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An improved method for preparing Limulus amoebocyte lysate (LAL) is described and compared with two other well-known procedures. In five Limuli, three different bleeding procedures and three different cell rupture methods were studied. Increased sensitivity of LAL was accomplished by avoidance of the anticoagulant N-ethylmaleimid (NEM) in the bleeding procedure, optimal methods for mechanical cell rupture and use of pyrogen free conditions. Reaction of LAL with as little as 10(-15) gram Lipopolysaccharide (LPS) per ml was generally obtained. To evaluate a possible explanation for the differences in reactivity of LAL between different Limuli, found in one experiment, six Limuli were bled, and the pH and the concentration of protein, Ca2+, Mg2+, Cu2+, Na+ and K+ were determined in LAL and cell-free hemolymph. Inter and intra batch variations were found in LAL, but there was no correlation between the sensitivity of LAL and the content of the above mentioned constituents in LAL and cell-free hemolymph. Experiments with use of NEM in the bleeding procedure and addition of NEM to homogenized cells in different concentrations, showed that NEM inhibits the reactivity of LAL to LPS. It is concluded that the modified method of producing LAL by bleeding without NEM and by using optimal methods for mechanical cell rupture is quick, simple and produces a very sensitive reagent for the Limulus test.

Six patients with chronic relapsing diarrhoea caused by Clostridium difficile were treated with rectal instillation of homologous faeces (one patient) or a mixture of ten different facultatively aerobic and anaerobic bacteria diluted in sterile saline (five patients). The mixture led to a prompt loss of Cl difficile and its toxin from the stools and to bowel colonisation by Bacteroides sp, which had not been present in pre-treatment stool samples. Strains of Escherichia coli, Cl bifermentans, and Peptostreptococcus productus in the mixture inhibited the in-vitro growth of Cl difficile, which in turn inhibited the growth of Bacteroides ovatus, Bacteroides vulgatus, and Bacteroides thetaiotaomicron. The finding that Bacteroides sp had been absent during the patients' illness but was present after recovery suggests that the absence of Bacteroides sp may result in chronic relapsing Cl difficile diarrhoea, and that its presence may prevent colonisation by Cl difficile.

A 23-year-old female with extreme hypercalcaemia was treated with calcitonin, mitramycin and parathyroidectomy and normocalcaemia was achieved after 4 weeks. Nevertheless, the patient later died of cutaneous necrosis, impaired circulation and multiple organ failure. Serum immunoreactive parathyroid hormone was in the normal range and parathyroid tissue normal. Mixed connective tissue disease was diagnosed on the basis of high titers of antibody to extractable nuclear antigen, moderately elevated levels of antibody to nuclear antigen and only marginal elevation of anti-double standed DNA. The role of Cl. difficile toxin in the blood and an acinic cell tumour is unclear.

Haemophilus segnis is a rarely recognised commensal in the oropharynx. We wish to report the first published case of endocarditis caused by H. segnis. The patient, a 76-year-old female did not recover until after 2 courses of ampicillin given for a total of 57 days. In the second course of treatment, ampicillin was combined with 10 days of netilmicin therapy.