4: Rapid Devices and Instruments for the Identification of Anaerobic Bacteria

Rapid Devices and Instruments for the Identification of Anaerobic Bacteria

Christopher L. Emery1, Maria D. Appleman2, Jean A. Siders3, and Thomas E. Davis3

1 Hahnemann University Hospital, Philadelphia, PA, USA

2 Microbiology Consultants, Inc., Dana Point, CA, USA

3 Indiana University, Indianapolis, IN, USA

4.1 Introduction and clinical considerations

From a practical standpoint, anaerobic bacteria are defined here as those bacteria that do not multiply on the surface of nutritionally adequate solid media incubated in air or in a CO2 incubator (10% CO2) but grow in environments without O2. They do not use molecular oxygen as a terminal electron acceptor. While many ferment a wide variety of carbohydrates, many others do not (i.e., they are asaccharolytic), obtaining their energy through fermentation of amino acids or various other organic compounds. Vegetative cells of anaerobic bacteria, but not the spores of clostridia, are killed by exposure to molecular oxygen in ambient air. However, as can be shown by aerotolerance testing, anaerobes vary considerably in their sensitivity or ability to tolerate oxygen [132, 140]. Some, referred to as “moderate obligate anaerobes (e.g., Bacteroides fragilis and Clostridium perfringens), ” will tolerate some oxygen exposure while others, referred to as “strict obligate anaerobes (e.g., Clostridium novyi type B and Clostridium hemolyticum),” cannot survive exposure to oxygen longer than a few minutes, and lastly, the “aerotolerant anaerobes” will have scant growth in the presence of O2 (e.g., Clostridium tertium, Clostridium carnis, and C. histolyticum). To successfully isolate and identify anaerobes in a specimen, the entire process is ideally done entirely under anaerobic conditions but if an anaerobic chamber is not available, the process should be done rapidly to minimize oxygen exposure.

There have been extensive changes in the taxonomic classification of anaerobes in recent years based on molecular genetic studies. The standard reference on bacterial taxonomy, Bergey’s Manual of Systematic Bacteriology, is organized using 16S rRNA gene sequence analysis [83]. Current genera with descriptions are available on-line: List of Prokaryotic Names with Standing in Nomenclature (http://www.bacterio.cict.fr/classifgenrafamilies.html). Summaries of recent taxonomic changes regarding anaerobic bacteria are available elsewhere [35, 74, 133, 137]. Keeping up with the many changes in the nomenclature and classification of anaerobes, with increased numbers of new genera and species in recent years, has been a challenge for manufacturers of commercial identification systems, as well as clinical microbiologists and clinicians alike [28, 77, 113, 130]. The databases of most of the commercial identification systems are somewhat out of date with respect to the current nomenclature for anaerobic bacteria.

The indigenous microflora of humans (e.g., upper respiratory tract, oral cavity, gastrointestinal tract, genitourinary tract and skin) is primarily composed of anaerobic bacteria [19, 21, 58, 77, 100, 131], and anaerobes take part in essentially all types of infections that involve bacteria. Although a few diseases involving anaerobes can be of exogenous origin (e.g., foodborne and wound botulism, tetanus), and pseudomembranous colitis (caused by Clostridium difficile), the vast majority of anaerobic infections are endogenous, arising from the body’s own microbial flora. These types of infections are often polymicrobial and commonly involve anaerobes mixed with aerobes, facultative anaerobes, or other anaerobes. However, some anaerobic infections involve only a single species. Anaerobic infections are often difficult to treat, life-threatening, and are associated with high mortality rates [14, 25, 79, 109].

Anaerobic infections probably are still among the most commonly underdiagnosed bacterial infections, although clinicians and microbiologists generally know more about anaerobic infections today than they did 30 years ago [18, 79, 99]. Factors that influence the selection of specimens submitted for anaerobic culture include the probability of whether or not anaerobes are likely to be a significant component of the infection and the perceived clinical importance of documenting the presence of anaerobes in the infectious process. Other important factors include the ability to collect an uncontaminated specimen, the cost of empirical treatment alone without anaerobic culture data, and the perceived cost benefit of having anaerobic culture data to aid in management of the patient. Several types of infections involve anaerobes relatively frequently. These include abscesses of all anatomic regions of the body (e.g., central nervous system, respiratory tract, intra-abdominal, pelvic, and other sites). In addition, anaerobes are commonly involved in aspiration pneumonia, appendicitis, bacteremia, chronic otitis media, chronic sinusitis, crepitant and noncrepitant cellulitis, dental-oral infections, endocarditis, endometritis, myonecrosis, necrotizing fasciitis, neutropenic enterocolitis (caused by Clostridium septicum), peritonitis, thoracic empyema, septic arthritis, subdural empyema, and many other types of infections [17, 25, 119]. Examples of infections in which anaerobes are seldom important include acute cholecystitis (although seen occasionally in diabetic patients), acute otitis media, acute osteomyelitis, acute sinusitis, bacterial meningitis, bronchitis, community-acquired pneumonia, cystitis, pharyngitis, pyelonephritis, superficial skin (surface) infections, and spontaneous (primary) peritonitis.

Some of the more frequently isolated anaerobes from properly selected and collected specimens are the Bacteroides fragilis group (which includes B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis, B. vulgatus and Parabacteroides distasonis as the most commonly isolated species), Prevotella and Porphyromonas species, Fusobacterium nucleatum and F. necrophorum, the anaerobic cocci (e.g., Peptostreptococcus anaerobius and Finegoldia [formerly Peptostreptococcus] magna), Actinomyces israelii, and certain Clostridium species (e.g., C. perfringens, C. ramosum, C. clostridioforme, C. septicum, and C. difficile ) [66, 99, 140].

