JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1975, p. 89-95 Copyright ( 1975 American Societv for Microbiology Vol. 1, No. 1 Printed in U.S.A. New Satellitism Test for Isolation and Identification of Haemophilus influenzae and Haemophilus parainfluenzae in Sputum NORA M. EVANS, S. M. BELL,* AND D. D. SMITH Bacteriology Laboratory, The Prince of Wales Hospital, Randwick, N.S. W., Australia Received for publication 1 November 1974 A new satellitism test designed to facilitate the isolation and identification of Haemophilus influenzae and Haemophilus parainfluenzae is described. In the basal medium, nicotinamide adenine dinucleotide is incorporated at a concentration of 0.2 ug per ml, an amount adequate for H. influenzae but not for H. parainfluenzae. Two disks are placed on the surface of the medium, one disk being impregnated with 60 Mg of hemin and the other with 15 Mg of nicotinamide adenine dinucleotide. Under these conditions, H. influenzae strains grow around the hemin disk only and the majority of H. parainfluenzae grow around the nicotinamide adenine dinucleotide disk. This procedure gives results which are more clear cut than other establised methods, especially in sputum culture. The traditional method of identifying Haemophilus influenzae is the demonstration of satellite growth of that organism around a colony of Staphylococcus aureus on blood agar but not on the basal medium without added blood (13). Despite the continued and widespread popularity of this method, a number of limitations have been recognized. For example, certain preparations used as the basal medium may contain traces of heme, so that the organism's requirement for this factor would not be demonstrated (2, 9, 14, 15). An earlier investigation (5) showed that over 30% of strains of H. influenzae would for this reason have been wrongly identified as Haemophilus parainfluenzae. On the other hand, using blood as a source of hemin and a hemin-free but otherwise unsuitable basal medium, some strains of H. parainfluenzae appeared to require both hemin and nicotinamide adenine dinucleotide (NAD) and therefore would have been mistakenly identified as H. influenzae. In this paper a new satellitism test is described. It is designed to facilitate and improve the reliability of identification of strains of H. influenzae and H. parainfluenzae and is particularly applicable and useful in the examination of specimens from the respiratory tract. MATERIALS AND METHODS Medium. The basal medium contained no detectable hemin and supported the growth of H. influenzae with the addition of hemin and NAD. It also supported the growth of H. parainfluenzae with the addition of NAD alone (N. M. Evans, D. D. Smith, and A. J. Wicken, J. Med. Microbiol., in press). The medium contained (wt/vol): proteose peptone (Difco), 2%; sodium chloride, 0.6%; sodium dithionite (Na2S2O4+H2O), 0.01%; and Oxoid ionagar no. 2, 1.2%. After autoclaving at 10 lb/in2 (0.7 kg per cm2) steam pressure for 20 min, glucose (0.2%). thiamin hydrochloride (1.0 ug per ml), sodium oleate (4.8,g per ml), and NAD (0.2 Mg per ml) (all final concentrations) were added to the medium. Each 85-mm dish contained 20 ml of medium; the prepared plates can be stored for 1 month at 4 C. Preparation of disks. Aqueous stock solution of NAD (#NAD, Sigma Chemical Co) and hemin (Koch- Light) each contained 2,000 ug per ml. Paper disks of 15-mm diameter (Ekwip Filter Paper, Industrial Equipment, Australia) or 12-mm diameter (W. Whatman and R. Balston Ltd., England) were impregnated with these solutions so as to contain approximately 15 gg of NAD or 60 ug of hemin. Dried paper disks containing NAD were stable for at least 2 months at 4 C. Moist disks containing hemin were used and could be stored for at least 1 month at 4 C in a 1-oz screw-capped bottle. Dried hemin disks were not used since they deteriorated on storage. Strains. A total of 191 strains were examined, including nine reference strains: H. influenzae no. NCTC4560 (noncapsulate), no. NCTC8465 (type a), no. NCTC7279 (type b), no. NCTC8469 (type c), no. NCTC8470 (type d), no. NCTC10479 (type e), no. NCTC8473 (type f), and H. parainfluenzae no. NCTC10665 and no. ATCC9796. The remaining 182 strains were freshly isolated from respiratory specimens, blood culture, and cerebrospinal fluid. Twenty of these were encapsulated strains of H. influenzae belonging to Pittman type b. All strains were identi- B9
90 EVANS, BELL, AND SMITH J. CLIN. MICROBIOL. fied on microscopic and cultural appearances together with NAD and hemin requirements determined by standard satellitism methods (5, 15). Inoculation of plates. A single colony from a 15- to 18-h plate culture was picked with a wire loop and spread over a small segment (7); without resterilizing, the flat side of the loop was then used to spread the inoculum over the entire surface of the plate using a series of parallel streaks at right angles to the original inoculum. In this way, the whole surface of the plate was covered four times. Disks of NAD and hemin were then placed on the surface of the inoculated plate with their edges 15 mm apart, approximately 55 mm from and opposite to the original inoculum. For primary cultures of sputum, the