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California Association
for
Medical Laboratory Technology
Distance Learning Program
ANAEROBIC BACTERIOLOGY
FOR THE CLINICAL LABORATORY
by
James I. Mangels, MA, CLS, MT(ASCP)
Consultant
Microbiology Consulting Services
Santa Rosa, CA
Course Number: DL-974
3.0 CE/Contact Hour
Level of Difficulty: Intermediate
© California Association for Medical Laboratory Technology.
Permission to reprint any part of these materials, other than for credit from CAMLT, must
be obtained in writing from the CAMLT Executive Office.
CAMLT is approved by the California Department of Health Services as a
CA CLS Accrediting Agency (#0021)
and this course is is approved by ASCLS for the P.A.C.E. ® Program (#519)
1895 Mowry Ave, Suite 112
Fremont, CA 94538-1766
Phone: 510-792-4441
FAX: 510-792-3045
Notification of Distance Learning Deadline
All continuing education units required to renew your license must be earned no later than
the expiration date printed on your license. If some of your units are made up of Distance
Learning courses, please allow yourself enough time to retake the test in the event you do
not pass on the first attempt. CAMLT urges you to earn your CE units early!.
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Outline
A. Introduction
B. What are anaerobic bacteria? Concepts of anaerobic bacteriology
C. Why do we need to identify anaerobes?
D. Normal indigenous anaerobic flora; the incidence of anaerobes at various body sites
E. Anaerobic infections; most common anaerobic infections
F. Specimen collection and transport; acceptance and rejection criteria
G. Processing of clinical specimens
1. Microscopic examination
2. Media: primary, selective, differential
3. Incubation systems
H. Isolation and identification
1. Provide identification to level needed by physician
2. Role of Gram stain and plate morphology
3. Presumptive grouping and identification using cost effective rapid tests
I. Anaerobic bacteriology cost containment concepts
Measurable Course Objectives
Upon completion of this course, the participant will be able to:
• Recognize the most important genera and species of clinically important anaerobes and
the infections they may cause
• Describe the normal anaerobic indigenous flora
• List appropriate techniques for specimen selection, collection and transport
• Describe initial processing techniques and the media employed
• Identify laboratory methods used for initial grouping, presumptive identification, and
definitive identification, and determine when each level is appropriate
• Identify techniques used for cost-effective clinical anaerobic bacteriology
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A. INTRODUCTION
Anaerobic bacteria cause a variety of infections in humans, including appendicitis,
cholecystitis, otitis media, dental and oral infections, endocarditis, endometritis, brain abscess,
myonecrosis, osteomyelitis, peritonitis, empyema, salpingitis, septic arthritis, liver abscess,
sinusitis, wound infections following bowel surgery and trauma, perirectal and tuboovarian
abscesses, and bacteremia (1). Many reports associate an incidence of at least 50% to 60% of
important infections due to anaerobic bacteria (Table 1).
Anaerobic bacteria are often overlooked or missed unless the specimen is properly
collected and transported to the laboratory. Next, the specimen must be subjected to appropriate
procedures for isolation, including the use of specialized media supplemented with growth
factors and the use of proper incubation methods. Anaerobes vary in their nutritional
requirements, but most isolates require vitamin K and hemin for growth. Anaerobes also vary in
their sensitivity to oxygen: a brief exposure (10 min.) to atmospheric oxygen is enough to kill
some organisms.
This course will discuss procedures for proper collection and transport of anaerobes;
appropriate specimen types for culture, processing, incubation, and isolation; and methods of
characterization of anaerobes from properly collected specimens. Practical schemes for isolating
the majority of clinically important anaerobes will be described, including their salient features
and cost-effective procedures for their work-up and identification.
Many laboratorians believe that the isolation and identification of anaerobes is difficult,
expensive, and time consuming. This course will present methods that will permit rapid, yet
cost-effective procedures for the recovery and identification of clinically significant anaerobes
for any clinical laboratory.
B. WHAT ARE ANAEROBIC BACTERIA?
Anaerobes are microorganisms that do not require oxygen for metabolism, reproduction
or growth. Most anaerobes are actually inhibited by oxygen or oxygen by-products, however
they vary as a group in their sensitivity to oxygen. An obligate or strict anaerobe (e.g.,
Porphyromonas spp., Fusobacterium spp., or Peptostreptococcus spp.) will grow only in an
absolute anaerobic environment (zero % O2). They are killed by exposure to air after only a few
minutes. A moderate anaerobe (e.g., Bacteroides fragilis grp.) can tolerate more exposure to air,
but damage can occur after 15-20 minutes of exposure to air. A microaerotolerant anaerobe
(e.g., Clostridium tertium) is an organism that is capable of growing in both an anaerobic and a
microaerophilic atmosphere. A microaerotolerant anaerobe may marginally grow when exposed
to air or in a CO2 incubator on a chocolate blood agar medium, but growth is best under
anaerobic conditions.
Molecular oxygen itself can be lethal to some anaerobes, however even more toxic
substances are produced when oxygen becomes chemically reduced. Initially, molecular oxygen
is reduced to superoxide anion (O2-), a highly reactive free radical capable of causing severe
damage to components of media, bacterial enzyme systems, proteins, lipids, and cell walls.
Further reduction of oxygen leads to the production of other toxic compounds of oxygen
(hydrogen peroxide {H2O2}, and hydroxyl radicals {OH-}) that can damage microorganisms or
the components of media on which they are to grow. Thus, oxygen, superoxide anions, hydroxyl
radicals, and hydrogen peroxides inhibit the growth of anaerobes and should be avoided to
permit their recovery in culture.
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All living creatures that use oxygen for metabolism have one or more enzymes to provide
protection from superoxide anions and their toxic derivates. These enzymes are known as
superoxide dismutases (SODs). Anaerobes have various amounts of SOD, ranging from none to
some, that presumably allow some anaerobes to tolerate oxygen. However, there is not a direct
correlation between levels of SOD and the anaerobe’s ability to tolerate oxygen. There are other
factors, such as the presence of catalase, which may play a role in the inability of anaerobic
organisms to tolerate oxygen (2).
C. WHY ISOLATE AND IDENTIFY ANAEROBES?
The recovery of anaerobes is very important because they are commonly resistant to
empiric antibiotic therapy (antibiotics that may be used prior to isolation of any organism), and
many anaerobes (including Bacteroides fragilis grp., the most commonly recovered anaerobe)
contain virulence factors that lead to abscess formation and chronic disease if not treated
correctly. The recovery of anaerobes aids the physician in making a specific diagnosis and may
provide the clinician with the potential source of the infection. Further, in this era of concern
about antibiotic resistance, the isolation and identification of anaerobes allows the clinician to
use appropriate antibiotic therapy instead of the “big gun”—the antibiotic with the broadest
spectrum which will inhibit both aerobes and anaerobes, but may also contribute to antibiotic
resistance. It has been shown that correctly employed specific therapy against anaerobes can
reduce mortality and morbidity, and reduce hospitalization (1).
There are some general concepts regarding anaerobic infections that are important to
mention now, but will be discussed in greater detail in this course.
• First, most anaerobic infections derive from our own indigenous microflora, so
specimen selection and collection are essential for quality results and to reduce
contamination.
• Second, anaerobic infections are often mixed, containing both aerobic and
anaerobic organisms. Employing an enriched primary medium as well as using
differential and selective media is essential to rapidly recover anaerobes from
specimens that contain a mixture of organisms.
• Third, despite the diversity of our normal indigenous flora (1, 2, 3, 4), most
infections are due to a relatively limited number of anaerobic isolates (Table 2):
almost 35% are members of B. fragilis group; 28% are Peptostreptococcus spp. or
other genera of anaerobic Gram-positive cocci; 6 % are pigmented Gram-negative
rods; and 8% are Fusobacterium spp. The recovery of Clostridium spp. is only
about 2%.
