Comparison of Four Selective Agars for the Isolation of

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1977, p pyright C 1977 American Society for Microbiology Vol. 33, No. 5 Printed in U.S.A. mparison of Four Selective Agars for the Isolation of Pseudomonads A. HART* AND PATRICIA E. KITE School of Pharmacy, Liverpool Polytechnic, Liverpool, L3 3AF, United Kingdom Received for publication 26 July 1976 Significant differences were found between Dettol(chloroxylenol) agar, nalidixic acid-cetrimide agar, and two other cetrimide-containing agars used for the isolation of small numbers of pseudomonads. Both the British Pharmacopoeia (BP) (1973, 1) ent broth no. 2 (Oxoid Ltd., London) plus cetrimide (.3%, wt/vol), and to 1 ml of fluid and the United States Pharmacopoeia (USP) (19th ed., 15) recommend the use of cetrimidecontaining media for the isolation of pseudomo- London) and incubated at 3 C. Subcultures soybean-casein digest medium (Oxoid Ltd., nads and Pseudomonas aeruginosa, respectively, from nonsterile pharmaceuticals. How- h of incubation, onto plates containing cetrim- were made from the broths, after 24, 48, and 72 ever, the detrimental effect of cetrimide-containing media against small numbers of pseu- ckeysville, Md.), Dettol (chloroxylenol;.3%, ide agar (BP formula [1]), Pseudosel (BBL, domonads has been reported (6, 8, 1, 13, 14). vol/vol) (Reckitt and lman Pharmaceutical This report describes a comparison of Dettol Division, Hull, U.K.) in nutrient agar (Oxoid (chloroxylenol) agar and nalidixic acid-cetrimide agar for the isolation of small numbers of cetrimide (.2%, wt/vol) agar similar to that Ltd., London), and nalidixic acid (15,ug/ml)- pseudomonads with the two cetrimide-containing agars of the BP and the USP (1, 15). cubated at 38 C and examined every 24 h for of Goto and Enomoto (6). All plates were in- The pseudomonads used in this study were the presence of bacterial growth, for a total selected from the classification for these organisms outlined in Bergey's Manual ofdetermi- examined by Gram stain and an oxidase test. incubation time of 72 h. Resulting growth was native Bacteriology (5). All organisms were After inoculation and subsequent incubation grown at 3 C, maintained on nutrient agar of fluid soybean-casein digest medium with the slopes, and stored at 4 C. The strains used 29 Pseudomonas strains tested, growth occurred with all strains, although in some in- were: group I-P. aeruginosa ATCC 927, NCTC 675, NCTC 7244, NCTC 874, and stances (e.g., P. cepacia, P. lemoignei, P. facilis, and Pseudomonas sp. NCIB 2581) abun- NCTC 6749, P. putida NCIB 115, NCIB 9494, NCIB 1432, NCIB 8249, and NCIB 8859, P. dant growth was only apparent after 72 h of fluorescens NCTC 169, P. chlororaphis NCIB incubation at 3. Only seven strains, P. 9392, P. aureofaciens NCTC 1686, P. syringae aeruginosa strains ATCC 927, NCTC 675, ATCC 1931, P. stutzeri NCTC 1475, and P. and NCTC 6749, P. putida strains NCIB 115 alcaligenes NCIB 9945; group II-P. pseudoalcaligenes NCIB 9946 and P. cepacia NCTC faciens, showed abundant growth in cetrimide and NCIB 1432, P. chlororaphis, and P. aureo- 1661; group III-P. lemoignei NCIB 9947, P. broth over the same incubation period. Visual testosteroni NCIB 8955, P. acidivorans NCIB assessment of growth in cetrimide broth was 9681, P. facilis ATCC 11228, and P. palleronii difficult because of the turbidity produced by ATCC 17724; group IV-P. maltophilia NCIB.3% (wt/vol) cetrimide, as reported previously 921 and P. diminuta NCIB 9393; addenda-p. (8). Calculated variance ratio F(5%) = fragi NCTC 1689, P. synxantha NCTC 1696, (critical value F5% = 4.45) indicated that there Pseudomonas sp. NCIB 2581, and Pseudomonas sp. NCIB 886. Methods used for the isola- the strains in cetrimide and fluid soybean-ca- is a significant difference between the growth of tion of pseudomonads were based on those outlined in the microbial contamination test of the domonas strains on various selective agars sein digest broths. Results of incubating Pseu- BP (1) for pseudomonads and in the microbial after prior incubation in cetrimide broth or limit test of the USP (15) for P. aeruginosa. fluid soybean-casein digest medium indicated Small numbers of viable cells (i.e., 1 to 5) of that the chance of detecting a greater number the test organisms prepared from a 24-h culture of Pseudomonas strains on cetrimide agar, in nutrient broth (Oxoid Ltd., London) were Pseudosel, and Dettol agar is enhanced if the added to 1 ml of cetrimide broth, i.e., nutri- primary incubation of the organism is carried 129 Downloaded from on May 12, 218 by guest

