Effect of storage time and temperature of equine feces on the subsequent enumeration of lactobacilli and cellulolytic bacteria

Authors: HARLOW, BRITTANY - University Of Kentucky; LAWRENCE, LAURIE - University Of Kentucky; Flythe, Michael

Submitted to: Proceedings of the Equine Science Society
Publication Type: Abstract Only
Publication Acceptance Date: 4/25/2015
Publication Date: 5/26/2016
Citation: Harlow, B.E., Lawrence, L.M., Flythe, M.D. 2016. Effect of storage time and temperature of equine feces on the subsequent enumeration of lactobacilli and cellulolytic bacteria. Proceedings of the Equine Science Society. Pgs. 411-412.

Interpretive Summary:

Technical Abstract: Cellulolytic bacteria and lactobacilli are beneficial microbes in the equine hindgut. There are several existing methodologies for the enumeration of these bacteria, which vary based on selective and differential media and sample handling procedures including storage time and temperature. The objectives of the current study were to: 1) compare media types and 2) evaluate the effects of storage time and temperature on the enumeration of cellulolytic bacteria and lactobacilli. Feces were collected on different days from 3 mature mares maintained on pasture. Fresh fecal samples (1g/tube) were placed in 16 pre-weighed, sterile tubes. The stoppers were replaced and the tubes were purged of air with CO2. The tubes were maintained at 37ºC and transported to the lab within 0.5 h of collection. The fecal samples were then assigned to 1 of 4 treatments including: initial (n = 1), 37ºC storage (37C; n = 5), room temperature (22­24ºC) storage (RT; n = 5), or 4ºC storage (4C; n = 5). The initial sample was enumerated 5 times immediately upon arrival to the lab to assess repeatability. A sample from each storage temperature was enumerated after 2, 4, 8, 12, and 24 h of incubation. At each time point the designated samples were reweighed, suspended in anaerobic PBS (1:10 w/w), and were serially diluted (10-fold w/w) to inoculate the following media types: Rogosa SL agar and MRS agar for lactobacilli enumeration and rich cellulolytic media (RM) and defined cellulolytic media (DM) for cellulolytic enumeration. Enumeration data were log transformed and analyzed using the mixed procedure of SAS (v 9.3). MRS was not sufficiently selective; therefore, no viable counts were reported for this media type. All media types were highly reproducible, with the same enumerations reported for both RM and DM. After only 2 h there were >100-fold fewer cellulolytics in RT and 4C, with 10-fold fewer observed with 37C in comparison to initial (P < 0.05, in all cases). By 24 h of storage, 105-106-fold fewer cellulolytics were observed with 37C and RT (P < 0.05, in all cases). There were no detectable cellulolytics in 4C at 24 h. After only 2 h of storage there were fewer lactobacilli in RT and 4C than in initial and continued to decrease until 8 h (P < 0.05, in all cases). In contrast, from 2 -4 h 37C were similar to initial. However, after 8 h 37C had more lactobacilli than initial that continued to increase through 12 h (P < 0.05, in all cases). These results demonstrate that the sample handling factors, storage time and temperature, are important to consider when enumerating lactobacilli and cellulolytics from equine feces.