Improved Aerobic Colony Count Technique for Hydrophobic Grid Membrane Filters
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1993, p. 2784-2789 Vol. 59, No. 9
0099-2240/93/092784-06$02.00/0
Copyright C 1993, American Society for Microbiology
Improved Aerobic Colony Count Technique for Hydrophobic
Grid Membrane Filters
LORNA J. PARRINGTON,* ANTHONY N. SHARPE, AND PEARL I. PETERKIN
Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health and Welfare Canada,
Tunney's Pasture, Ottawa, Ontario, Canada K1A OL2
Received 2 November 1992/Accepted 9 June 1993
The AOAC International official action procedure for performing aerobic colony counts on hydrophobic grid
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membrane filters (HGMFs) uses Trypticase soy-fast green FCF agar (FGA) incubated for 48 h. Microbial
growths are various shades of green on a pale green background, which can cause problems for automated as
well as manual counting. HGMFs which had been incubated 24 or 48 h at 35°C on Trypticase soy agar were
flooded underneath with 1 to 2 ml of 0.1% triphenyltetrazolium chloride (TTC) solution by simply lifting one
corner of the filter while it was still on the agar and adding the reagent. Microbial growths on HGMFs were
counted after color had been allowed to develop for 15 min at room temperature. With representative foods,
virtually all colonies stained pink to red. Automated electronic counts made by using the MI-100 HGMF
Interpreter were easier and more reliable than control HGMF counts made by the AOAC International official
action procedure. Manual counting was easier as well because of increased visibility of the microbial growths.
Except in the case of dairy products, 24-h TTC counts did not differ significantly from 48-h FGA counts,
whereas the FGA counts at 24 h were always significantly lower, indicating that for many food products the
HGMF TTC flooding method permits aerobic colony counts to be made after 24 h.
The aerobic colony count on Iso-Grid hydrophobic grid guishable from any intrinsic food background color. Re-
membrane filters (HGMFs) (QA Lifesciences Inc., San cently, the use of TSA plates containing 0.1% TTC (auto-
Diego, Calif.) has been an official final action procedure of claved with the medium) to produce automated HGMF
AOAC International and a standard method of the American counts of mesophilic aerobes in poultry abattoirs was found
Public Health Association for several years (3, 4, 9). The to be convenient and successful (10). However, for a tech-
procedure involves incubating the HGMF for 48 h on fast nique to be applied in routine food microbiology, there is a
green agar (FGA), which contains 0.025% fast green FCF need to circumvent two concerns: (i) TTC inhibits the
dye in Trypticase soy agar (TSA). Microbial growths de- growth of some organisms, and (ii) other organisms may not
velop hues ranging from pale green to intense blue-green. reduce TTC sufficiently to be detected (17, 7a). Our own
Growths within grid cells can result from potentially com- tests quickly confirmed that TTC incorporated into the
mingled microbial populations (15). Films of debris from medium can sometimes be inhibitory at concentrations high
some foods (e.g., corn and tuna [14]) adsorb the dye to enough to produce coloration of colonies suitable for the
produce a green background on the HGMF, which makes Interpreter.
discerning growths difficult. For manual counts, this can be It was surmised that if HGMFs were treated with TTC
an annoying, though minor, disadvantage of the method. after incubation on a standard nonselective medium such as
The automated HGMF counter (MI-100 HGMF Inter- TSA, then the first concern would be eliminated and the
preter, Richard Brancker Research Ltd., Ottawa, Ontario, intense metabolism of the colonies would provide an excel-
Canada) has been available for several years. In its simple lent environment for maximum reduction, minimizing the
counting mode, it inspects an HGMF placed under its second concern. This might then provide a stain more suited
camera and displays positive grid cells, the most probable than FGA to the Interpreter's needs.
