Performance Evaluation of Ready-to-Use Culture Media, Easy Plate SA
for Enumeration of Staphylococcus Aureus in a Broad Range of Foods
Shinichiro Sugiura1, Suzanne J. Jordan2, Kentaro Takenaka3, Mai Shimizu3 and Takeo Suzuki3(1)Kikkoman Biochemifa Company, (2)Campden BRI, (3)Kikkoman Corporation

1. Introduction

2. Summary

E-SA showed comparativeness to ISO 6888-1:2021 for a broad range offoods.

・For the enumeration of Staphylococcus aureus,
Baird-Parker agar (BPA) is traditionally usedaccording to the ISO 6888-1:2021: Enumerationof coagulase-positive staphylococci.

E-SA is found to be

1. Reliable method to enumerate S. aureus✓Comparative to the traditional ISO 6888-1 method
2. Applicable to a broad range of foods✓With reasonable repeatability and accuracy
3. Provide results in 24h✓Saves 24h compared with the traditional BPA method
4. Excels at differentiating S. aureus.
✓Clear colonies with greater selectivity to reducemisinterpretation

・A type of Ready-to-use (RTU) media, Easy Plate
SA (E-SA) (Kikkoman Biochemifa Company) canbe alternatively used to provide many benefits
including reduced time to result, simplicity ofuse and is plastic-saving compared to thetraditional method.

3. Methods & Results

3.2. Accuracy profile study

The Method validation study was done according to ISO 161402:2016, with ISO 6888-1 (2021) as the reference method. Analysis
using E-SA was performed following manufacturer’s instructions.

・ A total of 150 artificially contaminated samples across 5 categories

with 2 items per category was tested in this section. Each item
used was contaminated at 3 levels, with 5 replicates analyzed per level→ All 5 categories passed the 0.5log acceptability limits or therecalculated limits.

Figure 1. Method comparison work flow
ISO 6888-1

Sample dilution
according to ISO 6887
series3.3. Inclusivity and Exclusivity

E-SA・Inclusivity

Plating

Plating

Incubation

Incubation

sources.
→ E-SA was found to be comparative to the ISO method.
・Exclusivity was examined with 31 non-target isolates.
→ E-SA outperformed the ISO method.

24h

48h

Table 2. Summary results of inclusivity and exclusivity

37℃

37℃

Black colony withegg-yolk reaction

Blue colony

Inclusivity(Target)Confirmation test

3.1. Relative trueness study

・A total of 75 foods across 5 categories was tested. Each categorycontained 3 types and 15 items per category. Details of categories andtypes used are shown in Table 1.
→ No significant difference between E-SA and the ISO method
was noted as revealed in the scatter plot displayed in Figure 2below.

Table 1. Categories and types tested in Relative trueness study

ISO 6888-1
(BPA)
100%
(50/50)Exclusivity(Non-target)

E-SA

ISO 6888-1(BPA)*

E-SA

100%(50/50)71%(22/31)

97%(30/31)* Presumptive results recorded for exclusivity isolates

Table 3. Microorganisms tested for exclusivityNo.

Organism

Source1

Bacillus cereus

Dairy product2

Bacillus cereus

Unknown

3

Bacillus subtilis

Unknown

Identity
if available

Dairy

Raw milk and dairy products
Pasterurised milk and dairy products

and egg products
Fishery

Processed egg products

4Brochothrix thermospacta

Pork sausage

Industrial strain
ATCC 10876,
NCTC 7464
ATCC 6633,
NCTC 10400
NCTC 10822

Raw fish (unprocessed)
RTE/RTC/RTRH fish and seafoods
Crustaceans
Cut ready-to-eat vegetables/leafy greens and sprouts
Fresh fruit/Cut RTE fruit and vegetableproducts
Heat treated fruit and vegetables
Composite foods
with substantial raw ingredients
RTRH/RTE foods (chilled, frozen)
Mayonnaise based deli-salads
Cooked meat and poultry products
Fermented or dried products
Raw cured products

5

Flavobacterium species

Unknown

Industrial strain

6Enterobacter agglomerans

Raw mince

7

Enterococcus faecalis

Urine

8

Enterococcus faecalis

Unknown

Industrial Strain
NCIMB 13280,
ATCC 29212
NCTC 775

9Escherichia coli

Raw ground beef

Industrial Strain10

Lactobacillus brevisUnknown

NCTC13386

11Lactobacillus gasseri

Human source

NCIMB 13081

Ham

Industrial strain

Types(raw and processed)(raw, ready-to-eat,ready-to-reheat, ready-to-cook)

Produce and fruits(fresh and processed)

Multi-component foods
or meal components
Meat and poultry(ready-to-eat, ready-to-reheat)

Figure 2. Scatter plot for all categories
in the relative trueness study
87

log10 CFU/g E-SA

was examined with 50 S. aureus strains from various

65

12 Leuconostoc mesenteroides
13

Listeria monocytogenes

Soft cheese

14

Micrococcus luteus

Unknown

15Pediococcus pentosaceus

Brine

Industrial strain
NCTC 2665,
ATCC 13507
Industrial strain

16

Pseudomonas rhodesiae

Unknown

Industrial strain

17

Salmonella Enteritidis

Fish cakes

Industrial strain

18

Salmonella typhimurium

ChickenIndustrial strain

19

Staphylococcus caprae

Goat

Industrial strain

20

Staphylococcus carnosus Fermented sausage

21

Staphylococcus cohnii

Unknown

Industrial strain

22

Staphylococcus epidemidis

Human skin

Industrial strain

23

Staphylococcus hominis

Dried milk powder

Industrial strain

24

Staphylococcus hyicus

Pig skinIndustrial strain
NCTC 11048,
ATCC 29663

25 Staphylococcus intermedius

4

Industrial strain

Pigeons

27

Staphylococcus
piscifermentans
Staphylococcus sciuri

228

Staphylococcus simulans

Human skin

Industrial strain

29

Staphylococcus warneri

German salami

Industrial strain

130

Staphylococcus xylosus

Mettwurst sausage

Industrial strain

31

Streptococcus lactis

Milk powder

Industrial strain

26

300

1

2

3

45

log10 CFU/g BPA

678BPAISO
6888-1✓

E-SA✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓××××✓×✓✓✓✓✓✓×✓✓✓×✓

Unknown

Industrial strain×✓

Unknown

Industrial strain×✓×✓✓✓✓✓✓✓“×” indicates false-positive while “✓” indicates successfully suppressed.
NB: 8/9 presumptive positive colonies on BPA were confirmed as coagulase negative.