Dry botanicals stop microbes from growing, yet Salmonella and spores survive in them

• ⟨2021⟩ TAMC on soybean–casein digest agar, 48–72 h; TYMC on Sabouraud dextrose agar at 20–25 °C for 5–7 days; plates expected to give 30–300 colonies; preparatory testing must show more than 70% recovery of spiked organisms. ⟨61⟩ TAMC at 30–35 °C for 3–5 days; TYMC at 20–25 °C for 5–7 days; counts from plates below 250 (TAMC) or 50 (TYMC); suitability requires recovery within a factor of 2.
• ⟨2022⟩ Absence of Salmonella, E. coli, S. aureus and Clostridium species, normally in 10 g, with a 25 g retest for doubtful results. For Clostridium, one portion is heated to 80 °C for 10 min. ⟨62⟩ Bile-tolerant Gram-negative bacteria, E. coli, Salmonella, P. aeruginosa, S. aureus, Clostridia and C. albicans.
• ⟨2023⟩ Recommended limits by material (next figure), used when the monograph does not specify limits. ⟨1111⟩ Acceptance criteria by route; for nonaqueous oral drugs TAMC 10³, TYMC 10², absence of E. coli in 1 g; a criterion of 10¹ means a maximum acceptable count of 20, 10² means 200, and so forth.

Source: USP 38–NF 33, ⟨2023⟩ Tables 2 and 3. In USP 35–NF 30 (official May 1, 2012) the boiling-water category read TAMC 10⁵, TYMC 10³ and absence of E. coli in 10 g, so the chapter has changed before; the 2025 version could not be read without a subscription. AHPA's June 2022 comparison lists the same values as USP 38 for dried botanicals, powdered extracts and supplements with botanicals.

1. Suitability first. Inoculate 25–250 cfu of challenge strains; ⟨2021⟩ requires more than 70% recovery, ⟨61⟩ a count within a factor of 2 of the control. If the botanical inhibits growth, increase the diluent volume or add inactivators such as soy lecithin 0.5% and polysorbate 20 4.0%. FDA BAM states that "there are no known methods for neutralizing the toxicity" of allspice, cinnamon, cloves and oregano, and examines them at 1:100 (cloves 1:1000). Onion and garlic are pre-enriched with 0.5% K₂SO₃.
2. Count. Suspend 10 g in diluent to 100 mL (1:10), make tenfold dilutions and plate in duplicate. Report the mean in cfu/g, or "less than 10 per g" when the 1:10 plates are blank.
3. Absence. Pre-enrich in nonselective broth, transfer to selective broth (Rappaport Vassiliadis for Salmonella; MacConkey broth at 42–44 °C for E. coli), streak selective agar and confirm typical colonies.
4. Clostridia. Heat one portion to 80 °C for 10 min, culture both portions anaerobically, and look for catalase-negative Gram-positive rods.

FDA 2017 update, Table 1 (retail, 125 g) and Table 2 (import, 750 g). Imports and retail differ significantly for all spices except basil and dehydrated garlic. Spice risk profile 2013 draft: 14 outbreaks in Canada, Denmark, France, Germany, New Zealand, Norway, Serbia, the United Kingdom and the United States; Salmonella caused 10 (87% of illnesses) and Bacillus spp. 4 (13%).

FDA Fish and Fishery Products Hazards and Controls Guidance, Appendix 4, Table A-1 (downloaded October 23, 2026). Xerophilic fungi: some germinate at 0.650–0.605, and mycelial growth limits near 0.65 were lowered to 0.640 by adjusting solutes (Stevenson et al. 2015). Drying and particle engineering covers how to measure aw and choose packaging.

A vegetative cell forms a forespore, the mother cell lyses, and the free spore can stay dormant for years. Nutrient germinants act on receptors in the inner membrane; the core releases its dipicolinic acid, the cortex is degraded, and the core takes up water. The core holds DNA saturated with small acid-soluble proteins (SASP), which protect it. A lower core water content gives more wet-heat-resistant spores. Tests exploit this: ⟨2022⟩ and ⟨62⟩ heat one portion to 80 °C for 10 min before anaerobic culture. In the spice risk profile, Bacillus spp. caused 4 of 14 spice-linked outbreaks and 13% of illnesses.

D-values in phosphate-buffered saline (Table 1); log reductions in skim milk at inlet 150, 170 and 190 °C (Table 2). Alvarenga et al. 2018.

Foster and Hall grew Salmonella Typhimurium at pH 7.6, shifted it to pH 5.8 for one doubling, then challenged it at pH 3.3. Adapted cells were 100 to 1,000 times more resistant than unadapted cells. The response needed protein synthesis (12 proteins induced and 6 repressed) and the proton-translocating ATPase. The authors reported: “No cross protection was noted for hydrogen peroxide, SOS, or heat shock.” In E. coli, the glutamate system (GadA and GadB decarboxylases with the GadC antiporter) and the arginine system (AdiA with AdiC) each consume an intracellular proton per decarboxylation and export the product in exchange for fresh substrate. Lin et al. found that once induced, these systems remain active through prolonged cold storage at 4 °C.