There are a number of clinical clues that suggest anaerobes are probably involved in an infection [66]. For example, a putrid odor from an infected lesion or purulent discharge is strong evidence of infection with anaerobes. No other types of bacterial, mycobacterial, fungal, parasitic, or viral infections emit such foul odors. Presumably various products of anaerobic metabolism, including short-chain fatty acids (e.g., butyrate, isocaproate, and succinate), amines, hydrogen sulfide, and probably other sulfides can contribute to the unpleasant odors. However, in many if not the majority of cases, these types of odors go unnoticed. Thus, a lack of odor does not rule out infection with anaerobes. Lesions that arise in close proximity to mucosal surfaces where anaerobes reside within the flora, or from inocula derived from these sites (e.g., dental/oral infections, necrotizing pulmonary infections following aspiration of oral contents, and infections secondary to penetrating wounds to the abdomen or pelvis) are likely to involve anaerobes. Gas in tissue, underlying disease with tissue necrosis, tissues with blackened discoloration on bloody exudate, impaired blood supply, and chronic conditions such as diabetic foot infections and malignancies, gangrenous necrosis, abscess formation, previous antibiotic treatment, septic thrombophlebitis, infections following animal or human bites, and “sulfur granules” from suspected actinomycosis also are important clues. Anaerobic infections may be suspected when microorganisms are seen on Gram stain of the original specimen but its aerobic culture is negative.

The direct microscopic examination of Gram-stained smears frequently provides presumptive evidence consistent with anaerobic infections [66, 86–88]. Since anaerobic infections are commonly polymicrobial, the presence of different morphologic forms of bacteria within aspirated pus from a deep tissue site could represent polymicrobial infection involving a combination of anaerobes and facultative anaerobes or a variety of anaerobes. Pleomorphic, long Gram-negative rods with rounded ends are typical of Fusobacterium necrophorum. Pale, Gram-negative, slim rods with pointed ends, particularly in material from a lower respiratory tract infection, could suggest infection with F. nucleatum [87]. Boxcar-shaped, nonspore-forming, relatively large, broad, Gram-positive rods could suggest the presence of C. perfringens. Cell size may be used as an aid for identification, as with Parvimonas micra (formerly Micromonas micros) and F. magna (P. micra is between 0.3 and 0.7 μm in diameter while F. magna is between 0.7 and 1.2 μm in diameter) [48, 132]. In addition, the inspection of colonies on primary isolation plates following anaerobic incubation aids in the preliminary identification of a number of different anaerobes (e.g., Actinomyces species, the B. fragilis group, C. perfringens, F. nucleatum, and others) [66].

4.2 Steps in the diagnosis of anaerobic bacterial infections

The diagnosis of anaerobic bacterial infections involves the following steps:

  1. selection, collection, and transportation of specimens;
  2. rejection of unacceptable specimens; rapid processing of acceptable specimens;
  3. direct microscopic examination of clinical materials;
  4. selection and use of selective and non-selective primary isolation media;
  5. use of anaerobic systems for incubation;
  6. proper incubation conditions;
  7. inspection and subculture of colonies;
  8. identification of anaerobe isolates;
  9. antimicrobial susceptibility testing of significant isolates;
  10. issuing preliminary reports at appropriate times (e.g., after above steps, 3, and 7–9) prior to the final report.

Decisions made by physicians regarding which specimens to select and collect for anaerobic culture are extremely important for both optimal patient care and the laboratory. Anaerobes commonly cause pyogenic infections with purulent exudates. Abscesses may occur within any soft tissue, including brain, lung, liver, spleen, and other tissues within the peritoneal cavity. These abscesses are usually due to mixed organisms involving both obligate anaerobes and facultative anaerobes that reflect the microbial flora adjacent to mucosal surfaces. For example, abscesses in the lung commonly involve the flora of the oral cavity while those in the organs of the peritoneal cavity reflect the organisms of the intestinal tract. Specimens must be obtained in such a manner that only those organisms involved in the disease process are collected.

More information on collection, transport, and storage of specimens is provided in the section below. Upon receipt of the specimen in the laboratory, the direct microscopic examination of clinical materials can provide important information rapidly. The processing of specimens, selection and inoculation of media for primary isolation, use of anaerobic systems for incubation, procedures for inspection and subculture of colonies, identification, and anaerobic susceptibility testing, while important topics as well, are beyond the scope of this text. These topics are covered extensively elsewhere [11, 26, 32, 54, 66, 140], including CLSI document M56-A, principles and procedures for detection of anaerobes in clinical specimens; approved guideline, July 2014.

4.2.1 Collection, transport, and storage of specimens

The optimal laboratory diagnosis of anaerobic infections includes important considerations such as (i) selecting, collecting, and transporting appropriate specimens for microbiologic examination and (ii) processing and examining the specimens in the laboratory as rapidly as possible after they are received; general information on specimen collection, transport, and storage is provided by Baron and Thomson [13]. Additional detailed information on the selection, collection, and transport of specimens related to anaerobic infections is available elsewhere [12, 27, 29, 66, 132, 140].

Careful selection of materials to be examined for anaerobic bacteria is extremely important. The specimen should be collected from the active site of infection and should not be contaminated with extraneous flora [66, 140]. Because anaerobes are abundant components of the indigenous flora of mucous membrane surfaces and present on skin, several types of clinical materials should not be cultured for anaerobic bacteria. These include the following examples: throat or nasopharyngeal swabs, saliva, gingival swabs, expectorated sputum, bronchoscopic specimens not collected by a protective, double-lumen catheter [6, 139], gastric contents, small bowel contents (except in blind-loop and similar syndromes), feces (except in the workup of diseases due to C. difficile and C. botulinum), rectal swabs, colocutaneous fistulae, and colostomy stomata, surface material from decubitus ulcers, swab samples of other surfaces, sinus tracts, eschars, materials adjacent to skin or mucous membranes other than the above which have not been properly decontaminated, voided urine, and vaginal or cervical [66].

