Cannabis Testing

Evaluation of an Alternative Chromogenic Agar Plating Method for Detection
and Confirmation of Salmonella in Cannabis Flower, Cannabis-Infused Gummies,
Cannabis-Infused Chocolate, and Cannabis-Derived Concentrates
Mike Clark, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr, Hercules, CA 94547
Leo Horine, TEQ Analytical Labs, 12635 E Montview Blvd #175, Aurora, CO 80045

Abstract

The iQ-Check™ Salmonella II PCR Detection Kit is commonly used to screen for Salmonella in variousmatrices and has been validated by AOAC International for a broad range of foods. However, it is also
possible to screen for contamination on agar plates. Here we evaluated an alternative screening andconfirmation method for cannabis flower (10 g), cannabis-infused gummies (25 g), cannabis-infused
chocolate (25 g), and cannabis-derived concentrates (5 g) that consisted of a direct streak of primaryand secondary enrichment broths onto RAPID'Salmonella Agar and compared the results to Standard
Method Performance Requirements (SMPR) 2020.002 (AOAC 2020). Salmonella typically form magenta
colonies on RAPID'Salmonella Agar (Figure 1), allowing easy differentiation from competing organisms.
Statistical analysis demonstrated no difference between the alternative and SMPR methods.
Salmonella infection is characterized by acute gastroenteritis.
Symptoms include diarrhea, fever, abdominal cramps, andvomiting lasting 4 to 7 days in most people.

Fig. 1. Visual differences between Salmonella and competing organisms.
Salmonella typically form magenta colonies on RAPID'Salmonella Agar. Competingorganisms form blue or white colonies or are completely inhibited.

Introduction
Salmonella has been recognized as a primary cause of foodborne
illness worldwide. This genus of bacteria is classified into twospecies: S. enterica and S. bongori, the former containing themajority of serotypes associated with human disease. Salmonella
can contaminate a wide range of foods, including poultry, meat,eggs, dairy, fruit, and vegetables, as well as pet food. With thegrowing legalization of cannabis in the United States, many states
have enacted requirements for testing cannabis products for thepresence of Salmonella. In most cases, particularly in the U.S.,

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Methods
The inoculation procedure was followed as outlined in Appendix Jof AOAC’s Microbiology Validation Guidelines (Latimer 2023). Prior
to inoculation, a total aerobic plate count (APC) was performed foreach matrix following procedures outlined in the Bacteriological
Analytical Manual, Chapter 3: Aerobic Plate Count (U.S. Food andDrug Administration 2021). Matrices were prescreened for naturalcontamination of Salmonella species following culture procedures
outlined in SMPR 2020.002. No natural contamination was found inany of the matrices, so they were artificially contaminated, targetingthree levels, measured in colony forming units (CFU) per test portion:
noninoculated (0), low (0.2–5), and high (5–10).
For cannabis flower, lyophilized Salmonella Typhimurium American
Type Culture Collection (ATCC) 14028 pellets were crushed anddiluted with nonfat dry milk that previously screened negative for
Salmonella species. A bulk lot of cannabis flower was artificiallyinoculated with a low level expected to yield fractional positive results(5–15 positive results) and a high level expected to yield all positiveresults. For all other matrices, a single colony of a Salmonella strain
was transferred to Buffered Peptone Water Standard, dehydrated(Bio-Rad Laboratories, Inc., catalog #12013259) and incubatedEvaluation of an Alternative Chromogenic Agar Plating Method for Detection and Confirmation of Salmonella in Cannabis Products

overnight at 37°C. Cannabis-infused gummies were artificiallyinoculated with Salmonella Newport ATCC 6962, cannabis-infused
chocolate with Salmonella Typhimurium ATCC 14028, and cannabisderived concentrates with Salmonella Heidelberg ATCC 8326. The
freshly grown, nonstressed culture was diluted to an appropriate levelto achieve fractional positive results for the low level and all positive
results for the high level. Matrices were held at ambient temperature(20–25°C) for 2 weeks prior to analysis. A most probable number(MPN) analysis was performed on the day of testing to determine
inoculation levels.

