Effect of low-osmolality nutrient media on growth and culturability of Campylobacter species

Abstract

The growth and culturability of Campylobacter jejuni NCTC 11351 and other campylobacters were examined in media having different osmolalities at a range of temperatures (4, 25, and 42 degreesC). The medium osmolalities used ranged from the osmolality of full-strength nutrient medium (modified campylobacter broth having an osmolality of around 254 mosmol) down to 96 mosmol. The following two methods were used to produce media having different osmolalities: dilution of the nutrient medium with distilled water and reformulation of the medium such that the concentrations of various osmolytes were altered while the nutrient content of the medium was unchanged. The results obtained with the two experimental methods were similar, indicating that there was an osmotic threshold effect, such that none of the campylobacters examined (C. jejuni NCTC 11351 and ATCC 33291, Campylobacter lari, and Campylobacter coli) grew in media having osmolalities around 130 mosmol and at temperatures below at 42 degreesC. Conversely, growth occurred in media having osmolalities of around 175 mosmol and above. Osmolar concentrations can be expressed in terms of osmolarity or osmolality. Osmolality is easier to evaluate, is the more commonly used term, and was used in the current study. In nutrient media having low osmolalities (i.e., 130 mosmol and below), the number of CFUs per milliliter declined rapidly regardless of the temperature, and no cells were recovered after 24 h. However, at nongrowth temperatures (25 and 4 degreesC) in higher-osmolality media (175 mosmol and above) a significant population was recovered throughout the experiment (up to 96 h). In low-osmolality nutrient media, the cellular morphology was principally coccoid, while in the early stages of growth in full-strength media the morphology was predominantly rodlike. We propose that the formation of coccoid cells in these experiments was the result of osmotic stress in low-osmolality media. This osmotic effect was apparent regardless of the osmolyte used to reformulate the medium (NaCl, KCl, Na2SO4, NH4Cl, and glucose were used).

FIG. 1

Growth of C. jejuni NCTC 11351 at 42°C (A), 25°C (B), and 4°C (C) in media having differing osmolalities. The basal medium used was MCB, and media having differing osmolalities were produced by diluting MCB. Symbols: ■, 257 to 263 mosmol; •, 172 to 178 mosmol; ▴, 122 to 128 mosmol; ▾, 114 to 117 mosmol; ⧫, 93 to 99 mosmol.

FIG. 2

Growth of C. jejuni NCTC 11351 at 42°C (A), 25°C (B), and 4°C (C) in media having differing osmolalities. The basal medium used was MCB, and media having differing osmolalities were produced by altering the amount of NaCl in MCB. Symbols: ■, 251 to 257 mosmol; •, 168 to 174 mosmol; ▴, 127 to 133 mosmol; ▾, 111 to 114 mosmol; ⧫, 91 to 97 mosmol; ×, sterile distilled water (2 to 3 mosmol).

FIG. 3

CFU plate counts for C. jejuni NCTC 11351 at 42°C in MCB having differing osmolalities after 12 h (A) and 24 h (B). At each osmolality, MCB was reformulated by altering the amount of one osmolyte (salt or glucose). The CFU counts were zero after 24 h in media having osmolalities of 127 to 133 mosmol or less.

FIG. 4

CFU plate counts for different Campylobacter spp. after 12 h of incubation in MCB having differing osmolalities (osmalality was adjusted by using KCl as the osmolyte). A, 251 to 257 mosmol; B, 127 to 133 mosmol; C, 111 to 117 mosmol; D, 91 to 97 mosmol.

FIG. 5

Scanning electron micrographs of C. jejuni NCTC 11351 in a medium having an osmolality between 251 and 257 mosmol, showing predominantly rodlike cells (A), and in a medium having an osmolality of 111 to 117 mosmol, showing predominantly coccoid cells (B), after 18 h of incubation at 42°C. Bars = 1 μm.