JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1975, p. 89-95Copyright ( 1975 American Societv for Microbiology

Vol. 1, No. 1Printed in U.S.A.

New Satellitism Test for Isolation and Identification ofHaemophilus influenzae and Haemophilus parainfluenzae

in SputumNORA 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 ofHaemophilus influenzae and Haemophilus parainfluenzae is described. In thebasal medium, nicotinamide adenine dinucleotide is incorporated at a concentra-tion 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, onedisk being impregnated with 60 Mg of hemin and the other with 15 Mg ofnicotinamide adenine dinucleotide. Under these conditions, H. influenzae strainsgrow around the hemin disk only and the majority of H. parainfluenzae growaround the nicotinamide adenine dinucleotide disk. This proceduregives results which are more clear cut than other establised methods, especiallyin sputum culture.

The traditional method of identifying Hae-mophilus influenzae is the demonstration ofsatellite growth of that organism around acolony of Staphylococcus aureus on blood agarbut not on the basal medium without addedblood (13). Despite the continued and wide-spread popularity of this method, a number oflimitations have been recognized. For example,certain preparations used as the basal mediummay contain traces of heme, so that the orga-nism's requirement for this factor would not bedemonstrated (2, 9, 14, 15). An earlier investiga-tion (5) showed that over 30% of strains of H.influenzae would for this reason have beenwrongly identified as Haemophilus parainfluen-zae. On the other hand, using blood as a sourceof hemin and a hemin-free but otherwise unsuit-able basal medium, some strains of H. parain-fluenzae appeared to require both hemin andnicotinamide adenine dinucleotide (NAD) andtherefore would have been mistakenly identifiedas H. influenzae.

In this paper a new satellitism test is de-scribed. It is designed to facilitate and improvethe reliability of identification of strains of H.influenzae and H. parainfluenzae and is partic-ularly applicable and useful in the examinationof specimens from the respiratory tract.

MATERIALS AND METHODSMedium. The basal medium contained no detecta-

ble hemin and supported the growth of H. influenzaewith the addition of hemin and NAD. It also sup-ported 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). Themedium 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%). thiaminhydrochloride (1.0 ug per ml), sodium oleate (4.8 ,gper ml), and NAD (0.2 Mg per ml) (all final concentra-tions) were added to the medium. Each 85-mm dishcontained 20 ml of medium; the prepared plates canbe stored for 1 month at 4 C.

Preparation of disks. Aqueous stock solution ofNAD (#NAD, Sigma Chemical Co) and hemin (Koch-Light) each contained 2,000 ug per ml.

Paper disks of 15-mm diameter (Ekwip FilterPaper, Industrial Equipment, Australia) or 12-mmdiameter (W. Whatman and R. Balston Ltd., Eng-land) were impregnated with these solutions so as tocontain approximately 15 gg of NAD or 60 ug ofhemin. Dried paper disks containing NAD were stablefor at least 2 months at 4 C. Moist disks containinghemin were used and could be stored for at least 1month at 4 C in a 1-oz screw-capped bottle. Driedhemin disks were not used since they deteriorated onstorage.

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 182strains were freshly isolated from respiratory speci-mens, blood culture, and cerebrospinal fluid. Twentyof these were encapsulated strains of H. influenzaebelonging to Pittman type b. All strains were identi-

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fied on microscopic and cultural appearances togetherwith NAD and hemin requirements determined bystandard satellitism methods (5, 15).

Inoculation of plates. A single colony from a 15- to18-h plate culture was picked with a wire loop andspread over a small segment (7); without resterilizing,the flat side of the loop was then used to spread theinoculum over the entire surface of the plate using aseries of parallel streaks at right angles to the originalinoculum. In this way, the whole surface of the platewas covered four times. Disks ofNAD and hemin werethen placed on the surface of the inoculated plate withtheir edges 15 mm apart, approximately 55 mm fromand opposite to the original inoculum. For primarycultures of sputum, the same inoculating procedurewas adopted, but using instead of a colony a portion ofsaline-washed purulent material.

Incubation of cultures. All satellitism plates wereexamined after 15 to 18 h of incubation in anatmosphere of 5%3 CO2 in air (vol/vol) in a McIntoshand Fildes jar at 37 C.

