california association for medical laboratory technology … · 2016-05-06 · © california...

CAMLT Distance Learning Course # DL-974 1© California Association for Medical Laboratory Technology

California Associationfor

Medical Laboratory Technology

Distance Learning Program

ANAEROBIC BACTERIOLOGYFOR THE CLINICAL LABORATORY

by

James I. Mangels, MA, CLS, MT(ASCP)Consultant

Microbiology Consulting ServicesSanta Rosa, CA

Course Number: DL-9743.0 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 aCA CLS Accrediting Agency (#0021)

and this course is is approved by ASCLS for the P.A.C.E. ® Program (#519)

1895 Mowry Ave, Suite 112Fremont, CA 94538-1766

Phone: 510-792-4441FAX: 510-792-3045

Notification of Distance Learning DeadlineAll continuing education units required to renew your license must be earned no later thanthe expiration date printed on your license. If some of your units are made up of DistanceLearning 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!.


OutlineA. IntroductionB. What are anaerobic bacteria? Concepts of anaerobic bacteriologyC. Why do we need to identify anaerobes?D. Normal indigenous anaerobic flora; the incidence of anaerobes at various body sitesE. Anaerobic infections; most common anaerobic infectionsF. Specimen collection and transport; acceptance and rejection criteriaG. Processing of clinical specimens

1. Microscopic examination2. Media: primary, selective, differential3. Incubation systems

H. Isolation and identification1. Provide identification to level needed by physician2. Role of Gram stain and plate morphology3. 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 andthe 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


A. INTRODUCTIONAnaerobic 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 tuboovarianabscesses, and bacteremia (1). Many reports associate an incidence of at least 50% to 60% ofimportant infections due to anaerobic bacteria (Table 1).

Anaerobic bacteria are often overlooked or missed unless the specimen is properlycollected and transported to the laboratory. Next, the specimen must be subjected to appropriateprocedures for isolation, including the use of specialized media supplemented with growthfactors and the use of proper incubation methods. Anaerobes vary in their nutritionalrequirements, but most isolates require vitamin K and hemin for growth. Anaerobes also vary intheir sensitivity to oxygen: a brief exposure (10 min.) to atmospheric oxygen is enough to killsome organisms.

This course will discuss procedures for proper collection and transport of anaerobes;appropriate specimen types for culture, processing, incubation, and isolation; and methods ofcharacterization of anaerobes from properly collected specimens. Practical schemes for isolatingthe majority of clinically important anaerobes will be described, including their salient featuresand 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, yetcost-effective procedures for the recovery and identification of clinically significant anaerobesfor 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, howeverthey 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 anabsolute anaerobic environment (zero % O2). They are killed by exposure to air after only a fewminutes. 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 amicroaerophilic atmosphere. A microaerotolerant anaerobe may marginally grow when exposedto air or in a CO2 incubator on a chocolate blood agar medium, but growth is best underanaerobic conditions.

Molecular oxygen itself can be lethal to some anaerobes, however even more toxicsubstances are produced when oxygen becomes chemically reduced. Initially, molecular oxygenis reduced to superoxide anion (O2

-), a highly reactive free radical capable of causing severedamage 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 orthe components of media on which they are to grow. Thus, oxygen, superoxide anions, hydroxylradicals, and hydrogen peroxides inhibit the growth of anaerobes and should be avoided topermit their recovery in culture.


All living creatures that use oxygen for metabolism have one or more enzymes to provideprotection from superoxide anions and their toxic derivates. These enzymes are known assuperoxide dismutases (SODs). Anaerobes have various amounts of SOD, ranging from none tosome, that presumably allow some anaerobes to tolerate oxygen. However, there is not a directcorrelation between levels of SOD and the anaerobe’s ability to tolerate oxygen. There are otherfactors, such as the presence of catalase, which may play a role in the inability of anaerobicorganisms 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), andmany anaerobes (including Bacteroides fragilis grp., the most commonly recovered anaerobe)contain virulence factors that lead to abscess formation and chronic disease if not treatedcorrectly. The recovery of anaerobes aids the physician in making a specific diagnosis and mayprovide the clinician with the potential source of the infection. Further, in this era of concernabout antibiotic resistance, the isolation and identification of anaerobes allows the clinician touse appropriate antibiotic therapy instead of the “big gun”—the antibiotic with the broadestspectrum which will inhibit both aerobes and anaerobes, but may also contribute to antibioticresistance. It has been shown that correctly employed specific therapy against anaerobes canreduce mortality and morbidity, and reduce hospitalization (1).

There are some general concepts regarding anaerobic infections that are important tomention now, but will be discussed in greater detail in this course.

• First, most anaerobic infections derive from our own indigenous microflora, sospecimen selection and collection are essential for quality results and to reducecontamination.

• Second, anaerobic infections are often mixed, containing both aerobic andanaerobic organisms. Employing an enriched primary medium as well as usingdifferential and selective media is essential to rapidly recover anaerobes fromspecimens that contain a mixture of organisms.

• Third, despite the diversity of our normal indigenous flora (1, 2, 3, 4), mostinfections are due to a relatively limited number of anaerobic isolates (Table 2):almost 35% are members of B. fragilis group; 28% are Peptostreptococcus spp. orother genera of anaerobic Gram-positive cocci; 6 % are pigmented Gram-negativerods; and 8% are Fusobacterium spp. The recovery of Clostridium spp. is onlyabout 2%.

These three concepts of anaerobic bacteriology have a profound effect on how we isolateand identify anaerobes and should be part of your thought process during this course.