These three concepts of anaerobic bacteriology have a profound effect on how we isolate
and identify anaerobes and should be part of your thought process during this course.
D. NORMAL INDIGENOUS ANAEROBIC FLORA
Almost all surfaces of the human body are colonized by microorganisms referred to as
normal or indigenous microflora. These organisms normally inhabit the skin, mouth, nose,
throat, lower intestine, vagina, and outer portion of the urethra. Anaerobes colonizing these
regions are present in high numbers. For example, in the intestine anaerobes outnumber aerobic
bacteria 1,000 to 1.
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Under usual circumstances these organisms do no harm by their presence, and there is
considerable evidence that they are actually beneficial to their host. However, in cases where
host defenses are impaired or breaks in the normal skin or mucous membranes occur, or when
organisms are found in normally sterile sites after trauma or surgery, these organisms are capable
of producing serious infection.
Knowledge of the microflora composition at specific anatomic sites is useful for predicting
the particular organisms most apt to be involved in infectious processes that arise at or adjacent
to those sites. Because some anaerobes have fairly predictable susceptibility patterns, such
knowledge may also be of value to physicians considering empiric antimicrobial therapy prior to
isolation of organisms from clinical specimens and obtaining their susceptibility profile. In
addition, the finding of site-specific organisms at a distant and/or unusual site can serve as a clue
to the underlying origin of an infectious process. For example, the isolation of oral anaerobes
from a brain abscess may suggest communication between an oral lesion and the bloodstream.
Examples of the incidence of anaerobes at various body sites
Skin: The anaerobic microflora of the skin consists primarily of bacteria within the genera
of: Propionibacterium (usually P. acnes) and Peptostreptococcus and other anaerobic Grampositive cocci, and occasionally non-sporeforming Gram-positive bacilli in the genus
Eubacterium. Should a venipuncture site be inadequately disinfected before collection of a
specimen for blood culture, the specimen could become contaminated with skin flora, including
anaerobes.
Upper Respiratory Tract: In the upper respiratory tract, the number of anaerobes equals or
exceeds that of aerobic organisms obtained in specimens from nasal washings, saliva, and
gingival and tooth scrapings. Ninety percent of the bacteria present in saliva are anaerobes.
Because of the large numbers of anaerobes that live in the oral cavity, virtually all oral lesions
involve anaerobes, as do the majority of cases of aspiration pneumonia, and ear, nose and throat
(ENT) infections. A wide variety of anaerobes lives in the oral cavity, although their
concentrations and relative proportions vary from one microenvironment to another. Most often
Fusobacterium spp., Porphyromonas spp., Prevotella spp., anaerobic Gram-positive cocci,
Propionibacterium spp., Eubacterium spp., Lactobacillus spp. and Actinomyces spp. are
recovered from the oral cavity. Therefore, these particular anaerobes should be suspected as
participants in any infectious process from the respiratory tract.
Vagina: About 50% of the bacteria in cervical and vaginal secretions are anaerobes, the
most common being anaerobic Gram-positive cocci, Prevotella bivia, and Prevotella disiens,
some anaerobic lactobacilli, and Actinomyces spp. Other anaerobic organisms such as
Clostridium spp., Eubacterium spp., B. fragilis grp., Porphyromonas spp., and others may be
found in the indigenous microflora of the vagina because of its proximity to the anus. P. bivia
and P. disiens tend to dominate among the Gram-negative rods, but pigmented anaerobic Gramnegative bacilli, the B. fragilis group, and other Prevotella and Bacteroides species may be
recovered as well.
Whenever anaerobes are recovered from vaginal and cervical swabs, neither the
microbiologist nor the physician can distinguish the indigenous microflora contaminants from
organisms actually contributing to the patient’s infectious process. For this reason, genitourinary
tract swabs, including swabs of the vagina and cervix, are unacceptable for anaerobic
bacteriology.
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Intestine: Studies concerning the microflora of the intestine have found that anaerobes
outnumber aerobes by a factor of 1,000 to 1. Anaerobes occurring in the highest numbers in
intestinal flora are B. fragilis grp., Bifidobacterium, Clostridium, Eubacterium, Lactobacillus,
Peptostreptococcus and other anaerobic Gram-positive cocci, Prevotella spp., Porphyromonas
spp., and others. Intestine, intestinal contents, bowel, and other material such as rectal abscess,
may be unacceptable specimens unless collected properly to avoid the normal anaerobic
indigenous flora. The distal ileum may have counts of 104 to 105 colony forming units (CFU)/ml
and both coliforms and various anaerobes may be encountered. In the distal colon, total bacterial
counts average 1011 to 1012 CFU/g of feces, with anaerobes outnumbering the aerobes. Within
the B. fragilis group, the species that is most prevalent in the indigenous flora of the intestine is
Bacteroides thetaiotaomicron.
Beneficial Aspects of Indigenous Anaerobes
Many anaerobes of the indigenous microflora are beneficial and play an active role in
maintaining the health of humans and other animals. Anaerobes, together with other
microorganisms, provide a natural barrier to colonization of mucous membranes by pathogenic
organisms. Within the gastrointestinal tract, anaerobes provide a source of fatty acids, vitamins,
and cofactors that are used by the host and which degrade potentially toxic and/or oncogenic
(cancer-causing) compounds. Anaerobes also play a role in maturation of the immune system
during early development of neonates (1).
E. ANAEROBIC INFECTIONS
Anaerobes are key pathogens in brain abscess, oral/dental infections, aspiration
pneumonia, lung abscess, pelvic and abdominal infections, and soft tissue infections, but they
may cause any type of infection (Table 1). In a number of infections, anaerobic bacteria are the
predominant pathogen; in other infections they are often mixed with aerobic organisms and with
a variety of anaerobic organisms.
Anaerobes produce and possess a variety of virulence factors, including enzymes, toxins,
capsules, and adherence factors that are thought to play a role in pathogenicity. Certain clinical
hints may suggest the presence of anaerobes in a clinical specimen (1):
1. Foul odor of specimen
2. Location of infection in proximity to a mucosal surface
3. Infections secondary to human or animal bite
4. Gas in specimen
5. Previous antibiotic therapy with aminoglycoside antibiotics that may have failed
6. Tissue necrosis; abscess formation
7. Unique morphology on Gram stain
8. Failure of culture to grow aerobically when organisms were observed on original
Gram stain
Bacteroides fragilis grp. (34%), followed by anaerobic Gram-positive cocci (28%),
pigmented Gram-negative rods (Prevotella and Porphyromonas) (6.4%), and Fusobacterium
spp. (7.9%), are the most commonly recovered anaerobes from infections. Since B. fragilis grp.
can be forgiving in its tolerance toward oxygen, its physiological requirements of highly
enriched media, and its need of good transport and anaerobic environmental conditions,
laboratories may recover this group even if they use generally poor techniques. The anaerobic
Gram-positive cocci, pigmented Gram-negative rods, and Fusobacterium spp., however, are
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much more demanding and many laboratories do not frequently recover these organisms despite
their reported high incidence (3). See Table 2.
F. SPECIMEN COLLECTION AND TRANSPORT
Proper collection of specimens and prompt transport to the laboratory for processing are
imperatives. Specimens must be collected in a manner that will avoid contamination with
indigenous flora. The laboratory must reject specimens that have not been collected or
transported correctly or are likely to be contaminated. The saying “garbage in, garbage out”
certainly applies to the collection and transport of anaerobic specimens. If the specimen has
been improperly obtained or improperly transported, it may not provide information to the
clinician, and the laboratory may expend useless time and resources on an unsatisfactory
specimen. Indigenous anaerobes are often present in such large numbers on mucosal sites
(gastrointestinal, genital tract, oral cavity), that even minimal contamination with indigenous
flora will yield very misleading results and lead to much wasted effort by the laboratory.
