A MEDIUM developed for nutritional studies of Haemophilus influenzae proved satisfactory for a period of about 2 years @vans and Smith, 1972), but subsequently certain batches were found to be defective. The inoculum required to initiate growth increased from less than 10 cells to 107, the colonies were of irregular size and microscopically many filamentous forms were evident. The constituents of both satisfactory and unsatisfactory media appeared to be identical, and preliminary investigations suggested that the duration and temperature of autoclaving might adversely affect the medium.Inhibitory effects of media have been attributed to a number of agents including copper, colloidal sulphur, peroxides, fatty acids, certain amino acids and other substances (Burnet, 1925;Gordon and McLeod, 1926;O'Meara and Macsween, 1936;Ley and Mueller, 1946;Casman, 1947;Proom et al., 1950;Nieman, 1954;Woiwod, 1954;Traxler and Lankford, 1957;Dukes and Gardner, 1961). This paper describes an extension of earlier studies on proteose peptone as a basal medium for H. infuenzae and the value of dithionite and sodium oleate as additives to neutralise potential inhibitors.MATERIALS AND METHODS Media. Chocolate agar was prepared (Cruickshank, 1965) from Columbia Agar (Oxoid) with 7.5% horse blood. Proteose Peptone Agar for most experiments contained 2% Proteose Peptone (Difco Batch no. 560561) with 1.2% agar and 0.6% NaCl adjusted to pH 7-5; other proteose peptones used for comparison were Difco batch no. 542610, Oxoid no. Sterilisation. Agar and proteose peptone were autoclaved together in 20-ml amounts, or separately at double strength in 10-ml amounts, in Universal screw-capped containers.Chemicals and growth factors. These were sterilised as stock solutions by means of a Swinnex 0.45-p Millipore Filter Unit and added to the molten proteose peptone agar at 56°C. Haemin (Koch-Light), NAD (p-diphosphopyridine nucleotide, Sigma Chemical Co. Ltd) and glucose (Univar, Ajax Chemicals Ltd, Australia) were added to all plates to give final concentrations of 20 pg per ml, 1 pg per ml and 2000 pg per ml respectively. Sodium oleate (BDH Ltd) was used at a final concentration of 4 pg per ml. The reducing agents cysteine hydrochloride (BDH), sodium dithionite (BDH), sodium thioglycollate (Sigma), Cleland's reagent (Sigma) and glutathione (Sigma) were each incorporated in the medium at a final concentration of 100 pg per ml. Other compounds, impregnated in 6-mm Whatman AA disks, were manganese dioxide, catalase, bovine serum albumen, yeast extract, nucleic acids, Casamino Acids, purines, Fildes' extract, horse serum and a vitamin mixture.Organisms. A single laboratory strain of H . infuenzae, type b (strain West), isolated from cerebrospinal fluid, was initially used; 22 other strains were also used, six of which were type culture strains, nos. NCTC8467 (type b), NCTC8465 (type a), NCTC8469 (type c), NCTC8472 (type e), NCTC10479 (type e) and NCTC4560 (unencapsulated), and 16 strains, recently isolated from clinical material, of which four were enca...