D. NORMAL INDIGENOUS ANAEROBIC FLORAAlmost 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 theseregions are present in high numbers. For example, in the intestine anaerobes outnumber aerobicbacteria 1,000 to 1.


Under usual circumstances these organisms do no harm by their presence, and there isconsiderable evidence that they are actually beneficial to their host. However, in cases wherehost defenses are impaired or breaks in the normal skin or mucous membranes occur, or whenorganisms are found in normally sterile sites after trauma or surgery, these organisms are capableof producing serious infection.

Knowledge of the microflora composition at specific anatomic sites is useful for predictingthe particular organisms most apt to be involved in infectious processes that arise at or adjacentto those sites. Because some anaerobes have fairly predictable susceptibility patterns, suchknowledge may also be of value to physicians considering empiric antimicrobial therapy prior toisolation of organisms from clinical specimens and obtaining their susceptibility profile. Inaddition, the finding of site-specific organisms at a distant and/or unusual site can serve as a clueto the underlying origin of an infectious process. For example, the isolation of oral anaerobesfrom a brain abscess may suggest communication between an oral lesion and the bloodstream.

Examples of the incidence of anaerobes at various body sitesSkin: The anaerobic microflora of the skin consists primarily of bacteria within the genera

of: Propionibacterium (usually P. acnes) and Peptostreptococcus and other anaerobic Gram-positive cocci, and occasionally non-sporeforming Gram-positive bacilli in the genusEubacterium. Should a venipuncture site be inadequately disinfected before collection of aspecimen for blood culture, the specimen could become contaminated with skin flora, includinganaerobes.

Upper Respiratory Tract: In the upper respiratory tract, the number of anaerobes equals orexceeds that of aerobic organisms obtained in specimens from nasal washings, saliva, andgingival 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 lesionsinvolve 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 theirconcentrations and relative proportions vary from one microenvironment to another. Most oftenFusobacterium spp., Porphyromonas spp., Prevotella spp., anaerobic Gram-positive cocci,Propionibacterium spp., Eubacterium spp., Lactobacillus spp. and Actinomyces spp. arerecovered from the oral cavity. Therefore, these particular anaerobes should be suspected asparticipants in any infectious process from the respiratory tract.

Vagina: About 50% of the bacteria in cervical and vaginal secretions are anaerobes, themost common being anaerobic Gram-positive cocci, Prevotella bivia, and Prevotella disiens,some anaerobic lactobacilli, and Actinomyces spp. Other anaerobic organisms such asClostridium spp., Eubacterium spp., B. fragilis grp., Porphyromonas spp., and others may befound in the indigenous microflora of the vagina because of its proximity to the anus. P. biviaand P. disiens tend to dominate among the Gram-negative rods, but pigmented anaerobic Gram-negative bacilli, the B. fragilis group, and other Prevotella and Bacteroides species may berecovered as well.

Whenever anaerobes are recovered from vaginal and cervical swabs, neither themicrobiologist nor the physician can distinguish the indigenous microflora contaminants fromorganisms actually contributing to the patient’s infectious process. For this reason, genitourinarytract swabs, including swabs of the vagina and cervix, are unacceptable for anaerobicbacteriology.


Intestine: Studies concerning the microflora of the intestine have found that anaerobesoutnumber aerobes by a factor of 1,000 to 1. Anaerobes occurring in the highest numbers inintestinal flora are B. fragilis grp., Bifidobacterium, Clostridium, Eubacterium, Lactobacillus,Peptostreptococcus and other anaerobic Gram-positive cocci, Prevotella spp., Porphyromonasspp., and others. Intestine, intestinal contents, bowel, and other material such as rectal abscess,may be unacceptable specimens unless collected properly to avoid the normal anaerobicindigenous flora. The distal ileum may have counts of 104 to 105 colony forming units (CFU)/mland both coliforms and various anaerobes may be encountered. In the distal colon, total bacterialcounts average 1011 to 1012 CFU/g of feces, with anaerobes outnumbering the aerobes. Withinthe B. fragilis group, the species that is most prevalent in the indigenous flora of the intestine isBacteroides thetaiotaomicron.Beneficial Aspects of Indigenous Anaerobes

Many anaerobes of the indigenous microflora are beneficial and play an active role inmaintaining the health of humans and other animals. Anaerobes, together with othermicroorganisms, provide a natural barrier to colonization of mucous membranes by pathogenicorganisms. 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 systemduring early development of neonates (1).

E. ANAEROBIC INFECTIONSAnaerobes are key pathogens in brain abscess, oral/dental infections, aspiration

pneumonia, lung abscess, pelvic and abdominal infections, and soft tissue infections, but theymay cause any type of infection (Table 1). In a number of infections, anaerobic bacteria are thepredominant pathogen; in other infections they are often mixed with aerobic organisms and witha 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 clinicalhints may suggest the presence of anaerobes in a clinical specimen (1):

1. Foul odor of specimen2. Location of infection in proximity to a mucosal surface3. Infections secondary to human or animal bite4. Gas in specimen5. Previous antibiotic therapy with aminoglycoside antibiotics that may have failed6. Tissue necrosis; abscess formation7. Unique morphology on Gram stain8. Failure of culture to grow aerobically when organisms were observed on original

Gram stainBacteroides fragilis grp. (34%), followed by anaerobic Gram-positive cocci (28%),

pigmented Gram-negative rods (Prevotella and Porphyromonas) (6.4%), and Fusobacteriumspp. (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 highlyenriched media, and its need of good transport and anaerobic environmental conditions,laboratories may recover this group even if they use generally poor techniques. The anaerobicGram-positive cocci, pigmented Gram-negative rods, and Fusobacterium spp., however, are


much more demanding and many laboratories do not frequently recover these organisms despitetheir reported high incidence (3). See Table 2.