number of growth units (MPNGU) (on HGMFs, the GU is
equivalent to the more familiar CFU), and a value represent-
ing the reliability of the count (1, 15). The optical signal is MATERIALS AND METHODS
optimum and counts are most accurate and precise when the Media and reagents. Plates with 20 ml of TSA (Difco) or
range of hues produced by microbial growths inside grid FGA (TSA containing 0.025% fast green FCF [C.I.42053;
cells is limited. Growth on FGA frequently does not meet Sigma Chemical Co.]) were dried for 20 min in a laminar flow
this ideal, and we sought a technique more suited to the hood. Bottles with 90 ml of peptone-Tween (1.0% Tween 80
Interpreter. [Sigma] in 0.1% peptone [Difco]) for diluent (or decimal
Triphenyltetrazolium chloride (TTC) has been used in dilutions as necessary) were prepared. For flooding plates, a
microbiology as a vital stain for many years. Colorless in its 1.0% stock solution of 2,3,5-TTC (Sigma) in distilled water
oxidized form, it is reduced by bacterial metabolizing sys- was diluted to 0.1% as required. The 1% stock solution of
tems to an insoluble red triphenylformazan. TTC is an TTC was kept at 4°C, and the 0.1% working solution was
attractive reagent because even if adsorbed by food debris, it
does not create a colored background and because the red kept on the bench for up to 2 months. In a preliminary
produced on reduction by microorganisms is easily distin- experiment, TSA plates containing 0.001, 0.005, 0.01, 0.05,
and 0.1% TTC (added either before or after autoclaving)
were prepared.
Food samples and food preparation. Foods were obtained
*
Corresponding author. either from local retailers or from Health Protection Branch
2784VOL. 59, 1993 HGMF AEROBIC COLONY COUNTS 2785
TABLE 1. Effect of addition of TTC (before or after autoclaving) to TSA on MI-100 Interpreter countsa
Count with:
Food Incubation Autoclaved TTC at concn (%) Unautoclaved TIC at concn (%) Control culture (no
(h)0'
___1d_____________________1d____ TJC added during
0.1 0.05 0.01 0.005 0.001d 0.1 0.05 0.01 0.005 0*001d incubation)'
Chili powder 24 NGe 2.06 2.38 2.39 2.34 NG NG 2.46 2.33 2.37 2.49
48 0.48 2.29 2.43 2.41 2.40 NG NG 2.47 2.43 2.44 2.41
Onion soup mix! 24 0.70 2.64 3.17 3.16 3.35 0.30 0.30 3.10 3.09 3.33 3.53
Black pepperg 48 0.78 2.63 2.92 2.96 3.06 NG 0.48 2.83 2.92 3.00 3.41
Chicken rinse no. 1 24 2.36 2.46 2.59 2.82 2.85 1.74 2.35 2.53 2.75 2.81 3.08
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48 3.28 3.26 3.26 3.24 3.17 2.84 3.26 3.23 3.26 3.25 3.15
Chicken rinse no. 2 24 3.47 3.49 3.49 3.36 3.35 3.44 3.36 3.47 3.49 3.32 3.48
48 3.63 3.71 3.67 3.62 3.69 3.62 3.58 3.72 3.75 3.52 3.71
a
Counts are transformed to base log1o.
b All samples were incubated at 35'C.
I
Samples were incubated on TSA, and agar was flooded with 2 ml of 0.1% 1TC underneath HGMFs after incubation. 24- and 48-h HGMF colony counts are
from duplicate samples.
d Very faint stains were present with TJC at this concentration.
I
NG, no growth.
f Not counted at 48 h because of heavy mold growth.
g Not counted at 24 h; colonies too small.
laboratories from samples acquired during routine surveil- count to obtain a satisfactory display of positives (1, 13).
lance. Food samples were stored at 4°C and analyzed within Counts were verified by the operator before being stored by
24 h or held frozen at -20°C for later analyses. For each food the instrument and were later imported into a spreadsheet
sample, 10 g was agitated for 60 s in 90 ml of peptone-Tween program. HGMFs which were not countable by the Inter-
in a Colworth 400 Stomacher (Canlab, Mississauga, Ontario, preter were counted by using an HGMF line counter (15),
Canada). Enzyme treatment, when necessary to facilitate and the MPNGU were determined by using the standard
filtration of samples, was carried out according to the conversion table (2, 15).
