Anaerobes

Objectives for Anaerobes:

Students will be able to:

1. Demonstrate the significance and correct use of the media routinely used to setup
cultures for anaerobic microorganisms.
2. Demonstrate the ability to correctly follow protocols for colony workup.
3. Perform and interpret rapid biochemical testing.
4. Perform and interpret the special disk potency testing for anaerobes.
5. Demonstrate the ability to setup and read the anaerobic identification system.
6. Demonstrate the operation of the anaerobic chamber system.
7. Identify the major groups of common anaerobic bacteria.

ASSIGNMENTS:

Textbook – Koneman’s Color Atlas and Textbook of Diagnostic Microbiology

Sixth Edition

Read in Quality Link the following SOP’s:

Specimen Collection and Transport for Anaerobes (GEN-C-0121)

Anaerobic MIC System (MIC-C-0010)
Anaerobes, Examination and Isolation of (MIC-C-0011)
Culture Media for Anaerobes (MIC-C-0012)
RapID ANA II System (MIC-C-0013)
Cefinase (MIC-C-0016)

Koneman Chapter 16: The Anaerobic Bacteria

Read this chapter during your rotation in the anaerobe area. Study questions will be
provided to assist you. Test questions will be taken from the text chapter, your study
questions, the junior year handout material, assigned SOP’s and the senior year
syllabus.

Additional texts: Manual of Clinical Microbiology, 7th Edition, ASM Press.

Wadsworth Anaerobic Bacteriology Manual, 4th Edition
Principles and Practice of Clinical Anaerobic Bacteriology.
 Anaerobe culture

Anaerobic bacteria are overlooked or missed unless the specimen is properly collected
and transported to the laboratory and then subjected to appropriate procedures for
isolation. Anaerobes vary in their sensitivity to oxygen: a brief exposure (10 min) to
atmospheric oxygen is enough to kill some organisms. They also vary in their nutritional
requirements, but most isolates require vitamin K, and hemin for growth. Proper
collection, media, and incubation are important to the recovery of anaerobes.

Specimen collection

The best specimen for anaerobic culture is obtained by using a needle and syringe.
Tissue samples and biopsy samples are also very good specimens. When a swab must
be used to collect a specimen, use one of the systems available for the anaerobe swab
system. Special care must be taken to sample the active site of infection when a swab
is used.

Specimen transport

Transport time depends on the volume and nature of specimen. Large volumes of
purulent material and large pieces of tissue maintain the viability of anaerobes for many
hours. Swabs and small volumes of aspirated material, biopsy samples, or currettings
should be transported in an anaerobic transport device. Avoid extremes of heat or cold.
If delays are unavoidable, hold the specimen at room temperature until processing.

Site Acceptable Specimen Unacceptable Specimen

Head and neck Abscess aspirate Throat or nasopharyngeal

Biopsy material surgically swabs
Anaerobic swab surgically Gingival swabs
obtained when aspiration is Superficial material collected
not feasible with swabs

Lungs Transtracheal aspirate Expectorated sputum

Material from percutaneous Induced sputum
puncture Endotracheal aspirate
Bronchoscopic specimen
Thoracotomy specimen

Peritoneal fluid
Abdomen Abscess aspirate Aerobic swab
Bile

Urinary tract Suprapubic aspirate Void urine

Catheterized urine
Female genital tract Endometrial aspirate Vaginal or cervical swabs
Abscess aspirate
Biopsy material
IUD

Bone and joint Aspirate Superficial material collected

Biopsy material with swabs

Soft tissue Aspirate Superficial material collected

Biopsy material from skin surface or edges of
Deep aspirate of open wound wound
Large bowel Only for culture or toxin assay
for suspected involvement of
Clostridium difficile or
Clostridium botulinum

Suggested transport time for certain specimen volumes

Aspirated material
Very small vol (<1.0 ml) Less than 10 min
Small vol (~1.0 ml) Less than 30 min
Large vol (>2,0 ml) Less than 2-3 h

In anaerobic transport device Less than 2-3 h

Tissue or biopsy material

In sterile container Less than 30 min
Anaerobic transport device Less than 2-3 h

Anaerobic swabs
In tube with moist anaerobic atmosphere Less than 1 h
In anaerobic transport medium Less than 2-3 h

Anaerobic bag technique

In the laboratory we use this technique for our primary plate setup, Bio-Bag
Environmental Chamber Type A (Becton-Dickinson). The Bio-Bag consists of a
transparent bag (heat sealed), an anaerobic gas generator ampule, a catalyst,
and a resazurin indicator.

