN
staining using 1% sulphuric acid for decolourization
• gram positive hyphal elements with branching,
surrounded by a peripheral zone of swollen
radiating clubs
• sun ray appearance
•Sulphur granules and mycelia in tissue sections can
also be identified by direct fluorescence microscopy

57. Culture
•The sulphur granules inoculated on BHIA, BA and
thioglycollate broth
• Incubation is both aerobic and anaerobic with 5-10% CO
2

at 35-37°C for 14 days
• Colonies of A. israelii are 0.5-2mm in diameter, white or
grey white smooth, entire or lobulated resembling molar
tooth

58. Actinomycosis : Lab. Diagnosis
Identification
•Microscopy
•Direct fluorescent antibody tests
•Gel immunodiffusion
Biopsy : H & E staining
Molecular tests : DNA probes and PCR

59. Organism Growth in Spider

microcolony
Red
macrocolony
on BA
Starch
hydrolysis
(wide
zone)
Aesculin
hydrolysis
Propionic
acid
produced
Air Air
+CO2
ANO2+
CO2
A. israelli - Slight + + - - + -
A.
odontolyticus
+ + + - + - + / - -
A. eriksonii - - + - - - - -
A. bovis Slight + + - - + + -
A. naeslundii + + + - - - +/ - -
Arachnia
propionica

Slight
Slight
+
+ - - - +
SOME DIFFERENTIAL CHARACTERISTICS OF ACTINOMYCES AND ARACHNIA

60. Treatment
•Surgical excision
with Penicillin for 1 year.

61. Propionibacterium acnes
•Contamination of blood cultures
•Endocarditis,CNS shunt infection
•Diptheroid appeaance
•Anaerobic
•Produce indole, catalase & propionic acid

62. Lactobacillus species
•Vagina
•Endocarditis,peritonitis
•Many can grow aerobically
•Growth in rogosa’s selective tomato agar juice
•Gm + ve uniform bacilli in chains
•Catalase –ve
•(R) : Vancomycin

63. Anaerobic cocci
2
nd
most common group
encountered next to anaerobic GNR.

64. Anaerobic gram positive cocci
•FAMILY: Peptococcaceae
•GENUS: Peptococcus
Peptosreptococcus (most common)
Ruminococcus
Sarcina
Except for peptococcus niger all former species
of genus peptococcus were transferred to
genus peptostreptococcus

65. Clinically significant species
•Peptostreptococcus anaerobius (Ѳ by SPS)
pueperal sepsis,wound infection,abscess…
aerotolerant
grow well in 10 % CO2.
KANAMYCIN
Peptostreptococcus
anaerobius
R
Peptostreptococcus
asaccharolyticus
S

66. •Peptococcus niger (black pigment)
•Gemella morbillorum

67. Anaerobic gram negative cocci
•FAMILY: Veillonellaceae
•GENUS: Veillonella (most common)
Acidaminococcus
Megaspaera

68. Veillonella
•V. parvula (mc species)
life threatening Endocarditis

69. To be remembered regarding
Antibiotic susceptibility of anaerobic
cocci
•P. anaerobius (R) to Penicillin-G.
•Microaerophillic streptococci
Streptococcus intermedius (R) Metronidazole.
•Veillonella : (R) to Vancomycin
•Drugs C/I: Aminoglycosides, Aztreonam
Co-trimoxazole
Fluoroquinolones

70. Contd.. Drugs active against
•Metronidazole (a/e Strep.intermedius)
•Clindamycin
• β-lactam drugs including Penicillin
(except Cefperazone,Cefotaxime,Cefotetan)
•Chloramphenicol
•Imipenem
•Piperacillin – Tazobactam
•Newer Fluroquinolones (moxi/gati)
•Linezolid

71. Double zone β-
hemolysis
Catalase
C. perfringes + -
Propionibacterium - +
GRAM POSITIVE RODS

72. Gas- Liquid Chromatography
•Use of gas-liquid chromatography (GLC) to detect anaerobes in
exudates & body fluids has been developed.
•A major amount of butyric acid in a specimen that contains only
thin, pointed, gram-negative rods would suggest Fusobacterium
spp.
•A major peak of succinate & the presence of only gram-negative
rods would suggest Bacteroides spp., Prevotella spp.
•A major propionate peak in a positive blood culture containing
pleomorphic, non spore forming gram-positive rods would be most
consistent with Propionibacterium spp.
•However, direct GLC provides only presumptive clues, & should be
interpreted cautiously in polymicrobial infections.