Iso-Grid methods manual (2). For suspensions heavy with Statistical evaluation. The data were considered to have
debris, pipet-tip prefilters (Filtaflex, Almonte, Ontario, Can- come from a blocked, two-by-two factorial experiment, the
ada) were used to draw aliquots from the stomacher bag (19). factors of interest being time (24 versus 48 h) and the two
AOAC International FGA count procedure. Samples were stains (FGA and TTC). The analyses were done separately
analyzed by the AOAC International aerobic plate count for each of the five food categories.
method (3, 4). Duplicate 1.0-ml sample aliquots (of homoge- The usual assumptions for the analysis of variance are that
nate or dilutions) were filtered through HGMFs by using an (i) the experimental errors are random, independent, and
MF-10 Spreadfilter (Richard Brancker Research Ltd.). The normally distributed (Gaussian distribution) about a zero
HGMFs were laid on FGA and incubated at 35°C. HGMFs mean with a common variance and (ii) the treatment effects
were counted at both 24 + 3 h and 48 h. are additive (6). Normality was tested by Shapiro and Wilk's
TTC count procedure. Quadruplicate 1.0-ml sample ali- test (11) and homogeneity was tested by Cockran's test (5,
quots (of homogenate or dilutions) were filtered through 16). The dairy products category had an unequal number of
HGMFs by using an MF-10 Spreadfilter, laid on TSA plates, observations for each combination of time and stain factors.
and incubated at 350C. At 24 h (± 3 h), two TSA plates for The analyses of these data were done by the method for
each sample were stained by lifting the corner of the HGMF unbalanced designs, as outlined in reference 18. Because the
with forceps (Millipore [Canada], Mississauga, Ontario, design was unbalanced, the interaction terms were analyzed
Canada) applying 1.5 + 0.5 ml of 0.1% TTC solution to the by calculating the population marginal means (PMM) (12) for
agar, and re-laying the HGMF so that all of its undersurface each cell and the appropriate PMM were compared by the
was wetted. The plates were left at least 15 min on the bench Bonferroni method (8). For the other food categories, for
for color development before counting. The remaining plates which the design was balanced, the arithmetic means for
were removed from the incubator after 48 h of incubation each cell were equal to the PMM. Therefore, to simplify the
and stained with TTC in the same way. reporting of the results, the PMM were reported for all food
Counting. All HGMFs were counted by using a comput- categories and testing among cell differences was done by
erized counter, the MI-100 Interpreter (Richard Brancker the Bonferroni method (8).
Research Ltd.), which has been described in detail by
Sharpe and Peterkin (15). The Interpreter discriminates RESULTS AND DISCUSSION
between the reflectances of the grid cells that are positive
(growth) and those that are negative (no growth). It displays Effect of TTC in the agar on growth. Table 1 shows counts
positive grid cells, the MPNGU, and a value (width) repre- converted to log base 10 obtained for growth on TSA
senting the reliability of the count. When the Interpreter did containing various concentrations of TTC either added be-
not flag grid cells that were obviously positive, particularly fore autoclaving or added aseptically just before plates were
with FGA plates, the arrow keys were used to "nudge" the poured. Autoclaved TTC agar plates ranged from bright red2786 PARRINGTON ET AL. APPL. ENvIRON. MICROBIOL.
