Simple method for distinguishing gonococcal colony types.

Gonococcal colony types can be distinguished by a new procedure that makes use of a dissecting microscope with a concave mirror and a fluorescent lamp. Critical adjustment of the mirror angle results in illumination similar to that obtained in the dark-field microscope. When the concave mirror is set at a certain angle, colonies of the lenticular types 1 and 2 refract the light coming through them in such a way that an edge of the microscope stage is focused in each colony. By contrast, colonies of types 3 and 4, which are relatively flat, fail to refract incident light. Although distinguishable from each other by differences in color, type 3 and 4 colonies do not display the focusing effect typical for type 1 and 2 colonies and appear uniformly illuminated. This new technique permits the rapid identification and isolation of even a single type 1 or 2 colony in a field of type 3 or 4 colonies, making it possible to obtain and maintain competent colonies (type 1 or 2) for the genetic transformation assay for Neisseria gonorrhoeae strain identification as well as for other purposes.     

Superoxol and aminopeptidase tests for identification of pathogenicNeisseria species andMoraxella (Branhamella) catarrhalis

The superoxol test, and prolyl aminopeptidase and gammaglutamyl aminopeptidase tests were evaluated for the detection of pathogenicNeisseria spp. using 317 strains ofNeisseriaceae. The superoxol test was positive for all 116 gonococci and 62Moraxella (Branhamella) catarrhalis strains, but also for three strains ofNeisseria meningitidis, one strain ofNeisseria lactamica and eight saprophytic neisseriae. When using strains grown on Thayer-Martin medium, the positive and negative predictive values of the superoxol test for the identification ofNeisseria gonorrhoeae were 96.7 % and 100 % respectively. Meningococci were the only neisseriae growing on Thayer-Martin medium that showed gamma-glutamyl aminopeptidase activity. The prolyl aminopeptidase test showed low specificity.     

Radiometric selective inhibition tests for differentiation of Mycobacterium tuberculosis, Mycobacterium bovis, and other mycobacteria.

In the context of a busy reference laboratory, radiometric selective inhibition tests were evaluated for rapid differentiation of Mycobacterium tuberculosis and Mycobacterium bovis and of the M. tuberculosis complex from other mycobacteria. p-Nitro-alpha-acetylamino-beta-hydroxypropiophenone at 5 micrograms and hydroxylamine hydrochloride at 62.5 and 125 micrograms per ml of 7H12 medium were used to separate the M. tuberculosis complex from other mycobacteria (MOTT bacilli). Since it is important epidemiologically to distinguish M. tuberculosis from M. bovis, susceptibility to 1 microgram of thiophene-2-carboxylic acid per ml was also determined radiometrically. By using these three agents as selective inhibitors, M. tuberculosis, M. bovis, and MOTT bacilli were differentiated with a high degree of specificity by a BACTEC radiometric procedure. Results of tests performed on clinical isolates submitted on solid medium to our reference laboratory were available within 5 days.     

Response of several Limulus amoebocyte lysates to native endotoxin present in gonococcal and nongonococcal urethral exudates from human males.

Three Limulus amoebocyte lysate (LAL) preparations obtained from three different suppliers were comparatively evaluated for sensitivity to native endotoxin contained in urethral exudates from 28 men with gonococcal urethritis and 16 men with nongonococcal urethritis. One LAL preparation was not extracted with organic solvents during manufacture, whereas the other two were extracted with chloroform. All three LAL preparations had equivalent sensitivities (0.06 ng/ml) to an established reference endotoxin standard (EC-2), but significant differences in sensitivities were found among the different LAL preparations when testing clinical specimens. Dilution breakpoints of urethral samples for maximum sensitivity and specificity ranged from 1:400 to 1:1,600, depending on the LAL preparation. The nonextracted lysate was significantly more sensitive to the presence of endotoxin in gonococcal exudates than the other two preparations (P less than 0.001) but not significantly different from one LAL preparation (P greater than 0.05) in detecting endotoxin in nongonococcal exudates. An additional 116 men, 61 with culture-proven gonococcal urethritis and 55 with nongonococcal urethritis, were evaluated with three lots of nonextracted lysate with sensitivities ranging from 0.04 to 0.06 ng/ml, reference endotoxin EC-2. At a dilution breakpoint of 1:1,600, the sensitivity of the LAL test was 100%, and the specificity was 96%.     

Rationale for selective use of anaerobic blood cultures.

