Culture, Microscopic, and Sampling Methods
233
Mechanism of Repair
Pyruvate and catalase both act to degrade peroxides, suggesting that metabolically injured cells lack
this capacity. The inability of heat-damaged E. coli cells to grow as well when surface plated as when
pour plated with the same medium
47 may be explained by the loss of peroxides.
A large number of investigators have found that metabolic injury is accompanied by damage to
cell membranes, ribosomes, DNA, or enzymes. The cell membrane appears to be the most universally
affected.
55 The lipid components of the membrane are the most likely targets, especially for sublethal
heat injuries. Ribosomal damage is believed to result from the loss of Mg
2+ and not to heat effects per
se.
57 On the other hand, ribosome-free areas have been observed by electron microscopy in heat-injured
S. aureus cells.
64 Following prolonged heating at 50
◦ C, virtually no ribosomes were detected, and,
in addition, the cells were characterized by the appearance of surface blebs and exaggerated internal
membranes.
64 When S. aureus was subjected to acid injury by exposure to acetic, hydrochloric, and
lactic acids at 37
◦ C, coagulase and thermostable nuclease activities were reduced in injured cells.
132
Although acid injury did not affect cell membranes, RNA synthesis was affected. For more information
on cell injury and on methods of recovery, see reference 4).
VIABLE BUT NONCULTURABLE ORGANISMS
Under certain conditions and in some environments, standard plate count results suggest either an
absence of colony-forming units or numbers that may be considerably lower than the actual viable
population. Although this might appear to be the result of metabolic injury as outlined above, the viable
but nonculturable cells (VBNC) are in a state that sets them apart from injured cells. For example,
metabolically injured cells will repair when plated onto a nonselective medium that does not contain
inhibitors, but cells in the VBNC state will not.
The VBNC state was first noted with marine vibrios, which were difficult to culture from marine
waters during winter months. A downshift in temperature to around 5
◦ C is known to induce this
state. In an early study with Campylobacter jejuni, log phase cells were predominantly spiral shaped,
whereas late stationary phase cells were mainly coccoids.
109 The VBNC state was maintained at 4
◦ C
for >4 months. The cells in the VBNC state yielded low numbers by standard plate count, but by direct
viable count (DVC) and acridine orange direct count methods, viable cell numbers were found to be
about 7 logs higher; this phenomenon is illustrated in Figure 10–3.
Cells in the VBNC state are coccoid in shape, and in one study with V. vulnificus, this state was
induced in nutrient-limited artificial seawater after 27 days at 5
◦ C.
86 In another study, the VBNC state
was induced in V. vulnificus within 7 days following temperature downshift to 5
◦ C.
91 Resuscitation
normally occurs within 24 hours of return to temperatures around 21
◦ C.
92 Among internal cellular
changes known to occur as organisms enter the VBNC state are changes in cellular lipids and protein
synthesis. When the temperature was decreased from 23
◦ C to 13
◦ C for V. vulnificus, the generation
time increased from 3.0 to 13.1 hours and 40 new proteins were synthesized.
78 While in the VBNC
state, V. vulnificus has been shown to retain its virulence, although at reduced levels.
91 The VBNC state
has been demonstrated for Salmonella Enteritidis, Shigella, Vibrio cholerae, and enteropathogenic E.
coli, as well as those noted above. Although in one study evidence suggested that E. coli O157:H7
could enter the VBNC state in water,
130 investigators in another study were unable to induce the VBNC
state in a number of enteric bacteria, including E. coli.
18
Using a green fluorescent protein-tagged Pseudomonas fluorescens culture, cells that were stressed
at 37.5
◦ C and became VBNC fluoresced at an intensity of about 50% of nonstressed cells and those that
233
Mechanism of Repair
Pyruvate and catalase both act to degrade peroxides, suggesting that metabolically injured cells lack
this capacity. The inability of heat-damaged E. coli cells to grow as well when surface plated as when
pour plated with the same medium
47 may be explained by the loss of peroxides.
A large number of investigators have found that metabolic injury is accompanied by damage to
cell membranes, ribosomes, DNA, or enzymes. The cell membrane appears to be the most universally
affected.
55 The lipid components of the membrane are the most likely targets, especially for sublethal
heat injuries. Ribosomal damage is believed to result from the loss of Mg
2+ and not to heat effects per
se.
57 On the other hand, ribosome-free areas have been observed by electron microscopy in heat-injured
S. aureus cells.
64 Following prolonged heating at 50
◦ C, virtually no ribosomes were detected, and,
in addition, the cells were characterized by the appearance of surface blebs and exaggerated internal
membranes.
64 When S. aureus was subjected to acid injury by exposure to acetic, hydrochloric, and
lactic acids at 37
◦ C, coagulase and thermostable nuclease activities were reduced in injured cells.
132
Although acid injury did not affect cell membranes, RNA synthesis was affected. For more information
on cell injury and on methods of recovery, see reference 4).
VIABLE BUT NONCULTURABLE ORGANISMS
Under certain conditions and in some environments, standard plate count results suggest either an
absence of colony-forming units or numbers that may be considerably lower than the actual viable
population. Although this might appear to be the result of metabolic injury as outlined above, the viable
but nonculturable cells (VBNC) are in a state that sets them apart from injured cells. For example,
metabolically injured cells will repair when plated onto a nonselective medium that does not contain
inhibitors, but cells in the VBNC state will not.
The VBNC state was first noted with marine vibrios, which were difficult to culture from marine
waters during winter months. A downshift in temperature to around 5
◦ C is known to induce this
state. In an early study with Campylobacter jejuni, log phase cells were predominantly spiral shaped,
whereas late stationary phase cells were mainly coccoids.
109 The VBNC state was maintained at 4
◦ C
for >4 months. The cells in the VBNC state yielded low numbers by standard plate count, but by direct
viable count (DVC) and acridine orange direct count methods, viable cell numbers were found to be
about 7 logs higher; this phenomenon is illustrated in Figure 10–3.
Cells in the VBNC state are coccoid in shape, and in one study with V. vulnificus, this state was
induced in nutrient-limited artificial seawater after 27 days at 5
◦ C.
86 In another study, the VBNC state
was induced in V. vulnificus within 7 days following temperature downshift to 5
◦ C.
91 Resuscitation
normally occurs within 24 hours of return to temperatures around 21
◦ C.
92 Among internal cellular
changes known to occur as organisms enter the VBNC state are changes in cellular lipids and protein
synthesis. When the temperature was decreased from 23
◦ C to 13
◦ C for V. vulnificus, the generation
time increased from 3.0 to 13.1 hours and 40 new proteins were synthesized.
78 While in the VBNC
state, V. vulnificus has been shown to retain its virulence, although at reduced levels.
91 The VBNC state
has been demonstrated for Salmonella Enteritidis, Shigella, Vibrio cholerae, and enteropathogenic E.
coli, as well as those noted above. Although in one study evidence suggested that E. coli O157:H7
could enter the VBNC state in water,
130 investigators in another study were unable to induce the VBNC
state in a number of enteric bacteria, including E. coli.
18
Using a green fluorescent protein-tagged Pseudomonas fluorescens culture, cells that were stressed
at 37.5
◦ C and became VBNC fluoresced at an intensity of about 50% of nonstressed cells and those that