TABLE 2. Widths and log MPNGUs of organisms from HGMFs incubated on FGA or stained with 0.1% TJC after incubation at 35°Cc
FGA 1TC
Sample 24 h 48 h 24 h 48 h
Widthb MPNGUC Width MPNGU Width MPNGU Width MPNGU
Meats
Beef liver 2.5 2.18 5.5 2.58 5.0 2.61 8.5 2.62
Chicken pieces, frozen 10.5 3.27 20.0 3.52 6.0 3.09 26.5 3.54
Chicken skin 2.0 2.47 5.0 3.10 14.0 3.52 4.0 3.20
Ground beef no. 1 4.5 3.24 20.5 3.69 21.5 3.66 21.0 3.77
Ground beef no. 2 1.0 1.29 5.0 2.10 4.0 1.96 17.0 2.19
Ground beef no. 3, frozen 4.0 3.44 27.5 3.78 32.0 3.73 22.0 3.80
Ground chicken no. 1 6.5 3.47 25.0 3.79 32.0 3.75 13.0 3.80
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Ground chicken, frozen 2.5 2.07 6.0 3.04 5.5 3.00 9.0 3.17
Cognac pate no. 1 2.5 1.24 2.0 1.62 8.0 1.32 31.5 1.54
Cognac pate no. 2 1.0 1.82 6.0 2.39 3.0 2.35 29.0 2.25
Cognac pate, frozen 1.0 2.46 10.5 2.82 16.0 2.63 54.5 2.86
Pork hock no. 1 10.0 3.60 37.0 3.83 13.5 3.59 12.5 3.69
Pork hock no. 2 2.5 2.60 4.0 3.20 5.5 2.69 4.5 3.24
Pork hock no. 3 7.0 3.55 31.5 3.73 74.5 3.70 18.5 3.76
Pork hock no. 4 6.0 3.61 30.5 3.79 68.5 3.80 27.5 3.77
Pork liver no. 1 1.5 2.38 4.0 2.71 3.0 3.05 8.5 2.77
Pork liver no. 2 1.5 2.19 2.5 2.63 4.0 3.42 7.5 2.64
Stewing beef 2.0 2.45 4.0 3.02 5.5 3.28 18.5 3.69
Avg 3.8 2.63 13.7 3.07 17.9 3.06 18.5 3.13
Dairy products
Cottage cheese, 1% UCd NGe UC 1.1g 39.0 1.20 UC 1.73f
Curd cheese 1.5 1.53 9.5 2.54 2.5 2.13 52.5 2.46
Danish blue cheese UC 2.12f Uc 2.48 9.5 2.17 20.0 2.62
Medium cheddar cheese 4.0 1.54 3.0 2.12 3.0 2.10 3.0 2.56
Mild cheddar cheese no. 1 6.0 1.72 8.0 2.29 16.0 2.03 16.5 2.59
Mild cheddar cheese no. 2 UC 0.9of 6.0 1.71 UC 9.5 1.76
Mozzarella cheese shreds 1.0 0.78 4.0 1.64 9.5 1.16 22.0 1.98
Skim milk UC 7.0 1.87 UC 1.18f 18.0 1.97
Yogurt, natural style UC NG UC 1.22f 4.5 1.08 16.5 2.08
Avg 3.1 1.35 6.3 1.89 12.0 1.57 19.8 2.20
Vegetables
Green beans 4.0 2.72 7.0 2.87 39.5 2.84 11.5 2.76
Bean sprouts 6.0 3.34 18.5 3.51 40.5 3.53 18.5 3.54
Carrots no. 1 3.0 2.78 6.5 3.16 11.0 2.85 8.0 3.01
Carrots no. 2 4.5 1.94 18.0 2.74 7.5 2.44 17.5 2.79
Coleslaw no. 1 2.0 1.73 15.5 1.87 43.5 1.91 40.5 2.04
Coleslaw no. 2 6.5 3.53 18.5 3.57 50.5 3.64 12.5 3.56
Cucumber no. 1 7.5 3.64 24.5 3.70 48.0 3.85 13.0 3.78
Cucumber no. 2 6.0 2.65 13.0 3.00 19.0 2.86 13.5 2.94
Romaine lettuce no. 1 4.5 1.22 10.5 2.20 13.0 2.14 33.0 2.32
Romaine lettuce no. 2 5.5 3.61 29.5 3.84 75.0 3.91 24.0 3.73
Mushrooms, fresh no. 1 6.0 3.34 19.0 3.56 34.0 3.45 25.5 3.70
Mushrooms, fresh no. 2 3.5 1.48 3.5 2.25 11.0 1.86 10.0 2.62
Onion 3.5 1.11 6.5 1.81 14.0 1.40 19.0 1.97
Tomato 6.5 1.46 4.0 2.09 35.5 1.71 23.5 1.98
Avg 4.9 2.47 13.9 2.87 31.6 2.74 19.3 2.91
Seafood