Because of the declining frequency of anaerobic bacteremia, routinely using half the collected blood volume for anaerobic culture has been challenged. There is no data indicating whether more clinically relevant isolates would be recovered if all or most of the given blood sample were cultured aerobically. In this two-part study, we reviewed cases of anaerobic bacteremia to determine what proportion occurred in situations when anaerobes would be expected and then estimated the yield of different culture approaches by reanalyzing the data from a large prospective clinical blood culture study. The records of 61 patients who had an anaerobic isolate (excluding Propionibacterium species) recovered only from an anaerobic bottle were examined to define clinical settings in which such isolates occur. Fifty-six (92%) patients had clinically important isolates, and the source of infection was obvious at the time of culture in 47 of the 56 (84%). Of 56 patients, 36 (64%) had abdominal signs and symptoms, including 12 with recent abdominal surgery. Of nine patients without an obvious source of infection, six were on high-dose steroids. Relative yields were compared for (i) one aerobic bottle and one anaerobic bottle (5 ml to each) for all blood cultures, (ii) two aerobic bottles (5 ml to each), or (iii) two aerobic bottles plus an extra anaerobic bottle (only for clinically suspected anaerobic sepsis) (5 ml to each). The third approach had the highest yield (475 isolates), because the routine use of two aerobic bottles recovered more Candida spp., members of the family Enterobacteriaceae, and nonfermenters than did the first approach (448 isolates) (P < 0.02), and clinically directed culturing for anaerobes would recover anaerobes missed with the second approach (458 isolates). Our data suggest that the use of two aerobic bottles with selective culturing for anaerobes could increase the number of clinically relevant isolates by at least 6% compared with the current practice of inoculating an aerobic bottle and an anaerobic bottle with equal volumes of blood.     

Comparison of five selective media for beta-haemolytic streptococci.

Five selective media for beta-haemolytic streptococci were tested and compared with the conventional blood agar plate using 200 throat swabs from children with possible streptococcal pharyngitis. The medium described by Liebermeister and Braveny, which is based on the reduction of nutrients and enhancement of the haemolytic activity of beta-streptococci, was markedly superior to the other selective media containing inhibiting agents.     

Trimethoprim activity in media selective for Campylobacter jejuni.

The activity of trimethoprim (TMP) in two selective media used for isolation of Campylobacter jejuni was evaluated. The two selective media, Campy-BAP and Skirrow medium, contain TMP in addition to other antimicrobial agents. The minimal inhibitory concentrations of TMP in blood agar base (basal agar for Skirrow medium) or brucella agar (basal agar for Campy-BAP) for three sensitive control organisms were compared with those in Mueller-Hinton agar, which contains low levels of thymidine. TMP was inactive in both blood agar base and brucella agar, even when lysed horse blood or thymidine phosphorylase was added. TMP had activity when used in combination with the other antimicrobial agents normally included in Skirrow medium and Campy-BAP, probably indicating synergism between TMP and one or more of the other antimicrobial agents. Sheep blood could be substituted for lysed horse blood in Skirrow medium without compromising the activity of TMP.     

Comparison of two selective media for Actinobacillus actinomycetemcomitans.

Two selective media, malachite green-bacitracin (MGB) agar and tryptic soy-serum-bacitracin-vancomycin (TSBV) agar, were compared for the isolation of Actinobacillus actinomycetemcomitans. The highest recovery was found on TSBV agar plates cultured in air-5% CO2 both for plaque samples from periodontal pockets and for pure cultures.     

A rapid method for reading migration inhibition tests.

This article describes how the extent of cell-migration can be read directly using the Field iris of a microscope.     

Improved selective culture media for Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

By modifying the previously described media tryptic soy-serum-bacitracin-vancomycin (TSBV) agar and tryptic soy-serum-bacitracin-vancomycin-fluoride (TSBVF) agar, two improved selective culture media were developed for isolation and enumeration of Actinobacillus actinomycetemcomitans (A medium) and Haemophilus aphrophilus (H medium) in oral specimens. Both media were supplemented with fusidic acid and spiramycin, and carbenicillin was also added to A medium. The growth yields of pure cultures of A. actinomycetemcomitans on A medium and of H. aphrophilus on H medium were comparable with those on the reference media. Compared with blood agar, the selective media inhibited these species about 10-fold or less. In addition, A and H media suppressed the growth of pure cultures of Capnocytophaga spp. and Neisseria spp., commonly found as contaminants on TSBV and TSBVF, 10(5) times or more compared with that on blood agar. In samples from diseased periodontal pockets, the recoveries of A. actinomycetemcomitans on A medium and H. aphrophilus on H medium equaled those on TSBV and TSBVF, respectively. In about 50% of the cultures on the reference media, contaminating bacteria were detected at levels higher than 10(4) CFU/ml of sample. The corresponding value for both A and H media was about 2%.     

Growth of Streptococcus mutans on various selective media.

The ability of Streptococcus mutans to grow on mitis-salivarius (MS) agar, MC agar, mitis-sucrose-bacitracin (MSB), BCY agar, and MM10 sucrose agar was studied. Batch cultures of S. mutans serotype a demonstrated no growth on MSB agar. Certain serotype d and g strains did not grow on MC agar. The yield for most strains of other serotypes on these selective media was lower compared with that on MS agar. The number of total colony-forming units on BCY and MM10 sucrose agar was similar to the blood agar results. Similar data were obtained when fermenter-grown strains, harvested in the middle or the end of the logarithmic growth phase, were used for inoculation of the various media. Enumeration of S. mutans from plaque samples plated on MC and MSB agar yielded about 75% of the counts obtained on MS or the nonselective medium. When the proportions of S. mutans were expressed as a percentage of the total cultivable flora, the selective media (MC and MSB agar) showed approximately 10% lower values than the MS, BCY, and MM10 sucrose agar.     

