Culture, Microscopic, and Sampling Methods
231
freeze drying, drying, irradiation, aerosolization, dyes, sodium azide, salts, heavy metals, antibiotics,
essential oils, and other chemicals, such as ethylenediaminetetraacetic acid (EDTA) and sanitizing
compounds.
The recognition of sublethal stresses on foodborne microorganisms and their effect on growth under
varying conditions dates back to around 1900. However, a full appreciation of this phenomenon did
not come until the late 1960s. During the early 1960s, it was observed that an initial rapid decrease
in numbers of a metabolically injured organism was followed by only a limited recovery during
the resuscitation process (“Phoenix phenomenon”). The increased nutritional requirement of bacteria
that had undergone heat treatment was noted by Nelson
85 in 1943. (Nelson also reviewed the work
of others up to that time.) Gunderson and Rose
46 noted the progressive decrease in numbers of
coliforms from frozen chicken products that grew on VRBA with increasing storage time of products.
Hartsell
50 inoculated foods with salmonellae, froze the inoculated foods, and then studied the fate of the
organisms during freezer storage. More organisms could be recovered on highly nutritive nonselective
media than on selective media such as MacConkey, deoxycholate, or VRBA. The importance of
the isolation medium in recovering stressed cells was also noted by Postgate and Hunter
101 and by
Harris.
48 In addition to the more exacting nutritional requirements of foodborne organisms that undergo
environmental stresses, these organisms may be expected to manifest their injury via increased lag
phases of growth, increased sensitivity to a variety of selective media agents, damage to cell membranes
and tricarboxylic acid (TCA)-cycle enzymes, breakdown of ribosomes, and DNA damage. Although
damage to ribosomes and cell membranes appears to be a common consequence of sublethal heat
injury, not all harmful agents produce identifiable injuries.
Recovery/Repair
Metabolically injured cells can recover, at least in S. aureus, in no-growth media
59 and at a temperature of 15
◦ C but not 10
◦ C.
42 In some instances at least, the recovery process is not instantaneous,
for it has been shown that not all stressed coliforms recover to the same degree but that the process
takes place in a stepwise manner.
76 Not all cells in a population suffer the same degree of injury. Hurst
et al.
56 found dry-injured S. aureus cells that failed to develop on the nonselective recovery medium
(TSA), but did recover when pyruvate was added to this medium. These cells were said to be severely
injured in contrast to injured and uninjured cells. It has been found that sublethally heated S. aureus
cells may recover their NaCl tolerance before certain membrane functions are restored.
58 It is well
established that injury repair occurs in the general absence of cell wall and protein synthesis. It can
be seen from Figure 10–2 that the presence of chloramphenicol in the recovery medium had no effect
on the recovery of S. aureus from sublethal heat injury. The repair of cell ribosomes and membrane
appears to be essential for recovery, at least from sublethal heat, freezing, drying, and irradiation
injuries.
The protection of cells from heat and freeze injury is favored by complex media and menstra or
certain specific components thereof. Milk provides more protection than saline or mixtures of amino
acids,
81 and the milk components that are most influential appear to be phosphate, lactose, and casein.
Sucrose appears to be protective against heat injury
2,70 whereas glucose has been reported to decrease
heat protection for S. aureus.
81 Nonmetabolizable sugars and polyols such as arabinose, xylose, and
sorbitol have been found to protect S. aureus against sublethal heat injury, but the mechanism of this
action is unclear.
122
The consequences of not employing a recovery step have been reviewed by Busta.
20 The use of
trypticase soy broth (TSB) with incubations ranging from 1 to 24 hours at temperatures from 20
◦ C to
231
freeze drying, drying, irradiation, aerosolization, dyes, sodium azide, salts, heavy metals, antibiotics,
essential oils, and other chemicals, such as ethylenediaminetetraacetic acid (EDTA) and sanitizing
compounds.
The recognition of sublethal stresses on foodborne microorganisms and their effect on growth under
varying conditions dates back to around 1900. However, a full appreciation of this phenomenon did
not come until the late 1960s. During the early 1960s, it was observed that an initial rapid decrease
in numbers of a metabolically injured organism was followed by only a limited recovery during
the resuscitation process (“Phoenix phenomenon”). The increased nutritional requirement of bacteria
that had undergone heat treatment was noted by Nelson
85 in 1943. (Nelson also reviewed the work
of others up to that time.) Gunderson and Rose
46 noted the progressive decrease in numbers of
coliforms from frozen chicken products that grew on VRBA with increasing storage time of products.
Hartsell
50 inoculated foods with salmonellae, froze the inoculated foods, and then studied the fate of the
organisms during freezer storage. More organisms could be recovered on highly nutritive nonselective
media than on selective media such as MacConkey, deoxycholate, or VRBA. The importance of
the isolation medium in recovering stressed cells was also noted by Postgate and Hunter
101 and by
Harris.
48 In addition to the more exacting nutritional requirements of foodborne organisms that undergo
environmental stresses, these organisms may be expected to manifest their injury via increased lag
phases of growth, increased sensitivity to a variety of selective media agents, damage to cell membranes
and tricarboxylic acid (TCA)-cycle enzymes, breakdown of ribosomes, and DNA damage. Although
damage to ribosomes and cell membranes appears to be a common consequence of sublethal heat
injury, not all harmful agents produce identifiable injuries.
Recovery/Repair
Metabolically injured cells can recover, at least in S. aureus, in no-growth media
59 and at a temperature of 15
◦ C but not 10
◦ C.
42 In some instances at least, the recovery process is not instantaneous,
for it has been shown that not all stressed coliforms recover to the same degree but that the process
takes place in a stepwise manner.
76 Not all cells in a population suffer the same degree of injury. Hurst
et al.
56 found dry-injured S. aureus cells that failed to develop on the nonselective recovery medium
(TSA), but did recover when pyruvate was added to this medium. These cells were said to be severely
injured in contrast to injured and uninjured cells. It has been found that sublethally heated S. aureus
cells may recover their NaCl tolerance before certain membrane functions are restored.
58 It is well
established that injury repair occurs in the general absence of cell wall and protein synthesis. It can
be seen from Figure 10–2 that the presence of chloramphenicol in the recovery medium had no effect
on the recovery of S. aureus from sublethal heat injury. The repair of cell ribosomes and membrane
appears to be essential for recovery, at least from sublethal heat, freezing, drying, and irradiation
injuries.
The protection of cells from heat and freeze injury is favored by complex media and menstra or
certain specific components thereof. Milk provides more protection than saline or mixtures of amino
acids,
81 and the milk components that are most influential appear to be phosphate, lactose, and casein.
Sucrose appears to be protective against heat injury
2,70 whereas glucose has been reported to decrease
heat protection for S. aureus.
81 Nonmetabolizable sugars and polyols such as arabinose, xylose, and
sorbitol have been found to protect S. aureus against sublethal heat injury, but the mechanism of this
action is unclear.
122
The consequences of not employing a recovery step have been reviewed by Busta.
20 The use of
trypticase soy broth (TSB) with incubations ranging from 1 to 24 hours at temperatures from 20
◦ C to