Guidelines for collection of specimens for anaerobic culture are summarized in Table 4.1. Examples of acceptable specimens for isolation of anaerobes include an adequate sample volume of pus aspirated from an abscess or a deep wound (closed), tissue (biopsy, surgical removal, autopsy), normally sterile body fluids (e.g., cerebrospinal, pleural, pericardial, paracentesis, synovial), blood, bone marrow, fine needle aspirates of lung or other body sites, and “sulfur granules”. Aspiration of liquid samples by syringe and needle or biopsies of infected tissues are the methods of choice for collecting specimens to be processed for anaerobes. Endometrial specimens taken using double-sheathed swab devices are acceptable specimens. Swabs are generally not acceptable because they retain only a very small volume of sample, dry out, and subject anaerobes to undue oxygen exposure. If swabs must be used, it is best to use swabs made of synthetic fibers such as flocked nylon, polyester fiber, rayon, or calcium alginate, and not use cotton wool swabs, which contain inhibitory fatty acids. Prior to aspiration or biopsy of clinical material, the skin or mucous membrane surface should be properly decontaminated to prevent contamination with extraneous microorganisms at the time of specimen collection [13, 66, 140].

After aspiration of pus or other liquid, the sample should be carefully injected into an oxygen-free, anaerobic transport vial or anaerobic transport tube that contains reducing agents and redox indicator [27, 66]. The syringe and needle itself should not be transported to the laboratory because of the potential danger for needle-stick injury and because oxygen diffuses through the plastic in plastic syringes [30]. Tissue can be placed in a sterile container (capped loosely) and transported to the laboratory in an anaerobic bag that removes oxygen from the atmosphere inside the container [66]. Although swabs are the least desirable (of the methods described above for specimen collection), at times it is impossible to obtain an aspirate or tissue sample, and a swab may need to be used. Swabs are best transported in anaerobic transport containers that are commercially available for this purpose (Table 4.2) [66].

Table 4.1 Examples of specimens and collection procedures in anaerobic bacteriology

Source: Adapted from Jousimies-Somer et al. [66].

Site Specimens and methods of collection
Cardiovascular system Blood cultures
Central nervous system Abscess material, tissue obtained surgically, cerebrospinal fluid
Dental area, ear, nose, throat, and sinuses Carefully aspirated or biopsied material from abscesses after surface decontamination with povidone-iodine, needle aspirates, and surgical specimens from sinuses in chronic sinusitis patients
Pulmonary area Transtracheal aspiration, fine-needle aspirate of lung, thoracotomy specimen, thoracentesis (pleural fluid), bronchoscopic specimen obtained with protective, double-lumen catheter*
Abdominal area Paracentesis fluid, needle-and-syringe aspiration of deep abscesses under ultrasound or at surgery, surgical specimen if not contaminated with intestinal flora, bile
Female genital tract Culdocentesis after surface decontamination of the vagina with povidone-iodine; laparoscopy specimens, surgical specimens, endometrial cavity specimen with double-lumen catheter and microbiologic brush after cervical os is decontaminated
Urinary tract Suprapubic aspirate of urine
Bone and joint Aspirate of joint (in suppurative arthritis), deep aspirate of drainage material after surgery (e.g., in osteomyelitis)
Soft tissue Open wounds (deep aspirate of margin or biopsy specimen of the depths of wound only after careful surface decontamination with povidone-iodine); sinus tracts (aspiration by syringe and small plastic catheter after careful decontamination of skin orifice); deep abscess, anaerobic cellulitis, infected vascular gangrene, clostridial myonecrosis (needle aspirate after surface decontamination); surgical specimens, including curettings and biopsy material; decubiti and other surface ulcers (thoroughly cleanse area with povidone-iodine by surgical scrub technique, and aspirate pus from deep pockets or obtain biopsy specimen from deep tissue at margin)

*Reviewed elsewhere by Allen and Siders [6].

Technique uses a telescoping double-catheter assembly similar to that available for bronchoscopy. This procedure is inadequate to diagnose postpartum endometritis [66].

Table 4.2 Selected commercially available anaerobe transport devices: manufacturers and product numbers

Manufacturer Description Manufacturer’s number
Anaerobe Systems
San Jose, CA
Anaerobic Transport Medium (ATM) – 6 mL AS-911
ATM Sterile Surgery Pack – 6 mL AS-914
Wide Mouth ATM – 8 mL AS-915
Wide Mouth ATM Sterile Surgery Pack – 8 mL AS-919
Becton Dickinson
Franklin Lakes, NJ
BBL Vacutainer Anaerobic Specimen Collector 236500
BBL Port-A-Cu Tube with Swabs – sterile pack 221607
BBL Port-A-Cul Tube 221606
BBL Port-A-Cul Transport Jar 3” sterile pack screw cap jar with 1” wide mouth 221602
Copan Diagnostics
Murrieta, CA
Amies Agar Gel – double plastic swabs – blue cap 134C
ESwab – sterile single use sample collection pack 480C
Starplex Scientific
Etobicoke, Ontario, Canada
Anaerobic Transport System S120D
Modified Amies Clear Gel SP130X

Optimally, the viability and relative proportions of anaerobic bacteria or other microorganisms should not change while the specimen is being transported to the laboratory. In order to minimize loss of viability of anaerobes or overgrowth of aerobic microorganisms within the specimen container, rapid transportation is highly recommended. Under certain circumstances, some anaerobes lose viability during refrigeration (e.g., B. fragilis and C. perfringens at 4°C) [52, 128]. However, at room temperature or warmer temperatures, some aerobic or facultative anaerobic bacteria may overgrow (e.g., Escherichia coli or Pseudomonas species), making it difficult or impossible to recover any anaerobes that could originally have been present when the sample was collected [49, 52]. For these reasons, specimens are best maintained at 15–25°C during transportation to the laboratory.

4.2.2 Incubation, inspection and subculture of colonies

Once a properly collected and transported specimen has been received, appropriate selective and nonselective media are inoculated and incubated in an anaerobic system. Anaerobes grow best in freshly prepared, prereduced, anaerobically sterilized (PRAS) media [56, 85, 138]. The PRAS media are prepared by avoiding exposure to oxygen, which can oxidize the ingredients in the culture medium resulting in the formation of toxic oxygen intermediates. Commercial sources for PRAS media are listed in Table 4.3.