F. SPECIMEN COLLECTION AND TRANSPORTProper collection of specimens and prompt transport to the laboratory for processing are

imperatives. Specimens must be collected in a manner that will avoid contamination withindigenous flora. The laboratory must reject specimens that have not been collected ortransported 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 hasbeen improperly obtained or improperly transported, it may not provide information to theclinician, and the laboratory may expend useless time and resources on an unsatisfactoryspecimen. Indigenous anaerobes are often present in such large numbers on mucosal sites(gastrointestinal, genital tract, oral cavity), that even minimal contamination with indigenousflora 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 collectedspecimen to the laboratory in an approved anaerobic transport system, and that some specimensmay not be appropriate for anaerobic culture and may be rejected (5). Rejection of a clinicalspecimen can be a touchy subject to many clinicians. It works best to have meetings withphysicians and nurses prior to the initiation of any policy to reject specimens to explain rationaleand seek buy-in. Work with specific departments or physicians (surgery, OB, medicine,Pathologists, and Infectious Disease physician if your hospital has one) to explain informationabout the extent of normal anaerobic flora, contamination, and requirements to adequately isolateanaerobes. The clinical staff will understand that a quality specimen will reduce treatmentdelays and costs associated with working up improper specimens. Nurses in OR, ER, and ICUcan be particularly helpful because they often see the patient one-on-one and frequently obtainspecimens for culture. Patient care units, clinics, OR, and emergency rooms must be suppliedwith appropriate collection devices and complete instructions for their use. Goodcommunication between the clinical microbiology laboratory and the clinical staff will ensure thecollection 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 ora tissue sample (Table 3). Aspirated fluid collected by needle and syringe can be expelled inoxygen-free tubes or vials (Anaerobe Systems, BD, Hardy, Fisher Healthcare, and Remel) andthen promptly transported to the laboratory. Aspirated material should never be transported inthe syringe. Tissue samples or biopsy material are very satisfactory specimens and can be placedinto oxygen-free tubes or vials for immediate transport to the laboratory (5). All specimensshould be transported and held at room temperature. Do not place the transported specimen inthe incubator or in the refrigerator; incubator temperatures will cause overgrowth of somebacteria and loss of isolates, and cold temperatures will allow increased oxygen diffusion.Anaerobic transport vials may contain modified Cary-Blair or other media that containsubstances to scavenge excess oxygen (Anaerobe Systems, BD, Hardy, Fisher Healthcare, andRemel) and provide some moisture to the specimen.


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 thelaboratory at convenient times throughout the day without jeopardizing the recovery ofanaerobes. Purulent specimens contain numerous reducing compounds that also help protectanaerobes from the effects of oxygen.Least Desirable Specimens

The least desirable specimen for anaerobes is one collected by swab, and it should not becultured, even though swabs are the predominant specimen type collected by medical/nursingpersonnel. Many laboratories commonly reject swab specimens for anaerobic culture.Generally, the specimen volume when collected by a swab is small, reducing the probability ofisolating organisms. The specimen may be easily contaminated during collection. Manyorganisms adhere to the fibers of the swab and therefore are not recovered. Further, swabspecimens commonly produce smears of poor Gram stain quality, and the inherent dryness of aswab specimen reduces the viability of many anaerobes. If collecting a specimen by swab isunavoidable and is absolutely necessary, then collect as much specimen as possible and use acommercially available anaerobe transport swab system (Anaerobe Systems, BD, Copan, FisherHealthcare, and Remel). Take special care to sample the active site of infection to preventcontamination, and then place the swab deep into the agar butt. Break the stick off below whereit was handled and replace the cap quickly. The commercial anaerobe transport system thatcontains two glass tubes (tube within a tube) for swab specimens has been shown not to bereliable. Remember that if you supply only a swab anaerobic collection device to themedical/nursing units, you will certainly receive a swab back. Get around this by consistentlyproviding transport systems for collecting fluid or tissue. See Table 3 for appropriate specimensfor anaerobic culture.

G. PROCESSING CLINICAL SPECIMENS FOR ANAEROBIC CULTUREIdeally, 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 cannotbe inoculated onto media and placed immediately into an anaerobic atmosphere, it is best to holdspecimens 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 recoveryof anaerobes or their viability. Batch processing of media inoculation at convenient times ispreferred to processing specimens one at a time, which would require opening an anaerobicincubation 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 ofthe 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) onthe anaerobic transport vial to ensure even distribution of microorganisms. You may need togrind bone or tissue with thioglycollate (THIO) or chopped meat broth to permit inoculation ofspecimen onto solid media. Swab specimens (should you accept one) may require the additionof THIO or chopped meat broth to make a liquid specimen. Large volume specimens mayrequire centrifugation to produce the sediment needed to inoculate media and prepare a Gramstain.


Once the specimen has been prepared for culture, it should be inoculated onto theappropriate anaerobic media, placed in a liquid back-up broth, and onto a glass slide for a Gramstain. Once you begin processing the sample, you should complete it as quickly as possible, atleast within 15 minutes.Microscopic Examination

Always prepare a direct Gram stain from the clinical material. This is very important, forit often allows early presumptive evidence of the presence of anaerobes and provides informationabout the quality of the specimen. Direct smears for anaerobes are best fixed in absolutemethanol for 1 min, and then stained by standard Gram stain procedure and reagents (5). Evengentle 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 torecover all the morphotypes seen on the direct Gram stain smear may indicate a problem inspecimen collection, transportation or processing, or another problem that inhibited the growth ofanaerobic 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 mayappear Gram-negative with the same cell morphology as the Gram-positive rods.