73. PCR
•PCR amplification procedure appear promising, but are not well
commercialized.
•Anaerobes identified by colony PCR and sequencing of the 16S
rRNA gene using universal primers (LiPuma et al. 1999).

74. Rapid methods for diagnosis of anaerobes
•Two rapid systems are available for quick diagnosis of anaerobes.
1)RapID ANA by Innovative diagnostic systems
2)AnIDENT by Analytal Products, Inc.
•These both systems rely on preformed enzymes and only four
hours of aerobic incubation is required.
•Disadvantage is costly, and variable response.

75. Antibiotic susceptibility testing
•AST is not required in every anaerobic isolates but done in
1.Organism of known variability in susceptibility pattern, eg- B. fragilis
2.Organism isolated in pure culture.
3.Organism from seriously ill pt.
4.Organism from pt. undergoing long-term antibiotic therapy.
5.Organism from pt. failing to respond to empirical therapy.
6.For epidemiological purposes.

76. Pitfalls in anaerobic bacteriology
•Failure to bypass normal flora in collecting specimens.
•Failure to setup anaerobic culture promptly from specimens.
•Gram stain not prepared directly from clinical specimens
•Use of inadequate commercial media.
•Failure to use supplement in media eg.- Vitamin K1 for B. fragilis.
•Failure to use selective media.
•Failure to use a good anaerobic jar.
•Failure to monitor catalyst.
•Exposure of atmospheric gases during processing.
•Inaccurate identification & speciation.
•Failure to determine whether organism is a true anaerobes or
not etc.

77. Summary
•Many anaerobes grow more slowly than facultative or aerobic
bacteria & since clinical specimens yielding anaerobic bacteria
commonly contain several organisms.
•Limited knowledge of infections caused by anaerobes or
colonization of anaerobes.
•Limited labs. doing culture & identification.
•Culture is time consuming in most of the cases.
•Automated systems is costly for anaerobiosis.
•Except for few anaerobes, no rapid detection methods/systems is
available.
•No well formulated, universally accepted lab. protocol are available
except Wadsworth Anaerobic Bacteriology Manual (fourth ed.) 1986.
•This field of bacteriology should need more exploration.

78. Thank you

79. Gaspak
•Method of choice for preparing anaerobic jars. It is available as
disposable envelope, containing chemicals which generate H2 &
CO2 on addition of water.
•After the inoculated plates are kept in the jar, Gaspak envelope, with
water added, is placed inside & the lid screwed tight.
•Presence of a cold catalyst in the envelope permits combination of
H2 & O2 to produce an anaerobic environment.
•Gaspak is simple, effective, & eliminates the need for drawing a
vacuum & adding H2.
•Indicator should be used for verification of anaerobic environment.
•Reduced Methylene blue is used as indicator.

80. Anaerobic Jar Techniques- Jars are
used primarily with primary plated media or
subculture plates.
Oxoid jar has a metal lid, valves & a
pressure gauge.
It can be used either as an evacuation-
replacement jar or, it can be used with a
disposable gas generator (Gaspak).

81. Contd…
•Introduction of gas mixture containing H2 into a jar is followed by
catalytic conversion of the O2 in the jar with H2 to water, thus
establishing anaerobiosis.
•Catalyst composed of palladium-coated aluminum pellets. These
can be inactivated by excess moisture & H 2S produced by
anaerobic bacteria.
•So, they should be reactivated after each use by heating the basket
or sachet of pellets to 160°C in a drying oven for 1.5 to 2 hrs.

