sensitivity and specificity of the sanita-kun aerobic …...food biological contaminants sensitivity...

FOOD BIOLOGICAL CONTAMINANTS

Sensitivity and Specificity of the Sanita-kun Aerobic Count:Internal Validation and Independent Laboratory StudyHIROSHI MORITA

Chisso Corp., 13-1 Kachidoki 3 Chome, Chuo-ku, Tokyo 104-8555, JapanMASASHI USHIYAMA, SHIGEYUKI AOYAMA, and MIHOKO IWASAKI

Chisso Corp. Yokohama Research Center, 5-1 Okawa Kanazawa-ku, Yokohama 236-8605, Japan

The Sanita-kun Aerobic Count consists of a trans-parent cover film, an adhesive sheet, a layer ofnonwoven fabric, and a water-soluble compoundfilm, including a culture medium formula for detec-tion of aerobic microorganisms. The Sanita-kunsheet was validated for 14 food categories in an in-ternal study and an independent study was con-ducted on ground beef and hot dogs. Both studiesshowed no significant difference in performancebetween 5 or 8 replicates of the Sanita-kun sheetsand AOAC Method 966.23, excluding some lots offoods. The correlation coefficient to plate countagar in the internal accuracy study was 0.99. Theaverage relative standard deviation for repeatabil-ity of total foods was 0.26 and 0.19, respectively,excluding <10 average counts. The ruggednessstudy, which examined the influence of incubationtemperature and period, recommended incubationof the Sanita-kun sheet at 32.5 � 2.5�C for 46 � 2 h.Comparison of 3 lots of Sanita-kun sheets showedno decrease of performance in the older lot. Theshelf-life of the sheet is at least 14 months. TheSanita-kun Aerobic Counts has been grantedAOAC Performance Tested MethodSM status.

Performance Tested MethodSM certification is a valida-tion program administered by the AOAC Research In-stitute (AOAC RI). The goal of certification is to

validate the performance claims of commercial test kits. Datasupporting test kit performance claims, product literature, la-bels, manufacturing specifications, and QA/QC proceduresare submitted by the kit manufacturer. A laboratory study isdesigned by AOAC RI and conducted at an independent facil-ity. This report details the internal evaluation conducted by themanufacturer and the independent laboratory study of theSanita-kun Aerobic Count (Chisso Corp., Tokyo, Japan) per-formed at Silliker Laboratory Corporate Research Center(South Holland, IL).

The total aerobic count indicates the level of microorgan-isms in food. The official standard method for determining to-tal aerobic count uses plate count agar, which is prepared asfollows: dissolve the powder of plate count agar medium, ster-ilize, cool, store at 45 � 1�C, add to Petri dish, mix, and solid-ify. It is time-consuming and labor-intensive, and results inbulky waste. Sanita-kun (Figure 1), a ready-to-use sheet me-dium which features an easy test for food and environment isan easy swabbing test. It is quantitative, space saving, pro-duces little waste, and was developed as an alternative to platecount agar.

The Sanita-kun Aerobic Count device is a cultural mediumfor the enumeration of aerobic bacteria. It consists of anonwoven fabric on which a layer of microbial nutrients is de-posited in a film. The nutrient film, consisting of a water-solu-ble polymer, contains nutrients for microorganisms and2,3,5-triphenyltetrazolium chloride (TTC), which is reducedby bacterial metabolism to produce a visible red dye. Thenonwoven fabric and nutrient film are constructed on a basefilm (Figure 2). The basic device (fabric, nutrient, and basefilm) is inserted between an adhesive backing to stabilize thebasic device and a transparent film. The adhesive backing andthe transparent top film serve to deter evaporation and facili-tate inspection of the inoculated device.

Sample solutions containing bacteria are deposited on thefabric portion of the device. The sample solution then diffusesthroughout the entire pad to dissolve and release the nutrientcompound film, forming a highly viscous solution. Bacteriawill not permeate into the nutrient film layer because of itshigh viscosity. During the assimilation process of the dis-solved nutrient film and the nonwoven fabric, bacteria migrateto or near the surface of the fabric, where they metabolize thenutrients and TTC to produce a visible red dye after incuba-tion (Figure 2).

METHOD

Media and Reagents

(a) Sanita-kun Aerobic Count plate.—Chisso Corp. (To-kyo, Japan).

(b) Butterfield’s phosphate buffer (BPB).—Dissolve 34 gKH2PO4 in 500 mL water, adjust pH to 7.2 with 1N NaOHKH2PO4 34 g, and bring volume to 1000 mL with distilled wa-ter. Autoclave 15 min at 121�C. Dilute 1.25 mL of above solu-

MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 355

Received March 4, 2002. Accepted by AH August 14, 2002.Corresponding author’s e-mail: [email protected].

tion and bring volume to 1000 mL with distilled water. Auto-clave 15 min at 121�C.

(c) Physiological saline.—Dissolved 9.0 g NaCl in1000 mL water and autoclaved 15 min at 121�C.

Apparatus

(a) Incubator.—For maintaining 32.5 � 2.5�C.(b) Blender.—Waring, or equivalent, multispeed model

and 500 mL or 1 L metal blender jars with covers.(c) Stomacher.—IUL Instruments (Barcelona, Spain)

Masticater Type S, or equivalent with stomacher bags.(d) Pipets.—Gilson (Villier-le-Bel, France) Pipetman, or

equivalent, with sterilized or autoclaved chips.