Table 4.3 Selected commercially available sources for PRAS media

Manufacturer Packaging
Anaerobe Systems
15906 Concord Circle
Morgan Hill, CA 95037
Plate, tube
12076 Santa Fe Drive
Lenexa, KS 66215
Oxyrase, Inc.
P.O. Box 1345
Mansfield, OH 44901

Anaerobes vary considerably in their oxygen tolerance; some are extremely oxygen sensitive and are killed within minutes while others withstand longer oxygen exposure. Cultures need to be processed under anaerobic conditions or processed rapidly to minimize the negative effects of air exposure. Then the cultures require anaerobic incubation systems that protect them from oxygen exposure. More protective systems will grow more anaerobes and, especially, more of the oxygen sensitive organisms. Anaerobic incubation systems include anaerobic chambers, jars, boxes, and pouches. These vary in the time that they allow cultures to be exposed to oxygen, convenience, space requirements, and cost [66, 140]. Several commercially available sources of systems for anaerobic incubation are provided in Tables 4.4 and 4.5. Relatively current illustrations, discussions, and recommendations on the use of a number of these products are available elsewhere [66, 140].

Table 4.4 Selected commercially available anaerobic incubation systems: manufacturers and product numbers

Manufacturer Description Product number
Advanced Instruments Advanced Anoxomat Mark II System, Anaerobic Jar AJ9025
Advanced Anoxomat Mark II System, Anaerobic Jar (large capacity) AJ9028
Becton Dickinson
Franklin Lakes, NJ
Bio-Bag Environmental Chamber Type A: holds one 100 × 15 mm petri dish 261214
Bio-Bag Type A Multi-Plate: holds three 100 × 15 mm petri dishes 261216
GasPak EZ Gas Generating Pouch Systems 260683
GasPak EZ Standard Incubation Container 260671
GasPak EZ Large Incubation Container 260672
GasPak 100 System 260626
GasPak 100 System, Vented 260627
GasPak 150 Large Anaerobic System 260626
GasPak 150 System Large Anaerobic System, Vented 260627
Lenexa, KS
AnaeroPouch Bag R682001
AnaeroPack Rectangular Jar 2.5 L R684004
Lenexa, KS
AnaeroJar 2.5 L AG0025A

Table 4.5 Selected manufacturers/vendors* of anaerobe chambers and gas chromatographs

Anaerobe chambers Gas chromatographs
Anaerobe Systems
15906 Concord Circle
Morgan Hill, CA 95037
Phone: (408) 782-7557
Website: www.CoyLab.com
Agilent Technologies
5301 Stevens Creek Blvd Santa Clara, CA 95051
Phone: (800) 227-9770
Website: www.agilent.com
Baker Bioscience Solutions
P.O. Drawer E
Sanford, ME 04073
Phone: (877) 350-6414
Website: www.bakerbio.com
Buck Scientific, Inc.
58 Fort Point Street East Norwalk, CT 06855
Phone: (203) 853-9444
Website: www.bucksci.com
Coy Lab Products
14500 Coy Drive
Grass Lake, MI 49240
Phone: (734) 475-2200
Website: www.CoyLab.com
GOW-MAC Instrument Co.
277 Brodhead Road
Bethlehem, PA 18017
Phone: (610) 954-9000
Website: www.gow-mac.com
Sheldon Manufacturing, Inc.
P.O. Box 627
300 North 26th Avenue
Cornelius, OR 97113
Phone: (503) 640-3000
Website: www.shellab.com
Shimadzu Scientific Instruments
7102 Riverwood Drive
Columbia, MD 21046
Phone: (800) 477-1227
Website: www.ssi.shimadzu.com
Microbiology International
Suite H
5111 Pegasus Ct.
Frederick, MD 21704
Phone: (800) 396-4276
Website: www.800ezmicro.com
SRI Instruments
20720 Earl St.
Torrance, CA 90503
Phone: (310) 214-5092
Website: www.srigc.com

* Because of the numerous sizes and configurations of these kinds of equipment, it is suggested that the interested reader contact the manufacturer/vendor for sizes/models, other descriptive information, and prices.

4.2.3 Presumptive identification of anaerobes

Colonial morphology, cellular morphology from a Gram-stain examination, aerotolerance testing, and examination of growth on primary agar media are essential for initial presumptive identification of isolates. Many common anaerobes can be identified to genus or group by phenotypic characteristics [11, 66]. Because many anaerobes have distinctive colony characteristics, it can be useful to inspect and subculture colonies under a stereoscopic dissecting microscope (7–15× magnification). Photographs of anaerobe colonies on commercially available CDC Anaerobe Blood Agar, Brucella Blood Agar, or Bacteroides Bile Esculin Agar along with their microscopic features of anaerobe isolates are available in several manuals and atlases [66, 140]. The following characteristics are especially useful for presumptive identification [31]. The interpretation of these phenotypic characteristics for a presumptive identification is available in several recently published manuals such as the Manual of Clinical Microbiology [11] and Wadsworth-KLM Manual [66]:

  1. relation to oxygen (e.g., obligate anaerobe, aerotolerant anaerobe, facultative anaerobe);
  2. colony characteristics (e.g., size, form, elevation, margin, surface, consistency, pigment, fluorescence, pitting);
  3. microscopic features (e.g., Gram reaction, morphology, presence or absence of spores);
  4. presence of hemolysis on blood containing media;
  5. catalase production;
  6. reduction of nitrate to nitrite;
  7. indole production;
  8. reaction to special potency antibiotic disks that include vancomycin, kanamycin and colistin;
  9. inhibition by sodium polyanethol sulfonate;
  10. bile resistance;
  11. hydrolysis of esculin;
  12. lecithin and or lipase production on egg yolk agar;
  13. detection of short chain acid metabolic products by gas liquid chromatography (GLC).

Results of the phenotypic identification systems permit differentiation of most clinical isolates to the genus or a group level, and a few to the species level. Although such presumptive identifications may be cost effective and rapid, they may not always be sufficient for appropriate treatment or correlation with malignancy or other disease condition when a confirmatory or complete identification is needed.

