The Impact of Exposure Time and Fumigation Intensity of Dhoopa on Indoor Microbial Load

The environment is home to billions of microorganisms, and exposure through inhalation of contaminated air increases susceptibility to airborne diseases. Microorganisms such as bacteria, fungi, viruses, and parasites are responsible for transmitting a wide range of illnesses, posing a serious threat to human health ^[1]. The presence of these pathogens in ambient air highlights the need for effective methods of disinfection and sterilization, which remain a major concern ^[2]. In Indian mythology, Havana and Dhoopa fumigation practices have long been performed to reduce microbial loads to safer levels and prevent disease. However, modern synthetic sterilization methods, such as formalin gas fumigation, can cause occupational hazards and even potential carcinogenic effects in exposed groups ^[3]. This has created an urgent need for safer alternatives in infection control. Ayurveda describes Dhoopana Karma as an age-old practice where fumigation with medicinal plant-based materials is used for environmental purification ^[4]. Historically, fumigation has been incorporated into rituals like Homa and Havana, in which herbal substances are combusted, and the generated smoke helps disinfect vulnerable spaces ^[5,6,7]. While many herbal agents possess intrinsic antimicrobial activity, their combination is thought to enhance efficacy through synergistic effects ^[8].Two critical but underexplored factors in fumigation are f migation intensity and exposure time. Fumigation intensity refers to the concentration or dose of herbal material released per unit volume of air, while exposure time is the duration microbes remain in contact with the fumigant ^[9,10]. Research on chemical fumigants shows that microbial inactivation generally follows a dose–time pattern: higher doses and longer exposures increase microbial kill rates, though safety limits must be considered. Similar quantitative studies are lacking for herbal fumigants like Dhoopa. Evidence from chemical fumigants such as ethyl formate and formic acid further emphasizes the importance of studying both concentration and duration ^[11]. Addressing this gap, the present study investigates how varying Dhoopa intensity (g·ft?³) and exposure time affect bacterial and fungal reduction in indoor air. Fumigation experiments were performed in a standardized 1000-cubic-feet enclosed space using an electric burner.^[12].  For this purpose, two herbal formulations, DF-I and DF-II, were developed using cow’s ghee, camphor, and plant-derived materials including neem (Azadirachta indica), drumstick (Moringa oleifera), mustard seeds (Brassica juncea), and guggul resin (Commiphora wightii). These ingredients, long recognized in Ayurveda for their aromatic and antimicrobial properties, were combined to create a natural, safe, and cost-effective air-sanitizing alternative to chemical fumigants ^[13]. This study evaluates the effectiveness of the formulations by analyzing microbial load reduction under varying fumigation intensities and exposure times, providing scientific evidence for their potential as natural air sanitizers

2. MATERIALS AND METHODS

2.1 Collection and Certification of Plant Materials

Plant materials, such as Neem leaves and bark (Azadirachta indica) and Drumstick leaves and bark (Moringa oleifera) were collected from a medicinal garden at Jiwaji University campus. The identification of the plant materials was confirmed by a taxonomist from the CSIR–National Institute of Science Communication. Following identification, the plant materials were screened for quality before being used for preparation of the herbal Dhoopa. Cow's ghee, guggul, camphor, and mustard yellow seeds were also collected from the local market as additional materials.

2.2 Preparation of Dhoopa formulations

Dhoopa formulations were prepared using leaves and bark of Drumstick (Moringa oleifera), leaves and bark of Neem (Azadirachta indica), Guggul (Commiphora wightii), mustard yellow seeds (Brassica juncea), camphor (Cinnamomum camphora), and cow’s ghee. Initially, the plant materials were cleaned with running tap water, dried in the shade, and ground into a moderately coarse powder, which was then passed through a 60-mesh sieve. All ingredients were taken in equal proportions and thoroughly mixed. The formulation details are provided in Table 1 and Fig. 1.

Table 1 Ingredients Dhoopa Formulations (DF-I and DF-II)

Fig. 1 Ingredients of DF-I and DF-II

2.3 Fumigation Method

A room of 1000 cubic feet was fumigated by burning varying concentrations of Dhoopa (2 g, 4 g, 6 g, 8 g, 10 g, and 12 g) using an electric burner, after which the room was sealed. Air sampling was carried out before fumigation and subsequently at 1-hour intervals up to the 5th hour. To assess the effect of exposure time, 10 g of each Dhoopa formulation (DF-I and DF-II) was applied separately in the same room, with exposure durations ranging from 1 to 10 hrs., as shown in Fig. 2.

Fig. 2 Fumigation with Electric Burner

2.4 Media Preparation

Nutrient Agar (NA, 28 g/1000 mL) and Sabouraud Dextrose Agar (SDA, 65 g/1000 mL; Hi-Media) were prepared by dissolving the respective media in distilled water, followed by complete heating. Media were autoclaved for 15 minutes at 121°C to maintain 15 psi pressure, cooled to 45–50°C, and aseptically poured into sterile Petri dishes under a laminar air flow cabinet Fig. 3.