Typical growth on either XLD or RAPID'Salmonella Agar wastransferred to triple sugar iron (TSI) and lysine iron agar (LIA) slants.
Test portions with typical results for Salmonella were streaked
to tryptic soy agar (TSA) and confirmed using polyvalent O andpolyvalent H serology tests. Final identification was performed
using Analytical Profile Index (API) 20E. Figure 2 describes thestudy design.
Probability of detection (POD) statistical analysis was performed
using the AOAC Binary Workbook with Unpaired Reference, v5-2
(AOAC 2013; Wehling et al. 2011). MPN analysis values wereobtained using the Least Cost Formulations MPN Calculator(Least Cost Formulations Ltd 2016).

Each 10 g test portion of cannabis flower was added to 90 ml
Buffered Peptone Water (BPW), homogenized, and incubated for
18–22 hr at 37 ± 1°C. Each 25 g test portion of cannabis-infusedgummies and cannabis-infused chocolates was added to 225 ml
BPW (prewarmed, 37°C), homogenized, and incubated for
18–22 hr at 37 ± 1°C. Each 5 g test portion of cannabis-derived
concentrate was added to 45 ml BPW (prewarmed, 37°C),
homogenized, and incubated for 18–22 hr at 37 ± 1°C. Samples
were streaked to RAPID'Salmonella Agar (Bio-Rad, #3563963)
and incubated for 24 ± 2 hr at 37 ± 1°C.

Results
POD analysis results are presented in Table 1.
Cannabis FlowerThe APC result for the cannabis flower was 1.9 x 104 CFU/g. MPN
analysis resulted in 0.59 CFU/test portion for the low inoculationlevel and 3.26 CFU/test portion for the high inoculation level.
Of the five replicates analyzed at the noninoculated level, zeropresumptive positive results were observed. For the 20 samples
observed at the low inoculation level, seven presumptive positive
results were observed. Of the five replicates analyzed at thehigh inoculation level, five presumptive positive results wereobserved. No discrepancies were seen between the presumptive
and confirmed results, with confirmation results for all samples
matching the presumptive results. All RAPID'Salmonella plate
confirmation results matched SMPR 2020.002 confirmation results.

In addition, samples were subcultured to Rappaport-Vassiliadis(RV) and tetrathionate (TT) broths for confirmation according
to SMPR 2020.002. Each tube was streaked to xylose lysine
desoxycholate (XLD) and RAPID'Salmonella Agar plates. Typicalmagenta colonies on RAPID'Salmonella Agar were identified as
Salmonella and were easily selected for further confirmation.

Streak to XLD andRAPID'Salmonella+Isolation on TSI and LIA–Biochemical confirmation

Matrix inoculation andprimary enrichment in BPW

Subculture to secondary
enrichment in RV and TT broths

Streak to XLD andRAPID'Salmonella

Fig. 2. Study design. The workflow consisted first of inoculating and enriching the sample in BPW. Subsequent streaking on plates and subculturing of the sample, then isolation
on TSI and LIA, led to confirmation of Salmonella. BPW, buffered peptone water; LIA, lysine iron agar; RV, Rappaport-Vassiliadis; TSI, triple sugar iron; TT, tetrathionate; XLD,xylose lysine desoxycholate.

© 2023 Bio-Rad Laboratories, Inc.

