Protocol for Quantitative Estimation of Hydrogen Cyanide Production from Bacteria

Materials and reagents

Biological materials

1. Gram-positive isolates: Bacillus altitudinis, Bacillus amyloliquefaciens, and Bacillus haynesii

2. Gram-negative isolates: Pantoea agglomerans, Kosakonia cowanii, and Brucella intermedia

Reagents

1. Luria-Bertani (LB), Luria HiVeg broth, sterile powder (HiMedia, SKU: MV575G-500G)

2. Tryptone (HiMedia, SKU: CR014)

3. Yeast extract (HiMedia, SKU: RM027)

4. Sodium chloride (NaCl) (HiMedia, SKU: GRM031)

5. King’s B broth base, dehydrated, King’s Medium A Base (commonly called King’s B base) (HiMedia, SKU: M1543-500G)

6. Proteose peptone (HiMedia, SKU: RM005)

7. Glycerol (HiMedia, SKU: GRM081)

8. Potassium phosphate dibasic anhydrous (K₂HPO₄) (HiMedia, SKU: GRM3945)

9. Magnesium sulphate heptahydrate (MgSO₄·7H₂O) (HiMedia, SKU: PCT0008)

10. Glycine (cell culture–tested grade) (HiMedia, SKU: TC075-100G)

11. Picric acid, saturated, aqueous (HiMedia, SKU: S026-100ML)

12. Sodium carbonate anhydrous (Na₂CO₃) (HiMedia, SKU: MB253)

13. Potassium ferrocyanide (K₄[Fe(CN)₆]) (HiMedia, SKU: GRM1048)

14. Double-distilled water (ddH₂O)

Solutions

1. Luria-Bertani (LB) broth (see Recipes)

2. King’s B broth (supplemented with glycine) (see Recipes)

3. Alkaline picrate solution (see Recipes)

4. Potassium ferrocyanide standard solutions (see Recipes)

Recipes

1. Luria-Bertani (LB) broth

Adjust pH to 7.0. Autoclave at 121 °C for 15 min.

2. King’s B broth (supplemented with glycine)

Adjust pH to 7.0. Autoclave at 121 °C for 15 min. Add filter-sterilized glycine to a final concentration of 4.4 g/L after autoclaving. Dispense 10 mL into each sterile test tube under aseptic conditions.

3. Alkaline picrate solution

Mix both components thoroughly until fully dissolved. Store in a tightly closed amber bottle at room temperature.

4. Potassium ferrocyanide standard solutions

1. Use K₄[Fe(CN)₆] as a standard to prepare 0–50 ppm of CN^- ion equivalents.

b. Use the following molecular conversion:

422.4 g potassium ferrocyanide = 156 g of CN^-

c. Prepare standards in 10 mL of ddH₂O per tube (e.g., 0, 10, 20, 30, 40, and 50 ppm CN^- equivalents).

d. Cover immediately to prevent evaporation or contamination.

Laboratory supplies

Note: Prepare and autoclave sufficient double-distilled water (ddH₂O) and ensure availability of sterile test tubes, conical flasks, forceps, scissors, and pipettes.

1. Sterile culture tubes or conical flasks (HiMedia, catalog numbers: PW1233 and LA449

2. Sterile measuring cylinders, conical flasks, and beakers (HiMedia, catalog numbers: LA454, LA449, and LA450

3. Sterile filter paper (Whatman No. 1 or equivalent), cut into 12 × 1 cm strips (HiMedia, catalog number: MB059)

Note: For filter paper strip preparation, first cut Whatman No. 1 or equivalent filter paper into 12 × 1 cm strips, marking at 10 cm using a pencil. Sterilize strips via autoclaving (by keeping inside the autoclavable bag). Soak sterilized strips in alkaline picrate solution for 1–2 min. Let excess drip off in sterile conditions (e.g., under a laminar flow hood). Use immediately or store in sterile, sealed containers.

4. Sterile Suba seal or rubber stopper (HiMedia, catalog number: LA410)

5. 0.22 μm syringe filter (HiMedia, catalog number: ME038)

6. Parafilm, (HiMedia, catalog number: PW009)

7. Sterile forceps and scissors (HiMedia, catalog numbers: LA396 and LA394)

8. Sterile pipettes and tips (HiMedia, catalog numbers: PW1149 and MBP001–MBP003)

Equipment

1. Spectrophotometer (SHIMADZU, model: UV-3600 UV-VIS, catalog number: UV-3600-A112950)

2. Incubator (SANCO, model: B.O.D incubator JL-1028, catalog number: SAN-200)

3. Laminar airflow cabinet (JINAN BIOBASE BIOTECH, model: BBS-DDC, catalog number: BBS11V011402003D)

4. Autoclave (iGENE LABSERVE, model: LCD Vertical Autoclave, catalog number: PUR-96-LCD)

5. Orbital shaker/incubator shaker (YIHDER, model: LM-570RD, catalog number: LM-TW/2014)

6. Vortex mixer (TARSONS, model: SPINIX^TM Vortex Shaker, catalog number: 3020 SN 7903)

7. Computer (DELL, model: Intel^® Core^TM i5 system)

Procedure

A. Bacterial culture preparation

1. Inoculate the purified bacterial strain into pre-prepared LB broth and incubate overnight at 28 ± 2 °C in a shaker incubator (150–180 rpm) to allow active growth. Maintain a blank in the same conditions.