2 _*r 121 NOTES APPL. ENVIRON. MICROBIOL. C14 I x cls. c bi) 3._ csi o CO. 4-a X bd C-4 bio X. " cs X _4.. L. I I _ cu _ Q I cli._ X III -.C III cis v I= C- n. C. c.. C4 o *- I IIII C.- c Oc ; -.. h C- 4 2 e toooto CO. COC:O tq E- o U_ r QOZZZ m -E Z m ")'cq - m m u' -!4 - z r. co m V Z V = *4 Zt tz I-- Z, COD~ : Downloaded from on May 12, 218 by guest ; "I S (:- CZ P- I. ',,

3 VOL. 33, 1977 NOTES 1211 ± Nlo I. t C t a _ c Q Cl ± O _:; ~ ~~ a a CC. N. C". I I II k CS O IIO ~~~ ±- t t E Cl bc.n 11 1 ()s b, CC ~~~~~~~~~~~~~~~~~~~~b Cl I I Cl II -o.= CCZzZmmm cc C." bc. ~~~~~~~~~~~c" C C I± I. Cj~~~~~~~~~~~~ C)~E - - C)cqo " ~ ~~UUU- cc 4J qe c 'ZZZ C) ECO CC Cl IIZZ Z - I C. C.-Z"?:t5 CC~~~ -~~~~ -~~~ CC'C)t CC~~~~~ ~CLCaIIZ a a. I.ạ.Q..'aCC Downloaded from on May 12, 218 by guest

4 12 12 NOTES APPL. ENVIRON. MICROBIOL. O N ~ E q I E 3N t I I I.>X 4 I I 3 o= N.E F It O.= 3 v t cs II II r e l>,o o > X Q U: CS vo~~~~~~~~~~~~4 eesq H H Z o o ts t e 2. Ol O O O Z Z Z Z Z cl o u< O,C4X;PXX 4 o:~~~ Downloaded from on May 12, 218 by guest

5 VOL. 33, 1977 NOTES 1213 C,o E. - WC C,o L. *.. 4 C -o 4C L. L.b C.) C., m z.. r. r. I.. CO._ 4-4._._ c- 1- I.3.. es.. CO.... eq.. - C-I eq t. eq s... - I I I I o o- o COX CD E- E- E- <zzz ECCUe5 o) ot o o ix t -e C c co). b13 -Uq E~ 3 <5 =5 o CỌ _ 8=-s X cuq C.)S X X._.) Q~.. 5z- C.) -._ oc1 out in fluid soybean-casein digest medium rather than in cetrimide broth. Results for those Pseudomonas strains isolated on the selective agars after incubation in cetrimide broth or fluid soybean-casein digest medium are reported in Tables 1 through 4. The method of primary enrichment of microorganisms in nonselective media has been advocated (11), and our work appears to support this recommendation. Nalidixic acid-cetrimide agar has been used for the isolation of P. aeruginosa (6, 13). However, our results show little or no difference in the number of strains isolated when nalidixic acid-cetrimide agar was used, irrespective of whether primary enrichment was performed in cetrimide broth or fluid soybeancasein digest medium. The use of Dettol agar has been preferred to cetrimide-containing agars for the isolation of P. aeruginosa (7). Our results indicate that more Pseudomonas strains were detected on Dettol agar than with the other three selective agars after primary enrichment in fluid soybean-casein digest medium. Many of the strains detected on the four selective agars belonged to group I in Bergey's classification. Occasionally P. acidivorans (group II), P. synxantha (addenda), and P. fragi (addenda) were detected on the isolating agars, but only after primary incubation had been carried out in fluid soybean-casein digest medium. None of these organisms was isolated after primary incubation had been performed in cetrimide broth. P. aeruginosa NCTC 7244, P. stutzeri, P. pseudoalcaligenes, P. cepacia, P. lemoignei, P. testosteroni, P. facilis, P. acidivorans, P. maltophilia, P. diminuta, Pseudomonas sp. NCIB 2581, and Pseudomonas sp. NCIB 886 were never isolated on any of the four selective media. Calculated variance ratio F(5%) = (critical value F5% = 3.2) indicated that there is a significant difference among the four selective agars for the isolation of Pseudomonas strains. However, calculated variance ratio F(5%) = 3.35 (critical value F5% = 3.59) indicated that there is a small but not significant difference in the overall effect of increasing incubation times of the selective agars. Therefore, since pseudomonads and in particular P. aeruginosa have been isolated from contaminated pharmaceutical products (12), and, in addition to P. aeruginosa, the presence of other pseudomonads (e.g., P. putida, P. multivorans, P. maltophilia, and P. synxantha) is considered objectionable in nonsterile pharmaceutical products (2-4), it is essential that suitable microbial tests be available in order that a reproducible quality control procedure may be Downloaded from on May 12, 218 by guest