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Evaluation of an Alternative Chromogenic Agar Plating Method for Detection and Confirmation of Salmonella in Cannabis Products

Table 1. POD results. RAPID'Salmonella presumptive vs. confirmed results (paired) – POD results.
Matrix and Inoculation
Cannabis flower, 10 g
(Salmonella Typhimurium
ATCC 14028)

MPN/Test Portion

Nx

Presumptive
PODcp
95% Clx

Confirmed
PODcc
95% CldPODcp

95% Cl*NA500.00

0.00, 0.4300.00

0.00, 0.43

0.00(–0.47, 0.47)

0.59 (0.31, 1.01)

20

8

0.40

0.22, 0.61

8

0.40

0.22, 0.61

0.00(–0.13, 0.13)

3.26 (1.33, 7.99)

5

5

1.00

0.57, 1.00

5

1.00

0.57, 1.00

0.00(–0.47, 0.47)(–0.47, 0.47)

Cannabis infused chocolate,
25 g (Salmonella Typhimurium
ATCC 14028)

NA

5

0

0.00

0.00, 0.43

00.00

0.00, 0.43

0.00

0.65 (0.32, 1.15)

20

8

0.40

0.22, 0.61

8

0.40

0.22, 0.61

0.00(–0.13, 0.13)

4.07 (2.15, 234)

5

5

1.00

0.57, 1.00

51.00

0.57, 1.00

0.00(–0.47, 0.47)

Cannabis infused gummies,
25 g (Salmonella Newport
ATCC 6962)

NA

5

00.00

0.00, 0.43

00.00

0.00, 0.43

0.00(–0.47, 0.47)

0.83 (0.44, 1.44)

20

10

0.50

0.30, 0.70

10

0.50

0.30, 0.70

0.00(–0.13, 0.13)

4.65 (3.37, 234)

5

5

1.00

0.57, 1.00

51.00

0.57, 1.00

0.00(–0.47, 0.47)

Cannabis derived concentrate,
5 g (Salmonella Heidelberg
ATCC 8326)

NA500.000.00, 0.43

00.00

0.00, 0.43

0.00(–0.47, 0.47)

0.80 (0.42, 1.35)

20

90.45

0.26, 0.66

90.45

0.26, 0.66

0.00(–0.13, 0.13)

6.74 (3.18, 187)

55

1.00

0.57, 1.00

51.00

0.57, 1.00

0.00(–0.47, 0.47)* If the confidence interval of a dPOD does not contain zero, then 95% CI is the statistically significant difference at the 5% level.
dPODCP, difference between the candidate method–presumptive result and candidate method–confirmed result POD values; MPN, most probable number based on thePOD of culture confirmation of test portions using the Least Cost Formulations MPN Calculator, with 95% confidence interval; N, number of test portions; NA, not applicable;
POD, probability of detection; PODCC, candidate method–confirmed positive outcomes divided by the total number of trials; PODCP, candidate method–presumptive positiveoutcomes divided by the total number of trials; x, number of positive test portions; 95% CI, 95% confidence interval.

At the low inoculation level, the study data indicate with 95%
confidence that the true difference between the RAPID'Salmonella
method presumptive and confirmed results could be as small as–0.13 or as large as 0.13, with a best estimate of 0.00. At the highinoculation level, the true difference could be as small as –0.47 oras large as 0.47, with a best estimate of 0.00.

were seen between the presumptive and confirmed results, withconfirmation results for all samples matching the presumptive results.
All RAPID'Salmonella plate confirmation results matched SMPR
2020.002 confirmation results.
At the low inoculation level, the study data indicate with 95%
confidence that the true difference between the RAPID'Salmonella
method presumptive and confirmed results could be as small as–0.13 or as large as 0.13, with a best estimate of 0.00. At the highinoculation level, the true difference could be as small as –0.47 oras large as 0.47, with a best estimate of 0.00.

Cannabis-Infused Chocolate

The APC result for the cannabis-infused chocolate was 60 CFU/g.
MPN analysis resulted in 0.65 CFU/test portion for the low inoculationlevel and 4.07 CFU/test portion for the high inoculation level. Of thefive replicates analyzed at the noninoculated level, zero presumptive
positive results were observed. For the 20 samples observed atthe low inoculation level, eight presumptive positive results wereobserved. Of the five replicates analyzed at the high inoculation level,
five presumptive positive results were observed. No discrepancies
were seen between the presumptive and confirmed results, withconfirmation results for all samples matching the presumptiveresults. All RAPID'Salmonella plate confirmation results matched
SMPR 2020.002 confirmation results.