4.2.4 Phenotypic identification procedures

A systematic identification system for anaerobes was described by Holdeman and Moore in their 1972 Virginia Polytechnic Institute (VPI) Anaerobe Laboratory Manual [61]. This system was based on reactions in 20–50 sugars and other substrates in PRAS media, Gram-stain morphology, and analysis of end products by gas–liquid chromatography. Isolates were tested for their ability to use each sugar in PRAS peptone–yeast broth after incubation periods of 24 h to 1 week at 35°C. The pH of each different sugar broth was measured to see if it dropped by several log units as compared to its initial pH. Gas–liquid chromatography was used to determine the short-chain fatty organic acids and alcohols that are the end products from glucose and other substrate metabolism. Bacteria were grouped on the basis that related organisms with similar metabolism produced similar types and volumes of end products [9, 36, 61]. The volumes were reported as major, minor, or trace in a semi-quantitative fashion. Although labor intensive and time consuming, the VPI system was the basic guideline for conventional identification of anaerobes for decades. It continued to be periodically updated through the 1990s. The phenotypic testing scheme with an emphasis on clinical isolates was enhanced by addition of special potency disks and preformed enzyme tests as well as by incorporation of taxonomic changes into the identification tables as described in the Wadsworth-KTL Anaerobic Bacteriology Manual [66]. The PRAS biochemicals are available commercially or can be prepared according to the VPI manual [61].

Another system developed at the Centers for Disease Control (CDC) used growth characteristics on three quadrant plates (Presumpto Plates I, II, and III) to identify commonly encountered anaerobes [41]. The quadrant plates contained four differential agars per plate, with Lombard-Dowell agar as the basal medium. Following inoculation, the Presumpto plates were incubated anaerobically for 24–48 h. Characteristics used to identify anaerobic bacteria are listed in Table 4.6. Detailed instructions pertaining to the Presumpto Plates, along with identification tables, are available elsewhere [136].

Table 4.6 Media and characteristics of anaerobe isolates that can be determined using the CDC Presumpto Quadrant Plate procedure for anaerobe identification

Source: Modified from Winn et al. [140].

Media Characteristics
CDC anaerobe blood agar Relation to oxygen,* colony characteristics, hemolysis, pigment, fluorescence with ultraviolet light (Wood’s Lamp), pitting of agar, cellular morphology, Gram reaction, spores, motility (wet mount); inhibition by penicillin, rifampin, and kanamycin (disk tests)
Enriched thioglycolate medium Appearance of growth, rapidity of growth, gas production, odor, cellular morphology
Presumpto 1 plate
LD agar Growth on L-lysine decarboxylation (LD) medium, production of indole, indole derivative, catalase
LD esculin agar Esculin hydrolysis, hydrogen sulfide, catalas
LD egg yolk agar Lipase, lecithinase, proteolysis on egg yolk agar
LD bile agar Growth in presence of 20% bile (2% oxgall), insoluble precipitate under and immediately surrounding growth
Presumpto 2 plate
LD glucose agar Glucose fermentation; stimulation of growth by fermentable carbohydrate
LD starch agar Starch hydrolysis
LD milk agar Proteolysis of milk
LD DNA agar Deoxyribonuclease activity
Presumpto 3 plate
LD gelatin Gelatin hydrolysis
LD mannitol agar Mannitol fermentation
LD Lactose agar Lactose fermentation
LD rhamnose agar Rhamnose fermentation

* By comparing growth on anaerobe plate with blood agar (or chocolate agar) incubated in 5–10% CO2 incubator (or candle jar) or in room air.

The catalase test can be performed by adding 3% hydrogen peroxide to the growth on LD agar, but the reactions of catalase-positive cultures are more vigorous on LD esculin agar.

4.2.5 Alternative procedures for characterization of anaerobes

A variety of other methods for characterizing and identifying anaerobes have been proposed as practical alternatives to the laboratory methods described above (Table 4.7) [11, 54, 66]. Compared to the more traditional approaches for identifying anaerobes, the motivation for developing alternative procedures has often been to decrease materials costs, save time, simplify the work involved, or to find a more accurate and reproducible way to differentiate between anaerobe isolates. Among the approaches taken have been to test for certain key characteristics of isolates on differential agar media (e.g., Bacteroides Bile Esculin Agar; Anaerobe Systems, San Jose, CA), or to use relatively small volumes of media in small containers that can be easily and rapidly manipulated at the laboratory bench. Another convenient test format has been to use special potency disks to test for growth stimulation or growth inhibition and the use of disks or tablets for determining certain enzyme-substrate reactions. Supplies and other materials to perform several practical and simple tests are described in the Wadsworth-KTL Anaerobic Bacteriology Manual [66] and are commercially available (Table 4.8).

Table 4.7 Practical and simple manual methods for characterization and presumptive identification of anaerobes

Source: Modified from Jousimies-Somer et al. [66].