Comparative isolation of vaginal yeasts on selective and nonselective media.

The isolation of vaginal yeasts was compared on a selective medium, phosphomolybdic acid agar, and on starch agar, a nonselective differential medium used primarily to isolate Corynebacterium vaginale. The majority of the Candida albicans strains were isolated on starch agar, but the selective medium was required for isolating all yeasts from the greatest number specimens.     

Suitability of peracetic acid for sterilization of media for mycoplasma cultures.

The utility of peracetic acid for sterilization of serum and yeast extract additions to mycoplasma medium was studied by culturing six Mycoplasma species. Culture media containing additions that had been sterilized with peracetic acid proved to be as good as filtered components. The use of 0.05 to 0.1% peracetic acid is recommended to sterilize the serum and yeast extract additions since savings in time and equipment can be accomplished.     

Comparative efficacy of seven selective media for isolating Campylobacter jejuni.

Diarrheal stools from 263 patients were inoculated on seven selective media: Butzler selective medium, Blaser medium, Skirrow blood agar, Preston campylobacter selective medium, Preston campylobacter blood-free medium, Butzler Virion medium, and modified Preston medium (with amphotericin B [2 mg/liter]). A similar number of Campylobacter jejuni strains were isolated from all the media studied; nevertheless, the presence of competing fecal flora (FF) made the detection of suspect colonies difficult. Preston campylobacter blood-free medium with cefoperazone yielded the greatest number of C. jejuni isolations, and contaminating FF grew in only 9% of the plates showing C. jejuni growth; all the other media allowed the abundant growth of other FF, regardless of whether C. jejuni was isolated from them or not.     

Quantitative studies on competitive activities of skin bacteria growing on solid media.

Earlier quantitative investigations of antagonism between skin bacteria were based on the use of liquid cultures, but a more realistic model has now been devised, based on the use of the surfaces of solid media. Pure or mixed inocula were spread evenly over suitable agar media in Petri dishes marked out with a standard grid. Growth curves were constructed from viable counts of the surface bacteria after they had been removed from excised squares of the agar media and dispersed. The method was highly reproducible, and competitive interactions were revealed more clearly than in studies with liquid media. An antibiotic-producing strain of Staphylococcus epidermidis (S6+) readily suppressed strains of Micrococcus, Corynebacterium and Streptococcus species. However, a Staphylococcus aureus strain which was less sensitive to the antibiotic effect of S6+ interacted in a complex manner, depending on the absolute and relative size of the S6+ inoculum.     

Hemadsorption and hemadsorption inhibition tests for rubella virus

Summary A hemadsorption (HAd) reaction was demonstrated in rubella-infected cell cultures using erythrocytes of 1-day-old chickens, adult chickens, pigeons, geese, sheep or bovines. HAd occurred at 4°C, room temperature or 37°C, and at a pH of 6.2 or 7.4. BHK-21 cells were the most satisfactory for demonstrating HAd; the reaction could be demonstrated only in heavily-infected cultures of other cell types. Infected BHK-21 cell cultures generally had to attain infectivity titers of 10^–3.5 to 10^–4.0 InD₅₀ per 0.1 ml in order for HAd to be demonstrable. The HAd reaction could be used to detect unneutralized virus in neutralizing antibody assays, and the inhibition of HAd in infected cell cultures was a very sensitive method for assay of rubella antibodies. A procedure was developed for identification of rubella virus isolates by HAd inhibition.The work on which this report is based was supported by Grant AI-01475 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States Public Health Service, Department of Health, Education, and Welfare.     

Variability in growth of Neisseria polysaccharea on colistin-containing selective media for Neisseria spp.

In a prospective survey of 773 healthy schoolchildren in southern Alberta, Canada, Neisseria polysaccharea was isolated from the pharynxes of only 4 (0.5%) subjects, whereas Neisseria lactamica and Neisseria meningitidis were isolated from 110 (14%) and 15 (2%) children, respectively. These strains of N. polysaccharea, together with three other sporadic isolates from Alberta, Canada, were compared with the type strain from France and strains from Spain and Germany. All strains were phenotypically identical, except that the Canadian and German strains, for which the colistin MICs were 1 mg/liter, failed to grow on Thayer-Martin medium (TMM), whereas the type strain and the Spanish strains, for which the colistin MICs were greater than 7.5 mg/liter, were not inhibited. Multilocus enzyme electrophoresis indicated that six distinct electrophoretic types were present among the seven Canadian strains. Our results show that growth on gonococcal selective media which contain colistin is a variable feature of this taxon.