82. Newer anaerobic systems
•Recently, anaerobic gas-generating systems have been introduced
that don’t require either catalyst or the addition of water to activate
these systems.
•AnaeroPack, absorbs O2 and generates CO2, but doesn’t generate
H2.
•It appear to be an excellent alternative to the GasPak and other
established anaerobic incubation systems.
•Another type of commercially available catalyst free-system ie.
Anaerocult (Merck, Germany), makes use of iron filings in a sachet
to which water is added, producing an O2 free, CO2-rich atmosphere.

83. Anaerobic Glove Box System
•Self contained system that allows to process specimens & perform
test for isolation & identification without exposure to O2.
•Glove boxes suitable for cultivation, can be constructed from various
materials, including steel, acrylic plastic, vinyl plastic or fiberglass.
•Economical to operate b/c it permits the use of conventional plating
media & cost of gases for operation of the system is minimal.
•Once setup, the major expense is for the 85% N2, 10%H2, 5%CO2
gas mixture used to replace the air in the entry lock when materials
are passed into the glove box chamber.

84. Roll Streak System
•It uses PRAS media prepared in tubes with rubber stoppers.
•Tubes of agar media are cooled in a rolling machine after autoclaving,
which results in a thin coating of the inner surfaces of the tubes with
solidified medium.
•Both PRAS liquid media & roll streak tubes requires addition of a
reducing agent, such as L-cystine-hydrochloride, which is added just
before autoclaving to maintain a low oxidation-reduction potential.
•All inoculating & subculturing of the PRAS solid & liquid media are
performed under a stream of O2-free CO2, which minimizes exposure
to air & help to maintain a reduced oxidation-reduction potential in the
media before & after growth.

85. Anaerobic Disposable Plastic Bags
•Anaerobic bag system (BD Microbiology), and AnaeroPouch
(Mitsubishi), Anaerogen (Oxoid), & Anaerocult P (Merck) etc.
•Anaerobic Bag (BD) consists of a clear-plastic bag, an H2-CO2 gas
generator that generates an atmosphere when water is added to it,
cold palladium catalyst pellets, & a resazurin indicator.
•Bag is heat sealed following activation of the generator to permit
maintenance of anaerobic conditions.
•But in AnaeroPouch & Anaerocult achieve anaerobiosis, without
catalyst, to remove O2 from the atmosphere & generate CO2.
•O2 removed by combining with iron powder to form iron oxides.
•These are used as alternative to anaerobic jar or glove box system.

86. Anaerobic Holding Jar
•Convenient adjunct to the jar & glove box systems that allows
primary plating, inspection of cultures, & subcultures of colonies at
the bench with only minimal exposure to atmosphere.
•Inexpensive, commercial-grade N2 can be used in the holding jar
system.
•Open the small needle valve & set to gas tank regulator at
prescribed pressure.
•Alternatively, CO2 passed through a tube of heated copper catalyst
(Sargent furnace) can be used in the holding jars instead of N2.

87. Use of syringe methods for
anaerobiosis
•Tubes are almost same as used in roll streak method.
•Tubes containing prereduced medium are prepared by exclusion of
oxygen with the desired gas, and a standard quantity, 4.5 ml, of
reduced, anaerobic agar medium is dispensed into each tube.
•This permits a 10x dilution by the addition of 0.5 ml of inoculum.
•Relatively simple laboratory set-up is required.
•Tubes of melted agar used for culturing are held in a 46° C water
bath.
•Cylinder that delivers the desired gas, passed through a reduced
hot copper column (to remove oxygen).

88. Syringe methods for anaerobiosis
FIG. A) Removal of dead air space from syringe and needle.
B) Removal of clinical specimen in holding medium.