Cannabis-Derived Concentrate

The APC result for the cannabis-derived concentrate was <1 CFU/g.
MPN analysis resulted in 0.80 CFU/test portion for the low inoculationlevel and 6.74 CFU/test portion for the high inoculation level. Of thefive replicates analyzed at the noninoculated level, zero presumptive
positive results were observed. For the 20 samples observed at thelow inoculation level, nine presumptive positive results were observed.
Of the five replicates analyzed at the high inoculation level, five
presumptive positive results were observed. No discrepancies wereseen between the RAPID'Salmonella presumptive and confirmed
results, with confirmation results for all samples matching thepresumptive results. All RAPID'Salmonella plate confirmation results
matched SMPR 2020.002 confirmation results.

At the low inoculation level, the study data indicate with 95%
confidence that the true difference between the RAPID'Salmonella
method presumptive and confirmed results could be as small as–0.13 or as large as 0.13, with a best estimate of 0.00. At the highinoculation level, the true difference could be as small as –0.47 oras large as 0.47, with a best estimate of 0.00.

At the low inoculation level, the study data indicate with 95%
confidence that the true difference between the RAPID'Salmonella
method presumptive and confirmed results could be as small as–0.13 or as large as 0.13, with a best estimate of 0.00. At the highinoculation level, the true difference could be as small as –0.47 oras large as 0.47, with a best estimate of 0.00.

Cannabis-Infused Gummies

The APC result for the cannabis-infused gummies was <1 CFU/g.
MPN analysis resulted in 0.83 CFU/test portion for the low inoculationlevel and 4.65 CFU/test portion for the high inoculation level. Of thefive replicates analyzed at the noninoculated level, zero presumptive
positive results were observed. For the 20 samples observed at
the low inoculation level, ten presumptive positive results wereobserved. Of the five replicates analyzed at the high inoculation level,
five presumptive positive results were observed. No discrepancies© 2023 Bio-Rad Laboratories, Inc.

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Evaluation of an Alternative Chromogenic Agar Plating Method for Detection and Confirmation of Salmonella in Cannabis ProductsDiscussion
A chromogenic media was evaluated as part of the iQ-Check
Salmonella II AOAC validation extension for various cannabismatrices. The RAPID'Salmonella method successfully detected
target Salmonella species in cannabis flower (10 g), cannabisinfused gummies (25 g), cannabis-infused chocolate (25 g), andcannabis-derived concentrate (5 g). POD analysis proved that thestudy data did not demonstrate a statistically detectable difference
from zero between the RAPID'Salmonella method–presumptive
and reference method–confirmed results. The use of chromogenic
media has the advantage of simplifying the selection of typicalcolonies for detection and/or confirmation of target organisms.
This increases the overall ease of use of the method.

References
AOAC International (2013). AOAC Binary Data Interlaboratory Study Workbook.
aoac.org/wp-content/uploads/2019/08/09trad04_AOAC_binary-v2-3.xls, accessed
November 14, 2023.
AOAC International (2020). Standard Method Performance Requirements (SMPRs)
for Detection of Salmonella species in Cannabis and Cannabis Products. aoac.org/
wp-content/uploads/2020/07/SMPR-2020_002.pdf, accessed November 14, 2023.
Latimer GW (2023). Official Methods of Analysis: 22nd Edition (New York:
Oxford Academic).
Least Cost Formulations, Ltd. (2016) AOAC Binary Data Interlaboratory Study
Workbook, AOAC Interlaboratory Study Workbook - Binary Data, v2.0
U.S. Food and Drug Administration 2021 Bacteriological Analytical Manual (BAM)
Chapter 3: Aerobic Plate Count. fda.gov/food/laboratory-methods-food/bamchapter-3-aerobic-plate-count, accessed November 14, 2023.
Wehling P et al. (2011). Probability of Detection (POD) as a statistical model for thevalidation of qualitative methods. J AOAC Int 94, 335–347.

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