Communication and Supplies
The laboratory director or supervisor must provide the clinical staff (nurses, physicians,
etc.) with clear guidelines for the appropriate specimen types required for anaerobic culture
(Table 3). The clinical staff must be told to immediately transport the properly collected
specimen to the laboratory in an approved anaerobic transport system, and that some specimens
may not be appropriate for anaerobic culture and may be rejected (5). Rejection of a clinical
specimen can be a touchy subject to many clinicians. It works best to have meetings with
physicians and nurses prior to the initiation of any policy to reject specimens to explain rationale
and seek buy-in. Work with specific departments or physicians (surgery, OB, medicine,
Pathologists, and Infectious Disease physician if your hospital has one) to explain information
about the extent of normal anaerobic flora, contamination, and requirements to adequately isolate
anaerobes. The clinical staff will understand that a quality specimen will reduce treatment
delays and costs associated with working up improper specimens. Nurses in OR, ER, and ICU
can be particularly helpful because they often see the patient one-on-one and frequently obtain
specimens for culture. Patient care units, clinics, OR, and emergency rooms must be supplied
with appropriate collection devices and complete instructions for their use. Good
communication between the clinical microbiology laboratory and the clinical staff will ensure the
collection and transport of the best possible specimen for anaerobic culture (5).
Ideal specimens
The ideal specimens for anaerobic culture are fluid obtained using a needle and syringe or
a tissue sample (Table 3). Aspirated fluid collected by needle and syringe can be expelled in
oxygen-free tubes or vials (Anaerobe Systems, BD, Hardy, Fisher Healthcare, and Remel) and
then promptly transported to the laboratory. Aspirated material should never be transported in
the syringe. Tissue samples or biopsy material are very satisfactory specimens and can be placed
into oxygen-free tubes or vials for immediate transport to the laboratory (5). All specimens
should be transported and held at room temperature. Do not place the transported specimen in
the incubator or in the refrigerator; incubator temperatures will cause overgrowth of some
bacteria and loss of isolates, and cold temperatures will allow increased oxygen diffusion.
Anaerobic transport vials may contain modified Cary-Blair or other media that contain
substances to scavenge excess oxygen (Anaerobe Systems, BD, Hardy, Fisher Healthcare, and
Remel) and provide some moisture to the specimen.
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In a good transport medium, anaerobes survive for some time—usually up to 24 hours,
depending upon the nature of the specimen. This fact permits batching of specimens in the
laboratory at convenient times throughout the day without jeopardizing the recovery of
anaerobes. Purulent specimens contain numerous reducing compounds that also help protect
anaerobes from the effects of oxygen.
Least Desirable Specimens
The least desirable specimen for anaerobes is one collected by swab, and it should not be
cultured, even though swabs are the predominant specimen type collected by medical/nursing
personnel. Many laboratories commonly reject swab specimens for anaerobic culture.
Generally, the specimen volume when collected by a swab is small, reducing the probability of
isolating organisms. The specimen may be easily contaminated during collection. Many
organisms adhere to the fibers of the swab and therefore are not recovered. Further, swab
specimens commonly produce smears of poor Gram stain quality, and the inherent dryness of a
swab specimen reduces the viability of many anaerobes. If collecting a specimen by swab is
unavoidable and is absolutely necessary, then collect as much specimen as possible and use a
commercially available anaerobe transport swab system (Anaerobe Systems, BD, Copan, Fisher
Healthcare, and Remel). Take special care to sample the active site of infection to prevent
contamination, and then place the swab deep into the agar butt. Break the stick off below where
it was handled and replace the cap quickly. The commercial anaerobe transport system that
contains two glass tubes (tube within a tube) for swab specimens has been shown not to be
reliable. Remember that if you supply only a swab anaerobic collection device to the
medical/nursing units, you will certainly receive a swab back. Get around this by consistently
providing transport systems for collecting fluid or tissue. See Table 3 for appropriate specimens
for anaerobic culture.
G. PROCESSING CLINICAL SPECIMENS FOR ANAEROBIC CULTURE
Ideally, a specimen is processed immediately upon arrival to the laboratory and is
promptly incubated under anaerobic conditions to prevent further exposure to oxygen. However,
the operations of a busy laboratory may prevent this from happening. When specimens cannot
be inoculated onto media and placed immediately into an anaerobic atmosphere, it is best to hold
specimens in their transport containers and batch process them later (e.g., once in the morning,
and perhaps right before the day shift is ending, or at other convenient times throughout the day).
Holding the clinical specimen in an appropriate transport device will not jeopardize the recovery
of anaerobes or their viability. Batch processing of media inoculation at convenient times is
preferred to processing specimens one at a time, which would require opening an anaerobic
incubation jar each time, using expensive anaerobic incubation bags, or using up anaerobic gas.
Batch processing specimens for anaerobes clearly reduces costs and improves the efficiency of
the laboratory.
The specimen for anaerobic culture may require special preparation. For example,
grossly purulent specimens may require the use of a vortex mixer (avoid excessive aeration) on
the anaerobic transport vial to ensure even distribution of microorganisms. You may need to
grind bone or tissue with thioglycollate (THIO) or chopped meat broth to permit inoculation of
specimen onto solid media. Swab specimens (should you accept one) may require the addition
of THIO or chopped meat broth to make a liquid specimen. Large volume specimens may
require centrifugation to produce the sediment needed to inoculate media and prepare a Gram
stain.
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Once the specimen has been prepared for culture, it should be inoculated onto the
appropriate anaerobic media, placed in a liquid back-up broth, and onto a glass slide for a Gram
stain. Once you begin processing the sample, you should complete it as quickly as possible, at
least within 15 minutes.
Microscopic Examination
Always prepare a direct Gram stain from the clinical material. This is very important, for
it often allows early presumptive evidence of the presence of anaerobes and provides information
about the quality of the specimen. Direct smears for anaerobes are best fixed in absolute
methanol for 1 min, and then stained by standard Gram stain procedure and reagents (5). Even
gentle heat fixation can distort bacterial cell morphology, preventing clues in early identification.
A Gram stain reveals the types and relative numbers of microorganisms and host cells present,
and serves as a quality control measure for the adequacy of anaerobic technique. Failure to
recover all the morphotypes seen on the direct Gram stain smear may indicate a problem in
specimen collection, transportation or processing, or another problem that inhibited the growth of
anaerobic microorganisms.
The following are Gram stain clues for the presence of anaerobic organisms:
1. Large Gram-positive rods with boxcar-shaped cells and no spores usually indicate
Clostridium perfringens. Within the same microscopic field, organisms may
appear Gram-negative with the same cell morphology as the Gram-positive rods.
2. Gram-negative coccobacillary forms suggest Prevotella group or Porphyromonas
group.
3. Thin Gram-negative bacilli with tapered ends suggest Fusobacterium nucleatum.
4. Pleomorphic pale-staining Gram-negative bacilli, sometimes with vacuoles,
suggest Bacteroides fragilis spp.
Media
Efficient, cost-effective anaerobe recovery in the laboratory requires good media.
Skimping on media costs and using inferior media wastes time and money, as cultures may fail
to grow or yield inconclusive results and then have to be repeated. Use a highly enriched basal
medium for primary isolation, such as Brucella medium containing vitamin K and hemin, which
will support the growth of all anaerobes and aerobes. It has been shown that a PRAS (prereduced anaerobically sterilized) medium gives a faster growth rate and the ability to recover
more anaerobes within a shorter period of time. Anaerobe Systems is the sole source of PRAS
media, which have never been exposed to oxygen during any step of preparation. Therefore,
PRAS media have not been exposed to superoxide anions, or hydroxyl radicals which may
damage components of the media and prevent the growth of anaerobes. PRAS media also have a
prolonged shelf life compared to other anaerobic media.