Fig. 3 Petri plates Preparation

2.5 Collection of Air Samples Before and After Fumigation

Nutrient agar (NA) and Sabouraud dextrose agar (SDA) media were prepared for quantitative analysis of bacterial and fungal load, respectively. Fungal and bacterial samples were collected from the center of the room, placed on agar plates, followed by the passive settle plate technique at the time intervals. Both the pre- and post-fumigation sampling followed the standard method of the 1/1/1 schedule (Petri dishes left open to the air for 1 hr., 1 meter above the floor, and 1 meter from the wall/any obstacle) to obtain the appropriate surface density for counting the load for the time of exposure. Following exposure, collected samples were sealed with Para film, incubated in an upside-down position NA plates at 37°C for 24 hours for bacterial enumeration, and SDA plates at 25°C for 2 days for fungal enumeration^[14].

2.6 Assessment of Microbial Load

Air sampling was performed before fumigation and at hourly intervals post-fumigation. After incubation, bacterial and fungal colonies were enumerated as colony-forming units (CFU). The microbial load in indoor air (CFU/m³) was calculated for both pre- and post-fumigation samples using Omeliansky’s equation ^[15].

N=5aX104bt-1

Where N = Indoor air microbes in CFU/m³, a = Colonies number per Petri dish, b = surface area of dish (cm²), and t = Time of exposure (minutes).

3. Statistical Analysis

Statistical analysis of the intensity of fumigation (concentration of Dhoopa) and the exposure time of the Dhoopa Formulations (DF-I and DF-II) to evaluate percentage of Fumigation Effect or Mean Reduction of bacterial and fungal colony count CFU/m³ during different set of experiments with time intervals was done by One-Way ANOVA with post hoc analysis by Dunnett’s test to compare the mean of all controls to the means of all treatments with time intervals through GraphPad Prism Version 8.0.2 (263) and MS Excel 2019.

4. RESULTS

4.1 Impact of Fumigation Intensity (Conc.) of Dhoopa on indoor Bacterial and Fungal load

The fumigation intensity of Dhoopa was evaluated through Fumigation at different concentrations (2 g, 4 g, 6 g, 8 g, 10 g, and 12 g) in a 1000 cubic feet area of room, demonstrating a significant reduction statistically (P < 0.001) in both bacterial and fungal colony counts (CFU/m³). Pre-fumigation controls showed a mean bacterial colony count of 428.0±19.0 CFU/m³ and a fungal colony count of 192.1±11.6 CFU/m³. Following Dhoopa treatment, bacterial counts progressively decreased to 161.6±15.7 (1^st hr.), 96.1±11.6 (2^nd hr.), 52.4±7.6 (3^rd hr.), 34.9±4.4CFU/m³ (4^th hr.), and 65.5±7.6CFU/m³ (5^th hr.), representing the highest reduction at 4^th hr. (fumigation effect 92.26%). Similarly, fungal counts declined to 109.2±4.4 (1^st hr.), 78.6±7.6 (2^nd hr.), 39.3±7.6 (3^rd hr.), 8.7±4.4CFU/m³ (4^th hr.), and 17.5±8.6CFU/m³ (5^th hr.), achieving the highest reduction at 4^th hr. with 10g of Dhoopa concentration (fumigation effect 95.00%) as shown in Table 2, Table 3, and Fig.4.

Table 2 Impact of Fumigation Intensity (Conc.) of Dhoopa on Bacterial load

Table 3 Impact of Fumigation Intensity (Conc.) of Dhoopa on Fungal load

Fig. 4 Intensity of Dhoopa through Fumigation at different concentrations (2 g, 4 g, 6 g, 8 g, 10 g, and 12 g) in 1000 cubic feet area of room, demonstrating a Reduction of Mean Viable Bacterial and Fungal Colony Count CFU/m³ and Fumigation Effect of Dhoopa, Values are expressed as Mean ± SEM; *p<0.05; **p<0.002; ***p< 0.001, Dhoopa fumigation effect significant from 1^sthr. to 5^thhr. by repeated measures, One-way ANOVA followed by Dunnett’s multiple comparisons test.