2. Gram-negative coccobacillary forms suggest Prevotella group or Porphyromonasgroup.

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 failto grow or yield inconclusive results and then have to be repeated. Use a highly enriched basalmedium for primary isolation, such as Brucella medium containing vitamin K and hemin, whichwill support the growth of all anaerobes and aerobes. It has been shown that a PRAS (pre-reduced anaerobically sterilized) medium gives a faster growth rate and the ability to recovermore anaerobes within a shorter period of time. Anaerobe Systems is the sole source of PRASmedia, 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 maydamage components of the media and prevent the growth of anaerobes. PRAS media also have aprolonged shelf life compared to other anaerobic media.

Other manufacturers produce media for anaerobes that require pre-reduction (placing inanaerobic environment for 24 hrs. before use) or media that contain oxygen-scavengingsubstances (Oxyrase) or other reducing substances. It is best to perform side-by-side comparisontesting 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 acombination of selective and differential media for the recovery of anaerobes and forpresumptive identification (2). The following media are suggested for the isolation of anaerobicbacteria 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 andaerobic organisms.


• Phenylethyl alcohol-sheep blood agar (PEA) for the inhibition of enteric and certain otherfacultatively anaerobic Gram-negative bacilli that may overgrow anaerobes. PEA alsoreduces the spreading or swarming characteristic of certain Clostridium spp.

• Kanamycin-vancomycin-laked blood agar (KVLB or LKV) for the selection ofpigmented Prevotella and other Bacteroides spp.

• Bacteroides bile esculin agar (BBE) for the selection and presumptive identification ofBacteroides fragilis grp. and Bilophila wadsworthia. Fusobacterium mortiferum/variumgrp. is also resistant to bile and may occasionally grow on this medium.

• Thioglycollate medium without indicator, supplemented with vitamin K1, hemin, and amarble chip, for enrichment and back-up culture. Chopped meat broth with vitamin K1and hemin may also be used. Use either of these broths as backup only. If primary platesare positive, you may discard the backup broth. Do not subculture the broth. Subculturethe backup broth only if primary plates are negative and the broth is turbid.

Anaerobic Incubation SystemsThe 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/orreceives a rare anaerobic specimen after normal laboratory hours) may consider the use ofanaerobic bags or pouches. A clinical laboratory that receives perhaps 2-4 specimens per day foranaerobic culture may consider the use of anaerobic jars. The use of anaerobic jars may beeconomically employed if the laboratory batches the processing of specimens at convenienttimes rather than using one jar for one specimen. If the laboratory receives a specimen at oddtimes after jars have been closed, perhaps the new specimen may be incubated in a pouch or bagand then after 48 hrs. included in an anaerobic jar. A laboratory that may receive 3 or 4 or morespecimens per day should consider using an anaerobic chamber, the most economical way ofproducing an anaerobic atmosphere. The laboratory would need to consider the initial expenseand the space required for the chamber. The ability to examine cultures at 24 hrs. and report thepresence of anaerobes earlier (compared to jar and bag systems) may also be a patient-carebenefit for the hospital.

Whichever anaerobic system you use, the first step is to immediately place the inoculatedplates into the anaerobic environment, and incubate them at 35 to 37ºC for 24-48 hrs. Growingcultures must not be exposed to oxygen until after 48 hrs. of incubation in an anaerobic jar orpouch system, since anaerobes are most sensitive to oxygen during their log (early) phase ofgrowth. 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 resazurinchemical indicators. These indicators, initially blue and pink (respectively), change to colorlesswith low concentrations of oxygen.

The following is a more detailed description of the most common choices of anaerobicincubation systems.Anaerobic bag or pouch

Some anaerobic bag or pouch systems use a sachet that absorbs atmospheric oxygen withoutthe generation of hydrogen, without the addition of water, and without requiring a catalyst. Theresulting carbon dioxide level in these systems is generally higher than 10%. In other bag orpouch anaerobic atmospheric producing systems, a gas-generating envelope or ampoule provides


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 timethey are opened. There are some gas-generating systems that have a catalyst incorporated intothe 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 byusing special clamps. Check the anaerobic indicator through the clear plastic bag after a fewhours to see that the bag has not leaked. Incubate the bag at 35 to 37ºC in a standard incubatorfor 48 hrs. Examining plates before 48 hrs. is not recommended since any small colonies areparticularly susceptible to oxygen exposure at this stage and may not survive. At 48 hrs., removethe plates from the bag to examine them and work up the organisms as quickly as possible (thisprocess 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 donot take up a lot of space. However, the bags occasionally leak, and they are the most expensiveway 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 gas-generating system (usually an envelope, ampoule, or sachet) providing an atmosphere of 80 to90% nitrogen (N2), 5% hydrogen (H2), and 5 to 10% carbon dioxide (CO2); and an anaerobicindicator. If a sachet is employed, hydrogen is not produced and a catalyst is not required. (SeeAnaerobic Bag or Pouch from above).