Other manufacturers produce media for anaerobes that require pre-reduction (placing in
anaerobic environment for 24 hrs. before use) or media that contain oxygen-scavenging
substances (Oxyrase) or other reducing substances. It is best to perform side-by-side comparison
testing in your own laboratory to determine which type of media recovers more organisms.
In addition to using a highly enriched primary medium, it is also important to include a
combination of selective and differential media for the recovery of anaerobes and for
presumptive identification (2). The following media are suggested for the isolation of anaerobic
bacteria from clinical specimens:
• Brucella agar supplemented with 5% sheep blood and vitamin K1 (1µg/ml) and hemin
(5µg/ml) as a nonselective medium which supports the growth of both anaerobic and
aerobic organisms.
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Phenylethyl alcohol-sheep blood agar (PEA) for the inhibition of enteric and certain other
facultatively anaerobic Gram-negative bacilli that may overgrow anaerobes. PEA also
reduces the spreading or swarming characteristic of certain Clostridium spp.
• Kanamycin-vancomycin-laked blood agar (KVLB or LKV) for the selection of
pigmented Prevotella and other Bacteroides spp.
• Bacteroides bile esculin agar (BBE) for the selection and presumptive identification of
Bacteroides fragilis grp. and Bilophila wadsworthia. Fusobacterium mortiferum/varium
grp. is also resistant to bile and may occasionally grow on this medium.
• Thioglycollate medium without indicator, supplemented with vitamin K1, hemin, and a
marble chip, for enrichment and back-up culture. Chopped meat broth with vitamin K1
and hemin may also be used. Use either of these broths as backup only. If primary plates
are positive, you may discard the backup broth. Do not subculture the broth. Subculture
the backup broth only if primary plates are negative and the broth is turbid.
Anaerobic Incubation Systems
The choice of incubation system used for anaerobic specimens depends on the number of
anaerobic cultures performed, the cost of the system, and the space limitations of the laboratory.
In general, there are three methods for the incubation of anaerobes from clinical specimens:
anaerobic bags, anaerobic jars, and anaerobic chambers.
A clinical laboratory that receives very few requests for anaerobic culture (1 per day and/or
receives a rare anaerobic specimen after normal laboratory hours) may consider the use of
anaerobic bags or pouches. A clinical laboratory that receives perhaps 2-4 specimens per day for
anaerobic culture may consider the use of anaerobic jars. The use of anaerobic jars may be
economically employed if the laboratory batches the processing of specimens at convenient
times rather than using one jar for one specimen. If the laboratory receives a specimen at odd
times after jars have been closed, perhaps the new specimen may be incubated in a pouch or bag
and then after 48 hrs. included in an anaerobic jar. A laboratory that may receive 3 or 4 or more
specimens per day should consider using an anaerobic chamber, the most economical way of
producing an anaerobic atmosphere. The laboratory would need to consider the initial expense
and the space required for the chamber. The ability to examine cultures at 24 hrs. and report the
presence of anaerobes earlier (compared to jar and bag systems) may also be a patient-care
benefit for the hospital.
Whichever anaerobic system you use, the first step is to immediately place the inoculated
plates into the anaerobic environment, and incubate them at 35 to 37ºC for 24-48 hrs. Growing
cultures must not be exposed to oxygen until after 48 hrs. of incubation in an anaerobic jar or
pouch system, since anaerobes are most sensitive to oxygen during their log (early) phase of
growth. An obvious advantage of an anaerobic chamber is that it permits the processing,
inoculation of plates, and their examination at 24 hrs. or at any time under anaerobic conditions.
Any anaerobic environment needs to be monitored with a methylene blue strip or resazurin
chemical indicators. These indicators, initially blue and pink (respectively), change to colorless
with low concentrations of oxygen.
The following is a more detailed description of the most common choices of anaerobic
incubation systems.
Anaerobic bag or pouch
Some anaerobic bag or pouch systems use a sachet that absorbs atmospheric oxygen without
the generation of hydrogen, without the addition of water, and without requiring a catalyst. The
resulting carbon dioxide level in these systems is generally higher than 10%. In other bag or
pouch anaerobic atmospheric producing systems, a gas-generating envelope or ampoule provides
•
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an atmosphere of 80 to 90% nitrogen (N2), 5% hydrogen (H2), and 5 to 10% carbon dioxide
(CO2). Some heat is produced from these systems, and the bags require a new catalyst each time
they are opened. There are some gas-generating systems that have a catalyst incorporated into
the envelope.
The procedure is as follows: place the plates in the bag, activate the generating envelope,
ampoule, or sachet, add an anaerobic indicator, and seal the bag or pouch by heat-sealing or by
using special clamps. Check the anaerobic indicator through the clear plastic bag after a few
hours to see that the bag has not leaked. Incubate the bag at 35 to 37ºC in a standard incubator
for 48 hrs. Examining plates before 48 hrs. is not recommended since any small colonies are
particularly susceptible to oxygen exposure at this stage and may not survive. At 48 hrs., remove
the plates from the bag to examine them and work up the organisms as quickly as possible (this
process will be described in greater detail). Add a new anaerobic generating envelope, ampoule,
or sachet and reseal the bag or pouch. Bags and pouches are convenient, easy to use, and they do
not take up a lot of space. However, the bags occasionally leak, and they are the most expensive
way of producing an anaerobic environment (about $6.00 per bag). (BD Biosciences, Oxoid,
Mitsubishi Gas Chemical America, and Difco).
Anaerobic jars
Anaerobic conditions are maintained in a self-contained jar by using a catalyst; a gasgenerating system (usually an envelope, ampoule, or sachet) providing an atmosphere of 80 to
90% nitrogen (N2), 5% hydrogen (H2), and 5 to 10% carbon dioxide (CO2); and an anaerobic
indicator. If a sachet is employed, hydrogen is not produced and a catalyst is not required. (See
Anaerobic Bag or Pouch from above).
For most jar systems, the procedure is the same. Place the inoculated plates into the jar, add
an anaerobic indicator to the jar, add the anaerobic producing or catalyst system, close the jar,
and incubate it at 35 to 37ºC in a standard incubator for 48 hrs. before opening the jar. This
prevents exposure of smaller colonies to oxygen. The catalyst, composed of palladium-coated
alumina pellets, should be fresh or rejuvenated each time the jar is opened prior to use, unless the
catalyst is included in the gas pack envelope, or a water-less anaerobic generating system is used.
At 48 hrs., remove the plates from the jar to examine them and work up the organisms. Add
a new generating envelope, ampoule, or sachet system and reseal the jar. (BD Diagnostic
Systems, Hardy Diagnostics, PML Microbiologicals, and Remel). The recovery of anaerobes in
an anaerobic jar compares well to an anaerobic chamber if the plates are continuously incubated
for 48 hrs. Jars do not recover anaerobes well if plates are incubated for only 24 hrs. prior to
initial examination.
Anaerobic chamber
Anaerobic conditions are maintained in a gas-tight box or chamber by a gas mixture
containing 80-90% nitrogen (N2), 5 % hydrogen (H2), and 5 to 10% carbon dioxide (CO2), and
using a palladium catalyst. The hydrogen concentration should not exceed 5% to prevent
hazardous conditions.
Usually anaerobic chambers have a positive pressure inside to prevent oxygen from
coming into the chamber in case of a leak. The catalyst converts oxygen and hydrogen to water,
thus removing atmospheric oxygen from the chamber. Carbon dioxide is included because many
anaerobes require it for growth. Humidity is controlled by using silica gel crystals to absorb the
water formed in the catalytic conversion process. In other chambers, humidity is controlled with
a “cold spot” that condenses excess humidity and allows the water formed to be removed
through a drain. Plates are incubated at 35 to 37ºC and can be examined at any time within the
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chamber (generally at 24 to 48 hrs.) without removing them from the anaerobic environment
(Coy Laboratory Products, Forma Scientific, and Sheldon Manufacturing).
