232 Modern Food Microbiology
37
◦ C is widely used for various organisms. The enumeration of sublethally heated S. aureus strains
on various media has been studied.
14,37,56 In one of these studies, seven staphylococcal media were
compared on their capacity to recover 19 strains of sublethally heated S. aureus, and the Baird–Parker
medium was found to be clearly the best of those studied, including nonselective TSA. Similar findings
by others led to the adoption of this medium in the official methods of AOAC for the direct determination
of S. aureus in foods that contain ≥10 cells per gram. The greater efficacy of the Baird–Parker medium
has been shown to result from its content of pyruvate. The use of this medium following recovery in
an antibiotic-containing, nonselective medium has been suggested.
56 Although this approach may be
suitable for S. aureus recovery, some problems may be expected to occur with the widespread use
of antibiotics in recovery media to prevent cell growth. It has been shown that heat-injured spores
of C. perfringens are actually sensitized to polymyxin and neomycin,
10 and it is well established
that the antibiotics that affect cell wall synthesis are known to induce l-phase variations in many
bacteria.
Pyruvate is well established as an injury repair agent not only for injured S. aureus cells but also for
other organisms such as E. coli. Higher counts are obtained on media containing this compound when
injured by a variety of agents. When added to TSB containing 10% NaCl, higher numbers of both
stressed and nonstressed S. aureus were achieved,
14 and the repair–detection of freeze- or heat-injured
E. coli was significantly improved by pyruvate.
77
Catalase is another agent that increases recovery of injured aerobic organisms. First reported by
Martin et al.,
74 it has been found effective by many other investigators. It is effective for sublethally
heated S. aureus, Pseudomonas fluorescens, Salmonella Typhimurium, and E. coli.
74 It is also effective
for S. aureus in the presence of 10% NaCl,
14 and for water-stressed S. aureus.
37 Another compound,
shown to be as effective as pyruvate for heat-injured E. coli, is 3,3
-thiodipropionic acid.
77
Radiation injury of Clostridium botulinum type E spores by 4 kGy resulted in the inability to grow at
10
◦ C in the presence of polymyxin and neomycin.
110 The injured cells had a damaged postgermination
system and formed aseptate filaments during outgrowth, but the germination lytic system was not
damaged. The radiation injury was repaired at 30
◦ C in about 15 hours on tellurite polymyxin egg
yolk (TPEY) agar without antibiotics. When C. botulinum spores are injured with hypochlorite, the
l-alanine germination sites are modified, resulting in the need for higher concentrations of alanine
for repair.
39 The l-alanine germination sites could be activated by lactate, and hypochlorite-treated
spores could be germinated by lysozyme, indicating that the chloride removed spore coat proteins.
40
More detailed information on spore injury has been provided by Foegeding and Busta.
38
Sublethally heat-stressed yeasts are inhibited by some essential oils (spices at concentrations as low
as 25 ppm).
24 The spice oils affect colony size and pigment production.
Special plating procedures have been found by Speck et al.
123 and Hartman et al.
49 to allow for
recovery from injury and subsequent enumeration in essentially one step. The procedures consist of
using the agar overlay plating technique with one layer consisting of TSA, onto which are plated
the stressed organisms. Following a 1- to 2-hour incubation at 25
◦ C for recovery, the TSA layer is
overlaid with VRBA and incubated at 35
◦ C for 24 hours. The overlay method of Hartman et al. involved
the use of a modified VRBA. The principle involved in the overlay technique could be extended to
other selective media, of course. An overlay technique has been recommended for the recovery of
coliforms. By this method, coliforms are plated with TSA and incubated at 35
◦ C for 2 hours followed
by an overlay of VRBA.
In their comparison of 18 plating media and seven enrichment broths to recover heat-stressed
Vibrio parahaemolyticus, Beuchat and Lechowich
13 found that the two most efficient plating media
were water blue-alizarin, yellow agar and arabinose–ammonium–sulfate–cholate agar; arabinose-ethyl
violet broth was the most suitable enrichment broth.
