76 ◾ Fundamental Food Microbiology
surface, the size of the microcolony increases, and attachment between cells increases. Fimbriae
formation by the cells occurs faster at optimum temperature and pH of growth. Limited studies
also showed that when microorganisms, such as Pseudomonas fragi and Listeria monocytogenes, are
grown together they form a more complex biofilm than when either is grown separately. 1–4
The factors associated with the attachment of spoilage and pathogenic bacteria to meat and
carcass surfaces can be divided into three groups: associated with bacteria, associated with meat,
and extrinsic factors. 1–4 Many Gram-positive bacteria (Clostridium, Micrococcus, Staphylococcus,
Lactobacillus, and Brochothrix) and Gram-negative bacteria (Escherichia, Proteus, Pseudomonas,
Serratia, Salmonella, Enterobacter, Shewanella, and Acinetobacter spp.) can attach to skin and meat
surfaces of chicken, pork, beef, and lamb. Some studies showed that several Gram-negatives, such as
Proteus and Pseudomonas spp., attach more rapidly and in higher numbers than some Gram-positive
bacteria, such as Lactobacillus, Staphylococcus, and Micrococcus spp. However, there are differences in
observations, and some researchers think that the more negative charge the cells of species or strains
have, the faster and higher their ability to attach to muscle surfaces is. Similarly, results differ among
researchers with respect to the influence of types of food animals, birds, and microbial species to
attachment. Bacterial attachment to lean tissues is generally higher than to adipose tissues; however,
there is probably no difference in the attachment of both Gram-positive and Gram-negative bacterial species in the skin and muscle of different types of food animals and birds. Among the extrinsic
factors, the number of attached cells on meat surfaces is directly related to contact time and cell
concentrations used. At optimum growth temperature, a bacterial species shows a higher attachment
rate. Electrical stimulation of the carcasses may favor attachment, but opinions differ on this. 2–4
Intimate attachment of pathogens to fresh vegetables and fruits and their resistance to cleaning
and washing are thought to be responsible for increasing produce-related outbreaks. Escherichia
coli O157:H7 are found to attach to edges, grooves, or damaged tissues of lettuce, sprouts, or spinach. Salmonella enterica was also found on intact or broken skins of tomatoes, cantaloupes, and
apples and damaged areas of cilantro leaves. 6
Assessment of Biofilm Formation
Various methods are used to assess the formation of biofilms on solid surfaces. The direct microbiological plating method involves swabbing of the surface by using moistened cotton swabs or
sponges, and then diluted samples are spread on agar plates. To learn about the type of pathogens
involved, selective agar media are used. Swabs are also enriched in selective enrichment broths and
a polymerase chain reaction (PCR) is used for identification of the microorganisms. Contact plating is another method that allows direct imprinting of the surface onto the agar plate to determine
the microbes in biofilms. However, the rough surface of test materials, oils (lipids), pressure on
agar, and contact times may affect the result.
Indirectly, biofilm formation is also assessed by monitoring the ATP level of the surface swabs
by luminescence assay because ATP is present in living microbes (see Chapter 42). However, food
residues on the test surface may be a source of ATP, giving rise to false results. Traditional microscopic techniques involving both light microscopy and scanning electron microscopy are also used
to examine biofilm structure and organization of a microbial community of cells (Figure 7.1). In
addition, more sophisticated tools, such as laser scanning microscopy (LSM), magnetic resonance
imaging (MRI), and scanning transmission microscopy (STXM), are also used to assess structure
and composition of biofilms. 7
surface, the size of the microcolony increases, and attachment between cells increases. Fimbriae
formation by the cells occurs faster at optimum temperature and pH of growth. Limited studies
also showed that when microorganisms, such as Pseudomonas fragi and Listeria monocytogenes, are
grown together they form a more complex biofilm than when either is grown separately. 1–4
The factors associated with the attachment of spoilage and pathogenic bacteria to meat and
carcass surfaces can be divided into three groups: associated with bacteria, associated with meat,
and extrinsic factors. 1–4 Many Gram-positive bacteria (Clostridium, Micrococcus, Staphylococcus,
Lactobacillus, and Brochothrix) and Gram-negative bacteria (Escherichia, Proteus, Pseudomonas,
Serratia, Salmonella, Enterobacter, Shewanella, and Acinetobacter spp.) can attach to skin and meat
surfaces of chicken, pork, beef, and lamb. Some studies showed that several Gram-negatives, such as
Proteus and Pseudomonas spp., attach more rapidly and in higher numbers than some Gram-positive
bacteria, such as Lactobacillus, Staphylococcus, and Micrococcus spp. However, there are differences in
observations, and some researchers think that the more negative charge the cells of species or strains
have, the faster and higher their ability to attach to muscle surfaces is. Similarly, results differ among
researchers with respect to the influence of types of food animals, birds, and microbial species to
attachment. Bacterial attachment to lean tissues is generally higher than to adipose tissues; however,
there is probably no difference in the attachment of both Gram-positive and Gram-negative bacterial species in the skin and muscle of different types of food animals and birds. Among the extrinsic
factors, the number of attached cells on meat surfaces is directly related to contact time and cell
concentrations used. At optimum growth temperature, a bacterial species shows a higher attachment
rate. Electrical stimulation of the carcasses may favor attachment, but opinions differ on this. 2–4
Intimate attachment of pathogens to fresh vegetables and fruits and their resistance to cleaning
and washing are thought to be responsible for increasing produce-related outbreaks. Escherichia
coli O157:H7 are found to attach to edges, grooves, or damaged tissues of lettuce, sprouts, or spinach. Salmonella enterica was also found on intact or broken skins of tomatoes, cantaloupes, and
apples and damaged areas of cilantro leaves. 6
Assessment of Biofilm Formation
Various methods are used to assess the formation of biofilms on solid surfaces. The direct microbiological plating method involves swabbing of the surface by using moistened cotton swabs or
sponges, and then diluted samples are spread on agar plates. To learn about the type of pathogens
involved, selective agar media are used. Swabs are also enriched in selective enrichment broths and
a polymerase chain reaction (PCR) is used for identification of the microorganisms. Contact plating is another method that allows direct imprinting of the surface onto the agar plate to determine
the microbes in biofilms. However, the rough surface of test materials, oils (lipids), pressure on
agar, and contact times may affect the result.
Indirectly, biofilm formation is also assessed by monitoring the ATP level of the surface swabs
by luminescence assay because ATP is present in living microbes (see Chapter 42). However, food
residues on the test surface may be a source of ATP, giving rise to false results. Traditional microscopic techniques involving both light microscopy and scanning electron microscopy are also used
to examine biofilm structure and organization of a microbial community of cells (Figure 7.1). In
addition, more sophisticated tools, such as laser scanning microscopy (LSM), magnetic resonance
imaging (MRI), and scanning transmission microscopy (STXM), are also used to assess structure
and composition of biofilms. 7