Test or procedure Rationale, comments, commercial sources
Observations of colony characteristics on anaerobe blood agar and anaerobe selective media; Gram reactions and microscopic morphology Morphologic observations are necessary for identification (ID) of all anaerobes. Commercially prepared formulations of anaerobe blood agar are available from several manufacturers including Anaerobe Systems, BD Biosciences Microbiology Products, PML Microbiologicals, Remel, and Springs River Biologicals
Aerotolerance tests on anaerobe blood agar Necessary to define relationships to O2. See above for sources of prepared media
Egg yolk agar (EYA) reactions Lipase positive species include C. sporogenes, C. botulinum, C. novyi type A, P. intermedia and F. necrophorum. Lecithinase positive species include C. perfringens, C. sordellii, C. bifermentans, C. limosum, C subterminale,C. novyi A and C. baratii. Commercially prepared EYA is available from a number of manufacturers including Anaerobe Systems, BD Biosciences Micrrobiology Products, PML Microbiologicals, Remel, and Springs River Biologicals
Catalase test on hemin supplemented medium (e.g. 3% H2O2 on LD esculin agar, or slide catalase using 15% H2O2 with added tween 80) A positive catalase test is characteristic of some species of Bacteroides, Bilophila wadsworthia, some Anaerococcus prevotii, Staphylococcus saccharolyticus and Veillonella parvula, Propionibacterium spp. (P. propionicus is negative and an exception) and A. viscosus.
Spot indole test using p-dimethylaminocinnamaldehyde Useful for all groups of anaerobes. Reagent available from several sources including Anaerobe Systems, BD Biosciences Microbiology Products, and Remel.
Enhanced growth in presence of20% bile in medium or around disks containing bile Key characteristic of the B. fragilis group, Bilophila spp., Fusobacterium varium, F. ulcerans, Leptotrichia buccalis, and Mitsuokella multiacida. Available commercially in quadrant plates from Remel and Smith River Biologicals
Antibiotic differentiation disks (e.g. kanamycin1 mg; rifampin, 15 μg; penicillin, 2U; colistin, 10 μg; vancomycin, 5 μg.); used to detect growth in presence of drug or growth inhibition. Used to aid in distingushing between Gram-positive vs Gram-negative anaerobes and in presumptive identifications
Sodium polyanetholsulfonate (SPS) disk test SPS inhibits P. anaerobius; other anaerobic cocci are resistant to SPS. Alternatively, P. anaerobius is the only anaerobic coccus that produces a major isocaproic acid peak on GLC analysis. SPS disks available from Anaerobe Systems and BD Biosciences Microbiology Products
Disk test for nitrate reduction Most if not all Campylobacter ureolyticus(formerly Bacteroides ureolyticus), Veillonella spp. Eggerthella lenta (formerly Eubacterium lentum), Bilophila wadsworthia, Sutterella wadsworthensis are positive; test helpful for other cocci, non-sporing rods and clostridia as well. Available from Anaerobe Systems and BD
Urease test (various procedures can be used) A positive urease reaction is a key characteristic of C. ureolyticus, Bilophila wadsworthia, C. sordellii, Anaerococcus tetradius (formerly P. tetradius), S. saccharolyticus and certain Actinomyces spp. Available from Anaerobe Systems and BD Biosciences Microbiology Products
Rapid glutamic acid decarboxylase tube test Positive reactions given with most strains of the B. fragilis group, many strains of Fusobacterium spp., P. micra (53% of strains), most C. perfringens and C. sordellii, some C. difficile and all E. limosum tested. Available from Remel.
Esculin hydrolysis (on LD esculin agar or using a spot test with 1% ferric ammonium citrate to PY esculin broth) Useful for all groups of anaerobes. Available in quadrant plates from Remel and Smith River Biologicals.
Fluorescence under long-wave length (366-mm) UV light Useful for the pigmented Prevotella and Porphyromonas species. Porphyromonas gingivalis does not fluoresce whereas P. asaccharolytica is red or yellow; Prevotella intermedia is bright red. P.melaninogenica is red-orange. Also useful for Veillonella spp. (red). Observations made on laked blood agar
Lack of constitutive ß-glucosidase(esculinase) Fusobacterium species lack the enzyme but it is produced by Bacteroides spp. and Prevotella spp.

Table 4.8 Selected disk tests used in presumptive identification of anaerobes

Test Manufacturere’s number Package
SPS Disk AS-702 50 tests
Nitrate Disk AS-703 50 tests
Colistin Disk AS-705 50 tests
Kanamycin Disk AS-706 50 tests
Vancomycin Disk AS-707 50 tests

AS, Anaerobe Systems, Morgan Hill, CA.

Many anaerobic bacteria produce constitutive enzymes that rapidly hydrolyze various chromogenic substrates. WEE-TABS (Key Scientific Products, Round Rock, TX) are stand-alone chromogenic tests for several enzyme–substrate reactions which can be used as alternative and supplemental tests for differentiation of certain anaerobes (Table 4.9; Figure 4.1). Hudspeth and colleagues found WEE-TABS to be a rapid and accurate alternative for identification of Porphyromonas species isolated from infected dog and cat bite wounds in humans [62]. WEE-TABS are available in single test and dual test formats with nitrophenyl and naphthylamide bound substrates. According to the manufacturer’s instructions, inocula are prepared by harvesting a sufficient number of colonies into 1–2 mL of distilled water to make a suspension equal to a #5 McFarland standard. At least five or six drops of this suspension are placed into each tube containing a WEE-TAB tablet and the tube is mixed vigorously or vortexed to disintegrate the tablet and mix the suspension. The tubes are incubated aerobically at 37°C for 2 h. For the glycosidase tests, no change at 2 h is negative. The development of a yellow color at any time during the 2 h is considered a positive glycosidase test. After reading the glycosidase results (dual test format), or after incubating a single naphthylamide test, two drops of p-dimethylaminocinnamaldehyde reagent are added. The tube is then incubated for 15 min for color development. Positive tests will be red while negative tests will be yellow or a light peach-color. A triple test format is also available with nitrophenyl-, methylumbelliferyl-, and naphthylamide-bound substrates in each tube. Hydrolysis of methylumbelliferyl-bound substrates produces a bright, blue-green fluorescence that is detected using a long-wavelength UV light (366 nm). When only a small battery of enzyme tests is needed for characterizing an anaerobe isolate (e.g., six or fewer tests), WEE-TABS may be more cost effective than the prepackaged commercial kits for detecting various preformed enzymes (in terms of time and materials costs). Diagnostic tablets for bacterial enzyme testing are manufactured by Rosco Diagnostica (Taastrup, Denmark) and sold by Key Scientific Products, Round Rock, TX.

Photo of the WEE‐TABS stand‐alone chromogenic tests, with four tubes.

Figure 4.1 WEE-TABS stand-alone chromogenic tests. Each tube contains one tablet and a small volume of bacterial suspension.

(Courtesy of Key Scientific Products.)

Table 4.9 Selected tests available in single tablet format for assessing preformed enzymatic activity of anaerobes*,

Substrate and abbreviation Enzyme, comments
Glycosidase tests (WEE-TABS)
p-nitrophenyl-α-D-glucopyranoside (α-GLU) α-Glucosidase
p-nitrophenyl-ß-D-glucopyranoside (ß-GLU) ß-Glucosidase
o-nitrophenyl-α-D-glucopyranoside (α-GAL) α-Galactosidase
o-nitrophenyl-ß-D-galactopyranoside (ß-GAL or ONPG) ß-Galactosidase
p-nitrophenyl-ß-N-acetyl-d-glucosaminide (ß-NAG) ß-N-acetylglucosaminidase
p-nitrophenyl-α-L-fucopyranoside (α-FUC) α-Fucosidase
p-nitrophenyl phosphate (PNP) Alkaline phosphatase**
Naphythylamide-based aminopeptidase test tablets (ADD-A-TABS)§
Na-benzoyl-DL arginine ß-naphthylamine (BANA or TRY) Trypsin-like activity
N-glutaryll-gly-gly-phe ß-naphthylamide (CHY) Chymotrypsin-like activity
L-Phenylalanine α-naphthylamide (PAL)
L-Arginine α-naphthylamide (ARG)
L-Serine α-naphthylamide (SER)
L-Pyroglutamic acid α-naphthylamide (PYR)
L-proline α-naphthylamide (PRO)
Leucyl-glycine α-naphthylamide (GLY)

* In the United States, WEE-TABS and ADD-A-TABS are available commercially from Key Scientific Products, Round Rock, TX.

In Europe, tablets containing many of the substrates listed are available from Rosco Diagnostica, Taastrup, Denmark.

Glycosidases. When bound to nitrophenyl, hydrolysis of a colorless aryl-substituted glycoside substrate or phosphoester releases the nitrophenyl base. No color change at 2 h is negative; a yellow color at any time during the 2 h of aerobic incubation is a positive glycosidase test.

§ Naphthylamide. Enzyme hydrolysis of the arylamide in the tablet releases free α-naphthylamine, which is detected and shown by the color change after adding phosphoenol pyruvate (PEP) reagent (i.e., p-dimethylaminocinnamaldehyde in HCl). After reading the glycosidase test, two drops of PEP are added followed by 15 min incubation; positive tests are red while negative tests are yellow.

Test aids in differentiation of Porphyromonas spp.

** PNP: P. micra, A. hydrogenalis, and A. prevotii = positive; peptostreptococci = negative.

4.3 Commercial kit requiring 24 h of anaerobic incubation

The API 20A (bioMérieux; Figure 4.2; Table 4.10) micromethod kit has been marketed for the identification of anaerobes isolated from clinical materials. This miniaturized system, while smaller than the conventional biochemical identification systems discussed previously, requires turbid inocula and depends upon growth of anaerobes during incubation in an anaerobic system. The system is useful for identification of saccharolytic, fast-growing anaerobes but not those that are asaccharolytic or fastidious. Interpretations of the reactions for identifications must be made by comparisons to the manufacturer’s proprietary database.

Photo of API 20A test strip.

Figure 4.2 API 20A test strip. Miniaturized cupules are inoculated with a bacterial suspension and after 24–48 h of incubation, biochemical reactions are visualized.

(Courtesy of bioMérieux.)

Table 4.10 Selected miniaturized and rapid commercial packaged kit systems for identification of anaerobes: manufacturers/vendors

Kit type System Manufacturer’s number Package
Miniaturized API 20A 20300 25 tests/box
Rapid identification (≤4 h) RapID ANA II 83-11002 20 panels
Vitek 2 ANC ID Card 21347 20 tests/box
MicroScan Panels; Rapid anaerobe ID B1017-2 20 panels
Rapid ID 32 32300 25 panels
Crystal Anaerobe kit 245010 20 determinations

The API 20A consists of a plastic strip with 20 “microtubules” of dehydrated substrates to test for the following: indole, catalase, hydrolysis of urea, gelatin, and esculin, plus the fermentation of glucose and other carbohydrates. The microcupules of the strip are inoculated with a turbid suspension (i.e., ≥ #3 McFarland standard) of fresh colonies prepared in Lombard-Dowell broth and incubated anaerobically for 24–48 h. Databases exist for reading the strips at 24 h or 48 h. The indicator for the fermentation tests is bromcresol purple. An acid reaction is yellow (pH 5.2) or yellow-green; a negative reaction is purple. Various anaerobes (e.g., a number of Clostridium species) reduce the indicator to colorless, shades of green-brown or other colors, sometimes making reactions difficult to interpret. According to the manufacturer’s instructions, “other tests such as colonial and microscopic morphology, Gram stain, etc., should be performed and the results used to confirm or complete the identification.” Additional recommendations for use of the API 20A are supplied by bioMérieux along with identification tables and a numerical Analytical Profile Index.

An excellent review of the older literature by Stargel and colleagues [118] describes the development and evaluations of the packaged micromethod kit. A number of other reports have discussed the performance, advantages, and limitations of various systems [15, 55, 57, 59, 125]. It is important to note that one of the largest reports indicated that the API 20A system correctly identified only 50% of approximately 800 anaerobe isolates to the species level [125]. Although the system can accurately identify some commonly encountered species such as Bacteroides fragilis and Clostridium perfringens, many of the anaerobes that are less reactive or more fastidious (e.g., many species of Prevotella, Porphyromonas, Fusobacterium, other clostridia, anaerobic nonsporeforming rods, and most anaerobic cocci) require additional tests to achieve correct identification, which decreases the usefulness of the system and increases the time for identifications by another day or two. Thus, although the API 20A system can be set up relatively rapidly, it does not necessarily enable the user to identify anaerobes any faster than can be done using the conventional tests described previously. Additionally, at the time of this writing, the database for this system requires selective nomenclature updates.

4.4 Commercial enzyme kits for identification after four hours ofaerobic incubation

Commercially available microsystems in kit form that have been used for characterization and identification of anaerobes following aerobic incubation for 4 h include the following:

  1. RapID-ANA II (Remel/ThermoFisher)
  2. Vitek 2 ANC (bioMérieux)
  3. MicroScan Rapid Anaerobe Panel (Beckman Coulter)
  4. Rapid ID 32A (bioMérieux)
  5. BBL Crystal Anaerobe Identification System (Becton Dickinson Microbiology Systems).