For most jar systems, the procedure is the same. Place the inoculated plates into the jar, addan 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. Thisprevents exposure of smaller colonies to oxygen. The catalyst, composed of palladium-coatedalumina pellets, should be fresh or rejuvenated each time the jar is opened prior to use, unless thecatalyst 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. Adda new generating envelope, ampoule, or sachet system and reseal the jar. (BD DiagnosticSystems, Hardy Diagnostics, PML Microbiologicals, and Remel). The recovery of anaerobes inan anaerobic jar compares well to an anaerobic chamber if the plates are continuously incubatedfor 48 hrs. Jars do not recover anaerobes well if plates are incubated for only 24 hrs. prior toinitial examination.Anaerobic chamber

Anaerobic conditions are maintained in a gas-tight box or chamber by a gas mixturecontaining 80-90% nitrogen (N2), 5 % hydrogen (H2), and 5 to 10% carbon dioxide (CO2), andusing a palladium catalyst. The hydrogen concentration should not exceed 5% to preventhazardous conditions.

Usually anaerobic chambers have a positive pressure inside to prevent oxygen fromcoming 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 manyanaerobes require it for growth. Humidity is controlled by using silica gel crystals to absorb thewater formed in the catalytic conversion process. In other chambers, humidity is controlled witha “cold spot” that condenses excess humidity and allows the water formed to be removedthrough a drain. Plates are incubated at 35 to 37ºC and can be examined at any time within the


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 ANAEROBESIsolation:

After the plates—primary Brucella, PEA, BBE and LKU—have been incubated in ananaerobic 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 facultativeanaerobes (organisms that grow under either aerobic or anaerobic conditions) andmicroaerophilic organisms (organisms that grow in an atmosphere of reduced oxygen tension).Remember that facultative anaerobes and microaerophilic organisms will grow under anaerobicconditions, so you will need to exclude these from your workup. To determine which isolatesfrom the primary Brucella medium are anaerobes, test the organisms for aerotolerance using twomedia: Brucella agar incubated anaerobically, and chocolate blood agar incubated under 5-10%CO2 conditions. The facultative anaerobes and the microaerophilic organisms will grow on boththe 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 thechocolate blood agar.

Chocolate blood agar must be used for aerotolerance testing. You may incorrectly assumethat 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 Haemophilusspp., 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 plateincubated anaerobically, and do a Gram stain as well. The disks will help you identify theorganism 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 fasteridentification 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 bloodagar plate. Pick and subculture any colonies on the PEA, BBE and LKV plates that appeardifferent 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 betested for aerotolerance. Streak the Brucella agar plate for isolation. Label the Brucella plateand 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 Brucellamedium. Facultative and anaerobic bacteria may have similar colony appearances, so youneed to work up all colonial morphotypes you see on the primary media.

5. Incubate the Brucella plate anaerobically. Incubate the chocolate blood plate in anatmosphere of 5-10% CO2.

6. Observe after 24 hrs. Anaerobic organisms will grow only on the Brucella mediumincubated anaerobically, facultative anaerobes will grow on both the Brucella and chocolateblood agar.


7. Record a detailed description of each colony type from the anaerobic primary Brucellamedium that does not grow on chocolate. Describe such characteristics as pitting, swarming,hemolysis, pigment, “greening” of the medium, etc. These colony characteristics can provideclues to identify the isolates when used in conjunction with Gram stain and rapididentification tests (explained in the next section). (See Table 4. Anaerobic OrganismIdentification 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 Gramreaction of the isolate, you are ready to begin identification of the isolate. The extent ofidentification required may vary according to the type of isolate, the source of the specimen, theneeds of the physician, the clinical need, the patient’s type of illness, and the operational andfinancial issues of the laboratory.

In general, there are three different methods that can allow rapid and cost-efficientidentification of anaerobic isolates: Method 1: presumptive and preliminary grouping usingGram 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 anddisk tests; and Method 3: the use of commercially available identification systems. Theidentification 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 Method2 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 anaerobicisolate based upon the Gram stain and colonial morphology (See Table 4). Begin theidentification process by describing the colonial morphology in detail, including colony size,shape, edge, opacity, color and any other distinctive characteristic. Describe cellularmorphology, including size, shape, and Gram reaction. Examine colonies for hemolysis onBrucella agar. Examine colonies for pigment on Brucella or LKV. Test colonies forfluorescence 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 inseparating different anaerobic species and genera (See Table 5). Generally, Gram-positiveorganisms are sensitive to vancomycin and resistant to colistin, whereas the Gram-negativeorganisms are resistant to the vancomycin disk and variable to colistin. The special potencyantibiotic disks test is especially helpful with those clostridia that consistently stain Gram-negative, since their susceptibility to vancomycin disk confirms their “true” Gram reaction.

Place special-potency antibiotic disks of vancomycin, kanamycin, and colistin on aBrucella agar plate. If you know the organism is Gram-negative, also add a nitrate disk to theheavily inoculated section. Special potency antibiotic disks are not needed when the organismstains Gram-positive because they will all be vancomycin susceptible, and the colistin andkanamycin do not provide additional information on Gram-positive organisms.


After 24 hrs. anaerobic incubation, use the results obtained with special-potencyantibiotic disks for grouping or species identification (See Table 5). Examples of identificationof 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 nitratereductase positive.

3. Fusobacterium sp. are susceptible to special potency disks kanamycin and colistin, andresistant to vancomycin. These organisms are nitrate reductase negative.

4. Porphyromonas sp. are resistant to special potency disks kanamycin and colistin, and aresusceptible to vancomycin. They produce a black pigment.

5. Prevotella sp. are resistant to special potency disks kanamycin and vancomycin, and vary intheir susceptibility toward colistin. Some Prevotella sp. may have a special antibiotic diskpattern typical of the B. fragilis grp. (RRR), but these organisms do not grow in 20% bile oron BBE.