RESULTSTable 1 shows the satellite or other growth

pattern of the 191 strains of Haemophilus, whenexamined by the new satellitism test, togetherwith the identity of these strains. The strains ofHaemophilus examined showed three patternsof growth.

(i) Growth near the hemin disk only. The97 strains, requiring both hemin and NAD andincluding all type cultures of H. influenzae andtype b strains, grew in the vicinity of the hemindisk. Of these, 94 strains grew in a circular area,measuring about 30 mm in diameter with thehemin disk at its center (Fig. 1); with theremaining three strains, growth was restrictedto a sector near the hemin and opposite theNAD disk. The NAD required by these fewstrains was determined as 1.0 Ag per ml and not0.2 gg per ml characteristic of the majority ofstrains of this species (Evans et al., J. Med.Microbiol., in press).

All 97 strains with this growth pattern pre-sented microscopic and colonial appearancescharacteristic of H. influenzae (encapsulated or

TABLE 1. Distribution ofgrowth of H. influenzae andH. parainfluenzae on the new satellitism medium

Growth pattern No. of strains

Disk Region of growth H. in- H. parain-flueneae fluenzae

Hemin Circular area 94 OSector 3 O

NAD Circular area O 87

Neither Whole surface 7

noncapsulate) and required both hemin andNAD as determined by established methods (5,15).

(ii) Growth around the NAD disk only.Eighty-seven strains showed satellite growtharound the disk containing NAD only (Fig. 2),and zone sizes of these strains varied, thediameter measuring from 30 to 45 mm. Thesestrains were identif ied as H. parainfluenzae,requiring between 1 and 5 gg of NAD per ml.The diameter of the zone of growth variedinversely to the amount of NAD required.

(iii) Growth over the whole surface. Theseven remaining strains shown to require onlyNAD grew over the whole surface of the platewith no apparent increase in growth in the vi-cinity of either disk. These strains were alsoidentified as H. parainfluenzae but with a lowNAD requirement of 0.2 ,g per ml.

Isolation of H. influenzae from mixed cul-tures. The nondescript appearance and smallcolony size of H. influenzae on blood agar makesits isolation difficult and time consuming. Thisis particularly so when the organism is sought inthe midst of mixed flora characteristic of asputum culture. Figure 3 shows the distinctzone of growth of a strain of H. influenzaearound the hemin disk on a plate inoculateddirectly with a specimen of sputum.

This direct satellitism test overcame thedifficulty of isolating H. influenzae from spu-tum when it also contains H. parainfluenzae. Asboth species are identical in microscopic andcolonial appearances, colonies selected for theusual satellitism test could be either H. influen-zae or H. parainfluenzae. This satellitism test,as shown in Fig. 4, will readily demonstrate thepresence of either or both species in a mixedculture.

DISCUSSIONThe method described in this paper was

introduced primarily to assist in the identifica-tion and isolation of H. influenzae and H.parainfluenzae, particularly in sputum. Thepathogenic significance of H. influenzae is wellestablished, but identification and isolation,especially of noncapsulate strains of H. influen-zae and its differentiation from H. parainfluen-zae, is time consuming and presents technicaldifficulties (14). However, since there is noevidence that H. parainfluenzae can cause suchinfections 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 beattributed not only to NAD but also to catalase,which is produced by staphylococci and may

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substitute for hemin in the growth of H. influen-zae (3, 11). Furthermore, not all organismsshowing satellite growth in the vicinity of staph-ylococci belong to the genus Haemophilus (12).The use of yeast extract may also be criticizedas a source of NAD because it may containheme compounds (8, 10).Cooper and Attenborough (4) found satellit-

ism tests were too unreliable and proposed thathemin and NAD requirements be determined ina fluid medium. The disadvantages of such atechnique are the need for pure cultures and the

heavy inocula of between 107 and 108 cellsrequired to initiate growth of some Haemophi-lus species in fluid media (8). The plate methoddescribed by Everall (7) introduced certainimprovements, such as the use of hemin-impregnated strips instead of blood added tothe basal medium, but the limitations of thismethod were the possibility of contaminatingtraces of hemin in certain batches of nutrientagar and also in its use of yeast extract as asource of NAD.The new test described in this paper elimi-

FIG. 1. The growth of a typical strain of H. influenzae around the hemin disk (black) on a plate containing0.2 gg of NAD per ml.