Stages: (1) organic matter adsorbs to the surface; (2) cells attach loosely and can leave; (3) cells attach firmly and begin making EPS; (4) a microcolony grows inside the matrix; (5) a mature biofilm forms towers separated by water channels; (6) cells disperse to colonize new sites. The matrix is mainly polysaccharides, proteins, nucleic acids and lipids. With the Calgary Biofilm Device, eradicating biofilms of reference strains often needed 100 to 1,000 times the concentration that inhibits planktonic cells, for some antibiotics. Proposed mechanisms include slow growth, an rpoS-mediated stress response and exclusion of biocides by the matrix.

Joseph et al. 2001, abstract.

• Culturable cells grow into colonies on routine media.
• Sublethally injured cells result from heat, chilling, acid or alkali; they may need repair, for example in nonselective pre-enrichment broth, before growing on selective media.
• VBNC cells arise after starvation, low temperature, light, oxidative stress or chlorination; milk pasteurization and wastewater chlorination can push a subpopulation into this state. VBNC cells keep intact membranes, respire and transcribe genes; Listeria monocytogenes kept high ATP a year after entering the state. Resuscitation stimuli include temperature upshift, nutrients and resuscitation-promoting factor (Rpf) proteins.
• Persisters are dormant, non-dividing cells that tolerate antibiotics and regrow later; they arise by a spontaneous phenotypic switch.
• Endospores are formed only by spore formers and germinate on media.
• Dead cells have compromised membranes, but their DNA can persist and still be detected by PCR.

• Plate count: counts only cells that form colonies; spores germinate on the medium; no identity.
• Enrichment culture: nonselective pre-enrichment lets injured cells repair; some VBNC cells resuscitate in rich media; confirmation names only the target organism.
• qPCR on DNA: detects DNA from living, VBNC and dead cells alike; species-specific by primer design.
• PMA–qPCR: PMA penetrates cells with compromised membranes and removes their DNA from analysis, so intact VBNC cells still score; injured cells with damaged membranes may drop out.
• Flow cytometry with live/dead stains: separates intact from damaged membranes, with intermediate states; no species identity without added probes.
• ATP bioluminescence: tracks metabolically active cells and is rapid, but extracellular ATP inflates results, dormant spores are not detected, and it has no specificity.
• MALDI-TOF MS: identifies colonies already grown, if the spectral database contains the species.
• 16S rRNA gene sequencing: identifies DNA from any cell state; resolution below genus varies.

USP–NF 2025, Issue 2, includes chapters on respiration-based ⟨72⟩ and ATP bioluminescence-based ⟨73⟩ methods for detecting contamination in short-life products, with ⟨1071⟩ as the risk-based companion (USP–NF 2025 Issue 2 commentary, February 3, 2025).

Measured: Table 2 (dynamic conditions). Model parameters: Table 3, dynamic fit, D_{70 °C} = 16.11 min at aw 0.33, z_T = 16.25 °C, z_aw = 0.38 (reference aw 0.33). The isothermal fit gives D_{70 °C} = 17.61 min, z_T = 24.03 °C and z_aw = 0.44.

• Steam. Moist heat; the wet surface raises local water activity, which lowers D. Salmonella on chili at 65 °C: D 0.72 min at aw 0.97. Spores need far more heat.
• Dry heat. At aw 0.33 the same Salmonella had D 30 min at 65 °C. USP ⟨2023⟩ notes dry heat "may adversely affect stability and degradation of the raw material".
• Irradiation. 21 CFR 179.26 permits up to 30 kGy for microbial disinfection of dry or dehydrated aromatic vegetable substances "when used as ingredients in small amounts solely for flavoring or aroma" (culinary herbs, seeds, spices, vegetable seasonings and blends; turmeric and paprika also as color additives), and up to 10 kGy for dry enzyme preparations. Retail packages carry the radura logo and "Treated with radiation" or "Treated by irradiation". Gamma irradiation of S. Montevideo in black pepper and sesame gave 3–4 logs at 3 kGy and 5.5–6 logs at 5 kGy.
• Ethylene oxide. EPA tolerances (40 CFR 180.151): 7 ppm ethylene oxide and 940 ppm 2-chloroethanol in dried herbs and spices (except basil), licorice roots, dried peppermint and spearmint tops, sesame seed and dried vegetables. EPA's January 14, 2025 Interim Decision cancels use on specific dried herbs and spices where it is not critical, phases out others where alternatives exist, and notes use on about 30% of dried herbs and spices.

Whether a botanical used in a dietary supplement fits the 179.26 condition "in small amounts solely for flavoring or aroma" is a question for regulatory counsel.