Sample Preparation

Portions of 50 or 25 g sample are added to a stomacher bagor blender cup; 450 or 225 mL BPB or sterile physiological sa-line is then added to the stomacher bag or blender cup andstomached or homogenized for 2 min. Sample homogenate isdiluted with sterile physiological saline or BPB with decimaldilutions.

Analysis

Transparent cover film is opened, and 1 mL sample ho-mogenate or dilutions (1:100, 1:1000, 1:10 000 is applied tothe nonwoven fabric with pipet or equivalent. Transparentfilm is replaced to cover sample and will adhere to the sheet.The sheet is patted to ensure attachment; however, it shouldnot be squeezed. The sheet is incubated for 46 � 2 h at 32.5 �

2.5�C. Red colonies are counted with a marking pen or colonycounter. Used sheets are sterilized by autoclaving or boiling inhot water.

Reading and Interpreting Results

One red colony on the Sanita-kun sheet is counted as anaerobic colony. Colonies with a dim red color are also countedas aerobic colonies. When the number of red colonies is <300,one red colony is counted as 1 CFU. The CFU/g sample is de-termined by multiplying colonies by the dilution number.When the number of colonies is between 300 and 1000, colo-nies can be counted in the entire area or just one part.

Internal Laboratory Study

Precision

(a) Repeatability.—Several lots of 14 food types (frozenor raw pork, cabbage, roast pork, frozen or raw shrimp, icecream, fried egg, red pepper, buckwheat flour, raisins, udonnoodles, peanut butter, milk chocolate chips, dry cat food, andfrozen pizza) were obtained at local retail markets. The con-tamination level of each sample was determined by AOACOfficial Method 966.23. Several lots of each food type weremixed to prepare 4 target levels (<10, 10–25, ca 100, and ca250 CFU/mL). Each subsample (50 g) was homogenized with450 mL sterile physiological saline and diluted with sterilephysiological saline. Portions (1 mL) of homogenates were in-oculated to duplicate Sanita-kun and incubated at 35�C for48 h. Red colonies formed on the sheets were counted, andstandard deviations were calculated among 5 subsamples.

(b) Accuracy.—At least 3 lots each of 16 foods in 14 cate-gories (frozen or) raw pork, cabbage, roast pork, frozen andraw shrimp, ice cream, shelled eggs, red pepper, oregano,buckwheat flour, raisins, udon noodles, peanut butter, milkchocolate chips, dry cat food, frozen pizza, and frozen gyoza

356 MORITA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003

Figure 1. Sanita-kun Aerobic Counts package andsheet.

Figure 2. Structure of basic device of Sanita-kun sheet.

meat dumpling were obtained at local retail markets. Each50 g sample was homogenized with 450 mL BPB according toAOAC Official Method 966.23 except for shelled eggs, whichwere prepared according to AOAC Official Method 940.37.Sample dilutions (1:100, 1:1000, 1:10 000) were preparedwith sterile physiological saline. Samples of 1 mL homoge-nate and dilutions were plated on 5 replicates of Sanita-kunsheets according to package instruction, and with plate countagar according to Method 966.23. After incubation at 35�C for48 h, colonies formed on the plates, and Sanita-kun sheetswere counted.

(c) Ruggedness.—In a comparison of 24 and 48 h incuba-tion times, each 50 g sample was homogenized with 450 mLBPB according to AOAC Official Method 966.23 exceptshelled eggs, which were prepared according to AOAC Offi-cial Method 940.37. Sample dilutions (1:100, 1:1000,1:10 000) were prepared by adding sterile physiological sa-line. Portions (1 mL) of sample homogenate and dilutionswere applied on 5 replicate Sanita-kun sheets and incubated at35�C for 24 h. Colonies were counted after incubation. TheSanita-kun sheets were then incubated again at 35�C for 24 h,totaling 48 h. Colonies were counted again.

In an experiment to determine the effect of incubation tem-perature and time, each 50 g sample was added to a stomacherbag. To each bag, 450 mL sterile physiological saline wasadded, and the bag was stomached for 2 min. Sample dilutions(1:100, 1:1000, 1:10 000) were prepared by adding sterile phys-iological saline. Portions (1 mL) of sample homogenate and di-


Table 1. Precision study

Food

Targetlevel,

CFU/mL

Averagecount,a

CFU SDb RSDr

Pork <10 6 2 0.3

10–25 17 3 0.2

ca 100 118 15 0.13

ca 250 213 11 0.05

Cabbage <10 3 1 0.3

10–25 16 4 0.3

ca 100 107 23 0.21

ca 250 372 45 0.12

Frankfurter <10 5 1 0.2

10–25 23 5 0.2

ca 100 105 13 0.12

ca 250 288 16 0.06

Shrimp <10 2 1 0.5

10–25 9 3 0.3

ca 100 50 11 0.22

ca 250 141 25 0.18

Ice cream <10 4 1 0.3

10–25 25 1 0.04

ca 100 No sample

ca 250 No sample

Fried egg(Atsuyaki-tamago)

