Long-Term Survival of Streptococcus pneumoniae at Room Temperature on Dorset Egg Medium

Abstract

Forty-five isolates of Streptococcus pneumoniae were inoculated on Dorset egg and supplemented Columbia agar base media, incubated overnight at 37°C, and then kept at room temperature (RT; 21°C) or 4°C. Long-term viability was best at RT for both media, with all isolates remaining viable on Dorset egg medium for 44 days; viability was 90 and 57% on Columbia agar base medium after 7 and 30 days. We recommend the use of Dorset egg medium for the maintenance of pneumococci at RT.

Streptococcus pneumoniae causes a broad range of diseases, including pneumonia, meningitis, sepsis, sinusitis, and otitis media, and is a leading cause of morbidity and mortality in children worldwide (4). Since the mid-1960s, reports of antimicrobial resistance have increased, and resistant strains have been reported from all five continents (6). In 1978 a pneumococcal reference laboratory was established at the South African Institute for Medical Research to collect isolates from centers in South Africa, to monitor the prevalence of resistant isolates, and to serotype southern African strains of pneumococci. We currently receive strains for molecular analysis from many developing countries. Pneumococcal viability on the usual laboratory media is limited to 24 to 48 h, and delays in transport therefore lead to poor recovery of organisms. We have assessed the recoverability of pneumococci from different transport media.

A preliminary study, evaluating Dorset egg (DE), Robertson’s cooked meat, semisolid charcoal, semisolid brain heart infusion with blood, and diphasic charcoal media, found the best recoverability to be on DE medium (1). We therefore compared the long-term viability of 45 pneumococcal strains of varying serotypes and drug susceptibility patterns on DE medium and Columbia agar base (supplemented) (CABS) medium. The strains, described in Table 1, were previously isolated from South African patients with pneumococcal disease. Susceptibility was defined according to National Committee for Clinical Laboratory Standards criteria for microtiter dilution (7). The strains were inoculated onto Columbia agar base (Oxoid) supplemented with 5% horse blood and incubated overnight at 37°C in 5% CO₂. Overnight cultures were then streaked on duplicate 7-ml glass screw-cap bottles (Bijou bottles) containing a 4-ml slant of either DE medium, prepared by combining sterile normal saline solution and whole hen’s eggs (beaten) in a 1:3 ratio and inspissating the mixture in an electric inspissator at 80°C for 60 min (2), or CABS medium. The CABS medium was prepared by diluting 9.75 g of Columbia agar base (Oxoid) and 1.0 g of activated charcoal (Sigma Chemical Co.) in 250 ml of distilled water and autoclaving the mixture; after cooling to 50°C, 25 ml of defibrinated horse blood was added and the medium was chocolatized by immersion in 100°C water for 6 min (5). One bottle of each medium was left at room temperature (RT; 21°C), and the other was kept at 4°C. After 7 and 12 days, and weekly thereafter, the isolates were subcultured with a 0.001-ml calibrated loop onto Columbia blood agar to assess the viability of the strain. Cultures were recorded as viable (at least one visible colony) or nonviable. The results are shown in Fig. 1. After 7 days, recovery of organisms incubated at 4°C on DE medium was 95%, and recovery was 44% on CABS medium (Fig. 1A); by 30 days, viability was 93 and 9% on DE and CABS media, respectively. According to a proportional hazards model (8), survival on DE medium was significantly longer than on CABS medium (P = 0.0001). Storage of pneumococci at RT proved to be better (Fig. 1B). All of the isolates stored on DE medium remained viable for at least 44 days at RT, while half of the strains stored on CABS medium were no longer viable after 30 days. The survival curves for the two media were significantly different (P = 0.0001). Serotypes and serogroups remained stable at both storage temperatures and on both media, and there appeared to be no differences in maintenance of viability between isolates with differing antibiotic susceptibility patterns. Contamination rates at RT were higher for CABS medium than for DE medium (23 versus 2%). The costs of the media also differed, at approximately $0.28 per Bijou bottle of DE medium and $0.45 per Bijou bottle of CABS medium.

TABLE 1.

Serotypes and antibiotic susceptibility patterns of S. pneumoniae isolates used to test long-term viability on DE and CABS media

Serotype	No. of isolates tested	Antibiotic susceptibilitya
1	6	Susceptible
	2	T
	1	CT
3	1	Susceptible
5	7	T
	1	P
6	4	Susceptible
7	1	CT
13	1	Susceptible
	1	P
14	2	Susceptible
	2	P
	2	PCT
	1	PTR
16	1	PT
18	1	Susceptible
19f	2	Susceptible
	3	P
23f	1	P
	3	PCT
	2	PCTR

^aP, penicillin resistant; T, tetracycline resistant; C, chloramphenicol resistant; R, rifampin resistant. Susceptibility was based on National Committee for Clinical Laboratory Standards criteria for microtiter dilution (7). 

FIG. 1.

Percentage of S. pneumoniae isolates viable on DE (solid lines) and CABS (dotted lines) media after incubation at 4°C (A) and RT (B). Contaminated isolates were not included in calculations of viability, which in some instances resulted in increases in survival rates between time points.

In conclusion, the recovery of pneumococci varied at both RT and 4°C and on both media tested. CABS medium maintained the viability of pneumococci longer than Robertson’s cooked meat, semisolid charcoal, semisolid brain heart infusion with blood, or diphasic charcoal medium (1), but many isolates in our study failed to grow after 23 days at either 4°C or RT. Our contamination rate on CABS medium was consistent with that previously seen (5), where 7 of 24 strains (29%) were lost due to overgrowth of contaminants. The contamination appeared before 30 days and may have been introduced during the addition of blood to the medium after autoclaving.

In our study, recovery was best on DE medium kept at RT. DE medium is a modification of the whole-egg medium first described by Dorset (3) and is a nonselective solid medium most commonly used for maintenance of mycobacteria. This medium is inexpensive to make and maintains the viability of pneumococci for at least 30 days without the need for refrigeration, making it ideal for transporting pneumococci between laboratories. Additional studies are required to define the role of DE medium for the maintenance of pneumococci from primary swabs of the nasopharynx. DE medium should also be investigated as a possible transport medium for other respiratory pathogens.