6. Bilophila sp. are susceptible to special potency disks kanamycin and colistin and are resistantto vancomycin. Phenotypically this organism resembles the B. ureolyticus group and someFusobacterium sp. These organisms can be distinguished by their strong positive catalasereaction and resistance to 20% bile. In 3 to 4 days Bilophila wadswortha forms smallcolonies 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 mayuse other rapid tests to assist in anaerobe identification. One such rapid test is determining thefluorescence of anaerobes using a Woods Lamp at 366 nm. The presence and color offluorescing colonies can aid in the rapid detection and presumptive identification of certainanaerobic 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 isolateis a Gram-negative rod, use Table 8; if isolate is a Gram-positive rod with spores (Clostridiumspp.), 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 canuse the tables in this course or others listed in the Wadsworth KTL Anaerobic BacteriologyManual (2) or Clinical Microbiology Procedures Handbook (5). When typical morphology (celland colony) is apparent and is combined with rapid tests, the resulting preliminary identificationmay 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 distinctgroups: one genus of spore-formers (Clostridium spp.). and five genera of non-sporeformers(Actinomyces, Bifidobacterium, Eubacterium, Lactobacillus, and Propionibacterium). Theanaerobic 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 isrelatively 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 foridentification of some commonly isolated Clostridium spp. Clostridium spp. can cause acute,


severe, or chronic infections. Some Clostridium spp. are highly pathogenic or toxigenic, whileothers are rarely pathogenic. Some species are resistant to antimicrobial agents. A great sourceof confusion is that many Clostridium spp., and occasionally the non-sporeforming genera aswell, can stain Gram-negative. The use of the special antibiotic disks can help resolve thisproblem. 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 non-sporeforming bacilli (Actinomyces spp., Lactobacillus spp., and Propionibacterium spp.).

The identification of the anaerobic non-sporeforming Gram-positive bacilli can be achallenge for the Clinical Laboratory Scientist. In many instances the use of PRASbiochemicals, gas-liquid chromatography (GLC) and fatty acid analysis is necessary. Manylaboratories 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 fromeach other by their metabolic end products detected by GLC. The group is resistant to specialpotency disk of colistin, variable to kanamycin, and generally susceptible to vancomycin.However, there are rare strains of Lactobacillus and Clostridium spp. that may be vancomycinresistant (2).

The clinical laboratory may encounter Propionibacterium acnes occasionally from ablood culture and from wound sources as contaminants. However, these organisms have beenreported as causing chronic disease, so you need to rule this out before discarding the organismas 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, hybridizationdata, and cellular fatty acid profiles has permitted significant changes and reclassification amongspecies that were at one time in the genus Peptostreptococcus. Peptostreptococcushydrogenalis is now Anaerococcus hydrogenalis; Peptostreptococcus prevotii is nowAnaerococcus prevotti; Peptostreptococcus magnus is now Finegoldia magna;Peptostreptococcus micros is now Parvimonas micra , Peptostreptoccus asaccharolyticus is nowPeptoniphilus asaccharolyticus; Peptostreptococcus indolicus is now Peptoniphilus indolicus.The good news is that Peptostreptococcus anaerobius has not changed its name and issusceptible to the SPS (sodium polyanethanol sulfonate) disk which is useful for its rapididentification. Anaerobic cocci can be identified by Gram stain, colony morphology, spot testssuch as SPS disk and spot indole, and various biochemical preformed enzymatic reactions andcommercial systems (See Table 10). In some instances, PRAS biochemical, GLC, or fatty acidanalysis may be necessary for identification.Method 2: Rapid biochemical tests for identification

Many anaerobic isolates may be further identified using a variety of commerciallyavailable preformed-enzyme tests in conjunction with some of the rapid spot tests previouslydescribed in this course. Individual enzymatic biochemical tests may permit anaerobeidentification without excessive expense or delay. One example is the identification of somespecies of anaerobic Gram-positive cocci using the alkaline phosphatase enzyme test.

The combination rapid enzymatic tests are simple to perform and can be purchasedallowing two or more enzymatic tests to be performed in a single tube to detect enzymaticactivity visible by color change, or by detecting 4-methylumbelliferone fluorescent end productswhen 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 theinoculum, 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


this course, as well as in references # 2 and 5. Additional identification tables and informationare available from the manufacturer. (WeeTabs Package Insert. Key Scientific CorporationStamford, TX. www.keyscientific.com). Other rapid enzymatic test tablets are available fromRosco Diagnostica, Taastrup, Denmark.Method 3: Rapid Identification System Kits

Identification of anaerobes can be accomplished with commercially availablemicrosystem kits for the detection of preformed enzymes within a few hours followinginoculation: Vitek 2 ANI Anaerobe Card and Rapid ID 32A (bioMerieux, Inc.); Rapid AnaerobeID (Dade MicroScan); Crystal Anaerobe ID kit (BD Bioscience); and RapID-ANA (Remel).The systems allow the identification of many species not identified by previously describedmethods. The systems require 4 hrs. of aerobic incubation at 35º C. Each system has its owndatabase permitting identification. Tables in this course or other texts should not be used foridentification. These systems will not be discussed in any detail in this course. See themanufacturer’s insert for more details. Each system varies with specific QC, inoculum size, andtest procedures, including recommended media. The user needs to follow the manufacturer’srecommendations carefully. There are some distinct advantages and disadvantages of usingthese 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 otherconventional information, such as Gram stain, colonial morphology, and organism growthcharacteristics. Special potency antibiotic disks and other spot presumptive tests can be veryuseful in verifying and confirming the identification obtained using these kits. Results of allreactions must be considered. Do not automatically accept any answer from any identificationkit without comparing results to other methods described in this course. Keep in mind that eachidentification using these commercial systems costs about $6.00.