<10 1 1 1.0

10–25 2 1 0.5

ca 100 24 5 0.2

ca 250 187 10 0.05

Red pepper <10 5 3 0.6

10–25 23 6 0.3

ca 100 108 20 0.19

ca 250 264 36 0.14

Buckwheat flour <10 5 2 0.4

10–25 29 14 0.48

ca 100 91 26 0.29

ca 250 230 68 0.30

Raisins <10 3 1 0.3

10–25 6 2 0.3

ca 100 No sample

ca 250 No sample

Udon noodles <10 3 1 0.3

10–25 23 10 0.43

ca 100 118 14 0.12

ca 250 360 34 0.09

Peanut butter <10 8 2 0.3

10–25 No sample

ca 100 No sample

ca 250 No sample

Table 1. (continued)

Food

Targetlevel,

CFU/mL

Averagecount,a

CFU SDb RSDr

Milk chocolate chips <10 8 2 0.3

10–25 34 12 0.35

ca 100 No sample

ca 250 No sample

Dry cat food <10 6 2 0.3

10–25 24 4 0.2

ca 100 124 16 0.13

ca 250 289 23 0.08

Frozen pizza <10 5 1 0.2

10–25 16 4 0.3

ca 100 69 17 0.24

ca 250 173 21 0.12

All foods <10 5 0.38

10–25 19 0.30

ca 100 91 0.19

ca 250 229 0.12

a Average of counts of 5 subsamples.b SD = Standard deviation of counts of 5 subsamples.