H. ISOLATION AND IDENTIFICATION OF ANAEROBES
Isolation:
After the plates—primary Brucella, PEA, BBE and LKU—have been incubated in an
anaerobic pouch, jar or chamber, the next step is to isolate the anaerobes from other organisms.
The primary medium (Brucella) likely will have grown not only anaerobes, but also facultative
anaerobes (organisms that grow under either aerobic or anaerobic conditions) and
microaerophilic organisms (organisms that grow in an atmosphere of reduced oxygen tension).
Remember that facultative anaerobes and microaerophilic organisms will grow under anaerobic
conditions, so you will need to exclude these from your workup. To determine which isolates
from the primary Brucella medium are anaerobes, test the organisms for aerotolerance using two
media: Brucella agar incubated anaerobically, and chocolate blood agar incubated under 5-10%
CO2 conditions. The facultative anaerobes and the microaerophilic organisms will grow on both
the Brucella incubated anaerobically and the chocolate blood agar incubated under 5-10% CO2,
but the anaerobes will grow only on the Brucella incubated anaerobically and not on the
chocolate blood agar.
Chocolate blood agar must be used for aerotolerance testing. You may incorrectly assume
that you have isolated an anaerobe if you use only blood agar media for aerotolerance testing.
Use the chocolate blood agar media under 5-10% CO2 to permit organisms such as Haemophilus
spp., Actinobacillus spp., or other fastidious, slow-growing organisms to grow under “aerobic”
conditions.
When you set up the aerotolerance testing, also set up the special disks on the Brucella plate
incubated anaerobically, and do a Gram stain as well. The disks will help you identify the
organism once it shows growth (these disks are explained in detail in the next section,
“Identification”). Setting up the special potency disks at this time will permit faster
identification and reporting of the anaerobe. Here is the procedure:
1. Select a single, well-isolated colony of each morphotype seen from the primary set-up
medium (Brucella), and subculture it to a single Brucella agar plate and to a chocolate blood
agar plate. Pick and subculture any colonies on the PEA, BBE and LKV plates that appear
different from the colonies isolated on the anaerobic primary Brucella medium.
2. Divide the chocolate blood agar plate into quadrants so that 4 organisms at a time can be
tested for aerotolerance. Streak the Brucella agar plate for isolation. Label the Brucella plate
and the spot on the chocolate blood agar with the same identification number.
3. Add special potency antibiotic disks and a nitrate disk (as explained in the “Identification”
section below) to the heavy quadrant of the Brucella subculture plate.
4. Make a smear for Gram stain on each colony type you observed from the primary Brucella
medium. Facultative and anaerobic bacteria may have similar colony appearances, so you
need to work up all colonial morphotypes you see on the primary media.
5. Incubate the Brucella plate anaerobically. Incubate the chocolate blood plate in an
atmosphere of 5-10% CO2.
6. Observe after 24 hrs. Anaerobic organisms will grow only on the Brucella medium
incubated anaerobically, facultative anaerobes will grow on both the Brucella and chocolate
blood agar.
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7. Record a detailed description of each colony type from the anaerobic primary Brucella
medium that does not grow on chocolate. Describe such characteristics as pitting, swarming,
hemolysis, pigment, “greening” of the medium, etc. These colony characteristics can provide
clues to identify the isolates when used in conjunction with Gram stain and rapid
identification tests (explained in the next section). (See Table 4. Anaerobic Organism
Identification Clues from Colony Morphology).
Identification:
Once you know that you have isolated an anaerobic organism(s) from the clinical
specimen (growth on brucella medium, but no growth on chocolate), and you know the Gram
reaction of the isolate, you are ready to begin identification of the isolate. The extent of
identification required may vary according to the type of isolate, the source of the specimen, the
needs of the physician, the clinical need, the patient’s type of illness, and the operational and
financial issues of the laboratory.
In general, there are three different methods that can allow rapid and cost-efficient
identification of anaerobic isolates: Method 1: presumptive and preliminary grouping using
Gram stain information, colonial morphology (Table 4) and various rapid spot and disk tests;
Method 2: the use of a variety of individual preformed-enzyme tests along with rapid spot and
disk tests; and Method 3: the use of commercially available identification systems. The
identification of anaerobes using either one of the first two methods is less expensive (about 50¢
per isolate) than using the third method (commercial systems cost about $6.00 per identification).
The identification of anaerobic isolates to a group level using either Method 1 or Method
2 may be all that is necessary for many laboratories to provide clinically relevant information,
and to allow initiation of appropriate antibiotic therapy.
Method 1: Presumptive and Preliminary Grouping.
You may already have some significant information about the identity of the anaerobic
isolate based upon the Gram stain and colonial morphology (See Table 4). Begin the
identification process by describing the colonial morphology in detail, including colony size,
shape, edge, opacity, color and any other distinctive characteristic. Describe cellular
morphology, including size, shape, and Gram reaction. Examine colonies for hemolysis on
Brucella agar. Examine colonies for pigment on Brucella or LKV. Test colonies for
fluorescence on Brucella agar.
Next, determine susceptibility to special potency antibiotic disks (vancomycin 5 µg,
kanamycin 1,000 µg, and colistin 10 µg) (Anaerobe Systems, Becton Dickinson, Hardy, PML,
and Remel). The disks are used as an aid in determining the “true” Gram reaction and in
separating different anaerobic species and genera (See Table 5). Generally, Gram-positive
organisms are sensitive to vancomycin and resistant to colistin, whereas the Gram-negative
organisms are resistant to the vancomycin disk and variable to colistin. The special potency
antibiotic disks test is especially helpful with those clostridia that consistently stain Gramnegative, since their susceptibility to vancomycin disk confirms their “true” Gram reaction.
Place special-potency antibiotic disks of vancomycin, kanamycin, and colistin on a
Brucella agar plate. If you know the organism is Gram-negative, also add a nitrate disk to the
heavily inoculated section. Special potency antibiotic disks are not needed when the organism
stains Gram-positive because they will all be vancomycin susceptible, and the colistin and
kanamycin do not provide additional information on Gram-positive organisms.
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After 24 hrs. anaerobic incubation, use the results obtained with special-potency
antibiotic disks for grouping or species identification (See Table 5). Examples of identification
of Gram-negative isolates using special potency disks are as follows:
1. The B. fragilis grp. can be identified by the special potency antibiotic disk pattern showing
resistance to all three disks (RRR) and resistance to 20% bile or growth on BBE agar.
2. The Bacteroides ureolyticus grp. is susceptible to kanamycin and colistin special potency
disks, and resistant to vancomycin. These organisms reduce nitrate; they are nitrate
reductase positive.
3. Fusobacterium sp. are susceptible to special potency disks kanamycin and colistin, and
resistant to vancomycin. These organisms are nitrate reductase negative.
4. Porphyromonas sp. are resistant to special potency disks kanamycin and colistin, and are
susceptible to vancomycin. They produce a black pigment.
5. Prevotella sp. are resistant to special potency disks kanamycin and vancomycin, and vary in
their susceptibility toward colistin. Some Prevotella sp. may have a special antibiotic disk
pattern typical of the B. fragilis grp. (RRR), but these organisms do not grow in 20% bile or
on BBE.
6. Bilophila sp. are susceptible to special potency disks kanamycin and colistin and are resistant
to vancomycin. Phenotypically this organism resembles the B. ureolyticus group and some
Fusobacterium sp. These organisms can be distinguished by their strong positive catalase
reaction and resistance to 20% bile. In 3 to 4 days Bilophila wadswortha forms small
colonies on BBE that are clear with black centers, resembling “fish-eyes.”
Use the pure-culture growth on the brucella agar to perform additional tests as needed.