37
◦ C is widely used for various organisms. The enumeration of sublethally heated S. aureus strains
on various media has been studied.
14,37,56 In one of these studies, seven staphylococcal media were
compared on their capacity to recover 19 strains of sublethally heated S. aureus, and the Baird–Parker
medium was found to be clearly the best of those studied, including nonselective TSA. Similar findings
by others led to the adoption of this medium in the official methods of AOAC for the direct determination
of S. aureus in foods that contain ≥10 cells per gram. The greater efficacy of the Baird–Parker medium
has been shown to result from its content of pyruvate. The use of this medium following recovery in
an antibiotic-containing, nonselective medium has been suggested.
56 Although this approach may be
suitable for S. aureus recovery, some problems may be expected to occur with the widespread use
of antibiotics in recovery media to prevent cell growth. It has been shown that heat-injured spores
of C. perfringens are actually sensitized to polymyxin and neomycin,
10 and it is well established
that the antibiotics that affect cell wall synthesis are known to induce l-phase variations in many
bacteria.
Pyruvate is well established as an injury repair agent not only for injured S. aureus cells but also for
other organisms such as E. coli. Higher counts are obtained on media containing this compound when
injured by a variety of agents. When added to TSB containing 10% NaCl, higher numbers of both
stressed and nonstressed S. aureus were achieved,
14 and the repair–detection of freeze- or heat-injured
E. coli was significantly improved by pyruvate.
77
Catalase is another agent that increases recovery of injured aerobic organisms. First reported by
Martin et al.,
74 it has been found effective by many other investigators. It is effective for sublethally
heated S. aureus, Pseudomonas fluorescens, Salmonella Typhimurium, and E. coli.
74 It is also effective
for S. aureus in the presence of 10% NaCl,
14 and for water-stressed S. aureus.
37 Another compound,
shown to be as effective as pyruvate for heat-injured E. coli, is 3,3
-thiodipropionic acid.
77
Radiation injury of Clostridium botulinum type E spores by 4 kGy resulted in the inability to grow at
10
◦ C in the presence of polymyxin and neomycin.
110 The injured cells had a damaged postgermination
system and formed aseptate filaments during outgrowth, but the germination lytic system was not
damaged. The radiation injury was repaired at 30
◦ C in about 15 hours on tellurite polymyxin egg
yolk (TPEY) agar without antibiotics. When C. botulinum spores are injured with hypochlorite, the
l-alanine germination sites are modified, resulting in the need for higher concentrations of alanine
for repair.
39 The l-alanine germination sites could be activated by lactate, and hypochlorite-treated
spores could be germinated by lysozyme, indicating that the chloride removed spore coat proteins.
40
More detailed information on spore injury has been provided by Foegeding and Busta.
38
Sublethally heat-stressed yeasts are inhibited by some essential oils (spices at concentrations as low
as 25 ppm).
24 The spice oils affect colony size and pigment production.
Special plating procedures have been found by Speck et al.
123 and Hartman et al.
49 to allow for
recovery from injury and subsequent enumeration in essentially one step. The procedures consist of
using the agar overlay plating technique with one layer consisting of TSA, onto which are plated
the stressed organisms. Following a 1- to 2-hour incubation at 25
◦ C for recovery, the TSA layer is
overlaid with VRBA and incubated at 35
◦ C for 24 hours. The overlay method of Hartman et al. involved
the use of a modified VRBA. The principle involved in the overlay technique could be extended to
other selective media, of course. An overlay technique has been recommended for the recovery of
coliforms. By this method, coliforms are plated with TSA and incubated at 35
◦ C for 2 hours followed
by an overlay of VRBA.
In their comparison of 18 plating media and seven enrichment broths to recover heat-stressed
Vibrio parahaemolyticus, Beuchat and Lechowich
13 found that the two most efficient plating media
were water blue-alizarin, yellow agar and arabinose–ammonium–sulfate–cholate agar; arabinose-ethyl
violet broth was the most suitable enrichment broth.