Table 2. Accuracy study

Average count,a CFU RSDr APC, log10 CFU/g

Food Dilution Sanita-kunPlate count

agar Sanita-kunPlate count


agar

Frozen pork 1:1000 256a 237 0.04 0.03 5.41 5.38

1:10000 27 22 0.14 0.07

Frozen pork 1:10000 119 114 0.08 0.12 6.08 6.04

1:100000 10 12 0.36 0.46

Frozen pork 1:10000 68a 53 0.12 0.27 5.88 5.72

1:100000 7 5 0.31 0.60

Frozen pork 1:10000 189b 275 0.15 0.09 6.28 6.43

1:100000 16a 26 0.25 0.24

Raw pork 1:100000 109a 123 0.07 0.05 7.04 7.08

1:1000000 10 13 0.43 0.19

Raw pork 1:10000 364 417 0.04 0.12 6.57 6.62

1:100000 45 43 0.09 0.12

Cabbages 1:10000 47b 116 0.18 0.13 5.65 6.04

1:100000 3 9 0.80 0.40

Cabbages 1:10000 150 168 0.11b 0.02 6.18 6.23

1:100000 14 16 0.46 0.28

Cabbages 1:10000 136 222 0.11 0.06 6.15 6.34

1:100000 14 24 0.22 0.24

Roast pork 1:1000 94 157 0.18 0.07 4.97 5.20

1:10000 9 15 0.27 0.31

Roast pork 1:100 98 143 0.16 0.07 4.04 4.15

1:1000 25 16 0.37 0.10

Roast pork 1:1000 19 50 0.21 0.11 4.28 4.69

1:10000 1 4 0.37 0.61

Frozen shrimp 1:100 107 104 0.10 0.08 4.04 4.00

1:1000 10 11 0.40 0.13

Frozen shrimp 1:100 30 38 0.20 0.16 3.46 3.57

1:1000 2 3 0.80 0.30

Frozen shrimp 1:100 124a 140 0.07 0.06 4.08 4.15

1:1000 11 11 0.21 0.15

Frozen shrimp 1:1000 42b 75 0.04b 0.18 4.61 4.86

1:10000 2b 6 0.56 0.27

Frozen shrimp 1:1000 29b 84 0.19 0.17 4.46 4.91

1:10000 2b 7 0.59 0.14

Frozen shrimp 1:1000 50b 79 0.15 0.10 4.67 4.89

1:10000 2a 7 0.71 0.41

Ice cream 1:10 57 83 0.08 0.19 2.79 2.94

1:100 11 14 0.34 0.36

Ice cream 1:10 32 27 0.17 0.10 2.52 2.45

1:100 5 3 0.27 0.41

Ice cream 1:10 33 35 0.21 0.20 2.52 2.53

1:100 3 2 0.26 0.70

Shelled eggs 1:10 157 176 0.13 0.06 3.18 3.23

1:100 13 15 0.24 0.23







agar

Shelled eggs 1:10 33 33 0.19 0.19 2.48 2.48

1:100 3 3 0.62 0.34

Shelled eggs 1:10 91b 127 0.13 0.11 2.96 3.11

1:100 10 13 0.45 0.30

Oreganoc 1:1000 116 112 0.04 0.13 5.04 5.04

1:10000 6 9 0.46 0.36

Oreganoc 1:10000 113 135 0.07 0.13 6.04 6.11

1:100000 10 11 0.42 0.39

Oreganoc 1:10000 46a 61 0.09 0.14 5.66 5.79

1:100000 7 8 0.25 0.28

Red pepper 1:1000 88 77 0.07 0.19 4.94 4.89

1:10000 8 8 0.40 0.27

Red pepper 1:1000 90 87 0.15 0.14 4.96 4.94

1:10000 12 10 0.29 0.13

Red pepper 1:1000 94 89 0.08 0.08 4.99 4.95

1:10000 14 10 0.15 0.32

Buckwheat flour 1:1000 89 80 0.09 0.32 4.95 4.92

1:10000 9 11 0.32 0.31

Buckwheat flour 1:1000 74 77 0.10 0.19 4.87 4.88

1:10000 8 7 0.11 0.17

Buckwheat flour 1:1000 75 68 0.10 0.08 4.88 4.83

1:10000 8 7 0.28 0.23

Dry green laver 1:10 193 204 0.05 0.11 3.32 3.34

1:100 41 36 0.15 0.16

Raisins 1:100 31 22 0.31b 0.10 3.49 3.38

1:1000 2 4 0.54 0.34

Raisins 1:100 15a 18 0.07 0.09 3.18 3.28

1:1000 2 2 0.71 0.50

Raisins 1:100 5 8 0.45 0.46 2.64 2.88

1:1000 0 1 2.24 1.05

Udon noodles 1:1000 11 14 0.23 0.29 4.04 4.11

1:10000 1 0 0.56 2.24

Udon noodles 1:1000 236b 331 0.17a 0.04 5.38 5.52

1:10000 26 29 0.12 0.10

Udon noodles 1:100 42 46 0.15 0.12 3.62 3.66

1:100 4 4 0.39 0.50

Peanut butter 1:10 3 3 0.39 0.53 <2.34 <2.34

1:100 0 0 2.24 2.24

Peanut butter 1:10 7a 12 0.58 0.13 <2.34 <2.34

1:100 2 3 0.59 0.75

Peanut butter 1:10 16 15 0.51a 0.18 <2.34 <2.34

1:100 2 1 0.71 0.81

Milk chocolate 1:10 44 52 0.19 0.39 2.63 2.75

1:100 4b 10 0.47 0.20

lutions were applied on 5 replicate Sanita-kun sheets and incu-bated at 30, 32.5, 35, and 37�C for 24–48 h. Colonies werecounted at 24, 27–30, 41, 44–45 and/or 48 h after incubation.

(d) Limit of quantitation.—Sample homogenate or dilutionwas prepared to contain the aerobic microorganism at ca1 CFU/mL. Sample homogenate or dilution was inoculated to 10replicate Sanita-kun sheets and incubated at 35�C for 48 h. Col-onies on each Sanita-kun sheet were counted after incubation.

(e) Lot-to-lot study.—The 3 lots of Sanita-kun sheets were9909TC (manufactured in September 1999), 00918TC (Sep-tember 2000), and 001124TC (November 2000). Elevenfoods were prepared as homogenates according to AOAC Of-ficial Method 966.23. Sample dilutions (1:100, 1:1000,1:10 000) were prepared by adding sterile physiological sa-line. Sample homogenates and dilutions were applied on

duplicate Sanita-kun sheets and incubated at 35�C for 48 h. Col-onies on each Sanita-kun sheet were counted after incubation.

Independent Laboratory Study

Comparative Recovery

(a) Sanita-kun Aerobic Count vs AOAC Official Method966.23 Methodology.—A single strain of Escherichia coli wasused in the study. It was obtained from the Silliker Labora-tories Research (SLR) culture collection and labeled asSLR 1392. The original source of the isolate was AmericanType Culture Collection (ATCC) 11229. The strain was culti-vated in trypticase soy broth and incubated at 35�C for 24 h. Af-ter incubation, serial dilutions of the strain were made and ana-lyzed for viable counts by the pour plate technique using







agar

Milk chocolate chips 1:10 2 5 0.46 0.58 <2.34 <2.34

1:100 0 1 2.24 1.05


1:100 0 0


1:100 0 0

Dry cat food 1:10 14 16 0.46 0.27 <2.34 <2.34

1:100 1 0 1.00

Dry cat food 1:10 8 6 0.31 0.50 <2.34 <2.34

1:100 0 0

Dry cat food 1:10 33 37 0.32 0.54 2.53 2.57

1:100 5 3 0.55 0.64

Frozen pizza 1:1000 406b 285 0.02 0.09 6.61 6.46

1:10000 48b 33 0.08 0.10

Frozen pizza 1:10000 53a 86 0.09 0.18 6.75 6.94

1:100000 8 10 0.19 0.44

Frozen pizza 1:10000 171b 120 0.05 0.13 6.23 6.08

1:100000 21b 10 0.11 0.26

Frozen gyoza meatdumpling

1:10 21 13 0.20 0.55 2.32 2.15

1:100 2 2 0.50 0.95


1:10 21 13 0.20 0.55 2.32 2.15

1:100 2 2 0.50 0.95


1:10 12 17 0.40 0.28 2.11 2.23

1:100 2 2 0.55 0.40

a Significantly higher or lower (p < 0.05).b Significantly higher or lower (p < 0.01).c The colonies in oregano by Sanita-kun were counted after 24 h incubation.

trypticase soy agar incubated at 35�C for 24 h. The strain washeld at 4�C until viable plate counts were determined. A por-tion of the appropriate culture dilution was inoculated into thefoods to prepare the target level sample considering indige-nous populations.

(b) Food.—Ground beef and hot dogs, purchased from lo-cal grocery stores, were used.

