Effect of temperature on transport and plating media for enteric pathogens

Abstract

The effect of wide variations in incubation temperatures and long periods of incubation on transport and enrichment broths and plating media was determined by exhaustive analysis of 132 diarrheal stools for salmonellae and shigellae. Homogenized stools were streaked onto eosin methylene blue (EMB), Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) agar plates, and into saline, Cary-Blair (CB) transport medium, and Selenite F and gram-negative (GN) enrichment broths. Incubation temperatures were compared at 20 C, 35 C, 40 C and ambient, and over a range of 4 to 52 C for media incubated in an insulated picnic cooler in an auto trunk. At 1, 2, 4, and 7 days the plates were observed, and the broths were subcultured. Each stool was streaked to 12 plates for 48 observations and pickings, and to 48 tubes, subcultured to 192 plates, for a total of 240 observations for pathogens. Analysis of data from 6,246 Salmonella-positive plates showed direct streaking to be most effective after 2 days of incubation, but broths were equally effective at 1 or 2 days. By day 4 many plates were overgrown, and both plates and broths showed diminution of positives by about 10% and at day 7, 19%. The 2,434 Shigella-positive plates were more demanding in all times and temperatures of incubation than salmonellae. Although at day 2 best results were obtained on direct streaking, shigellae die-offs in broths were excessive, with positive declining 23.7% by day 2, 49% by day 4, and 60% by day 7. Direct plating of both pathogens was poor at 20 C with about 48% success, but salmonellae preferred higher temperatures (35 and 40 C), whereas shigellae chose 35 C and ambient, which averaged 28 C for the 10-month study. Temperature was immaterial to salmonellae in broths with ambient slightly better than 35 C, but shigellae preferred 20 C and showed a 50% failure rate at 40 C, ambient being equal to 35 C. The preferential rank of broths in efficacy was GN greater than selenite greater than saline greater than CB greater than direct for salmonellae; for shigellae, GN greater than saline greater than direct greater than CB greater than selenite, with selenite proving to be unsuitable for shigellae. Plating media preferences were XLD greater than EMB greater than SS. Ten of 39 shigellae strains could not be recovered from the selenite and SS media combination, the many replications notwithstanding. The effectiveness of salmonellae and shigellae detection at ambient temperatures in Louisiana during the 10-month study period, as compared to controlled incubation temperatures, indicates that satisfactory enteric bacteriology can be done in warm climates without constant temperature incubators.