Once the true Gram stain reaction is known from the special potency disks, the laboratorian may
use other rapid tests to assist in anaerobe identification. One such rapid test is determining the
fluorescence of anaerobes using a Woods Lamp at 366 nm. The presence and color of
fluorescing colonies can aid in the rapid detection and presumptive identification of certain
anaerobic bacteria. Fluorescence disappears when black pigment has developed. See Table 6.
Additional spot tests may include spot indole, catalase, SPS, bile test, lipase, lecithinase,
pigment, and urease. See Table 7 for tests for the rapid identification of anaerobes. If the isolate
is a Gram-negative rod, use Table 8; if isolate is a Gram-positive rod with spores (Clostridium
spp.), use Table 9; and if the isolate is an anaerobic Gram-positive coccus, use Table 10.
For guidance on further tests to permit rapid identification, the clinical laboratorian can
use the tables in this course or others listed in the Wadsworth KTL Anaerobic Bacteriology
Manual (2) or Clinical Microbiology Procedures Handbook (5). When typical morphology (cell
and colony) is apparent and is combined with rapid tests, the resulting preliminary identification
may be useful until more exhaustive tests are completed or are needed by the clinician.
Anaerobic Gram-positive bacilli of human clinical relevance are divided into two distinct
groups: one genus of spore-formers (Clostridium spp.). and five genera of non-sporeformers
(Actinomyces, Bifidobacterium, Eubacterium, Lactobacillus, and Propionibacterium). The
anaerobic Gram-positive bacilli are part of the normal microbiota of the oral cavity,
gastrointestinal and genitourinary tracts, and skin.
Currently there are 130 species of clostridia. Fortunately for the clinical microbiologist,
the percentage of clostridial isolates commonly recovered in properly collected specimens is
relatively small (Table 2). Clostridium perfringens is the most common clostridial isolate,
followed by C. clostridioforme, C. innocuum, and C. ramosum (2, 4, 5). See Table 9 for
identification of some commonly isolated Clostridium spp. Clostridium spp. can cause acute,
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severe, or chronic infections. Some Clostridium spp. are highly pathogenic or toxigenic, while
others are rarely pathogenic. Some species are resistant to antimicrobial agents. A great source
of confusion is that many Clostridium spp., and occasionally the non-sporeforming genera as
well, can stain Gram-negative. The use of the special antibiotic disks can help resolve this
problem. There are a few aerotolerant strains of clostridia (C. tertium, C. carnis, C. histolyticum)
that will grow marginally under aerobic conditions, and also a few aerotolerant strains of nonsporeforming bacilli (Actinomyces spp., Lactobacillus spp., and Propionibacterium spp.).
The identification of the anaerobic non-sporeforming Gram-positive bacilli can be a
challenge for the Clinical Laboratory Scientist. In many instances the use of PRAS
biochemicals, gas-liquid chromatography (GLC) and fatty acid analysis is necessary. Many
laboratories do not have access to these methods, and they will not be discussed in this course.
The non-sporeforming Gram-positive bacilli comprise several genera that are differentiated from
each other by their metabolic end products detected by GLC. The group is resistant to special
potency disk of colistin, variable to kanamycin, and generally susceptible to vancomycin.
However, there are rare strains of Lactobacillus and Clostridium spp. that may be vancomycin
resistant (2).
The clinical laboratory may encounter Propionibacterium acnes occasionally from a
blood culture and from wound sources as contaminants. However, these organisms have been
reported as causing chronic disease, so you need to rule this out before discarding the organism
as a “contaminant.” P. acnes has a typical Gram stain appearance of clubbing, palisading, and
“Chinese character.” P. acnes is nitrate, catalase, and spot indole positive.
For identification of the Gram-positive cocci, the use of DNA composition, hybridization
data, and cellular fatty acid profiles has permitted significant changes and reclassification among
species that were at one time in the genus Peptostreptococcus. Peptostreptococcus
hydrogenalis is now Anaerococcus hydrogenalis; Peptostreptococcus prevotii is now
Anaerococcus prevotti; Peptostreptococcus magnus is now Finegoldia magna;
Peptostreptococcus micros is now Parvimonas micra , Peptostreptoccus asaccharolyticus is now
Peptoniphilus asaccharolyticus; Peptostreptococcus indolicus is now Peptoniphilus indolicus.
The good news is that Peptostreptococcus anaerobius has not changed its name and is
susceptible to the SPS (sodium polyanethanol sulfonate) disk which is useful for its rapid
identification. Anaerobic cocci can be identified by Gram stain, colony morphology, spot tests
such as SPS disk and spot indole, and various biochemical preformed enzymatic reactions and
commercial systems (See Table 10). In some instances, PRAS biochemical, GLC, or fatty acid
analysis may be necessary for identification.
Method 2: Rapid biochemical tests for identification
Many anaerobic isolates may be further identified using a variety of commercially
available preformed-enzyme tests in conjunction with some of the rapid spot tests previously
described in this course. Individual enzymatic biochemical tests may permit anaerobe
identification without excessive expense or delay. One example is the identification of some
species of anaerobic Gram-positive cocci using the alkaline phosphatase enzyme test.
The combination rapid enzymatic tests are simple to perform and can be purchased
allowing two or more enzymatic tests to be performed in a single tube to detect enzymatic
activity visible by color change, or by detecting 4-methylumbelliferone fluorescent end products
when exposed to a Wood’s Lamp at 366 nm. (WeeTabs, Key Scientific Co., Stamford, TX).
The tablet is inoculated heavily from fresh 24 hr. growth from Brucella medium; the heavier the
inoculum, the better (>2.0 McFarland turbidity). Incubate for at least 2 hrs. at 37ºC.
Identification tables of some anaerobes using the rapid preformed enzymatic tests are included in
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this course, as well as in references # 2 and 5. Additional identification tables and information
are available from the manufacturer. (WeeTabs Package Insert. Key Scientific Corporation
Stamford, TX. www.keyscientific.com). Other rapid enzymatic test tablets are available from
Rosco Diagnostica, Taastrup, Denmark.
Method 3: Rapid Identification System Kits
Identification of anaerobes can be accomplished with commercially available
microsystem kits for the detection of preformed enzymes within a few hours following
inoculation: Vitek 2 ANI Anaerobe Card and Rapid ID 32A (bioMerieux, Inc.); Rapid Anaerobe
ID (Dade MicroScan); Crystal Anaerobe ID kit (BD Bioscience); and RapID-ANA (Remel).
The systems allow the identification of many species not identified by previously described
methods. The systems require 4 hrs. of aerobic incubation at 35º C. Each system has its own
database permitting identification. Tables in this course or other texts should not be used for
identification. These systems will not be discussed in any detail in this course. See the
manufacturer’s insert for more details. Each system varies with specific QC, inoculum size, and
test procedures, including recommended media. The user needs to follow the manufacturer’s
recommendations carefully. There are some distinct advantages and disadvantages of using
these kits. Interpretation of colors can be difficult, but is critical for obtaining accurate,
reproducible results. Rapid enzymatic test kits should be used in conjunction with other
conventional information, such as Gram stain, colonial morphology, and organism growth
characteristics. Special potency antibiotic disks and other spot presumptive tests can be very
useful in verifying and confirming the identification obtained using these kits. Results of all
reactions must be considered. Do not automatically accept any answer from any identification
kit without comparing results to other methods described in this course. Keep in mind that each
identification using these commercial systems costs about $6.00.
There is one caveat: As with aerobic identification systems, it is often difficult for the
manufacturer of anaerobic identification systems to keep up with the explosion of taxonomic
name changes and the need for additional biochemical tests. Often the name listed by the
manufacturer for identification may be out-of-date and you may need to change the identification
accordingly.
I. METHODS FOR COST EFFECTIVE ANAEROBIC BACTERIOLOGY
Methods for cost effective anaerobic bacteriology depend upon the following:
1. Accept only appropriate specimens. Educate the clinical staff so they are aware of what
specimens are appropriate and how to collect and transport specimens for anaerobes. It all
begins here: if you receive a bad specimen that is contaminated and that is transported
incorrectly, you will spend the laboratory’s resources working up a useless specimen.