(c) Procedure.—Each 25 g of inoculated or uninoculatedsample was added to 400 mL stomacher bags; 225 mL BPBwas added to each stomacher bag and samples were stom-ached for 2 min. Sample dilutions (1:100, 1:1000, 1:10 000)were prepared by adding BPB. Sample homogenate and dilu-tions were plated on 8 replicates of Sanita-kun according topackage instruction and with plate count agar according toAOAC Official Method 966.23.

(d) Statistical analyses.—The difference of mean aerobicplate counts was compared by using the t-test, and the differ-ence of standard deviation was compared with the F-ratio test.

Results

Internal Laboratory Study

(a) Repeatability.—The average counts and the relativestandard deviations for repeatability (RSDr) of 5 replicates(5 subsamples) of 4 target contamination levels (<10, 10–25,about 100, about 250 CFU/mL) of 14 categories are shown inTable 1. The contamination level, >100 CFU/mL, was notfound in ice cream, raisins, and milk chocolate chips. In pea-nut butter, even >10 CFU/mL level contamination was notfound. In all foods, the RSDr in higher contamination levelswas relatively small, and large RSDr values were observed incontamination levels <10 CFU/mL. The average RSDr values

of frozen or raw pork, cabbage, roast pork, frozen or rawshrimp, ice cream, fried egg, red pepper, buckwheat flour, rai-sins, udon noodles, peanut butter, milk chocolate chips, drycat food, and frozen pizza were 0.17 (0.13), 0.23 (0.21), 0.15(0.13), 0.30 (0.20), 0.17 (0.04), 0.44 (0.13), 0.31 (0.21), 0.37(0.36), 0.3, 0.24 (0.21), 0.3, 0.33 (0.35), 0.18 (0.14), and 0.22(0.22), respectively. (Shown in parentheses are the averageRSDr values, excluding those in the contamination level<10 CFU/mL.) The average RSDr values of total foods were0.38, 0.30, 0.19, and 0.12 in <10, 10–25, about 100, and about250 CFU/mL target levels, respectively. The average RSDr oftotal foods, except those <10 average counts, was 0.19.

(b) Accuracy.—Most aerobic plate counts (APCs) offoods with Sanita-kun and plate count agar were similar (Ta-ble 2). The APCs for one lot each of frozen pork, cabbage,shelled eggs, and udon noodles and one set of 3 lots of frozenshrimp were significantly lower (p < 0.01) with Sanita-kunthan with plate count agar, and one lot each of raw pork,frozen shrimp, and frozen pizza was significantly lower (p <0.05). The APCs for 2 lots of frozen pork and frozen pizzawere significantly higher (p < 0.05 and p < 0.01, respectively)with Sanita-kun than with plate count agar.

The log10 APC results of both Sanita-kun sheets and platecount agar were plotted on logarithmic paper. The regressionanalysis is shown in Figure 3. The correlation coefficient (r2)of the Sanita-kun sheets compared to the plate count agar wascalculated as 0.99.

The average RSDr values of both Sanita-kun sheets andplate count agar on the calculated lower dilutions in each foodtype were 0.08 and 0.11 (frozen and raw pork), 0.13 and 0.07(cabbage), 0.18 and 0.08 (roast pork), 0.13 and 0.13 (frozenshrimp), 0.15 and 0.16 (ice cream), 0.15 and 0.12 (shelledeggs), 0.07 and 0.13 (oregano), 0.10 and 0.14 (red pepper),0.10 and 0.20 (buckwheat flour), 0.28 and 0.22 (raisins), 0.18and 0.15 (udon noodles), 0.10 and 0.28 (peanut butter), 0.46and 0.58 (milk chocolate), 0.36 and 0.44 (dry cat food), 0.05and 0.13 (frozen pizza), and 0.28 and 0.34 (frozen gyoza). Theaverage RSDr values of total foods were 0.18 (Sanita-kunsheets) and 0.21 (plate count agar). Significant difference ofthe RSDr between Sanita-kun sheets and plate count agar wasobserved in some foods. The RSDr values of one lot each ofcabbage and raisins were significantly higher (p < 0.01) withSanita-kun sheets than with plate count agar. For one lot offrozen shrimp, the RSDr for the Sanita-kun sheets was signifi-cantly lower (p < 0.01).

(c) Ruggedness.—In many foods, the APC between 24and 48 h incubation was similar (Table 3). For pork, cabbage,shrimp, and udon noodles, the APCs at 24 h incubation werelower than those at 48 h by approximately one-half.

In all foods, no significant differences of APCs between44 and 45 h and 48 h incubation at 32.5 and 35�C were ob-served. For raw pork, buckwheat flour, and dry cat food, nosignificant differences of APCs between 24 and 48 h incuba-tion at 35�C were observed (Table 4). No significant differ-ences were observed between 32.5 and 35�C incubation, ex-cept for cabbage, frozen shrimp, and fried egg. The APCs ofcabbage, frozen shrimp, and fried egg at 32.5�C were signifi-


Figure 3. Regression analysis of Sanita-kun sheets toplate count agar.