4.2 Impact of Exposure Time (1 to 10 hours) of Dhoopa Formulation-I (DF-I) on Bacterial Load

The Impact of Exposure Time of Dhoopa Formulation-I (DF-I) was evaluated through Fumigation with different Time Intervals (1 to 10 hrs.) in a 1000 cubic feet area of room, demonstrating a significant reduction statistically (P < 0.001) in both bacterial and fungal colony counts (CFU/m³). Pre-fumigation controls showed a mean bacterial colony count of 1782.0±30.3 CFU/m³ and a fungal colony count of 91.7±7.6 CFU/m³. Following Dhoopa treatment, bacterial counts progressively decreased to 611.4±19.0 (1^st hr.), 476.1±26.6 (2^nd hr.), 310.1±43.0 (3^rd hr.), 65.5±7.6 CFU/m³ (4^th hr.),100.4±11.6 (5^th hr.), 139.7±15.7 (6^th hr.), 179.0±11.6 (7^th hr.), 196.5±7.6 (8^th hr.), 214.0±15.7 (9^th hr.) and, 231.5±11.6 (10^th hr.) CFU/m³. Representing the highest reduction at 4^th hr. (fumigation effect 96.30%). Similarly, fungal counts declined to 61.1±4.4 (1^st hr.), 30.6±4.4 (2^nd hr.), 13.1±0.0 (3^rd hr.), 4.4±4.4 CFU/m³ (4^th hr.), 8.7±14.4 CFU/m³ (5^th hr.), 13.1±0.0 CFU/m³ (6^th hr.), 13.1±0.0 CFU/m³ (7^th hr.), 17.5±4.4 CFU/m³ (8^th hr.), 17.5±4.4 CFU/m³ (9^th hr.) and 17.5±4.4 CFU/m³ (10^th hr.) achieving the highest reduction at 4^th hr. (fumigation effect 95.20%). as shown in Table 4, Fig.5 Graph (A) & (B), Fig.7.

Table 4 Impact of Exposure Time (1 to 10 hours) of DF-I on Bacterial and Fungal Load

Fig. 5 The Impact of Exposure Time of Dhoopa Formulation-I (DF-I) through Fumigation at different Time Intervals (1to10 hrs.) in 1000 cubic feet area of room, demonstrating a Reduction of Mean Viable Bacterial and Fungal Colony Count CFU/m³ and Dhoopa Formulation-I (DF-I), Values are expressed as Mean ± SEM; *p<0.05; **p<0.002; ***p< 0.001, Dhoopa fumigation effect significant from 1^sthr. to 10^thhr. by repeated measures, One-way ANOVA followed by Dunnett’s multiple comparisons test.

4.3 Impact of Exposure Time (1 to 10 hours) of Dhoopa Formulation-II (DF-II) on Bacterial Load

The Impact of Exposure Time of Dhoopa Formulation-II (DF-II) was evaluated through Fumigation with different Time Intervals (1hr. to 10hr.) in a 1000 cubic feet area of room, demonstrating a significant reduction statistically (P < 0.001) in both bacterial and fungal colony counts (CFU/m³). Pre-fumigation controls showed a mean bacterial colony count of 1127±30.3 CFU/m³ and a fungal colony count of 104.8±7.6 CFU/m³.Following Dhoopa treatment, bacterial counts progressively decreased to 436.8±34.1 (1^st hr.), 314.5±15.1 (2^nd hr.), 209.6±27.3 (3^rd hr.), 78.6±7.6 CFU/m³ (4^th hr.), 144.1±7.6 (5^th hr.), 174.7±4.4 (6^th hr.), 240.2±11.6 (7^th hr.), 288.3±15.1 (8^th hr.), 371.3±24.3 (9^th hr.) and, 432.4±27.3 (10^th hr.) CFU/m³ representing the highest reduction at 4^th hr. (fumigation effect 93.00%). Similarly, fungal counts declined to 69.87±15.7 (1^st hr.), 39.3±7.6 (2^nd hr.), 26.2±7.6 (3^rd hr.), 4.4±4.4 CFU/m³ (4^th hr.), 8.7±14.4 CFU/m³ (5^th hr.), 13.1±0.0 CFU/m³ (6^th hr.), 17.47±4.4 CFU/m³ (7^th hr.), 17.5±4.4 CFU/m³ (8^th hr.), 17.5±4.4 CFU/m³ (9^th hr.) and 30.6±4.4 CFU/m³ (10^th hr.) achieving the highest reduction at 4^th hr. (fumigation effect 95.83%). as shown in Table 5, Fig.6 Graph (A) & (B), Fig.7.

Table 5 Impact of Exposure Time (1 to 10 hours) of DF-II on Bacterial and Fungal Load

Fig. 6 The Impact of Exposure Time of Dhoopa Formulation-II (DF-II) through Fumigation at different Time Intervals (1to10 hrs.) in 1000 cubic feet area of room, demonstrating a Reduction of Mean Viable Bacterial and Fungal Colony Count CFU/m³ and Dhoopa Formulation-II (DF-II), Values are expressed as Mean ± SEM; *p<0.05; **p<0.002; ***p< 0.001, Dhoopa fumigation effect significant from 1^st to 10^thhr. by repeated measures, One-way ANOVA followed by Dunnett’s multiple comparisons test

Fig.7 Antibacterial and Antifungal efficacy of Dhoopa Formulations (DF-I & DF-II) on Nutrient Agar (NA) & Sabouraud Dextrose Agar (SDA) Plates with Time Intervals

4. DISCUSSION