L~P O L D (1953) reported the isolation of a hitherto undescribed organism, a small, non-motile, non-capsulated, pleomorphic Gramnegative rod, from the genito-urinary tract of both men and women. On Casman's blood agar (essentially a proteose peptone, tryptose, beef extract agar with 5 per cent. human red blood cells and small amounts of corn starch, nicotinamide, para-aminobenzoic acid and glucose) incubated at 37" C. in air containing 10 per cent. C02, it grew slowly, forming tiny colonies surrounded by zones of haemolysis.Leopold did not attempt to classify the organism either on this or on a subsequent occasion (1956). Gardner and Dukes (1955) in the U.S.A. and Lutz, Grooten and Wurch (1955) in France reported frequent hlations of a similar micro-organism from women with vaginitis and from their consorts; Gardner and Dukes proposed the name Haemphilus vagittalis for it. Both groups of workers considered that the small bacillus they isolated belonged to the genus Huenwphilus because they could grow it only on media containing at least 5 per Cent. blood and because of its microscopical appearance.Gardner, Dampeer and Dukes (1957) described the clinical condition in which they found the organism, but added little to the bacteriological information already published. Brewer, Halpern and Thomas (1957) commented that H. vugindis organisms consistently assume a striking but vague and indescribable pattern that makes differentiation from small, pleomorphic, Gram-negatively staining diphtheroids difficult, and that many transplanted strains become Gram-variable, some to the point of being Gram-positive and looking very much like small diphtheroids.Amies and Jones (1957) described micro-organisms that were different in many respects from those of Leopold and of Gardner and Dukes, and appeared to belong to a different species (Edmunds, 1959, 196oa), more closely related to the genus Huemophillus than is the bacillus of Gardner and Dukes (Lapage, 1961).Four further reports appeared in 1959 and 1960, two of which (Bret and &hen-Debray; Ritwfeld and Kiimmel) gave descriptions very similar to those of Gardner and Dukes, except that they did not observe haemolysis; however, they used sheep's blood which according to Edmunds (19604 is not haemolysed. The third report (Heltai and Taleghany) stated that an organism similar to that described by Gardner and D u k e s was seen and cultured, but never in pure culture, and that no association could be established between the presence of this organism and vaginitis. The last of t h w four papers (Edmunds, 1959) mentions a tendency of some strains to be Gram-positive. A year later, Edmunds (1960~) gave a fuller description of the microscopical appearance of his organism which suggested that it belonged to a genus other than Haemphilus. He stated (p. 279 that " It has 1 . PATH. BAm. --YOL 85 (1963) 213 0 2

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O N account of the confusion existing at present over the taxonomic position of Haemophilus aphrophilus it was decided to re-examine Khairat's (1 940) original strain.H. aphrophilus was first described and named by Khairat, who isolated it from a fatal case of endocarditis. Characteristic features of the organism were that it was a Gramnegative coccobacillus that required a high concentration (5 per cent.) of C02 for optimum growth and failed to grow on solid medium in air unless a very heavy inoculum was employed. When first isolated, the organism required X factor but not V factor. After continued subculture it became adapted to grow better in air and showed less dependence upon X factor.King and Tatum (1962) examined 34 strains of H. aphrophilus from 23 states of the USA and judged them to be identical with Khairat's strain and another strain (Hunka) that had been isolated by Toshach and Bain (1958) from a case of aortic sinus aneurysm complicating subacute bacterial endocarditis. They found that all strains grew without the addition of either X factor or V factor on Alexander's medium (Dubos, 1952) free from accessory growth factors and that the addition of X and V factors gave little stimulation of growth. Moreover, all but one of the strains were described as gas producers when tested by a " rather unorthodox method ". MATERIALS AND METHODSStrains. Cultures of Haemophilus aphrophilus were obtained through the courtesy of the National Collection