89. SpeciesAesculin
hydrolysi
s
GluMaltLactSucGrowt
h in
bile
Resistant to

Other
Penic-
illin
Kana-
mycin
Rifam-
picin
B. fragilis + + + + + + -/+ + + +
B.
melaninog
enicus
-/ ++/ -+/ -+/-+/- - +/- - + - Prote
olytic,
black
fluore
scent
colon
y
B. oralis + + + + + - - - + -
B.
capillosus
+ + - - - + - + + -
B.
praeacutu
s
- - - - - + - +
B.
corrodens
- - - - - + - - - - Oxid
ase
+ve;
pittin
g of
agar
DIFFERENTIAL CHARACTERISTICS OF SOME BACTEROIDES SPECIES

90. Contd…
•Spectrum of infection from deep seated abscesses (Abdominal,
pelvic, brain, thoracic etc.), soft tissue infection (Bite wound, diabetic
ulcer, cutaneous abscess, decubitus ulcer, gas gangrene, breast
abscess, perirectal abscess), dental abscess, periodontal abscess,
aspiration pneumonia, bronchiectasis, vulvovaginal abscess, septic
abortion, bacteremia, otitis media, neck space infection, and to
ocular infection etc.

91. Collection & transport of specimens
•Decontaminate skin & mucus membrane properly before sample
collection from these sites.
•Surgical soap scrub should be used, followed by application of 70%
ethyl or isopropyl alcohol, then iodine for 1 min.
•A needle & syringe should be used whenever possible for collecting
specimens for anaerobic culture.
•Once collected, precaution should be taken to protect them from
oxygen exposure & deliver them promptly to lab.

92. Isolation of Anaerobic bacteria
•Proper selection, collection, and transport : most important
step
Selection of specimens for culture- With few exceptions, all
materials collected from sites not harboring an indegenous flora,
such as body fluids other than urine, exudates from deep
abscesses, FNAs, and tissue biopsies, should be cultured for
anaerobic bacteria.
•However, since anaerobes normally inhabit the skin and mucous
membranes as a part of the normal indigenous flora, following
samples should not be accepted for anaerobic culture.

93. Specimens that should not be cultured
for anaerobic bacteria
•Gingival swabs
•Throat or nasopharyngeal swabs
•Sputum or bronchoscopic specimens
•Gastric contents, small bowel contents, feces, rectal swabs etc.
•Surfaces of decubitus ulcers, swab samples of encrusted wall of
abscesses, mucosal lining etc.
•Material adjacent to skin or mucus membranes other than the above
•Voided urine
•Vaginal or cervical swabs.
•Wound or lesions that will respond to I & D.

94. A) Direct examination of clinical
materials
•A foul odor, purulent appearance of fluid specimens, & the
presence of necrotic tissue & gas or sulfur granules are
valuable for suspicion of anaerobes.
•Background & cellular characteristics of smear, Gram reaction;
size, shape, number, arrangement of bacteria, presence of
spores, & their position, filaments with spherical bodies,
pointed ends, and granular forms recorded.
•Acridine orange stains are useful for detecting bacteria in
blood cultures, CSF, pleural fluid, joint fluid, and exudates.

95. Methods for diagnosis of anaerobic infections
A) Direct examination of specimens and staining
B) Culture
C) Metabolic product detection by gas-liquid chromatography
D) Molecular methods like PCR etc.
E) Rapid systems.

96. B) Anaerobic culture techniques
•Anaerobic bacteria differ in their requirements of & sensitivity to
oxygen.
Robertson’s cooked meat medium- Probably the most widely
used fluid medium for anaerobes.
•It contains fat free-minced cooked bullock meat in broth (either
peptone infusion/thioglycollate broth). It indicates saccharolytic (eg.
Clostridium perfringens.) or proteolytic (C. histolyticum, C. tetani)
activities, by the meat turned red or black, respectively.
•Other Culture media. Schaedler Broth (Oxoid), Brain-Heart
Infusion Broth (Oxoid), Wilkins-Chalgren Anaerobe Broth/agar
(Oxoid) and Thioglycollate Broth.