Table 3. Ruggedness study (comparison of 24 and 48 h incubation)


Food Dilution 24 h 48 h 24 h 48 h 24 h 48 h

Raw pork 1:10000 291 364 0.09 0.04 6.46a 6.57

1:100000 25 45 0.27 0.09

Cabbage 1:10000 10 47 0.15 0.18 5.00a 5.65

1:100000 1 3 1.05 0.80

Cabbage 1:10000 90 150 0.16 0.11 5.95a 6.18

1:100000 8 14 0.41 0.46

Frozen shrimp 1:10000 52 74 0.12 0.11 5.72a 5.91

1:100000 6 8 0.63 0.61

Frozen shrimp 1:1000 22 42 0.19 0.04 4.32a 4.61

1:10000 1 2 1.05 0.56

Frozen shrimp 1:1000 27 50 0.17 0.15 4.40a 4.67

1:10000 1 2 0.91 0.71

Ice cream 1:10 31 33 0.21 0.21 2.49 2.52

1:100 3 3 0.26 0.26

Ice cream 1:10 30 32 0.16 0.17 2.49 2.52

1:100 4 5 0.34

Shelled eggs 1:10 110 157 0.16 0.13 3.00 3.15

1:100 1 2 0.96 0.71

Shelled eggs 1:10 24 33 0.22 0.19 2.36 2.52

1:100 1 3 0.91 0.62

Red pepper 1:10000 78 90 0.17 0.15 5.90 5.94

1:100000 10 12 0.36 0.29

Red pepper 1:10000 81 88 0.08 0.07 5.90 5.96

1:100000 7 8 0.41 0.40

Red pepper 1:10000 81 94 0.08 0.08 5.92 5.99

1:100000 10 14 0.24 0.15

Dry tangle 1:10 9 12 0.74 0.5 <2.34 <2.34

1:100

Dry green laver 1:10 135 193 0.05 0.05 3.18 3.32

1:100 26 41 0.14 0.15

Buckwheat flour 1:1000 61 89 0.16 0.09 4.79b 4.95

1:10000 7 9 0.31 0.32

Buckwheat flour 1:1000 67 74 0.11 0.10 4.81 4.87

1:10000 5 8 0.27 0.11

Buckwheat flour 1:1000 46 75 0.20 0.10 4.65 4.88

1:10000 4 8 0.35 0.28

Udon noodles 1:1000 111 236 0.09 0.14 5.04b 5.38

1:10000 9 26 0.48 0.12

Udon noodles 1:1000 19 42 0.31 0.15 3.26 3.62

1:10000 1 4 1.73 0.39

Peanut butter 1:10 10 16 0.60 0.51 <2.34 <2.34

1:100 0 1 0.96 0.71

Peanut butter 1:10 6 7 0.68 0.58 <2.34 <2.34

1:100 1 2 1.09 0.59

Milk chocolate 1:10 28 44 0.26 0.19 2.43 2.63

1:100 2 4 0.46 0.47

Dry cat food 1:10 24 33 0.25 0.32 2.41 2.531:100 4 5 0.54 0.55

a Significantly low (p < 0.01).b Significantly low (p < 0.05).


Table 4. Ruggedness study (effect of incubation temperature and time)

Food Incubation time, h

APC, log10 CFU/g

Temperature, �C

30 32.5 35 37

Raw pork 24 5.48a 5.49a 5.36a 4.89a

29 5.58b 5.53b 5.43a 4.96a

45 5.80 5.73 5.60 5.26a

48 5.81 5.75 5.60 5.28a

Cabbage 24 6.20a 6.20a 5.85a 5.72a

30 6.34a 6.30a 6.15a 5.76a

48 6.63 6.62 6.53a 6.28a

Roast pork 24 4.56a 4.70a 4.64a 4.60a

29 4.82a 4.81a 4.74a 4.70a

45 5.04 5.04 5.04 4.90a

48 5.08 5.08 5.04 4.92a

Frozen shrimp 24 3.96a 3.92a 3.86a 3.80a

27 3.99a 3.96a 3.88a 3.83a

48 4.60 4.54 4.36a 4.00a

Frozen shrimp 2 24 3.69a 3.62a

41 3.89 3.78a

44 3.90 3.79a

48 3.93 3.83 b

Fried egg 24 3.94c 3.56b 3.23a 2.95a

29 4.00c 3.65 3.34a 2.98a

45 4.04c 3.76 3.38a 3.11a

48 4.08c 3.78 3.40a 3.11a

Red pepper 24 5.95b 5.99 6.00 5.95a

27 5.97 6.00 6.00 5.97b

48 6.04 6.00 6.04 5.98b

Red pepper 2 24 5.96 5.93a

41 6.00 5.97

44 6.04 5.99

48 6.04 6.00

Buckwheat flour 1 24 4.30a 4.36 4.34 4.52

27 4.36b 4.38 4.40 4.54

48 4.41 4.46 4.43 4.56

Buckwheat flour 2 24 4.32a 4.36a 4.40 4.40

44 4.40 4.40 4.41 4.41

48 4.40 4.41 4.43 4.43

Udon noodles 24 1.34a 1.46a 2.11a 1.88a

44 2.93d 2.74 2.67 b 2.41a

48 2.96d 2.77 2.68 2.41a

Dry cat food 24 5.04a 5.23b 5.23 5.26

29 5.20a 5.26 5.26 5.26

45 5.26 5.28 5.28 5.28

48 5.26 5.28 5.28 5.28

Frozen pizza 24 2.18a 2.22a 2.26a 2.28a

30 2.28a 2.23a 2.30a 2.32a

48 3.15c 2.94 2.91 2.78

Frozen pizza 2 24 2.43a 2.45a

41 2.74a 2.71a

44 2.79b 2.74a

48 2.83 2.77b

a Significantly lower (p < 0.01) than incubation at 32.5�C for 48 h.b Significantly lower (p < 0.05) than incubation at 32.5�C for 48 h.c Significantly higher (p < 0.01) than incubation at 32.5�C for 48 h.d Significantly higher (p < 0.05) than incubation at 32.5�C for 48 h.