Besugo 7.5 1.82 26.5 1.92 20.0 1.85 12.5 2.29
Conch no. 1 14.0 3.50 35.0 3.78 74.5 3.79 41.5 3.82
Conch no. 2 5.5 3.27 17.0 3.39 15.0 3.28 28.5 3.30
Hasa 1.0 2.03 6.0 3.03 4.0 2.56 8.5 3.15
Marlin loins 2.0 2.51 5.0 2.90 8.0 2.71 4.0 2.68
Shrimp, raw 2.0 2.17 2.5 2.96 3.0 2.80 5.0 2.82
Shrimp, peeled and cooked 10.5 2.16 19.0 2.24 32.5 2.13 53.0 2.33
Sole no. 1 16.0 3.34 20.0 3.36 27.5 3.51 25.5 3.55
Sole no. 2 13.0 2.85 20.5 2.88 43.5 2.82 22.0 2.89
Whiting 2.0 2.58 3.5 3.08 3.0 3.27 8.5 3.39
Avg 7.4 2.62 15.5 2.95 23.1 2.87 20.9 3.02
Continued on following pageVOL. 59, 1993 HGMF AEROBIC COLONY COUNTS 2787
TABLE 2-Continued
FGA TTC
Sample 24 h 48 h 24 h 48 h
Widthb MPNGUC Width MPNGU Width MPNGU Width MPNGU
Miscellaneous
Carrot muffin batter 6.5 0.81 11.0 1.26 29.5 1.08 7.0 1.18
Black pepper 12.5 3.16 19.5 3.22 44.5 3.21 29.0 3.29
Potato salad 5.5 1.20 6.5 1.51 7.0 1.36 9.5 1.41
Turkey-vegetable pie no. 1 3.5 1.90 5.0 2.31 41.5 2.27 27.5 2.44
Turkey-vegetable pie no. 2 3.5 1.49 13.0 1.70 18.0 1.72 24.0 1.78
Zucchini muffin batter 3.0 1.64 5.0 2.24 10.5 1.91 13.0 2.21
Avg 5.8 1.70 10.0 2.04 25.2 1.93 18.3 2.05
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Total avge 5.0 12.8 22.4 19.3
a
Values are averages for duplicate plates.
b Value representing the reliability of the count.
c Converted to log1o.
d UC, uncountable by HGMF Interpreter.
' NG, no growth.
f Counted manually.
g Average for all countable samples.
(0.1%) to palest pink (0.001%), indicating some reduction of cheeses). Many foods (e.g., chicken, pork hock, shrimp,
the TTC; there was no coloration of the agar when the TIC marlin, and vegetables) produced very pale growths which
was added aseptically after autoclaving. There was signifi- tended to match the HGMF background on FGA at 24 h (the
cant inhibition of growth from chili powder, onion soup mix, HGMFs adsorbed the dye when laid on the agar). The color
and black pepper at TTC concentrations of 0.05 and 0.1%, of the growths intensified at 48 h for FGA. Table 2 shows
though autoclaved TTC, at 0.05%, was less inhibitory. At 48 that at 24 h, width values for TTC-flooded HGMFs were
h, soup mix plates showed heavy mold growth and HGMFs greater than those for HGMFs on FGA at either 24 or 48 h.
were not counted. For two chicken rinses, inhibition at 24 h At 48 h, widths for some TIC-flooded samples (particularly
was less noticeable than with other foods (particularly for vegetables) actually decreased slightly because intense TTC
autoclaved TTC) and not significant at 48 h. At 0.001% TIC, stains at the bases of the thicker colonies were masked by
there was little inhibition but colors were very faint. unstained growth at the upper surfaces of the colonies.
