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SYNOPSISThe sensitivity of Haemophilus influenzae to penicillins in vitro, determined either by serial antibiotic dilution in broth or by the disc method on agar, is apparently profoundly influenced by inoculum size if the results are read by macroscopic inspection. Microscopic inspection of the growth, however, reveals that the turbidity in heavily inoculated broth containing concentrations higher than the minimal inhibitory concentration is the product of L forms which have failed to succumb to osmotic lysis. Similarly, minute colonies appearing in the 'inhibition zone' of disc tests are composed of L forms. In both broth and agar tests reduction of the osmolality of the medium from 340 to 144 mOsm per kg failed to bring about lysis of organisms exposed either to ampicillin or amoxycillin. The significance of this remarkable osmotic stability of haemophilus L forms is discussed in relation both to testing of sensitivity of this organism to penicillins and to persistence of chronic haemophilus infections of the lower respiratory tract.It is generally accepted that inoculum size must be carefully regulated when testing the sensitivity of a bacterial species to an antibacterial drug, since too large an inoculum may give rise to apparent resistance of an organism which is in fact fully sensitive. The importance of the 'inoculum effect', however, varies in relation to different drugs, being minimal in the testing of the sensitivity of non-penicillinaseproducing organisms to penicillin. This is illustrated, for example, by the demonstration by Rolinson and Stevens (1961) that the minimal inhibitory concentration (MIC) of ampicillin for Salmonella typhi varied only between 0 6 ,ug per ml and 0 25 ,ug per ml when the inoculum size was varied between 1/10 and 1/100 000 of an overnight broth culture. In contrast, a similar variation of inoculum size caused a tenfold change in the MIC of tetracycline.In the course of extensive testing of the sensitivity of strains of Haemophilus influenzae to ampicillin and, more recently, amoxycillin, we have become aware that apparent resistance can readily result from the use of an inoculum only moderately in excess of that usually regarded as appropriate for general sensitivity testing, and microscopic inspection of the organisms surviving in drug concentrations higher than the MIC has shown them to be

The isolation rate of Haemophilus influenzae from patients with persistent production of purulent sputum has been increased by the routine use of a selective medium. Nevertheless, some purulent sputum still fails to yield a pathogen. The selective medium was supplemented with N-acetyl-D-glucosamine to encourage primary isolation of colony forming, spheroplastic H influenzae, which reverted to normal forms on subculture.

Nontypeable Haemophilus influenzae commonly causes infections in the lower and upper respiratory tract, although the mechanisms of its colonization and persistence in the airways are unclear. Culture filtrates from six clinical isolates of this bacterium were assessed for their abilities to influence neutrophil function in vitro. Each culture filtrate was assessed on six separate occasions with neutrophils obtained from six different donors. During the log and early stationary phases of growth (0 to 18 h), culture filtrates contained primarily neutrophil chemokinetic activity but no activity affecting neutrophil migration toward the chemotactic factors N-formyl-L-methionyl-L-leucyl-L-phenylalanine and leukotriene B4. In contrast, filtrates obtained after 24 h of culture contained factors which inhibited neutrophil migration toward both of these chemotactic factors. This chemotaxis-inhibitory activity persisted between 24 and 72 h of bacterial culture, and it was not associated with the presence of either chemotactic or chemokinetic activity as assessed by checkerboard analysis. Gel filtration of pooled 72-h filtrates yielded three major peaks of chemotaxis-inhibitory activity. Endotoxin was present together with two other low-molecular-mass hydrophobic factors of approximately 8 and 2 kDa. These low-molecular-mass factors are chloroform insoluble and heat stable, and they are inactivated by protease, periodate, and diborane reduction. Activity was completely retained on a wheat germ agglutinin column, and it could be eluted with N-acetyl-D-glucosamine. These data suggest that inhibitory activity is associated with N-acetyl-D-glucosamine-containing glycopeptides, possibly derived from the bacterial cell wall. The production of these compounds may contribute to the persistence of this bacterium in vivo by inhibiting neutrophil chemotaxis in the microenvironment of the respiratory mucosa.