222 Modern Food Microbiology
incubation for Gram positives.
107 The microcolonies that develop must be viewed with a microscope.
For coliforms, pseudomonads, and staphylococci, as few as 10
3 /g could be detected within 8 hours.
107
In another variation, a microcolony epifluorescence microscopy method that combined DEFT with
hydrophobic grid membrane filter (HGMF) was devised.
106 By this method, nonenzyme detergenttreated samples are filtered through Nucleopore polycarbonate membranes, which are transferred to
the surface of a selective agar medium and incubated for 3 or 6 hours for Gram-negative or Grampositive bacteria as for microcolony-DEFT. The membranes are then stained with acridine orange,
and the microcolonies are enumerated by epifluorescence microscopy. The method allows results to
be obtained in <6 hours without a repair step for injured organisms, and in about 12 hours when a
repair step was employed.
106
Hydrophobic Grid Membrane Filter (HGMF)
The hydrophobic grid membrane filter (HGMF) technique was advanced by Sharpe and
Michaud,
118,119 and it has since been further developed and used to enumerate microorganisms from
a variety of food products. The method employs a specially constructed filter that consists of 1600
wax grids on a single membrane filter that restricts growth and colony size to individual grids. On
one filter, from 10 to 9 × 10
4 cells can be enumerated by an MPN procedure, and enumeration can
be automated.
19 The method can detect as few as 10 cells per gram, and results can be achieved in 24
hours or so.
116 It can be used to enumerate all cfus or specific groups such as indicator organisms,
8,15,33
fungi,
17 salmonellae,
32 and pseudomonads.
66 It has been given AOAC approval for total coliforms,
fecal coliforms, salmonellae, and yeasts and molds. The ISO-GRID method for fungi employs a special plating medium that contains two antibacterial antibiotics and trypan blue. The latter gives fungal
colonies a blue color, and as few as 10 cfu can be detected in 48 hours.
In a typical application, 1 ml of a 1:10 homogenate is filtered through a filter membrane, followed
by the placing of the membrane on a suitable agar medium for incubation overnight to allow colonies
to develop. The grids that contain colonies are enumerated, and the MPN is calculated. The method
allows the filtering of up to 1 g of food per membrane.
117 The ISOGRID method employing SD-39 agar
has been shown to be more versatile than ISO-GRID with lactose monensin glucuronate (LMG) agar
in conjunction with buffered MUG (4-methylumbelliferyl-β-d-glucuronide) agar for the detection of
E. coli in foods since it enables the simultaneous detection of E. coli O157:H7 and β-glucuronidasepositive E. coli.
34 The SD-39 agar method provides results in about 24 hours with a sensitivity of <10,
while LMG requires about 30 hours.
When compared to a five-tube MPN for coliforms, the HGMF method, employing a resuscitative step, produced statistically equivalent results for coliforms and fecal coliforms.
19 In the latter
application, HGMF filters were placed first on trypticase soy agar for 4–5 hours at 35
◦ C (for resuscitation of injured cells) followed by removal to m-FC agar for additional incubation. An HGMF-based
enzyme-labeled antibody (ELA) procedure has been developed for the recovery of E. coli O157:H7
(hemorrhagic colitis, HC) strains from foods.
126 The method employs the use of a special plating
medium that permits HC strains to grow at 44.5
◦ C. The special medium, HC agar, contains only
0.113% bile salt #3 in contrast to 0.15%. With its use, about 90% of HC strains could be recovered
from ground beef.
124 The HGMF-ELA method employs the use of HC agar incubated at 43
◦ C for 16
hours, washing of colony growth from membranes, exposure of membranes to a blocking solution,
and immersion in a horseradish peroxidase-protein A-monoclonal antibody complex. By the method,
ELA-positive colonies stain purple, and 95% of HC strains could be recovered within 24 hours with
a detection limit of 10 HC strains per gram of meat.
incubation for Gram positives.
107 The microcolonies that develop must be viewed with a microscope.
For coliforms, pseudomonads, and staphylococci, as few as 10
3 /g could be detected within 8 hours.
107
In another variation, a microcolony epifluorescence microscopy method that combined DEFT with
hydrophobic grid membrane filter (HGMF) was devised.
106 By this method, nonenzyme detergenttreated samples are filtered through Nucleopore polycarbonate membranes, which are transferred to
the surface of a selective agar medium and incubated for 3 or 6 hours for Gram-negative or Grampositive bacteria as for microcolony-DEFT. The membranes are then stained with acridine orange,
and the microcolonies are enumerated by epifluorescence microscopy. The method allows results to
be obtained in <6 hours without a repair step for injured organisms, and in about 12 hours when a
repair step was employed.
106
Hydrophobic Grid Membrane Filter (HGMF)
The hydrophobic grid membrane filter (HGMF) technique was advanced by Sharpe and
Michaud,
118,119 and it has since been further developed and used to enumerate microorganisms from
a variety of food products. The method employs a specially constructed filter that consists of 1600
wax grids on a single membrane filter that restricts growth and colony size to individual grids. On
one filter, from 10 to 9 × 10
4 cells can be enumerated by an MPN procedure, and enumeration can
be automated.
19 The method can detect as few as 10 cells per gram, and results can be achieved in 24
hours or so.
116 It can be used to enumerate all cfus or specific groups such as indicator organisms,
8,15,33
fungi,
17 salmonellae,
32 and pseudomonads.
66 It has been given AOAC approval for total coliforms,
fecal coliforms, salmonellae, and yeasts and molds. The ISO-GRID method for fungi employs a special plating medium that contains two antibacterial antibiotics and trypan blue. The latter gives fungal
colonies a blue color, and as few as 10 cfu can be detected in 48 hours.
In a typical application, 1 ml of a 1:10 homogenate is filtered through a filter membrane, followed
by the placing of the membrane on a suitable agar medium for incubation overnight to allow colonies
to develop. The grids that contain colonies are enumerated, and the MPN is calculated. The method
allows the filtering of up to 1 g of food per membrane.
117 The ISOGRID method employing SD-39 agar
has been shown to be more versatile than ISO-GRID with lactose monensin glucuronate (LMG) agar
in conjunction with buffered MUG (4-methylumbelliferyl-β-d-glucuronide) agar for the detection of
E. coli in foods since it enables the simultaneous detection of E. coli O157:H7 and β-glucuronidasepositive E. coli.
34 The SD-39 agar method provides results in about 24 hours with a sensitivity of <10,
while LMG requires about 30 hours.
When compared to a five-tube MPN for coliforms, the HGMF method, employing a resuscitative step, produced statistically equivalent results for coliforms and fecal coliforms.
19 In the latter
application, HGMF filters were placed first on trypticase soy agar for 4–5 hours at 35
◦ C (for resuscitation of injured cells) followed by removal to m-FC agar for additional incubation. An HGMF-based
enzyme-labeled antibody (ELA) procedure has been developed for the recovery of E. coli O157:H7
(hemorrhagic colitis, HC) strains from foods.
126 The method employs the use of a special plating
medium that permits HC strains to grow at 44.5
◦ C. The special medium, HC agar, contains only
0.113% bile salt #3 in contrast to 0.15%. With its use, about 90% of HC strains could be recovered
from ground beef.
124 The HGMF-ELA method employs the use of HC agar incubated at 43
◦ C for 16
hours, washing of colony growth from membranes, exposure of membranes to a blocking solution,
and immersion in a horseradish peroxidase-protein A-monoclonal antibody complex. By the method,
ELA-positive colonies stain purple, and 95% of HC strains could be recovered within 24 hours with
a detection limit of 10 HC strains per gram of meat.