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nates these potential sources of error by using ahemin-free basal medium and purified heminand NAD (5). The medium also contains thia-min and glucose which are essential for abun-dant growth of Haemophilus species, and cer-tain inhibitors in agar and peptone are neutral-ized by the inclusion of sodium oleate and thereducing agent dithionite (6). In this satellitismtest, the amount of NAD incorporated in thebasal medium allows the growth of all thestrains of H. influenzae around the hemin diskonly. With three out of 97 strains of H. influen-zae, the zone of growth was limited to a sector

around the hemin disk and opposite the NADdisk, and this pattern was found to be charac-teristic of those strains which had a higher NADrequirement, that is, 1 ,ug per ml rather than0.2 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 zgper ml, grow around the disk supplying addi-tional NAD. The introduction of a quantita-tive element into testing has in our experiencegiven results that are easier to read than withother methods which are purely qualitative.

FIG. 2. The growth of a typical strain of H. parainfluenzae around the NAD disk (white) on a platecontaining 0.2 ,ug of NAD per ml.

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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 canoften be identified in primary cultures of clini-cal specimens even when H. parainfluenzae isalso present. H. influenzae will grow around thehemin disk only and H. parainfluenzae aroundthe NAD. This differentiation cannot beachieved using the three disks or strips contain-ing NAD, hemin, and NAD plus hemin (Oxoidor Baltimore Biological Laboratories) on a basalmedium without added NAD. In that test, H.influenzae satellites around the NAD plushemin disk only and H. parainfluenzae aroundthe 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 ofH. influenzae. Another incidental advantage isthat Streptococcus viridans and Streptococcuspneumoniae, which are both commonly presentin sputum, are inhibited on this basal mediumbecause of the inhibitory effect of the oleate (1),and also because of its lack of enrichment withblood.This test was not designed or intended to

determine the requirements of all species ofHaemophilus but has been found economicaland accurate in the preliminary isolation and

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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 theNAD (white).

identification of H. influenzae and H.parainfluenzae in the bacteriological examina-tion 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 ofHaemophilus species grown with graded amounts of

hemin. J. Bacteriol. 81:380-384.3. Brumfitt, W. 1959. Some growth requirements of Hae-

mophilus influenzae and Haemophilus pertussis. J.Pathol. Bacteriol. 77:95-100.

4. Cooper, R. G., and I. D. Attenborough. 1968. An indicatormethod for the detection of bacterial X and V factordependence. Aust. J. Exp. Biol. Med. Sci. 46:803-806.

5. Evans, N. M., and D. D. Smith. 1972. The effect of themedium and source of growth-factors on the satellitismtest for haemophilus species. J. Med. Microbiol.5:509-514.

6. Evans, N. M., and D. D. Smith. 1974. The inhibition ofHaemophilus influenzae by certain agar and peptonepreparations. J. Med. Microbiol. 7:305-310.

7. Everall, P. H. 1953. A plate method for demonstrating thegrowth factor requirements of the genus Haemophilus.J. Med. Lab. Technol. 11:181-184.

8. Gilder, H., and S. Granick. 1947. Studies on the hemoph-ilus group of organisms. Quantitative aspects of growthon variotus porphin compounds. J. Gen. Physiol.

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31:103-117.9. Koser, S. A. (ed.). 1968. Vitamin requirements of bac-

teria and yeasts, p. 282-285. Charles C Thomas,Springfield, Illinois.

10. Neter, E. 1947. Yeast autolysate: a culture medium forHaemophilus influenzae. Science 106:350-352.

11. Pickett, M. J., and R. M. Stewart. 1953. Identification ofhemophilic bacilli by means of the satellite phenome-non. 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 influen-

zae. Its clinical importance, p. 10-12. English Univer-sities Press Ltd., London.

15. Zinnemann, K. 1960. Haemophilus influenzae and itspathogenicity. Ergeb. Mikrobiol. Immunitaetsforsch.Exp. Ther. 33:307-368.

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