Table 5. Ruggedness study (effect of incubation time at 32.5�C)

Food

APC, log10 CFU/g

Incubation time, h

24 27 29 30 41 44 45 48

Raw pork 5.49a — 5.53b — — — 5.73 5.75

Cabbage 6.20a — — 6.30a — — — 6.66

Roast pork 4.70a — 4.81a — — — 5.04 5.08

Frozen shrimp 3.92a 3.96a — — — — — 4.54

Frozen shrimp 2 3.69a — — — 3.89 3.90 — 3.93

Fried egg 3.56a — 3.65 — — — 3.76 3.78

Red pepper 5.99 6.00 — — — — — 6.00

Red pepper 2 5.96 — — — 6.00 6.04 — 6.04

Buckwheat flour 1 4.36a 4.38a — — — — — 4.48

Buckwheat flour 2 4.36a — — — — 4.40 — 4.41

Udon noodles 1.46a — — — — 2.74 — 2.77

Dry cat food 5.23b — 5.26 — — — 5.28 5.28

Frozen pizza 2.22a — — 2.23a — — — 2.94

Frozen pizza 2 2.43a — — — 2.74 2.79 — 2.83

a Significantly lower (p < 0.01) than 48 h incubation.b Significantly lower (p < 0.05) than 48 h incubation.

Table 6. Ruggedness study (effect of temperature at 48 h incubation)

Food

APC, log10 CFU/g

Incubation temperature, �C

30 32.5 35 37

Raw pork 5.81 5.75 5.60 5.28a

Cabbage 6.63 6.62 6.53a 6.28a

Roast pork 5.08 5.08 5.04 4.92a

Frozen shrimp 4.60 4.54 4.36a 4.00a

Frozen shrimp 2 — 3.93 3.83b —

Fried egg 4.08a 3.78 3.40a 3.11a

Red pepper 6.04 6.00 6.04 5.98b

Red pepper 2 — 6.04 6.00 —

Buckwheat flour 1 4.41 4.46 4.43 4.56

Buckwheat flour 2 4.40 4.40 4.43 4.43

Udon noodles 2.96a 2.77 2.68 2.41a

Dry cat food 5.26 5.28 5.28 5.28

Frozen pizza 3.15b 2.94 2.91 2.78

Frozen pizza 2 — 2.83 2.77 —

a Significantly higher or lower than 32.5�C (p < 0.01).b Significantly higher or lower than 32.5�C (p < 0.05).

cantly higher than at 35�C. The APCs at 30�C incubation weresignificantly higher than those at 35�C incubation except forroast pork, buckwheat flour, and dry cat food; no significantdifferences were seen between 30 and 32.5�C incubation ex-cept for udon noodles and fried egg. The APCs at 37�C incu-bation were significantly lower than those at 35�C, except forfrozen pizza, buckwheat flour, and dry cat food.

From these results (Tables 4–6) we can recommend incubationat 32.5�C with �2.5�C allowance, for 46 h with �2 h allowance.

(d) Limit of quantitation.—On nearly 1 CFU/mL, the av-erage counts of 10 replicate Sanita-kun sheets were 1.0–2.2and the RSDr values were 0.56–1.30 (Table 7).

Lot-to-lot study.—In all tested foods, APCs were similarfor the 3 lots of Sanita-kun sheets. RSDr values ranged from

0.03 (fried egg) to 0.20 (frozen shrimp) as shown in Table 8.The average RSDr was calculated as 0.13. A performance de-crease in the older lot of Sanita-kun sheets was not recognizedand the shelf-life of the sheet was suggested to be at least14 months.

Independent Laboratory Study

When the target level was <10 CFU/g, all samples were<10 CFU/g by Sanita-kun sheets, and all samples except onewere <10 CFU/g by the AOAC Official Method. In ground beef,no significant difference was observed among the APCs of theAOAC Method and 20 and 48 h incubation of Sanita-kun sheets.In the target levels of 100–250 and 1000 CFU/g hot dogs, theAPCs of Sanita-kun sheets were significantly higher (p < 0.05)


Table 9. Independent validation study

AOAC Method Sanita-kun, 20 ± 2 h Sanita-kun, 48 h

Food Target level, CFU/g Average APC, CFU/g RSDr Average APC, CFU/g RSDr Average APC, CFU/g RSDr

Hot dogs <10 <10 except 1 replicate <10 <10

100–250 400 0.14 510a 0.16 500a 0.14

1000 1100 0.14 1300a 0.11 1300a 0.11

2500 4100 0.17 4400 0.14 4500 0.14

Ground beef <10 <10 <10 <10

100–250 410 0.13 380 0.16 400 0.15

1000 920 0.26 780 0.31 820 0.24

2500 3500 0.12 3900 0.16 4200 0.19

a Significantly higher (p < 0.05) than AOAC Method.