Anaerobic chamber technique

The glove-free chamber (Anaerobe Systems) utilizes sleeves with cuffs that seal around
the forearm.
The system allows materials to be passed in and out of the chamber through an
interchange. The glove-free system has the advantage of permitting passage of small
materials through the cuff (sleeve). The interchange is a rigid compartment with an inner
and outer door and is attached to the chamber by a gas-tight seal. To minimize expense
the interchange may be initially evacuated and refilled by a series of flushes with an
inexpensive oxygen-free gas such as N2. The final fill is made with a gas mixture
containing 5% H2, 5% CO2 and 90% N2. Three evacuations and replacements are
usually sufficient. Anaerobiosis is maintained by the palladium catalysts and the H 2.
The H2 is needed to rid the chamber of any oxygen that may slowly leak through the
chamber walls and it also can serve as an electron donor stimulating the growth of
certain anaerobes such as the Bacteriodes ureolyticus group. The CO2 is included since
many anaerobes require it for growth. The balance of N 2 is chosen for its low cost and
low flammability/explosion properties.
Methylene blue or resazurin indicator strips should be opened within the chamber every
1 to 2 days. The chamber has a cold spot which condenses excess humidity and allows
the water formed to be removed through an external drain.
Media used are a combination of enriched, nonselective, selective and differential for
isolation and presumptive identification of bacteria from clinical material. The following
media are used in this laboratory.

Primary Set up Media

Brucella agar (BA) supplemented with 5% sheep blood and vitamin K (1

ug/ml) and hemin (5 ug/ml) as a nonselective medium t support the growth
of all bacteria.

Bacteroides bile esculin (BBE) agar for the selective isolation of the
Bacteroides fragilis group.

Kanamycin-Vancomycin laked blood (LKV) agar for the selection of

pigmented and other Prevotella spp. and Bacteroides spp.

Phenylethyl alcohol sheep blood (PEA) agar to inhibit facultative gram

negative rods and to inhibit swarming of certain Clostridia spp.

Thioglycolate medium without indicator, supplemented with vitamin K,

hemin, and a marble chip, for enrichment and back-up culture.

Incubation of Cultures

Plates incubated in the chamber or anaerobic pouches can be inspected for

growth after 24 hours or earlier, because they do not need to be
removed from the anaerobic environment. Nonselective blood agar plates should
not be opened before 48 hours. Hold negative culture plates for a minimum of
seven days before final examination. Inspect the thioglycolate broth
accompanying a set of negative plates daily. If the thioglycolate broth becomes
turbid and the primary plates remain “negative”, subculture a few drops onto a
BRU (anaerobic incubation) and onto a chocolate agar plate for CO 2 incubation.
Rapid Enzymatic system

Rapid identification of anaerobes can be accomplished with this commercially

available microsystem for the detection of preformed enzymes within a few hours
following incubation, obviating the need for growth of the isolates. The RapID-
ANA II (Innovative Diagnostic Systems) requires only 4 h of aerobic incubation
after inoculating with a turbid culture suspension.

Rapid disk and Spot tests for the Identification of anaerobes

Betalactamase (Cefinase) Spot indole Catalase

Special-Potency disks;

Vancomycin 5μg Sodium polyanethol sulfonate (SPS)

Kanamycin 1,000μg Colistin 10μg

Organism Colistin Kanamycin Vancomycin

(10 ug) (1,000 ug) (5 ug)
All gram positive R S
Gram negative S S R
cocci
Bact. fragilis group R R R
Other Bacteroides V R R
sp
Bact ureolyticus grp S S R
Fusobacterium spp. S S R
Porphyromonas R R S
Prevotella spp V R R
Special potency antibiotic disks for the identification of anaerobic bacteria
R-resistant; S-susceptible; V-variable
Work flow for Anaerobes: