A Dichotomous Key for the Identification of Miscellaneous Gram-Negative Bacteria

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1 A Dichotomous Key f the Identification of Miscellaneous Gram-Negative Bacteria Tech Com: Microbiology by Jim Romeo, MT(ASCP)SM Many clinical labaties in the United States now use the Center f Disease Control (CDC) bacterial identification key 2 one of its various modifications. 1 ' 3-4 After the Gram stain and growth characteristics on MacConkey agar are evaluated, and the oxidase and OF glucose reactions are d e t e r m i n e d, the choices f identification of an ganism are reduced to a group of f r o m one to 22 ganisms. Various tables and written descriptions of the ganisms direct one to the additional substrates tests needed f final identification, a process that can be very time-consuming and occasionally misleading. To simplify this process, a supplemental dichotomous key was developed and has been used in our labaty f the past t w o years. This key was developed using the data presented in the CDC key2 and crosschecked with other references. 1,5,fi The confidence level f the branch points in the key is 90% greater, except where indicated otherwise. It is not suggested that this key should replace tables currently in use that the number of tests required f final identification of a particular isolate should necessarily be reduced. This key usually leads one to a rapid preliminary identification and facilitates selection of culture media f further testing and confirmaty reactions. Materials and Methods The Center f Disease Control recommends the following media and tests f the identification of Jim Romeo, MT(ASCP)SM, is with the Labaty of the King Faisal Specialist Hospital and Research Centre in Riyadh, Saudi Arabia. ganisms included in this key. The reader is referred to the references cited f me detailed descriptions and fmulas. 1 ' 2 The oxidase test is perfmed by adding one t w o drops of 0.5% aqueous tetramethyl-p-phenylenediamine dihydrochlide to colonies on a culture plate. The Kovacs oxidase method is also satisfacty. MacConkey agar slants are used to detect growth () no growth ( - ). Three different substrates are used to determine carbohydrate reactions f glucose, xylose, mannitol, lactose, sucrose, maltose and levulose. Semisolid OF medium of King 1 with phenol red indicat and 1 % carbohydrate is used f oxidative bacteria. Liquid peptone basal medium with Andrade's indicat is recommended f fermentative bacteria, and cystine tryptophan agar medium (CTA) is used f. In the dichotomous key, OF CTA precedes the name of the carbohydrate where these substrates are used. Fermentative substrates may be assumed when a carbohydrate is named w i t h o u t a prefix. Other Media and Tests 1. Blood agar plate (heart infusion agar w i t h 5% defibrinated rabbit blood at 35 C in 3% C 0 2 ). 2. Catalase test perfmed by flooding tryptoneglucose-yeast extract (TGY) agar slants with 3% hydrogen peroxide. 3. Christensen's urea agar slant. 4. Esculin agar. 5. Leifson flagella stain to determine number and arrangement of flagella. 6. Loeffler media f pigment production of certain ganisms as indicated in the key. 7. L-lysine decarboxylase, ninhydrin (Carlquist ninhydrin base) is recommended f cepacia. 8. Methyl red-voges-proskauer m e d i u m. 9. Moeller decarboxylase broths f lysine and nithine decarboxylase and arginine dihydrolase. 0007/5027/79/0900/547 $01.10 American Society of Clinical Pathologists In recent years the development of rapid identification systems along with the availability of various new media have greatly simplified the identification of c o m m o n clinically significant bacteria. The identification of certain less c o m m o n clinical isolates still poses problems f many labaties. 1 The isolation of u n c o m m o n bacteria, some currently classified only by letters and numbers, provides a challenge and sometimes frustration f the clinical microbiologist faced with the task of identifying t h e m. 547

2 Table 1 Organisms Listed in the CDC Key (August 1974) which Appear under a Different Name in the Eighth Edition of Bergey's Manual of Determinative Bacteriology CDC Key Bergey's Manual Achromobacter species Achromobacter xylosoxidans Alcaligenes denitrificans Alcaligenes odans HB-1 {Eikenella crodens) catarrhalis fiava perflava Pasteurella gallinarum Vibrio extquens Vibrio fetus "Related Vibrios" Alcaligenes species Alcaligenes species Alcaligenes faecalis Alcaligenes faecalis Bacteroides crodens Branhamella catarrhalis subflava subflava Pasteurella multocida Methylamonas species Campylobacter fetus Campylobacter fetus ss. jejuni 19. SS agar. 20. Triple sugar iron agar slant (TSI) % tryptone broth f indole production (incubate 48 hours and extract with xylol befe addition of Ehrlich-Boehme reagent). 22. Tryptone-glucose-yeast extract (TGY) agar slants are incubated at 25 C, 35 C and 42 C to determine optimal growth temperatures. (Thioglycollate broth is used f Campylobacter species; i.e., Vibrio fetus.) It is suggested that the preliminary w o r k u p of an ganism to be identified include an evaluation of: 1) Gram stain reaction and mphology, 2) TSI (triple sugar iron agar) reaction, 3) OF glucose reaction, 4) Table II Organisms Listed as Nomina Incertae Sedis in the Eighth Edition of Bergey's Manual of Determinative Bacteriology Actinobacillus actinomycetem comitans Actinobacillus suis Comamonas terrigena kingii lactamicus Pasteurella anatipestifer denitrificans putrefaciens 548 LABORATORY MEDICINE VOL. 10. NO. 9. SEPTEMBER 1979 oxidase reaction, and 5) growth on MacConkey agar. After making these preliminary assessments, which may be enlarged upon at the discretion of the user, other substrates tests may be prudently selected from the dichotomous key. Final confirmation of bacterial identification may require further testing. It should be emphasized that with this group of ganisms, many of the observed reactions may be weak delayed f several days. Therefe it is often necessary to hold the test media and observe reactions daily f a week and occasionally longer. The OF reactions in particular may be slow, sometimes adding to the difficulty in making an identification; f exam pie, an ganism that appears to be a glucose oxidizer initially may actually be a "weak glucose fermenter." Recent revisions of the CDC key 2 have discounted the use of the term "weak fermenters," and the ganisms previously included under this heading are now classed as " o x i d i z e r s. " While this change has the advantage of simplifying the interpretation of OF glucose reactions, the suggestion of using fermentative substrates has been eliminated. Whichever way this reaction is interpreted, the technologist is required to select fermentative substrates at the appropriate times. The term "weak f e r m e n t e r " is used in the key presented here w i t h a footnote indicating that the user should consider this group whenever a positive reaction is observed only in the open tube of the OF glucose test. Nomenclature of the Bacteria in the Key The wide spectrum of bacteria included in this key encompasses many familiar and well-established ganisms of clinical significance. It also includes a number of species and groups which have been classified only recently, and the taxonomic position of many of these ganisms is still being debated. All of the ganisms included have been isolated f r o m human sources and are considered to be potentially opptunistic pathogens. Some of the groups have 10. Motility test by the hanging drop m e t h o d. 11. Motility m e d i u m. 12. Nitrate medium (infusion base incubated 48 hours). 13. Nitrate medium (peptone base incubated 48 hours). 14. Nitrite reduction b r o t h. 15. Nutrient gelatin to determine gelatin liquefaction. 16. Phenylalanine deaminase agar. 17. agars f detection of pyocyanin and fluescein (A and B media of King). 18. Simmon's citrate agar.

3 Table III List of CDC Groups of Unclassified Gram-Negative Bacilli -I ike bacteria. Possibly members of the genus possibly of the genus Xanthomonas Vb-2 Vb-3 -like bacteria resembling stutzeri except f a positive arginine reaction HB-1 Eikenella crodens (Bacteroides crodens) HB-5 A group of fermentative microganisms with unifm biochemical characteristics IVe Resemble Alcaligenes species in some respects EF-4 Unnamed fermentative bacteria M-3 M-4 M-4f M-5 M-6 Bacteria that resemble members of the genus lie lie llf llh in iij Nonmotile ganisms which demonstrate weak indole production. Similarities to Flavobacterium species TM-1 Unnamed group of ganisms which have been isolated frequently on Thayer-Martin media inoculated with pharyngeal swabs not been named and are assigned letter-number test, g r o w t h on MacConkey agar and the oxidase symbols at CDC. The nomenclature used in this key reaction. Numbers in parentheses indicate the addi- cresponds to that used in the CDC key. 2 tional figures in this article w h i c h must be consulted Tables I, II and III are provided to clarify the nomenclature used in the d i c h o t o m o u s key. Table I lists those ganisms w h i c h appear in the eighth edit i o n of Bergey's Manual of Determinative Bacteriology* under a new name. The taxonomic position of some of these is still contested by the CDC g r o u p. 1 Achromobacter, f example, is not recognized in Bergey's Manual; rather, it is included in the discussion of Alcaligenes species. At CDC the genus Achromobacter is defined as oxidase-positive peritrichously flagellated gram-negative rods w h i c h are strictly aerobic and attack carbohydrates oxidatively. Alcaligenes species have similar characteristics but are nonsaccharolytic. Similarly the CDC g r o u p suggests that the microaerophilic bacteria fmerly designated HB-1 and later named Eikenella crodens should be recognized as a separate genus f r o m the strict anaerobe Bacteroides crodens. Table IV lists the abbreviations used in Figs Legends f Figs appear on page 553. Use of the Key Fig. 1 shows the reactions of four maj groups of ganisms as divided by the use of the OF glucose to differentiate the ganisms included in these f o u r Table I V - -Abbreviations Used in the Identification Keys (Figs. 1-12) 90% 90% me of strains positive 90% me of strains negative ARG arginine beta beta hemolysis around isolated colonies cd coccoid, diplococcoid and plump bacillary fms db diplobacilli, some cocci in chains dc diplococci diplococci lavender-green colation under heavy Ig ig growth (proteolysis) large lr large rods, cell diameter ao.9 >0.9 (JL /x LYS lysine lysine ORN nithine PD phenylalanine phenylalanine deaminase sht to medium length pale-staining rods srps sy slight slight yellow tan tiny coccoid rods tr TSI Reactions triple sugar iron agar N/N neutral slant and butt NN K/A alkaline slant, acid butt K/N alkaline slant, neutral butt vlg very light growth - Ve 1 Ve 2 Va Ilk type 1 Ilk type 2 LABORATORY MEDICINE VOL. 10, NO. 9, SEPTEMBER

4 OXIDIZERS MacCONKEY OXIDASE OXIDASE I NONOXIDIZERS MacCONKEY OXIDASE - OXIDASE I (2) (2) (2) (3) (4) (5) (6) (V) FERMENTERS MacCONKEY WEAK FERMENTERS MacCONKEY Fig. 1 - OXIDASE - OXIDASE OXIDASE - OXIDASE (8) (9) HB-5 & Enterobacteriaceae (10) Ilk type 1 (11) None (12) *WEAK FERMENTERS are classed as OXIDIZERS in recent publications of the CDC key. Organisms which give a positive reaction in only the open tube of OF glucose media may actually belong in the weak fermenter group. Refer to the discussion in the accompanying text. Ilk type 1 kingii ARGININE CATALASE Brucella species mallei MOTILITY Vibrio ex tquens (1 FLAGELLA) (PINK PIGMENT) Fig. 2 MOTILITY FLAGELLA STAIN OFMANNITOL LYSINE' Acinetobacter calcoaceticus var. anitratum cepacia ARGININE ESCULIN cepacia Ve2 Ve1 *L-lysine decarboxylase, ninhydrin is recommended LABORATORY MEDICINE VOL. 10, NO. 9, SEPTEMBER 1979

5 FLAGELLA STAIN peritrichous polar FLUORESCENT PIGMENT OF MALTOSE "Achromobacter sp. Type 1 GROWTH A T 42 C 'Achromobacter sp. Type 2 fluescens (PYOCYANIN-) putida aeruginosa (USUALLY PYOCYANIN) ARGININE OF MALTOSE LYSINE" cepacia (ONPG ) '- Vb-2 ( & & OF LACTOSE OF LACTOSE NEGATIVE) stutzeri pseudomallei Vb-3 OF SUCROSE Va vesicularis Ilk type 2 Refer t o Table I. *L-lysine decarboxylase, ninhydrin is recommended. Fig. 3 groups. The user of the key is cautioned to recognize that ganisms classified as weak fermenters may appear initially to be oxidizers. Also, in certain instances it may be necessary to enrich the OF basal media by adding t w o drops of rabbit serum to each tube of media to enhance the growth of fastidious ganisms. mallei and Ilk type 1 are the only oxidative gram-negative rods which characteristically do not grow on MacConkey agar and are oxidase negative. As shown in Fig. 2a, they may be differentiated by the arginine dihydrolase test. Ilk type 1 colonies exhibit a deep yellow pigment. Oxidizers which do not grow on MacConkey agar and are oxidase positive (Fig. 2b) can be easily differentiated by Gram stain characteristics, by colonial pigmentation and by the motility and catalase tests. Brucella species are small gram-negative rods, usually coccobacillary, and produce small nonpigmented colonies. Vibrio extquens (Methylamonas) mphologically are curved branched, and the colonies have a pink pigment due to the presence of carotenoid. Oxidizers which grow on MacConkey agar and are oxidase negative can be differentiated first by motility (Fig. 2c). Nonmotile strains of cepacia may be differentiated from/4c/nefo6acferca/coaceticus by flagella stains and OF mannitol reactions. Acinetobacter is characteristically pleomphic on Cram stain, while species show straight slightly curved rods. Lysine decarboxylase separates P. cepacia from the Ve g r o u p. SS Oxidizers which grow on MacConkey agar and are oxidase positive (Fig. 3) include species, -like bacteria and Achromobacter species. Flagella stains are very useful and are sometimes required f final identification of this group of GRAM STAIN cd Bdetella parapertussis Acinetobacter caicoaceticus var. Iwofii (Mima polympha) Ir Bacillus species Fig. 4 LABORATORY M E D I C I N E VOL. 10, NO. 9, SEPTEMBER

6 Ol Ol to I- > CD o > UREA CATALASE BETA HEMOLYSIS PD PD INDOLE *HB-1 (Eikenella crodens) bo vis NITRATE _ MOTILITY dc GRAM STAIN db 1 flavescens I ***Pasteurella phenylpyruvica anatipestifer catarrhal is phenylpyruvica Vibrio extquens Bacillus species GRAM STAIN Ir MOTILITY Bacillus species GRAM STAIN db dc srps tr GRAM STAIN db "Vibrio extquens flavescens WITH ADDED SERUM *HB-1 (Eikenella crodens) Bacillus antfiracis Brucella species osloensis HEMOLYSIS lq beta NITRATE** lacunata NITRATE llf db GRAM STAIN osleonsis nonliquefaciens * * 'Pasteurella anatipestifer lacunata bovis Bacillus cereus * Refer to Table I. ** M. osloensis is 28% nitrate positive. ** P. anatipestifer is shown as urea positive in the CDC key and urea negative in Bergey's Manual. Fig. 5

7 _ cd OF MALTOSE tr "1 maltophilia GRAM STAIN Acinetobacter calcoaceticus var. Iwoffi (gram-negative, coccoid, some rod fms, some chains) Bdetella parapertussis (gram-negative, sht rods) LYS ARGORN- Fig. 6 The three ganisms listed in Fig. 4 are easily differentiated by colony and Gram stain mphology. Acinetobacter species are coccoid, diplococcoid and p l u m p bacillary fms, and occasionally long thick filaments w i t h large swollen areas are seen. Typically, rods ( /xm by ^im) are seen during the logarithmic stage of g r o w t h. Brucella species are tiny coccobacilli ( /xm by /xm) occurring singly, in pairs rarely in sht chains. Bacillus species are large straight rods ( /xm by /xm). These ganisms are usually gram-positive during early growth stages, but are frequently gramnegative in older cultures. Bacillus species are included here and elsewhere in the key because they are occasionally isolated (usually as contaminants) in the clinical labaty and may stain gramnegative. Fig. 5 includes a group of ganisms difficult to identify, requiring careful observations of Gram stain mphology and hemolytic reactions on blood agar Fig. 1. Four maj groups of gram-negative ganisms as divided by OF glucose reactions. Numbers in parentheses refer to the figure numbers of the identification keys. Fig. 2. Identification keys f ganisms with the following characteristics: oxidizers that do not grow on MacConkey agar and are oxidase negative (Fig. 2a); oxidizers that do not grow on MacConkey agar and are oxidase positive (Fig. 2b); and oxidizers that grow on MacConkey agar and are oxidase negative (Fig. 2c). Fig. 3 Identification key f ganisms that are oxidizers, grow on MacConkey agar and are oxidase positive. Fig. 4 Identification key f ganisms that are nonoxidizers, do not grow on MacConkey agar and are oxidase negative. Fig. 5 Identification key f ganisms that are nonoxidizers, do not grow on MacConkey agar and are oxidase positive. Fig. 6 Identification key f ganisms that are nonoxidizers, grow on MacConkey agar and are oxidase negative. Fig. 7 Identification key f ganisms that are nonoxidizers, grow on MacConkey agar and are oxidase positive. Fig. 8 Identification key f ganisms that are fermenters, do not grow on MacConkey agar and are oxidase negative. Fig. 9 Identification key f ganisms that are fermenters, do not grow on MacConkey agar and are oxidase positive. Fig. 10 Identification key f ganisms that are fermenters, grow on MacConkey agar and are oxidase positive. ganisms. A flagella stain should be perfmed on any ganism w h i c h has the characteristics of this group and fails to produce fluescein (fluescent pigment). aeruginosa, fluescens and putida, w h e n grown on appropriate media, are the only ganisms in the key that produce a fluescent pigment w h e n viewed under ultraviolet light. O f these three ganisms, P. aeruginosa is the most frequent isolate but, since P. fluescens and P. putida are occasionally isolated f r o m clinical specimens, confirmaty tests should be p e r f o r m e d. Demonstration of the presence of pyocyanin may be used to confirm the identification of P. aeruginosa; but, when this pigment is absent, P. fluescens and P. putida are possibilities. As shown in Fig. 3, the best tests to differentiate the three ganisms are growth at 42 C and gelatin liquefaction. Susceptibility patterns are also helpful in recognizing these ganisms. P. aeruginosa generally is susceptible to carbenicillin and resistant to kanamycin and tetracycline. This pattern generally is reversed w i t h P. putida and P. fluescens.5 Legends f Figures Fig. 11 Identification key f ganisms that are weak fermenters, do not grow on MacConkey agar and are oxidase positive. Fig. 12 Identification key f ganisms that are weak fermenters, grow on MacConkey agar and are oxidase positive. (heart infusion agar containing 5% defibrinated rabbit b l o o d is recommended). species are typically sht, p l u m p rods ( /xm by /xm) approaching coccoid shape, usually in pairs sht chains, and pleomphic fms are often seen. species are spherical cells seen in pairs masses with flattened adjacent sides. The Gram stain mphology of Brucella species and Bacillus LABORATORY MEDICINE VOL. 10, NO. 9, SEPTEMBER

8 ** osloensis M-3 Alcaligenes faecalis phenylpyruvlca * Alcaligenes odans F R UITY ODOR * Alcaligenes denitrificans OF X Y L O S E N03-*gas N02-^gas putrefaciens H2S(TSI) diminuta pseud oalcaligenes Refer to Table I. Confidence level of branchpoint less than 90%. alcaligenes * denitrificans M-4 (rarely osloensis) 1-2 polar flagella M-5 - ** OF L E V U L O S E "Related V i b r i o s " GROWTH at 42 C 'SLIGHT YELLOW PIGMENT 1-2 polar flagella microaerophilic * Vibrio fetus (Campylobacter species) M-6 'CATALASE CITRATE PD UREA MOTILITY MlbN03 N 0 2 *Achromobacter xylosoxidans Ilia N 0 3 N 0 2 UREA gas IVe SS Bdetella bronchiseptica Comamonas terrigena polar t u f t w i t h me than 2 flagella F L A G E L L A STAIN peritrichous flagella M-4f

9 NITRATE Haemophilus vaginalis (Cynebacterium vaginale) (INDOLE & C A T A L A S E -, TSI A/A) INDOLE NG N/N A/AK/A T! [ HB-5 (TSI A/A, CATALASE - ) Actinobacillus actinomycetem<omitans Bacillus species (Both CATALASE ) Haemophilus aphrophilus (CATALASE - weak) 8 Nonoxidizers that grow on MacConkey agar and are oxidase negative (Fig. 6) can be differentiated by OF maltose reaction and Gram stain mphology as described. The large group of nonoxidizers in Fig. 7 is first divided by the motility test into a subgroup of species and a larger heterogenous group of motile ganisms possessing flagella. Motility tests commonly employed f the identification of the Enterobacteriaceae are not satisfacty. The reader should refer to references cited f me detailed discussion on motility testing. Comamonas terrigena is the only ganism in Fig. 7 which possesses polar tufts of three me flagella; this is the single most imptant identifying characteristic of this ganism. The ganisms with peritrichous flagella are differentiated by urease prod u c t i o n, OF-xylose reaction and the reduction of nitrates and nitrites. The species included in Fig. 7 are characterized by one to t w o polar flagella. putrefaciens is easily recognized by the production of H_,S on TSI agar. The other four species are not easily separated. Positive gelatin liquefaction separates diminuta, but only about 75% exhibit this characteristic. denitrificans is listed in Bergey's Manual1'' as species incertae sedis and is not described. There is no clear differentiation of this ganism from alcaligenes by the reactions used in this key. The Campylobacter species are unique compared to the other ganisms included in Fig. 7 because of their microaerophilic properties. Best growth is obtained in a reduced oxygen atmosphere. Occa- sionally some strains grow slightly under aerobic conditions. Campylobacter species grow well in thioglycollate b r o t h, and tests f growth at 42 C should be perfmed in this m e d i u m. Nitrate reduction, indole, TSI and catalase reactions differentiate the four fermenters in Fig. 8. Accding to Bergey's Manual,B Haemophilus vaginalis does not belong in the genus Haemophilus but its taxonomic position is not yet settled. Because it is widely identified by this name, it still appears in the manual. In the group of fermenters that are oxidase positive and fail to grow on MacConkey agar (Fig. 9), species are easily separated from other ganisms in this group by Gram stain mphology. Cardiobacterium hominis is separated by a negative catalase test. CTA carbohydrate substrates are used f identification of the species. Loeffler slants are useful f demonstrating the yellow pigment of the following species: N. lactamicus, N. sicca, N. flava, N. subflava, and N. mucosa. species was previously described. Pasteurella anatipestifer appears in Sergey's Manual" under species incertae sedis and is "probably a species." Pasteurella "gas" is possibly a strain of Pasteurella multocida which is urea positive and usually produces gas f r o m glucose. The nithine decarboxylase reaction is negative, separating it from Pasteurella pneumotropica accding to the CDC key. Urea, indole, Moeller decarboxylase and carbohydrate fermentations speciate the nitrate-positive ganisms in Fig. 9. Fermenters which grow on MacConkey agar and are oxidase negative (Fig. 1) include the Enterobacteriaceae and a group of fermentative ganisms designated as HB-5. The latter may show light growth on MacConkey agar and produce acid in the slant and butt on TSI. Indole production by HB-5 is demonstrable only after extraction w i t h xylol. Catalase, LABORATORY MEDICINE VOL 10. NO. 9, SEPTEMBER

10 10 '* ' flava I sicca lactamicus EF-4 tr-cd EF-4 G R A M STAIN CTA LACTOSE dc 'Pasteurella gallinarum Bacillus species SUCROSE MALTOSE tr-cd G R A M S T A I N HB-5 Pasteurella multocida LYSINE Pasteurella ureae Bacillus species SUCROSE mucosa dc INDOLE UREA Vibrio cholera ' '* Pasteurella haemoly tica HB-5 (CATALASE weak ) Actinobacillus equui SUIS Actinobacillus ESCULIN I I - slow Vibrio cholera A N T 1,0:1 LACTOSE Aeromonas shigelloides * * Non-cholera Vibrio Vibrio alginolyticus Aeromonas hydrophila ARGININE SUCROSE Vibrio parahaemoly ticus MANNITOL Chromobacterium violaceum (oxidase weak ) Providencia species LYSINE Motile by polar flagella except Chromobacterium (polar and lateral flagella) and Providence (peritrichous flagella). Refer to text. EF-4 (CATALASE) LEVULOSE SUCROSE - TREHALOSE Actinobacillus lignieresii UREA 'MOTILITY Pasteurella pneumotropica ORINITHINE Pasteurella "gas" (multocida) INDOLE Refer to Table I. N. mucosa usually produces a yellow slight yellow pigment on Loeffler media. Mucoid characteristics vary with the media used. subflava - CTA SUCROSE CTA LEVULOSE Cardiobacterium hominis CATALASE NITRATE

11 dc GRAM STAIN r( deb) SPECIAL GROWTH REQUIREMENTS CTA DEXTROSE I CTASUCROSE CTA MALTOSE - NITRATE YELLOW PIGMENT and - CATALASE INDOLE YELLOW PIGMENT and gonrhoeae - CTA LACTOSE mucosa Ilk type 1 PIGMENT slight yellow very yellow - CTA LEVU LOSE I lac tarnicus MALTOSE Flavobacterium Flavobacterium meningosepticum sp. lib meningitidis GROWTH IN * - NUTRIENT BROTH flava subflava sicca " pert lava TM-1 kingii SUCROSE MALTOSE lie Hi ESCULIN lie llh Refer to Table II. Fig. 11 INDOLE and GLUCOSE LACTOSE SUCROSE MALTOSE PIGMENT slight yellow very yellow Ilk type 1 Ilk type 2 Flavobacterium meningosep ticum Flavobacterium sp. lib Fig. 12

12 urea, citrate, arginine, lysine, nithine and H2S (TSI) are negative. Few carbohydrates are fermented. Providence species are included w i t h fermenters that grow on MacConkey agar and are oxidase positive (Fig. 10) because members of this species occasionally give a weak positive oxidase reaction. Organisms in this group which may resemble members of the family Enterobacteriaceae are easily separated by their positive oxidase reaction; this suggests the need f routine use of the oxidase test even w h e n isolates appear to be enteric ganisms. A group of -like ganisms designated Ilk type 1 are the only weak fermenters which are oxidase negative and fail to grow on MacConkey agar (Fig. 1). Weak fermenters which do not grow on MacConkey agar and are oxidase positive (Fig. 11) include two groups of ganisms. species are differentiated f r o m Flavobacterium species and other fermenters by Gram stain. species are gramnegative cocci occurring singly but me often in pairs. O n Gram stain, flavobacteria are long thin gram-negative rods, usually with bulbous ends. Ilk type 1 ganisms are long rods. Gram stains of kingii and TM-1 demonstrate mphologic characteristics as described earlier f species. Indole production is an imptant characteristic of Flavobacterium species; however, the reaction is weak and the indole must be extracted with xylol befe addition of Ehrlich-Boehme reagent. The four ganisms classed as weak fermenters that grow on MacConkey agar and are oxidase positive (Fig. 12) are differentiated by indole, gelatin, pigment and carbohydrate fermentation reactions. Ilk type 1 is easily differentiated from Ilk type 2 in that the fmer fails to ferment any of the four carbohydrates shown and the latter ferments all of t h e m LABORATORY MEDICINE VOL. 10, NO. 9, SEPTEMBER 1979 The key presented here contains over 100 species of bacteria which are subdivided into groups, providing adequate infmation f presumptive identification, although it is recommended in many i n stances that further confirmaty tests be p e r f o r m e d. The primary advantages of the key are to quickly eliminate certain species from a group and to allow the rapid selection of tests to be p e r f o r m e d. Once a preliminary identification is achieved, the microbiologist can refer to detailed descriptions and select additional tests as needed. The ganisms included in the key fm a large heterogenous group, with the only c o m m o n characteristic being their gram-negative staining reaction. Many of the ganisms show only subtle differences in biochemical reactions and mphology. Publications discussing the numbers of ganisms received at CDC indicate that many of the groups species presented have been characterized by studies on fewer than 50 ganisms. Obviously, changes in taxo n o m y may be expected in the f u t u r e, and clinical microbiologists must continue to update their identification schema. The challenge f the labaty wker is to make very careful observations and identify ganisms to the fullest extent possible, referring to qualified reference state labaties when necessary f identification of unusual ganisms. References 1. Lennette. E.H., Spaulding, E.H., and Truant. J.P., Manual of Clinical Microbiology. 2nd edition. Washington. D.C.. American Society f Microbiology. 2. Weaver, R.E., Tatum, H.W.. and Hollis, D.G The Identification of Unusual Pathogenic Gram Negative Bacteria (E.O. King). Atlanta, Gegia, Special Bacteriology Section, Bacteriology Division. Center f Disease Control 3. Wolf, P L, Russell, B., and Shimoda, A., Practical Clinical Microbiology and Mycology. New Yk, John Wiley and Sons, pp Frankel, S.. Reitman. S.. and Sonnenwirth. A C 1970 Gradwohl's Clinical Labaty Methods and Diagnosis. 7th edition. St. Louis, C.V Mosby, pp Finegold, S.M., Martin, W.J., and Scott, E.G Bailey and Scott's Diagnostic Microbiology. 5th edition. St. Louis. C.V. Mosby. 6. Buchanan, R.E., and Gibbons, N.E Bergey's Manual of Determinative Bacteriology. 8th edition. Baltime, Williams and Wilkins In Fig. 10, "non-cholera v i b r i o " refers to strains of Vibrio cholera which belong to antigenic O groups other than the 0 : 1 group to which cholera vibrios belong." Classic cholera is always associated with strains belonging to serogroup 0 : 1. Strains belonging to the other groups produce a milder f o r m of the disease. Discussion