of Type Cultures (NCTC 5886 and 5907; both Khairat strains), Dr Margaret Pittman {Khairat strains PM5 (NCTC5908) and 320 (NCTC5906)) and Dr Sheila Toshach (the Hunka strain). They were received as freeze-dried cultures and were reconstituted in Levinthal's (1918) broth and subcultured on chocolate (10 per cent. heated horse blood) agar. They were maintained on chocolate agar by subculturing single colonies twice weekly and incubating at 37°C in air with added C02.The following Haemophilus species, some of which are our own laboratory strains, were used in control tests: (1) capsulated H. infzienzae type e2 (no. 8455), (2) H. parainguenzae (MB), and (3) H . haemoglobinophilus (canis) (NCTC8540). The Heatley Oxford strain of Staphylococcus aureus was used as a feeder strain to supply V factor in the identification method of determining X and V factor requirements described by Zinnemann (1960).Media. Determinations of growth factor requirements were carried out on solid media so that colony counts could be performed and contaminants could be more easily recognised. These media were 0.3 per cent. yeastrel agar (YA) (Zinnemann), 2 per cent. proteose peptone no. 3 (Difco) agar containing 0.6 per cent. NaCl (PPA), and a medium containing 1 part yeast extract to 9 parts of proteose no. 3 agar (Difco) (YEA), prepared as recommended by

From a high proportion of children sent to hospital H. influenzae can be isolated if suitable culture media are used. A number of H. influenzae strains were isolated from unusual sites, such as (1) blood cultures after tonsillectomy or tonsillotomy in five cases; (2) urine or the urinary tract in eight cases; (3) the lumen of appendices removed at operation in 11 cases (4 %); (4) osteomyelitis or pyarthrosis in six cases; (5) miscellaneous infections including two perianal abscesses, three cases of paronychia, one infected thyroglossal cyst, and several skin infections.It is suggested that infections of the skeletal system and the urinary tract arise from haematogenous spread of H. influenzae, as demonstrated by positive blood cultures after tonsillectomy and in two cases of skeletal infection. Infection of the appendix, perianal abscesses, paronychia, and skin infections probably arise by the direct route, either by immediate contact or by passage of viable organisms through the alimentary canal.The routine bacteriological techniques that are used in the Birmingham Children's Hospital allow ffaemophilus influenzae to be identified in any specimen except faeces. The species of Haemophilus that are the subject of this communication were isolated from children. There was no opportunity to examine similar material from adults. The purpose of this paper is to draw attention to the presence of species of Haemophilus in sites and lesions where these organisms are not usually expected or found. Methods and MaterialsMethod of Isolation.-Specimens of pus, from any site, were seeded on heated blood (chocolate) agar plates, as well as on the "composite" blood agar plate described by Rogers and Heslop (1948). Heated blood agar plates were prepared by adding 10% of oxalated horse blood to heart infusion agar at about 60°C.; after gentle mixing, the temperature was raised slowly to 75-80' C. and maintained at that temperature for 10 minutes. (The colour of the mixture should then be that of milk chocolate and there should be no gross granularity or clumping.) The molten mixture was again mixed gently and poured into Petri dishes.All inoculated heated blood agar plates were placed in large tin containers inside which a lighted small wax candle or night light was placed to supply a higher C02 tension than found in normal atmosphere. The lid was sealed to the tins by a 2 in. wide rubber elastic band. Pneumococci may require increased tension C02 for growth (Gladstone and Fildes, 1940; Fleming, 1941), and the presence of this gas does not impede the isolation of H. influenzae. This simple device was employed for every routine culture normally incubated aerobically, except those on desoxycholate citrate agar, MacConkey's, Hoyle's tellurite, or Sabouraud's media. Early subcultures of strains of H. influenzae from an unusual situation were sent to Leeds for detailed identification, and typing where appropriate.For the determination of X and V requirements, an autoclaved fresh or heated blood agar and an autoclaved yeastrel agar pla...