2. Dispense 10 mL of King’s B broth (with glycine) into sterile test tubes under aseptic conditions.

3. Transfer 1% (v/v) of the overnight-grown culture into each test tube containing King’s B broth and incubate until the culture reaches an OD₆₀₀ ≈ 1.0, indicating idiophase (secondary metabolite production phase).

B. Paper strip handling and setup

1. Using sterile forceps, take the alkaline picrate–soaked filter strips and hang them vertically inside each test tube, ensuring 10 cm of the strip remains inside the headspace above the bacterial broth without touching the liquid (Figure 1).

2. Seal the tubes tightly with Suba seals or rubber stoppers and further wrap the seals with Parafilm to prevent gas leakage.

3. Incubate the sealed tubes at 28 ± 2 °C for 14 days without disturbance.

Figure 1. Insertion of the strip and sealing it with Suba seals and Parafilm

C. Standard curve assay (parallel setup)

1. Place pre-treated alkaline picrate filter strips inside sterile test tubes containing 10 mL of potassium ferrocyanide standard solutions (ranging from 0 to 50 ppm CN^- equivalents).

2. Seal and incubate these tubes under the same conditions as for the test culture (28 ± 2 °C for 14 days).

3. After incubation, carefully cut each strip at the 10 cm mark using sterile scissors and place it into 10 mL of ddH₂O in a clean tube or beaker.

4. Gently shake or vortex the tubes to fully elute the color from the strip, forming the isopurpurate compound.

D. Color elution from bacterial samples

1. Repeat the same cutting and elution process (as in step C4) for strips recovered from bacterial culture tubes and the blank.

2. Ensure equal volume and shaking conditions are maintained for all samples (Figure 2).

Figure 2. Hydrogen cyanide (HCN) production assays. (A) Color development for different isolates. (B) Color elution from filter paper at different concentrations of potassium ferrocyanide. (C) Eluted color of isopurpurate.

E. Measurement and quantification

1. Measure the absorbance (OD) at 625 nm for each eluted solution using a spectrophotometer.

2. Plot a standard curve using OD values from the potassium ferrocyanide standards against known CN^- concentrations.

3. Use this curve to estimate the HCN concentration in each bacterial sample by correlating its OD value.

4. Apply the derived linear regression formula (e.g., x = y/0.0013-0.0076) to calculate precise HCN production in ppm, where x = HCN concentration and y = OD₆₂₅.

Data analysis

A. Preparation of the 50 ppm stock of CN- ion from potassium ferrocyanide

1. The molecular weight of potassium ferrocyanide trihydrate (K₄[Fe(CN)₆]·3H₂O) is approximately 422.39 g/mol.

2. Each molecule of potassium ferrocyanide contains six cyanide ions (CN^-).

3. The molar mass of one CN^- ion is 26.02 g/mol.

4. Therefore, one mole of potassium ferrocyanide provides 6 × 26.02 = 156.12 g of cyanide.

5. The proportion of cyanide in potassium ferrocyanide is 156.12/422.39 ≈ 0.3695 or 36.95%.

6. A concentration of 50 ppm corresponds to 50 mg of cyanide per liter of solution.

7. To find out how much potassium ferrocyanide is needed to provide 50 mg of cyanide, divide 50 mg by 0.3695.

8. This gives approximately 135.3 mg of potassium ferrocyanide trihydrate.

9. Therefore, dissolving 135.3 mg of potassium ferrocyanide in 1 L of water will yield a 50 ppm cyanide (CN^-) solution.

B. Quantification of the CN- ion

The quantitative estimation of hydrogen cyanide (HCN) is based on the relationship between optical density (OD) and cyanide concentration derived from a standard curve using potassium ferrocyanide (Figure 3). The standard curve equation obtained from linear regression is:

y = 0.0013x + 0.0076

Where:

y = measured absorbance at 625 nm

x = concentration of HCN (in ppm)

C. Calculation of HCN concentration

To determine the HCN concentration in a bacterial culture:

1. Measure the absorbance in terms of OD of the eluted isopurpurate compound from the filter strip at 625 nm.

2. Use the standard curve equation and rearrange it to solve for x:

x = y-0.00760/0013

Example calculation:

If the observed OD is 0.073, the calculation becomes:

x = 0.073-0.0076/0.0013

x = 0.0654/0.0013 = 50.31 ppm

The sample contains approximately 50.31 ppm of HCN.

Note: Perform all measurements in triplicate to ensure reliability. Calculate the mean ± standard deviation for each isolate.

Validation of protocol

1. The absorbance values were compared with the standard curve of potassium ferrocyanide. The calculation of HCN concentration is based on the dissociation of potassium ferrocyanide. It dissociates into four K⁺ ions and one [Fe (CN)₆]^-4 ion, which further dissociates into six CN- ions in an aqueous condition [7].

