Enumeration and Detection of Bacillus cereus Using a ...

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1Herbalife Manufacturing, LLC, Quality Control, 20481 Crescent Bay Drive, Lake Forest, CA 92630. 2Herbalife International of America, 950 West 190th Street, ...
Enumeration and Detection of Bacillus cereus Using a modified FDA Bacteriological Analytical Method Devin Salyer1, Jacqueline Tam1, Christopher Thompson1, Veronica Zarraga1, Prashant Ingle2, Quanyin Gao1*, Peter Chang2, and Gary Swanson2 1Herbalife

Manufacturing, LLC, Quality Control, 20481 Crescent Bay Drive, Lake Forest, CA 92630 2Herbalife International of America, 950 West 190th Street, Torrance, CA 90502

Abstract

Accuracy – Enumeration and Recovery

Precision using 10 CFU of B. cereus

An increased incubation temperature from 30°C (BAM method) to 32.5 – 37.5°C (adjusted BAM method) was adapted into incubators available in most laboratory performing BAM micro test methods. This adjusted method was successfully validated for specificity, accuracy, precision, limit of detection, and ruggedness for different matrices in food and dietary supplement finished products and raw materials. This work is significant and important for micro laboratories in food and supplement industry as this method can be consolidated to maximize incubator space usage without compromising accuracy, specificity and other parameters for method performance.

BACARA (CFU/plate) 33 31 34 34 42 41 38 35 25 30 34.3 5.1 14.9%

Plate 1 2 3 4 5 6 7 8 9 10

Bacillus cereus is an aerobic spore-forming bacteria and is widely present in the environment. Contaminated food products with B. cereus at certain level will result in food poisoning that is evident to having diarrhea or vomiting. FDA BAM has several media including a chromogenic agar (Bacara) that could detect B. cereus. We have conducted method validation for the enumeration and detection of B. cereus with modified BAM method using Bacara agar.

average stdev RSD

10^1 Concentration PCA (CFU/plate) 56 57 54 34 50 PCA Average: 50.2

Microbiological testing laboratory plays an important role in food and dietary supplement industry for product quality control. The lab is required to have established test methods to detect and quantitate microbes in raw materials and finished products for the absence of these microbes or enumerate the level to assure product quality. Bacillus cereus is one of the microbes for a microbiological testing lab to have the capability to assure the absence or enumerate if detected. In our lab, method validation was performed for adopting FDA BAM method for quantitation and detection of Bacillus cereus with adjusted incubation temperature for different product matrices. The method performance under the adjusted incubation temperature is validated successfully.

Equipment and Materials Equipment and Materials: BACARA agar, Butterfield’s Phosphate Buffer Water , Tryptic Soy Broth, balance, sterile pipettes, incubator, B. cereus as positive control

Samples and Results for Detection

Analyst 2

Plates

Results in BACARA

Results in BACARA

Replicate 01

Growth

Growth

Replicate 02

Growth

Growth

Replicate 03

Growth

Growth

Replicate 04

Growth

Growth

Replicate 05

Growth

Growth

Positive Control

Growth

Growth

Negative Control

No Growth

No Growth

Sample plate Fig. 1: Plate positive with B. cereus

Plate 2

1) Sample 1

Growth

Growth

2) Sample 2

Growth

Growth

3) Sample 3

Growth

Growth

4) Sample 4

Growth

Growth

5) Smaple 5

Growth

Growth

6) Sample 6

Growth

Growth

Negative Control

No Growth

Positive Control

Growth

Note: Samples tested here with B. cereus inoculation include both raw materials and finished products range from different types of matrices

No Growth

B. spizizenii Replicate 2

No Growth

B. spizizenii Replicate 3

No Growth

B. spizizenii Replicate 4

No Growth

BACARA / PCA *100 = 51.2/50.2*100=102.0% recovery vs. PCA

B. spizizenii Replicate 5

No Growth

B. cereus Positive Control

Growth

Negative Control

No Growth

BACARA (CFU/plate) 455 699 503 369 393 BACARA Average: 483.8

BACARA (CFU/plate)

Note: B. cereus produced an orangish-pink streak surrounded by an opaque precipitate, while B. spizizenii showed no growth. This indicates that BACARA agar is selective for B. cereus that is capable of preventing false positive results.

BACARA / PCA *100 = 483.8/436.4*100 =110.9% recovery vs. PCA Note: More than 70% recovery (cfu/plate) in BACARA plate inoculated with 10 CFU and 100 CFU B. cereus

Precision/Limit of detection 10^1 Concentration PCA (CFU/plate)

BACARA (CFU/plate) 33 31 34 34 42 41 38 35 25 30 BACARA Average: 34.3

35

38 PCA Average: 36.5 Acceptable Range: +/- 1 log of 36.5 Result = Pass

BACARA Streak Plate from Enriched TSB solutions inoculated with