6 1214 NOTES adopted for the determination of these organisms. Fluid soybean-casein digest medium is a nonselective medium and appears to be more suitable for promoting the initial growth of Pseudomonas strains than the selective cetrimide broth. Whereas none of the four selective agars tested appeared to be suitable for the isolation of these organisms, recent work (9) indicates the inhibition ofpseudomonas species on selective agars that are recommended for the isolation of these organisms, and therefore, in our opinion, the use of selective media for the isolation of small numbers of pseudomonads must be regarded with caution. We thank K. McKelvie of the Department of Mathematics, Liverpool Polytechnic, for his help with the statistical analysis of the results. LITERATURE CITED 1. British Pharmacopoeia Her Majesty's Stationary Office, London. 2. Bruch, C. W Microbiological products of topical quality. Types vs numbers of micro-organisms. Drug smet. Ind. 19:26-3, Bruch, C. W Possible modifications of USP microbial limit tests. Drug smet. Ind. 11:32-37, Bruch, C. W Objectionable micro-organisms in non-sterile drugs and cosmetics. Drug smet. Ind. 111:51-54, Doudoroff, M., and N. J. Palleroni Genus I. Pseudomonas, p InR. E. Buchanan and N. APPL. ENVIRON. MICROBIOL. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Williams., Baltimore. 6. Goto, S., and S. Enomoto Nalidixic acid-cetrimide agar. A new selective plating medium for the selective isolation of Pseudomonas aeruginosa. Jpn. J. Microbiol. 14: Gould, J. C., and J. W. McLeod A study of the use of agglutinating sera and phage lysis in the classification of strains of Pseudomonas aeruginosa. J. Pathol. Bacteriol. 79: Hart, A., K. E. Moore, and D. Tall A comparison of the British Pharmacopoeia (1973) and United States Pharmacopoeia (XIX revision) methods for detecting pseudomonads. J. Appl. Bacteriol. 41: Hecker, W., M. T. Heintz, X. Buhlmann, and M. Gay Isolation and differentiation of clinically important Pseudomonas species in the microbiological testing of pharmaceutical products. Pharm. Ind. 38: Highsmith, A. K., and R. L. Abshire Evaluation of a mosprobable-number technique for the enumeration of Pseudomonas aeruginosa. Appl. Microbiol. 3: Hoadley, A. W., and C. M. Cheng The recovery of indicator bacteria on selective media. J. Appl. Bacteriol. 37: Kallings, L. O.,. Ringertz, L. Silverstolpe, and F. Enerfeldt Microbiological contamination of medical preparations. Acta Pharm. Suecica. 33: Lilly, H. A., and E. J. L. Lowbury Cetrimidenalidixic acid agar as a selective medium for P. aeruginosa. J. Med. Microbiol. 5: Lowbury, E. J Improved culture methods for the detection ofp. pyocyanea. J. Clin. Pathol. 4: United States Pharmacopoeia, 19th ed Mack Publishing., Easton, Pa. Downloaded from on May 12, 218 by guest