Reaction equation:

[ F e ( C N ) 6 ] K 4 · 3 H 2 O → [ F e ( C N ) 6 ] - 4 + 4 K +

[ F e ( C N ) 6 ] - 4 ⇔ H + F e + 2 + 6 C N - → H + 6 H C N

2. It was observed that two-thirds of the cyanide ions were involved in the formation of isopurpurate, while one-third went into the formation of carbonate ions [8]. This can be represented by the equation:

C 6 H 3 N 3 O 7 + 2 H C N + N a 2 C O 3 → C 8 H 2 O 6 N a N 5 + H 2 C O 3 + N a O H

Therefore, taking OD without prior calculation of CN- ion from the standard compound would not show the real value of produced HCN.

3. The OD value obtained from the quantitative assay represents the amount of isopurpurate compound formed, which was directly related to the concentration of CN- ion calculated from potassium ferrocyanide, which represented the value of HCN produced (Table 1, Supplementary Table S1).

Figure 3. Standard curve of hydrogen cyanide (HCN) produced from potassium ferrocyanide

3. The above-mentioned methodology is the next advancement in the old traditional method to quantify HCN production by bacterial isolates. This method includes the colorimetric test for the eluted color of the isopurpurate compound in double-distilled water. Through the standard curve of potassium ferrocyanide, we can get the exact concentration of cyanide for different intensities of color development. The obtained OD of eluted color with respect to the isolates can give a quantitative value of their HCN-producing ability by applying the formula x = y/0.0013 – 0.0076, where x is the concentration of the HCN, and y is the OD.

4. As HCN is a volatile compound [9], it is critical to check whether the seal was airtight or not. Any minor leakage may alter the results. For safety, it is advised to use Parafilm tape even after tightening with the cork. As in the protocol, the airtight tube may hinder the growth of aerobic bacterial isolates. To overcome this, a pre-grown culture at OD = 1 is pre-requested to take for analysis. Being a secondary metabolite, HCN production will start to be produced in the idiophase of cell growth [10], so we can give it enough time for incubation. It has been observed that as the days pass, the color of the strip starts to fade, so it is recommended to take the reading at 14 days, as this is the optimum incubation time, as proved by various trials [12,13].

5. The poor dissociation constant of potassium ferrocyanide makes this compound nearly ideal for estimating HCN production from bacterial isolates, as the slow release of the cyanide ions takes a proper incubation period. Other strong cyanide salts like KCN may cause errors in the value because of the strong dissociation constant and may release some HCN before capping of a tube. In this experiment, the dimensions of the strips were taken very precisely to avoid any error and allow equal surface area to produce the isopurpurate compound.

Table 1. Quantitative value of HCN production of different isolates

General notes and troubleshooting

1. Ensure the bacterial culture reaches OD₆₀₀ ≈ 1.0 before inserting the picrate strip to allow HCN production during the idiophase.

2. Lack of color change may indicate non-HCN-producing strains or inadequate incubation time; verify using a positive control.

3. Always suspend the picrate strip (not less than 10 cm) above the broth surface without contact to prevent smearing or underexposure.

4. Incomplete sealing with Suba seal or Parafilm may lead to HCN gas escape and false negative results.

5. Uneven or faint color development can occur due to poor strip soaking or unequal strip dimensions; maintain consistent strip preparation.

6. Elute the isopurpurate compound only after a full 14-day incubation; premature elution can cause underestimation.

7. Shake elution tubes gently but thoroughly; insufficient mixing can cause incomplete color release and variability in OD readings.

8. Use sterile, autoclaved filter paper and aseptic techniques during strip preparation and handling to avoid contamination.

9. Always calibrate the spectrophotometer before measurement and ensure cuvettes are clean and aligned correctly.

10. Prepare standard solutions freshly using potassium ferrocyanide and incubate them in identical conditions as the test samples.

11. Avoid using cyanide salts like KCN, which dissociate too quickly and can lead to inaccurate standard curves.

12. Store picric acid only in aqueous form and in tightly sealed amber bottles to prevent drying and explosion hazard.

13. Perform all steps involving picric acid and potential HCN exposure under a fume biosafety cabinet.

14. Use triplicate measurements for each sample and include both positive and negative controls in every experiment.

15. Dispose of used strips and bacterial culture broth waste according to hazardous chemical waste disposal protocols.

Supplementary information

The following supporting information can be downloaded here:

1. Table S1: HCN production by bacterial isolates

Acknowledgments

Contributions of each author: Conceptualization was done by A.S. Investigation and writing of the original draft was done by D.P. Writing–Review & Editing was done by P.S., A.S., and V.G. There are no funding sources that supported the work. This work has been modified from a previously published article by Marag et al. (2018). We acknowledge ICAR-IARI and Dr RPCAU for providing scholarships and scientific support to handle the work, respectively.

Competing interests

The authors declare no conflicts of interest.

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