There is one caveat: As with aerobic identification systems, it is often difficult for themanufacturer of anaerobic identification systems to keep up with the explosion of taxonomicname changes and the need for additional biochemical tests. Often the name listed by themanufacturer for identification may be out-of-date and you may need to change the identificationaccordingly.

I. METHODS FOR COST EFFECTIVE ANAEROBIC BACTERIOLOGYMethods for cost effective anaerobic bacteriology depend upon the following:

1. Accept only appropriate specimens. Educate the clinical staff so they are aware of whatspecimens are appropriate and how to collect and transport specimens for anaerobes. It allbegins here: if you receive a bad specimen that is contaminated and that is transportedincorrectly, 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 goodprimary, selective, and differential media. It may seem that you are spending too muchmoney on media, but good media will save you time and expense in the long run. Poormedia results in poor growth or growth that is delayed, which may mean the laboratoryfinally 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 processingat convenient times and reduces the cost of setting up anaerobic cultures and improves theefficiency of the laboratory.

4. Provide rapid identification to the level needed by the physician to make a diagnosis and toguide appropriate therapy. You may not need to identify the isolate to its exact genus andspecies to enable the physician to treat the patient correctly. Costs can be controlled simply


by identifying an organism according to the physician’s needs, and to the extent determinedby the specimen source and the type of organism recovered. Many laboratories do this nowwith aerobic organisms by having abbreviated identification systems for swarming Proteusspp., lactose fermenting organisms from MacConkey, etc. The same practice should apply toanaerobic organisms as well.

5. Use rapid, spot and presumptive tests as needed. The rapid tests may permit earlyidentification that may allow the physician to use appropriate therapy, and the cost of theidentification will be about 50¢. Use commercial identification kits wisely—remember theycost $6.00 each.

6. Finally, communicate with the physician frequently. CLSs don’t often like to do this, but bycommunicating with the physician you will be able to determine what his/her needs are, andwhat 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 inappropriatelylabeled and was really obtained from a superficial wound and further workup can stop. Youneed 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 rapidlyisolate and identify anaerobes in a cost-efficient manner.

J. REFERENCES1. 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 ClinicalMicrobiology, 7th ed. Washington, DC: ASM Press; 1999.

4. Engelkirk PG, Duben-Engelkirk J, Dowell VR, Jr. Principles and Practice of ClinicalAnaerobic Bacteriology. Belmont, CA: Star Publishing Co.; 1992.

5. Mangels JI, ed. Section 4, Anaerobic Bacteriology. In: Isenberg H. Clinical MicrobiologyProcedures Handbook. 2nd ed. Washington, DC: ASM Press; 2004.


Table 1. Incidence of anaerobic bacteria in various infectionsType of Infection Incidence (%) of anaerobic bacteriaCentral Nervous System

Brain abscess 89Head and Neck

Chronic sinusitisChronic otitis mediaPeriodontal abscessOther oral infections

5030-60100

94-100Pleuropulmonary

Aspiration pneumoniaLung abscessNecrotizing pneumoniaEmpyema

85-90938576

Intra-abdominalPeritonitisLiver abscess

90-95>50

Female Genital TractSalpingitis, pelvic peritonitisTubo-ovarian abscessVulvovaginal abscessSeptic abortion

>55927473

Soft TissueGas gangrene (myonecrosis) 100

Adapted from: Manual of Clinical Microbiology, ASM Press, 5th Edition

Table 2. Incidence of anaerobic bacteria in clinical specimensOrganism No. of isolates % of all anaerobes recoveredBacteriodes fragilis grp.

B. fragilisB. thetaiotaomicronB. vulgatusB. distasonisB. ovatusUnidentified

141771210106

23

34.819.03.02.42.41.55.7

Pigmented GNR 26 6.4Other 45 11.1Fusobacterium spp. 32 7.9Peptostreptococcus spp. 117 28.9Clostridium spp. 9 2.2Non-sporeforming GPB 20 4.9Gram-negative cocci 15 3.7

Adapted from: Manual of Clinical Microbiology, ASM Press, 5th Edition.GNR = Gram-negative rodsGPB = Gram-positive bacilli


Table 3. Specimen Types for Anaerobic CultureAcceptable Not Acceptable

Abscess Cervical or vaginal secretionsDeep Wounds Sputum, throat, naso-pharyngeal

Body fluid FecesTissue Gingival material

Catheterized urine Small bowel contentsNormally sterile site Gastric contents

Lung Superficial skin lesionsAspirate Ulcers

Voided urineSurface wounds

Bronchial washings (except by doublelumen catheter)

Table 4. Anaerobic Organism Identification Clues from Colony MorphologyColony morphology Possible identificationAgar 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


Table 5. Identification by means of special-potency antibiotic disksResponse to antibiotic diska:

Organism Kanamycin 1,000 µg Vancomycin 5 µg Colistin 10 µgGram-positive Sb 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 AnaerobesOrganism ColorPorphyromonas 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


Table 7. Tests for Rapid Identification of AnaerobesTest Principle of useSpecial 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 hydrogenperoxide is preferred.

SPS disk Sodium polyanethanol sulfanate. Used to differentiatePeptostreptococcus 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 thepresence of bile.

Fluorescense Some anaerobes are capable of fluorescing different colors whenexposed to UV light (Woods Lamp 366nm).

Lipase Fats in egg yolk medium are broken down by lipase enzyme and appearas a surface iridescent layer. F. necrophorum is lipase positive.