97. Contd…
Blood culture techniques- The mortality associated with an
anaerobic bloodstream infection is high.
Liquid media- Some commercially available media contain Sodium
polyanethol sulfonate (SPS), which has been reported to enhance
the recovery of anaerobes, but may be inhibitory to
Peptosreptococcus anaerobius. This effect can be overcome by
addition of 1.2% gelatin.
•eg- Tryptic soy broth, thiol broth, Columbia broth, trypticase soy
broth, and thioglycollate medium, PRAS with brain-heart infusion
yeast extract broth, supplemented peptone broth, radiometric
method.

98. Contd…
•Various automated anaerobic blood culture bottles ; Bactec Plus
Anaerobic/F, Bactec Lytic/10 Anaerobic F, Bactec Anaerobic F,
BacT/Alert, BacT/Alert FN, ESP 80N Anaerobic Broth, ESP 40N
Anaerobic Broth.

99. Selection & use of media
•Media used for recovering anaerobes from specimens should
include nonselective, selective, and enrichment types.
1) CDC Anaerobe blood agar- Trypticase soy agar, 5% sheep blood;
yeast extract, hemin, vitamin K1, L-cystine for anaerobes requiring
additional growth factors. Acts as nonselective BA plating media for
primary isolation of all anaerobes found in clinical specimens.
2) Phenylethyl alcohol blood agar- Above contents + 2.5 gm/L of
Phenylethyl alcohol (for inhibition of swarming of Proteus spp.).
Used for selective isolation of anaerobes from infected material
containing mixture of bacteria.
3) Kanamycin-vancomycin blood agar- Above + 100 mg/L of
kanamycin & 7.5 mg/L of vancomycin. Used for selective isolation of
most Bacteroides spp., Prevotella spp., Fusobacterium spp.,
and Veillonella spp. from specimens containing mixed aerobes
& anaerobes.

100. Contd…
4) Paromomycin-vancomycin blood agar- Above (3) + 100mg/L of
paromomycin substituted for kanamycin. Used for selective isolation
of B. fragilis group (pig. & nonpigmented ie. Prevotella spp.),
Fusobacterium spp., Veillonella spp.
5) Cycloserine-cefoxitin fructose agar- Trypticase soy base,
fructose, neutral red as indicator, cycloserine (500mg/L), and
cefoxitin (16mg/L). Used for selective isolation of C. difficile from
stool specimen.
6) Enriched thioglycollate medium- BBL-0135C formula
thioglycollate medium (without indicator) with hemin and vitamin K1.
Used for primary isolation of Actinomycetes.

101. Anaerobic systems for cultivation
•Anaerobic jars with disposable gas generators
• Evacuation replacement jars
•Anaerobic gloves box techniques,
•Roll tube & roll streak tube with prereduced anaerobically sterilized
(PRAS) media etc.
•Anoxamat

102. Anoxomat

103. Anaerobic Culture Methods
•Anaerobic
jar
•Chemical
reactions
remove
oxygen

104. McIntosh & Fildes Jar GasPak Jar
System

105. Anaerobic Glove Box System

106. Roll Streak System
FIG. A) Side view of roll tube, butyl rubber closure and
screw-on cap. B) Top view of closed roll tube.

107. Anaerobic Disposable Plastic Bags

108. presumptive tests

109. Peptostreptococcus
•P.anaerobius : (R) to peniciillin-G

110. Peptococcus + Coccal Singly, pairs,
short chains
and clumps
Peptostrept
ococcus
+ Coccal Singly, pairs
and in chains
Veillonella _ Coccal Pairs, short
chains and
irregular
clumps

111. Methods used for AST
1.Broth dilution methods-
a) Macrodilution method
b) Microdilution method
2.Broth disk method
3.Agar dilution method
a) Wadsworth method
b) Approved reference method
4.beta-lactamase testing- Required in Bacteroides fragilis
Pigmented Bacteroides
F. nucleatum

112. Gas- Liquid Chromatography
Gas-liquid chromatograms of bile acids in the nonamidate and glycine-conjugate
fractions after a piperidinohydroxypropyl dextran gel (PHP GEL) column.