The effect of TTC-containing agar on HGMF counts However, this did not affect the ease of counting growths on
agrees with previous indications regarding possible inhibi- these plates. The vegetable samples had many heavily pig-
tion. However, the results obtained with chicken rinses mented growths at 48 h which did not appear to reduce the
support the decision made by McNab and coworkers (10) to TTC but which were still easily countable both with the
incorporate and autoclave TTC in the agar for analyses Interpreter and manually.
carried out on abattoir premises to obtain comparative Comparison of TTC flooding and FGA. Color development
results in tightly defined conditions. in colonies flooded with TTC at concentrations below 0.1%
Effect of TTC on ease and reliability of counting. Reflec- was fairly slow; at 0.5% or greater, stains quickly became
tances (luminances) of positive and negative grid cells fall very dark, which can sometimes cause image recognition
within a bi- or multimodal distribution on the histogram of problems. The concentration of 0.1% TTC chosen for flood-
number of grid cells possessing a certain reflectance versus ing gave both a convenient time and a color intensity suitable
reflectance displayed by the MI-100 Interpreter (1, 13, 15). for the Interpreter. The colony colors in samples flooded
Ideally, the distribution describing dark (positive) grid cells with TTC were much more uniform than those in samples
is well separated from the distribution describing light (neg- incubated on FGA. This was reflected in the width values,
ative) ones. The Interpreter calculates the reflectance (lumi- which reflect the reliability and reproducibility of the counts
nance) value separating negative and positive HGMF grid (13, 15). Table 2 shows the comparison of widths and log
cells by analyzing the shape of the histogram. In the Health MPNGUs for TTC and FGA, arranged according to time of
Protection Branch version (15) of the program used in this incubation and food category. The averages of the widths for
study, the Interpreter reports the separation of the distribu- TTC at 24 and 48 h were 22.4 and 19.3, respectively, while
tions as width (the number of reflectance units for a 5% the values for FGA at 24 and 48 h were 5.0 and 12.8. For five
change in apparent count). Well-separated distributions that of the dairy products on FGA, growths at 24 h were
are easily distinguished yield widths of 10 units or more. For uncountable by the Interpreter (i.e., there was no growth,
widths less than 10, the reproducibility of Interpreter counts the color was too pale, or the background color was too
decreases rapidly. While this is not serious because the intense), and for three, growths were still uncountable at 48
instrument alerts the operator, it increases the likelihood h. In contrast, growths on two of the dairy samples flooded
that nudging will be needed. with TTC were uncountable at 24 h (the growths were too
Many food types produce background color on the HG- small for the Interpreter to recognize a change in reflectance)
MFs, either intrinsically (such as chili powder and tomato) and only one sample had uncountable growth at 48 h
or through adsorption of the dye fast green FCF (most (overgrowth of grid cell walls).
noticeable in this case were the Danish blue and cheddar Table 3 shows the comparison of cell means, calculated2788 PARRINGTON ET AL. APPL. ENvIRON. MICROBIOL.
TABLE 3. Comparison of cell means of widths and MPNGUs from HGMFs incubated on FGA or stained with 0.1% TTC
after incubationa
Mean log width' Mean log MPNGUC
Food FGA TIC FGA 1TC
24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
Meat 0.46 0.96 A 1.02 A 1.17 A 2.63 3.07 B 3.06 B 3.13 B
Dairy products 0.50 C 0.87 CD 0.86 CD 1.28 D 1.05 1.89 E 1.57 2.20 E
Vegetables 0.67 1.06 F 1.41 G 1.24 FG 2.47 2.87 H 2.74 H 2.91 H
Seafood 0.70 1.05 I 1.14 I 1.18 I 2.63 2.95 J 2.87 J 3.02 J
Miscellaneous 0.70 K 0.94 K 1.31 L 1.20 L 1.70 2.04 M 1.93 M 2.05 M
a Values followed by the same letter are not significantly different at a pairwise significance level of 1% (calculated from PMM).
b Value representing the reliability of the count transformed to log1o to correct for lack of homogeneity.