These growth-independent systems are designed to test for various miniaturized conventional reactions and preformed enzymes (e.g., aminopeptidases and glycosidases). All of them require that inocula be prepared using dense cell suspensions from the surfaces of purity plate cultures. The short incubation time of 4 h or less for each of the systems is in sharp contrast to the longer incubation times required for the growth-dependent identification systems discussed previously (Table 4.10). All of the packaged systems listed above provide numerical codes or computerized databases that aid in identification of isolates characterized using these systems.

4.4.1 RapID ANA II

The RapID ANA II tests for 18 preformed enzymes using 10 wells (Figure 4.3). The dehydrated substrates included in these wells are listed in Table 4.11.

Photo of the Thermo Scientific RapID ANA II panel.

Figure 4.3 Thermo Scientific™ RapID™ ANA II System. This 4-h rapid test system for the identification of anaerobes contains 10 wells for testing the performance of 18 preformed enzymatic tests.

(Photo courtesy of ThermoFisher Scientifc, copying is prohibited.)

Table 4.11 Summary of biochemical test reactions or substrates available in the 4-h commercial packaged microsystems

RapID ANA II MicroScan Rapid Anaerobe Panel Rapid ID 32 A Crystal Vitek 2 ANC card
Urea p-Nitrophenyl-β-D-galactopyranoside Urease L-Arginine-7-amino-4-methylcoumarin (AMC) D-Galactose
p-Nitrophenyl-β, D -lactoside p-Nitrophenyl-α-D-galactopyranoside Arginine dihydrolase L-Histidine-AMC Leucine arylamidase
p-Nitrophenyl-α, L-arabinoside bis-p-Nitrophenyl phosphate α-Galactosidase 4-Methylumbelliferon (MU)-α-D-mannoside Ellman
Leucyl-glycyl-β-naphthylamide p-Nitrophenyl-N-acetyl-β-D-glucosaminide β-Galactosidase L-Serine-AMC Phenylalanine arylamidase
o-Nitrophenyl-β, D -galactoside p-Nitrophenyl-α-D-glucopyranoside β-Galactosidase-6- phosphatase L-Isoleucine-AMC L-proline arylamidase
glycyl-β-napthylamide o-Nitrophenyl-β-D-glucopyranoside Glucosidase 4MU-β-D-mannoside L-pyrrolidonyl-arylamidase
p-Nitrophenyl-α, D -glucoside p-Nitrophenyl phosphate β-Glucosidase Glycine-AMC D-Cellobiose
Prolyl-β-naphthylamide p-Nitrophenyl-α-L-fucopyranoside α-Arabinosidase L-alanine-AMC Tyrosine arylamidase
p-Nitrophenyl-β, D-glucoside p-Nitrophenyl-α-D-mannopyranoside β-Glucuronidase 4MU-N-acetyl-β-D-galactosaminide Ala-phe-pro-arylamidase
Phenylalanyl-β-naphthylamide L-Leucine-β-naphthylamide β-N-acetyl-glucosaminidase L-Pyroglutamic acid D-Glucose
p-Nitrophenyl-α, D-galactoside DL-Methionine-β-naphthylamide Mannose fermentation L-Lysine-AMC D-Mannose
Arginyl-β-naphthylamide L-Lysine-β-naphthylamide (alkaline) Raffinose fermentation L-Methionine-AMC D-Maltose
p-Nitrophenyl-α, L-fucoside L-lysine-β-naphthylamide (acid) Glutamic acid decarboxylase 4MU-β-D-cellobiopyranoside Saccharose/sucrose
Seryl-β-naphthylamide Glycylglycine-β-naphthylamide α-Fucosidase 4MU-β-D-xyloside Arbutin
p-Nitrophenyl-N-acetyl-β, D-glucosaminide Glycine-β-naphthylamide Nitrate L-Phenylalanine-AMC N-Acetyl-D-glucosamine
Pyrrolidonyl-β-naphthylamide L-Proline-β-naphthylamide Indole L-Leucine-AMC 5-Bromo-4-chloro-3-indoxyl-beta-glucoside
p-Nitrophenyl phosphate L-Arginine-β-naphthylamide Alkaline phosphatase Ecosyl Urease
Tryptophane L-Pyrrolidonyl-β-naphthylamide Arginine arylamidase Disaccharide 5-Bromo-4-chloro-3-indoxyl-β-glucuronide

L-Tryptophan-β-naphthylamide Proline arylamidase Furanose β-Galactopyranosidase indoxyl

3-Indoxyl-phosphate Leucyl glycine arylamidase Pyranose alpha-Arabinosidase

Trehalose Phenylalanine arylamidase p-Nitrophenyl-α-D-galactoside 5-Bromo-4-chloro-3-indoxyl-α-galactoside

Urea Leucine arylamidase p-Nitrophenyl-β-D-galactoside β-mannosidase

Indole Pyroglutamic acid arylamidase p-Nitrophenyl phosphate Arginine GP

Nitrate Tyrosine arylamidase p-Nitrophenyl-α-D-glucoside Pyruvate

Alanine arylamidase p-Nitrophenyl-N-acetyl-glucosaminide Maltotriose

Glycine arylamidase L-Proline-p-nitroanilide Esculin hydrolysis

Histidine arylamidase p-Nitrophenyl-α-L-fucoside β-D-fucosidase

Glutamyl glutamic acid arylamidase p-Nitrophenyl-β-D-glucoside 5-Bromo-4-chloro-3-indoxyl-beta-N-acetyl glucosamide

Serine arylamidase L-Alanyl-L-alanine-p-nitroanilide 5-Bromo-4-chloro-3-indoxyl-α-mannoside




D-ribose 2


α- L-arabinofuranoside

D-xylose Preparation of inocula: important considerations

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Dec 10, 2017 | Posted by in MICROBIOLOGY | Comments Off on 4: Rapid Devices and Instruments for the Identification of Anaerobic Bacteria

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