2. Once a good specimen has been received, use good environmental conditions and good
primary, selective, and differential media. It may seem that you are spending too much
money on media, but good media will save you time and expense in the long run. Poor
media results in poor growth or growth that is delayed, which may mean the laboratory
finally recovers and identifies the anaerobe, only to discover the patient has gone home.
3. Batch process specimens for anaerobic culture. A good transport system permits processing
at convenient times and reduces the cost of setting up anaerobic cultures and improves the
efficiency of the laboratory.
4. Provide rapid identification to the level needed by the physician to make a diagnosis and to
guide appropriate therapy. You may not need to identify the isolate to its exact genus and
species to enable the physician to treat the patient correctly. Costs can be controlled simply
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by identifying an organism according to the physician’s needs, and to the extent determined
by the specimen source and the type of organism recovered. Many laboratories do this now
with aerobic organisms by having abbreviated identification systems for swarming Proteus
spp., lactose fermenting organisms from MacConkey, etc. The same practice should apply to
anaerobic organisms as well.
5. Use rapid, spot and presumptive tests as needed. The rapid tests may permit early
identification that may allow the physician to use appropriate therapy, and the cost of the
identification will be about 50¢. Use commercial identification kits wisely—remember they
cost $6.00 each.
6. Finally, communicate with the physician frequently. CLSs don’t often like to do this, but by
communicating with the physician you will be able to determine what his/her needs are, and
what extent of identification is needed. Perhaps the patient is doing fine, perhaps the B.
fragilis grp. is all that is necessary for treatment, or maybe the specimen was inappropriately
labeled and was really obtained from a superficial wound and further workup can stop. You
need to verbally communicate at times instead of just sending out reports.
In summary, I hope the material in this course has provided you the tools to rapidly
isolate and identify anaerobes in a cost-efficient manner.
J. REFERENCES
1. Finegold SM, George WL. Anaerobic Infections in Humans. New York: Academic Press,
Inc.; 1989.
2. Jousimies-Somer HR, Summanen P, Citron DM, Baron E J, Wexler HM, Finegold SM.
Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed. Belmont, CA: Star Publishing Co.;
2002.
3. Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, eds. Manual of Clinical
Microbiology, 7th ed. Washington, DC: ASM Press; 1999.
4. Engelkirk PG, Duben-Engelkirk J, Dowell VR, Jr. Principles and Practice of Clinical
Anaerobic Bacteriology. Belmont, CA: Star Publishing Co.; 1992.
5. Mangels JI, ed. Section 4, Anaerobic Bacteriology. In: Isenberg H. Clinical Microbiology
Procedures Handbook. 2nd ed. Washington, DC: ASM Press; 2004.
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Table 1. Incidence of anaerobic bacteria in various infections
Type of Infection
Incidence (%) of anaerobic bacteria
Central Nervous System
Brain abscess
89
Head and Neck
Chronic sinusitis
50
Chronic otitis media
30-60
Periodontal abscess
100
Other oral infections
94-100
Pleuropulmonary
Aspiration pneumonia
85-90
Lung abscess
93
Necrotizing pneumonia
85
Empyema
76
Intra-abdominal
Peritonitis
90-95
Liver abscess
>50
Female Genital Tract
Salpingitis, pelvic peritonitis
>55
Tubo-ovarian abscess
92
Vulvovaginal abscess
74
Septic abortion
73
Soft Tissue
Gas gangrene (myonecrosis)
100
Adapted from: Manual of Clinical Microbiology, ASM Press, 5th Edition
Table 2. Incidence of anaerobic bacteria in clinical specimens
Organism
No. of isolates
% of all anaerobes recovered
Bacteriodes fragilis grp.
141
34.8
B. fragilis
77
19.0
B. thetaiotaomicron
12
3.0
B. vulgatus
10
2.4
B. distasonis
10
2.4
B. ovatus
6
1.5
Unidentified
23
5.7
Pigmented GNR
26
6.4
Other
45
11.1
Fusobacterium spp.
32
7.9
Peptostreptococcus spp.
117
28.9
Clostridium spp.
9
2.2
Non-sporeforming GPB
20
4.9
Gram-negative cocci
15
3.7
Adapted from: Manual of Clinical Microbiology, ASM Press, 5th Edition.
GNR = Gram-negative rods
GPB = Gram-positive bacilli
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Table 3. Specimen Types for Anaerobic Culture
Acceptable
Not Acceptable
Abscess
Cervical or vaginal secretions
Deep Wounds
Sputum, throat, naso-pharyngeal
Body fluid
Feces
Tissue
Gingival material
Catheterized urine
Small bowel contents
Normally sterile site
Gastric contents
Lung
Superficial skin lesions
Aspirate
Ulcers
Voided urine
Surface wounds
Bronchial washings (except by double
lumen catheter)
Table 4. Anaerobic Organism Identification Clues from Colony Morphology
Colony morphology
Possible identification
Agar pitting
Bacteroides ureolyticus grp.
Black or tan pigmentation
Porphyromonas spp. or pigmented Prevotella spp.
Double-zone of beta hemolysis
Clostridium perfringens
“Fried egg”
Fusobacterium necrophorum, or F. varium
“Greening” of medium
Fusobacterium spp.
Large with irregular margin
Clostridium spp.
“Medusa-head”
Clostridium septicum
“Molar tooth”
Actinomyces spp.
Pink to red colony (Gram-positive rod)
Actinomyces odontolyticus
Speckled or “breadcrumb”
Fusobacterium nucleatum
Swarming growth
Clostridium septicum, C. sordelli, or C. tetani
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Table 5. Identification by means of special-potency antibiotic disks
Response to antibiotic diska:
Organism
Kanamycin 1,000 µg Vancomycin 5 µg
Colistin 10 µg
b
Gram-positive
S
R
Gram-negative
V
R
V
Bacteroides fragilis grp.
R
R
R
Bacteroides ureolyticus grp.
S
R
S
Fusobacterium spp.
S
R
S
Porphyromonas spp.
R
Sc
R
Prevotella spp.
R
R
V
Veillonella spp.
S
R
S
Adapted from: Wadsworth-KTL Anaerobic Bacteriology Manual, 6th Edition, 2002.
a.
S= Sensitive is zone of inhibition ≥12mm. R= resistant. V= variable in reaction.
b.
Rare strains of Lactobacillus sp. and Clostridium sp. may be vancomycin resistant.
c.
Porphyromonas spp. is vancomycin-sensitive
Table 6. Fluorescence of Anaerobes
Organism
Color
Porphyromonas gingivalis
No fluorescence
Other Porphyromonas spp.
Red, orange
Pigmented Prevotella spp.
Red
Fusobacterium spp.
Chartreuse
Veillonella spp.
Red or no fluorescence
Clostridium difficile
Chartreuse
Clostridium innocuum
Chartreuse
Clostridium ramosum
Red
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Table 7. Tests for Rapid Identification of Anaerobes
Test
Principle of use
Special potency disks
Used as an aid in determining the Gram reaction as well as in
preliminary ID of some Gram-negative genera and species.
Spot Indole test
Used to group and identify many anaerobes.
Must use p-dimethylcinnamaldehyde (DMAC) reagent.
Nitrate disk
Use to test nitrate reduction. Useful for separating B. ureolyticus grp.
from Fusobacterium grp.
Catalase test
Some anaerobic bacteria possess catalase. A 15% solution of hydrogen
peroxide is preferred.
SPS disk
Sodium polyanethanol sulfanate. Used to differentiate
Peptostreptococcus anaerobius which produces a zone >12 mm.