Lecithinase Lecithin in egg yolk medium is split by lecithinase enzyme resulting inopaque halo surrounding an organism. C. perfringens is lecithinasepositive.

Pigment production Some anaerobic gram-negative rods, namely Porphyromonas spp. andsome Prevotella, produce a dark pigment on sheep or rabbit blood agarmedia. 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 yellowto red. B. ureolyticus is positive.


REVIEW QUESTIONSCourse DL-974Choose the one best answer

1. Anaerobic bacteria are generally not involved with one of the following types of infection:a. appendicitisb. bacteremiac. bladder infectiond. liver abscess

2. Which statement best describes superoxide anions?a. causes damage to media, bacterial cell walls and enzyme systemsb. promotes growth of anaerobesc. causes damage to RNAd. neutralizes oxygen

3. An example of an appropriate specimen for anaerobic culture is:a. voided urineb. vaginal swabc. lung tissued. superficial wounds

4. The common indigenous anaerobic flora of the oral cavity does not include:a. anaerobic Gram-positive coccib. Actinomyces spp.c. Porphyromonas spp.d. Clostridium spp.

5. The most frequently isolated anaerobe from anaerobic infections is:a. Propionibacterium acnesb. 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 possibleanaerobic infection?a. location of infection in proximity to mucoid surfaceb. vomitingc. abscess formationd. secondary to human or animal bite

7. What is an important reason to identify anaerobes from clinical specimens?a. commonly resistant to empiric antibiotic therapyb. risk to health care workersc. provide documentation in the event of legal actiond. improves use of CPT codes


8. Which of the following are Gram stain clues for the presence of Bacteroides fragilis grp.?a. Gram-negative rod with tapered endsb. pale staining pleomorphic Gram-negative rods often with vacuolesc. pleomorphic Gram-positive coccobacillid. large Gram-positive box car shaped rods

9. To best monitor an anaerobic environment, which chemical indicator should be used?a. congo redb. crystal violetc. safranind. methylene blue

10. The primary goal of using selective and differential media for the recovery of anaerobesincludes:a. early detection and recovery of clinically important isolatesb. improves the growth of clostridiac. decreases need for quality controld. decreases need for aerotolerance testing

11. Which one of the following is necessary for aerotolerance testing of clinical isolates?a. BBE agarb. chocolate agarc. use of strict anaerobic conditionsd. 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 morphologyb. provides early clues to susceptibility testingc. determines true Gram stain reactiond. provides information concerning obligate anaerobes

13. The term PRAS media stands for:a. pre reductive anaerobically sensitive mediab. post reduction aerobically sterilized mediac. pre reduced anaerobically sterilized mediad. 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 formationd. prevents swarming of Clostridium spp.


15. What is the correct reason swab specimens are an inferior specimen type and should not beused?a. excessive moisture associated with swabs, easy to collect, hard to contaminateb. difficult to inoculate media, easy to contaminate, infection control principlesc. difficult to use, easy to inoculate media, hard to contaminated. 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 perfringensc. Porphyromonas spp.d. Propionibacterium acnes

17. What is an example of moderate anaerobe?a. Peptostreptococcus anaerobiusb. Bacteroides fragilis grp.c. Fusobacterium nucleatumd. Clostridium tertium

18. The term SOD means:a. superoxide dismutaseb. sensitive oxide dimerc. superoxide dimerd. 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 nucleatumd. 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 acnesc. Fusobacterium nucleatumd. Clostridium perfringens

21. One benefit of the normal anaerobic microflora is:a. the production of antioxidantsb. the production of vitamins and co-factorsc. a source of mineralsd. increases absorption of water


22. Porphyromonas spp. is a:a. Gram-negative rod, bile resistantb. pigmented Gram-negative rod sensitive to vancomycinc. Gram-positive non sporeforming rod with chartreuse fluorescenced. pigmented Gram-negative rod sensitive to kanamycin

23. What anaerobe does not show red fluoresce under a Wood’s Lamp?a. Fusobacterium nucleatumb. some Prevotella sp.c. Veillonellad. Porphyromonas asaccharolyticus

24. How can you determine bile sensitivity of anaerobes?a. sensitivity to special potency disksb. preformed enzymatic testsc. reaction on egg yolk mediumd. BBE medium

25. How are SPS disks used in anaerobic bacteriology?a. selects for certain Gram-positive rodsb. identification of Clostridia perfringensc. identification of Peptostreptococcus anaerobiusd. presumptive identification of Bacteroides fragilis grp.

26. Which of the following are the three special potency antibiotic disks for anaerobeidentification?a. cephalotin, kanamycin, colistinb. clindamycin, penicillin, vancomycinc. kanamycin, colistin, vancomycind. penicillin, vancomycin, colistin

27. Which is not a correct principle for cost effective anaerobic bacteriology?a. collect anaerobic specimens by swabb. provide identification to level needed by physicianc. provide a good transport and environmental systemd. 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 bileb. sensitive to all three special potency disks, sensitive to bilec. sensitive to all three special potency disks, resistant to biled. resistant to all three special potency disks, sensitive to bile


29. Which is the correct identification profile of Fusobacterium spp.?a. resistant to kanamycin and colistin disks, nitrate negativeb. sensitive to kanamycin and colistin disks, nitrate positivec. resistant to kanamycin and colistin disks, nitrate positived. 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 positiveb. Gram-positive clubbing rod, indole positive, nitrate negative, catalase negativec. Gram-positive clubbing rod, indole negative, nitrate positive, catalase negatived. Gram-positive clubbing rod, indole positive, nitrate positive, catalase positive


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