Table 8. Lot-to-lot study

Food

APC, log10 CFU/gRSDr

of APC9909TC 00918TC 001124TC Avg

Raw pork 5.91 5.89 5.93 5.91 0.05

Frankfurter 5.93 5.79 5.83 5.85 0.18

Frozen shrimp 4.96 4.80 4.86 4.87 0.20

Ice cream 2.48 2.54 2.49 2.50 0.08

Fried egg 3.49 3.46 3.49 3.48 0.03

Red pepper 5.95 5.86 5.99 5.93 0.15

Oregano 6.58 6.48 6.48 6.51 0.14

Buckwheat flour 5.15 5.15 5.18 5.16 0.04

Udon noodles 4.20 4.20 4.26 4.22 0.07

Peanut butter 2.08 2.00 2.11 2.06 0.13

Dry cat food 5.04 5.00 4.95 5.00 0.10

Table 7. Limit of quantitationa

FoodAverage count,

CFU SD RSDr

Raw pork 1.6 1.4 0.88

Cabbage 2.0 1.9 0.95

Frankfurter 1.4 0.84 0.60

Raw shrimp 1.4 1.8 1.29

Ice cream 1.7 0.95 0.56

Shelled eggs 1.8 1.0 0.56

Red pepper 1.7 2.2 1.29

Buckwheat flour 1.7 1.1 0.65

Udon noodles 2.2 1.3 0.59

Peanut butter 1.7 1.3 0.76

Milk chocolate chips 1.3 0.82 0.63

Dry cat food 1.0 0.94 0.94

Frozen pizzas 1.5 1.3 0.87

a SD = Standard deviation; RSDr = relative standard deviation forrepeatability.

than those of the AOAC Method (Table 9). In the average APCand RSDr of each target level of Sanita-kun sheets, no significantdifference was observed between 20 and 48 h incubation.

The analyst pointed out that the Sanita-kun sheets wereeasy to use and read.

Discussion

We did not detect aerobic microorganisms in food dyes(data not shown). Food dyes interrupted the color visualiza-tion of the colonies on the Sanita-kun sheets. Therefore, wewithdrew food dyes from the claims of the food matrixes.

In the repeatability study, the average RSDr of fried eggwas higher (0.57). This was caused by the low actual meancounts (1 and 2) in the lower 2 target levels (<10, 10–25).

In the accuracy study, the higher average RSDr values ofmilk chocolate and dry cat food by the Sanita-kun and platecount method were caused by low counts. The APCs of sev-eral lots were significantly higher or lower (p < 0.05 orp < 0.01) than those of the plate count agar. However, the dif-ference was small; therefore, we believe there will be practi-cally no problem in daily monitoring of such foods.

In the independent validation study, the analyst pointed outthat the Sanita-kun sheets were easy to use and read. She alsopointed out, however, that the colony was sometimes morediffuse, with string-like spreading at 48 h; therefore, coloniesthat had been counted as 2 at 20 h may have been counted asone at 48 h. However, she reported quite similar counts be-tween 20 and 48 h (Table 9). In the independent validationstudy, BPB was used to prepare the homogenate and sampledilutions. We often observed spread colonies on theSanita-kun sheets after 48 h incubation when buffers or solu-tions without 0.5–0.9% NaCl were used for sample prepara-tion and dilution. Spread colonies were not observed on the

sheets when buffers or solutions containing 0.5–0.9% NaClwere used.

We recognized increases in colonies on the Sanita-kunsheets at 48 h from 24 h incubation in several types of foods(Tables 3–6). To obtain precise numbers of colonies on theSanita-kun sheets, 48 h incubation is preferable. From resultsof further ruggedness study, we recommend incubation at32.5 � 2.5�C for 46 � 2 h. The 24 h incubation is enough in adaily monitoring of food contamination except for udon noo-dles, because the differences of log10 counts between 24 and48 h incubation at 32.5�C was within 1.

Sanita-kun Aerobic Counts sheets contain TTC as growthindicator; the Petri-film AC plate also uses the same indicator.The performance of Sanita-kun Aerobic Counts and thePetri-film AC plate was compared on about 300 foods (datanot shown). The APCs of both methods were similar and thecorrelation coefficient (r2) was calculated as 0.99. At times,degradation of the Petri-film AC plate and liquefaction of thegel by bacteria and diffused colonies were observed. The deg-radation of water-soluble polymer or the nonwoven fabric inthe Sanita-kun sheets was never observed.

Sanita-kun sheets are not required for laborious mediumpreparation, and the sheets have the advantages of being spacesaving and causing small waste when compared with conven-tional agar medium. The Sanita-kun aerobic count was similarto that with plate count agar. We believe that the Sanita-kunsheets provide an alternative method to plate count agar. TheSanita-kun Aerobic Counts has been granted AOAC Perfor-mance Tested MethodSM status.

Acknowledgements

We thank Wendy Lepper and Ann M. Schulz (SillikerLaboratory Corporate Research Center) for their extensiveworks in independent laboratory study.


sensitivity and specificity of the sanita-kun aerobic …...food biological contaminants sensitivity...

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