The Use of Bromthymol Blue Agar and Phenol Red Mannitol Agar for the Isolation of Pathogenic Types of Staphylococci

It was shown¹ that, on alkaline proteose lactose agar containing 0.017 per cent bromthymol blue, 98.5 per cent of pathogenic staphylococci grow luxuriantly in 48 hours while 94 per cent of non-pathogenic staphylococci grow poorly or not at all. The medium was proposed for the isolation of probable pathogenic staphylococci from mixed cultures.

The basic principles of the medium were: (a) selective inhibition of non-pathogenic staphylococci by bromthymol blue; and (b) enhancement of growth of pathogenic staphylococci by lactose which is fermented by pathogenic and a few non-pathogenic strains. While certain facts suggested that pathogenic strains were also inhibited by this medium, the parallel results obtained by selecting and testing various types of colonies from rabbit blood agar plates seemed to disprove this idea. However, the impression persisted and an alternate isolation medium was sought. Carbohydrate fermentation media were considered.

Numerous workers have studied the relation between fermentation of carbohydrates and other properties of staphylococci. The conclusion to be drawn from most of the reports is that certain carbohydrates are fermented by most pathogenic staphylococci but that this property is also shared by some non-pathogenic strains. Recent workers have centered their attention on mannitol. Fermentation of this alcohol was found to be parallel with in vitro and pathogenic properties of staphylococci² and mannitol agar was selected for further study.

Crude cultures suspected of containing pathogenic staphylococci were plated on Bacto phenol red mannitol agar and incubated overnight. Acid-producing colonies were isolated and tested. Most of them proved to be pathogenic types, the results being qualitatively similar to those obtained with bromthymol blue agar. However, many phenol red mannitol agar plates showed better growth of in vitro positive staphylococci than the corresponding bromthymol blue agar plates. An occasional bromthymol blue agar plate gave better results. This suggested that both media should be used to complement each other.

Because they contain a large number of different bacteria, cultures from the nose, throat, sputum and gum margins of patients with chronic infections serve as a severe test of the efficiency of a medium for the isolation of pathogenic staphylococci. A series of 111 samples was inoculated on both media, spread by means of glass spreaders and incubated. Acid-producing colonies could be detected on phenol red mannitol agar within 18 to 24 hours. Occasionally the yellow zone was not apparent until later, while in other cultures the initial yellow zones disappeared after 24 to 48 hours incubation. Bromthymol blue agar plates required 36 to 48 hours incubation.

Typical or suspicious colonies were isolated from both media and tested by the following methods:

Pigment: Incubate on proteose lactose agar 48 hours. Scrape a loopful from the plate and examine it in a good light. Orange, lemon yellow, yellow and light yellow are considered positive (+) while cream, white and doubtful colors are considered negative (0).

Hemolysis: Spread a minute amount of culture on rabbit blood agar plates and incubate overnight. If the colonies have varying degrees of hemolysis, separate the hemolytic types and test again after the variants have been purified. A wide or moderate hemolytic zone is considered positive (+), while slight hemolysis or none at all is considered negative (0).

Coagulase: Mix 1 loopful of the culture from solid medium with 0.5 cc. of fresh human or rabbit plasma. Shake thoroughly and let stand. At the end of 3 hours, and again overnight, tilt the tube to a horizontal position and note the presence of a solid clot or a softer jelly-like mass which may appear as a swollen tapioca grain rising slightly above the surface, or as an opaque disc. In these instances the result is considered positive (+).

Crystal violet agar: Take a loopful of the culture from solid medium and make a streak about 2 cm. long on a plate of crystal violet agar. Incubate 36 hours and observe the color of the growth. Violet or orange growths are considered positive (+), while white or pale violet growths are considered negative (0). Some of the white growths may have violet borders. Some non-pathogenic strains may fail to grow.

Bromthymol blue agar: Spread a loopful of the culture over the surface of a plate of bromthymol blue agar and incubate 36 to 48 hours. Strains producing luxuriant growths are considered positive (+) while those producing poor or no growth are considered negative (0).

Mannitol fermentation: Spread the culture on a petri dish of Difco phenol red mannitol agar and incubate overnight. Colonies surrounded by yellow zones are mannitol positive (+), while those with no yellow zones are negative (0).

The criteria of Chapman, Berens, Peters and Curcio³ were used in interpreting the results of hemolysis and coagulase tests which, in most cases, were parallel with the other in vitro and in vivo tests. Any coagulating strain, also hemolytic aureus strains (i.e. pigment, hemolysis and coagulase tests of +++0++, +0+, 00+ and ++0) were considered positive. A few strains which were negative to the hemolysis and coagulase tests (i.e 000, +00 or 0+0) gave positive reactions to the crystal violet agar, bromthymol blue agar and mannitol fermentation tests. For reasons which will be discussed elsewhere2 they were considered only feebly pathogenic. As a rule non-pathogenic strains reacted negatively to all six tests.

The results of comparison of the 111 cultures are detailed in table 1. For simplicity, the reactions to the 6 tests just described were recorded only when the strain did not react positively to all. It will be seen that both media gave similar results in 58.5 per cent of the cultures. Phenol red mannitol agar gave slightly better results in 20.7 per cent, moderately better in 11.7 per cent, and definitely better in 8.1 per cent of the cultures. Bromthymol blue agar gave better results in 1 culture (0.9 per cent).

 

During the course of these studies, it was noted that, in addition to their selective ability, both media had peculiarities which influenced their usefulness. From the summary of these factors in table 2 it is evident that, for best results, a combination of both methods should be used to isolate the maximum number of pathogenic staphylococci.

Phenol red mannitol agar is useful for rapid isolation of probable pathogenic staphylococci, 98.3 per cent of the colonies isolated proving to be of the pathogenic type. Bromthymol blue agar is more suitable when a large number of different bacteria are present or in those occasional instances where a pathogenic staphylococcus does not ferment mannitol. The chance of missing a pathogenic strain by the combined use of these two media is in proportion to the number of strains that fail to grow on bromthymol blue agar and fail to ferment mannitol. Of 367 hemolysis-coagulase-positive strains from pathologic sources, only 2 reacted negatively to these 2 tests.

For best results it is suggested that the scheme outlined in table 3 be used for the isolation of probable pathogenic staphylococci.

Conclusion

Probable pathogenic staphylococci may be isolated from mixed cultures by using bromthymol blue agar and phenol red mannitol agar for initial isolation.

 

References Cited:

1. Chapman, G. H., Lieb, C. W., Berens, C. and Curcio, L. G.: “The isolation of probable pathogenic staphylococci.” Jour. Bact. 33: 533-543. 1937.
2. Chapman, G. H., Berens, C., Nilson, E. L. and Curcio, L. G.: “Differentiation of pathogenic staphylococci from non-pathogenic types.” Jour. Bact. In press. 
3. Chapman, G. H., Berens, C., Peters, A. and Curcio, L. G.: “Coagulase and hemolysin tests as measures of the pathogenicity of staphylococci.” Jour. Bact. 28: 343-363. 1934.