We describe a new calcium ion-selective electrode for measurement of the substance concentration of free calcium ion [Ca2+] in the plasma phase of whole blood and in serum at 37 degrees C. A sample volume of 50 microliter suffices to obtain simultaneous values of pH and [Ca2+]. We found the within-series analytical standard deviation for serum to be 0.013 mmol/litre (CV, 1.1%) and day-to-day precision to be 0.022 mmol/litre (CV, 1.7%). The reference interval for [Ca2+] (at pH 7.40) in serum was found to be 1.184 +/- 0.054 mmol/litre (2 SD) from measurements on sera from 121 healthy blood donors. Measurements on capillary blood from 29 healthy volunteers gave a mean (+/- 2 SD) value for [Ca2+] (at pH 7.40) of 1.22 +/- 0.072 mmol/litre.
A simple iodometric spot test for detecting beta-lactamase activity in Haemophilus influenza was compared with the capillary procedure for detecting betalactamase and the Bauer-Kirby disk susceptibility procedure. Isolates were classified similarly by all three procedures.
A new technique for detecting penicillinase production in Haemophilus influenzae and Staphylococcus aureus was compared with the capillary procedure for detecting beta-lactamase and the Bauer-Kirby disk susceptibility procedure. Isolates were classified similarly by all three procedures.Ampicillin-resistant strains of Haemophilus influenzae have appeared with increasing frequency since reported by Thomas et al. in 1974 (13). Khan et al. (5) showed that these resistant strains produced beta-lactamase (penicillinase). Subsequently, a variety of assays for betalactamase have been used to detect ampicillinresistant strains ofH. influenzae. These assays have involved the use of an iodometric technique with a starch indicator (3, 8), chromogenic cephalosporin as the enzymatic substrate (4, 6), or other colorimetric methods (1,11,14 The streak method was performed by using heavy inocula of the test organism picked from pure culture with a bacteriological loop. The streak patterns were made in two ways. (i) Unknown and non-penicillinase-producing control organisms were streaked in a cross pattern, but disconnected at the center to avoid contamination (Fig. 1A).(ii) Unknown and control organisms were streaked about 8 to 10 mm apart in a parallel line as shown in Fig. 1B. Antibiotic disks were placed as illustrated, and the plates were incubated at 37°C for 4 h or until growth was clearly visible. The inactivation of ampicillin was well demonstrated in crossstreaked patterns by inhibition zones that resembled a pair of kidney beans (Fig. 1A). The Bacillus subtilis grew along the streak of the resistant isolate due to the inactivation of ampicillin. On the other hand, if two isolates were streaked in parallel, the pattern illustrated in Fig. 1B
A simple technique with antibiotic-impregnated disks has been developed to demonstrate the antibiotic synergism of penicillins and aminoglycosides against enterococci. The two antibiotic disks are placed on inoculated plates so that the distance between their centers is less than the sum of the two radii of their previously determined inhibitory zones. After 5 h of incubation, ,8-lactamase powder is dusted onto the susceptibility plate with a sterile cotton swab. After overnight incubation, the synergistic effect of the antibiotics is demonstrated by a clear area of no growth in between the two disks. There is good correlation between the synergism determined by this procedure and the susceptibility of the organisms to 2 mg of streptomycin per ml. This technique is simple and can be employed by any laboratory which performs Kirby-Bauer susceptibility procedures.Combined antibiotic therapy has proven to be effective for serious infections such as enterococcal endocarditis (2-4) and listerial meningitis (1). However, the method for determining antibiotic interactions in vitro is too tedious and time consuming to perform as a routine service by most laboratories. The new method described here can be applied even in a small laboratory, offering useful information readily for the clinicians.The most frequently employed antibiotic combination has been the use of a penicillin plus one of the aminoglycosides (2). Their synergistic action on bacteria is demonstrated after use of the neutralizing activity of ,B-lactamase on the penicillin. This paper describes a unique technique with carefully spaced antibiotic susceptibility disks and direct application of ,8-lactamase powder onto the disk susceptibility plate. The effect of the combined antibiotics is then observed after overnight incubation of the plate. MATERIALS AND METHODS,B-Lactamase was purchased from Schering Corporation, Bloomfield, N.J. Twenty-five strains of streptococci had previously been obtained from the National Center for Disease Control.A routine Kirby-Bauer antibiotic sensitivity test (5) was performed to determine the inhibitory zone sizes of selected antibiotics. Next, the organism was swabbed onto Mueller-Hinton agar, as similarly done in the Kirby-Bauer procedure. Then disks of the two antibiotics expected to react synergistically were placed on the plate. The distance between the disks was measured so that margins of the two antibiotic inhibitory zones would meet or slightly overlap. For example, if disk A had a zone diameter size of 20 mm, and disk B of 10 mm, then the distance between the centers of the two disks should be less than 15 mm ( [20 + 10 Y2). After allowing the disks to diffuse 5 h on the agar plate at 37 C, ,-lactamase powder was dusted over the area with a sterile, dry cotton swab (Fig. 1)
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