Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.

An actinomycete, isolated as a contaminant of a culture medium containing 25% NaCl, has been classified as Actinopolyspora halophila gen. et sp. nov. in the family Nocardiaceae. The morphology and biochemical characteristics of this organism distinguish it from other members of the family Nocardiaceae and other genera possessing a type IV cell wall. It requires high NaCl concentrations for growth and can grow in saturated NaCl. The lowest concentration permitting growth in liquid medium is 12%, and on solid medium, 10%. Colonies developing at lower salt concentrations contain holes resembling viral plaques. No growth occurred in a medium containing 30% KCl instead of NaCl. This organism can grow in simple media with NH4+ salts as nitrogen source and different sugars and other compounds as carbon source. Though it has a salt requirement almost as great as the extremely halophilic rods and cocci, it differs from these in containing diaminopimelic acid and in sensitivity to lysozyme; both properties suggest that it has a mucopeptide cell wall. It also contains some phospholipids common to other actinomycetes, but does not contain any phytanyl ether linked lipids characteristic of other extremely halophilic bacteria.

The sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans (LRGs) and lipid A was tested by using commercially available and experimentally formulated LAL reagents. The glucans included two kinds of 0-(1,3)-D-glucans, laminarin and curdlan, and cellulosic material, LAL-reactive material (LAL-RM), extracted from a hollow-fiber (Cuprophan) hemodialyzer. LAL-RM loses its LAL activity when it is digested with cellulase and thus appears to be a 3-(1,4)-D-glucan or a mixed glucan containing a substantial proportion of P-(1,4) linkages. All LAL reagents tested were at least 1,000-fold more sensitive to endotoxin than to LRGs. The presence of the surfactant Zwittergent was shown to interfere with reactivity to LRGs; LAL reagents without added Zwittergent reacted more strongly to LRGs than did the same reagents containing Zwittergent. Chloroform extraction of LAL increased the reagents' sensitivity to both endotoxin and LRGs, but it was not responsible for LRG reactivity. The addition of Zwittergent significantly reduced the sensitivity of LAL reagents to lipid A. LAL without the surfactant was equally sensitive to endotoxin and lipid A. Both curdlan and LAL-RM amplified or enhanced the LAL response to endotoxin. Kinetic turbidimetric studies demonstrated that the enhancement was dependent on the glucan concentration. The Limulus amebocyte lysate (LAL) test is extremely sensitive to endotoxin and has been considered to be specific for lipopolysaccharide (LPS). However, a few polymeric forms of glucose have been shown to be LAL reactive when they are present in sufficient quantities, i.e., nanogram to microgram amounts per milliliter. These glucans include

Clinical predictions alone are insufficiently accurate to identify patients with specific types of bloodstream infection; laboratory assays might improve such predictions. Therefore, we performed a prospective cohort study of 356 episodes of sepsis syndrome and did Limulus amebocyte lysate (LAL) assays for endotoxin. The main outcome measures were bacteremia and infection due to gram-negative organisms; other types of infection were secondary outcomes. Assays were defined as positive if the result was > or = 0.4 enzyme-linked immunosorbent assay units per milliliter. There were positive assays in 119 (33%) of 356 episodes. Assay positivity correlated with the presence of fungal bloodstream infection (P < .003) but correlated negatively with the presence of gram-negative organisms in the bloodstream (P = .04). A trend toward higher rates of mortality in the LAL assay-positive episodes was no longer present after adjusting for severity. Thus, results of LAL assay did not correlate with the presence of bacteremia due to gram-negative organisms or with mortality after adjusting for severity but did correlate with the presence of fungal bloodstream infection.

NOVITSKY: LIMITATIONS OF THE LAL TEST 417FIGURE 1. Biochemistry of the LAL "cascade" showing endotoxin and glucan pathways.