same inoculating procedure was adopted, but using instead of a colony a portion of saline-washed purulent material. Incubation of cultures. All satellitism plates were examined after 15 to 18 h of incubation in an atmosphere of 5%3 CO2 in air (vol/vol) in a McIntosh and Fildes jar at 37 C. RESULTS Table 1 shows the satellite or other growth pattern of the 191 strains of Haemophilus, when examined by the new satellitism test, together with the identity of these strains. The strains of Haemophilus examined showed three patterns of growth. (i) Growth near the hemin disk only. The 97 strains, requiring both hemin and NAD and including all type cultures of H. influenzae and type b strains, grew in the vicinity of the hemin disk. Of these, 94 strains grew in a circular area, measuring about 30 mm in diameter with the hemin disk at its center (Fig. 1); with the remaining three strains, growth was restricted to a sector near the hemin and opposite the NAD disk. The NAD required by these few strains was determined as 1.0 Ag per ml and not 0.2 gg per ml characteristic of the majority of strains of this species (Evans et al., J. Med. Microbiol., in press). All 97 strains with this growth pattern presented microscopic and colonial appearances characteristic of H. influenzae (encapsulated or TABLE 1. Distribution of growth of H. influenzae and H. parainfluenzae on the new satellitism medium Growth pattern No. of strains Disk Region of growth H. in- H. parainflueneae fluenzae Hemin Circular area 94 O Sector 3 O NAD Circular area O 87 Neither Whole surface 7 noncapsulate) and required both hemin and NAD as determined by established methods (5, 15). (ii) Growth around the NAD disk only. Eighty-seven strains showed satellite growth around the disk containing NAD only (Fig. 2), and zone sizes of these strains varied, the diameter measuring from 30 to 45 mm. These strains were identif ied as H. parainfluenzae, requiring between 1 and 5 gg of NAD per ml. The diameter of the zone of growth varied inversely to the amount of NAD required. (iii) Growth over the whole surface. The seven remaining strains shown to require only NAD grew over the whole surface of the plate with no apparent increase in growth in the vicinity of either disk. These strains were also identified as H. parainfluenzae but with a low NAD requirement of 0.2,g per ml. Isolation of H. influenzae from mixed cultures. The nondescript appearance and small colony size of H. influenzae on blood agar makes its isolation difficult and time consuming. This is particularly so when the organism is sought in the midst of mixed flora characteristic of a sputum culture. Figure 3 shows the distinct zone of growth of a strain of H. influenzae around the hemin disk on a plate inoculated directly with a specimen of sputum. This direct satellitism test overcame the difficulty of isolating H. influenzae from sputum when it also contains H. parainfluenzae. As both species are identical in microscopic and colonial appearances, colonies selected for the usual satellitism test could be either H. influenzae or H. parainfluenzae. This satellitism test, as shown in Fig. 4, will readily demonstrate the presence of either or both species in a mixed culture. DISCUSSION The method described in this paper was introduced primarily to assist in the identification and isolation of H. influenzae and H. parainfluenzae, particularly in sputum. The pathogenic significance of H. influenzae is well established, but identification and isolation, especially of noncapsulate strains of H. influenzae and its differentiation from H. parainfluenzae, is time consuming and presents technical difficulties (14). However, since there is no evidence that H. parainfluenzae can cause such infections as are associated with H. influenzae, the distinction of the two species is essential. The satellite growth of Haemophilus species around a colony of Staphylococcus can be attributed not only to NAD but also to catalase, which is produced by staphylococci and may
VOL. 1, 1975 substitute for hemin in the growth of H. influenzae (3, 11). Furthermore, not all organisms showing satellite growth in the vicinity of staphylococci belong to the genus Haemophilus (12). The use of yeast extract may also be criticized as a source of NAD because it may contain heme compounds (8, 10). Cooper and Attenborough (4) found satellitism tests were too unreliable and proposed that hemin and NAD requirements be determined in a fluid medium. The disadvantages of such a technique are the need for pure cultures and the NEW TEST FOR H. INFLUENZAE ISOLATION heavy inocula of between 107 and 108 cells required to initiate growth of some Haemophilus species in fluid media (8). The plate method described by Everall (7) introduced certain improvements, such as the use of heminimpregnated strips instead of blood added to the basal medium, but the limitations of this method were the possibility of contaminating traces of hemin in certain batches of nutrient agar and also in its use of yeast extract as a source of NAD. The new test described in this paper elimi- 91 FIG. 1. The growth of a typical strain of H. influenzae around the hemin disk (black) on a plate containing 0.2 gg of NAD per ml.