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c Converted to log1o.
from the PMM for TTC and FGA for width and MPNGU. for log MPNGU, whereas only the stain was significant for
The results of the residuals for width indicated a lack of log width in the miscellaneous category.
homogeneity, which was corrected by a log transformation This postincubation staining by TTC requires very little
to the base 10. There were no significant differences among extra manipulation, since one does not have to remove the
HGMFs flooded with TTC at both 24 and 48 h and those HGMFs from the surface of the agar but simply lifts a corner
grown on FGA for the categories meat, vegetables, seafood, of the filter and applies TTC under it so that its undersurface
and miscellaneous for MPNGU. The value for FGA at 24 h is wetted. Unlike earlier, more-cumbersome staining meth-
was significantly lower than those for the other three com- ods in which the HGMFs required a destaining step to
binations for both MPNGU and width. For the dairy prod- remove excess stain (7), 1TC does not impart any color to
ucts, there was no significant difference between values for the HGMF and only those grids that contain growth are
T1C and FGA at 48 h, and both of these were significantly colored. It should be pointed out that while this study was
higher than that for TTC at 24 h, which was significantly aimed at providing a better technique for the automated
higher than the value for FGA at 24 h. For width, the value counter, growths on HGMFs stained with TTC were easier
for TTC at 24 h was not significantly different from its value and more pleasant to count manually than those on FGA.
at 48 h for all food categories. In conclusion, automated counts made by the MI-100
In general, the results for TTC were higher than those for HGMF Interpreter by the 1TC flooding method were easier
FGA; also, they were higher at 48 h than at 24 h for both and more reliable than control HGMF colony counts on
width and MPNGU. The significant elements from the FGA, and manual counts were made easier. The data show
analysis of variance table are reported in Table 4. For the that with the exception of dairy products, there is no
dairy products, both the time and the stain were significant significant difference between the TTC procedure at both 24
and 48 h and the 48-h AOAC International fast green HGMF
procedure for estimating mesophilic aerobic bacterial loads.
TABLE 4. Analyses of variance of widths and MPNGUs from
HGMFs incubated on FGA or stained with 0.1% TTC ACKNOWLEDGMENT
after incubationa We are grateful to Patrick J. Laffey, Biostatistics and Computer
Applications Division, HPB, for performing statistical analyses of
Food category Response
factorb Effect P value the data.
Meat Width Interactionc 0.01 REFERENCES
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Dairy products Width Timed 0.015 tario, Canada.
Width Staind 0.017 2. Anonymous. 1989. Iso-GridT' methods manual, 3rd ed. QA
MPNGU Time 0.0001 Lifesciences Inc., San Diego, Calif.
MPNGU Stain 0.0001 3. Association of Official Analytical Chemists. 1986. Aerobic plate
count in foods, hydrophobic grid membrane filter method. First
Vegetables Width Interaction 0.001 action. J. Assoc. Off. Anal. Chem. 69:376-378.
MPNGU Interaction 0.028 4. Association of Official Analytical Chemists. 1990. Official meth-
ods of analysis of the Association of Official Analytical Chem-
Seafood Width Interaction 0.006 ists, 15th ed. Association of Official Analytical Chemists, Ar-
MPNGU Time 0.0005 lington, Va.
MPNGU Stain 0.014 5. Barnett, V., and T. Lewis. 1984. Outliers in statistical data, 2nd
ed. John Wiley & Sons Ltd., New York.
Miscellaneous Width Stain 0.0001 6. Box, G. E. P., W. G. Hunter, and J. S. Hunter. 1978. Statistics
MPNGU Interaction 0.017 for experimenters. John Wiley & Sons Ltd., New York.
a Time and stain are the significant elements. 7. Brodsky, M. H., P. Entis, M. P. Entis, A. N. Sharpe, and G. A.
b Width and MPNGU values were converted to log1o. Jarvis. 1982. Determination of aerobic plate and yeast and mold
c Interaction implies a multiplicative effect between time and stain; values counts in foods using an automated hydrophobic grid-membrane
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