Bile test
Bile disks or BBE agar plates. B. fragilis grp., F. mortiferium, F.
varium and Bilophila wadsworthia are capable of growing in the
presence of bile.
Fluorescense
Some anaerobes are capable of fluorescing different colors when
exposed to UV light (Woods Lamp 366nm).
Lipase
Fats in egg yolk medium are broken down by lipase enzyme and appear
as a surface iridescent layer. F. necrophorum is lipase positive.
Lecithinase
Lecithin in egg yolk medium is split by lecithinase enzyme resulting in
opaque halo surrounding an organism. C. perfringens is lecithinase
positive.
Pigment production
Some anaerobic gram-negative rods, namely Porphyromonas spp. and
some Prevotella, produce a dark pigment on sheep or rabbit blood agar
media. Some isolates produce pigment in 4 to 6 days.
Urease
Some organisms are capable of hydrolysis of urea, releasing ammonia.
The resulting pH change causes phenol indicator to change from yellow
to red. B. ureolyticus is positive.
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REVIEW QUESTIONS
Course DL-974
Choose the one best answer
1. Anaerobic bacteria are generally not involved with one of the following types of infection:
a. appendicitis
b. bacteremia
c. bladder infection
d. liver abscess
2. Which statement best describes superoxide anions?
a. causes damage to media, bacterial cell walls and enzyme systems
b. promotes growth of anaerobes
c. causes damage to RNA
d. neutralizes oxygen
3. An example of an appropriate specimen for anaerobic culture is:
a. voided urine
b. vaginal swab
c. lung tissue
d. superficial wounds
4. The common indigenous anaerobic flora of the oral cavity does not include:
a. anaerobic Gram-positive cocci
b. Actinomyces spp.
c. Porphyromonas spp.
d. Clostridium spp.
5. The most frequently isolated anaerobe from anaerobic infections is:
a. Propionibacterium acnes
b. Clostridium spp.
c. Fusobacterium spp.
d. Bacteroides fragilis grp.
6. Which one of the following is not commonly a clinical clue for the presence of a possible
anaerobic infection?
a. location of infection in proximity to mucoid surface
b. vomiting
c. abscess formation
d. secondary to human or animal bite
7. What is an important reason to identify anaerobes from clinical specimens?
a. commonly resistant to empiric antibiotic therapy
b. risk to health care workers
c. provide documentation in the event of legal action
d. improves use of CPT codes
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8. Which of the following are Gram stain clues for the presence of Bacteroides fragilis grp.?
a. Gram-negative rod with tapered ends
b. pale staining pleomorphic Gram-negative rods often with vacuoles
c. pleomorphic Gram-positive coccobacilli
d. large Gram-positive box car shaped rods
9. To best monitor an anaerobic environment, which chemical indicator should be used?
a. congo red
b. crystal violet
c. safranin
d. methylene blue
10. The primary goal of using selective and differential media for the recovery of anaerobes
includes:
a. early detection and recovery of clinically important isolates
b. improves the growth of clostridia
c. decreases need for quality control
d. decreases need for aerotolerance testing
11. Which one of the following is necessary for aerotolerance testing of clinical isolates?
a. BBE agar
b. chocolate agar
c. use of strict anaerobic conditions
d. blood agar plate under CO2 conditions
12. What is the best reason for testing anaerobes using special potency antibiotic disks?
a. determines if organism is a coccus shaped or rod shaped morphology
b. provides early clues to susceptibility testing
c. determines true Gram stain reaction
d. provides information concerning obligate anaerobes
13. The term PRAS media stands for:
a. pre reductive anaerobically sensitive media
b. post reduction aerobically sterilized media
c. pre reduced anaerobically sterilized media
d. post reduced anaerobically sterilized media
14. Why is BBE agar important to use on anaerobes?
a. selective for B. fragilis grp.
b. selective for Fusobacterium spp.
c. promotes pigment formation
d. prevents swarming of Clostridium spp.
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15. What is the correct reason swab specimens are an inferior specimen type and should not be
used?
a. excessive moisture associated with swabs, easy to collect, hard to contaminate
b. difficult to inoculate media, easy to contaminate, infection control principles
c. difficult to use, easy to inoculate media, hard to contaminate
d. small volume, organisms adhere to fibers of swab, easy to contaminate
16. What is an example of a strict or obligate anaerobe?
a. Bacteroides fragilis grp.
b. Clostridium perfringens
c. Porphyromonas spp.
d. Propionibacterium acnes
17. What is an example of moderate anaerobe?
a. Peptostreptococcus anaerobius
b. Bacteroides fragilis grp.
c. Fusobacterium nucleatum
d. Clostridium tertium
18. The term SOD means:
a. superoxide dismutase
b. sensitive oxide dimer
c. superoxide dimer
d. super oxygen dismutase
19. Which is the correct statement regarding the use of PEA agar for anaerobes?
a. provides detection of Proteus spp.
b. provides presumptive evidence of B. fragilis grp.
c. selective medium for Fusobacterium nucleatum
d. inhibits enteric and certain facultatively anaerobic Gram-negative bacilli
20. The most common indigenous normal flora anaerobe on the skin surface is:
a. B. fragilis grp.
b. Propionibacterium acnes
c. Fusobacterium nucleatum
d. Clostridium perfringens
21. One benefit of the normal anaerobic microflora is:
a. the production of antioxidants
b. the production of vitamins and co-factors
c. a source of minerals
d. increases absorption of water
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22. Porphyromonas spp. is a:
a. Gram-negative rod, bile resistant
b. pigmented Gram-negative rod sensitive to vancomycin
c. Gram-positive non sporeforming rod with chartreuse fluorescence
d. pigmented Gram-negative rod sensitive to kanamycin
23. What anaerobe does not show red fluoresce under a Wood’s Lamp?
a. Fusobacterium nucleatum
b. some Prevotella sp.
c. Veillonella
d. Porphyromonas asaccharolyticus
24. How can you determine bile sensitivity of anaerobes?
a. sensitivity to special potency disks
b. preformed enzymatic tests
c. reaction on egg yolk medium
d. BBE medium
25. How are SPS disks used in anaerobic bacteriology?
a. selects for certain Gram-positive rods
b. identification of Clostridia perfringens
c. identification of Peptostreptococcus anaerobius
d. presumptive identification of Bacteroides fragilis grp.
26. Which of the following are the three special potency antibiotic disks for anaerobe
identification?
a. cephalotin, kanamycin, colistin
b. clindamycin, penicillin, vancomycin
c. kanamycin, colistin, vancomycin
d. penicillin, vancomycin, colistin
27. Which is not a correct principle for cost effective anaerobic bacteriology?
a. collect anaerobic specimens by swab
b. provide identification to level needed by physician
c. provide a good transport and environmental system
d. use good media, including selective and differential agar
28. Which is the correct identification profile of Bacteroides fragilis grp.?
a. resistant to all three special potency disks, resistant to bile
b. sensitive to all three special potency disks, sensitive to bile
c. sensitive to all three special potency disks, resistant to bile
d. resistant to all three special potency disks, sensitive to bile
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29. Which is the correct identification profile of Fusobacterium spp.?
a. resistant to kanamycin and colistin disks, nitrate negative
b. sensitive to kanamycin and colistin disks, nitrate positive
c. resistant to kanamycin and colistin disks, nitrate positive
d. sensitive to kanamycin and colistin disks, nitrate negative
30. Which is the correct identification profile of Propionibacterium acnes?
a. Gram-positive clubbing rod, indole negative, nitrate negative, catalase positive
b. Gram-positive clubbing rod, indole positive, nitrate negative, catalase negative
c. Gram-positive clubbing rod, indole negative, nitrate positive, catalase negative
d. Gram-positive clubbing rod, indole positive, nitrate positive, catalase positive
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CAMLT Distance Learning Course DL-974
Anaerobic Bacteriology for the Clinical Laboratory
3.0 CE Credits
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