92 EVANS, BELL, AND SMITH J. CLIN. MICROBIOL. nates these potential sources of error by using a hemin-free basal medium and purified hemin and NAD (5). The medium also contains thiamin and glucose which are essential for abundant growth of Haemophilus species, and certain inhibitors in agar and peptone are neutralized by the inclusion of sodium oleate and the reducing agent dithionite (6). In this satellitism test, the amount of NAD incorporated in the basal medium allows the growth of all the strains of H. influenzae around the hemin disk only. With three out of 97 strains of H. influenzae, the zone of growth was limited to a sector around the hemin disk and opposite the NAD disk, and this pattern was found to be characteristic of those strains which had a higher NAD requirement, that is, 1,ug per ml rather than 0.2 Ag per ml (Evans et al., J. Med. Microbiol., in press). Most strains of H. parainfluenzae with their higher NAD requirement, namely, 1 to 5 zg per ml, grow around the disk supplying additional NAD. The introduction of a quantitative element into testing has in our experience given results that are easier to read than with other methods which are purely qualitative. FIG. 2. The growth of a typical strain of H. parainfluenzae around the NAD disk (white) on a plate containing 0.2,ug of NAD per ml.
VOL. 1, 1975 NEW TEST FOR H. INFLUENZAE ISOLATION 93 FIG. 3. A zone of growth around the hemin disk on a plate culture of a sputum containing other flora, including Streptococcus viridans and Neisseria. A further advantage is that H. influenzae can often be identified in primary cultures of clinical specimens even when H. parainfluenzae is also present. H. influenzae will grow around the hemin disk only and H. parainfluenzae around the NAD. This differentiation cannot be achieved using the three disks or strips containing NAD, hemin, and NAD plus hemin (Oxoid or Baltimore Biological Laboratories) on a basal medium without added NAD. In that test, H. influenzae satellites around the NAD plus hemin disk only and H. parainfluenzae around the NAD as well as the NAD plus hemin disk and thus, in a mixed culture of the two species, it would not be possible to detect the presence of H. influenzae. Another incidental advantage is that Streptococcus viridans and Streptococcus pneumoniae, which are both commonly present in sputum, are inhibited on this basal medium because of the inhibitory effect of the oleate (1), and also because of its lack of enrichment with blood. This test was not designed or intended to determine the requirements of all species of Haemophilus but has been found economical and accurate in the preliminary isolation and
94 EVANS, BELL, AND SMITH J. CLIN. MICROBIOL. FIG. 4. The growth obtained on a satellitism plate inoculated with a mixture of a strain of H. influenzae, which has grown around the hemin disk (black), and a strain of H. parainfluenzae, which has grown around the NAD (white). identification of H. influenzae and H. parainfluenzae in the bacteriological examination of sputum. LITERATURE CITED 1. Avery, 0. T. 1918. A selective medium for B. influenzae. Oleate-hemoglobin agar. J. Amer. Med. Ass. 71:2050-2051. 2. Biberstein, E. L., and M. Gills. 1961. Catalase activity of Haemophilus species grown with graded amounts of hemin. J. Bacteriol. 81:380-384. 3. Brumfitt, W. 1959. Some growth requirements of Haemophilus influenzae and Haemophilus pertussis. J. Pathol. Bacteriol. 77:95-100. 4. Cooper, R. G., and I. D. Attenborough. 1968. An indicator method for the detection of bacterial X and V factor dependence. Aust. J. Exp. Biol. Med. Sci. 46:803-806. 5. Evans, N. M., and D. D. Smith. 1972. The effect of the medium and source of growth- factors on the satellitism test for haemophilus species. J. Med. Microbiol. 5:509-514. 6. Evans, N. M., and D. D. Smith. 1974. The inhibition of Haemophilus influenzae by certain agar and peptone preparations. J. Med. Microbiol. 7:305-310. 7. Everall, P. H. 1953. A plate method for demonstrating the growth factor requirements of the genus Haemophilus. J. Med. Lab. Technol. 11:181-184. 8. Gilder, H., and S. Granick. 1947. Studies on the hemophilus group of organisms. Quantitative aspects of growth on variotus porphin compounds. J. Gen. Physiol.
VOL. 1, 1975 NEW TEST FOR H. INFLUENZAE ISOLATION 95 31:103-117. 9. Koser, S. A. (ed.). 1968. Vitamin requirements of bacteria and yeasts, p. 282-285. Charles C Thomas, Springfield, Illinois. 10. Neter, E. 1947. Yeast autolysate: a culture medium for Haemophilus influenzae. Science 106:350-352. 11. Pickett, M. J., and R. M. Stewart. 1953. Identification of hemophilic bacilli by means of the satellite phenomenon. Amer. J. Clin. Pathol. 23:713-715. 12. Selwyn, S., and H. Ellis. 1972. Skin bacteria and skin disinfection reconsidered. Brit. Med. J. 1:136-140. 13. Stokes, E. J. 1962. Clinical bacteriology, 2nd ed. p. 116-117. Edward Arnold Ltd., London. 14. Turk, D. C., and J. R. May. 1967. Haemophilus influenzae. Its clinical importance, p. 10-12. English Universities Press Ltd., London. 15. Zinnemann, K. 1960. Haemophilus influenzae and its pathogenicity. Ergeb. Mikrobiol. Immunitaetsforsch. Exp. Ther. 33:307-368.